Microbial inoculant composition comprising nitrogen fixing, phosphorus solubilizing, and potassium solubilizing bacteria

Abstract

A composition for a non-leguminous plant comprising a microbial inoculant comprising at least one or more live nitrogen fixing bacteria, Methylobacterium aminovorans and / or Methylobacterium radiotolerans, at least one or more live potassium solubilizing bacteria, and at least one or more live phosphorus solubilizing bacteria. Methods for producing and using the same are also disclosed.

Description

DESCRIPTIONMICROBIAL INOCULANT COMPOSITION COMPRISING NITROGEN FIXING, PHOSPHORUS SOLUBILIZING, AND POTASSIUM SOLUBILIZING BACTERIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of Indian Provisional Application No. 202541005116, filed January 22, 2025, the contents of which is incorporated into the present application by reference in its entirety.BACKGROUNDA. Field of the invention

[0002] The invention generally concerns microbial inoculant compositions and methods of making the same. In particular embodiments, the composition comprises nitrogen fixing bacteria, phosphorus solubilizing bacteria, and potassium solubilizing bacteria.B. Description of related art

[0003] Soil nutrients, such as nitrogen, phosphorus, potassium, and sulfur, as well as trace elements such as iron, zinc, copper, and magnesium, are useful for achieving thriving agriculture and growth of plants. Upon repeated planting cycles, the quantity of these nutrients in the soil may be depleted, resulting in reduced plant growth and decreased production. To counter this effect, fertilizers have been developed to help replace the depleted vital nutrients. Single-nutrient fertilizers and multi-nutrient fertilizers, such as fertilizer blends, have been developed to meet the varied needs of crop production worldwide. Continuous use of fertilizers leads to loss of soil fertility and nutrient balance. To increase the crop yield and satisfy the growing need of increasing population, more fertilizers are being used. In addition, large application or usage of urea and urea’s rapid hydrolysis and nitrification in the soil is causing deterioration of soil health and environmental issues such as greenhouse emissions and ground water contamination.

[0004] Soil does not comprise just nutrients, it also comprises mineral substances, organic matter, and microorganisms. The role and activities of microorganisms in the soil is important for nutrient uptake by plants (e.g., via the root system), as beneficial microorganisms in the soil can directly participate in formation of soil fertility, e.g., conversion of substances and energy in the soil, formation and decomposition of humus and / or other organic material, release and / or fixation of trace elements and / or nutrients, fixation of nitrogen, etc.

[0005] However, in a purely natural state, the quantity of beneficial microorganisms in the soil may decrease and / or be insufficient for the agricultural yields anticipated by modem agrarian methods. Therefore, similar to the replenishment of soil using fertilizers comprising nutrients, soil may also need to be replenished with beneficial microorganisms.

[0006] In addition, the increasing need for environmentally friendly agricultural practices is driving the use of fertilizers based on beneficial microorganisms. Beneficial microorgansism belong to a wide array of genera, classes, and phyla, ranging from bacteria to yeasts and fungi, which can support plant nutrition with different mechanisms.

[0007] For decades, scientists and agronomists have identified biological nitrogen fixation, a function of certain prokaryotes and archaea called diazotrophs, as an alternative source of nitrogen with lower production costs and greater use efficiency. Nitrogen fixation was first characterized in Rhizobiales, an order of proteobacteria which are able to catalyze the reduction of atmospheric nitrogen via the enzyme nitrogenase, within highly specialized symbiotic root nodules of leguminous plants. Since then, diverse bacteria have shown to perform nitrogen fixation in symbiosis with non-leguminous plants (endophytic fixation) and as free-living populations in soil and aquatic environments (associative fixation).SUMMARY

[0008] A discovery has been made that provides a solution to at least some of the problems associated with production, characteristics of, and / or use of composition for non-leguminous plants comprising microorganisms and at least some of the problems associated with loss of beneficia microorganisms, decreasing soil health, and decreasing plant production. In particular, disclosed herein are microbe-enhanced composition comprising live nitrogen fixing bacteria, live phosphorus solubilizing bacteria, and live potassium solubilizing bacteria^ Nonlimiting examples include use of compatible microbial consortium containing at least Methylobacterium aminovorans and / or Methylobacterium radiotolerans , at least one or more potassium solubilizing bacteria, and at least one or more phosphorus solubilizing bacteria. The formulation is designed to have improved shelf-life and provide enhanced performance and enhanced nitrogen, potassium, and phosphorous availability for plants, such as non-leguminous plants. As an example, shelflife of the microbes have been demonstrated at 4 to 6 months under ambient conditions for a solid fertilizer containing the microbes and for 1 to 2 years for a liquid fertilizer containing the microbes.

[0009] In some aspects, the composition comprises or consists of at least one or more potassium solubilizing bacteria, and at least one or more phosphorus solubilizing bacteria, and at least one or more of Methylobacterium aminovorans and / or Methylobacterium radiotolerans .

[0010] In some instances, the composition comprises the potassium solubilizing bacteria comprises Bacillus mucilaginosus and / or Frateuria aurantia. In some instances, the phosphorus solubilizing bacteria comprise Bacillus megaterium var. phosphaticum, Paenibacillus polymyxa. and / or Bacillus subtilis. In some aspects, the composition further comprising Azotobacter chroococcum and / or Azospirillum brasilense.

[0011] In some aspects, the composition further comprises a nutrient for bacteria, a stabilizer, and / or a dispersant. In some aspects, the nutrient for bacteria comprises a monosaccharide, di-saccharide, amino acid, enzyme, vitamin, plant growth hormone, micronutrient, humic acid, humate, and / or skim milk. In some aspects, the stabilizer comprises polyethylene glycol (PEG), polyvinyl alcohol (PVA), and / or ethanol. In some aspects, the dispersant comprises humic acid, a humate, talc, and / or a clay.

[0012] In some aspects, the composition further comprises glycerol, carboxy methyl cellulose (CMC), polyvinyl pyrrolidone (PVP), gum arabic, guar gum, and / or a CMC disaccharide. In some aspects, the composition further comprising a carrier. In certain aspects, the carrier comprises a organic, inorganic, and / or polymeric carrier. In certain aspects, the carrier comprises an alginate, activated carbon, biochar, and / or a starch.

[0013] In some aspects, the composition further comprises a spore inducing compound. In some aspects, at least a portion of the live nitrogen fixing bacteria, the live potassium solubilizing bacteria, and / or the live phosphorus solubilizing bacteria are in a spore form.

[0014] In certain aspects, the composition contains at least any one of, at most any one of, equal to any one of, or between any two of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 wt.% of a combination of the live nitrogen fixing bacteria, the live potassium solubilizing bacteria, and the live phosphorus solubilizing bacteria based on the total weight of the composition or a portion thereof, such as a coating, or any range thereof or number therein. In some aspects, the composition contains between 80 to 99, 85 to 99, 90 to 99, 95 to 99, 98 to 99, 80 to 95, 80 to 90, 80 to 85, 90 to 98, 95 to 98, or 85 to 98 wt. %, of a combination of the live nitrogen fixing bacteria, the live potassium solubilizing bacteria, and the live phosphorus solubilizing bacteria based on the total weight of the composition or aportion thereof, such as a coating, or any range or number therein. In some aspects, the concentration of the live nitrogen fixing bacteria, the live potassium solubilizing bacteria, and the live phosphorus solubilizing bacteria in the composition, or any portion thereof, is greater than any one of, equal to any one of, or between any two of IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12colony forming units (CFU) per mL, or any range thereof or number therein. In some aspects, the composition, or any portion thereof, contains between IxlO5to IxlO10, IxlO6to IxlO10, IxlO7to IxlO10, IxlO8to IxlO10, IxlO5to IxlO9, IxlO5to IxlO8, IxlO6to IxlO9, IxlO6to IxlO8, IxlO10to IxlO12CFU per mL, or any range thereof or number therein.

[0015] In some aspects, the composition is a solid. In some aspects, the composition is a slurry. In some aspects, the composition is a liquid. In some aspects, the composition is capable of being, or is, blended, coated on, or distributed with a fertilizer and / or other microbial compositions. In some aspects, the composition is capable of being used in fertigation. In some aspects, the composition is capable of being used in a foliar spray.

[0016] Certain aspects are directed to a method for improving nutrient use efficiency of a non-leguminous plant. In some aspects, the method comprises applying a disclosed composition described herein to a soil wherein the non-leguminous plant and / or seed is growing, to a non-leguminous plant, to a non-leguminous seed, to water for the non-leguminous plant, and / or to a fertilizer for the non-leguminous plant. In some instances, the composition is applied to the fertilizer for the non-leguminous plant. In some instances, the non-leguminous plant includes a rice, corn, soy bean, tomato, lettuce, barley, wheat, and / or grass.

[0017] In some embodiments, methods of applying the composition to a plant, soil, water, seed, or combination thereof are described. A method can include applying the composition to a portion of a soil, a crop, water, seed, or any combination of the soil, the water, and the crop. In some embodiments, the soil is at least partially or fully submerged under water (e.g., rice paddy crops) and the granules sink in the water to contact the soil.

[0018] Certain embodiments of the present invention are characterized through the following aspects.

[0019] Aspect 1 concerns a composition for a non-leguminous plant comprising a microbial inoculant comprising:at least one or more live nitrogen fixing bacteria comprises Methylobacterium aminovorans and / or Methylobacterium radiotolerans',at least one or more live potassium solubilizing bacteria; andat least one or more live phosphorus solubilizing bacteria.

[0020] Aspect 2 concerns the composition of aspect 1, wherein the potassium solubilizing bacteria comprises Bacillus mucilaginosus and / or Frateuria aurantia and / or wherein the phosphorus solubilizing bacteria comprise Bacillus megaterium var. phosphaticum, Paenibacillus polymyxa, and / or Bacillus subtilis.

[0021] Aspect 3 concerns the composition of any one of aspects 1 to 2, further comprising a nitrogen fertilizer, wherein the nitrogen fertilizer is at least partially coated with the microbial inoculant.

[0022] Aspect 4 concerns the composition of any one of aspects 1 to 3, further comprising Azotobacter chroococcum and / or Azospirillum brasilense.

[0023] Aspect 5 concerns the composition of any one of aspects 1 to 4, further comprising:a nutrient for bacteria;a stabilizer; and / ora dispersant.

[0024] Aspect 6 concerns the composition of aspect 5, wherein the nutrient for bacteria comprises a monosaccharide, di-saccharide, amino acid, enzyme, vitamin, plant growth hormone, micronutrient, humic acid, humate, and / or skim milk, wherein the stabilizer comprises polyethylene glycol (PEG), polyvinyl alcohol (PVA), and / or ethanol, and / or wherein the dispersant comprises humic acid, a humate, talc, and / or a clay.

[0025] Aspect 7 concerns the composition of any one of aspects 3 to 6, wherein the composition comprises at least one coating at least partially between the microbial inoculant and the nitrogen fertilizer, wherein the at least one coating comprises an oil, a wax, humate, and / or a humic acid.

[0026] Aspect 8 concerns the composition of any one of claims 5 to 7, wherein the nutrient for bacteria is at least partially in contact with the microbial inoculant, the stabilizer is at least partially between the nitrogen fertilizer and the microbial inoculant, the microbial inoculant isat least partially between the stabilizer and the nitrogen fertilizer, and / or the microbial inoculant is at least partially between the dispersant and the nitrogen fertilizer.

[0027] Aspect 9 concerns the composition of any one of aspects 1 to 8, further comprising glycerol, carboxy methyl cellulose (CMC), polyvinyl pyrrolidone (PVP), gum Arabic, guar gum, a CMC disaccharide, arabic gum, and / or guar gum.

[0028] Aspect 10 concerns the composition of any one of aspects 1 to 9, further comprising a carrier.

[0029] Aspect 11 concerns the composition of aspect 10, wherein the carrier comprises an alginate, activated carbon, biochar, and / or a starch.

[0030] Aspect 12 concerns the composition of any one of aspects 1 to 11, further comprising a spore inducing compound.

[0031] Aspect 13 concerns the composition of any one of aspects 1 to 12, wherein at least a portion of the nitrogen fixing bacteria, the potassium solubilizing bacteria, and / or the phosphorus solubilizing bacteria are comprised in a spore.

[0032] Aspect 14 concerns the composition of any one of aspects 1 to 13, comprising 95 to 99 wt. % of a combination of the nitrogen fixing bacteria, the potassium solubilizing bacteria, and the phosphorus solubilizing bacteria based on the total weight of the composition.

[0033] Aspect 15 concerns the composition of any one of aspects 1 to 14, wherein the microbial inoculant comprises IxlO7to IxlO12colony forming units (CFU) per mL of a combination of the live nitrogen fixing bacteria, the live potassium solubilizing bacteria, and the live phosphorus solubilizing bacteria

[0034] Aspect 16 concerns the composition of any one of aspects 1 to 15, wherein the composition is a liquid composition.

[0035] Aspect 17 concerns the composition of any one of aspects 1 to 15, wherein the composition is a solid composition.

[0036] Aspect 18 concerns a method for improving nutrient use efficiency of a non-leguminous plant, the method comprising applying a composition of any one of aspects 1 to 17 to a soil wherein the non-leguminous plant and / or seed is growing, to a non-leguminous plant,to a non-leguminous seed, to water for the non-leguminous plant, and / or to a fertilizer for the non-leguminous plant.

[0037] Aspect 19 concerns the method of aspect 18, wherein the composition is applied to the fertilizer for the non-leguminous plant.

[0038] Aspect 20 concerns the method of any one of aspects 18 to 19, wherein the non-leguminous plant is a rice, corn, soy bean, tomato, lettuce, barley, wheat, and / or grass.

[0039] The following includes definitions of various terms and phrases used throughout this specification.

[0040] The term “fertilizer” is defined as a material applied to soils or to plant tissues to supply one or more plant nutrients essential or beneficial to the growth of plants and / or stimulants or enhancers to increase or enhance plant growth. Non-limiting examples of fertilizers include materials having one or more of urea, ammonium nitrate, calcium ammonium nitrate, urea calcium sulfate adduct, one or more superphosphates, binary NP fertilizers, binary NK fertilizers, binary PK fertilizers, NPK fertilizers, molybdenum, zinc, copper, boron, cobalt, and / or iron. In some embodiments, fertilizers include agents that enhance plant growth and / or enhance the ability for a plant to receive the benefit of a fertilizer, such as, but not limited to bio stimulants, urease inhibitors, and nitrification inhibitors.

[0041] The term “microbe” or “microorganism” can include bacteria, fungi, protists, and / or archaea.

[0042] The term “micronutrient” is defined as a chemical element or substance used in trace amounts for the normal growth and development of a plant. Non-limiting examples of micronutrients include B, Cu, Fe, Mn, Mo, Zn, Se, and Si or compounds thereof.

[0043] The term “secondary nutrient” is defined as a chemical element or substance used in moderate amounts for plant growth and are less likely to limit crop growth in comparison to N, P, and K. Non-limiting examples of secondary nutrients include Ca, Mg, and S.

[0044] The term “organic agent” is defined as a substance that is produced by or part of an organism. Non-limiting examples of organic agents suitable for a fertilizer include neem oil, seaweed extract, bio- stimulants, char, bio waste, ashes from incineration of animal waste or animal tissues, and diatomaceous earth.

[0045] The term “granule” can include a solid material. A granule can have a variety of different shapes, non-limiting examples of which include a spherical, a puck, an oval, a rod, an oblong, or a random shape.

[0046] The terms “about” or “approximately” are defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment, the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

[0047] The terms “wt.%,” “vol.%,” or “mol.%” refers to a weight percentage of a component, a volume percentage of a component, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, that includes the component. In a non-limiting example, 10 grams of component in 100 grams of the material is 10 wt.% of component.

[0048] The term “substantially” and its variations are defined to include ranges within 10%, within 5%, within 1%, or within 0.5%.

[0049] The terms “inhibiting” or “reducing” or “preventing” or “avoiding” or any variation of these terms, when used in the claims and / or the specification, includes any measurable decrease or complete inhibition to achieve a desired result.

[0050] The term “effective,” as that term is used in the specification and / or claims, means adequate to accomplish a desired, expected, or intended result.

[0051] The use of the words “a” or “an” when used in conjunction with any of the terms “comprising,” “including,” “containing,” or “having” in the claims, or the specification, may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0052] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0053] The microbial inoculant and methods of producing or using the microbial inoculant of the present invention can “comprise,” “consist essentially of,” or “consist of’ particular ingredients, components, compositions, steps, etc. disclosed throughout the specification. Withrespect to the transitional phase “consisting essentially of,” in one non-limiting aspect, a basic characteristic of the inoculant of the present invention is the presence of a combination of nitrogen fixing bacteria, phosphorus solubilizing bacteria, and potassium solubilizing bacteria in the inoculant.BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Advantages of the present invention may become apparent to those skilled in the art with the benefit of the following detailed description and upon reference to the accompanying drawings.

[0055] FIG. 1: (A) depicts bacterial populations in root zone soil 30 days after Methylobacterium aminovorans inoculation of seeds and soil. (B) depicts bacterial populations in root zone soil 30 days after Methylobacterium aminovorans inoculation of seeds and soil and application of NPK fertilizer. (C) depicts bacterial populations in root zone soil 50 days after Methylobacterium aminovorans inoculation of seeds and soil. (D) depicts bacterial populations in root zone soil 50 days after Methylobacterium aminovorans inoculation of seeds and soil and application of NPK fertilizer.

[0056] FIG. 2: depicts properties of com plant yields for treated and control plants.

[0057] FIG. 3: depicts properties of com plants for treated and control plants.

[0058] FIG. 4: depicts cross-streak assay of bacteria. Vertically streaked from left to right: B. Subtilis', B. mucilaginosus', B. megateriunv, Paenibacillus polymyxa', M. aminovorans', Frateuria aurantia. Horizontally streaked: M. aminovorans (Top); M. radiotolerans (Bottom).

[0059] FIG. 5: depicts a non-limiting example of a fertilizer coated with a microbial inoculant and other optional coatings.

[0060] FIG. 6: depicts the effects of soil application of liquid biofertilizer on yield of sweet com.

[0061] FIG. 7: depicts the effects of soil application of liquid biofertilizer on stover yield of sweet corn.

[0062] FIG. 8: depicts an aerial view of crop plots.

[0063] FIG. 9: depicts the effects of urea coated with liquid biofertilizer on yield of sweet com.

[0064] FIG. 10: depicts the effects of urea coated with liquid biofertilizer on stover yield of sweet corn.

[0065] FIG. 11: depicts an aerial view of crop plots.

[0066] While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings. The drawings may not be to scale.DETAILED DESCRIPTION

[0067] Disclosed herein, among other things, is a microbial inoculant composition, and a method of producing and using the same. The microbes can include live nitrogen fixing bacteria, live phosphorus solubilizing bacteria, and live potassium solubilizing bacteria. The microbial inoculant composition enables improved higher efficiency / yield performance in the non-leguminous plants. The microbial inoculant composition can have an increased microorganism and / or fertilizer shelf-life and / or synergistic effect on nutrient uptake, plant health, plant growth, soil health, etc. A composition comprising the microbial inoculant composition can further include a fertilizer, a carrier, a nutrient for the microbes, etc.

[0068] The methods and / or compositions of the current disclosure provide an economically efficient means to produce and / or utilize a stable and high quality microbial inoculant compositions. These and other non-limiting aspects of the present invention are discussed in further detail in the following sections.A. Microbial Inoculant Compositions

[0069] The fertilizer composition can comprised of 80 wt. % to 99 wt. % or at least any one of, equal to any one of, or between any two of 80 wt. %, 81 wt. %, 82 wt. %, 83 wt. %, 84 wt. %, 85 wt. %, 86 wt. %, 87 wt. %, 88 wt. %, 89 wt. %, 90 wt. %, 91 wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %, 96 wt. %, 97 wt. %, 98 wt. %, and 99 wt. %99 wt. %, 99.1 wt. %, 99.2 wt. %, 99.3 wt. %, 99.4 wt. %, 99.5 wt. %, 99.6 wt. %, 99.8 wt. %, 99.9 wt. %, 99.99 wt. % and 99.999 wt. % of the a combination of nitrogen fixing bacteria, phosphorus solubilizing bacteria, and potassium solubilizing bacteria based on the total weight of the composition. In some aspects, the composition can contain 5 wt. to 90 wt. % or at least any one of, equal to any one of, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90 wt. % ofthe nitrogen fixing bacteria. In some aspects, the composition can contain 5 wt. to 90 wt. % or at least any one of, equal to any one of, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90 wt. % of the phosphorus solubilizing bacteria. In some aspects, the composition can contain 5 wt. to 90 wt. % or at least any one of, equal to any one of, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90 wt. % of the potassium solubilizing bacteria.

[0070] In certain aspects, the composition may include 80 wt. % to 99.9 wt. % of a nitrogen fertilizer, or at least any one of, equal to any one of, or between any two of 80 wt. %, 81 wt. %, 82 wt. %, 83 wt. %, 84 wt. %, 85 wt. %, 86 wt. %, 87 wt. %, 88 wt. %, 89 wt. %, 90 wt. %, 91 wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %, 96 wt. %, 97 wt. %, 98 wt. %, 99 wt. %, 99.1 wt. %, 99.2 wt. %, 99.3 wt. %, 99.4 wt. %, 99.5 wt. %, 99.6 wt. %, 99.8 wt. %, 99.8 wt. %, 99.9 wt. % of the nitrogen fertilizer based on the total weight of the composition. In certain aspects, the composition may include 80 wt. % to 99.9 wt. % of a phosphorus fertilizer, or at least any one of, equal to any one of, or between any two of 80 wt. %, 81 wt. %, 82 wt. %, 83 wt. %, 84 wt. %, 85 wt. %, 86 wt. %, 87 wt. %, 88 wt. %, 89 wt. %, 90 wt. %, 91 wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %, 96 wt. %, 97 wt. %, 98 wt. %, 99 wt. %, 99.1 wt. %, 99.2 wt. %, 99.3 wt. %, 99.4 wt. %, 99.5 wt. %, 99.6 wt. %, 99.8 wt. %, 99.8 wt. %, 99.9 wt. % of the phosphorus fertilizer based on the total weight of the composition. In certain aspects, the composition may include 80 wt. % to 99.9 wt. % of a potassium fertilizer, or at least any one of, equal to any one of, or between any two of 80 wt. %, 81 wt. %, 82 wt. %, 83 wt. %, 84 wt. %, 85 wt. %, 86 wt. %, 87 wt. %, 88 wt. %, 89 wt. %, 90 wt. %, 91 wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %, 96 wt. %, 97 wt. %, 98 wt. %, 99 wt. %, 99.1 wt. %, 99.2 wt. %, 99.3 wt. %, 99.4 wt. %, 99.5 wt. %, 99.6 wt. %, 99.8 wt. %, 99.8 wt. %, 99.9 wt. % of the potassium fertilizer based on the total weight of the composition. In some aspects, the composition contains 0.1 wt. % to 20 wt. % of the combination of nitrogen fixing bacteria, phosphorus solubilizing bacteria, and potassium solubilizing bacteria based on the total weight of the composition, such as at least any one of, equal to any one of, or between any two of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 wt. % of the combination based on the total weight of the composition. In certain aspects, the composition comprises urea, diammonium phosphate, and / or a nitrogen, phosphorous, and potassium fertilizer. In certain aspects, the fertilizer is at least partially coated with the microbial inoculant. In certain aspects, the composition comprises at least one coating at least partially between the microbial inoculant and the fertilizer. In certain aspects, the at least one coating comprises an oil, a wax, humate, and / or a humic acid.

[0071] In certain aspects, the composition can contain 0.1 wt. % to 20 wt. %, or at least any one of, at most any one of, equal to any one of, or between any two of 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 wt. % of a nutrient for bacteria based on the total weight of the composition. In certain aspects, the nutrient for bacteria can be a monosaccharide, di-saccharide, amino acid, enzyme, vitamin, plant growth hormone, micronutrient, humic acid, humate, and / or skim milk. In certain aspects, the composition can contain 0.001 wt. % to 20 wt. %, or at least any one of, at most any one of, equal to any one of, or between any two of 0.001, 0.005, 0.01, 0.03, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 wt. % of a stabilizer based on the total weight of the composition. In certain aspects, the stabilizer can be a starch, agar, alginate, chitosan, polyethylene glycol (PEG), polyvinyl alcohol (PVA), and / or ethanol. In certain aspects, the composition can contain 0.001 wt. % to 20 wt. %, or at least any one of, at most any one of, equal to any one of, or between any two of 0.001, 0.005, 0.01, 0.03, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 wt. % of a dispersant based on the total weight of the composition. In certain aspects, the dispersant can be humic acid, a humate, talc, glycerol, carboxy methyl cellulose, polyvinyl pyrrolidone, gum arabic, guar gum, a diatomaceous earth, and / or a clay.

[0072] In some aspects, the nutrient for a bacteria in the composition is at least partially in contact with the microbial inoculant. In some aspects, the stabilizer in the composition is at least partially between the fertilizer and the microbial inoculant. In some aspects, the microbial inoculant in the composition is at least partially between the stabilizer and the fertilizer, and / or the microbial inoculant is at least partially between the dispersant and the fertilizer.

[0073] In some aspects, the composition may contain 0.1 to 20 wt. % of a carrier or at least any one of, at most any one of, equal to any one of, or between any two of 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 wt. % of the carrier based on the total weight of thecomposition. In some aspects, the carrier may be organic, inorganic, and / or a polymeric carrier. In some aspects, the carrier can be an alginate, activated carbon, biochar, and / or a starch.

[0074] In some aspects, the composition may contain 0.001 to 5 wt. % or at least any one of, at most any one of, equal to any one of, or between any two of 0.001, 0.005, 0.01, 0.03, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, or 5 wt. % of a spore inducing compound based on the total weight of the composition. In some aspects, at least a portion of the live nitrogen fixing bacteria, the live potassium solubilizing bacteria, and / or the live phosphorus solubilizing bacteria are comprised in a spore state. In some instances, the spore state is induced by nutrient depletion and / or high cell density.

[0075] The composition can be of any suitable shape or can be amorphous or a liquid or slurry. Non-limiting shapes include spherical, cuboidal, cylindrical, puck shape, oval, and oblong shapes. In some aspects, the composition can be of cylindrical shape with a circular, elliptical, ovular, triangular, square, rectangular, pentagonal, or hexagonal cross section, although cylindrical shaped compositions having a cross-section of other shapes can also be made. In some aspects, the compositions can have a dimension such as length, width, height and / or cross-sectional diameter between 0.5 mm to 5 mm or at least any one of, equal to any one of, or between any two of 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, and 5 mm. In some particular aspects, the compositions can have a substantially spherical shape with an average diameter 1 mm to 5 mm or at least any one of, equal to any one of, or between any two of 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, and 5 mm.

[0076] In some other aspects, the composition can form a coating over at least a portion of an outer surface of a carrier or fertilizer, such as a fertilizer granule / powder / particle. In some aspects, at least a portion of the microorganisms are comprised in a coating of a fertilizer composition.

[0077] In some embodiments, a microorganism that can be included in the composition can be a bacteria, archaea, fungi, or a protist. In some instances, the microorganism can include an endophyte, rhizosphere microbe, and / or phyllosphere microbe. In some embodiments, the microorganism can included more than one species of microorganism. In some embodiments, more than one species, genus, phylum, class, and / or kingdom can be represented in a group of microorganisms included in a composition. In some embodiments, a microorganism can be cultured and / or grown in a laboratory. In some embodiments, a microorganism can be obtainedfrom a natural source. In some embodiments, a microorganism can be a spore / cyst forming microorganism. In some embodiments, a microorganism can be a spore / cyst forming bacteria.

[0078] In some instances, the compositions further comprise additional fertilizers, micronutrients, primary nutrients, urea, additional nitrogen nutrients, insecticides, herbicides, or fungicides, or combinations thereof.

[0079] A fertilizer granule can be coated with or can have distributed within the fertilizer a microbial inoculant composition described herein. In some instances, the fertilizer granules can have desirable physical properties such as desired levels of abrasion resistance, granule strength, pelletizability, hygroscopicity, granule shape, and size distribution, which are important properties for the fertilizer.

[0080] The microbial inoculant composition described herein can be comprised in a composition useful for application to soil, water, and / or a crop.

[0081] The microbial inoculant composition described herein can be coated, distributed in, or combined with urea, monoammonium phosphate (MAP), diammonium phosphate (DAP), muriate of potash (MOP), monopotassium phosphate (MKP), triple super phosphate (TSP), rock phosphate, single super phosphate (SSP), ammonium sulfate, and the like.

[0082] As a non-limiting example, referring to FIG. 5, a fertilizer composition 100 according to an example of the present invention is shown. The fertilizer composition 100 contains a core fertilizer such as urea and / or NPK 101, an optional protective coating layer 102 coating an outer surface of the core, such as a coating with oils, wax, and / or humic acid, and a microbial inoculant coating layer 103 containing live microbes. In some aspects, the microbial inoculant coating layer may comprise at least one or more nitrogen fixing bacteria, Methylobacterium aminovorans and / or Methylobacterium radiotolerans , at least one or more potassium solubilizing bacteria, and at least one or more phosphorus solubilizing bacteria covering an outer surface of the protective coating layer 102. The microbial inoculant coating layer 103 may optionally be covered with a nutrient layer 104 for bacteria, such as sucrose, dextrose, starch, and / or agar. An optional external protection coating layer 105 is represented as covering the entire outer surface of the nutrient coating layer. The external protection coating layer 105 may comprise binders such as bentonite clay, diatomaceous earth, and / or polymers such as chitosan. The layers may be rearranged in a different order, may only partially or may completely coat the underlying layers, and / or may be found in the composition distributed throughout the composition instead of, or in addition to, being in the form of a layer. In someinstances, the components of the composition are distributed in a homogenous composition, or some of the components are combined into one or more layer and / or core.B. Microorganisms

[0083] In some embodiments, the microorganisms that can be included in a microbial inoculant composition can be at least one or more live nitrogen fixing bacteria, at least one or more the live potassium solubilizing bacteria, and at least one or more live phosphorus solubilizing bacteria. In some aspects, the at least one or more live nitrogen fixing bacteria comprises Methylobacterium aminovorans, Methylobacterium radiotolerans, Azotobacter chroococcum and / or Azospirillum brasilense. In some aspects, the at least one or more live potassium solubilizing bacteria comprises Bacillus mucilaginosus and / or Frateuria aurantia. In some aspects, the at least one or more live phosphorus solubilizing bacteria comprises Bacillus megaterium var. phosphaticum, Paenibacillus polymyxa, and / or Bacillus subtilis.

[0084] In some embodiments, the composition can include an additional bacteria, archaea, fungi, or a protist. In some instances, the additional microorganism can include an endophyte, rhizosphere microbe, and / or phyllo sphere microbe. In some embodiments, the composition can included more than one species of microorganism. In some embodiments, more than one species, genus, phylum, class, and / or kingdom can be represented in a group of microorganisms included in the microbial inoculant.

[0085] In some embodiments, a microorganism can be, but is not limited to, a diazotrophic bacteria, Azospirillum species, Azotobacter species, Frateuria aurantia, Bacillus species, endophytes, nitrogen fixing bacteria, methylotrophs, comammox (e.g., (COMplete AMMonia OXidation) an organism that can convert ammonia into nitrite and then into nitrate through the process of nitrification), phosphorus solubilizing, nitrite oxidizing, Nitrospira species, Methylobacterium species, and / or pink pigmented facultative methylotrophs (PPFM-trophs).

[0086] In some embodiments, the microorganism can be cultured and / or grown in a laboratory prior to addition into the composition. In some embodiments, a microorganism can be obtained from a natural source. In some embodiments, the microorganism can be concentrated prior to addition to the composition.

[0087] In some embodiments, a microorganism can be a spore / cyst forming microorganism. In some embodiments, a microorganism can be a spore / cyst forming bacteria. In some embodiments, a microorganism can be induced to form spores / cysts prior to addition to the composition. In some embodiments, a microorganism is not induced to form spores / cysts priorto addition to the composition. In some embodiments, a microorganism is not chemically induced to form spores / cysts prior to addition to the composition. In some embodiments, a microorganism has been selected for heat tolerance. In some embodiments, a microorganism has not been selected for heat tolerance.

[0088] In some embodiments, microorganisms can be cultured and / or concentrated to greater than, or equal to, approximately IxlO4to IxlO13colony forming units (CFU) per mL prior to addition to the composition or in the composition. In some embodiments, microorganisms can be cultured and / or concentrated to greater than, or equal to, approximately IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13CFU per mL. In some embodiments, microorganisms can be cultured and / or concentrated to greater than, or equal to, approximately IxlO5to IxlO10, IxlO6to IxlO10, IxlO7to IxlO10, IxlO8to IxlO10, IxlO5to IxlO9, IxlO5to IxlO8, IxlO6to IxlO9, IxlO6to IxlO8, or IxlO8to IxlO9CFU per mL prior to addition to the composition or in the composition.

[0089] Prior to coating a fertilizer, microorganisms in the composition may be protected. In some embodiments, microorganism protection may comprise any one or more of encapsulation, physical protection, and / or engineering methods. In some embodiments, microorganism protection is by contact with a protectant. In some instances, microorganism protection is by encapsulation in a protectant. In some embodiments, a protected microorganism is protected by addition of one or more physical protectants, engineering methods, encapsulating agents, water-soluble additives, stabilizer additives, and / or dispersants.

[0090] In some embodiments, microorganism protection can comprise encapsulation with a stabilizer protectant. In some embodiments, a stabilizer comprises one or more of clay, diatomaceous earth, starch, agar, alginate, chitosan, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyacrylic acid, ethanol, humic acid, humates, talc, clay, peat, lignite, vermiculite, perlite and / or chemically modified versions of the same.

[0091] In some embodiments, chemical modification of a stabilizer can comprise, but is not limited to, one or more of esterification, alkylation, acetylation, phosphorylation, hydrophobic modification, sulfation, sulfomethylation, methylation, amidation, amination, protonation, halogenation, nitration, copolymerization, and / or physical or covalent cross -linking.

[0092] In some embodiments, protection of a microorganism can also comprise addition of a water-soluble additive protectant. In some embodiments, a water soluble additive can be butis not limited to, glycerol, carboxy methyl cellulose (CMC), polyvinyl pyrrolidone (PVP), gum Arabic, guar gum, and / or mono and / or disaccharide based CMC / Arabic gum / guar gum.

[0093] In some embodiments, microorganism protection can comprise improved stickiness, stabilization, and surfactant and dispersal abilities. In some embodiments, such characteristics can be provided by protectants / inducers and nutrients (e.g. Alginates / Glycerol / polyvinyl alcohol, PEG / PVP, Clay / humate, Mono and disaccharides, CMC / Arabic gum / guar gum).

[0094] In some embodiments, protection can comprise inclusion of certain stabilizers and / or additives at set proportions, including but not limited to 1:0.05, 1:0.10, 1:0.15, 1:0.20, 1:0.25, 1:0.30, 1:0.35, 1:0.40, 1:0.45, 1:0.50, 1:0.55, 1:0.60, 1:0.65, 1:0.70, 1:0.75, 1:0.80, 1:0.85, 1:0.90, 1:0.95, 1:1, 1:1.05, 1:1.10, 1:1.15, 1:1.20, 1:1.25, 1:1.30, 1:1.35, 1:1.40, 1:1.45, 1:1.5, 1:1.55, 1:1.60, 1:1.65, 1:1.70, 1:1.75, 1:1.80, 1:1.85, 1:1.90, 1:1.95, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:1,000, 1:10,000, 1:100,000, 1:1,000,000, or any range derivable therein.

[0095] In some embodiments, physical protection and / or engineering methods facilitate pelleting and / or layering of microorganisms as a liquid solution at the core, distributed in a fertilizer, or around a core of a fertilizer. In some embodiments, a bolus of concentrated microbes in a liquid carrier are protected with a soluble additive, such as in a slurry.

[0096] In some embodiments, physical protection of microorganisms may comprise addition of protectants that are molecules and / or enzymes derived from thermophiles (e.g., heat tolerant bacteria), these molecules and / or enzymes may contribute to the thermo-protection phenotypes observed in thermophiles (see e.g., Pedro Lamosa et al., Thermo stabilization of Proteins by Diglycerol Phosphate, a New Compatible Solute from Hyperthermophile Archaeoglobus fulgidus. Applied and Environmental Microbiology, Vol. 66, No. 5, 01 May 2000). Molecules and / or enzymes derived from thermophiles include proteins, lipids, saccharides, nucleic acids, small molecules, and / or inorganic compounds. Thermophiles may include bacteria, archaea, protists, and / or fungi. Thermophile microorganisms may include microorganisms that can thrive, divide, and / or survive at temperatures of 50 °C or greater. A non-limiting example of a thermophile is Archaeoglobus fulgidus.

[0097] In some embodiments, engineering methods for the protection of microorganisms may comprise spray drying and / or freeze-drying of the microorganisms. Freeze-drying can be performed by freezing the microorganisms or a composition containing the microorganisms, exposing the frozen microorganism or composition containing the microorganism to pressuresbelow atmospheric pressures, and removing ice from or surrounding the frozen microorganism or composition. The composition containing the microorganism can contain, in some instances, a cryoprotectant, encapsulating agent, water-soluble additive, stabilizer additive, and / or a dispersant.

[0098] In some embodiments, a microorganism is contacted with a protectant using a spray, liquid stream, semi-solid, or solid (such as a powder) comprising said protectant. In some embodiments, a protectant is contacted with a microorganism using a spray, liquid stream, semi-solid, or solid (such as a powder) comprising said microorganism.

[0099] In some embodiments, a protected microorganism is concentrated (e.g., settlement, centrifugation, affinity capture, selective growth media, etc.,) prior to contact with a protectant and / or prior to protecting. In some embodiments, a protected microorganism is contacted with the protectant or with the fertilizer at a concentration of higher than 1012cells per gram of the protectant. In some embodiments, a protectant is comprised in a liquid, suspension, and / or dried powder.

[0100] In some embodiments, a protected microorganism can contain low amounts of moisture. In some embodiments, a free-moisture content of a protected microorganism can be less than 0.6 wt.%, less than 0.5 wt.% water or 0.25 wt.% to less than 0.6 wt.% water. In some instances, the free moisture content is 0.5, 0.4, 0.3, 0.2, 0.1, or 0 wt.%.C. Methods of Making Microbial Inoculant Compositions

[0101] A method of producing the microbial inoculant composition is disclosed. In some aspects, the method comprises one or more of the following steps:(a) culturing a nitrogen fixing microbe, a potassium solubilizing microbe, and / or a phosphorous solubilizing microbe;(b) combining in a composition the nitrogen fixing microbe, the potassium solubilizing microbe, and the phosphorous solubilizing microbe to form a combination;(c) optionally combining additional compounds / compositions to the combination before, during, or after the nitrogen fixing microbe, the potassium solubilizing microbe, and the phosphorous solubilizing microbe are combined;(d) optionally preparing the combination for storage;(e) optionally coating or combining the combination with a fertilizer to form a microbe- enhanced fertilizer.

[0102] In some instances, the method does not require step (a) be performed by the same entity as any of the other steps. In some instances, in step (a), two or more of the nitrogen fixing microbe, the potassium solubilizing microbe, and / or the phosphorous solubilizing microbe are cultured together. In some instances, in step (a), the nitrogen fixing microbe, the potassium solubilizing microbe, and the phosphorous solubilizing microbe are cultured separately before step (b).

[0103] In some embodiments, the microbe-enhanced fertilizer comprises any granulatable fertilizer (e.g., granular fertilizer). In some embodiments, a microbe-enhanced fertilizer comprises or excludes one or more granular fertilizers, preferably but not limited to, one or more of urea, single super phosphate (SSP), triple super phosphate (TSP), ammonium sulfate, monoammonium phosphate (MAP), diammonium phosphate (DAP), muriate of potash (MOP), sulfate of potash (SOP), potassium sulfate, binary NP fertilizers, binary NK fertilizers, binary PK fertilizers, and / or a nitrogen phosphorus potassium (NPK) mix.

[0104] In some embodiments, step (e) comprises granulation with the fertilizer. In some instances, fertilizer granulation comprises chemically reacting reactants to form the fertilizer. In some embodiments, the fertilizer is formed from or is provided in a solution. In some embodiments, the fertilizer is formed or is provided in a fertilizer melt. The fertilizer melt can be formed, in some instances by evaporating a fertilizer solution. In some instances, the fertilizer is formed from or is provided in a solidified fertilizer. The solidified fertilizer can be formed, in some instances by cooling a fertilizer melt. In some instances, the fertilizer is formed from or is provided in a granulated fertilizer. The granulated fertilizer can be formed, in some instances by granulating the solidified fertilizer melt or a cooling fertilizer melt. In some embodiments, a microbe-enhanced fertilizer is produced when a microorganism is contacted with the fertilizer before or during granulation. In some embodiments, water or an aqueous solution, such as steam and / or a scrubber solution, can be combined with a fertilizer composition in granulator to facilitate granulation of a fertilizer composition.

[0105] In some embodiments, a dryer for a fertilizer or for the combination can be heated by steam, such as in a steam jacketed dryer. In some embodiments, a dryer can be or can be part of a rotating dryer. In some instances, a dryer can be a freeze dryer. In some instances, a dryer is a dehydrator that does not increase a temperature in the dryer above 75 °C. In some embodiments, a granulator can be separate from a dryer. In some embodiments, a dryer and granulator can be the same vessel or part of a same vessel. In some embodiments, a granulator, may include a rotatable section, a rotatable internal container, and / or a section that vibrates. Insome embodiments, the rotatable section and / or rotatable internal container may contain internal flights and / or be rotated to induce movement of a fertilizer composition in the granulator. In some embodiments, a granulator can be or can be part of a granulation drum, pugmill, pan granulator, etc.

[0106] In some embodiments, the combination or a microorganism is contacted with a fertilizer by spraying onto a fertilizer particle and / or granule, by mixing into a fertilizer, by spraying a fertilizer onto the microorganism or combination, by coating a fertilizer, by being coated by a fertilizer, by being encapsulated in a fertilizer matrix, by encapsulating a fertilizer to form a matrix of the microorganism(s), etc.

[0107] In some embodiments, a microorganism or the combination is contacted with a fertilizer using a spray, liquid stream, semi-solid, or solid (such as a powder) comprising said microorganism(s). In some embodiments, the microorganism(s) is contacted with a fertilizer using a dosage pump or a spray head.

[0108] In some embodiments, a microorganism or the combination is concentrated (e.g., settlement, centrifugation, affinity capture, selective growth media, etc.) prior to contact with a fertilizer to form a microbe-enhanced fertilizer. In some embodiments, the microorganism is contacted with the fertilizer at a concentration of IxlO4to IxlO12CFU per gram of the fertilizer, such as at IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13CFU per gram of the fertilizer. In some preferred embodiments, a microorganism or the combination is contacted with the fertilizer at a concentration of IxlO8to IxlO9CFU per gram of the fertilizer. In some embodiments, a microorganism is comprised in a liquid, suspension, and / or dried powder.

[0109] In some embodiments, a microbe-enhanced fertilizer particle can have a crush strength of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 kgf / particle, or more, or any amount there between or range thereof, preferably 2 kgf / particle to 5 kgf / particle.

[0110] In some embodiments, a microbe-enhanced fertilizer can contain a coating on the surface of one or more particles of a microbe-enhanced fertilizer can contain additional ingredients. In some instances, the coating can include nutrients for a plant, inhibitors of urea hydrolysis and / or nitrification, agents to slow or increase the rate of degradation of the granule and / or fertilizers, agents to repel moisture and / or provide a hydrophobic layer, agents that decrease or increase the reactivity of the granule and / or fertilizers, agents that provide additional benefits to plants, agents that increase the stability and / or crush strength of thegranule and / or fertilizers, pH buffering agents, drying agents, a binder, carrier, etc. or any combination thereof. The coating can be a commercially available coating, an oil, a fertilizer, a micronutrient, talc, a seaweed and / or seaweed extract, a wax, etc. In some instances, the coating can contain surfactants. In some instances, the coating contains a wax, surfactants, and / or an amine -based compound.

[0111] In some instances, the inhibitors include nitrification inhibitors and / or urease inhibitors. Suitable nitrification inhibitors include, but are not limited to, 3,4-dimethylpyrazole phosphate (DMPP), dicyandiamide (DCD), thiourea (TU), 2-chloro-6-(trichloromethyl)-pyridine (Nitrapyrin), 5-ethoxy-3-trichloromethyl-l,2,4-thiadiazol, which is sold under the tradename Terrazole®, by OHP Inc., USA, 2-amino 4-chloro 6-methyl pyrimidine (AM), 2-mercaptobenzothiazole (MBT), or 2-sulfanilamidothiazole (ST), and any combination thereof. In one aspect, a nitrification inhibitor can comprise DMPP, DCD, TU, nitrapyrin, 5-ethoxy-3-trichloromethyl-l,2,4-thiadiazol, AM, MBT, or ST, or a combination thereof. In some instances, the urease inhibitor may include, but are not limited to, triamide, such as N-(n-butyl) thiophosphoric triamide (NBTPT) and N-(n-propyl) thiophosphoric triamide. In some embodiments, a fertilizer composition can comprise NBTPT, DMPP, TU, DCD, phenylphosphorodiamidate (PPDA), nitrapyrin, 5-ethoxy-3-trichloromethyl-l,2,4-thiadiazol, AM, MBT, ST, or a combination thereof.

[0112] In some instances, the pH buffers include MgO, KH2PO4, NaHCCh, chalk powder, aluminum, magnesium hydroxide, aluminum hydroxide / magnesium hydroxide co-precipitate, aluminum hydroxide / sodium bicarbonate co-precipitate, calcium acetate, calcium bicarbonate, calcium borate, calcium carbonate, calcium bicarbonate, calcium citrate, calcium gluconate, calcium hydroxide, dibasic sodium phosphate, dipotassium hydrogen phosphate, dipotassium phosphate, disodium hydrogen phosphate, magnesium acetate, magnesium borate, magnesium bicarbonate, magnesium carbonate, magnesium hydroxide, magnesium lactate, magnesium oxide, magnesium phosphate, magnesium silicate, magnesium succinate, magnesium tartrate, potassium acetate, potassium carbonate, potassium bicarbonate, potassium borate, potassium citrate, potassium metaphosphate, potassium phthalate, potassium phosphate, potassium polyphosphate, potassium pyrophosphate, potassium succinate, potassium tartrate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium gluconate, sodium hydrogen phosphate, sodium hydroxide, sodium lactate, sodium phthalate, sodium phosphate, sodium polyphosphate, sodium pyrophosphate, sodium tartrate, sodium tripolyphosphate, synthetic hydrotalcite, tetrapotassium pyrophosphate, tetrasodiumpyrophosphate, tripotassium phosphate, trisodium phosphate, or trometamol, or combinations thereof.

[0113] In some aspects, the carrier comprises an organic solvent, plaster of paris, flour, chalk powder, starch, gluten, kaolin, bentonite, colloidal silica, silica, dried distillers grains with solubles, lignin, a synthetic polymer, a wax, chitin, glycoaminoglycans, pectins, hyaluronic acid, chondroitin sulphate, dermatan sulphate, alginic acid, polymannuronic acid, polyguluronic acid, polyglucuronic acid, amylose, amylopectin, callose, chitosan, polygalactomannan, dextran, xanthan, keratan sulphate, MgO, CaO, bone mill powder, rice husk, CaCCh, NaiCCh, K2CO3, KH2PO4, NaHCCh, and / or MgCCh. In some embodiments, the synthetic polymer comprises poly butylene succinate adipate, poly lactic acid, poly butylene succinate, cellulose triacetate, cellulose diacetate, cellulose acetate, a starch acetate, poly (caprolactone), a poly(butylene terephthalate adipate), hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, methylcellulose, ethylcellulose, ethyl methyl cellulose, hydroxy ethyl cellulose, hydroxypropyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl cellulose, polyethylene glycol, or cyclodextrin, or a combination thereof. In some aspects, the organic solvent comprises one or more of an alcohol, an alkane, an acid, a hydroxide, an oil, a polyol solvent, a polyamine solvent, a primary amine solvent, a secondary amine solvent, an alkanol solvent, a protic solvent, etc.

[0114] Exemplary binders include a phosphate, a polyphosphate, a biodegradable polymer, or a wax, or a combination thereof. Suitable waxes include, but are not limited to, vegetable waxes, high melt waxes, ethylene bis(stearamide) wax, paraffin waxes, polyethylene based waxes, and olefin waxes. Suitable phosphates include, but are not limited to, diammonium phosphate and / or monoammonium phosphate. Suitable polyphosphates include, but are not limited to, ammonium polyphosphate. Suitable biodegradable polymers include, but are not limited to, polyacrylamide, polyacrylic acid, polyacrylonitrile, biodegradable polylactic acid, or other biodegradable polymeric material such as polylactic acid, poly(3 -hydroxypropionic acid), polyvinyl alcohol, polyethylene glycol, poly e-caprolactone, poly L-lactide, poly butylene succinate, or biodegradable starch based polymers. The binder can include plaster of Paris, flour, starch, gluten, kaolin, bentonite, colloidal silica, a cyclodextrin, or combinations thereof. Suitable flours include, but are not limited to, rice flour, wheat flour, and / or bleached wheat flour. Suitable starches include, but are not limited to, dextrin modified starches.D. Methods of Using Microbial Inoculant Compositions

[0115] In some embodiments, the microbial inoculant compositions of the present disclosure can be used in methods of increasing the amount of one or more nutrients and one or more microorganisms in soil, and of enhancing plant growth. In some embodiments, methods can include applying to the soil an effective amount of a microbial inoculant compositions of the present disclosure. In some embodiments, methods may include increasing the growth and yield of crops, trees, ornamentals, etc. such as, for example, palm, coconut, rice, wheat, com, barley, oats, tomatoes, and soybeans. In some embodiments, methods can include applying the microbial inoculant composition of the present disclosure to at least one of a soil, an organism, a liquid carrier, a liquid solvent, a fertilizer, etc. (e.g., a target substrate).

[0116] In some embodiments, a microbial inoculant composition can be stored. In some embodiments, the microbe-enhanced fertilizer can be stored for any amount of time, such as 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 1 day, 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more, or any amount of time or range thereof or there between without 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % of the microorganisms in the microbial inoculant composition dying. In some embodiments, the microorganisms and / or fertilizer components of the microbial inoculant composition have an extended shelf life relative to microbe-enhanced fertilizers created through traditional methods. In some instances, the microbes may be alive for a solid fertilizer containing the microbes for 4 to 6 months, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or any amount of time or range thereof or there between. In some instances, the microbes may be alive for a liquid fertilizer containing the microbes for 1 to 2 years, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 months, or any amount of time or range thereof or there between.

[0117] Non-limiting examples of plants that can benefit from the microbial inoculant composition of the present invention include vines, trees, shrubs, stalked plants, fems, etc. The plants may include orchard crops, vines, ornamental plants, food crops, timber, and harvested plants. The plants may include Gymnosperms, Angiosperms, and / or Pteridophytes. The Gymnosperms may include plants from the Araucariaceae, Cupressaceae, Pinaceae,Podocarpaceae, Sciadopitaceae, Taxaceae, Cycadaceae, and Ginkgoaceae families. The Angiosperms may include plants from the Aceraceae, Agavaceae, Anacardiaceae, Annonaceae, Apocynaceae, Aquifoliaceae, Araliaceae, Arecaceae, Asphodelaceae, Asteraceae, Berberidaceae, Betulaceae, Bignoniaceae, Bombacaceae, Boraginaceae, Burseraceae, Buxaceae, Canellaceae, Cannabaceae, Capparidaceae, Caprifoliaceae, Caricaceae, Casuarinaceae, Celastraceae, Cercidiphyllaceae, Chrysobalanaceae, Clusiaceae, Combretaceae, Cornaceae, Cyrillaceae, Davidsoniaceae, Ebenaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae, Grossulariaceae, Hamamelidaceae, Hippocastanaceae, Illiciaceae, Juglandaceae, Lauraceae, Lecythidaceae, Lythraceae, Magnoliaceae, Malpighiaceae, Malvaceae, Melastomataceae, Meliaceae, Moraceae, Moringaceae, Muntingiaceae, Myoporaceae, Myricaceae, Myrsinaceae, Myrtaceae, Nothofagaceae, Nyctaginaceae, Nyssaceae, Olacaceae, Oleaceae, Oxalidaceae, Pandanaceae, Papaveraceae, Phyllanthaceae, Pittosporaceae, Platanaceae, Poaceae, Polygonaceae, Proteaceae, Punicaceae, Rhamnaceae, Rhizophoraceae, Rosaceae, Rubiaceae, Rutaceae, Salicaceae, Sapindaceae, Sapotaceae, Simaroubaceae, Solanaceae, Staphyleaceae, Sterculiaceae, Strelitziaceae, Styracaceae, Surianaceae, Symplocaceae, Tamaricaceae, Theaceae, Theophrastaceae, Thymelaeaceae, Tiliaceae, Ulmaceae, Verbenaceae, and / or Vitaceae family.

[0118] In some embodiments, the effectiveness of compositions comprising microbial inoculant compositions of the present invention can be ascertained by measuring the amount of particular nutrients in the soil at various times after applying the microbial inoculant composition to the soil. In some embodiments, the effectiveness of compositions comprising microbial inoculant composition of the present invention can be ascertained by measuring the amount of the microorganism in the soil at various times after applying the microbial inoculant composition to the soil. It is understood that different soils have different characteristics, which can affect the stability of nutrients and microorganisms in the soil. In some embodiments, effectiveness of a microbial inoculant composition can be directly compared to other microbial or fertilizer compositions by doing a side-by-side comparison in the same soil under the same conditions.

[0119] In some embodiments, a microbial inoculant composition according to the present disclosure can have a density that is greater than water. This can allow the composition to sink in water rather than float. This can be especially beneficial in instances where application is intended to a crop that is at least partially or fully submerged in water. A non-limiting example of such a crop is rice, as the ground in a rice paddy is typically submerged in water. Thus,application of microbial inoculant composition to such crops can be performed such that the granules and / or fertilizer are homogenously distributed on the ground that is submerged under water.EXAMPLES

[0120] The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results Example 1

[0121] Efficiency and yield performance of tomato was determined for tomato plants that had their seeds inoculated with nitrogen fixing bacteria. The nitrogen fixing bacteria tested included Methylobacterium aminovorans, Azotobacter chroococcum, and Azospirillum brasilense.

[0122] Azotobacter chroococcum (A. chroococcum) is a free-living plant growth-promoting rhizobacterium (PGPR) that can fix atmospheric nitrogen under aerobic conditions. A. chroococcum is rod shaped and is a Gram negative bacterium. It produces auxins, cytokinins, and gibberellic acid like substances that enhance plant growth. Azotobacter chroococcum was grown in Norri’s medium, a nitrogen free media. See Table 1.

[0123] Methylobacterium aminovorans (M. aminovorans) is a pink-pigmented facultatively methylotrophic (PPFM) soil bacteria. Methylobacterium aminovorans is rod shaped and is a Gram negative bacterium. Methylobacterium species are reported to be PGPR and influence plant growth by fixing nitrogen. They can grow on single-carbon compounds such as formate, formaldehyde and methanol as sole source of carbon and energy as well as on a wide range of multi-carbon growth substrates. Methylobacterium aminovorans was grown on Glycerol Peptone medium. See Table 1.

[0124] Azospirillum brasilense (A. brasilense) is a Gram-negative, free-living bacterium. Azospirillum brasilense associates with plant roots, excretes plant hormones, and fixes nitrogen. This bacterium can increase the growth of numerous agricultural crops worldwide through the excretion of various hormones and due to its ability to fix nitrogen. Azospirillum brasilense was grown on Modified Nutrient medium. See Table 1.Table 1: Growth Mediums and Optimal Growth Times and Temperatures

[0125] Multiplication of bacteria: A. chroococcum, A. brasilense, and M. aminovorans were multiplied by inoculating each bacterium in respective medium in a conical flask aseptically. Inoculated conical flasks were incubated in a microbiological incubator at optimum temperature for 3-7 days. Fully grown cultures were centrifuged in a cold centrifuge to collect bacterial pellets of each respective bacterium. The collected bacterial pellets were mixed in phosphate buffer solution (“PBS”) and used for inoculation. Initial population load was determined by serial dilution plate method using respective media and confirmed to have colony forming units (CFU) of greater than IxlO8per mL of PBS.

[0126] Raising seedlings in pro trays and first inoculation: Pro trays with 98 cells were filled with seedling growing substrate medium (vermiculite mix). A seeding hole was made in the middle of the each cell using a glass rod. Three tomato seeds were sown in each cell and respective bacterial cultures at 2 mL per each cell was inoculated according to the treatments, 2 ml of Azotobacter chroococcum for group 2, 2 ml of Azospirillum brasilense for group 3, 2 ml of Methylobacterium aminovorans for group 4, and a combination of 1 ml of Azotobacter chroococcum and 1 mL of Methylobacterium aminovorans for group 5. Seeds were covered and watering was done whenever necessary. 25 day old seedlings grown in pro trays were used for transplanting to polybags. The groups of treated seeds were:1. Uninoculated control2. Inoculation with Azotobacter chroococcum3. Inoculation with Azospirillum brasilense4. Inoculation with Methylobacterium aminovorans5. Inoculation with A. chroococcum + M. aminovorans

[0127] Transport to polybags, additional inoculation, growth, and recording: Polybags of size 27 cm x 14 cm with holding capacity of 4 kg substrate were filled with a soil: sand: compost substrate mixture in the ratio of 1:1:0.25 (V / V / V). A planting hole was made at the middle of the substrate mixture in the polybag and respective bacterial inoculated seedlings were placed in the planting hole. Eight replicates were made for each treatment. A second dose of inoculant at 3 mF per each polybag was inoculated and the planting hole was closed with the substrate and watered whenever necessary. The substrate sample was outsourced for analysis to determine macro and micro nutrient status. 50% of a half recommended dose of fertilizers for tomato crop was added 10 days after transplantation (DAT) of seedlings to all the polybags including the controls (urea at 0.3 g, single super phosphate at 0.9 g, and muriate of potash at 0.25 g). The remaining 50% of the half recommended dose of fertilizers was added during the flowering stage, e.g., around 35 DAT. A first reading of growth parameters, e.g., plant height, stem diameter, and biovolume index, was recorded at 40 DAT to assess the inoculation’s initial effects.

[0128] Plant growth parameters recorded at harvest (95 DAT): A second reading of growth parameters was recorded at harvest, 95 DAT. Plant height (cm / plant) was recorded using standard measuring tape by measuring from the soil surface to the growing tip of each plant. Stem diameter (mm / plant) was measured for each plant one cm above the soil surface using digital Vernier Calipers. Biovolume index (BI) (which depicts the total volume of a plant) was calculated by multiplying plant height (ht.) and stem diameter (dia.) [Biovolume index = Plant height (cm) x Stem diameter (mm)]. Fruits (g / plant) plucked when matured from each plant after the fruit reached physiological maturity was weighed using a standard weighing balance. Shoot and root parts were separated and collected in a paper cover individually, dried in a hot air oven at 60 °C to a constant weight, and the weight (g / plant) was recorded using standard weighing balance. Total dry biomass of each plant was calculated and recorded. The plant samples were analyzed for macro (N, P, K, Ca, Mg) and micro (Zn, Fe, Cu, Mn and Bo) nutrients.

[0129] Results: In general, inoculation with all the bacteria resulted in increased plant growth, yield, and dry weight of the plants. Methylobacterium aminovorans significantly increased the plant growth, fruit yield, and dry weight of the plants compared to other treatments. The next most significant improvement was seen in plants treated with Azotobacter chroococcum and Azospirillum brasilense both being on par with each other. It was observed that a consortia with Azotobacter chroococcum and Methylobacterium aminovorans resultedin the third best treatment, indicating a possible negative compatibility of these bacteria. Uninoculated control plants recorded the least of the parameters. See Tables 2 and 3.Table 2: Effect of different N-fixing bacteria on plant height, stem diameter and biovolume index of tomatoValues superscribed with identical letters within each column do not differ significantly at p < 0.05; SEd = standard error deviation; CD = critical differenceTable 3: Effect of different N-fixing bacteria of fruit yield and dry biomass of tomatoValues superscribed with identical letters within each column do not differ significantly at p < 0.05; SEd = standard error deviation; CD = critical differenceExample 2

[0130] The effect of chemical fertilizers on microbes was tested on tomato seeds inoculated with nitrogen-fixing bacterium in root zone soil. The population of inoculated Methylobacterium aminovorans was tested in the following groups.1. M. aminovorans (inoculated at the time of sowing)2. M. aminovorans (inoculated at the time of sowing) + 50% recommended NPK in the form of chemical fertilizers added 10 days after sowing

[0131] Procedure: Plastic pots were filled with vermiculite substrate mix with three replicates for each treatment. Centrifuge collected M. aminovorans was added (5mL of 109CFU / mL / pot) to the seeding hole before sowing tomato seeds. Chemical fertilizers (nitrogen,phosphorous, and potassium (NPK)) were added at 50% of the recommended dose (RDF) 10 days after sowing.

[0132] At the sowing, the initial population of the bacterium was greater than IxlO9CFU / mL of soil in the root zone. Subsequent population enumeration was done at 30 days and 50 days after sowing by serial dilution plate method.

[0133] Results: Analysis of bacterial population in root zone soil of both treatments revealed reduced population in chemical fertilizer treated plants. The experiment showed clearly a decrease in the M. aminovorans population with addition of 50% RDF both at 30 and 50 days after application. See FIG. 1 where (A) depicts bacterial populations in root zone soil 30 days after Methylobacterium aminovorans inoculation of seeds and soil; (B) depicts bacterial populations in root zone soil 30 days after Methylobacterium aminovorans inoculation of seeds and soil and application of NPK fertilizer; (C) depicts bacterial populations in root zone soil 50 days after Methylobacterium aminovorans inoculation of seeds and soil; and (D) depicts bacterial populations in root zone soil 50 days after Methylobacterium aminovorans inoculation of seeds and soil and application of NPK fertilizer.Example 3

[0134] Efficiency and yield performance on baby corn by a microbial consortia (MC) containing nitrogen-fixer bacterium, phosphorus solubilizing bacterium, and potassium solubilizing bacterium, Methylobacterium radiotolerans, Bacillus subtilis, and Bacillus mucilaginosus, respectively, versus the nitrogen-fixer bacterium, Methylobacterium radiotolerans, alone (MR) or a control (C) with no microbes added. The microbes were provided to the baby corn soil by either inoculation of 5 ml of a liquid inoculant to the seeds and soil or by coating 5 ml of the liquid inoculant on urea (Co), which was then applied to the soil at the time of sowing. The plants were fertilized with different percentages of RDF of NPK as well at the time of sowing. Urea fertilizer was applied in two split doses (at the time of sowing and at the time of flowering). The full dose of phosphorous and potassium, was applied at the time of sowing. The coated urea contributed to the total nitrogen provided as part of the percent of RDF, and did not provide additional nitrogen over the percent RDF for each group. These tests were performed as pot cultures under glasshouse conditions. The tested groups were as follows, with three replicates for each.1. 100% RDF of NPK, no microbes (100:C)2. 85% RDF of NPK, no microbes (85:C)3. 75% RDF of NPK, no microbes (75:C)4. 100% RDF of NPK, inoculation with MR (100:MR)5. 100% RDF of NPK, inoculation with Microbial consortia (100:MC)6. 100% RDF of NPK, Microbial consortia coated on urea (100:MC:Co)7. 85% RDF of NPK, inoculation with Microbial consortia (85:MC)8. 85% RDF of NPK, Microbial consortia coated on urea (85:MC:Co)9. 75% RDF of NPK, inoculation with Microbial consortia (75:MC)10. 75% RDF of NPK, Microbial consortia coated on urea (75:MC:Co)

[0135] Results: Com cob yield (g) for all of the plants in each treatment (Total Cob Yield (g) / trt), Com kernel yield (g) for all of the plants in each treatment (Total Corn Yield (g) / trt), average cob mass (g) per plant in each treatment (Avg Cob Yield (g) / plant (g)), average com yield per plant for each treatment (Avg Com Yield (g) / plant), average height per plant (cm) in each treatment (Ht (cm)), average girth (mm) per plant in each treatment (Girth or Diameter (mm)), and average bio volume index per plant in each treatment (BI). See Table 4 and Figs. 2 and 3.Table 4: Effect of different microbial consortia (MC) containing nitrogen-fixer bacterium, phosphorus solubilizing bacterium, and potassium solubilizing bacterium on height, yield and biomass of cornExample 4

[0136] Compatibility of selected nitrogen-fixer bacterium, phosphorus solubilizing bacterium, and potassium solubilizing bacterium with Methylobacterium radiotolerans and Methylobacterium aminovorans in a culture as a consortia was tested. Specifically, Bacillus subtilis, Bacillus megaterium, and Paenibacillus polymyxa of the phosphorus solubilizing bacterium and Bacillus mucilaginosus and Frateuria aurentia of the potassium solubilizing bacterium were tested for compatibility on a cross-streak assay method.

[0137] Each bacteria was grown in their respective growth medium, Gylcerol Peptone (GP) Medium for the nitrogen-fixing bacterium, Nutrient Medium for the phosphorus solubilizing bacterium, and Glucose Yeast extract Calcium Carbonate (GYCC) Medium for the potassium solubilizing bacterium. Each bacterium was streaked on a Modified Nutrient agar (MNA) medium plate. The grown cultures on MNA were cross streaked against each other on an MNA plate. After incubation and growth, compatibility was checked based on growth where the streaks crossed between each bacterium.

[0138] Results: All the test bacterium were able to grow on modified nutrient agar (MNA) and all the test bacterium were compatible with M. radiotolerans and M. aminovorans. See Table 5 and FIG. 4.Table 5: Compatibility of bacteria with M. radiotolerans and M. aminovoransExample 5

[0139] Efficiency of potassium solubilizing bacteria was tested on inoculated tomato seeds. Inoculation with Bacillus mucilaginosus and Frateuria aurentia were tested for changes to plant height, stem diameter, and biovolume index of tomato plants.

[0140] Bacillus mucilaginosus is a Gram- negative, spore forming rod- shaped bacteria commonly found in soil. It is most commonly found in the rhizosphere region of soils and has the ability to solubilize potassium from minerals in soil. Bacillus mucilaginosus was grown on Glucose Yeast Extract Calcium Carbonate (GYCC) medium.

[0141] Frateuria aurentia is a Gram-negative, rod-shaped soil bacteria which is known to solubilize potassium minerals in the soil and also mobilize to plant roots. Frateuria aurentia was grown on Glucose Yeast Extract Calcium Carbonate (GYCC) medium.

[0142] GYCC is prepared with the following: Glucose 20g; Yeast extract 3g; CaCOs 5g; Distilled water 1 hr, with a final pH of 7.0+0.2. The optimum temperature and time for growth is 30-35°C for 1-2 days.

[0143] Multiplication of bacteria: B. mucilaginosus and F. aurantia were multiplied by inoculating each bacterium in respective broth medium in a conical flask aseptically. Inoculated conical flasks were incubated in a microbiological incubator at optimum temperature for 1-2 days. Fully grown cultures were centrifuged in a cold centrifuge to collect the respective bacterial pellets. The collected bacterial pellets were mixed in phosphate buffer solution (PBS) and used for inoculation. Initial population load was determined by serial dilution plate method using respective media and confirmed to have colony forming units (CFU) to be greater than IxlO8per mL of PBS.

[0144] Raising seedlings in pro trays and first inoculation: Methods as described in Example 1 were followed except with the bacteria tested here. The groups of treated seeds were:1. Uninoculated control2. Inoculation with Bacillus mucilaginosus3. Inoculation with Frateuria aurantia4. Inoculation with B. mucilaginosus + F. aurantia (Bm + Fa)

[0145] Transport to polybags, additional inoculation, growth, and recording: Methods as described in Example 1 were followed except with the bacteria tested here.

[0146] Plant growth parameters recorded at harvest (95 DAT): Methods as described in Example 1 were followed except with the bacteria tested here.

[0147] Results: In general inoculation with all the bacteria resulted in increased plant growth, fruit yield between 65 to 80 days after sowing (when the tomato was ready for harvest for each plant), and dry weight of the plants. Plant growth, fruit yield, and dry weight was significantly increased with treatment of Bacillus mucilaginosus compared to other treatments. Frateuria aurentia was on par with Bacillus mucilaginosus in increasing dry weight of the plant. Plant growth was poor in plants treated with the combination of B. mucilaginosus + F. aurantia compared to single inoculation with either of the bacteria. This indicates a possible negative compatibility between the two bacteria. Uninoculated control plants had the least of the measurements. See Table 6.Table 6: Effect of different K- solubilizing bacteria on plant height, stem diameter and biovolume index of tomatoValues superscribed with identical letters within each column do not differ significantly at p < 0.05; SEd = Standard error deviation; CD = critical differenceExample 6

[0148] Efficiency of phosphorous solubilizing bacteria was tested on inoculated tomato seeds. Inoculation with Bacillus megaterium, Paenibacillus polymyxa, and Bacillus subtilis were tested for changes to plant height, stem diameter, and biovolume index of tomato plants.

[0149] Bacillus megaterium is a rod-like, Gram-positive, mainly aerobic spore forming bacterium found in diverse habitats. These are commonly found in soils and are members of the microbiome of several plant hosts worldwide. The strain phosphaticum is a well known phosphorus solubilizer. Bacillus megaterium was grown on Nutrient medium.

[0150] Paenibacillus polymyxa is a Gram-positive bacterium and is a PGPR. It is found in soil, plant tissues, marine sediments and hot springs. It has been reported to possibly have arole in forest ecosystems and potential future applications as a biofertilizer and biocontrol agent in agriculture. Some findings show it can solubilize phosphorus. Paenibacillus polymyxa was grown on Nutrient medium.

[0151] Bacillus subtilis, also known as hay bacillus or grass bacillus, is a Gram-positive, spore forming, catalase-positive bacterium found in soil. It is a well known PGPR used in sustainable agriculture system in crops like vegetables, soybeans, cotton, and peanuts, and in flowers and ornamental seeds. Bacillus subtilis may colonize and protect the root systems of plants and suppress soil-borne pathogens. Bacillus subtilis was grown on Nutrient medium.

[0152] Nutrient medium is prepared with the following: Beef extract 1g; Yeast extract 2g; Peptone 5g; Distilled water 1 hr, with a final pH of 7.0+0.2. The optimum temperature and time for growth is 30-35 °C for 1-2 days.

[0153] Multiplication of bacteria: B. megaterium, P. polymyxa, and B. subtilis were multiplied by inoculating aseptically each bacterium in nutrient broth medium in a conical flask separately. Inoculated conical flasks were incubated in a microbiological incubator at optimum temperature for 1-2 days. Fully grown cultures were centrifuged in a cold centrifuge to collect the respective bacterial pellets. The collected bacterial pellets were mixed in phosphate buffer solution (PBS) and used for inoculation. Initial population load was determined by serial dilution plate method using respective media and confirmed to have colony forming units (CFU) to be greater than IxlO8per mL of PBS.

[0154] Raising seedlings in pro trays and first inoculation: Methods as described in Example 1 were followed except with the bacteria tested here. The groups of treated seeds were:1. Uninoculated control2. Inoculation with Bacillus megaterium3. Inoculation with Paenibacillus polymyxa4. Inoculation with Bacillus subtilis5. Inoculation with Bacillus megaterium + Paenibacillus polymyxa

[0155] Transport to polybags, additional inoculation, growth, and recording: Methods as described in Example 1 were followed except with the bacteria tested here.

[0156] Plant growth parameters recorded at harvest (95 DAT): Methods as described in Example 1 were followed except with the bacteria tested here.

[0157] Results: In general inoculation with all the bacteria significantly increased plant growth, yield, and dry weight of the plants compared to uninoculated control. Plant growth parameters plant height (Height) and biovolume index were higher in all the inoculated plants and all were on par with each other. Stem girth measurements (Diameter) however was not significant over the control. Fruit yield and dry weight were significantly increased in plants treated with Bacillus subtilis and was on par with the second best treatment, Bacillus megaterium. The third best treatment was the combination of B. megaterium + P. polymyxa. Uninoculated control plants recorded the least of the parameters tested. See Tables 7 and 8.Table 7: Effect of different P-solubilizing bacteria on plant height, stem diameter and biovolume index of tomatoValues superscribed with identical letters within each column do not differ significantly at p < 0.05; SEd = Standard error deviation; CD = critical differenceTable 8: Effect of different P-solubilizing bacteria on fruit yield and dry weight of tomatoValues superscribed with identical letters within each column do not differ significantly at p < 0.05; SEd = Standard error deviation; CD = critical differenceExample 7

[0158] Evaluation of microbial consortia coated on urea (wax coated) under pot culture conditions

[0159] The experiment was conducted to check the performance of microbial consortia (A / . radiotolerans (N-fixer) + B. subtilis (P- solubilizer) + B. mucilaginosus (K- solubilizer)) coated on urea (wax coated) for a baby corn crop with Syngenta G5417 variety under pot culture conditions.

[0160] Experimental details

[0161] Treatments1. 100 NPK2. 100 NPK + Methylobacterium radiotolerans + B. subtilis + B. mucilaginosus (Regular Inoculation)3. 100 NPK + Methylobacterium radiotolerans + B. subtilis + B. mucilaginosus (Coated on urea)4. 85 NPK5. 85 NPK + Methylobacterium radiotolerans + B. subtilis + B. mucilaginosus (Regular Inoculation)6. 85 NPK + Methylobacterium radiotolerans + B. subtilis + B. mucilaginosus (Coated on urea)7. 100 NPK + Azotobacter chroococcum + B. subtilis + B. mucilaginosus (Regular Inoculation)8. 100 NPK + Azospirillum brasilense + B. subtilis + B. mucilaginosus (Regular Inoculation)9. 100 NPK + Rhizobium sp. + B. subtilis + B. mucilaginosus (Regular Inoculation) 10. 85 NPK + Azotobacter chroococcum + B. subtilis + B. mucilaginosus (Regular Inoculation)11. 85 NPK + Azospirillum brasilense + B. subtilis + B. mucilaginosus (Regular Inoculation)12. 85 NPK + Rhizobium sp. + B. subtilis + B. mucilaginosus (Regular Inoculation)

[0162] Bacterial cultures used

[0163] Methylobacterium radiotolerans (N-fixer), Bacillus subtilis (P-solubilizer), and Bacillus mucilaginosus (K- solubilizer) as well as:

[0164] Azotobacter chroococcum: is a free-living plant growth-promoting rhizobacterium (PGPR) fixing atmospheric nitrogen under aerobic conditions. A. chroococcum is rod shaped Gram negative bacterium. It produces auxins, cytokinins, and gebberlic acid like substances that enhance plant growth. (Media: Norris N-free media).

[0165] Azospirillum brasilense: is a Gram-negative, free-living bacterium that associates with plant roots, excretes plant hormones and fixes nitrogen and increase the growth of numerous agricultural crops worldwide through the excretion of various hormones and by its ability of nitrogen fixation. (Media: Modified nutrient broth).

[0166] Rhizobium sp.:Rhizobium is a genus of Gram-negative soil bacteria that fixes nitrogen. Rhizobium species form an endosymbiotic nitrogen-fixing association with roots of legumes. The bacteria colonize plant cells to form root nodules, where they convert atmospheric nitrogen into ammonia using the enzyme nitrogenase. The ammonia is shared with the host plant in the form of organic nitrogenous compounds such as glutamine or ureides. The plant, in turn, provides the bacteria with organic compounds made by photosynthesis. (Media: Yeast extract mannitol broth).

[0167] Multiplication of bacteria: All the bacterial cultures listed above were multiplied by inoculating each bacterium in their respective broth medium in a conical flask aseptically. Inoculated conical flasks were incubated in a microbiological incubator at optimum temperature. Fully grown cultures were centrifuged and inoculated into the soil directly or coated on urea with talc based substrate and then added to soil as per the treatment. Initial population load in the broth culture was confirmed to have colony forming units (CFU) of > 1 x 108per ml of culture using serial dilution method grown on respective media.

[0168] Seed sowing in polybags: Polybags of size 25 cm x 25 cm with 4 kg substrate holding capacity of were filled with the soil, sand compost substrate mixture in the ratio 1:1:0.25 (V / V / V). There were 12 treatments and 3 replications and hence 36 polybags were prepared. Two seeding holes per polybag was made in the substrate. Broth inoculum @ 5 ml per polybag was centrifuged and added directly or through coated DAP as per the treatment. 4 seeds were sown per polybag and watered whenever necessary. After seed germination only 2 seedlings were allowed to grow per polybag. RDF per plant was calculated to be Urea (5.4g),SSP (10.4) and MoP (2.8g) which will be added in two split doses. According to NPK levels of different treatment first split dose of urea and urea coated with bacterial consortia were added 5 days after sowing to control and respective treatments. Regular inoculation to respective treatments were added @ 5ml per polybag (Table 9). Later first split does of SSP and MoP were added 10 days after sowing (DAS) and second dose of NPK during cob initiation stage (around 45-50 DAS).Table 9: Quantity of fertilizer, inoculum added, and inoculation method followed.&& &Note: Mr: Methylobacterium radiotolerans', Ac: Azotobacter chroococcimr, Ab: Azospirillum brasilense, Rh: Rhizobium sp., *l.lg contains centrifuged inoculum of 5ml raw broth inoculum

[0169] Plant growth parameters recorded at harvest (95 DAT): Plant height (cm / plant) was recorded using standard measuring tape by measuring from soil surface to the growing tip of the each plant. Stem diameter (mm / plant) was measured one cm above the soil surface using digital Vernier Callipers. Biovolume index (BI) (which depicts the total volume of a plant) was calculated by multiplying plant height (ht.) and stem diameter (dia.) [Biovolume index = Plant height (cm) x Stem diameter (mm)]. Cobs were harvested as indicated and when matured. Number of cobs per plant, cob weight and corn weight per plant was compiled and recorded. Shoots were cut, collected in paper cover individually, dried in a hot air oven at 60°C up to a constant weight and weight recorded using standard weighing balance. The powdered plant samples were outsourced for macro (N, P, K, Ca, Mg) and micro (Zn, Fe, Cu, Mn and Bo) nutrient analysis.

[0170] Results: Overall, plants inoculated with the microbial consortium exhibited significantly greater plant growth (height, shoot diameter, and biovolume index), shoot biomass, and yield compared to the control treatments (100% RDF) without inoculants. Plants with regular inoculation and urea coating also showed a notably higher yield than their respective controls. This trend was consistent at varying NPK levels (85% RDF) as well (Tables 10 & 11). Further inoculations with other nitrogen-fixers, such as Azotobacter chroococcum, Azospirillum brasilense, and Rhizobium spp., did not enhance plant growth as effectively as Methylobacterium radiotolerans. Macronutrients (N, P, and K) and micronutrients (Fe and Mn) were higher in inoculated plants at both nutrient levels (100% and 85% RDF) (Table 3). However, there were no significant differences in Ca, Mg, S, Zn, Cu, and B levels.Table 10: Response of baby corn to talc based microbial consortia (M. radiotolerans + B. subtilis + B. mucilaginosus') coated on urea along with other N-fixers Azotobacter chroococcum, Azospirillum brasilense and Rhizobium sp.) with 100 and 85% RDF on baby corn plant height, stem diameter, biovolume index and dry weight under glasshouse conditions.""Note: RI: Regular inoculation; Mr: Methylobacterium radiotolerans', Bs: Bacillus subtilis', Bm: Bacillus mucilaginosus', Ac: Azotobacter chroococcum; Ab: Azospirillum brasilense', Rh: Rhizobium sp.; Values superscribed with identical letters within each column do not differ significantly at p < 0.01; SEd = Standard error deviation; CD = Critical DifferenceTable 11: Response of baby corn to talc based microbial consortia {M. radiotolerans + B. subtilis + B. mucilaginosus') coated on urea along with other N-fixers Azotobacter chroococcum, Azospirillum brasilense and Rhizobium sp.) with 100 and 85% RDF on baby corn yield (cob and corn) under glasshouse conditions.Note: RI: Regular inoculation; Mr: Methylobacterium radiotolerans', Bs: Bacillus subtilis', Bm: Bacillus mucilaginosus', Ac: Azotobacter chroococcum; Ab: Azospirillum brasilense', Rh: Rhizobium sp.; Values superscribed with identical letters within each column do not differ significantly at p < 0.01; SEd = Standard error deviation; CD = Critical DifferenceTable 12: Response of baby corn to talc based microbial consortia {M. radiotolerans + B. subtilis + B. mucilaginosus) coated on urea along with other N-fixers {Azotobacter chroococcum, Azospirillum brasilense and Rhizobium sp.) with 100 and 85% RDF on plant macro (%) and micro nutrients (ppm) under glasshouse conditions.Note: RI: Regular inoculation; Mr: Methylobacterium radiotolerans', Bs: Bacillus subtilis', Bm: Bacillus mucilaginosus', Ac: Azotobacter chroococcum; Ab: Azospirillum brasilense', Rh: Rhizobium sp.; Values superscribed with identical letters within each column do not differ significantly at p < 0.01; SEd = Standard error deviation; CD = Critical Difference; NS: Not significantExample 8Evaluation of liquid biofertilizer and urea coated with NPK consortia on sweet corn crops

[0171] Products tested:1. Liquid nitrogen fixing biofertilizer (as soil application);2. Liquid NPK Consortia Biofertilizer (mixture of N-fixing, Phosphorus solubilizing, and Potassium solubilizing microbes);3. 1 above coated on Urea; and4. 2 above coated on Urea

[0172] RDF = Recommended Dose of Fertilizer (NPK) = 150:75:40 Kg / Hectare. Source of nitrogen (N), phosphorous (P), and potassium (K) fertilizers as RDF were: urea as a source for N; Single Superphosphate fertilizer (SSP) as a source of P; and Muriate Of Potash (MOP) as a source of K.

[0173] RDN = Recommended Dose of Nitrogen (N) = 150 Kg / Hectare.

[0174] N-Fixing microbe = Methylobacterium radiotolerans.

[0175] P-Solubilizer microbe = Bacillus subtilis.

[0176] K-Solubilizing Microbe = Bacillus mucilaginosus.

[0177] Coated fertilizers comprises 80 mL of microbial inoculant and 200 grams of talc per kg of fertilizer.

[0178] Land Operations: A layout of 48 plots was used for growing sweet corn crops. Land preparation included ploughing, harrowing, cultivating, levelling, and rotavating. Sowing operations included sowing, thinning, gap filling, and applying fertilizer. Crop activities included weeding, irrigating, protecting crops, and watching & warding for birds. Harvesting activities included harvesting, winnowing, threshing, and packaging.Table 13: Experimental details.Table 14: Number of samples for soil analysis at 30 days after sowing (DAS), 60 DAS, and harvest.&Table 15: Treatment details.&&&&&&&&

[0179] In treatments that specify N-fixer, the only bacteria used was the N-fixing microbe. In treatments that specify NPK microbial consortia, the bacteria used included the N-fixing microbe, P-solubilizer microbe, and K-solubilizing microbe. In T3-T8, the recommended dose of P and K remained constant (100% P&K) while the dose of N varied (75-100%). In T9-T14, the dose of each of N, P, and K varied according to the specified RDF. For example, in T9 the dose of N, P, and K was 75% of the recommended dose. See Table 16 for more details.Table 16: Fertilizer applications for each treatment.&&&&&&&Table 17: Organization of plots and treatments.

[0180] Results: Addition of NPK microbial consortia and N-fixing microbes resulted in significantly higher yields than controls. For example, crops receiving 100% RDF through Sabie urea coated with NPK microbial consortia (T11) recorded significantly higher sweet corn yield (28.21 tons ha1) followed by 100% RDN through Sabie urea coated with N fixer + 100% P&K (T8; 26.22 tons ha1) compared with RDF (T2; 25.02 tons ha-1). Similar trends were observed in all yield parameters. Results are shown in Tables 18-21 and summarized in FIGs.6-11.Table 18: Effect of liquid biofertilizer (NPK consortia) on yield of sweet com.&&&&Table 19: Effect of soil application of liquid biofertilizer on growth attributes of sweet corn.&&&&Table 20: Effect of urea coated with NPK consortia on yield attributes of sweet corn.&&&&Table 21: Effect of urea coated with NPK consortia on growth attributes of sweet com.&&&&

Claims

1. CLAIMSWe claim:

1. A composition for a non-leguminous plant comprising a microbial inoculant comprising:at least one or more live nitrogen fixing bacteria comprising Methylobacterium aminovorans and / or Methylobacterium radiotolerans',at least one or more live potassium solubilizing bacteria; andat least one or more live phosphorus solubilizing bacteria.

2. The composition of claim 1, wherein the potassium solubilizing bacteria comprises Bacillus mucilaginosus and / or Frateuria aurantia; and / or wherein the phosphorus solubilizing bacteria comprise Bacillus megaterium var. phosphaticum, Paenibacillus polymyxa, and / or Bacillus subtilis.

3. The composition of any one of claims 1 to 2, further comprising a nitrogen fertilizer, wherein the nitrogen fertilizer at least partially coated with the microbial inoculant.

4. The composition of any one of claims 1 to 3, further comprising Azotobacter chroococcum and / or Azospirillum brasilense.

5. The composition of any one of claims 1 to 4, further comprising:a nutrient for bacteria;a stabilizer; and / ora dispersant.

6. The composition of claim 5, wherein the nutrient for bacteria comprises a monosaccharide, di-saccharide, amino acid, enzyme, vitamin, plant growth hormone, micronutrient, humic acid, humate, and / or skim milk, wherein the stabilizer comprises polyethylene glycol (PEG), polyvinyl alcohol (PVA), and / or ethanol, and / or wherein the dispersant comprises humic acid, a humate, talc, and / or a clay.

7. The composition of any one of claims 3 to 6, wherein the composition comprises at least one coating at least partially between the microbial inoculant and the nitrogen fertilizer, wherein the at least one coating comprises an oil, a wax, humate, and / or a humic acid.

8. The composition of any one of claims 5 to 7, wherein the nutrient for bacteria is at least partially in contact with the microbial inoculant, the stabilizer is at least partially between the nitrogen fertilizer and the microbial inoculant, the microbial inoculant is at least partially between the stabilizer and the nitrogen fertilizer, and / or the microbial inoculant is at least partially between the dispersant and the nitrogen fertilizer.

9. The composition of any one of claims 1 to 8, further comprising glycerol, carboxy methyl cellulose (CMC), polyvinyl pyrrolidone (PVP), gum Arabic, guar gum, a CMC disaccharide, arabic gum, and / or guar gum.

10. The composition of any one of claims 1 to 9, further comprising a carrier.

11. The composition of claim 10, wherein the carrier comprises an alginate, activated carbon, biochar, and / or a starch.

12. The composition of any one of claims 1 to 11, further comprising a spore inducing compound.

13. The composition of any one of claims 1 to 12, wherein at least a portion of the nitrogen fixing bacteria, the potassium solubilizing bacteria, and / or the phosphorus solubilizing bacteria are comprised in a spore.

14. The composition of any one of claims 1 to 13, comprising 95 to 99 wt. % of a combination of the nitrogen fixing bacteria, the potassium solubilizing bacteria, and the phosphorus solubilizing bacteria based on the total weight of the composition.

15. The composition of any one of claims 1 to 14, wherein the microbial inoculant comprises IxlO7to IxlO12colony forming units (CFU) per mL of a combination of the live nitrogen fixing bacteria, the live potassium solubilizing bacteria, and the live phosphorus solubilizing bacteria.

16. The composition of any one of claims 1 to 15, wherein the composition is a liquid composition.

17. The composition of any one of claims 1 to 15, wherein the composition is a solid composition.

18. A method for improving nutrient use efficiency of a non-leguminous plant, the method comprising applying a composition of any one of claims 1 to 17 to a soil wherein the non-leguminous plant is growing, to a non-leguminous plant, to a non-leguminous seed, to water for the non-leguminous plant, and / or to a fertilizer for the non-leguminous plant.

19. The method of claim 18, wherein the composition is applied to the fertilizer for the non-leguminous plant.

20. The method of any one of claims 18 to 19, wherein the non-leguminous plant is a rice, com, soy bean, tomato, lettuce, barley, wheat, and / or grass.

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