WO2024006500A1

WO2024006500A1 - Compositions and methods for stabilizing biological materials

Info

Publication number: WO2024006500A1
Application number: PCT/US2023/026684
Authority: WO
Inventors: Daniel Joo; Neta SCHWARTZ; Natalia SHESTAKOVA; Brice TUGBENYOH; Gregory Linshiz; Karen V. Ambrose
Original assignee: Patternag, Inc.
Priority date: 2022-07-01
Filing date: 2023-06-30
Publication date: 2024-01-04
Also published as: WO2024006500A4

Abstract

The present invention relates to stabilizing compositions of microorganisms for food, agricultural, or pharmaceutical use. These stabilizing compositions maintain viability and activity for up to 36 months of the microorganisms contained therein and may be applied as a solution or in a desiccated form. Wherein the stabilizing composition has an agricultural use, the composition may be a seed coating or applied to soil or plant parts or may be applied in furrow. Methods for preparing the agricultural compositions are provided.

Description

COMPOSITIONS AND METHODS FOR STABILIZING BIOLOGICAL MATERIALS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of U.S. Provisional Application No. 63/358,075, filed July 1 , 2022, the contents of which are hereby incorporated by reference.

FIELD OF INVENTION

[0002] The present invention relates to stabilizing compositions for microorganisms used in agricultural, industrial, and therapeutic spaces. The microorganisms retain viability and activity between 10-50x longer than microorganisms in traditional media.

BACKGROUND OF THE INVENTION

[0003] The benefits of advantageous microorganisms are well known across the agricultural, industrial and therapeutic spaces. However, the ability to cultivate, manufacture, and apply microorganisms is challenging. Challenges arise when microorganisms are processed for commercial use, they often are not viable, or are unable to be reconstituted at the site of application.

[0004] The viability, activity, and long-term stability of microorganisms may be affected by a number of environmental factors; for example, temperature, pH, the presence of water/humidity and oxygen or oxidizing or reducing agents. It is well known that, in an aqueous phase, bacteria instantly lose their activity during storage at ambient temperatures (AT) and lose viability quickly. Generally, bacteria are dried before or during mixing with other components. The drying process can often result in a significant loss in activity from mechanical, chemical, and osmotic stresses induced by the drying process. Loss of viability and activity may occur at many different stages, including drying during initial manufacturing, preparation (upon exposure to high temperature, high humidity, oxygen and high pressure), transportation and long-term storage (temperature, oxygen, and humid exposure), and after the intended application (e.g., agricultural, industrial, or therapeutic). Production of products with live cell organisms is particularly challenging because the bacteria are very sensitive to oxygen, temperature and moisture, all of which are typically present at the site of production and place of use.

[0005] It is well known that most microorganisms exhibit their optimal benefit when they are alive. Hence, they need to survive the manufacturing process, transport, and shelf life before application. To compensate for losses due to environmental conditions, an excessive quantity of microorganism is generally included in products, in hopes that a portion of those will reach the intended target. In addition to the questionable shelf-life viability of those products, these practices are not cost-effective. Furthermore, such practices lead to variability in dosage, which is undesirable.

[0006] Various formulations and compositions have been devised to overcome these challenges. For instance, using multiple coating layers and stabilizing bacteria through oxygen scavengers have been used in the food industry. U.S. Patent Application Publication 2016/0360777. Others have created compositions in a “glassy state” through the addition of sugars and salts. U.S. Patent No. 9,504,750. While these, and other, publications include the use of cellulose derivatives as thickeners, its properties as a stabilizer of the microorganism are not taught.

[0007] There is a need for a stabilizing composition that is useful for a broad range of microorganisms and provides superior stabilization and preservation of microorganisms over extended periods of time, variations in temperature and humidity, such as can be encountered during shipping and storage of materials, while still retaining a significant amount of viability and activity upon use or rehydration. See Ying Ma (2019). Seed coating with beneficial microorganisms for precision agriculture, Biotechnology Advances, 37(7), 107423, ISSN 0734- 9750 and Digat, B. (1989). STRATEGIES FOR SEED BACTERIZATION Acta Hortic. 253, 121-130, DOI: 10.17660/ActaHortic.1989.253.12.

SUMMARY OF THE INVENTION

[0008] The present invention relates to stabilizing compositions of microorganisms for food, agricultural, or pharmaceutical use. These stabilizing compositions maintain viability and activity for up to 36 months of the microorganisms contained therein and may be applied as a solution or in a desiccated form. Wherein the stabilizing composition has an agricultural use, the composition may be a seed coating or applied to soil or plant parts or may be applied in furrow. The disclosure also provides methods for preparing agricultural compositions.

[0009] The solution of a stabilizing composition of microorganisms is a significant advancement in preserving microorganism viability and activity before those microorganisms are used at their desired destination (e.g., field, greenhouse, therapeutic center, or distribution center).

[0010] According to the present invention the stabilizing composition is a 0.5% MC if fully soluble amorphous viscous solution in water and at least one microorganism. In other embodiments the stabilizing composition is desiccated into a granule or dust form. In yet other embodiments the stabilizing composition is rchydratcd from a granule or dust form. In yet other embodiments, the stabilizing composition consists of 0.15%, 0.33%, or 1% of MC. In some embodiments, the microorganism is a bacterium. In some of those embodiments, the bacteria are present in an amount of 10⁸ - 10¹⁰ CFU/ml.

[0011] In other embodiments, the stabilizing composition is a 1% of PEG4,000 in a fully soluble amorphous viscous solution in water and a microorganism. In yet other embodiments, the stabilizing composition consists of 0.8, 1.0, or 1.2% of PEG4,000. In some embodiments, the microorganism is a bacteria. In some of those embodiments, the bacteria is present in an amount of 10⁵ - 10¹⁰ CFU/ml.

[0012] In some embodiments the 0.5% MC solution is made with MQ water, wherein MQ water is water that has been purified using an ion exchange cartridge. The purity of the water is monitored by measuring the conductivity. The higher the resistance, the fewer ions in the water. The MQ water in preferred embodiments has a conductivity of - < 100 S/c @ 25 °C (as NaCI).

[0013] In other embodiments the 0.5% MC solution is made with DI water. In yet other embodiments, the water grade is ultrapure water type 2.

[0014] In some embodiments the microorganism is one type of microbe. In other embodiments the microorganism is more than one type of microbe. In those embodiments the more than one type of microbe is a consortia or consortium, which are used interchangeably herein.

[0015] In some preferred embodiments the microorganism is one type of bacterium. In some preferred embodiments the bacterium is a phosphate solubilizing bacterium. In some of those embodiments the bacterium is Pseudomonas, Burkholderia, Rhanella, Serratia, or Pantoea . In other of those embodiments the bacteria are Burkholderia cenocepacia, Pantoea agglomerans, Yersinia frederiksenii, Pseudomonas simiae, Rhizobium rhizogenes, Rahnella aquatilis, Pseudomonas fluorescens, Serratia marcescens, Serratia bockelmannii, or Stenotrophomonas sp. In some preferred embodiments the bacterium is a nitrogen fixing bacteria. In some of those embodiments the bacterium is Azospirillum .

[0016] In other embodiments, the microorganism of the present invention is a bacteria and may be: Proteobacteria, Gammaproteobacteria, Enterobacterales, Erwiniaceae, Pantoea, Betaproteobacteria, Burkholderiales, Burkholderiaceae, Burkholderia, Enterobacterales, Yersiniaceae, Yersinia, Rahnella, Pseudomonadales, Pseudomonadaceae, Pseudomonas, Enterobacteriaceae, Enterobacter, Xanthomonadales, Xanthomonadaceae, Xanthomonas, Xanthomonadales, Xanthomonadaceae, Stenotrophomonas, Moraxellales, Moraxellaceae, Acinetobacter, Alphaproteobacteria, Hyphomicrobiaceae, Rhizobiaceae, Rhizobium/Agrobacterium, Bacteroidetes, Rhanella, Serratia, Sphingobacteriia, Sphingobacteriales, Sphingobacteriaceae, Sphingobacterium, Alcaligenaceae, Achromobacter, Comamonadaceae, Comamonas, Firmicutes, or Bacilli..

[0017] In some embodiments, the microorganism is a bacterial strain that may be selected from the group consisting of Chryseobacterium daecheongense, Chryseobacterium rhizosphaerae, Frigidibacter albus, Arthrobacter nicotinovorans, Pseudomonas helmantic ensis, Agrobacterium fabrum, Exiguobacterium sibiricum, Exiguobacterium anlarcticum, Leifsonia lichenia, and Tumebacillus permanentifrigoris .

[0018] In some embodiments the stabilizing composition further comprises a plant-promoter, biostimulant, or a biofertilizer. In other embodiments, the stabilizing composition further comprises an herbicide, pesticide, or fertilizer. In yet other embodiments the composition may provide a coating on a plant-promoter, biostimulant, biofertilizer, herbicide, pesticide, fertilizer, or nanoparticle.

[0019] These and other objects and features of the invention will become more fully apparent from the following detailed description of the invention and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a graph showing the sustained viability of gram-negative bacteria in the stabilizing composition using MC over 250 days, as described in the present invention.

[0021] Figure 2 is a graph showing the sustained viability of two gram-negative bacteria in the stabilizing compositions using MC over 250 days, as described in the present invention.

[0022] Figure 3 is a graph showing the sustained viability of a strain of gram-negative bacteria in the stabilizing composition using MC over 250 days, as described in the present invention.

[0023] Figure 4 is a graph showing another sustained viability of the Figure 3 strain of gramnegative bacteria in the stabilizing composition using MC over 200 days, as described in the present invention.

[0024] Figure 5 is a graph showing sustained viability of the two strains of gram-negative bacteria in the stabilizing composition using 0.33% MC over 12 weeks as compared to those strains in water, as described in the present invention.

[0025] Figure 6 is a graph demonstrating sustained viability of multiple PSB strains in 0.15% MC over 36 weeks, as in described herein.

[0026] Figure 7 is a graph demonstrating sustained viability of multiple PSB strains in 0.33% MC over 36 weeks, as in described herein.

[0027] Figure 8 is a graph demonstrating sustained viability of multiple PSB strains in 0.5% MC over 36 weeks, as in described herein.

[0028] Figure 9 is a graph demonstrating sustained viability of multiple PSB strains in 1.0% MC over 36 weeks, as in described herein. [0029] Figure 10 is a graph demonstrating sustained viability of multiple PSB strains in 1.0% PEG 400 over 36 weeks, as in described herein.

[0030] Figure 11 is a graph demonstrating sustained viability of multiple PSB strains in 1.0% PEG 4,000 over 36 weeks, as in described herein.

[0031] Figure 12 is a graph demonstrating survival of three strains subjected to desiccation over 8 weeks in water, as described herein.

[0032] Figure 13 is a graph demonstrating survival of three strains subjected to desiccation over 8 weeks in 0.5% MC, as described herein.

[0033] Figure 14 is a picture of a plant grown in the presence of the stabilizing composition further comprising MC, showing the improved effect of the compositions with a microorganism relative to an MC solution alone wherein the plants received supplemental Cas/PCL , as described in the present invention.

[0034] Figure 15 is a depiction of root tips of microorganisms in the stabilizing compositions relative to controls, as described in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

[0035] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. [0036] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof.

[0037] “Ambient” room temperatures or conditions, as used herein, are those at any given time in a given environment. Typically, the ambient room temperature is about 22-25°C, the ambient atmospheric pressure is about 760 Torr or 1 atmosphere, and the ambient relative humidity is about 30-50% in winter and about 40-65% in summer, which can be readily measured and may vary depending on the time of year, weather and climatic conditions, altitude, etc. All measurements (e.g., water activity) are made under ambient conditions (i.e., ambient temperature, ambient atmospheric pressure, and ambient humidity) unless indicated otherwise.

[0038] As used herein, “biofertilizers” arc microbial fertilizers that supply the plant with nutrients and thereby can promote plant growth in the absence of chemical fertilizers. Nonlimiting examples of microbial isolates that can directly promote plant growth and/yield include Nz-fixing bacteria Rhizobium and Bradyrhizobium species that, through symbiotic nitrogen fixation, can form nodules on roots of leguminous plants, in which they convert atmospheric N2 into ammonia which, in contrast to atmospheric N2, can be used by the plant as a nitrogen source. Other examples include Azospirillum species, which are free-living N2-fixers that can fertilize and increase yield of cereal crops such as wheat, sorghum, and maize. Despite Azo spirillum's N2- fixing capacity, the yield increase caused by inoculation by Azospirillum is often attributed to increased root development and thus to increased rates of water and mineral uptake. In this respect, several rhizobacteria like Azotobacter spp. have been reported to be capable of producing a wide array of phytohormones (e.g., auxins, cytokinins) and enzymes (e.g., pectinase). Many of these phytohormones and enzymes have been shown to be intimately involved in the infection process of symbiotic bacteria-plant associations which have a regulatory influence on nodulation by Rhizobium. Biofertilizers can also affect the plant growth and development by modifying nutrient uptake. They may alter nutrient uptake rates, for example, by direct effects on roots, by effects on the environment which in turn modify root behavior, and by competing directly for nutrients (Gaskin et al., Agricult. Ecosyst. Environ. 12: 99-116, 1985). Some factors by which Biofertilizers may play a role in modifying the nutrient use efficiency in soils include, for example, root geometry, nutrient solubility, nutrient availability by producing plant congenial ion form, partitioning of the nutrients in plant and utilization efficiency. For example, a low level of soluble phosphate can limit the growth of plants. Some plant growth-promoting microbes are capable of solubilizing phosphate from either organic or inorganic bound phosphates, thereby facilitating plant growth. Several enzymes of microbial origin, such as nonspecific phosphatases, phytases, phosphonatases, and C-P lyases, release soluble phosphorus from organic compounds in soil. For example, an increased solubilization of inorganic phosphorus in soil has been found to enhance phosphorus uptake in canola seedling using Pseudomonas putida as well as increased sulfur-oxidation and sulfur uptake (Grayston and Germida, Can. J. Microbiol. 37: 521-529, 1991; Banerjee, Phytochemicals and Health, vol. 15, May 18, 1995).

[0039] “Biostimulants", as used herein, can produce substances that stimulate the growth of plants in the absence of pathogens. For example, the production of plant hormones is a characteristic of many plant-associated microorganisms. Some microorganisms can also produce secondary metabolites that affect phytohormone production in plants. Probably, the best-known example is hormone auxin, which can promote root growth. Other examples include pseudomonads which have been reported to produce indole acetic acid (IAA) and to enhance the amounts of IAA in plants, thus having a profound impact on plant biomass production (Brown, Annual Rev. Phytopathology, 68: 181-197, 1974). For example, Tien et al. Applied Environmental Microbiol., 37:1016-1024, 1979) reported that inoculation of nutrient solutions around roots of pearl millet with Azospirillum brasiliense resulted in increased shoot and root weight, an increased number of lateral roots, and all lateral roots were densely covered with root hairs. Plants supplied with combinations of IAA, gibberellins and kinetin showed an increase in the production of lateral roots similar to that caused by Azospirilla. Additionally, some rhizobacteria, such as strains of the bacterial species B. subtilis, B. amyloliquefaciens, and Enterobacter cloacae, promote plant growth by releasing volatile organic compounds, VOCs. The highest level of growth promotion has been observed with 2,3-butanediol and 3-hydroxy-2- butanone (also referred to as acetoin) as elicitors of induced systemic resistance. The cofactor PQQ has been described as a plant growth promoter, which acts as an antioxidant in plants. [0040] “Colony Forming Units” or “CFUs” is a measurement of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell, often expressed in orders of magnitude (e.g., 6.7 x 10^s)

[0041] “Crop” is a plant that can be grown and harvested extensively for profit or subsistence. Crops may refer either to the harvested parts or to the harvest in a more refined stale. Most crops are cultivated in agriculture, aquaculture, or vertical/indoor farming. Important non-food crops include horticulture, floriculture and industrial crops. Horticulture crops include plants used for other crops (e.g. fruit trees). Crops include row crops (e.g., wheat, corn, sugar beets, etc.) and specialty crops (e.g., leafy greens, berries, tomatoes).

[0042] “Dry” and variations thereof refer to a physical state that is dehydrated or anhydrous, i.c., substantially lacking liquid. Drying includes for example, spray drying, fluidized bed drying, lyophilization, and vacuum drying. A material such as a formulation is dry if its water activity is low, for example, no more than about 0.4, 0.35, 0.3, 0.2, or 0.1.

[0043] As used herein “fertilizer”, which generally are classified according to their NPK content. NPK is common terminology used in the fertilizer industry and stands for: (1) N — the amount of nitrogen in the formulation as N; (2) P — the amount of phosphorus in the formulation as P2O5; and (3) K — the amount of potassium in the formulation as K2O. In other words, the N refers to nitrogen-containing compounds that are added to the soil and are utilized by the particular plant to satisfy its nitrogen requirement. The P refers to phosphorus -containing compounds that are added to the soil and are utilized by the particular plant to satisfy its phosphorus requirement (a nutrient required for plant growth). K refers to potassium-containing compounds that are added to the soil and are utilized by the particular plant to satisfy its potassium requirement (another nutrient essential for plant growth). Besides these nutrients, namely nitrogen, phosphorus and potassium, which are normally provided by the addition of fertilizers that typically are known as NPK fertilizers, other nutrients can also be provided by the addition of fertilizers to the soil. Typical nutrients are calcium, magnesium, sulfur, iron, zinc, manganese, copper, boron and molybdenum. The term “fertilizer” as used herein, unless expressly indicated otherwise, refers to NPK fertilizers, that is, fertilizers that include one or more of the nutrients (nitrogen, phosphorus and potassium). [0044] An “herbicide,” as used herein, is any substance used to kill, destroy, or mitigate the growth of an unwanted seed, plant, or plant part.

[0045] “Media” as described herein is adequate to grow selected bacteria. Media may be R2A, NB, LB, or TSB. As is known by those skilled in the art, the media may be used in a variety of concentrations such as lx, O.lx, 0.2x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, and the like.

[0046] Methyl Cellulose (or MC) is a chemical compound derived from cellulose. It is sold under a variety of trade names and is used as a thickener and emulsifier in various food and cosmetic products, and also as a bulk-forming laxative. Like cellulose, it is not digestible, not toxic, and not an allergen. In preferred embodiments, MC is used as a stabilizing agent for microorganisms.

[0047] “Microorganism” means bacteria (e.g. gram negative, gram positive, etc.), microbes, phage or viruses. Microorganisms may be live or not as referred to herein.

[0048] “Pesticide”, as used herein, is any substance used to kill, destroy, mitigate, remove, repel or any other similar action against any pest on a seed, plant, or plant part.

[0049] Polyethylene glycol (or PEG) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide or polyoxyethylene, depending on its molecular weight. PEG as used herein may be PEG 400, PEG 1,000, PEG 4,000, PEG 6,000 and PEG 8,000 in molecular weight. In some embodiments, PEG as used herein is between 200-35,000 in molecular weight. In some embodiments, PEG is present at 0.5%, or 1% wt/vol concentration. In other embodiments, PEG is present at between 0.2- 1.5% wt/vol concentration. In preferred embodiments, PEG is used as a stabilizing agent for microorganisms.

[0050] Plant or plant part includes all parts of the plant, including: root, stem, meristem, seed, leaf, cotyledons, and the like.

[0051] A “stable” formulation or composition, as used herein, is one in which the microorganism therein essentially retains its physical stability, chemical stability, and/or biological activity upon storage. The terms “formulation,” “formula” and “compositions” are used herein interchangeability. Stability can be measured at a selected temperature and humidity conditions for a selected time period. Trend analysis can be used to estimate an expected shelf life before a material has actually been in storage for that time period. In a particular aspect of the present invention, live microorganisms such as bacteria, parasites, and viruses, stability is defined based on the loss of viability of live microorganisms, for example, about 1 or a predetermined log of colony forming units per gram CFU/g for bacteria and/or plaque forming units per gram (PFU/g) for viruses, in a formulation or on seeds under predefined conditions, for example, temperature, humidity, and time period.

[0052] As used herein, a “stable” microorganism is one that when coated onto a seed, as taught herein, is viable for 1 month. In particular embodiments, the stable microorganism is coated onto a seed and remains viable for at least 6 months. In other particular embodiments the stable microorganism is coated onto a seed and remains viable for 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. In still other particular embodiments the stable microorganism is coated on to a seed and remains viable for up to one year. In still other particular embodiments the stable microorganism is coated on to a seed and remains viable for at least 3 months, hr more particular embodiments a stable microorganism when coated on to a seed remains viable for 3 to 6 months. In still more particular embodiments, a stable microorganism when coated on to a seed remains viable for 6 to 12 months.

[0053] “Viability” with regard to the microorganism wherein the microorganism is a bacteria, refers to the ability to form a colony (CFU or Colony Forming Unit) on a nutrient media appropriate for growth of the microorganism (e.g., bacteria). Viability with regard to a microorganism that is a virus, refers to their ability to infect and reproduce in suitable host cells, resulting in the formation of a plaque (PFU or Plaque Forming Unit) on a lawn of host cells.

[0054] “Water activity” or “Aw” in the context of dried formulation compositions, refers to the availability of water and represents the energy status of the water in a system. It is defined as the vapor pressure of water above a sample divided by that of pure water at the same temperature. Pure distilled water has a water activity of exactly one, i.e., Aw=1.0. [0055] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0056] No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0057] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and embodiments will be apparent to those of skill in the art upon review of this disclosure.

COMPOSITION OF THE INVENTION

[0058] The present invention, in a preferred embodiment, consists of 0.5% methyl cellulose (“MC”) and a microorganism. The surprising discovery has been made that wherein the microorganism is a bacteria, bacteria in the MC suspension retain viability for at least 6 months and up to 18 months relative to bacteria suspended in water or other media. Bacterial suspension in 0.15%, 0.5% and 1% MC have maintained viability for at least 6 months and up to 18 months. In other preferred embodiments, the stable composition consists of either 1% PEG 400 or PEG 4,000 and a microorganism. The equally surprising discovery has been made that wherein the microorganism is a bacteria, the bacteria in the PEG suspension retain viability for 6 months relative to bacteria suspended in water or other media. In preferred embodiments, microorganisms in the stable composition are selected to be beneficial for one of the following plant functions: phosphate solubilization, potassium solubilization, and/or nitrogen fixation. In some most preferred embodiments, the bacteria of the present invention are phosphorus solubilizing. Examples of bacteria that are contemplated in the present invention include Pseudomonas, Acinetobacter, Burkholderia, Serratia, Rahnella, or Pantoea. In some embodiments the bacteria are Burkholderia cepacia, Pantoea agglomerans, Yersinia frederiksenii, Pseudomonas simiae, Rhizobium rhizogenes, Acinetobacter calcoaceticus, Rahnella aquatilis, Pseudomonas fluorescens, Serratia marcescens, or Stenotrophomonas sp. In a preferred embodiment of the invention the phosphorus solubilizing bacteria in the stabilizing composition demonstrate five times the activity relative to the same bacteria in other compositions. In other embodiments the phosphorus solubilizing bacteria in the stabilizing composition demonstrate 10, 15, 20, 25, 30, 25, 40, 45, or 60 times the activity relative to bacteria in other compositions such as water and the like. Moreover, bacteria in the stabilizing composition retain their viability, CFUs decrease at a lower rate, relative to those in other compositions known to those skilled in the art.

[0059] As seen in Figure 1, the phosphorus solubilizing bacteria maintain viability for 250 days in the stabilizing compositions. Figure 2 provides a closer look at the stability of a Burkholderia cepacia and a Pseudomonas fluorescens over 250 days. Figures 3 and 4 demonstrate the stability of another Burkholderia cepacia .

[0060] As described, the stabilizing compositions provide extended viability to a multitude of phosphorus solubilizing bacteria relative to those bacteria suspended in water, as demonstrated in Figure 5. This graph demonstrates stability over 12 weeks of two strains in 0.33% MC relative to those strains in water. In water, the strains are not viable after 1 week (Tl), but the strains retain viability and nearly the original level through 12 weeks (T12).

[0061] Figure 6 demonstrates extended viability over 36 weeks of 9 strains of phosphorus solubilizing bacteria in 0.15% MC. These strains vary with a viability of approximately 10¹⁰ at 0 weeks, to approximately 10⁶ at 36 weeks. The same 9 strains in 0.33% MC show a similar pattern of viability as demonstrated in Figure 7. Figure 8 depicts the same 9 strains in 0.5% MC, demonstrating better retention of viability for some strains such as B19 (Serratia marscens).

Figure 9 depicts the 9 strains retention of viability in 1.0% MC.

[0062] Figure 10, as in one embodiment, demonstrates poor viability of the 9 strains of phosphorus solubilizing bacteria over 36 weeks when formulated in 1% PEG 400. Whereas Figure 11 demonstrates extended viability over 36 weeks in 1% PEG 4,000.

[0063] Moreover, when the stabilizing composition comprising MC of the present invention is dried, the microorganisms therein retain viability for surprisingly longer duration than those microorganisms contained in other compositions known to those skilled in the art. [0064] This is demonstrated in Figures 12 and 13. Figure 12 shows three bacteria in water over a longitudinal study for 3 weeks, wherein significant viability was lost in the B96 and B107 strains. Those strains when formulated with 0.5% MC show a significant improvement in viability.

[0065] In another surprising discovery, the phosphate solubilizing bacteria (PSB) microorganisms of the stable composition, when desiccated and subsequently rehydrated, have an improved effect on plant growth relative to the PSB in MC suspension. This surprising discovery is also observed with nitrogen-fixing bacteria.

[0066] In other embodiments, the bacteria of the present invention may be: Proteobacteria, Gammaproteobacteria, Enterobacterales, Erwiniaceae, Pantoea, Proteobacteria, Betaproteobacteria, Burkholderiales, Burkholderiaceae, Burkholderia, Enterobacterales, Yersiniaceae, Yersinia, Yersiniaceae, Rahnella, Pseudomonadales, Pseudomonadaceae, Pseudomonas, Enterobacteriaceae, Enterobacter, Xanthomonadales, Xanthomonadaceae, Xanthomonas, Xanthomonadales, Xanthomonadaceae, Stenotrophomonas, Moraxellales, Moraxellaceae, Acinetobacter, Alphaproteobacteria, Hyphomicrobiales, Rhizobiaceae, Rhizobium/Agrobacterium, Bacteroidetes, Sphingobacteriia, Sphingobacteriales, Sphingobacteriaceae, Sphingobacterium, Alcaligenaceae, Achromobacter, Comamonadaceae, Comamonas, Firmicutes, or Bacilli.

[0067] In some embodiments, an isolated bacterial strain may be selected from the group consisting of Chryseobacterium daecheongense, Chryseobacterium rhizosphaerae, Frigidibacter albus, Arthrobacter nicotinovorans, Pseudomonas helmanticensis, Agrobacterium fabrum, Exiguobacterium sibiricum, Exiguobacterium antarcticum, Exiguobacterium antarcticum, Leifsonia lichenia, and Tumebacillus permanentifrigoris .

[0068] In yet other preferred embodiments, the bacteria are isolated from soil. Those isolated bacteria are identified by 16S sequencing. Note, some isolated bacteria contain more than one strain, as is used in some embodiments of the present invention. For example, the present invention contemplates the use, either alone or in combination, of the following PSB: Table t:

[0069] In some embodiments, the amount of one or more of the microorganisms in the stable compositions of the present invention can vary depending on the final formulation as well as the type of plant or seed utilized. Preferably, one or more of the microorganisms with the compositions are present in about 2 x 10⁹ CFUs. In other embodiments the amount of bacteria is 9xl0⁸ to 2.5X10⁹CFUS. [0070] In some embodiments of the present invention the stable compositions are applied as a seed treatment, in furrow, or in granule or dust form. Particularly preferred methods include inoculation of growth medium or soil with suspensions of microorganism cells and the coating of plant seeds with microorganism cells and/or spores.

[0071] Most stable compositions of the invention are chemically inert; hence they are compatible with substantially any other constituents of an application schedule. They may also be used in combination with plant growth affecting substances, such as fertilizers, plant growth regulators, and the like, provided that such compounds or substances are biologically compatible. They can also be used in combination with biologically compatible pesticidal active agents as for example, herbicides, nematocides, fungicides, insecticides, and the like.

[0072] When used as biofertilizers in their commercially available formulations and in the use forms, prepared from these formulations, the active microorganism strains in the stable compositions according to the present invention can furthermore be present in the form of a mixture with synergists. Synergists are compounds by which the activity of the active compositions is increased without it being necessary for the synergist added to be active itself.

[0073] When used as biofertilizers in their commercially available formulations and in the use forms, prepared from these formulations, the active microorganism in the stable compositions according to the invention can furthermore be present in the form of a mixture with inhibitors which reduce the degradation of the stable compositions after application in the habitat of the plant, on the surface of parts of plants or in plant tissues.

[0074] The active microorganism strains in the stable compositions according to the invention, as such or in their formulations, can also be used as a mixture with known fertilizers, acaricides, bactericides, fungicides, insecticides, microbicides, nematicides, pesticides, or combinations of any thereof, for example in order to widen the spectrum of action or to prevent the development of resistances to pesticides in this way. In many cases, synergistic effects result, i.e., the activity of the mixture can exceed the activity of the individual components. A mixture with other known active compounds, such as growth regulators, safeners and/or semiochemicals is also contemplated. [0075] In a preferred embodiment of the present invention, the stable compositions may further include at least one chemical or biological fertilizer. The amount of at least one chemical or biological fertilizer employed in the compositions can vary depending on the final formulation as well as the size of the plant and seed to be treated. Preferably, the at least one chemical or biological fertilizer employed is about 0.1% w/w to about 60% w/w based on the entire formulation. More preferably, at least one chemical or biological fertilizer is present in an amount of about 1% w/w to about 60% w/w and most preferably about 10% w/w to about 50% w/w.

[0076] The present invention also provides method of treating a plant by application of the stable composition in any of a variety of customary formulations in an effective amount to either the soil (i.e., in-furrow), a portion of the plant (i.e., drench) or on the seed before planting (i.e., seed coating or dressing). Customary formulations include solutions, emulsifiable concentrate, wettable powders, suspension concentrate, soluble powders, granules, coatings for granules, suspension-emulsion concentrate, natural and synthetic materials impregnated with active compound, and very fine control release capsules in polymeric substances. In certain embodiments of the present invention, the stable compositions are formulated in powders that are available in either a ready-to-use formulation or are mixed together at the time of use. In either embodiment, the powder may be admixed with the soil prior to or at the time of planting. In an alternative embodiment, one or both of either the plant growth-promoting agent or biocontrol agent is a liquid formulation that is mixed together at the time of treatment. One of ordinary skill in the art understands that an effective amount of the inventive compositions depends on the final formulation of the composition as well as the size of the plant or the size of the seed to be treated.

[0077] In a particularly preferred embodiment, the stable compositions of the present invention are formulated as a seed treatment. It is contemplated that the seeds can be substantially uniformly coated with one or more layers of the stable compositions disclosed herein using conventional methods of mixing, spraying or a combination thereof through the use of treatment application equipment that is specifically designed and manufactured to accurately, safely, and efficiently apply seed treatment products to seeds. Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mists or a combination thereof. Liquid seed treatments such as those of the present invention can be applied via either a spinning “atomizer” disk or a spray nozzle which evenly distributes the seed treatment onto the seed as it moves through the spray pattern. Preferably, the seed is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying. The seeds can be primed or unprimed before coating with the inventive compositions to increase the uniformity of germination and emergence. In an alternative embodiment, a dry powder formulation can be metered onto the moving seed and allowed to mix until completely distributed.

[0078] The formulation that is used to treat the seed in the present invention can be in the form of a suspension; emulsion; slurry of particles in an aqueous medium (e.g., water); wettable powder; wettable granules (dry flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation is preferably about 0.5% to about 99% by weight (w/w), preferably 5-40% or as otherwise formulated by those skilled in the art.

METHODS OF MAKING THE COMPOSITION

[0079] Preparation of the MC solution. Suspensions of Methyl Cellulose (MC) weight per volume (wt/vol) were prepared by adding, with stirring, MC powder to distilled water. In some embodiments the water is distilled, Milli-Q, or HPLC water. The following amounts of MC were used to attain the following percentages: 0.15g for 0.15%, 0.33g for 0.33%, 0.5g for 0.5%, and 1g for 1%. The suspension was slowly brought to a boil and boiled for 5 min until small amorphous aggregates of MC formed. In other embodiments the suspension is brought to a boil, and boiled for 4 minutes, 3 minutes, 6 minutes, or 7 minutes. The MC suspension was then autoclaved for 15 min at 121 lb/in², which caused it to become a colloidal gel. In other embodiments the MC suspension is autoclaved for 20 min at 121 °C, causing the suspension to become a colloidal gel. The suspension is cooled at room temperature until completely solubilized. In some embodiments the suspension is cooled at 20°C for 12 hours until completely solubilized. In other embodiments the MC solution is at a concentration of 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% MC weight per volume. Preparation of the PEG solution.

[0080] 1% PEG 4,000 and PEG 8,000 (wt/vol) were prepared by adding, with stirring, PEG powder to distilled water. The suspension was slowly brought to a boil and boiled for 5 min until PEG was completely solubilized. In other embodiments the suspension is brought to a boil, and boiled for 4 minutes, 3 minutes, 6 minutes, or 7 minutes. The PEG solution was then autoclaved for 15 min at 121 lb/in2. In other embodiments the PEG solution is autoclaved for 20 min at 121°C. In some embodiments the solution is cooled at 20°C for 12 hours until completely solubilized. In other embodiments the PEG solution is at a concentration of 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 1% or 2% PEG weight per volume.

[0081] Preparation of microorganism biomass with MC suspension. In a preferred embodiment, frozen stock of bacteria may be used as a seed culture to inoculate liquid media and grow out targeted bacteria. In some embodiments the target bacteria is Pseudomonas, Burkholderia, Rahnella, Serratia, or Pantoea. In other embodiments the bacteria may be Burkholderia cenocepacia, Pantoea agglomerans, Yersinia frederiksenii, Pseudomonas simiae, Rhizobium rhizogenes, Rahnella aquatilis. Pseudomonas fluorescens, Serratia marcescens, Stenotrophomonas sp. The bacterial frozen stock, in a preferred embodiment, is approximately 1 xlO⁹ cfu/ml. The liquid media, in one embodiment is R2A at a lx concentration. In other embodiments the liquid media may be NB, LB, or TSB at a concentration of lx, 0.5x or O.lx. The inoculation is grown at 28°C for about 48 hours. In other embodiments, the inoculation is grown at 30°C for 24 hours. Microbial cultures are then centrifuged, media supernatant is removed and cell pellet resuspended in the MC solution. In preferred embodiments, the MC solution is 0.33%. In some embodiments the resuspended solution has a volume of approximately 20mL. In other embodiments the resuspended solution has a volume of lOmL, 30mL, 50mL, 75mL, 100ml, 250ml or IL of media.

[0082] The microorganism biomass may be contained in a frozen stock. The microorganism biomass may be resuspended in a media compliant with the microorganism of interest, as is known by those skilled in the art.

[0083] Desiccation of the resuspended solution. The resuspended solution may be dried, to further stabilize the microorganism biomass of the present invention. The microorganism biomass is concentrated by being spun down in a centrifuge and resuspended. In a preferred embodiment the microorganism biomass is resuspended in 1 ml of 0.5% MC per strain per condition. In other embodiments the microorganism biomass is resuspended in 1 ml of 0.15% MC, 1ml of 0.33% MC, 1ml of 1% MC, or 1ml of 1% PEG 4,000. The microorganism could be resuspended in more than 1 ml of MC, such as 0.5mL, 2 ml, 5 ml, 10 ml or 20 ml. In many embodiments, the microorganism could be suspended in between 0.3 - 50ml MC. In other embodiments the microorganism could be suspended in an MC solution that is between 0.25 - 5% MC. In other embodiments the microorganism biomass is resuspended in 1 ml of 1% PEG400. The microorganism could be resuspended in more than 1 ml of PEG400, such as 0.5mL, 2 ml, 5 ml, 10 ml or 20 ml. In many embodiments, the microorganism could be suspended in between 0.3 - 50ml PEG400. In other embodiments, the percentage of PEG400 is between 0.5-3.0%. Tn yet other preferred embodiments, the microorganism biomass is resuspended in 1 ml of 1% PEG 4,000. The microorganism could be resuspended in more than 1 ml of PEG 4,000, such as 0.5mL, 2 ml, 5 ml, 10 ml or 20 ml. In many embodiments, the microorganism could be suspended in between 0.3 - 50ml PEG4,000. In other embodiments the percentage of PEG4,000 is between 0.5 -3.0%.

[0084] The suspension is then dried in a preferred embodiment of the present invention. In some embodiments the suspension is dried at room temperature for 7 days. In yet other embodiments the suspension is dried at room temperature for 5, 14 or 21 days. In other embodiments the suspension is dried by spray drying, fluidized bed drying, lyophilization, and vacuum drying. The present invention may then be used to treat a seed, plant, or plant part.

[0085] In some embodiments the suspension after being dried for 7 days was re-suspended in 100 ul of sterile DI water. This was repeated every three (3) weeks, wherein the suspension was dried and then rehydrated. After 3 rounds of rehydration with DI water, the suspension was finally rehydrated in lx R2A medium.

[0086] The resuspended solution may be desiccated in the presence of charcoal powder in the present invention. In one embodiment the microorganism biomass is concentrated by being spun down in a centrifuge and resuspended, as disclosed. In another embodiment, the microorganism biomass is resuspended in 1ml of 0.33% MC and 0.01g charcoal powder. In other embodiments the amount of charcoal powder is between 0.01 and 1 gram. In another embodiment, the microorganism biomass is resuspended in 1ml of 1.0% PEG400 and 0.01g charcoal powder. In other embodiments the amount of charcoal powder is between 0.01 and 1 gram. In another embodiment, the microorganism biomass is resuspended in 1ml of 1.0% PEG4,000 and 0.01g charcoal powder. In other embodiments the amount of charcoal powder is between 0.01 and 1 gram. In this embodiment the suspension is dried. The present invention may then be used to treat a seed, plant, or plant part.

[0087] Treatment. A plant may be treated with the present invention in either the biomass MC suspension or the desiccated solution. In a preferred embodiment, the biomass MC suspension is applied to the growth substrate in an amount of about 2ml. In some embodiments the biomass MC suspension is a bacteria at a concentration of approximately 9xl0⁸ to 2.5xl0⁹. In other embodiments the concentration of bacteria is 2 x 10⁹. The surprising discovery was made that bacterial MC suspensions are able to retain viability and activity for far longer periods than the same bacteria suspended in water. For example, the bacterial MC suspensions retain viability and activity for more than 12 months, wherein bacteria suspended in water survive for a period of 1 week. This represents over a lOOx increase in viability for the bacterial MC suspensions. Moreover, the MC desiccated solution provides similar viability and activity preservation as the biomass MC suspension. The MC desiccated solution can be applied in a rehydrated or dry form.

[0088] A plant may be treated with the present invention in either the biomass PEG4000 suspension. In a preferred embodiment, the biomass PEG4000 suspension is applied to the growth substrate in an amount of about 2ml. In some embodiments the biomass PEG4000 suspension is a bacteria at a concentration of approximately 9xl0⁸ to 2.5xl0⁹. In other embodiments the concentration of bacteria is 2 x 10⁹. The surprising discovery was made that bacterial PEG4000 suspensions are able to retain viability and activity for far longer periods than the same bacteria suspended in water. For example, the bacterial PEG4000 suspensions retain viability and activity for more than 6 months, wherein bacteria suspended in water survive for a period of 1 week. This represents over a 20x increase in viability for the bacterial MC suspensions. [0089] Also provided are stable compositions that include a bacterial strain of the invention or a culture thereof. Such bacterial compositions according to some preferred embodiments may comprise an agriculturally effective amount of an additional compound or composition, in which the additional compound or composition may be a fertilizer, an acaricide, a bactericide, a fungicide, an insecticide, a microbicide, a nematicide, or a pesticide. In some other preferred embodiments, the stable compositions may further include a carrier. In yet other preferred embodiments, the carrier may be a plant seed. In certain embodiments of this aspect, the stable composition is prepared as a formulation that can be an emulsion, a colloid, a dust, a granule, a pellet, a powder, a spray, an emulsion, or a solution. In some other preferred embodiments, the stable compositions may be seed coating formulations. In yet another aspect, plant seeds that are coated with a stable composition in accordance with the present invention are also provided.

[0090] In another aspect, there arc provided methods for treating plant seeds. Such methods include exposing or contacting the plant seeds with a microorganism strain according to the present invention or a culture thereof.

PLANTS SUITABLE FOR THE INVENTION

[0091] In principle, the methods and compositions according to the present invention can be deployed for any plant species. Monocotyledonous as well as dicotyledonous plant species are particularly suitable. The methods and compositions are preferably used with plants that are important or interesting for agriculture, horticulture, for the production of biomass used in producing liquid fuel molecules and other chemicals, and/or forestry.

[0092] Thus, the invention has use over a broad range of plants, preferably higher plants pertaining to the classes of Angiospermae and Gymnospermae. Plants of the subclasses of the Dicolylodenae and the Monocotyledonae are particularly suitable. Dicotyledonous plants belong to the orders of the Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Illiciales, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumbaginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales, and Violates. Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plants belonging to the class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales.

[0093] Suitable species may include members of the genus Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

[0094] The methods and compositions of the present invention are preferably used in plants that are important or interesting for agriculture, horticulture, biomass for the production of biofuel molecules and other chemicals, and/or forestry. Non-limiting examples include, for instance, Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet), Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp.. Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bcrmudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. eucalyptus), Triticosecale spp. (triticum — wheat X rye), Bamboo, Carthamus tinctorius (safflower), Jatropha curcas (Jat opha). Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (king palm), Syagrus romanzoffiana (queen palm), Linum usitatissimum (flax), Brassica juncea, Manihot esculenta (cassava), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Ahelmoschus esculentus (okra), Solanum melongena (eggplant), Papaver somniferum (opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (ryegrass), Phleum pratense (timothy), and conifers. Of interest are plants grown for energy production, so called energy crops, such as cellulose-based energy crops like Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord- grass), Medicago sativa (alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. eucalyptus , Triticosecale spp. (triticum-wheat X rye), and Bamboo; and starch-based energy crops like Zea mays (corn) and Manihot esculenta (cassava); and sugar- based energy crops like Saccharum sp. (sugarcane), Beta vulgaris (sugarbeet), and Sorghum bicolor (L.) Moench (sweet sorghum -, and biofuel-producing energy crops like Glycine max (soybean), Brassica napus (canola), Helianthus annuus (sunflower), Carthamus tinctorius (safflower), Jatropha curcas Jatropha}, Ricinus communis (castor), Elaeis guineensis (African oil palm), Elaeis oleifera (American oil palm), Cocos nucifera (coconut), Camelina saliva (wild flax), Pongamia pinnata (Pongam), Olea europaea (olive), Linum usitatissimum (flax), Crambe abyssinica (Abyssinian-kale), and Brassica juncea.

[0095] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and embodiments will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0096] It should also be understood that the following examples are offered to illustrate, but not limit, the invention.

[0097] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and embodiments will be apparent to those of skill in the art upon review of this disclosure and are to be included within the spirit and purview of this application.

[0098] It should also be understood that the following examples are offered to illustrate, but not limit, the invention.

EXAMPLES

EXAMPLE 1 PREPARATION OF THE COLLOIDAL COMPOSITION

[0099] The following protocol was followed to make the colloidal composition.

1 . To 500 mL glassware add 300 mL sterilized milliQ water

2. Place stir bar and begin stirring on hot plate, set heat to 380°C 3. Add 0.99 g methyl cellulose slowly to stirring water

4. Bring to boil and lower temp to continue boiling but not to cause bubbles and spillage

5. Boil for 5-10 minutes

6. Autoclave for 20 minutes at 121°C

7. Leave on bench top to cool overnight

EXAMPLE 2 PREPARATION OF THE MICROORGANISM BIOMASS

[00100] Wherein the microorganism is a bacteria, the following protocol was followed to prepare the biomass:

1 . Use lOul of frozen stock to inoculate 10ml of lx R2A medium

2. Incubate for 24 hours at 28°C at 200 RPM shaking

3. Transfer 10ml of seed bacterial culture to 90ml of R2A medium in Erlenmeyer flask

4. Incubate 45-48 hours at 28°C with 200rpm shaking

5. Centrifugate microbial cultures at 5,000 rpm x 20 min

6. Remove supernatant and resuspend cell pellet in 20ml of 0.33% Methyl Cellulose solution

EXAMPLE 3 DESICCATION OF MICROORGANISM BIOMASS

[00101] Microbial biomass was spun down and resuspended in 1 ml of 0.33% MC. The suspension was then dried out for 7 days at room temperature. The same protocol was used to dry microbial suspension in 0.33% MC with addition of 0.01 g charcoal powder. After desiccation, dried microbial biomass was re-suspended in 1ml of R2A medium and recovered overnight at room temperature. The suspension was then plated onto O.lx R2A/Gellan medium with serial dilutions (see protocol for CFU plating below). The numbers of colonies were counted, and CFU/ml was calculated per each treatment.

EXAMPLE 4 HIGH THROUGHPUT SCREENING FOR VIABILITY AFTER DESICCATION

[00102] Microbial culture collection (80 x 96 isolates) was screened with MC to test bacterial stability for desiccation. lOOul of each bacterial culture growing in R2A medium was transferred flat bottom 96 well plate and mixed with 50ul of 0.33% MC. Plate was covered with breathable tape and incubated at room temperature for 7 days. After 7 days of incubation bacterial cultures were dried out and then resuspended in 150ul of R2A medium. The suspension was kept overnight for bacterial recovery and then stamped to solid media to determine bacterial viability.

EXAMPLE 5 MICROBIAL VIABILITY AFTER DESICCATION

[00103] The following PSB bacteria were grown, Burkholderia cenocepacia, Panloea agglomerans, Yersinia frederiksenii, Pseudomonas simiae, Rhizobium rhizogenes, Rahnella aquatilis, Burkolderia cepacia, Settaita marcescens, Pseudomonas fluorescens, or Stenotrophomonas sp. in R2A media at 28°C for over 4 weeks. Viability was assessed to determine which day of growth is most viable and functionally active. Burkholderia cenocepacia, Pantoea agglomerans, Yersinia frederiksenii, Pseudomonas simiae, Rhizobium rhizogenes, Rahnella aquatilis, Burkolderia cepacia, Settaita marcescens, Pseudomonas fluorescens, or Stenotrophomonas sp. microbes were grown to early stationary stage, spun down, media supernatant was removed and cell pellet was resuspended in 0.33% MC. After this point bacterial cells stopped cell division and stayed on the stage where they were collected and were preserved in the MC solution. The bacterial suspension in MC was stored at room temperature and tested for its viability (CFU plating) every 14 days. The bacteria maintained viability at nearly the same level from the first time point and during 4 months of testing. The control was the first time point (TO CFU) that was being compared with CFU count of the bacterial suspension in the MC solution after 4 months. Growth rate depended on bacterial growth stage such as laq, log, stationary or death, and also growth conditions (media, aeration, temperature etc.). Different rates during incubation period, upon and after hydration were observed. See the following protocol:

1 . Take out prepared 0. lx R2A Gellan media plate to get to room temperature

2. Label plates with date of original soil inoculation, sample number, dilution factor.

3. Add 90 pl of lx R2A medium into the each well of the 96 well round bottom plate

4. Add 10 pl of your microbe into row A on designated area (Pre mix microbe using vortex)

5. Using the multi-channel pipette (Mix well before transfer) grab 10 ul from row A and transfer to row B 6. Grab new tips, mix row B, transfer 10 pl to row C

7. Repeat until finished

8. Premix and transfer 5 pL from wells diluted 10⁵ to 10⁸ onto the media plate in its corresponding location.

9. Incubate for 2 days at 28°C.

10. Count each colony for every sample corresponding with the dilution factor and fill in the datasheet.

11 . Calculate CFU/mL = number of colonies/(dilution factor * volume plated)

12. Repeat once/week.

EXAMPLE 6 STABILITY IN LIQUID FORMULATION

[00104] As illustrated in Figure 1. Microbes were prepared according to the protocol in Example 2 and tested for viability. As is seen, microbes retained viability for at least 36 weeks in the stabilizing compositions described herein.

EXAMPLE 7 STABILITY IN DRY FORMULATION (DESICCATION)

[00105] As illustrated in Figure 13, this Example shows the stability of various microbes from Table 1 in dry formulation (after desiccation). In this Example, microbes were suspended in various formulations of the stable compositions described herein according to Example 3.

EXAMPLE 8 BACTERIAL EFFICACY MEASUREMENTS IN SOLUTION AND AFTER DESICCATION

[00106] The phosphate solubilizing activity of bacteria was measured after suspension with MC or after desiccation of the MC solution. To test the bacterial activity, the bacteria are placed on a solid media. The solid media has insoluble phosphate substrate (such as CaPO4, phytate, Rock phosphate etc) that is white in appearance and makes culture media cloudy. When microbes grow and solubilize it, they convert insoluble phosphate to its soluble form and make a zone of clearance on the surface (ZOC). The zones of clearance are detectable from solid media background. The size of zones of clearance were calculated by subtracting a diameter of colony size from the diameter of zone of clearance. The zones of clearance have different sizes. [00107] In particular, the size of zones of clearance varied from 1 to 2.5 mm after one week of growth at 28°C for wild type of phosphate solubilizing microorganisms. Desiccated microbes were not measured for the diameter of zones of clearance.

EXAMPLE 9 TREATMENT OF SEED WITH THE MICROORGANISM BIOMASS MC

SUSPENSION

[00108] The following protocol was used to treat seeds with the stabilizing compositions of the present invention:

1 . Use lOul of frozen stock to inoculate 10ml of lx R2A medium

2. Incubate for 24 hours at 28°C at 200 RPM shaking

4. Incubate 45-48 hours at 28°C with 200rpm shaking

5. Centrifugate microbial cultures at 5,000 rpm x 20 min

7. For inoculation use 2ml/pot. Apply on top of the com seed and all the way around it.

EXAMPLE 10 TREATMENT OF PLANTS WITH THE MICROORGANISM BIOMASS MC

SUSPENSION

[00109] The following protocol was used in the treatment of plants with the stabilizing compositions of the present invention:

1 . Wet -500 g of soil in a 6 inch pot with 50 mL lukewarm tap water.

2. In 2.5 cm hole in the soil, place 2 seeds of com. Keep the hole uncovered.

3. Following suspension of bacterial biomass with methyl cellulose, using a micropipette, add 2 mL of resuspended bacteria to each pot directly to the seed.

4. Cover seeds with soil gently to ensure no compaction.

5. Irrigate soil every day for 15 seconds for 1.5 weeks. After 1.5 weeks, irrigate soil every other day for 15 seconds. EXAMPEE 11 IN PLANTA DESICCATION RESULTS

[00110] This Example is illustrated in Figure 14. An increase in root tips was observed in seeds treated with bacterium that were in an MC suspension, desiccated, and rehydrated before application. In this experiment a bacteria biomass of ~2xl0⁹ CFU/mL was cultivated in 100ml of lx R2A media. The biomass was spun down by centrifugation at 5,000 g for 20 minutes, and the supernatant removed. The supernatant was resuspended in 40 mF 0.33% MC. The MC suspension was pipetted onto seed in soil with 2mE of the MC suspension (containing the biomass). For the positive control, lOmE undiluted solution of soluble phosphate fertilizer (e.g., Neptune’s Harvest Fish and Seaweed Fertilizer) and 480mE of water was combined. After mixing, 50mE of the diluted fertilizer was applied to the pot. A negative control (MC) was comprised of 2mE 0.33% MC inoculated directly to seed in soil. The positive control (SF) was the soluble commercial fertilizer (e.g., Neptune’s Harvest) with NPK in a ratio of 2:3:1. Sample B10 is a lead microbe Pseudomonas simiae after desiccation and rehydration. B10 sample was grown out in R2A medium, spun down, resuspended in 2ml of 0.33% MC and the suspension was directly added to the seed. Sample B8 is the lead of wild type of the microbes. The sample was grown out in R2A medium, spun down, resuspended in 2ml of 0.33% MC and the suspension was directly added to the seed. And Sample B8D (desiccated B8) contained the lead microbe after desiccation. The sample was grown out in R2A medium, spin down, resuspended in 2ml of 0.33% MC and cell suspension was directly added to the seed.

[00111] A number of embodiments of the invention have been described. Nevertheless, it will be understood that elements of the embodiments described herein can be combined to make additional embodiments and various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments, alternatives and equivalents are within the scope of the invention as described and claimed herein.

[00112] Headings within the application are solely for the convenience of the reader, and do not limit in any way the scope of the invention or its embodiments.

[00113] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically can individually indicated to be incorporated by reference.

Claims

CLAIMS:

1 . A stabilizing composition comprising: a stabilizer, wherein the stabilizer is methyl cellulose (MC) and the MC is in a concentration of at least 0.1%, or at least 0.2%, or at least 0.3% percent, or at least 0.4%, or at least 0.5%, or at least 1%, or preferably at approximately 0.5% measured in weight per volume; and a microorganism, wherein the viability of the microorganism is retained for up to 18 months.

2. The stabilizing composition of claim 1, wherein the microorganism is a bacteria, and wherein the composition is dried, the bacteria may be reconstituted up to 6 months after drying wherein the bacteria may then grow at a rate of less than two log loss of CFU/g relative to the same bacteria growing in media.

3. The stabilizing composition of claim 2, wherein the composition is dried by air drying, vacuum-drying, or fluid bed drying.

4. The stabilizing composition of claim 1, wherein the microorganism is a bacteria and the bacteria is selected from the group consisting of Pseudomonas, Burkholderia, Serratia, Rahnella, or Pantoea.

5. The stabilizing composition of claim 1, wherein the microorganism is a phosphate solubilizing bacterium.

6. The stabilizing composition of claim 1, wherein the composition is applied to a plant, or plant part.

7. A dry stabilizing composition comprising a microorganism and methyl cellulose at 0.2-

1 % weight by volume wherein the microorganism is a bacterium and wherein the composition exhibits less than two log loss of CFU/g after 30 days at room temperature relative to day 1 of growth, and wherein the composition is prepared by a method comprising: making a solution of 0.2-l%MC by weight per volume of water, heating the solution to a boil, raising the temperature to 121°C, and cooling the suspension to a colloidal gel; and preparing a bacterial biomass with the solution by centrifugation of bacterial culture, removing supernatant, and re-suspending cell pellet in the MC solution wherein the amount of bacteria in the resulting stabilizing composition is between 9xl0⁸ to 2.5xl0⁹, or is 2.0xl0⁹CFU/g. A stabilizing composition comprising: a stabilizer, wherein the stabilizer is polyethylene glycol (PEG) and the PEG is in a concentration of at least 0.5%, or at least 0.7%, or at least 0.8% percent, or at least 0.9%, or preferably at approximately 1.0% measured in weight per volume; and a microorganism, wherein the PEG stabilizes said microorganism such that said microorganism’s viability is unaffected for up to twelve months. The stabilizing composition of claim 8 or 9, wherein the composition is dried and the bacteria may be reconstituted up to 12 months after drying wherein the bacteria retain viability and activity. The stabilizing composition of claim 8 or 9, wherein the composition is dried by air drying, vacuum-drying, fluid bed drying, or spray drying. The stabilizing composition of claim 9, wherein the PEG is of 400 or 4,000 molecular weight. The stabilizing composition of claim 8 or 9, wherein the microorganism is a bacteria and the bacteria is selected from the group consisting of Pseudomonas, Burkholderia, or Pantoea. The stabilizing composition of claim 8 or 9, wherein the microorganism is a phosphate solubilizing bacterium. The stabilizing composition of claim 8 or 9, wherein the composition is applied to a plant, or plant part.