Encapsulated microbial compositions and methods of making same

Abstract

For example, encapsulated microbial compositions with increased stability that can be applied to plant seeds to promote growth and / or provide pest control are described, as well as methods for preparing such encapsulated microbial compositions with increased stability. The methods can include combining a microorganism with at least one hydrogel, such as an alginate, to form a precursor mixture, solidifying the precursor mixture with a crosslinker comprising a divalent cation, a divalent cation salt, or a combination thereof to form an intermediate microbial composition, and drying the intermediate microbial composition to form an encapsulated microbial composition.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 172,898, filed April 9, 2021, the disclosure of which is incorporated herein by reference in its entirety. Technical Field The present disclosure relates generally to encapsulated microbial compositions having increased stability for applications such as plant seed treatments. The present disclosure further relates generally to methods of preparing such encapsulated microbial compositions having increased stability, and methods of applying the encapsulated microbial compositions to plant seeds. [Background technology]

[0002] background This section provides background information related to the present disclosure that is not necessarily prior art.

[0003] The microbiota surrounding plants is highly diverse, including bacteria, fungi, yeasts, and algae. The microbes (also called microorganisms) that make up the plant microbiome originate from a variety of sources, including seeds, plants, soil, pollinators and other animals, and the environment. Some of these microbes are beneficial to their plant hosts and can promote plant growth and crop productivity, while others are disease-causing agents, often referred to as pathogens. There is growing interest in the use of microbial agents in agriculture, horticulture, forestry, and environmental management to improve plant traits. Many microbes known to reside in the zone of soil surrounding the roots of plants, commonly known as the rhizosphere and root plane, have been noted for their ability to promote plant growth. Furthermore, it has been shown that microbes can be used as biological control agents to increase agricultural productivity and efficiency, for example, by combating plant pathogens and / or improving plant growth. Some microorganisms can enhance the adaptive capacity of their hosts through many mechanisms, such as the fixation of molecular nitrogen, the mobilization of soil nutrients (e.g., iron, phosphorus, sulfur, etc.), the synthesis of plant hormones and vitamins, and the decomposition of plant material in soil, which can increase soil organic matter. By inoculating plant seeds and / or soil with certain beneficial microorganisms, plants can be provided with important nutrients or beneficial factors that control pests and / or enhance their growth and development. However, microorganisms typically need to remain viable to positively affect plant health and traits, and may not be stable in storage or in the environment, or on plants or plant seeds. Summary of the Invention

[0004] Therefore, there is a need for improved methods and compositions that include such beneficial microorganisms, which can be incorporated, for example, into seed treatment formulations, coatings and / or biofertilizers or other agricultural applications, such as for long-term storage and later use. However, preserving the viability, structure and function of microorganisms during storage, transportation and application to plants or plant seeds, especially at various temperatures and humidities, has proven difficult. Although methods and compositions have been developed for stabilizing biological materials, there is still a need for improved compositions and methods for formulating microorganisms, such as bacteria or fungi, for agricultural applications. Indeed, many non-sporulating bacterial and fungal strains are particularly sensitive to storage and formulation conditions and may not remain viable during storage and agricultural applications using existing formulation approaches and strategies. Therefore, there is a further need for methods to produce more stable compositions for storage of a wide range of biological materials over long periods of time at various temperatures and humidities, such as those encountered during transportation and storage of biological materials. In another aspect, there is a need for a microorganism-containing composition that remains stable and active before and after application to plants, plant parts or seeds. overview

[0005] This section provides a general overview of the disclosure, but is not an exhaustive disclosure of its entire scope or all of its features.

[0006] Novel and useful encapsulated microbial compositions and methods for their production are set forth in the appended claims. Exemplary embodiments are also provided to enable one of ordinary skill in the art to make and use the claimed subject matter.

[0007] In one aspect, a method for preparing an encapsulated microbial composition is described. The method generally includes combining a microorganism with at least one hydrogel to form a precursor mixture, solidifying the precursor mixture with a crosslinker to form an intermediate microbial composition, and drying the intermediate microbial composition to form an encapsulated microbial composition. The encapsulated microbial composition can have a moisture content of about 10% or less, or about 5% or less. The at least one hydrogel generally includes an alginate salt, and the crosslinker generally includes a divalent cation, a divalent cation salt, or a combination thereof. Drying is performed at a drying temperature of about 15° C. or greater and at a drying speed of about 25,000 g / hr / m 2 The following evaporation rates can be achieved:

[0008] In another aspect, a method for preparing an encapsulated microbial composition is described. The method generally includes combining a microorganism with at least one hydrogel to form a precursor mixture, solidifying the precursor mixture with a crosslinker to form an intermediate microbial composition, and drying the intermediate microbial composition to form an encapsulated microbial composition. The at least one hydrogel generally includes an alginate salt, and the crosslinker generally includes a divalent cation, a divalent cation salt, or a combination thereof. The encapsulated microbial composition can have a tap density of about 0.5 g / mL or more to about 1.5 g / mL or less.

[0009] The alginate may be selected from the group consisting of sodium alginate, potassium alginate, barium alginate, calcium alginate, magnesium alginate, strontium alginate, and combinations thereof.

[0010] The at least one hydrogel may further comprise carboxymethylcellulose, carrageenan, cellulose acetate phthalate, chitosan and chitosan derivatives, collagen, gelatin, glycosaminoglycans, guar gum, acacia gum, hyaluronic acid, hydroxyethylcellulose, hydroxypropylmethylcellulose, locust bean gum, methylcellulose, pectin, polyethylene oxide, polyglycolic acid, polyurethane, starch, modified starch, xanthan gum, or combinations thereof.

[0011] The at least one hydrogel may be present in the precursor mixture in an amount of about 5% to about 50% by weight, based on the total weight of the precursor mixture.

[0012] The precursor mixture may include a nitrogen-containing stabilizer, a sugar-containing stabilizer, or a combination thereof. The nitrogen-containing stabilizer includes ammonia, ammonium hydroxide, urea, ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, gluten, hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, malt extract, milk powder, whey powder, yeast extract, and combinations thereof. The sugar-containing stabilizer includes, for example, a disaccharide, a sugar alcohol, or a combination thereof. For example, the sugar-containing stabilizer may be selected from the group consisting of mannitol, sorbitol, xylitol, trehalose, sucrose, lactose, and combinations thereof.

[0013] The nitrogen-containing stabilizer and the sugar-containing stabilizer may each be present in the precursor mixture in an amount of about 5% to about 50% by weight, based on the total weight of the precursor mixture.

[0014] The microorganism may be a non-spore-forming bacterium. For example, the microorganism may be a Pseudomonas (e.g., Pseudomonas fluorescens), Bradyrhizobium (e.g., Bradyrhizobium japonicum), Herbaspirillum (e.g., Herbaspirillum frisingense, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, etc.), Phytobacter (e.g., Phytobacter diazotrophicus, Phytobacter ursingii, etc.), or a Phytobacter species (e.g., Phytobacter purpurea ... ursingii, Phytobacter palmae, etc.), Pseudacidovorax (e.g., Pseudacidovorax intermedius), Mitsuaria, Azospirillum, Burkholderia, Chryseomonas, Aeromonas, Acinetobacter, Stenotrophomonas, Chromobacterium, Agrobacterium, Chryseobacterium, Xenorhabdus, Photorhabdus, and combinations thereof.

[0015] The precursor mixture may further include a filler such as starch, cellulose, biochar, pumice, silica, clay, zeolite, perlite, or combinations thereof.

[0016] The filler may be present in the precursor mixture in an amount of about 0.1% to about 90% by weight, based on the total weight of the precursor mixture.

[0017] The divalent cation may be a barium cation, a calcium cation, a magnesium cation, a strontium cation, a cobalt cation, a nickel cation, a zinc cation, a manganese cation, or a combination thereof. The divalent cation salt may be a barium salt, a calcium salt, a magnesium salt, a strontium salt, a cobalt salt, a zinc salt, a manganese salt, or a combination thereof.

[0018] The crosslinker may further comprise a solvent.

[0019] Solidifying may include contacting droplets of the precursor mixture with a cross-linking agent to form the intermediate microbial composition in the form of cross-linked beads having an average particle size of, for example, about 1 mm to about 20 mm.

[0020] Additionally or alternatively, solidifying may include flowing the precursor mixture through an opening having a diameter of, for example, about 100 μm to about 500 μm, and contacting the precursor mixture with a cross-linking agent to form an intermediate microbial composition in the form of cross-linked microbeads having an average particle size of, for example, about 100 μm to about 20 mm.

[0021] Divalent cations may be present in an amount of about 0.1% to 10% of the total dry weight of the encapsulated microbial composition.

[0022] Additionally or alternatively, solidifying can include high shear mixing of the precursor mixture with the crosslinker. For example, the high shear mixing can be carried out at a tip speed of at least about 3.5 m / s (e.g., from about 3.5 m / s to about 25 m / s) for at least about 5 seconds (e.g., from about 5 seconds to about 20 minutes) and at a temperature of from about 4° C. to about 30° C.

[0023] Additionally or alternatively, solidification may include combining the precursor mixture with a crosslinker to form an intermediate microbial composition, where the crosslinker comprises a divalent cation salt, the divalent cation salt comprising a divalent cation carbonate, such as barium carbonate, calcium carbonate, magnesium carbonate, strontium carbonate, cobalt carbonate, nickel carbonate, zinc carbonate, manganese carbonate, or combinations thereof, particularly calcium carbonate. The divalent cation may be present in an amount corresponding to about 0.001% to about 25% of the total dry weight of the encapsulated microbial composition.

[0024] The solidifying may further include adding an acidic buffer. For example, the acidic buffer may be added (i) to the precursor mixture and / or the crosslinker before the crosslinker is combined with the precursor mixture; (ii) to the precursor mixture substantially simultaneously with the crosslinker; (iii) to a mixture including the precursor mixture and the crosslinker; or to any combination of (i), (ii), and (iii). The mixture may have a pH of about 4 to about 6.5.

[0025] The combining step can be carried out for about 5 minutes or more (eg, from about 5 minutes to about 48 hours) and / or at a temperature of about 4° C. or more (eg, from about 4° C. to about 40° C.).

[0026] The combining step may include mixing the precursor mixture with a cross-linking agent.

[0027] The method may further include applying the intermediate microbial composition to a substrate, for example, the intermediate microbial composition is in the form of a film on the substrate. The film can have a thickness of about 1 mm to about 1 cm.

[0028] In this method, the drying temperature may be from about 15° C. to about 60° C., and / or the drying time may be from about 15 minutes or more (eg, from about 15 minutes to about 200 hours).

[0029] The evaporation rate is approximately 15,000g / hr / m 2 Below, approximately 10,000g / hr / m 2Below, approximately 5,000g / hr / m 2 Less than or equal to 1g / hr / m 2 ~About 500g / hr / m 2 , about 50g / hr / m 2 ~About 500g / hr / m 2 , about 1g / hr / m 2 ~About 250g / hr / m 2 Or about 1g / hr / m to about 100g / hr / m 2 may be also possible.

[0030] The method may further include processing the encapsulated microbial composition to form encapsulated microbial particles and / or reducing the size of the encapsulated microbial particles, e.g., by grinding the encapsulated microbial particles, to have an average particle size of about 200 μm or less.

[0031] The encapsulated microbial composition may have less than a 2 log loss or less than a 1 log loss in colony forming units per gram (CFU / g) after 4 weeks, 8 weeks, or 12 weeks at 25° C. and 65% relative humidity (RH).

[0032] The method may further comprise applying the encapsulated microbial composition to one or more of: a plant seed, a plant, a plant part, and a plant environment. The plant seed may further comprise a protectant, a herbicide, a pesticide, a fungicide, or a combination thereof. The plant seed may include, for example, a vegetable seed, a fruit seed, a grain seed, and a combination thereof.

[0033] The method may further include combining at least one hydrophobic additive with the microorganism and the at least one hydrogel to form a precursor mixture.

[0034] The method may further include drying the intermediate microbial composition by one or more of air drying the intermediate microbial composition, vacuum drying the intermediate microbial composition, and / or drying the intermediate microbial composition in a fluidized bed to form an encapsulated microbial composition.

[0035] In a further aspect, another method for preparing an encapsulated microbial composition is described. The method generally includes combining a microorganism with at least one hydrogel to form a precursor mixture, solidifying the precursor mixture with a crosslinking agent to form an intermediate microbial composition in the form of crosslinked beads, and drying the intermediate microbial composition to form an encapsulated microbial composition. Solidifying generally includes contacting droplets of the precursor mixture with a crosslinking agent. The encapsulated microbial composition can have a moisture content of about 10% or less. The at least one hydrogel generally includes an alginate, and the crosslinking agent generally includes a divalent cation, a divalent cation salt, or a combination thereof.

[0036] In a further aspect, another method for preparing an encapsulated microbial composition is described. The method generally includes combining a microorganism with at least one hydrogel to form a precursor mixture, solidifying the precursor mixture with a crosslinking agent to form an intermediate microbial composition in the form of crosslinked microbeads, and drying the intermediate microbial composition to form an encapsulated microbial composition. The solidifying generally includes flowing the precursor mixture through an opening having a diameter of about 100 μm to about 500 μm to contact the precursor mixture with the crosslinking agent. The encapsulated microbial composition can have a moisture content of about 10% or less. The at least one hydrogel generally includes an alginate, and the crosslinking agent generally includes a divalent cation, a divalent cation salt, or a combination thereof.

[0037] In a further aspect, another method for preparing an encapsulated microbial composition is described. The method generally includes combining a microorganism with at least one hydrogel to form a precursor mixture, solidifying the precursor mixture with a crosslinker to form an intermediate microbial composition, and drying the intermediate microbial composition to form an encapsulated microbial composition. Solidifying generally includes high shear mixing of the precursor mixture with a crosslinker. The encapsulated microbial composition can have a moisture content of about 10% or less. The at least one hydrogel generally includes an alginate, and the crosslinker generally includes a divalent cation, a divalent cation salt, or a combination thereof.

[0038] In a further aspect, another method for preparing an encapsulated microbial composition is described. The method generally includes combining a microorganism with at least one hydrogel comprising an alginate to form a precursor mixture, solidifying the precursor mixture with a crosslinking agent comprising a divalent cation salt to form an intermediate microbial composition, and drying the intermediate microbial composition to form an encapsulated microbial composition. The solidifying generally includes combining the precursor mixture with a crosslinking agent, and the divalent cation salt can be a divalent cation carbonate. The encapsulated microbial composition can have a moisture content of about 10% or less.

[0039] In another embodiment, an encapsulated microbial composition is described. The encapsulated microbial composition generally comprises a microorganism embedded in a polymer matrix comprising at least one hydrogel bound together with a divalent cation. The at least one hydrogel generally comprises an alginate. The encapsulated microbial particle composition can have a moisture content of about 10% or less, or about 5% or less.

[0040] In another embodiment, an encapsulated microbial composition is described. The encapsulated microbial composition generally includes a microorganism embedded in a polymer matrix that includes at least one hydrogel bound together with a divalent cation. The at least one hydrogel generally includes an alginate. The encapsulated microbial particle composition can have a tap density of about 0.5 g / mL or more to about 1.5 g / mL or less.

[0041] The alginate may be selected from the group consisting of sodium alginate, potassium alginate, barium alginate, calcium alginate, magnesium alginate, strontium alginate, and combinations thereof.

[0042] The at least one hydrogel may further comprise carboxymethylcellulose, carrageenan, cellulose acetate phthalate, chitosan and chitosan derivatives, collagen, gelatin, glycosaminoglycans, guar gum, acacia gum, hyaluronic acid, hydroxyethylcellulose, hydroxypropylmethylcellulose, locust bean gum, methylcellulose, pectin, polyethylene oxide, polyglycolic acid, polyurethane, starch, modified starch, xanthan gum, or combinations thereof.

[0043] The at least one hydrogel may be present in the composition in an amount of about 5% to about 50% by weight, based on the total weight of the composition.

[0044] The polymer matrix may further comprise a nitrogen-containing stabilizer, a sugar-containing stabilizer, or a combination thereof. The nitrogen-containing stabilizer includes ammonia, ammonium hydroxide, urea, ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, gluten, hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, malt extract, milk powder, whey powder, yeast extract, and combinations thereof. The sugar-containing stabilizer includes, for example, a disaccharide, a sugar alcohol, or a combination thereof. For example, the sugar-containing stabilizer can be selected from the group consisting of mannitol, sorbitol, xylitol, trehalose, sucrose, lactose, and combinations thereof.

[0045] The nitrogen-containing stabilizer and the sugar-containing stabilizer may each be present in the composition in an amount of about 5% to about 50% by weight, based on the total weight of the composition.

[0046] The microorganism may be a non-spore-forming bacterium. For example, the microorganism may be a Pseudomonas (e.g., Pseudomonas fluorescens), Bradyrhizobium (e.g., Bradyrhizobium japonicum), Herbaspirillum (e.g., Herbaspirillum frisingense, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, etc.), Phytobacter (e.g., Phytobacter diazotrophicus, Phytobacter ursingii, etc.), or a Phytobacter species (e.g., Phytobacter purpurea ... ursingii, Phytobacter palmae, etc.), Pseudacidovorax (e.g., Pseudacidovorax intermedius), Mitsuaria, Azospirillum, Burkholderia, Chryseomonas, Aeromonas, Acinetobacter, Stenotrophomonas, Chromobacterium, Agrobacterium, Chryseobacterium, Xenorhabdus, Photorhabdus, and combinations thereof.

[0047] The polymer matrix may further include a filler such as starch, cellulose, biochar, pumice, silica, clay, zeolite, perlite, or combinations thereof.

[0048] The filler may be present in the composition in an amount of about 0.1% to about 90% by weight, based on the total weight of the composition.

[0049] The divalent cations may be barium cations, calcium cations, magnesium cations, strontium cations, cobalt cations, nickel cations, zinc cations, manganese cations, or combinations thereof.

[0050] The encapsulated microbial composition may be in the form of particles having a particle size of, for example, about 300 μm or less.

[0051] The encapsulated microbial composition may have less than a 2 log loss or less than a 1 log loss in colony forming units per gram (CFU / g) after 4 weeks, 8 weeks or 12 weeks at 25° C. and 65% relative humidity (RH).

[0052] The polymer matrix may further include at least one hydrophobic additive.

[0053] In another aspect, a seed composition is described.The seed composition generally comprises a plant seed and the encapsulated microbial composition described herein present on at least a portion of the surface of the plant seed.The plant seed includes, for example, vegetable seed, fruit seed, grain seed, and combinations thereof.Further examples of plant seed include canola seed, corn seed, soybean seed, wheat seed, and combinations thereof.

[0054] The plant seed may further include a protectant, herbicide, pesticide, fungicide, or combination thereof.

[0055] The seed composition may further comprise a hydrophobic coating.

[0056] Further areas of applicability will become apparent from the description provided herein.The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS The drawings described herein are only for purposes of illustrating selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure. [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 shows the stability of alginate-encapsulated Pseudomonas fluorescens beads on seeds at 25°C and 65% relative humidity (RH); [Diagram 2] FIG. 2 shows the stability of alginate-encapsulated Pseudomonas fluorescens beads on seeds at 10°C and 50% RH; [Diagram 3] FIG. 3 shows the stability of microbial powder (seeds removed) in alginate-encapsulated Pseudomonas fluorescens beads at 25°C; [Figure 4] FIG. 4 shows the stability of alginate-encapsulated Bradyrhizobium japonicum beads on seeds at 25°C and 65% RH; [Diagram 5] FIG. 5 shows the stability of alginate-encapsulated Bradyrhizobium japonicum beads on seeds at 10°C and 50% RH; [Figure 6]FIG. 6 shows the stability on seeds of alginate-encapsulated Pseudomonas fluorescens prepared by shear mixing method at various calcium chloride concentrations at 25°C and 65% RH; [Figure 7] FIG. 7 shows the stability on seeds of alginate-encapsulated Pseudomonas fluorescens prepared by shear mixing method with added hydrogel or biochar at 25°C and 65% RH; [Figure 8] Figure 8 (FIG. 8) shows the average CFU / g after 24 and 96 h of incubation at different soil moisture concentrations; [Figure 9] FIG. 9 shows dynamic water vapor sorption (DVS) data for different cross-linked alginate preparations containing Pseudomonas fluorescens; [Figure 10] FIG. 10 shows the effect of alginate encapsulation on the stability of Herbaspirillum frisingense; [Figure 11] FIG. 11 shows the effect of alginate encapsulation on the stability of Pseudacidovorax intermedius; [Figure 12] FIG. 12 shows the dynamic water vapor sorption (DVS) data of alginate-encapsulated Phytobacter diazotrophicus containing different additives; [Figure 13] FIG. 13 shows the effect of alginate encapsulation with different additives on the stability of Phytobacter diazotrophicus; [Figure 14]FIG. 14 shows dynamic water vapor sorption (DVS) data for alginate-encapsulated Pseudomonas fluorescens with a hydrophobic coating; and [Figure 15] FIG. 15 shows dynamic water vapor sorption (DVS) data for alginate-encapsulated Bradyrhizobium japonicum with a hydrophobic coating. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS The following description of exemplary embodiments provides information to enable one skilled in the art to make and use the subject matter described in the appended claims, but may omit certain details already known in the art. Thus, the following detailed description should be interpreted as illustrative and not limiting. That said, exemplary embodiments will be more fully described with reference to the accompanying drawings.

[0059] Described herein are encapsulated microbial compositions with increased stability that can be applied to plant seeds to promote growth and / or provide pest control, and methods for preparing such encapsulated microbial compositions with increased stability. The compositions can include microorganisms, e.g., non-sporulating bacteria, embedded in a polymer matrix that includes at least one hydrogel, such as alginate, bound together with a divalent cation. The compositions can have a generally (or relatively) lower moisture content, e.g., less than about 10%; a generally (or relatively) increased tap density, e.g., about 0.5 g / mL to about 1.5 g / mL; and / or an average particle size of about 200 μm or less (including, e.g., about 150 μm or less, about 100 μm or less, etc.). Typically, encapsulated microbial compositions have been produced by a process that includes a fast drying step, such as a spray drying step. However, these conventional compositions have difficulty maintaining long-term storage stability. Surprisingly, it has been discovered that encapsulated microbial compositions with increased stability may be achieved by the methods described herein that include a slower drying step, for example, by utilizing slower evaporation rates at lower temperatures, as discussed more fully below. I. Encapsulated Microbial Compositions

[0060] Provided herein is an encapsulated microbial composition.The composition can include microorganisms embedded in a polymer matrix that includes at least one hydrogel that is bound together with divalent cations.The composition advantageously has a lower moisture content and increased stability that makes them more suitable for long-term storage, for example, for plant seed application and treatment.

[0061] In any embodiment, the encapsulated microbial composition can be formed away from (or off) the seed and stored, for example, as a powder, prior to application to the seed (e.g., as part of a seed treatment for the seed). For example, in powder form, the microbial composition can be applied as a dry powder or as a powder dispersed in a suitable solvent, for example, a water-miscible solvent such as polyethylene glycol. Alternatively, in any embodiment, the encapsulated microbial composition can be applied directly to the seed and / or formed on the seed, for example, as a treatment on the seed. A. Microorganisms

[0062] The term "microbe" refers to any species or type of microorganism, including, but not limited to, bacteria, archaea, viruses, fungi, prions, protozoa, algae, and parasites. The term "microorganism" as used herein is intended to encompass both individual microorganisms and combinations or preparations that include any number of microorganisms. The terms "microbe" and "microorganism" are used interchangeably herein. The terms "bacteria" and "bacterium" refer to unicellular prokaryotic microorganisms. The term is intended to encompass all microorganisms considered to be bacteria, including mycoplasma. All forms of bacteria are encompassed herein, including cocci, bacilli, spirochetes, spheroplasts, protoplasts, and the like. The term also includes gram-negative and gram-positive bacteria, as well as spore-forming and non-spore-forming bacteria. The term "fungus" or "fungi" refers to eukaryotic microorganisms from the kingdom Fungi, such as molds, yeasts, and mushrooms.

[0063] In any embodiment, the microorganism may be a non-sporulating bacterium, a sporulating bacterium, including vegetative cells of sporulating bacteria, a fungus, or a combination thereof. For example, in any embodiment, the microorganism herein may include vegetative cells of any bacterium, fungus, or a combination thereof. Furthermore, the microorganism may be a vegetative cell of a sporulating bacterium, in which case the sporulating bacterium has been cultured in a manner that results in a complete lack of sporulation or partial sporulation, such that the cultured cells that are formulated are all or partly vegetative cells.

[0064] Examples of bacterial microorganisms include, but are not limited to, bacteria from the following genera: Achromobacter, Acinetobacter, Aeromonas, Agrobacterium, Ampelomyces, Aureobasidium, Azospirillum, Azotobacter, Bacillus, Beauveria, Bradyrhizobium, Burkholderia, Candida, Chaetomium, Chromobacterium, Chryseobacterium, Chryseomonas, Cordyceps, Cryptococcus, Dabaryomyces, Delftia, Erwinia ia), Exophilia, Gliocladium, Herbaspirillum, Lactobacillus, Mariannaea, Microccocus, Mitsuaria, Paecilomyces, Paenibacillus, Pantoea, Photorhabdus, Phytobacter acter, Pichia, Pseudomonas, Pseudacidovorax, Rhizobium, Saccharomyces, Sporobolomyces, Stenotrophomonas, Streptomyces, Talaromyces, Trichoderma, Xenorhabdus,and combinations thereof. For example, the microorganism can be a bacterium selected from the group consisting of Pseudomonas, Bradyrhizobium, Herbaspirillum, Phytobacter, Pseudacidovorax, Mitsuaria, Azospirillum, Burkholderia, Chryseomonas, Aeromonas, Acinetobacter, Stenotrophomonas, Chromobacterium, Agrobacterium, Chryseobacterium, Xenorhabdus, Photorhabdus, and combinations thereof. ,

[0065] In any embodiment, the microorganism can be a bacterium belonging to the Pseudomonas genus, Bradyrhizobium genus, Phytobacter genus, or a combination thereof.Examples of Pseudomonas species include, but are not limited to, the Pseudomonas aeruginosa group (e.g., P. aeruginosa, P. alcaligenes, P. anguilliseptica, etc.), the Pseudomonas chloroaphis group (e.g., P. aurantiaca, P. aureofaciens, P. chlororaphis, etc.), the Pseudomonas fluorescens group (e.g., P. fluorescens ... fluorescens group (e.g., P. azotoformans, P. fluoroscens, P. migulae, etc.), Pseudomonas pertucinogena group (e.g., P. denitrificans, P. pertucinogena), Pseudomonas putida group (e.g., P. cremoricolorata, P. entomophila, P. fulva, etc.), Pseudomonas stutzeri group (e.g., P. stutzeri, P. fluores ... pertucinogena, P. pertucinogena), Pseudomonas pertucinogena group (e.g., P. pertucinogena, P. pertucinogena), Pseudomonas pertucinogena group (e.g., P. pertucinogena, P. pertucinogena), Pseudomonas pertucinogena group (e.g., P. Examples of suitable species include species of the Pseudomonas stutzeri group (e.g., P. balearica, P. luteola, P. stutzeri), Pseudomonas syringae group (e.g., P. amygdali, P. avellanae, P. caricapapayae), and combinations thereof.Examples of Bradyrhizobium species include, but are not limited to, Bradyrhizobium arachidis, Bradyrhizobium betae, Bradyrhizobium canariense, Bradyrhizobium diazoefficiens, Bradyrhizobium erythrophlei, Bradyrhizobium ferriligni, Bradyrhizobium icense, Bradyrhizobium ingae, Bradyrhizobium japonicum, and the like. Examples of Phytobacter species include, but are not limited to, species of Phytobacter diazotrophicus, Phytobacter ursingii, Phytobacter palmae, Phytobacter massilienses, and combinations thereof.Examples of Herbaspirillum species include, but are not limited to, species of Herbaspirillum frisingense, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, and combinations thereof. In some embodiments, the microorganism may be Pseudomonas fluorescens, Bradyrhizobium japonicum, Phytobacter diazotrophicus, Herbaspirillum frisingense, or combinations thereof.

[0066] Additionally or alternatively, the microorganism may be a fungus. Examples of fungi include, but are not limited to, Muscodor species, Aschersonia aleyrodis, Beauveria bassiana ("white muscarine"), Beauveria brongniartii, Chladosporium herbarum, Cordyceps clavulata, Cordyceps entomorrhiza, Cordyceps facis, Cordyceps gracilis, Cordyceps melolanthae, Cordyceps militaris, Cordyceps myrmecophila, Cordyceps spp. ... myrmecophila, Cordyceps ravenelii, Cordyceps sinensis, Cordyceps sphecocephala, Cordyceps subsessilis, Cordyceps unilateralis, Cordyceps variabilis, Cordyceps washingtonensis, Culicinomyces clavosporus, Entomophaga grylli, Entomophaga maimaiga, Entomophaga musca muscae, Entomophaga praxibulli, Entomophthora plutellae, Fusarium lateritiumlateritium, Hirsutella citriformis, Hirsutella thompsoni, Metarhizium anisopliae ("green muscarine"), Metarhizium flaviride, Muscodor albus, Neozygites floridana, Nomuraea rileyi, Paecilomyces farinosus, Paecilomyces fumosoroseus, Pandora neoaphidis, Tolypocladium cylindrosporum cylindrosporum, Verticillium lecanii, Zoophthora radicans, mycorrhizal species such as Laccaria bicolor, and combinations thereof. B. Hydrogels

[0067] In various embodiments, at least one hydrogel can comprise a hydrophilic polymer, a polysaccharide, a gum, a resin, a hydrolyzed protein, or a combination thereof. Examples of suitable hydrogels include, but are not limited to, alginates, carboxymethylcellulose, carrageenan, cellulose acetate phthalate, chitosan and chitosan derivatives, collagen, gelatin, glycosaminoglycans, guar gum, acacia gum, hyaluronic acid, hydroxyethylcellulose, hydroxypropylmethylcellulose, locust bean gum, methylcellulose, pectin, polyethylene oxide, polyglycolic acid, polyurethane, starch, modified starch, xanthan gum, or a combination thereof.

[0068] In any embodiment, at least one hydrogel can include an alginate salt, for example, the alginate salt can be selected from the group consisting of sodium alginate, potassium alginate, barium alginate, calcium alginate, magnesium alginate, strontium alginate, and combinations thereof.

[0069] In any embodiment, the at least one hydrogel can comprise an alginate as described herein, as well as an additional hydrogel selected from the group consisting of carboxymethylcellulose, carrageenan, cellulose acetate phthalate, chitosan and chitosan derivatives, collagen, gelatin, glycosaminoglycans, guar gum, acacia gum, hyaluronic acid, hydroxyethylcellulose, hydroxypropylmethylcellulose, locust bean gum, methylcellulose, pectin, polyethylene oxide, polyglycolic acid, polyurethane, starch, modified starch, xanthan gum, and combinations thereof.

[0070] The at least one hydrogel may be present in the composition in an amount of about 2.5% by weight or more, about 5% by weight or more, about 10% by weight or more, about 20% by weight or less, about 60% by weight or less, about 50% by weight or less, about 40% by weight or less, or about 30% by weight or less, or about 5% by weight to about 60% by weight, about 5% by weight to about 50% by weight, about 5% by weight to about 40% by weight, about 5% by weight to about 30% by weight, about 5% by weight and about 20% by weight, about 10% by weight to about 60% by weight, about 10% by weight to about 50% by weight, about 10% by weight to about 40% by weight, about 10% by weight to about 30% by weight, or about 10% by weight and about 20% by weight, etc. In any embodiment, the at least one hydrogel may be present in the composition in an amount of, for example, about 5% by weight to about 50% by weight, based on the total weight of the composition. C. Divalent cations

[0071] Suitable divalent cations include metal divalent cations capable of crosslinking the hydrogel. Non-limiting examples of suitable divalent cations include barium (Ba 2+) cation, calcium (Ca 2+ ) cation, magnesium (Mg 2+ ) cation, strontium (Sr 2+ ) cation, cobalt (Co 2+ ) cation, nickel (Ni 2+ ) cation, zinc (Zn 2+ In any embodiment, the divalent cation may be, for example, a calcium cation. D. Stabilizers

[0072] In any embodiment, the polymer matrix can further include one or more stabilizers. For example, the stabilizer can be a nitrogen-containing stabilizer, a sugar-containing stabilizer, or a combination thereof.

[0073] Examples of suitable nitrogen-containing stabilizers include, but are not limited to, ammonia, ammonium hydroxide, urea, ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, gluten, hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, malt extract, milk powder, whey powder, yeast extract, and combinations thereof.

[0074] Examples of suitable sugar-containing stabilizers include, but are not limited to, disaccharides, sugar alcohols, and combinations thereof. For example, the sugar-containing stabilizer may be selected from the group consisting of mannitol, sorbitol, xylitol, trehalose, sucrose, lactose, and combinations thereof.

[0075] Stabilizers, such as nitrogen-containing stabilizers and sugar-containing stabilizers, alone or in combination, may be present in the composition in an amount of about 2.5% by weight or more, about 5% by weight or more, about 10% by weight or more, about 20% by weight or more, about 60% by weight or less, about 50% by weight or less, about 40% by weight or less, or about 30% by weight or less, or about 5% by weight to about 60% by weight, about 5% by weight to about 50% by weight, about 5% by weight to about 40% by weight, about 5% by weight to about 30% by weight, about 5% by weight and about 20% by weight, about 10% by weight to about 60% by weight, about 10% by weight to about 50% by weight, 10% by weight to about 40% by weight, about 10% by weight to about 30% by weight, or about 10% by weight and about 20% by weight, etc., based on the total weight of the composition. In any embodiment, the nitrogen-containing stabilizers and sugar-containing stabilizers, either alone or in combination, may be present in the composition in an amount of, for example, about 5% to about 50% by weight, based on the total weight of the composition. E. Additional Ingredients

[0076] In any embodiment, the polymer matrix may further comprise a filler (also called an inert carrier). Examples of fillers include, but are not limited to, starch, modified starch, maltodextrin, gum, diatomaceous earth, loam, cellulose, biochar, pumice, silica, silicates, talc, kaolin, limestone, line, chalk, clay (e.g., mectite, hectorite, bentonite, montmorillonite, celite, illite, and combinations thereof), dolomite, zeolite, perlite, and combinations thereof. In any embodiment, the filler may comprise biochar. The term "biochar" refers to a carbonaceous solid formed from the pyrolysis of biomass under anoxic or anaerobic conditions.

[0077] The filler is, based on the total weight of the composition, about 0.1% by weight or more, about 1% by weight or more, about 2.5% by weight or more, about 5% by weight or more, about 10% by weight or more, about 20% by weight or more, about 30% by weight or more, about 40% by weight or more, about 50% by weight or more, about 95% by weight or less, about 90% by weight or less, about 80% by weight or less, about 70% by weight or less, or about 60% by weight or less, or about 0.1% by weight to about 95% by weight, about 0.1% by weight to about 90% by weight, about 0.1% by weight to about 80% by weight, about 0.1% by weight to about 70% by weight, about 0.1% by weight to about 60% by weight, about 0.1% by weight to about 50% by weight, about 0.1% by weight to about 40% by weight, about 0.1% by weight to about 30% by weight, about 0.1 % to about 20% by weight, about 0.1% to about 10% by weight, about 0.1% to about 5% by weight, about 0.1% to about 2.5% by weight, about 10% to about 95% by weight, about 10% to about 90% by weight, about 10% to about 80% by weight, about 10% to about 70% by weight, about 10% to about 60% by weight, about 10% to about 50% by weight, about 10% to about 40% by weight, about 10% to about 30% by weight, about 10% to about 20% by weight, about 50% to about 95% by weight, about 50% to about 90% by weight, about 50% to about 80% by weight, about 50% to about 70% by weight, or about 50% to about 60% by weight. In any embodiment, the filler may be present in the composition in an amount of, for example, from about 0.1% by weight to about 90% by weight based on the total weight of the composition.

[0078] Additionally or alternatively, the composition may optionally include one or more additional components (or additives), such as, but not limited to, surfactants, dispersants, emulsifiers, wetting agents, adjuvants, solubilizers, penetration enhancers, protective colloids, adhesives, thickeners, humectants, repellents, attractants, feeding stimulants, compatibilizers, antifreeze agents, antifoam agents, colorants, tackifiers, binders, and / or hydrophobic additives. In particular, in any embodiment, the additional components may include one or more hydrophobic additives, and thus the composition may include one or more hydrophobic additives that are hydrophobic, water soluble and / or dispersible, microbial compatible, film forming, and / or moisture barrier when dry. In such embodiments, the one or more hydrophobic additives may include, for example, silk fibroin solutions, wax-based dispersions, dispersions of ethylcellulose or other modified celluloses, alkalized shellac solutions, modified chitosan solutions, polyvinyl alcohol, modified acrylate or methacrylate-based solutions, or combinations thereof. Additionally, in such embodiments, the one or more hydrophobic additives may enhance, extend, etc., the stability of the composition on the seed (e.g., by reducing, limiting, inhibiting, etc., water uptake by the matrix of the composition). F. Composition properties

[0079] As mentioned above, the composition can advantageously exhibit increased stability. For example, the increased stability of the composition can be demonstrated by the continued viability of the microorganisms present in the composition after an extended period of time, for example, at elevated temperature and elevated relative humidity (RH). This continued viability of the microorganisms in the composition can be demonstrated by, for example, a minimal loss of microbial colony forming units per gram (CFU / g) after a period of time at a range of temperatures and relative humidity after the composition is applied to a seed, plant, or part of a plant. For example, the encapsulated microbial composition can exhibit a loss of about 4 logs or less of CFU / g, a loss of about 3 logs or less of CFU / g, a loss of about 2 logs or less of CFU / g, or a loss of about 1 log or less of CFU / g. This measured loss CFU / g of microorganisms may be measured and / or occur about 1 week, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, about 20 weeks, or about 24 weeks, or from about 1 week to about 24 weeks, from about 1 week to about 20 weeks, from about 1 week to about 16 weeks, from about 1 week to about 12 weeks, from about 1 week to about 8 weeks, from about 1 week to about 4 weeks, from about 4 weeks to about 24 weeks, from about 4 weeks to about 20 weeks, from about 4 weeks to about 16 weeks, from about 4 weeks to about 12 weeks, or from about 4 weeks to about 8 weeks, etc., after application of the composition to the seed, plant, or part of the plant. Additionally or alternatively, this measured loss CFU / g of microorganisms may be measured and / or occur at a temperature (e.g., ambient temperature, composition temperature, etc.) of about 15° C. or more, about 20° C. or more, about 25° C. or more, about 40° C. or less, about 35° C. or less, about 30° C. or less, or about 15° C. to about 40° C., about 15° C. to about 35° C., about 15° C. to about 30° C., about 15° C. to about 25° C., or about 15° C. to about 20° C. Additionally or alternatively, this measured loss CFU / g of microorganisms may be measured and / or occur at an RH of about 50% or more, about 60% or more, about 65% or more, about 90% or less, about 80% or less, about 70% or less, or about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, or about 50% to about 65%, etc. In particular, the encapsulated microbial composition may have less than 2 log or less than 1 log loss in colony forming units / g (CFU / g) after 4 weeks, 8 weeks or 12 weeks at 25° C. and 65% relative humidity (RH).

[0080] Additionally, the composition may have a lower moisture content. For example, the composition may have a moisture content of about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, or about 0.1% or less, or about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, or about 0.1% to about 1%, etc. The moisture content of the composition may be measured using an infrared (IR) moisture analyzer. For example, to measure the moisture content of a sample, the starting weight of the sample may be recorded, after which a halogen lamp or other infrared emitter may heat and dry the sample, while an integrated balance continuously records the sample weight. When the sample no longer loses weight, the IR moisture analyzer is stopped and the moisture content may be calculated based on the total loss in weight, as will be understood by those skilled in the art.

[0081] In any embodiment, the composition may have a diameter of about 1 μm or more, about 10 μm or more, about 25 μm or more, about 50 μm or more, about 100 μm or more, about 150 μm or more, about 200 μm or more, about 250 μm or more, about 300 μm or more, about 20 mm or less, about 1 mm or less, about 500 μm or less, about 300 μm or less, about 200 μm or less, about 100 μm or less, about 75 μm or less, about 50 μm or less, about 25 μm or less, about 10 μm or less, about 5 ... The particle size may be about 25 μm to about 20 mm, about 25 μm to about 1 mm, about 25 μm to about 500 μm, about 25 μm to about 300 μm, about 25 μm to about 200 μm, about 25 μm to about 150 μm, about 25 μm to about 100 μm, about 25 μm to about 50 μm, about 50 μm to about 300 μm, about 50 μm to about 200 μm, about 50 μm to about 150 μm, or about 50 μm to about 100 μm.

[0082] In any embodiment, the composition comprises about 1×10 8 CFU / gram or more, approximately 3×10 8 CFU / gram or more, approximately 5×10 8 CFU / gram or more, approximately 8 x 10 8 CFU / gram or more, approximately 1×10 9 CFU / gram or more, approximately 1×1010 CFU / gram or less, approximately 8 x 10 9 CFU / gram or less, approximately 5 x 10 9 or less, or about 3 x 10 9 CFU / gram or less, or approximately 1 x 10 8 CFU / gram ~ approx. 1 x 10 10 CFU / gram, approximately 1 x 10 8 CFU / gram ~ approx. 8 x 10 9 CFU / gram, approximately 1 x 10 8 CFU / gram ~ approx. 5 x 10 9 CFU / gram, approximately 1 x 10 8 CFU / gram ~ approx. 3 x 10 9 CFU / gram, approximately 1 x 10 8 CFU / gram ~ approx. 1 x 10 9 CFU / gram, approximately 1 x 10 8 CFU / gram ~ approx. 8 x 10 8 CFU / gram, approximately 1 x 10 8 CFU / gram ~ approx. 5 x 10 8 CFU / gram, or approximately 1 x 10 8 CFU / gram ~ approx. 3 x 10 8 It may contain CFU / gram.

[0083] Additionally, the compositions may have a generally (or relatively) higher tap density (e.g., compared to conventionally formed compositions (e.g., compositions formed using conventional spray drying techniques, etc.). As used herein, tap density may refer to the increased bulk density achieved after mechanically tapping a container containing the composition. For example, the initial volume or mass of the composition may be determined, and then the container holding the composition may be mechanically tapped (e.g., by raising the container and composition and dropping it under its own weight, a particular distance, etc.). Then, after each tapping, further volume or mass readings of the composition may be determined again until little or no (or minimal) further volume or mass change is observed. It has been found that such higher tap density of the compositions herein may be associated with and correspond to a reduced water uptake of the composition, etc. For example, the composition may have a tap density of about 0.5 g / mL or more, about 0.6 g / mL or more, about 0.7 g / mL or more, about 0.8 g / mL or more, about 1.5 g / mL or less, about 1.4 g / mL or less, about 1.3 g / mL or less, about 1.2 g / mL or less, about 1.1 g / mL or less, about 1 g / mL or less, or about 0.9 g / mL or less; or about 0.5 g / mL or more to about 1.5 g / mL or less, about 0.5 g / mL or more to about 1.4 g / mL or less, about 0.5 g / mL or more to about 1.3 g / mL, about 0.5 g / mL or more to about 1.2 g / mL or less, about 0.7 g / mL or more to about 1.5 g / mL or less, about 0.7 g / mL or more to about 1.2 g / mL or less, about 0.7 g / mL or more to about 1.1 g / mL or less, etc. II. Seed composition

[0084] Also provided herein is a plant seed composition, which comprises a plant seed and an encapsulated microbial composition as described herein present on at least a portion of the surface of the plant seed, for example, as a coating on the seed. In any embodiment, the encapsulated microbial composition can be formulated as (or as part of) a seed treatment. It is contemplated herein that the seed can be substantially uniformly coated with one or more layers of the encapsulated microbial composition as described herein using conventional methods of mixing, spraying, or combinations thereof, through the use of a treatment application device designed and manufactured to precisely, safely, and efficiently apply the seed treatment product to the seed. Such devices use various types of coating techniques, such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mist, or combinations thereof. Thus, in any embodiment, the encapsulated microbial composition can be applied to the seed, for example, in liquid form, either through a rotating "atomizer" disk or spray nozzle (or multiple spray nozzles, each associated with a different component of the encapsulated microbial composition, e.g., when formed on the seed), which distributes the composition evenly on the seed as it moves through the spray pattern. Preferably, the seeds are then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying. The seeds may or may not be primed prior to coating with the encapsulated microbial composition to increase germination and emergence uniformity. In an alternative embodiment, a dry powder formulation may be metered (e.g., applied, etc.) onto the moving seeds and mixed until completely distributed.

[0085] Additionally or alternatively, the plant seeds and / or seed treatments comprising the encapsulated microbial composition may further contain protective agents that can protect the seeds from the deleterious effects of selective herbicides, such as activated carbon, nutrients (fertilizers), and other agents that can improve the germination and quality of the product, or combinations thereof.

[0086] Various additives can be added to the seed treatment formulation containing the encapsulated microbial composition. Binders may be added, including those that are preferably composed of adhesive polymers that do not have phytotoxic effects on the seed being coated and can be natural or synthetic. Binders can be selected from polyvinyl acetate, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, celluloses including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose, polyvinylpyrrolidone, starches, modified starches, dextrins, maltodextrins, polysaccharides including alginates and chitosan, fats, oils, proteins including gelatin and zein, gum arabic, shellac, vinylidene chloride and vinylidene chloride copolymers, calcium lignosulfonate, acrylic copolymers, polyvinyl acrylates, polyethylene oxide, acrylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomers, and polychloroprene, or combinations thereof.

[0087] The colorants may include any of a variety of colorants such as organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo, and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene; or combinations thereof. Other additives that may be added include micronutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, zinc, or combinations thereof. Polymers or other dust control agents may be applied to retain the treatment on the seed surface.

[0088] Additionally or alternatively, the coating may further comprise an adhesive layer. Examples of suitable adhesives include, but are not limited to, polyvinyl acetate, polyvinyl acetate copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, celluloses such as methylcellulose, hydroxymethylcellulose, and hydroxymethylpropylcellulose, dextrins, alginates, sugars, molasses, polyvinylpyrrolidone, polysaccharides, proteins, fats, oils, gum arabic, gelatin, syrups, starches, and combinations thereof.

[0089] Various additives, such as dispersants, surfactants, and nutrients and buffer components, can also be included on the seed and / or in the seed treatment formulation. Other conventional seed treatment additives include, but are not limited to, coating agents, wetting agents, buffers, and polysaccharides. At least one agriculturally acceptable carrier, such as water, solid or dry powder, can be added to the seed treatment formulation. Dry powders can be derived from various materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon, various phosphorus compounds, and / or combinations thereof.

[0090] In any embodiment, the seed and / or seed treatment formulation may contain at least one filler, which is an organic or inorganic, natural or synthetic component that combines the active ingredients to facilitate application to the seed. Preferably, the filler is an inert solid, such as clay, natural or synthetic silicate, silica, resin, wax, solid fertilizer (e.g. ammonium salt), natural soil minerals, such as kaolin, clay, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earth, or a synthetic mineral, such as silica, alumina or silicate, in particular aluminum or magnesium silicate.

[0091] The seed and / or seed treatment formulation may further comprise one or more of the following ingredients: pesticides, including compounds that act only underground; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of these materials; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxy, urea, and benzoic acid; herbicide safeners, such as benzoxazines, benzhydryl derivatives, N,N-diallyldichloroacetamides, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanones, naphthalic anhydride compounds, and oxime derivatives; chemical fertilizers; and biological fertilizers. These ingredients may be added as a separate layer on the seed or as part of the seed coating composition described herein.

[0092] Preferably, the amount of encapsulated microbial composition or other component used in a seed treatment should not inhibit germination of the seed or cause phytotoxic damage to the seed.

[0093] The formulations used to treat the seeds may be in the form of suspensions; emulsions; slurries of particles in an aqueous medium (e.g., water); wettable powders (dusts); wettable powders (dry flowables); and dry granules.

[0094] As mentioned above, other conventional inactive or inert ingredients can be incorporated into the formulation. Such inactive ingredients include, but are not limited to: conventional adhesives; dispersants such as methylcellulose, for example, polyvinyl alcohol, lecithin, polymeric dispersants (e.g., polyvinylpyrrolidone / vinyl acetate), which act as a mixed dispersant / adhesive for use in seed treatment, thickeners (e.g., clay thickeners to improve viscosity and reduce settling of particle suspensions), emulsion stabilizers; surfactants; antifreeze compounds (e.g., urea), dyes, colorants, etc.

[0095] The microbial encapsulation compositions and coating formulations described herein can be applied to seeds by a variety of methods, including, but not limited to, mixing in a container (e.g., a bottle or bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inactive materials can be used to contact the seeds with the microbial compositions described herein, such as conventional film coating materials, including, but not limited to, water-based film coating materials such as SEPIRET™ (Seppic, Inc., NJ) and OPACOAT™ (Berwind Pharm. Services, PA). It is also contemplated herein that the microbial encapsulation compositions and coating formulations described herein can be applied to plants, plant parts, plant environments, or combinations thereof. For example, the seeds can be coated with a polymer-based or sugar-based binder, and the dry encapsulated microbial composition can be applied to the seeds. The seed(s) and the dry encapsulated microbial composition (e.g., in powder form) can be mixed in a seed treater for at least about 1 minute to ensure uniform distribution.

[0096] The amount of the composition used for seed treatment may vary depending on the type of seed and the type of active ingredient, but the treatment includes contacting the seed with an agriculturally effective amount of the encapsulated microbial composition.As used herein, an "effective amount" is generally an amount sufficient to produce a beneficial or desired result.An effective amount can be applied in one or more doses.

[0097] In addition to the coating layer, the seeds may be treated with one or more of the following ingredients: fungicides and other pesticides, including herbicides, herbicide safeners, fertilizers, and / or biocontrol agents. These ingredients may be added as separate layers or may be added to the coating layer.

[0098] The encapsulated microbial composition and / or seed coating formulation can be applied to the seeds using various techniques and machines, such as fluidized bed technology, roller mill method, rotostatic seed treater, and drum coater. Other methods, such as spouted bed, can also be useful. The seeds can be pre-sized before coating. After coating, the seeds are typically dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.

[0099] The encapsulated microbial compositions described herein may also be wrapped in a film overcoating (e.g., before application to seeds, etc.) to protect the coating, and such overcoatings may be applied using fluidized bed and drum film coating techniques. For example, in any embodiment, one or more hydrophobic additives may be additionally applied to the encapsulated microbial composition (e.g., during drying as part of the fluidized bed technique, etc.). In such an embodiment, the hydrophobic additive may provide a coating around the composition, for example, during drying.

[0100] In any embodiment, the encapsulated microbial composition described herein can be introduced onto a seed by using solid matrix priming. For example, a quantity of the encapsulated microbial composition can be mixed with a solid matrix material, and then the seed can be contacted with the solid matrix material for a period of time that allows the composition to be introduced into the seed. The seed can then be optionally separated from the solid matrix material and stored or used, or the mixture of the solid matrix material and the seed can be directly stored or planted. Solid matrix materials useful herein can include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material that can absorb or adsorb the composition herein for a period of time and release the composition into or onto the seed. In various examples, it can be useful for the composition herein and the solid matrix material to be compatible with each other. For example, the solid matrix material can be selected to be able to release the composition at a reasonable rate, for example, over a period of minutes, hours, or days.

[0101] It is contemplated herein that any plant seed that can germinate to form a plant, such as vegetable seeds, fruit seeds, grain seeds, and combinations thereof, can be treated as described above.Suitable seeds include cereals, coffee, cole crops, fiber crops, flowers, fruits, legumes, oil crops, trees, tuber crops, vegetables, and other plant seeds of monocotyledonous and dicotyledonous species.Nevertheless, exemplary crop seeds that can be coated include, but are not limited to, bean, canola, carrot, corn, cotton, boxwood, lettuce, peanut, peanut, pepper, potato, rapeseed, rice, rye, sorghum, soybean, sugar beet, sunflower, tobacco, tomato, and wheat seeds. III. Methods for Preparing Encapsulated Microbial Compositions

[0102] Methods for preparing the encapsulated microbial compositions described herein are also provided. The methods may include (a) combining a microorganism described herein with at least one hydrogel described herein to form a precursor mixture; (b) solidifying the precursor mixture with a crosslinker to form an intermediate microbial composition; and (c) drying the intermediate microbial composition to form an encapsulated microbial composition. The methods described herein can surprisingly achieve a more stable encapsulated microbial composition with increased storage stability. For example, as described above, the encapsulated microbial compositions formed by the methods provided herein can exhibit minimal loss of microbial colony forming units per gram (CFU / g) as described herein. For example, the encapsulated microbial compositions can have less than 2 log loss or less than 1 log loss of colony forming units / gram (CFU / g) after 4 weeks, 8 weeks, or 12 weeks at 25° C. and 65% RH. Additionally, the encapsulated microbial compositions formed by the methods provided herein can have a lower moisture content, e.g., a moisture content of about 10% or less, or about 5% or less, as described herein. Additionally, the encapsulated microbial compositions formed by the methods provided herein can have an increased tap density, for example, a tap density of about 0.5 g / mL or more to about 1.5 g / mL or less, as described herein.

[0103] In any embodiment, the at least one hydrogel may comprise an alginate as described herein and may optionally comprise additional hydrogels as described herein. The at least one hydrogel may be present in the precursor mixture in an amount of about 2.5% by weight or more, about 5% by weight or more, about 10% by weight or more, about 20% by weight or less, about 60% by weight or less, about 50% by weight or less, about 40% by weight or less, or about 30% by weight or less, or about 5% to about 60% by weight, about 5% to about 50% by weight, about 5% to about 40% by weight, about 5% to about 30% by weight, about 5% and about 20% by weight, about 10% to about 60% by weight, about 10% to about 50% by weight, about 10% to about 40% by weight, about 10% to about 30% by weight, or about 10% and about 20% by weight, based on the total weight of the precursor mixture. In any embodiment, the at least one hydrogel may be present in the precursor mixture in an amount of about 5% to about 50% by weight, based on the total weight of the precursor mixture.

[0104] Additionally or alternatively, the precursor mixture may further include a stabilizer as described herein, such as a nitrogen-containing stabilizer as described herein, a sugar-containing stabilizer as described herein, or a combination thereof. Stabilizers, e.g., nitrogen-containing stabilizers and sugar-containing stabilizers, alone or in combination, may be present in the precursor mixture in an amount such as about 2.5% by weight or more, about 5% by weight or more, about 10% by weight or more, about 20% by weight or more, about 60% by weight or less, about 50% by weight or less, about 40% by weight or less, or about 30% by weight or less, or between about 5% by weight and about 60% by weight, between about 5% by weight and about 50% by weight, between about 5% by weight and about 40% by weight, between about 5% by weight and about 30% by weight, between about 5% by weight and about 20% by weight, between about 10% by weight and about 60% by weight, between about 10% by weight and about 50% by weight, between about 10% by weight and about 40% by weight, between about 10% by weight and about 30% by weight, or between about 10% by weight and about 20% by weight, based on the total weight of the precursor mixture. In any embodiment, the nitrogen-containing stabilizer and the sugar-containing stabilizer, alone or in combination, may be present in the precursor mixture in an amount of about 5% to about 50% by weight, based on the total weight of the precursor mixture.

[0105] Additionally or alternatively, the precursor mixture may further comprise a filler as described herein, the filler being present in an amount of about 0.1% by weight or more, about 1% by weight or more, about 2.5% by weight or more, about 5% by weight or more, about 10% by weight or more, about 20% by weight or more, about 30% by weight or more, about 40% by weight or more, about 50% by weight or more, about 95% by weight or less, about 90% by weight or less, about 80% by weight or less, about 70% by weight or less, or about 60% by weight or less, or between about 0.1% by weight and about 95% by weight, between about 0.1% by weight and about 90% by weight, between about 0.1% by weight and about 80% by weight, between about 0.1% by weight and about 70% by weight, between about 0.1% by weight and about 60% by weight, between about 0.1% by weight and about 50% by weight, between about 0.1% by weight and about 40% by weight, between about 0.1% by weight and about 30% by weight, between about 0.1% by weight and about 50% by weight, between about 0.1% by weight and about 60% by weight, or between about 0.1% by weight and about 70% by weight, between about 0.1% by weight and about 60% by weight, or between about 0.1% by weight and about 95% by weight, between about 0.1% by weight and about 90% by weight, between about 0.1% by weight and about 80% by weight, between about 0.1% by weight and about 70% by weight, between about 0.1% by weight and about 60% by weight, between about 0.1% by weight and about 50% by weight, between about 0.1% by weight and about 40% by The precursor mixture may be present in an amount of from about 0.1% to about 20% by weight, from about 0.1% to about 10% by weight, from about 0.1% to about 5% by weight, from about 0.1% to about 2.5% by weight, from about 10% to about 95% by weight, from about 10% to about 90% by weight, from about 10% to about 80% by weight, from about 10% to about 70% by weight, from about 10% to about 60% by weight, from about 10% to about 50% by weight, from about 10% to about 40% by weight, from about 10% to about 30% by weight, from about 10% to about 20% by weight, from about 50% to about 95% by weight, from about 50% to about 90% by weight, from about 50% to about 80% by weight, from about 50% to about 70% by weight, or from about 50% to about 60% by weight. In any embodiment, the filler may be present in the precursor mixture in an amount of about 0.1% to about 90% by weight, based on the total weight of the precursor mixture.

[0106] Additionally or alternatively, the precursor mixture may further comprise one or more additional components such as, but not limited to, surfactants, dispersants, emulsifiers, wetting agents, adjuvants, solubilizers, penetration enhancers, protective colloids, adhesives, thickeners, humectants, repellents, attractants, feeding stimulants, compatibilizers, antifreeze agents, antifoam agents, colorants, tackifiers, hydrophobic additives, and / or binders.

[0107] The components of the precursor mixture, e.g., microorganisms, hydrogel, stabilizer, optional filler, and optional additional components, can be combined in any suitable manner and order. For example, the microorganisms can be combined with the hydrogel and added to an aqueous protectant solution, e.g., including stabilizer, optional filler, and any other optional additional components described herein. It is contemplated herein that the hydrogel can be present as an aqueous solution that can be mixed prior to combination with the microorganism. Alternatively, the hydrogel and the microorganisms can be added separately to the aqueous protectant solution and then combined. The aqueous protectant solution can be mixed prior to the addition of the microorganisms and / or hydrogel. A. Solidification of the Precursor Mixture

[0108] The crosslinking agent may include a divalent cation, a divalent cation salt, or a combination thereof, as described herein. For example, the divalent cation may be barium (Ba 2+ ) cation, calcium (Ca 2+ ) cation, magnesium (Mg 2+ ) cation, strontium (Sr 2+ ) cation, cobalt (Co 2+ ) cation, nickel (Ni 2+ ) cation, zinc (Zn 2+ ) cation, manganese (Mn 2+ ) cations, and combinations thereof. Examples of divalent cation salts include, but are not limited to, barium salts, calcium salts, magnesium salts, strontium salts, cobalt salts, zinc salts, manganese salts, or combinations thereof. Examples of salts include, but are not limited to, chloride salts, fluoride salts, bromide salts, iodide salts, carbonate salts, citrate salts, and combinations thereof. In any embodiment, the crosslinker may be calcium chloride (CaCl2), calcium carbonate (CaCO3), or combinations thereof. Additionally or alternatively, the crosslinker may be in solution with a solvent, e.g., water. i. BEAD solidification method

[0109] In any embodiment, the solidification step may include contacting the droplets of the precursor mixture with a cross-linking agent to form an intermediate microbial composition in the form of cross-linked beads. Any suitable device may be used to generate the droplets of the precursor mixture, for example, the precursor mixture may be pumped or flowed through a tube having a suitable cross-section to generate droplets that may then be contacted with a cross-linking agent, for example, an aqueous solution containing the cross-linking agent. Optionally, the cross-linked beads may be filtered and / or washed to remove any excess divalent cations.

[0110] The crosslinked beads may have an average particle size of about 500 μm or more, about 1 mm or more, about 10 mm or more, about 15 mm or more, about 20 mm or less, about 50 mm or less, about 40 mm or less, about 30 mm or less, or about 25 mm or less, or from about 500 μm to about 50 mm, from about 500 μm to about 30 mm, from about 500 μm to about 20 mm, from about 1 mm to about 50 mm, from about 1 mm to about 40 mm, from about 1 mm to about 30 mm, from about 1 mm to about 20 mm, from about 1 mm to about 25 mm, or from about 1 mm to about 15 mm, etc.

[0111] Thus, in various aspects, methods of preparing an encapsulated microbial composition described herein are provided, where the methods generally include: (a) combining a microorganism described herein with at least one hydrogel, e.g., an alginate, described herein to form a precursor mixture described herein; (b) solidifying the precursor mixture with a crosslinker described herein to form an intermediate microbial composition in the form of crosslinked beads, where solidification involves contacting droplets of the precursor mixture with the crosslinker; and (c) drying the intermediate microbial composition to form an encapsulated microbial composition described herein.

[0112] In an alternative embodiment, the solidification step may include flowing the precursor mixture through an opening having a suitable diameter and contacting the precursor mixture with a cross-linking agent as it exits the opening to form an intermediate microbial composition in the form of cross-linked microbeads. The openings may have diameters of about 50 μm or more, about 100 μm or more, about 150 μm or more, about 650 μm or less, about 500 μm or less, about 450 μm or less, or about 300 μm or less; or about 50 μm to about 650 μm, about 50 μm to about 500 μm, about 50 μm to about 450 μm, about 50 μm to about 250 μm, about 50 μm to about 150 μm, about 100 μm to about 650 μm, about 100 μm to about 500 μm, about 100 μm to about 450 μm, about 100 μm to about 250 μm, or about 100 μm to about 150 μm, etc. Examples of devices through which the precursor mixture can flow include any commercially available encapsulation device having an opening (e.g., a nozzle) with the diameters described above. The nozzle can be vibrated, and the bead size can be varied by nozzle size, vibration frequency, and flow rate, as well as hydrogel viscosity (temperature). Commercially available encapsulation devices include, for example, Encapsulator B-390 (available from BUECHI Labortechnik AG). As the precursor mixture exits the encapsulation device, it can be injected into a crosslinker, for example, an aqueous solution containing a crosslinker. Optionally, the crosslinked microbeads may be filtered and / or washed to remove any excess divalent cations.

[0113] The crosslinked microbeads may have an average particle size of about 50 μm or more, about 100 μm or more, about 250 μm or more, about 500 μm or more, about 750 μm or more, about 30 mm or less, about 20 mm or less, about 15 mm or less, about 10 mm or less, or about 1 mm or less, or from about 50 μm to about 30 mm, from about 50 μm to about 20 mm, from about 50 μm to about 1 mm, from about 100 μm to about 30 mm, from about 100 μm to about 20 mm, from about 100 μm to about 10 mm, from about 100 μm to about 1 mm, from about 100 μm to about 750 μm, from about 100 μm to about 500 μm, or from about 100 μm to about 250 μm, etc.

[0114] Thus, in various aspects, methods are provided for preparing an encapsulated microbial composition as described herein, the methods generally comprising: (a) combining a microorganism as described herein with at least one hydrogel, e.g., alginate, as described herein, to form a precursor mixture as described herein; (b) solidifying the precursor mixture with a crosslinking agent as described herein to form an intermediate microbial composition in the form of crosslinked microbeads as described herein, where solidifying comprises flowing the precursor mixture through an opening having a diameter, e.g., about 100 μm to about 500 μm, and contacting the precursor mixture with the crosslinking agent; and (c) drying the intermediate microbial composition to form an encapsulated microbial composition as described herein. ii. High shear mixing solidification method

[0115] Additionally or alternatively, the solidification step can include high shear mixing of the precursor mixture described herein with a crosslinker described herein. For example, the precursor mixture can be combined with the crosslinker, e.g., as an aqueous solution containing the crosslinker, and the combination can be subjected to high shear mixing. The term "high shear mixing" refers to mixing at temperatures of up to 1000 s -1 High shear mixing refers to mixing or mixing steps involving shear rates of more than or equal to 1000 rpm. High shear mixing can be achieved using any suitable high shear mixing device, such as, but not limited to, vortex mixers and homogenizers. Any suitable commercially available vortex mixer or homogenizer can be used. An example of a commercially available homogenizer is the Kinematica POLYTRON® homogenizer (available from KINEMATICA AG).

[0116] In any embodiment, the high shear mixing can be carried out at a mixing tip speed of about 2 m / s or more, about 3.5 m / s or more, about 5 m / s or more, about 7.5 m / s or more, about 10 m / s or more, about 30 m / s or less, about 25 m / s or less, about 20 m / s or less, or about 15 m / s or less, or from about 2 m / s to about 30 m / s, from about 2 m / s to about 25 m / s, from about 2 m / s to about 20 m / s, from about 3.5 m / s to about 30 m / s, from about 3.5 m / s to about 25 m / s, from about 3.5 m / s to about 20 m / s, from about 3.5 m / s to about 15 m / s, or from about 3.5 m / s to about 10 m / s, etc.

[0117] Additionally or alternatively, the high shear mixing can be carried out at about 1,000 rpm or more, about 2,500 rpm or more, about 5,000 rpm or more, about 7,500 rpm or more, about 10,000 rpm or more, about 15,000 rpm or less, or about 12,500 rpm or less, or from about 1,000 rpm to about 15,000 rpm, from about 1,000 rpm to about 12,500 rpm, from about 10,000 rpm to about 10,000 rpm, from about 1,000 rpm to about 7,500 rpm, from about 1,000 rpm to about 5,000 rpm, or from about 1,000 rpm to about 2,500 rpm, etc.

[0118] Additionally or alternatively, the high shear mixing can be carried out for about 2.5 seconds or more, about 5 seconds or more, about 30 seconds or more, about 1 minute or more, about 5 minutes or more, about 25 minutes or less, about 20 minutes or less, about 15 minutes or less, or about 10 minutes or less, or from about 2.5 seconds to about 25 minutes, from about 2.5 seconds to about 20 minutes, from about 2.5 seconds to about 15 minutes, from about 2.5 seconds to about 10 minutes, from about 2.5 seconds to about 5 minutes, from about 2.5 seconds to about 1 minute, from about 2.5 seconds to about 30 seconds, from about 5 seconds to about 25 minutes, from about 5 seconds to about 20 minutes, from about 5 seconds to about 15 minutes, from about 5 seconds to about 10 minutes, from about 5 seconds to about 5 minutes, from about 5 seconds to about 1 minute, or from about 5 seconds to about 30 seconds, etc. In any embodiment, high shear mixing can be carried out at a tip speed of about 3.5 m / s or greater, such as from about 3.5 m / s to about 25 m / s, for about 5 seconds or greater, such as from about 5 seconds to about 20 minutes.

[0119] Additionally or alternatively, the high shear mixing may be carried out at a temperature of, for example, about 1° C. or more, about 4° C. or more, about 10° C. or more, about 15° C. or more, about 35° C. or less, about 30° C. or less, about 25° C. or less, or about 20° C. or less, or from about 1° C. to about 35° C., from about 1° C. to about 30° C., from about 1° C. to about 25° C., from about 1° C. to about 20° C., from about 1° C. to about 15° C., from about 1° C. to about 10° C., from about 1° C. to about 4° C., from about 4° C. to about 35° C., from about 4° C. to about 30° C., from about 4° C. to about 25° C., from about 4° C. to about 20° C., from about 4° C. to about 15° C., or from about 4° C. to about 10° C., such as ambient temperature and / or the temperature of the mixture of the precursor mixture and the crosslinker.

[0120] Thus, in various aspects, methods for preparing an encapsulated microbial composition as described herein are provided, wherein the methods generally include: (a) combining a microorganism as described herein with at least one hydrogel, e.g., an alginate, as described herein, to form a precursor mixture as described herein; (b) solidifying the precursor mixture with a crosslinker as described herein to form an intermediate microbial composition, wherein solidifying comprises high shear mixing of the precursor mixture with the crosslinker as described herein; and (c) drying the intermediate microbial composition to form an encapsulated microbial composition as described herein. iii. Carbonate solidification method

[0121] Additionally or alternatively, the solidification step may include combining the precursor mixture with a cross-linking agent to form an intermediate microbial composition, where the cross-linking agent comprises a divalent cation salt, and the divalent cation salt may be a divalent cation carbonate. For example, the divalent cation carbonate may be barium carbonate, calcium carbonate, magnesium carbonate, strontium carbonate, cobalt carbonate, nickel carbonate, zinc carbonate, manganese carbonate, or combinations thereof. In any embodiment, the divalent cation carbonate may be calcium carbonate.

[0122] Additionally or alternatively, the solidification may further include adding an acidic buffer to the precursor mixture and / or the crosslinker. Any suitable acidic buffer having a pH of about 7 or less can be added, including, but not limited to, acetate buffer (e.g., a solution of acetic acid and a salt of acetic acid (e.g., sodium acetate)), glucono-delta-lactone buffer, weak acids (e.g., citric acid, formic acid, oxalic acid, hydrofluoric acid, nitrous acid, sulfurous acid, phosphoric acid, etc.), and combinations thereof.

[0123] The acidic buffer can be added in a variety of ways and orders. For example, the acidic buffer can be added: (i) to the precursor mixture and / or the crosslinker before the crosslinker is combined with the precursor mixture; (ii) to the precursor mixture substantially simultaneously with the crosslinker; (iii) to the mixture including the precursor mixture and the crosslinker; or to any combination of (i), (ii), and (iii). The mixture including the crosslinker, precursor mixture, and any acidic buffer can have and / or be maintained at a pH of about 2 or more, about 4 or more, about 5 or more, about 7 or less, about 6.5 or less, or about 6 or less, or about 2 to about 7, about 2 to about 6.5, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 4 to about 7, about 4 to about 6.5, about 4 to about 6, or about 4 to about 5, etc.

[0124] In any embodiment, the precursor mixture, crosslinker, and optional acidic buffer may be combined for about 5 minutes or more, about 30 minutes or more, about 2 hours or more, about 6 hours or more, about 12 hours or more, about 18 hours or more, about 48 hours or less, about 42 hours or less, about 36 hours or less, about 30 hours or less, or about 24 hours or less, or for about 5 minutes to about 48 hours, about 5 minutes to about 42 hours, about 5 minutes to about 36 hours, about 5 minutes to about 30 hours, about 5 minutes to about 24 hours, about 5 minutes to about 18 hours, about 5 minutes to about 12 hours, about 5 minutes to about 6 hours, about 5 minutes to about 2 hours, or about 5 minutes to about 30 hours, etc. During this aforementioned period of time, the mixture including the precursor mixture, crosslinker, and optional acidic buffer may be mixed in a suitable mixer for any of the above-mentioned periods of time, and / or the mixture may be allowed to stand without further mixing for any of the above-mentioned periods of time. For example, a mixture containing the precursor mixture, the crosslinker, and any acidic buffering agent may be mixed for about 5 minutes to about 6 hours, and then allowed to stand for an additional about 18 hours to about 30 hours.

[0125] Additionally or alternatively, the precursor mixture, crosslinker, and optional acidic buffer can be combined at a temperature of about 1° C. or higher, about 4° C. or higher, about 10° C. or higher, about 15° C. or higher, about 40° C. or lower, about 30° C. or lower, about 25° C. or lower, or about 20° C. or lower, or between about 1° C. and about 40° C., between about 1° C. and about 30° C., between about 1° C. and about 25° C., between about 1° C. and about 20° C., between about 1° C. and about 15° C., between about 1° C. and about 10° C., between about 1° C. and about 4° C., between about 4° C. and about 35° C., between about 4° C. and about 40° C., between about 4° C. and about 25° C., between about 4° C. and about 20° C., between about 4° C. and about 15° C., or between about 4° C. and about 10° C., e.g., ambient temperature and / or the temperature of the mixture of the precursor mixture, crosslinker, and optional acidic buffer. In any embodiment, the precursor mixture, crosslinker, and optional acidic buffer may be combined for about 5 minutes or more, e.g., from about 5 minutes to about 48 hours, and may be carried out at a temperature of about 4° C. or more, e.g., from about 4° C. to about 40° C.

[0126] Thus, in various aspects, methods are provided for preparing an encapsulated microbial composition as described herein, the methods generally comprising: (a) combining a microorganism as described herein with at least one hydrogel as described herein, e.g., an alginate, to form a precursor mixture as described herein; (b) solidifying the precursor mixture with a cross-linking agent comprising a divalent cation salt as described herein, e.g., a divalent cation carbonate salt, to form an intermediate microbial composition, wherein solidifying comprises combining the precursor mixture with the cross-linking agent; and (c) drying the intermediate microbial composition to form an encapsulated microbial composition as described herein.

[0127] When any of the above solidification steps (e.g., bead solidification, high shear mixing, carbonate solidification, etc.) are performed, the divalent cations are about 0.001% or more, about 0.1% or more, about 1% or more, about 2.5% or more, about 5% or more, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 7.5% or less, or about 0.001% to about 25%, about 0.001% to about 20%, of the total dry weight of the encapsulated microbial composition. , about 0.001% to about 15%, about 0.001% to about 10%, about 0.001% to about 7.5%, about 0.001% to about 5%, about 0.001% to about 2.5%, about 0.001% to about 1%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 7.5%, about 0.1% to about 5%, about 0.1% to about 2.5%, or about 0.1% to about 2.5%, etc. For example, when any of the bead solidification steps are performed, the divalent cations may be present in an amount of about 0.1% to about 10% of the total dry weight of the encapsulated microbial composition. Additionally, when either the high shear mixing or carbonate solidification step is performed, divalent cations may be present in an amount of about 0.001% to about 25% of the total dry weight of the encapsulated microbial composition.

[0128] For example, it is also contemplated herein that the intermediate microbial composition formed after the high shear mixing solidification step or the carbonate solidification step may be applied to any suitable substrate, and the intermediate microbial composition may be in the form of a film disposed on the substrate. The film may have a thickness of about 1 mm or more, 2.5 mm or more, 5 mm or more, about 7.5 mm or more, about 5 cm or less, about 2.5 cm or less, or about 1 cm or less, or about 1 mm to about 5 cm, about 1 mm to about 2.5 cm, about 1 mm to about 1 cm, about 1 mm to about 7.5 mm, about 1 mm to about 5 mm, or about 1 mm to about 2.5 mm, etc. B. Drying of the Intermediate Microbial Composition

[0129] As noted above, it has surprisingly been discovered that encapsulated microbial compositions with increased stability can be achieved by the methods described herein that include a slower drying step, for example, by utilizing slower evaporation rates at lower temperatures.

[0130] Slower drying can be achieved, for example, by maintaining a slower evaporation rate of the intermediate microbial composition. Such drying can be achieved (or carried out, or completed, etc.) in (or by the use of) air (e.g., air drying, ambient air drying, forced or directional air drying), vacuum drying, fluidized bed drying, etc., by (or by the use of) a dryer, oven, humidity control chamber, or any combination thereof. For example, drying can be achieved at a rate of about 30,000 g / hr / m 2 Below, approximately 25,000g / hr / m 2 Below, approximately 20,000g / hr / m 2 Below, approximately 15,000g / hr / m 2 Below, approximately 10,000g / hr / m 2 Below, approximately 5,000g / hr / m 2 Below, approximately 1,000g / hr / m 2 Below, about 500g / hr / m 2 Below, about 100g / hr / m 2 Below, about 50g / hr / m 2 Below, about 10g / hr / m 2 Below, about 1g / hr / m2 Above, about 10g / hr / m 2 Above, about 25g / hr / m 2 Above, about 50g / hr / m 2 Above, about 100g / hr / m 2 Above, about 250g / hr / m 2 Above, about 500g / hr / m 2 or more, or approximately 1,000 g / hr / m 2 or more, or approximately 1 g / hr / m 2 ~About 25,000g / hr / m 2 , about 10g / hr / m 2 ~About 25,000g / hr / m 2 , about 25g / hr / m 2 ~About 25,000g / hr / m 2 , about 50g / hr / m 2 ~About 25,000g / hr / m 2 , about 1g / hr / m 2 ~About 20,000g / hr / m 2 , about 50g / hr / m 2 ~About 20,000g / hr / m 2 , about 1g / hr / m 2 ~About 15,000g / hr / m 2 , about 50g / hr / m 2 ~About 15,000g / hr / m 2 , about 1g / hr / m 2 ~About 10,000g / hr / m 2 , about 50g / hr / m 2 ~About 10,000g / hr / m 2 , about 1g / hr / m 2 ~Approx. 5,000g / m / hr / m 2 , about 50g / hr / m 2 ~Approx. 5,000g / hr / m 2 , about 1g / hr / m 2 ~Approx. 1,000g / m / hr / m 2 , about 50g / hr / m 2 ~Approx. 1,000g / hr / m 2 , about 1g / hr / m 2 ~About 500g / hr / m 2 , about 50g / hr / m 2 ~About 500g / hr / m 2 , about 1g / hr / m2 ~About 250g / hr / m 2 , about 50g / hr / m 2 ~About 250g / hr / m 2 , or about 1 g / hr / m 2 ~About 100g / hr / m 2 , about 50g / hr / m 2 ~About 100g / hr / m 2 , about 1g / hr / m 2 ~About 50g / hr / m 2 , about 1g / hr / m 2 ~About 30g / hr / m 2 In any embodiment, the evaporation rate is about 1 g / hr / m 2 ~Approx. 150g / hr / m, approx. 50g / hr / m 2 ~ about 150 g / hr / m, etc.

[0131] In any embodiment, drying may be performed at a drying temperature of about 10° C. or higher, about 15° C. or higher, about 20° C. or higher, about 60° C. or lower, about 50° C. or lower, about 40° C. or lower, about 30° C. or lower, or about 25° C. or lower, or about 10° C. to about 50° C., about 10° C. to about 40° C., about 10° C. to about 30° C., about 10° C. to about 25° C., about 10° C. to about 20° C., about 10° C. to about 15° C., about 15° C. to about 50° C., about 15° C. to about 40° C., about 15° C. to about 30° C., about 15° C. to about 25° C., or about 15° C. to about 20° C. In any embodiment, the drying temperature may be about 15° C. to about 50° C.

[0132] Additionally or alternatively, drying may be performed for a drying time of about 10 minutes or more, about 15 minutes or more, about 30 minutes or more, about 1 hour or more, about 12 hours or more, about 24 hours or more, about 48 hours or more, about 72 hours or more, about 288 hours or less, about 240 hours or less, about 200 hours or less, about 170 hours or less, about 144 hours or less, or about 96 hours or less, or from about 10 minutes to about 288 hours, from about 15 minutes to about 288 hours, from about 15 minutes to about 240 hours, from about 15 minutes to about 200 hours, from about 15 minutes to about 170 hours, from about 15 minutes to about 144 hours, from about 15 minutes to about 96 hours, from about 15 minutes to about 72 hours, from about 15 minutes to about 48 hours, from about 15 minutes to about 24 hours, from about 15 minutes to about 12 hours, from about 15 minutes to about 1 hour, or from about 15 minutes to about 30 minutes. In any embodiment, the drying time can be from about 15 minutes to about 200 hours.

[0133] As described herein, it is contemplated that drying of the intermediate microbial composition may be performed in (or by use of) air (e.g., air drying, ambient air drying, forced or directional air drying), vacuum drying, fluidized bed drying, etc.), a dryer, an oven, a humidity controlled chamber, or in (or by use of) any combination thereof. For example, the intermediate microbial composition may be placed in air at room temperature (e.g., about 15°C to about 25°C) for the drying time described above (e.g., about 24 hours to about 72 hours), and then optionally further dried in a humidity controlled chamber at a temperature of about 20°C to about 30°C for about 15 minutes to about 72 hours, and optionally subsequently further dried in a dryer (e.g., a fluidized bed dryer) at a temperature of about 40°C to about 60°C for about 15 minutes to about 72 hours.

[0134] In any embodiment, the intermediate microbial composition can be placed in air at room temperature (e.g., about 15° C. to about 25° C.) for the drying time described above (e.g., about 24 hours to about 72 hours). In doing so, the tap density of the resulting composition formed by air drying of the intermediate microbial composition as described above can be relatively higher than the tap density of the composition formed by spray drying, etc. For example, in any embodiment, the tap density of the composition formed by air drying of the intermediate microbial composition as described above can be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or about 300%, or about 5% to about 300%, etc. higher than the tap density of the composition formed by spray drying.

[0135] In any embodiment, drying of the intermediate microbial composition may be performed, accomplished, etc., by air drying, whereby, for example, Crosslinked Alginate Microbe Mixtures (CAMMs) are generated by either the shear mixing method or the bead / microbead method herein (see, e.g., Example 3, etc.), spread onto a sheet or surface (e.g., a 9" x 13" baking sheet, another sheet or surface, etc.), then placed in a biohood with air recirculation, and the mixture is allowed to air dry at room temperature (e.g., about 18°C ​​to about 25°C (about 64°F to about 78°F), etc.) for 24 to 72 hours. After 24-72 hours at room temperature, the CAMMS (on the surface) can be moved to a humidity controlled chamber set at a typically (or relatively) low relative humidity (RH) (e.g., about 10% to about 20%) or to a drying chamber with a dry air purge (e.g., RH less than about 5%) to facilitate complete drying (e.g., less than about 5% moisture content in the dried alginate).

[0136] In any embodiment, drying of the intermediate microbial composition may be performed, accomplished, etc., by fluidized bed drying. In this regard, for example, Crosslinked Alginate Microbe Mixtures (CAMMs) may be produced by the bead / microbead method herein (see, e.g., Example 3, etc.) and loaded into a fluidized bed dryer, e.g., Sherwood Scientific 50100201 Programmable Fluid Bed Dryer from Sherwood Scientific Ltd. or Freund Vector VFC-Lab Micro Flo-Coater from Freund-Vector Corporation. The fluidized bed dryer may then be operated at a temperature between about 20°C and about 60°C for about 1 hour to about 8 hours until the moisture content of the microbeads is less than about 5%. Alternatively, the microbeads may be removed from the fluidized bed dryer, not completely dried but still as a free-flowing powder, and then dried in a vacuum oven at about 20°C to about 40°C until their moisture content is less than about 5%.

[0137] In any embodiment, drying of the intermediate microbial composition may be performed, accomplished, etc., by vacuum drying. In this regard, for example, Crosslinked Alginate Microbe Mixtures (CAMMs) may be produced by the bead / microbead method herein (see, e.g., Example 3, etc.) and loaded into a vacuum dryer, e.g., a vacuum paddle dryer, an agitator vacuum dryer, or a conical screw vacuum dryer. The vacuum dryer may then be operated at a temperature of about 20°C to about 50°C for about 1 hour to about 8 hours until the moisture content of the microbeads is less than about 5%. C. Optional Further Processing

[0138] The encapsulated microbial composition may be subjected to any further treatment before further application, for example, use as a seed treatment and / or application to seeds.Further treatment may include, for example, grinding the encapsulated microbial composition to form encapsulated microbial particles and / or to further reduce the size of the encapsulated microbial particles.For example, any suitable grinding process known in the art, such as ball mill grinding, freeze grinding, or a combination thereof, may be used to form the encapsulated microbial particles.

[0139] In any embodiment, the encapsulated microbe particles can have a diameter of about 1 μm or more, about 15 μm or more, about 25 μm or more, about 50 μm or more, about 75 μm or more, about 100 μm or more, about 500 μm or less, about 350 μm or less, or about 200 μm or less, or from about 1 μm to about 500 μm, from about 1 μm to about 350 μm, from about 1 μm to about 200 μm, from about 1 μm to about 100 μm, from about 1 μm to about 75 μm, from about 1 μm to about 50 μm, from about 1 μm to about 25 μm, or from about 1 μm to about 15 μm, etc. EXAMPLES

[0140] Working Example The following non-limiting examples are provided to further illustrate the present disclosure. Non-limiting examples of microbial formulations and methods of making and testing such formulations are provided that may be useful for encapsulation and improved stability of microorganisms, particularly fungal and non-sporulating bacterial strains. Example 1. Preparation of microbial strains Microbial strains and growth conditions

[0141] Bradyrhizobium japonicum (OPTIMIZE® XC) was stored at 4°C before use.

[0142] Pseudomonas fluorescens, Phytobacter diazotrophicus, Herbasprillum frisingense, and Pseudacidovorax intermedius fermentations were freshly prepared for each experiment from frozen stocks at -80 °C. Cells were grown in rich complex medium (3 g / L beef extract, 5 g / L peptone water, 6 g / L mannitol, 30 g / L glucose, 18.9 g / L trehalose, 15 mM HEPES buffer, pH 7.2) in shake flasks for 18–48 h at 28 °C. Typically, the optical density of the cultures measured at 600 nm rose from 0.05 to within the range of 1.5 to 2.5 during the overnight fermentation. CFU (colony forming unit) measurement

[0143] The cultures were serially diluted in PBS buffer to obtain the desired amount of colony forming units (CFU) per milliliter. Bacteria were quantified by plating appropriate dilutions on agar plates and incubating overnight at 30°C. Colonies grown on the plates were counted. Final CFU / mL was calculated by multiplying the colony count by the dilution factor. CFU determinations of the strains may be performed before encapsulation, after encapsulation, and after different storage periods to determine the loading rate and stability profile. Example 2. Preparation of Alginate Microbial Mixture (AMM)

[0144] Mixtures containing protectant, microorganism of interest and alginate were prepared from the following solutions: The prepared microbial culture (20 mL, undiluted) and alginate solution were added to the protectant solution to produce Alginate Microbe Mixtures (AMMs) which were used to prepare Cross-Linked Alginate Microbe Mixtures (CAMMs). Protectant Solution

[0145] A protectant solution was prepared as follows: 200 uL of 1000X trace nutrients consisting of 4 grams of soluble starch, 10 grams of skim milk powder, 10 grams of sucrose, 5 grams of maltodextrin, 0.0012 g / mL of magnesium sulfate, 0.018 g / mL of mannitol, 0.117 g / L of betaine, 0.342 g / mL of trehalose, 0.167 g / mL of dipicolinic acid, and 2 drops of Antifoam 204 were added to 180 grams of MilliQ water (i.e., purified and deionized water) and homogenized in a high shear mixer. Alginate solution

[0146] An alginate solution was prepared as follows: 8.0 grams of sodium alginate was added to 192 grams of MilliQ water to form a 4% w / w sodium alginate mixture. The mixture was heated to 55° C., mixed on a shaker until homogenous, and cooled to room temperature.

[0147] The following reagents were used without further purification: "Std" sodium alginate from brown algae (Sigma Aldrich W201502); sodium alginate from brown algae, low viscosity (Sigma Aldrich A1112); sodium alginate from brown algae, medium viscosity (Sigma Aldrich A2033); sodium alginate, high viscosity (Alfa Aesar J61887); water soluble chitosan (Carbosynth YC58325); pectin (from apple, Sigma-Aldrich 93854); and gelatin (from bovine skin type B, Sigma Aldrich G9391). Example 3. Preparation of Cross-Linked Alginate-Microbial Mixtures (CAMMs)

[0148] Different types of cross-linked alginate-microbial mixtures (CAMMs) were prepared by the following method. CAMM Beads

[0149] Cross-linked alginate microbial mixture (CAMM) beads were prepared as follows: 300 mL of the AMM preparation in Example 2 above was pumped through 1.6 mm ID tubing (Masterflex HV-06508-14) at 57 rpm (Watson-Marlow pump, model: 120U / DM3) to generate droplets of the AMM preparation. The droplets were directed into 1 liter of 1.0 w / w% CaCl2 solution. Upon contact with calcium ions in the solution, solid cross-linked alginate beads were formed. The beads were filtered and rinsed with water to remove excess calcium ions.

[0150] For example, a typical preparation of Pseudomonas fluorescens CAMM beads after drying contains 1 × 10 8 From 1×10 9 CFU / gram. Beads produced by this method were typically greater than 1 centimeter in diameter. CAMM Microbeads

[0151] Cross-linked alginate microbial mixture (CAMM) microbeads were prepared as follows: hydrogel microbeads were prepared by injecting 300 mL of the AMM preparation from Example 2 above into 1 L of 1.0 w / w% CaCl2 solution using a commercial encapsulation unit (Encapsulator B-390, BUCHI, Switzerland). The encapsulation device was operated under fixed conditions: frequency = 1500 Hz; electrode potential = 1000 V; and operating pressure = 500-1000 mbar. Nozzle sizes of 150, 200, and 450 μm produced final wet microbead sizes of 560 μm, 750 μm, and >1 mm, respectively, after encapsulation.

[0152] For example, a typical preparation of Pseudomonas fluorescens CAMM microbeads after drying contains 1 × 10 9 From 3×10 9CFU / g. Microbeads made by this method for use according to these examples were typically greater than 1 millimeter in diameter. Shear Mixing

[0153] A shear-mix cross-linked alginate-microbial preparation was prepared as follows: 2.5 mL of 1% w / w calcium chloride solution was added to 100 mL of the alginate-microbial mixture prepared as in Example 2 above. The combination was mixed in a Kinematica POLYTRON® homogenizer (available from KINEMATICA AG, Luzernerstrasse 147A CH-6014 Littau, Switzerland) at 11,000 rpm for 20-30 seconds. The consistency of the cross-linked alginate became thick and paste-like. Calcium carbonate

[0154] Instead of calcium chloride, calcium carbonate solution was used for cross-linking. Cross-linked alginate-microbial preparations were prepared as follows: 0.09 grams of calcium carbonate was added to 100 grams of alginate-microbial mixture (AMM). The mixture was mixed vigorously in a horizontal shaker for 4 hours and left at room temperature for an additional 24 hours. The calcium carbonate was neutralized by adding 10 mL of either 100 mM acetate buffer (AC) or 100 mM glucono-delta-lactone (GDL), mixed vigorously in a horizontal shaker for 4 hours and left at room temperature for an additional 24 hours. Example 4. Drying of cross-linked alginate-microbial mixture

[0155] Spread the cross-linked alginate-microbe mixtures (CAMMs) onto a 9" x 13" baking sheet, place in a biohood with air recirculation, and air-dry the mixture at room temperature for at least 2 days until the moisture content reaches 5-10%. For example, air-dry CAMM beads or microbeads for 3 days at room temperature.

[0156] Alternatively, the cross-linked alginate-microbe mixtures (CAMMs) were spread onto a 9” x 13” baking sheet and placed in a biohood with air recirculation to allow the mixture to air dry at room temperature for 24 hours and then placed in a humidity controlled chamber at 23°C and 20% relative humidity (RH) to reduce the moisture content to 5-7%.

[0157] To further reduce the moisture content, the crosslinked alginate particles were dried in a Sherwood Scientific 50100201 Programmable Fluid Bed Dryer fluid bed dryer at 45-55° C. until the moisture content was reduced to approximately 3%. Example 5. Grinding of cross-linked alginate-microbial mixture

[0158] The cross-linked alginate-microbe mixture (CAMM) was milled to produce powder particles suitable for long-term storage and seed treatment. The cross-linked alginate-microbe mixture was freeze-milled using liquid nitrogen (Spex Sample Prep Model 6970D) with the following milling parameters: cycle: 3 min, pre-cool: 0 min, run time: 7 min, inter-run cooling: 1 min, speed 13 cycles per second (cps).

[0159] Alternatively, the material was reduced in size using a ball mill by filling 10 grams of dry material into a 125 mL jar with a 3×20 mm metal ball as the grinder. The container was shaken vigorously for 1 hour at room temperature using a horizontal shaker.

[0160] A typical milled preparation yielded particle size distributions of D10=8-15 μm, D50=75-100 μm, and D90=350-500 μm. Example 6. Spray drying method

[0161] Cross-linked alginate-microorganism mixtures (CAMMs) were prepared by the following spray drying method: Encapsulation and drying were completed using a BUECHI® Mini Spray Dryer B-290 (BUECHI Labortechnik AG, Flawil, Switzerland). Two-Fluid Nozzle Spray Drying and Polymer Encapsulation

[0162] CAMM formulations were spray dried using a BUECHI® B-290 laboratory spray dryer equipped with a standard two-fluid nozzle. The spray drying procedure for producing crosslinked alginate microparticles was as follows: air flow was set at 40 mm and air pressure was set at 90 psi. Suction was set at 100%, dehumidifier was set to "on", and pump speed was calibrated to ~10 mL / min with water and set at 32%. Inlet temperature was 110-170°C resulting in outlet temperatures ranging from 50-87°C. Three-fluid nozzle spray drying and polymer encapsulation

[0163] CAMM formulations were spray dried using a BUECHI® B-290 laboratory spray dryer equipped with a three-fluid nozzle. The spray drying procedure for producing crosslinked alginate microparticles was as follows: air flow was set to maximum, air pressure was set to 90 psi. Suction was set to 100%, dehumidifier was set to "on", and a WATSON MARLOW® 120U multichannel pump with 1.42 mm x 0.8 mm tubing (part number 983.0142.000) internal feed pump was calibrated to ~5 mL / min with water and set to 57%. Internal fluid contained equal amounts of microbial ferment and aqueous 4% sodium alginate / 2% soluble starch solution unless otherwise stated, external (BUECHI®) feed pump = 17% (calibrated to ~5 mL / min with water). External fluid: dilute aqueous calcium chloride (typically 0.125-0.25% by weight) unless otherwise stated. Cold water from a mains water supply was pumped through the spray nozzle assembly with inlet temperatures ranging from 110-120°C resulting in outlet temperatures ranging from 50-87°C. Example 7. Seed treatment

[0164] The microbial alginate formulations were applied to seeds using the following procedure: The formulations were applied to soybean seeds that were untreated or treated with crop protection chemicals.

[0165] To apply the wet or dry cross-linked alginate formulation to seeds, 250 grams of soybean seeds and 1.0 gram of seed treatment sugar solution (if used) were placed in a gallon-sized bag, sealed, and vigorously shaken. The bag was reopened, 1.5 grams of the cross-linked alginate formulation was added, the bag was resealed, and vigorously shaken to ensure an even coating. The sugar solution makes the surface of the seed "sticky" so that the alginate formulation powder coats and adheres to the surface of the seed.

[0166] Treated seeds were transferred to breathable lined paper bags (3.375 in. × 2.5 in. × 10.25 in.; Midco TTX591) for long-term storage at 25°C / 65% RH and / or 10°C / 50% RH. Example 8. Recovery of Pseudomonas fluorescens encapsulated in dry cross-linked alginate using the bead method

[0167] Pseudomonas fluorescens was encapsulated by the CAMM bead method, dried, and ground as described above in Examples 3, 4, and 5. The calcium chloride concentration used to gel the beads was varied from 0.5% to 5% to determine its effect on CFU recovery.

[0168] Table 1 As shown in Figure 1, CFU recovery of Pseudomonas fluorescens from CAMM beads immediately after encapsulation was highest at the lower calcium chloride concentrations tested (0.5% CaCl2 had the highest CFU recovery). CFU recovery was determined by plating on agar plates. Table 1: Initial CFU recovery of Pseudomonas fluorescens encapsulated in dried CAMM beads made with different concentrations of calcium chloride. [Table 1] Example 9. Recovery of Pseudomonas fluorescens encapsulated in dry cross-linked alginate prepared by shear mixing method

[0169] Pseudomonas fluorescens was encapsulated by the CAMM shear mixing method described in Example 3 above with or without the addition of different combinations of hydrogels (chitosan, pectin, gelatin) and / or inert carriers (biochar) to the alginate-microbe mixture prior to crosslinking. When added, the ratio of alginate to added hydrogel was 1:1 and the total hydrogel concentration was 2%. When no additional hydrogel was added, the alginate concentration was 2%, whereas when additional hydrogel was added, the alginate and hydrogel concentrations were 1% each (combined for a total of 2%). When added, the ratio of alginate to added biochar was 2:5 by dry weight. Crosslinked preparations were dried and ground as described in Examples 4 and 5 above.

[0170] All combinations with or without hydrogel or inert carrier were at 1 × 10 8 Many combinations yielded recoveries in excess of CFU / gram. Table 2 1 x 10 of Pseudomonas fluorescens strain as indicated. 9 Recovery was greater than CFU / gram, demonstrating the flexibility of the hydrogel type for the stabilization methods described herein. Table 2: Initial CFU recovery of Pseudomonas fluorescens encapsulated in cross-linked alginate produced by shear mixing method. [Table 2] Example 10. Recovery of Pseudomonas fluorescens encapsulated in dry cross-linked alginate prepared by calcium carbonate method

[0171] Pseudomonas fluorescens was cultured using different alginate materials by the calcium carbonate method described in Example 3 above, and by the method described in Examples 9 and 10 above. Table 2The alginate-microbe mixture was encapsulated with or without the addition of hydrogel (chitosan, pectin, gelatin) prior to crosslinking in the combinations provided in. The ratio of alginate to additional hydrogel was 1:1, with a total hydrogel concentration of 2% as above. The crosslinked formulations were dried and ground as described above in Examples 4 and 5.

[0172] CAMMs prepared in various amounts of acetate buffer (AC) or glucono-delta-lactone (GDL) were 1 × 10 8 Recovery of CFU / gram was achieved in excess of 100%. Many preparations Table 3 As shown in 9 The recovery of CFU / gram exceeded 100%, demonstrating the flexibility of the amount of acidifying agent for the stabilization method described herein using calcium carbonate as a crosslinker. CAMMs made with different types of hydrogels were Table 4 As shown in 9 Recoveries of greater than CFU / gram were achieved, demonstrating the flexibility of hydrogel types for the various alginate stabilization methods described herein. Table 3: Initial CFU recovery of Pseudomonas fluorescens encapsulated in cross-linked alginate prepared with different amounts of acidifier by calcium carbonate method. [Table 3] Table 4: Initial CFU recovery of Pseudomonas fluorescens encapsulated in cross-linked alginate prepared with added hydrogels by the calcium carbonate method. [Table 4] Example 11. Recovery of Pseudomonas fluorescens encapsulated in dried cross-linked alginate prepared by three-fluid nozzle spray drying

[0173] Shear-crosslinked alginate containing Pseudomonas fluorescens was produced and spray dried using a three-fluid nozzle spray dryer as described in Example 3 above with different amounts of calcium chloride. Initial CFU determinations (starting titers) of the spray-dried CAMM powder were Table 5 As shown in. Table 5: In-Situ Crosslinked Spray-Dried Formulations Containing P. fluorescens [Table 5] Example 12. On-Seed Viability of Pseudomonas fluorescens with Various Cross-Linked Alginate Preparations Alginate cross-linking before spray drying

[0174] Cross-linked alginate containing Pseudomonas fluorescens was produced using the shear mixing method as described above in Example 3, spray dried using a two-fluid nozzle spray dryer as described above in Example 6, and applied to soybean seeds as provided above in Example 7. As shown in FIG. 1, Pseudomonas fluorescens encapsulated in the shear cross-linked alginate mixture and applied to seeds maintained a population of 1×10 for over 8 weeks. 5 Viability remained above CFU / seed. Direct seed treatment with cross-linked alginate paste

[0175] Pseudomonas fluorescens was encapsulated in alginate by the shear mixing method described in Example 3 above and applied to soybean seeds as described herein, except that the cross-linked alginate paste was not spray-dried prior to application to the seeds. In this test, the initial microbial titer was 1.7 x 10 for the cross-linked alginate paste. 9 CFU / g. The effects of chemical (AI), bisulfite treatment (to remove biocides in the chemical slurry), and osmoprotectant solution on microbial stability were determined. The theoretical maximum of CFU / seed during seed treatment, assuming no CFU loss, was 1 × 10 6 The actual initial CFU / seed measured for all samples in this study was 10 4 ~10 5 CFU / seed, and after 1 week on the seeds, the CFU / seed experienced a 1-3 log reduction ( Table 6 (see ).

[0176] The stability of Pseudomonas fluorescens on seeds after direct seed treatment was poor when encapsulated in an alginate paste and applied to seeds. Pseudomonas fluorescens applied to seeds as a cross-linked alginate paste that was not spray dried reached 1 × 10 2 Viability fell to less than CFU / seed (i.e., at least a 2-3 log drop in CFU / seed after just one week on the seeds) (see FIG. 1). Table 6: Stability of Pseudomonas fluorescens on seeds at 25 °C / 65% RH from direct seed treatment with cross-linked alginate paste. [Table 6] Effect of encapsulation method and drying rate on the stability of encapsulated Pseudomonas fluorescens at different temperatures and humidities

[0177] Pseudomonas fluorescens was encapsulated in cross-linked alginate via shear mix or bead methods, crushed, and placed on seeds as described above. As shown in Figure 1, the decay rate of CFU on seeds / seed was 0.15 log / week for seeds stored at 25 °C and 65% RH, in contrast to the nearly 2 log drop per week for the spray dried liquid ferment applied to seeds. Seeds treated with the wet shear mixed alginate formulation without spray drying had a 1 × 10 2 The results showed a reduction in viability of the microbial strains to less than CFU / seed. These results indicate that a slower drying method for the cross-linked alginate mixture provides a more stable microbial formulation for application on seeds.

[0178] As further shown in FIG. 2, the number of CFU per seed remained virtually unchanged over the 23-week test period at 10°C and 50% RH. In a further study, 10 grams of alginate-encapsulated Pseudomonas fluorescens were stored in 20 mL glass scintillation vials, 2 grams of desiccant packages were added, the vials were capped and stored in a chamber at 25°C. Samples were taken periodically to track their long-term viability, as shown in FIG. 3. Effect of calcium concentration on stability on seeds

[0179] Pseudomonas fluorescens was encapsulated in alginate by shear mixing, ground, and placed on seeds as described herein. The amount of calcium chloride used to cross-link the alginate was varied. Pseudomonas fluorescens encapsulated in alginate using the shear mixing method showed good stability within the first 4 weeks. For samples cross-linked with 2.5 mL of 1% w / w calcium loading (for 100 mL of alginate-microbe mixture), the decay rate averaged 0.15 log / week, as shown in Figure 6. Stability on seeds with additional hydrogel or biochar

[0180] Pseudomonas fluorescens was encapsulated by the CAMM shear mixing method with the addition of hydrogels (chitosan, pectin, gelatin) and inert carriers (biochar) to the alginate-microbe mixture prior to crosslinking. The ratio of alginate to added hydrogel was 1:1, with a total hydrogel concentration of 2%. The ratio of alginate to added biochar was 2:5 by dry weight. The crosslinked formulation was dried, ground as above, placed on seeds, stored in breathable paper bags, and exposed to warm temperatures and humidity of 25 °C / 65% RH. Encapsulation with alginate as the only hydrogel yielded the greatest stability. The addition of biochar as a solid excipient further improved stability, with less than a 1 log decrease from weeks 3–7, indicating that biochar acts as a stabilizer as shown in Figure 7. Stability of spray-dried Pseudomonas fluorescens formulations on seeds

[0181] Pseudomonas fluorescens was encapsulated by the three-fluid nozzle spray drying CAMM shear mixing method, placed on the seeds, stored in a breathable paper bag, and stored for the following period: Table 7 The seeds were exposed to warm temperatures and humidity at 25°C / 65%RH as shown in Figure 1. The stability results on those seeds are shown in Figure 1. Drying and calcium chloride loading did not affect the stability trends. The inclusion of soluble starch also had no significant effect. Table 7: In-situ cross-linked spray-dried formulations containing Pseudomonas fluorescens used in the stability study on seeds. [Table 7] Example 13. Survival of Bradyrhizobium japonicum on seeds with different cross-linked alginate preparations Effect of encapsulation method and drying rate on the stability of encapsulated Bradyrhizobium japonicum at 25°C / 65% RH and 10°C / 50% RH

[0182] Bradyrhizobium japonicum was encapsulated in alginate by the shear mixing and beading method, crushed, and placed on seeds as described herein. In contrast, Bradyrhizobium japonicum was also spray dried and processed on seeds to compare the effect of drying rate on stability. As shown in FIG. 4, at 25°C / 65% RH, the CFU decay rates on seeds for both the beading and shearing methods were comparable at 0.26-0.31 log / week. This is in contrast to the approximately 1.5 log drop per week for spray-dried Bradyrhizobium. FIG. 5 shows that at 10°C / 50% RH, CFU decay on seeds is substantially reduced to 0.7 log over 23 weeks. Stability of spray-dried Bradyrhizobium japonicum formulations on seeds

[0183] Bradyrhizobium japonicum was encapsulated by the twin-fluid nozzle spray drying CAMM shear mixing method, placed on the seeds, stored in breathable paper bags, and exposed to warm temperature and humidity of 25 °C / 65% RH. table The formulations were compared to a commercial peat-based formulation as shown in Figure 1. The CFU / seed values ​​of these formulations were evaluated over several weeks and log 10 was used to determine the rate of decay. Table 8: Initial CFU / seed values ​​and decay rates on seeds (per week) calculated from storage stability tests at 25°C / 65% RH for various Bradyrhizobium japonicum preparations. [Table 8] Example 14. Release studies of encapsulated microorganisms

[0184] Alginate-encapsulated Pseudomonas formulated in a bead method with 0.5% CaCl2 was used as a model encapsulant to evaluate the release of calcium cross-linked alginate-encapsulated microorganisms. Studies were conducted to determine how readily this preparation could deliver viable cells to the environment under simulated planting conditions as a function of time and soil moisture content.

[0185] Farm soil collected from Greenville, MS was oven-dried overnight at 65 °C and sterilized by autoclaving (121 °C, 15 psi, 30 min). Five gram aliquots of the sterilized soil were dispensed into sterile 50 mL centrifuge tubes, and samples with moisture contents ranging from about 0% to about 30% were created by the addition of sterile water. Soybean seeds were sown into these samples, Table 9 Under these conditions, the bacteria were assessed for their ability to germinate after 96 h at 22°C as shown in . Table 9: Composition of soil samples and resulting effects on soybean germination. [Table 9]

[0186] The alginate-encapsulated microbial preparations used in this study were approximately 1 × 10 9 This material was combined with dry sterile field soil and mixed to determine approximately 2.4 x 10 CFU / g. 3 Fresh soil preparations containing CFU / g were made by distributing 5 g aliquots of the soil + alginate-encapsulated P. fluorescens mixture into sterile 50 mL centrifuge tubes, and samples with moisture content ranging from approximately 0% to approximately 30% were made in triplicate by adding sterile water as described above.

[0187] Each sample was incubated at 22°C after the addition of water. Aliquots were taken at 24 and 96 hours and assayed for CFU / g using serial dilution plating and colony counts on R2A growth medium. These counts were Table 10 This is summarized in Figure 8. Table 10: CFU / g after 24 and 96 hours of incubation at various soil moisture contents. [Table 10]

[0188] The results of this study show that microbial populations are readily released from this alginate encapsulation and grow vigorously to high titers after 96 h of incubation in soil with a moisture content of 10-30%. Microbial release and subsequent growth is slower in drier soils, as shown by the initial lack of growth after 24 h of incubation in soil with a moisture content of 10%. Concentrations of viable cells were approximately 2.4 x 10 3 The encapsulated material appears to be stable over this time frame in dry soil as the original baseline titers of CFU / g remained. Example 15. Tap density measurements and moisture sorption characteristics of various cross-linked alginate preparations containing Pseudomonas fluorescens

[0189] Various dry alginate formulations containing Pseudomonas fluorescens were produced by the CAMM shear mixing method in Example 3 and the spray drying method in Example 6. The tap densities of these powder formulations were determined using a manual method using 2 mL cryogenic vials as measuring vessels and were calculated as follows: Table 11 The moisture uptake of these formulations was compared after drying overnight at 40° C. in a vacuum oven in a Dynamic Vapor Sorption (DVS) system (such as the DVS system from Surface Measurement Systems) using the following program: 0.5 hours at 0% RH (then mass adjusted to 100%), 10 hours at 90% RH, and 10 hours at 0% RH. The DVS data (FIG. 9) show that the denser formulations absorb less moisture under the same % RH. The higher the moisture uptake rate, the less stable the spray dried formulations shown in FIG. 1 are on seeds. Table 11 [Table 11] Example 16. Survival of Herbaspirillum frisingense on seeds in cross-linked alginate preparations Effect of formulation additives on the stability of encapsulated Herbaspirillum frisingense at 25°C / 65% RH compared to liquid fermentation

[0190] Herbaspirillum frisingense was encapsulated in alginate by shear mixing, dried in sheets, crushed by freeze grinding, and placed on seeds as described herein. Herbaspirillum frisingense fermentation was applied directly onto the seeds to compare the stability of the encapsulated formulations to that of the liquid formulations on the seeds. The fermentation was applied in a similar manner to the seed treatment described in Example 7, with 1 mL of fermentation broth applied directly onto the seeds without a binder solution. The treated seeds were transferred to poly-lined paper bags (3.375 in. x 2.5 in. x 10.25 in.; Midco BA TT CW-6L) for long-term storage at 25°C / 65% RH. As shown in FIG. 10, at 25°C / 65% RH, the stability of all encapsulated formulations on the seeds is significantly better than the liquid fermentation applied directly onto the seeds. Example 17. Survival of encapsulated Pseudacidovorax intermedius on seeds compared to liquid fermentation

[0191] Pseudacidovorax intermedius was encapsulated in alginate by shear mixing, dried in sheets, crushed by freeze grinding, and placed on seeds as described herein. Pseudacidovorax intermedius fermentation was also applied directly onto seeds to compare the stability of the encapsulated formulations with the liquid ones on seeds. The fermentation was applied in a similar manner to the seed treatment described in Example 7, using 1 mL of fermentation broth and without adding binder solution, and applied directly onto the seeds. These treated seeds were transferred to poly-lined paper bags (3.375 in. x 2.5 in. x 10.25 in.; Midco BA TT CW-6L) for long-term storage at 25°C / 65% RH. As shown in Figure 11, at 25°C / 65% RH, the stability of all encapsulated formulations on seeds is significantly better than the liquid fermentation applied directly onto the seeds. Example 18. Survival of Phytobacter diazotrophicus on seeds in different cross-linked alginate preparations Effect of formulation additives on the stability of encapsulated Phytobacter diazotrophicus at 25°C / 65% RH compared to liquid fermentation

[0192] Phytobacter diazotrophicus was encapsulated in alginate by shear mixing method, dried on sheets, crushed by freeze grinder and placed on seeds as described herein. Table 12 Several commercially available hydrophobic additives, as shown in Figure 1, were included in the AMM before crosslinking at 1.5 wt / vol% in the mixture to determine the effect of these additives on the water uptake and on-seed stability of the powder formulation before seed treatment. Table 12 [Table 12]

[0193] After drying and milling, the powder formulations were further dried in a vacuum oven at 40° C. for 24 hours. Moisture uptake was assessed at 65% RH via a Dynamic Water Vapor Sorption (DVS) instrument (Surface Measurement Systems DVS Advantage). The DVS program was as follows: 6 hours at 0% RH (then mass adjusted to 100%), 40 hours at 65% RH, and 20 hours at 0% RH. FIG. 12 shows that the incorporation of hydrophobic additives reduces the mass change upon exposure to 65% RH, with additives #2 and #3 having the greatest reduction in moisture uptake.

[0194] Phytobacter diazotrophicus fermentation was applied directly onto the seeds to compare the stability of the different encapsulated formulations with that of the liquid fermentation on the seeds. The fermentation was applied in a similar manner to the seed treatment described in Example 7, and 1 mL of the fermentation broth was applied directly onto the seeds without any binder. These treated seeds were transferred to poly-lined paper bags (3.375 in. x 2.5 in. x 10.25 in.; Midco BA TT CW-6L) for long-term storage at 25°C / 65% RH. As shown in FIG. 13, the on-seed stability of all encapsulated formulations is significantly better than the liquid fermentation applied directly onto the seeds at 25°C / 65% RH. The formulations made with additives #2 and #3 had the best on-seed stability. These formulations had the lowest water uptake in the DVS data shown in FIG. 12. Example 19. Incorporation of a hydrophobic additive as an external coating Adding a hydrophobic coating in a spray dryer

[0195] Two CAMMs containing Pseudomonas fluorescens were made using the three-fluid nozzle spray drying procedure listed in Example 6. The first formulation used 0.125 wt% calcium chloride in the external fluid of the spray dryer to create in situ crosslinked alginate microparticles. The second formulation used 0.125 wt% calcium chloride + 1 wt% silk fibroin (supplied as 5 wt% by Sigma Aldrich; catalog #5154-20ML). All other spray dryer settings were the same. The hygroscopicity of these formulations was compared in a DVS instrument after drying overnight at 40°C in a vacuum oven using the following program: 0% RH for 0.5 hours (then mass adjusted to 100%), 90% RH for 10 hours, and 0% RH for 10 hours. The DVS data (Figure 14) show that the formulation with the hydrophobic coating absorbs less water at the same % RH. These formulations were applied onto seeds as described in Example 7. At 25 °C / 65% RH, the spray-dried in situ cross-linked alginate formulation showed a decay rate of ~2.5 logs per week, while the formulation containing silk fibroin in the external fluid was more stable on seeds with a decay rate of ~1.5 logs per week. Adding a hydrophobic coating in a fluid bed dryer

[0196] CAMM microbeads containing Bradyrhizobium japonicum were produced using a 200 μm nozzle as described in Example 3. These microbeads were dried in a fluid bed dryer at 40° C. with or without 10 wt. % SURELEASE® (Colorcon; 25 wt. % aqueous dispersion of ethylcellulose) sprayed onto the wet microbeads during drying. The coating load was ∼5% of the weight of the dry CAMM microbeads without coating. The hygroscopicity of these formulations was compared in a DVS instrument after drying overnight at 40° C. in a vacuum oven using the following program: 65% RH for 24 hours, followed by 90% RH for 12 hours. The DVS data (FIG. 15) show that the formulation with the hydrophobic coating absorbs much less moisture at the same % RH.

[0197] Numerous specific details are described, such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that the exemplary embodiments may be embodied in many different forms, and neither should be construed as limiting the scope of the present disclosure. In some exemplary embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0198] The terms used herein are for the purpose of describing exemplary embodiments only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" may be intended to include the plural unless the context clearly indicates otherwise. The terms "comprises," "comprising," "including," and "having" are inclusive and thus specify the presence of a stated feature, element, composition, step, integer, operation, and / or component, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The open-ended term "comprising" should be understood as an open-ended term used to describe and claim the various embodiments described herein, although in certain aspects the term may instead be understood as a more restrictive and limiting term such as "consisting of" or "essentially of." Thus, for any given embodiment that recites compositions, materials, components, elements, features, integers, operations, and / or process steps, the disclosure also specifically includes embodiments that consist of, or consist essentially of, such recited compositions, materials, components, elements, features, integers, operations, and / or process steps. Whereas "consisting of" alternative embodiments exclude any additional compositions, materials, components, elements, features, integers, operations, and / or process steps, where "consisting essentially of" excludes from such embodiments any additional compositions, materials, components, elements, features, integers, operations, and / or process steps that do not substantially affect the basic and novel characteristics, but may be included in the embodiments.

[0199] Any method steps, processes, and operations described herein should not be construed as necessarily requiring their performance in the particular order described or illustrated, unless specifically identified as an order of performance, and it should be understood that additional or alternative steps may be used, unless otherwise indicated.

[0200] Throughout this disclosure, numerical values ​​represent approximate measures or limits for a given value and ranges encompassing embodiments having slight deviations from and about the stated value, as well as embodiments having the exact stated value. Except for the examples provided at the end of the detailed description, all numerical values ​​of parameters (e.g., of amounts or conditions) in this specification, including the appended claims, should be understood to be modified in all cases by the term "about", regardless of whether "about" actually appears before the numerical value. "About" indicates that the stated numerical value allows for some slight imprecision (with some approach to the precision of the value; approximately or reasonably close to the value; approximately). If the imprecision provided by "about" is not otherwise understood in the art in this ordinary sense, "about" as used herein indicates at least the variation that may result from ordinary methods of measuring and using such parameters. For example, "about" can include a variation of 5% or less, optionally 4% or less, optionally 3% or less, optionally 2% or less, optionally 1% or less, optionally 0.5% or less, and in certain aspects, optionally 0.1% or less.

[0201] Additionally, the disclosure of ranges includes the disclosure of all values, as well as further divided ranges within that entire range, including the endpoints and subranges given for the range.

[0202] The above detailed description is provided to assist those skilled in the art in practicing the present disclosure. However, the disclosure described and claimed herein is not limited in scope by the specific embodiments disclosed herein, since these embodiments are intended as illustrations of some aspects of the present disclosure. Any equivalent embodiments are intended to be within the scope of the present disclosure. Various modifications of the present disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description without departing from the spirit or scope of the present disclosure. Also, such modifications fall within the scope of the appended claims.

Claims

1. 1. A method for preparing an encapsulated microbial composition comprising: (a) combining a microorganism with at least one hydrogel to form a precursor mixture, the at least one hydrogel comprising an alginate; (b) solidifying the precursor mixture with a cross-linking agent comprising a divalent cation, a divalent cation salt, or a combination thereof to form an intermediate microbial composition; and (c) drying the intermediate microbial composition to form an encapsulated microbial composition having a moisture content of about 10% or less, said drying being performed at a drying temperature of about 15° C. or greater and at a drying rate of about 25,000 g / hr / m ２ and performing the method at an evaporation rate of:

2. 1. A method for preparing an encapsulated microbial composition comprising: (a) combining a microorganism with at least one hydrogel to form a precursor mixture, the at least one hydrogel comprising an alginate; (b) solidifying the precursor mixture with a cross-linking agent comprising a divalent cation, a divalent cation salt, or a combination thereof to form an intermediate microbial composition; and (c) drying the intermediate microbial composition to form an encapsulated microbial composition having a tap density of about 0.5 g / mL or more and about 1.5 g / mL or less.

3. 3. The method of claim 1 or claim 2, wherein the alginate is selected from the group consisting of sodium alginate, potassium alginate, barium alginate, calcium alginate, magnesium alginate, strontium alginate, and combinations thereof.

4. 3. The method of claim 1 or claim 2, wherein the at least one hydrogel further comprises carboxymethylcellulose, carrageenan, cellulose acetate phthalate, chitosan and chitosan derivatives, collagen, gelatin, glycosaminoglycans, guar gum, acacia gum, hyaluronic acid, hydroxyethylcellulose, hydroxypropylmethylcellulose, locust bean gum, methylcellulose, pectin, polyethylene oxide, polyglycolic acid, polyurethane, starch, modified starch, xanthan gum, or combinations thereof.

5. 3. The method of claim 1 or claim 2, wherein the at least one hydrogel is present in the precursor mixture in an amount of about 5% to about 50% by weight, based on the total weight of the precursor mixture.

6. The method of claim 1 or claim 2, wherein the precursor mixture includes a nitrogen-containing stabilizer.

7. 7. The method of claim 6, wherein the nitrogen-containing stabilizer is selected from the group consisting of ammonia, ammonium hydroxide, urea, ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, gluten, hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, malt extract, milk powder, whey powder, yeast extract, and combinations thereof.

8. The method of claim 6, wherein the nitrogen-containing stabilizer is present in the precursor mixture in an amount of about 5% to about 50% by weight, based on the total weight of the precursor mixture.

9. The method of claim 1 or claim 2, wherein the precursor mixture includes a sugar-containing stabilizer.

10. 10. The method of claim 9, wherein the sugar-containing stabilizer is a disaccharide or a sugar alcohol.

11. 10. The method of claim 9, wherein the sugar-containing stabilizer is selected from the group consisting of mannitol, sorbitol, xylitol, trehalose, sucrose, lactose, and combinations thereof.

12. 10. The method of claim 9, wherein the sugar-containing stabilizer is present in the precursor mixture in an amount of about 5% to about 50% by weight, based on the total weight of the precursor mixture.

13. The method of claim 1 or claim 2, wherein the microorganism is a non-spore-forming bacterium.

14. The microorganism is selected from the group consisting of Pseudomonas, Bradyrhizobium, Herbaspirillum, Phytobacter, Pseudoacidovorax, Mitsuria, Azospirillum, Burkholderia, Chryseomonas, Aeromonas, and the like. monas, Acinetobacter, Stenotrophomonas, Chromobacterium, Agrobacterium, Chryseobacterium, Xenorhabdus, Photorhabdus, and combinations thereof.

15. 3. The method of claim 1 or claim 2, wherein the microorganism is selected from the group consisting of Pseudomonas fluorescens, Bradyrhizobium japonicum, Phytobacter diazotrophicus, Herbaspirillum frisingense, and combinations thereof.

16. The method of claim 1 or claim 2, wherein the precursor mixture further comprises a filler.

17. 17. The method of claim 16, wherein the filler is selected from the group consisting of starch, cellulose, biochar, pumice, silica, clay, zeolite, perlite, or combinations thereof.

18. The method of claim 16, wherein the filler is present in the precursor mixture in an amount of about 0.1% to about 90% by weight, based on the total weight of the precursor mixture.

19. 3. The method of claim 1 or claim 2, wherein the divalent cation is a barium cation, a calcium cation, a magnesium cation, a strontium cation, a cobalt cation, a nickel cation, a zinc cation, a manganese cation, or a combination thereof.

20. 3. The method of claim 1 or claim 2, wherein the divalent cation salt is a barium salt, a calcium salt, a magnesium salt, a strontium salt, a cobalt salt, a zinc salt, a manganese salt, or a combination thereof.

21. The method of claim 1 or claim 2, wherein the crosslinker further comprises a solvent.

22. 3. The method of claim 1 or claim 2, wherein the solidifying comprises contacting droplets of the precursor mixture with the cross-linking agent, and the intermediate microbial composition is in the form of cross-linked beads.

23. 23. The method of claim 22, wherein the crosslinked beads have an average particle size of about 1 mm to about 20 mm.

24. 3. The method of claim 1 or claim 2, wherein the solidifying comprises flowing the precursor mixture through an opening having a diameter of about 100 μm to about 500 μm and contacting the precursor mixture with the cross-linking agent, and the intermediate microbial composition is in the form of cross-linked microbeads.

25. 25. The method of claim 24, wherein the crosslinked microbeads have an average particle size of about 100 μm to about 20 mm.

26. 23. The method of claim 22, wherein the divalent cation is present in an amount from about 0.1% to about 10% of the total dry weight of the encapsulated microbial composition.

27. The method of claim 1 or claim 2, wherein the solidifying comprises high shear mixing of the precursor mixture with the crosslinker.

28. 28. The method of claim 27, wherein the high shear mixing is carried out at a tip speed of at least about 3.5 m / s for at least about 5 seconds.

29. 30. The method of claim 28, wherein the high shear mixing is carried out at a tip speed of about 3.5 m / s to about 25 m / s for about 5 seconds to about 20 minutes.

30. 28. The method of claim 27, wherein the high shear mixing is carried out at a temperature of from about 4°C to about 30°C.

31. 3. The method of claim 1 or claim 2, wherein the solidifying comprises combining the precursor mixture with the cross-linking agent to form the intermediate microbial composition, the cross-linking agent comprising the divalent cation salt, and the divalent cation salt is a divalent cation carbonate.

32. 32. The method of claim 31 , wherein the divalent cation carbonate is barium carbonate, calcium carbonate, magnesium carbonate, strontium carbonate, cobalt carbonate, nickel carbonate, zinc carbonate, manganese carbonate, or a combination thereof.

33. 32. The method of claim 31 , wherein the divalent cation carbonate is calcium carbonate.

34. 32. The method of claim 31, wherein the solidifying further comprises adding an acidic buffer.

35. 35. The use of claim 34, wherein the acidic buffer is added (i) to the precursor mixture and / or the crosslinker before the crosslinker is combined with the precursor mixture; (ii) to the precursor mixture substantially simultaneously with the crosslinker; (iii) to a mixture comprising the precursor mixture and the crosslinker; or to any combination of (i), (ii), and (iii).

36. 36. The method of claim 35, wherein the mixture has a pH of about 4 to about 6.

5.

37. 32. The method of claim 31 , wherein the combining is carried out for about 5 minutes or more and / or at a temperature of about 4° C. or more.

38. 38. The method of claim 37, wherein the combining is carried out for about 5 minutes to about 48 hours and / or at a temperature of about 4°C to about 40°C.

39. 32. The method of claim 31 , wherein said combining comprises mixing said precursor mixture with said crosslinker.

40. 28. The method of claim 27, wherein the divalent cation is present in an amount equivalent to about 0.001% to about 25% of the total dry weight of the encapsulated microbial composition.

41. 30. The method of claim 27, further comprising the step of applying the intermediate microbial composition to a substrate, wherein the intermediate microbial composition is in the form of a film on the substrate.

42. The method of claim 41, wherein the film has a thickness of about 1 mm to about 1 cm.

43. The method of claim 1 or claim 2, wherein the drying temperature is from about 15°C to about 60°C.

44. 3. The method of claim 1 or claim 2, wherein the drying time is about 15 minutes or more.

45. 45. The method of claim 44, wherein the drying time is from about 15 minutes to about 200 hours.

46. The evaporation rate is about 15,000 g / hr / m ２ 3. The method of claim 1 or claim 2, wherein:

47. The evaporation rate is about 10,000 g / hr / m ２ 3. The method of claim 1 or claim 2, wherein:

48. The evaporation rate is about 5,000 g / hr / m ２ 3. The use according to claim 1 or claim 2, wherein:

49. The evaporation rate is about 1 g / hr / m ２ ~Approx. 500g / hr / m ２ The method according to claim 1 or claim 2, wherein

50. 3. The method of claim 1 or claim 2, further comprising processing the encapsulated microbial composition to form encapsulated microbial particles.

51. 51. The method of claim 50, further comprising reducing the size of the encapsulated microorganism particles to have an average particle size of about 200 μm or less.

52. 52. The method of claim 51, wherein reducing the size of the encapsulated microorganism particles comprises crushing the encapsulated microorganism particles.

53. 3. The method of claim 1 or claim 2, wherein the encapsulated microbial composition has a moisture content of about 5% or less.

54. 3. The method of claim 1 or claim 2, wherein the encapsulated microbial composition has a loss of colony forming units per gram (CFU / g) of less than 2 log after 4 weeks, 8 weeks, or 12 weeks at 25° C. and 65% relative humidity (RH).

55. 3. The method of claim 1 or claim 2, wherein the encapsulated microbial composition has a loss of colony forming units per gram (CFU / g) of less than 1 log after 4 weeks, 8 weeks or 12 weeks at 25° C. and 65% relative humidity (RH).

56. 3. The method of claim 1 or claim 2, further comprising applying the encapsulated microbial composition to one or more of a plant seed, a plant, a plant part, and a plant environment.

57. 57. The method of claim 56, wherein the plant seed further comprises a protectant, a herbicide, a pesticide, a fungicide, or a combination thereof.

58. 57. The method of claim 56, wherein the plant seed is selected from the group consisting of a vegetable seed, a fruit seed, a cereal seed, and combinations thereof.

59. 3. The method of claim 1 or claim 2, further comprising combining at least one hydrophobic additive with said microorganism and said at least one hydrogel to form said precursor mixture.

60. 3. The method of claim 1 or claim 2, wherein drying the intermediate microbial composition to form the encapsulated microbial composition comprises one or more of air drying the intermediate microbial composition, vacuum drying the intermediate microbial composition, and / or drying the intermediate microbial composition in a fluidized bed.

61. 1. A method for preparing an encapsulated microbial composition, the method comprising: (a) combining a microorganism with at least one hydrogel to form a precursor mixture, the at least one hydrogel comprising an alginate; (b) solidifying the precursor mixture with a cross-linking agent to form an intermediate microbial composition in the form of cross-linked beads, the solidifying comprising contacting droplets of the precursor mixture with a cross-linking agent, the cross-linking agent comprising a divalent cation, a divalent cation salt, or a combination thereof; and (c) drying the intermediate microbial composition to form an encapsulated microbial composition having a moisture content of about 10% or less.

62. 1. A method for preparing an encapsulated microbial composition, the method comprising: (a) combining a microorganism with at least one hydrogel to form a precursor mixture, the at least one hydrogel comprising an alginate; (b) solidifying the precursor mixture with a cross-linking agent to form an intermediate microbial composition in the form of cross-linked microbeads, wherein the solidifying comprises flowing the precursor mixture through an opening having a diameter of about 100 μm to about 500 μm to contact the precursor mixture with the cross-linking agent, and wherein the cross-linking agent comprises a divalent cation, a divalent cation salt, or a combination thereof; and (c) drying the intermediate microbial composition to form the encapsulated microbial composition having a moisture content of about 10% or less.

63. 1. A method for preparing an encapsulated microbial composition, the method comprising: (a) combining a microorganism with at least one hydrogel to form a precursor mixture, the at least one hydrogel comprising an alginate; (b) solidifying the precursor mixture with a cross-linking agent to form an intermediate microbial composition, the solidifying comprising high shear mixing the precursor mixture with the cross-linking agent, and the cross-linking agent comprising a divalent cation, a divalent cation salt, or a combination thereof; and (c) drying the intermediate microbial composition to form the encapsulated microbial composition having a moisture content of about 10% or less.

64. 1. A method for preparing an encapsulated microbial composition, the method comprising: (a) combining a microorganism with at least one hydrogel to form a precursor mixture, the at least one hydrogel comprising an alginate; (b) solidifying the precursor mixture with a cross-linking agent comprising a divalent cation salt to form an intermediate microbial composition, the solidifying comprising combining the precursor mixture with the cross-linking agent, and the divalent cation salt is a divalent cation carbonate; and (c) drying the intermediate microbial composition to form the encapsulated microbial composition having a moisture content of about 10% or less.

65. An encapsulated microbial composition comprising: a microorganism embedded in a polymer matrix comprising at least one hydrogel bound together with a divalent cation, wherein the at least one hydrogel comprises an alginate; and the encapsulated microbial composition has a moisture content of about 10% or less.

66. An encapsulated microbial composition comprising: a microorganism embedded in a polymer matrix comprising at least one hydrogel associated with a divalent cation, wherein the at least one hydrogel comprises an alginate; and the encapsulated microbial composition has a tap density of about 0.5 g / mL or more and about 1.5 g / mL or less.

67. 67. The composition of claim 65 or claim 66, wherein the alginate is selected from the group consisting of sodium alginate, potassium alginate, barium alginate, calcium alginate, magnesium alginate, strontium alginate, and combinations thereof.

68. 67. The composition of claim 65 or claim 66, wherein the at least one hydrogel further comprises carboxymethylcellulose, carrageenan, cellulose acetate phthalate, chitosan and chitosan derivatives, collagen, gelatin, glycosaminoglycans, guar gum, acacia gum, hyaluronic acid, hydroxyethylcellulose, hydroxypropylmethylcellulose, locust bean gum, methylcellulose, pectin, polyethylene oxide, polyglycolic acid, polyurethane, starch, modified starch, xanthan gum, or combinations thereof.

69. 67. The composition of claim 65 or claim 66, wherein the at least one hydrogel is present in an amount of about 5% to about 50% by weight, based on the total weight of the composition.

70. 67. The composition of claim 65 or claim 66, wherein the polymer matrix further comprises a nitrogen-containing stabilizer.

71. 71. The composition of claim 70, wherein the nitrogen-containing stabilizer is selected from the group consisting of ammonia, ammonium hydroxide, urea, ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, gluten, hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, malt extract, milk powder, whey powder, yeast extract, and combinations thereof.

72. 71. The composition of claim 70, wherein the nitrogen-containing stabilizer is present in an amount of about 5% to about 50% by weight, based on the total weight of the composition.

73. 67. The composition of claim 65 or claim 66, wherein the polymer matrix further comprises a sugar-containing stabilizer.

74. 74. The composition of claim 73, wherein the sugar-containing stabilizer is a disaccharide or a sugar alcohol.

75. 74. The composition of claim 73, wherein the sugar-containing stabilizer is selected from the group consisting of mannitol, sorbitol, xylitol, trehalose, sucrose, lactose, and combinations thereof.

76. 74. The composition of claim 73, wherein the sugar-containing stabilizer is present in an amount of about 5% to about 50% by weight, based on the total weight of the composition.

77. 67. The composition of claim 65 or claim 66, wherein the microorganism is a non-spore-forming bacterium.

78. The microorganism is selected from the group consisting of Pseudomonas, Bradyrhizobium, Herbaspirillum, Phytobacter, Pseudoacidovorax, Mitsuria, Azospirillum, Burkholderia, Chryseomonas, Aeromonas, and the like. onas, Acinetobacter, Stenotrophomonas, Chromobacterium, Agrobacterium, Chryseobacterium, Xenorhabdus, Photorhabdus, and combinations thereof.

79. 67. The composition of claim 65 or claim 66, wherein the microorganism is selected from the group consisting of Pseudomonas, Bradyrhizobium, Phytobacter, Herbaspirillum, Mitsuria, and combinations thereof.

80. 67. The composition of claim 65 or claim 66, wherein the microorganism is selected from the group consisting of Pseudomonas fluorescens, Bradyrhizobium japonicum, Phytobacter diazotrophicus, Herbaspirillum frisingense, and combinations thereof.

81. 67. The composition of claim 65 or claim 66, wherein the divalent cation is a barium cation, a calcium cation, a magnesium cation, a strontium cation, a cobalt cation, a nickel cation, a zinc cation, a manganese cation, or a combination thereof.

82. 67. The composition of claim 65 or claim 66, wherein the polymer matrix further comprises a filler.

83. 83. The composition of claim 82, wherein the filler is selected from the group consisting of starch, cellulose, biochar, pumice, silica, clay, zeolite, perlite, or combinations thereof.

84. 83. The composition of claim 82, wherein the filler is present in an amount of about 0.1% to about 90% by weight, based on the total weight of the composition.

85. 67. The composition of claim 65 or claim 66, wherein the encapsulated microbial composition is in the form of particles having a particle size of about 300 μm or less.

86. 67. The composition of claim 65 or claim 66, wherein the encapsulated microbial composition has a moisture content of about 5% or less.

87. 67. The composition of claim 65 or claim 66, wherein the encapsulated microbial composition has a loss of colony forming units per gram (CFU / g) of less than 2 log after 4 weeks, 8 weeks or 12 weeks at 25° C. and 65% relative humidity (RH).

88. 67. The composition of claim 65 or claim 66, wherein the encapsulated microbial composition has a loss of colony forming units per gram (CFU / g) of less than 1 log after 4 weeks, 8 weeks or 12 weeks at 25° C. and 65% relative humidity (RH).

89. 67. The composition of claim 65 or claim 66, wherein the polymer matrix further comprises at least one hydrophobic additive.

90. 67. A seed composition comprising: a plant seed; and an encapsulated microbial composition according to claim 65 or claim 66 present on at least a portion of a surface of the plant seed.

91. 91. The seed composition of claim 90, wherein the plant seed further comprises a protectant, a herbicide, a pesticide, a fungicide, or a combination thereof.

92. 91. The seed composition of claim 90, wherein the plant seed is selected from the group consisting of a vegetable seed, a fruit seed, a grain seed, and combinations thereof.

93. 91. The seed composition of claim 90, wherein the plant seed is selected from the group consisting of a canola seed, a corn seed, a soybean seed, a wheat seed, and combinations thereof.

94. 91. The seed composition of claim 90, further comprising a hydrophobic coating.

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