Whole-cell methanotroph-based biostimulant compositions, methods and applications thereof

Abstract

The present disclosure relates to whole-cell-based biostimulant compositions and methods for improving agricultural productivity. In particular, the compositions disclosed herein include a microbial consortium having gammaproteobacterial methanotrophs. As a result of these methanotrophs, the biostimulant compositions improve plant performance, enable methane utilization, and promote improved plant nitrogen fixation. The compositions also help reduce the need for exogenous chemical fertilizers for plant growth, development, performance, and / or survival.

Description

[Technical Field]

[0001] The present disclosure is in the field of biosciences, with a particular focus on biotechnology, agricultural science, and environmental science. The disclosure particularly relates to whole cell-based biostimulant compositions and methods for improving agricultural productivity. In particular, the compositions disclosed herein include a microbial consortium having gammaproteobacterial methanotrophs. As a result of the effects of these methanotrophs, the biostimulant compositions improve plant performance, enable methane utilization, and promote improved nitrogen fixation in plants. The compositions also help reduce the need for exogenous chemical fertilizers for plant growth, development, performance, and / or survival. [Background technology]

[0002] There is an urgent need to increase agricultural production in today's world. The world's population is projected to reach 9.6 billion by 2050. To meet the needs of this growing population, agricultural production will have to increase by approximately 60-70% from current levels, which is no small feat.

[0003] Growing scarcity of natural resources, including land, water, and energy resources, highlights the fact that global agriculture must address the impacts of climate change, extreme temperatures, unpredictable rainfall, and other stressors. While agriculture adapts to changing times, increasing agricultural productivity presents additional challenges and side effects. For example, global consumption of NPK—nitrogen (N), phosphorus (P2O5), and potassium (K2O)—was 292 million tons in 2016 and is expected to increase to 318 million tons by 2022. Demand for NPK grew at an average annual rate of 2.2% from 2015 to 2020 (FAO 2019; World fertilizer trends and outlook to 2022; Rome). However, the continued and increasing use of synthetic fertilizers poses serious environmental threats. For example, long-term use of chemical fertilizers alters soil pH and microflora, leading to an increase in pests and plant pathogens. This, in turn, negatively impacts the beneficial microbial community in the soil. Furthermore, because chemical fertilizers are highly soluble in water, they leach into groundwater and pollute the water table. Excessive use of chemical fertilizers also depletes essential nutrients in the soil. Food crops produced in such soils generally have low vitamin and mineral content. Thus, the use of NPK and other such chemical fertilizers has multiple side effects, and addressing this issue is therefore an ongoing challenge. Therefore, there is a need to curb this excessive use of chemical fertilizers.

[0004] However, one of the obstacles in doing so is finding alternative and efficient methods and means that can help continuously improve nitrogen availability / fixation in plants / crops so that the use of chemical fertilizers can be significantly reduced.While progress has been made in the use of nitrogen-fixing microorganisms to improve nitrogen availability / fixation in plants / crops, better approaches are continually needed.

[0005] Another global concern today is environmental methane emissions, one of the major drivers of climate change. Over the past 20 years, methane has become a potent greenhouse gas with a global warming potential 84 times greater than that of CO2. While methane is produced through several naturally occurring processes, it is anthropogenic production (caused by human activities) that accounts for the majority of methane emissions and is of concern. Agricultural activities are one such area of concern, as they account for a significant proportion of anthropogenic methane emissions. Globally, agricultural activities account for approximately 40% of methane emissions (Agriculture and Climate Change, McKinsey & Company, April 2020). While researchers worldwide are increasingly focusing their efforts on combating climate change, there is an urgent need to reduce methane emissions during agricultural activities. More importantly, there is an increasing need to utilize, channelize, and recycle these agriculturally related methane emissions in an efficient, environmentally friendly, and sustainable manner.

[0006] Therefore, there is a great need to address some of the above-mentioned key concerns / challenges, especially to reduce atmospheric methane levels and increase agricultural productivity in an environmentally friendly and sustainable manner. The present disclosure addresses said needs through a novel / unique approach. Summary of the Invention

[0007] The present disclosure relates to a biostimulant composition comprising a microbial consortium of whole cells, wherein the consortium comprises at least 50% whole cells of Gammaproteobacteria methanotrophs.

[0008] In some embodiments, the composition further comprises at least one metabolite, at least one nutrient derived from the medium, and optionally at least one agriculturally acceptable excipient.

[0009] In some embodiments, the microbial consortium of whole cells is about 1 x 10 per gram or per milliliter of the composition. 3 cells ~ approx. 5 x 10 10 Contains cells.

[0010] In some embodiments, the microbial consortium in the composition utilizes methane, and the composition comprises: a. Improve or enhance plant performance; b. increasing the availability or efficient use of at least one of nitrogen, phosphorus, and potassium by plants; c. reducing the need for external addition of at least one nutrient selected from nitrogen, phosphorus and potassium, either individually or as part of a fertilizer; or Any combination of da to c.

[0011] Thus, the present disclosure also relates to a method of improving or enhancing plant performance comprising contacting or applying the biostimulant composition to the plant.

[0012] In some embodiments, the composition improves or enhances plant performance, including stimulating or promoting quantitative or qualitative plant attributes selected from the group including biomass production, yield, photosynthetic activity, nutritional value, secondary metabolites and nutrient utilization efficiency, or any combination thereof.

[0013] Additionally, the present disclosure also relates to a method for simultaneously promoting methane utilization and nitrogen fixation in plants, comprising contacting or applying the biostimulant composition to the plants.

[0014] In some embodiments, nitrogen fixation is promoted by increasing the expression of nitrogenase genes selected from the group including nifA, nifD, nifH, and nifK, or any combination thereof, in microbial whole cells present in the biostimulant.

[0015] The present disclosure also provides a method for reducing the need for exogenous addition of at least one nutrient or nutrient-carrying fertilizer for plant growth, development, performance, and / or survival, comprising contacting or applying the biostimulant composition to a plant.

[0016] In some embodiments, the nutrients are selected from the group including nitrogen, phosphorus, and potassium, or any combination thereof.

[0017] In some embodiments, the composition improves or enhances plant performance by either increasing the availability or efficient utilization of at least one of nitrogen, phosphorus, and potassium by the plant, or both.

[0018] The present disclosure also provides a method for preparing a biostimulant composition, comprising combining a consortium comprising at least 50% whole cells of a gammaproteobacterial methanotroph, together with at least one metabolite and nutrient from the medium, and optionally at least one agriculturally acceptable excipient.

[0019] In some embodiments, uses of the biostimulant compositions herein are also provided. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows the effect of methanotroph-based whole cell compositions on improving spinach yield. Foliar and soil applications of methanotroph-based whole cell compositions resulted in a 23-36% increase in crop biomass. [Figure 2] Figure 2. Effect of methanotroph-based whole cell composition on guar pea. Foliar application of methanotroph-based whole cell composition resulted in an improvement in pod yield of ~22%. [Figure 3]Figure 3 shows the effect of methanotroph-based whole cell compositions on nutrient uptake in spinach. Soil application of methanotroph-based whole cell compositions resulted in a significant improvement in plant NPK uptake. [Figure 4] Figure 4. Effect of methanotroph-based whole cell composition on coriander grown under various fertilizer levels. Foliar application of methanotroph-based whole cell composition resulted in a ~42% increase in biomass compared to the 100% NPK control. [Figure 5] FIG. 5. Effect of methanotroph-based whole cell compositions on improving the SPAD index. [Figure 6] Figure 6 shows Nif gene expression analysis in methanotroph cell populations. [Figure 7] Figure 7. Effect of methanotroph-based whole cell composition on broad bean. Foliar application of methanotroph-based whole cell composition resulted in an improvement in pod yield of ~15% compared to the commercial control. [Figure 8] Figure 8. Effect of methanotroph-based whole cell composition on guar pea. Foliar application of methanotroph-based whole cell composition resulted in an ~8% improvement in pod yield compared to the commercial control. [Figure 9] Figure 9. Effect of methanotroph-based whole cell composition on coriander. Foliar application of methanotroph-based whole cell composition resulted in a ~28% increase in biomass compared to the commercial control. [Figure 10] Figure 10. Effect of methanotroph-based whole cell composition on spinach. Foliar application of methanotroph-based whole cell composition resulted in a biomass improvement of -13% compared to the commercial control. [Figure 11] Figure 11. Effect of methanotroph-based whole cell composition on chili pepper. Foliar application of methanotroph-based whole cell composition resulted in improved biomass compared to the commercial control. [Figure 12]Figure 12. Effect of methanotroph-based whole cell composition on spinach. Foliar application of methanotroph-based whole cell composition resulted in improved biomass compared to the commercial control. [Figure 13] Figure 13: Effect of methanotroph-based whole cell composition on early seedling establishment of rice. Seed treatment with methanotroph-based whole cell composition improved root and shoot length of rice seeds compared to the control, thus aiding in early seedling establishment. [Figure 14] Figure 14. Effect of methanotroph-based whole cell composition on carrot. Foliar application of methanotroph-based whole cell composition resulted in a 7-10% improvement in taproot compared to the control. [Figure 15] Figure 15 shows the effect of a methanotroph-based whole cell composition on improving the yield of spinach in hydroponic culture. Foliar application of the methanotroph-based whole cell composition resulted in a 43% increase in biomass compared to the control. [Figure 16] FIG. 16. Effect of methanotroph-based whole cell compositions on dietary fiber and protein in spinach. [Figure 17] FIG. 17. Effect of whole cell composition of methanotrophic bacteria on spinach biomass. [Figure 18] FIG. 18. Effect of whole cell composition of methanotrophic bacteria on root and shoot biomass of Radish. [Figure 19] FIG. 19. Effect of whole cell composition of methanotrophic bacteria on fruit number in tomato. [Figure 20] FIG. 20. Effect of relative levels of methanotroph-based whole cell composition on seed germination. DETAILED DESCRIPTION OF THE INVENTION

[0021] In view of the limitations discussed above, and to address the need in the art for an efficient method to reduce the levels of methane generated due to agricultural activities and to improve agricultural practices, the present disclosure provides a biostimulant composition comprising a whole-cell microbial consortium that not only improves or enhances plant performance, but does so while reducing the carbon footprint generated by methane.

[0022] Therefore, the present disclosure aims to address the need to increase agricultural productivity with technological solutions in an environmentally friendly and sustainable approach.

[0023] More specifically, the present disclosure provides an efficient method for reducing atmospheric methane levels and increasing agricultural productivity in an environmentally friendly and sustainable manner by providing a biostimulant comprising whole cells of Gammaproteobacteria methanotrophs, which, when applied to plants, not only increases plant performance but also, due to the presence of Gammaproteobacteria methanotrophs, efficiently utilizes methane.

[0024] Therefore, the present disclosure addresses the problem of effectively utilizing methane emissions generated from agricultural activities or other sources. In other words, the objective of the present disclosure is to reduce the carbon footprint (total greenhouse gas emissions) caused by methane emitted during agricultural activities, while utilizing that same methane to result in increased agricultural productivity.

[0025] One way to achieve this objective of the present disclosure is by making plants more efficient in utilizing nutrients such as nitrogen, phosphorus, and potassium for their growth. Increasing nitrogen fixation is one example, allowing for greater availability of nitrogen to plants. Therefore, another objective of the present disclosure is to improve nitrogen fixation in plants and / or increase the availability of nitrogen to plants.

[0026] Combining this aspect of increased nitrogen fixation with better methane utilization therefore forms another object of the present disclosure, which is to promote methane utilization and nitrogen fixation in an environmentally friendly / biological manner. In particular, the object is to simultaneously promote atmospheric methane utilization and nitrogen availability / fixation in an environmentally friendly / biological manner as a means to improve agricultural productivity.

[0027] Another way to achieve more efficient agricultural production is by reducing the amount of chemical / synthetic fertilizers used in plant growth. Typically, such fertilizers have a negative impact on the soil, the environment, and the water table, thereby affecting the entire ecosystem. Providing biostimulants that reduce plant dependency on chemical / synthetic fertilizers is one example of an innovative solution to this problem. Therefore, another objective of the present disclosure is to reduce the use / application of chemical / synthetic fertilizer-based inputs for agricultural activities.

[0028] It is another object of the present disclosure to combine this aspect of reduced fertilizer use with better methane utilization and nutrient uptake / nitrogen fixation. The object is to promote methane utilization, more efficient nutrient use, better nitrogen availability / fixation, and reduced chemical fertilizer use in an environmentally friendly / biological manner. In particular, the object is to simultaneously promote methane utilization, nitrogen fixation, and reduced chemical fertilizer use in an environmentally friendly / biological manner as a means of improving agricultural productivity.

[0029] Therefore, the present disclosure aims to provide a simple, economical, and sustainable solution to simultaneously address the aforementioned needs of a) utilizing methane generated due to methane emissions from agricultural activities or other sources, b) improving plant nutrient availability / nitrogen fixation, c) reducing the use of chemical fertilizers, and d) providing products and methods for improving agricultural productivity.

[0030] Before proceeding in more detail, the following definitions are provided for some terms that will be used throughout this disclosure.

[0031] As used in this disclosure, the terms "methanotroph(s)" or "methanotroph" or "methanotrophs" refer to prokaryotic cells that use methane as their primary and sole carbon and energy source. In some embodiments of the present disclosure, the methanotroph uses methane as its sole carbon and energy source. In some embodiments of the present disclosure, the methanotroph comprises a methanotrophic bacterium.

[0032] As used in this disclosure, the term "gammaproteobacterial methanotroph" refers to a methanotroph belonging to the class Gammaproteobacteria, which consists of Type I / X methanotrophs. In some embodiments of the present disclosure, the gammaproteobacterial methanotroph includes a member of the order Methylococcales. One example of such a gammaproteobacterial methanotroph is Methylococcus capsulatus (also referred to herein as M. capsulatus).

[0033] As used in this disclosure, the terms / phrases "improving plant performance," "enhancing plant performance," "promoting plant growth," and the like, refer to stimulating / facilitating one or more plant attributes important to plant growth, development, performance, and / or survival, selected from, but not limited to, biomass production, yield, photosynthetic activity, nutritional value, secondary metabolites, and nutrient utilization efficiency, or any combination thereof. Improving or enhancing plant performance or plant growth includes having a stimulatory / promoting effect on the plant as measured by a result selected from, but not limited to, an increase in the number, size, or quality of below-ground or above-ground biomass selected from the group including, but not limited to, roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits, and seeds, or any combination thereof; an increase in photosynthetic activity or chlorophyll content; an increase in protein, dietary fiber, beta-carotene, or essential oil content; an increase in plant-specific metabolites, or any combination thereof; or efficient absorption or utilization of available or externally supplied nutrients or minerals.

[0034] As used in this disclosure, the terms "increase," "increased," "increasing," "enhance," "enhanced," "enhancing," "promote," "promoted," "promoting," "improve," "improved," or "improving," or their commonly known synonyms, are used interchangeably and refer to their ordinary meanings as known in the art. In the context of attributes related to plant growth, development, performance, and / or survival, these terms are used herein to highlight the positive effect that the composition(s) or method(s) of the present disclosure have on the plant, causing the plant to grow or survive better, as compared to a previous setting that did not use the composition(s) or method(s) of the present disclosure.

[0035] As used in this disclosure, the term "metabolite" refers to a metabolic intermediate, precursor, or end product. In embodiments of the present disclosure, metabolites include products produced during metabolic reaction(s) of methanotroph(s). In some embodiments of the present disclosure, metabolites include products produced by metabolic reaction(s) of methanotrophic bacteria during cultivation of said methanotrophic bacteria.

[0036] As used in this disclosure, the terms "cell" or "whole cell" can be used interchangeably and refer to a collection or mass of microorganisms. In some embodiments of the present disclosure, a cell refers to a collection of methanotrophic bacterial cells. In some embodiments of the present disclosure, a cell refers to a collection of methanotrophic bacterial cells alone or in combination with plant growth-promoting microorganism(s).

[0037] As used in this disclosure, the terms "microbial consortium," "bacterial consortium," or "consortium of microorganisms," or simply "consortium," are all used interchangeably in this disclosure and include one or more microorganisms that function symbiotically or independently, wherein at least one microorganism is a methanotroph. In some embodiments of the present disclosure, the microbial consortium includes a combination of one or more species of microorganisms that function symbiotically or independently, wherein at least one microorganism is a methanotroph. In other embodiments of the present disclosure, the microbial consortium includes a combination of at least one methanotrophic bacterium and at least one plant growth-promoting microorganism. In the context of the present disclosure, plant growth-promoting microorganisms include nitrogen-dissolving microorganisms, phosphorus-dissolving microorganisms, mineral-dissolving microorganisms, plant hormone-secreting microorganisms, organic acid-secreting bacteria, and plant beneficial microorganisms, or any combination thereof. In the context of the present disclosure, these terms at least highlight the fact that the consortium comprises one or more Gammaproteobacterial methanotrophs, and thus the consortium may be composed entirely of Gammaproteobacterial methanotrophs.

[0038] As used in this disclosure, the terms "optionally," "optional," and the like mean that the component or feature may or may not be present as part of the composition(s) or method(s) of the present disclosure.

[0039] As used in this disclosure, the terms "plant," "crop," and the like are used interchangeably and generally refer to plants under the kingdom Plantae. In preferred embodiments, the plants are agricultural plants, horticultural plants, cereal crops, cash crops, indoor plants, floriculture plants, plantation crops, spice crops, and combinations thereof.

[0040] As used in this disclosure, the terms "whole cell composition," "methanotroph-based whole cell composition," "methanotroph-based composition," "methanotroph-based product," "biostimulant," "biostimulant composition," "whole cell biostimulant composition," "plant biostimulant composition," "plant biostimulant composition comprising whole cell methanotroph," and the like are used interchangeably and refer to the product(s) of the present disclosure. Thus, the term biostimulant, with or without other accompanying terms, is meant to provide the same meaning, which is generally known to those skilled in the art, for example, a biological or biologically derived composition used in plants to enhance their properties, productivity, or efficiency. Similarly, compositions comprising microorganisms that, when applied to plants, seeds, or the rhizosphere, stimulate natural processes that benefit nutrient uptake, nutrient utilization efficiency, and / or crop quality, regardless of their nutrient content, are also encompassed. In particular, in the context of the present disclosure, the term biostimulant means any composition comprising at least one microorganism that is useful to a plant and provides at least one benefit that affects the overall performance, productivity or efficiency of the plant.

[0041] As used in this disclosure, the terms "nitrogen availability" or "nitrogen fixation" and the like are intended to refer to the general meaning of the terms as known in the art. In the context of this disclosure, the terms include increasing the availability of nitrogen by plants or enabling better utilization of nitrogen in the form of ammonia, nitrate, nitrite, proteins, amino acids, peptides, nucleic acids, alone or in combination with other nitrogen-fixing microorganisms, through compositions such as the whole cell biostimulant compositions described herein, and / or from other sources such as amino acids, glutamine, ammonium, urea, sulfur-coated urea, methylene urea, polymer-coated urea, isobutylidenediurea, nitrate, nitrite, ammonium-, nitrate-, or nitrite-containing molecules, or any combination thereof.

[0042] As used in this disclosure, the term "phosphorous availability" and the like is intended to refer to the general meaning of the term as known in the art. In the context of this disclosure, the term includes increasing the availability or enabling better utilization of phosphorus to plants, either alone or in combination with other phosphorus-solubilizing microorganisms, through compositions such as the whole-cell methanotroph compositions described herein, and / or from sources including diammonium phosphate, monoammonium phosphate, single superphosphate, ammonium dihydrogen phosphate, ammonium phosphate, superphosphate, tricalcium phosphate, or any combination thereof, as a source of phosphate.

[0043] As used in this disclosure, the term "potassium availability" and the like is intended to refer to the general meaning of the term as known in the art. In the context of this disclosure, the term includes increasing the availability or enabling better utilization of potassium to plants in the form of potassium from compositions such as the whole-cell methanotroph compositions described herein, alone or in combination with other potassium solubilizing microorganisms, and / or from sources including, but not limited to, potassium chloride (muriate of potash), potassium sulfate, potassium nitrate, potassium magnesium sulfate, kainite, or any combination thereof, as a source of potassium.

[0044] Thus, to reiterate, the present disclosure provides a biostimulant composition comprising a gammaproteobacterial methanotroph. When applied to a plant, the biostimulant composition improves or enhances plant performance. Furthermore, because the composition comprises a gammaproteobacterial methanotroph, a method for simultaneously promoting methane utilization and nitrogen fixation in a plant is also provided, the method comprising contacting or applying the biostimulant composition to the plant. As a result, the biostimulant composition improves the overall efficiency of the plant, thus allowing for a reduction in the external addition of at least one nutrient or nutrient-containing fertilizer for plant growth, development, performance, and / or survival.

[0045] The composition(s), their use(s), and related method(s) of the present disclosure are further described in more detail in the following embodiments. For the sake of brevity, the same embodiment will not be repeated for each of the different composition(s), use(s), or method(s) described herein. However, any combination of an embodiment captured anywhere in this disclosure with another embodiment captured elsewhere in this disclosure falls fully within the scope of the present disclosure. Thus, the full meaning of the aspects described herein can be derived by considering such combinations.

[0046] Biostimulant Composition The present disclosure provides a biostimulant composition based on a microbial consortium comprising methanotrophs, preferably methanotrophic bacteria, for improving plant performance, methane utilization, and nitrogen fixation / availability in plants. The composition also allows for a reduction in the use of chemical or artificial fertilizers typically used in agricultural practices.

[0047] As mentioned above, since one of the objectives of the present disclosure is to improve or enhance plant performance while utilizing greenhouse gases as methane, it is important that the corresponding compositions include microorganisms that can efficiently utilize methane produced by agricultural activities and other sources to benefit plants, which is achieved by the presence of a microbial consortium including Gammaproteobacteria methanotrophs in the compositions of the present disclosure.

[0048] The gamma proteobacterial methanotrophs total about 1 x 10 per gram or milliliter of the composition of the present disclosure. 3 Total cells ~ approx. 5 x 10 10 It exists in a consortium containing all cells and includes all values and ranges therein.

[0049] In some embodiments, the microbial consortium comprises a total of about 5 x 10 3Total cells ~ approx. 5 x 10 10 In another embodiment, the microbial consortium comprises about 1 x 10 total cells, including all values and ranges therein. 3 Total cells ~ approx. 1 x 10 10 It includes all cells and all values and ranges within them.

[0050] In some embodiments, Gammaproteobacterial methanotrophs are microorganisms that can use methane as a primary or sole carbon and / or energy source.

[0051] Accordingly, the present disclosure provides a biostimulant composition comprising a microbial consortium of whole cells, including whole cells of a gammaproteobacterial methanotroph.

[0052] These gammaproteobacterial methanotrophs are known to efficiently utilize methane for growth and survival, and the compositions of the present disclosure use methanotrophs not only to utilize methane, but to do so while providing beneficial effects to the plants to which the compositions are applied. Thus, gammaproteobacterial methanotrophs form the most important part of the compositions of the present disclosure. Thus, they form the majority of the microorganisms present in the compositions.

[0053] Accordingly, the present disclosure provides a biostimulant composition comprising a microbial consortium of whole cells, wherein the consortium comprises at least 50% Gammaproteobacteria methanotroph whole cells.

[0054] In some embodiments, the microbial consortium within the biostimulant composition comprises at least about 60% to about 100% Gammaproteobacteria methanotrophic whole cells, including all values and ranges therein.

[0055] In some embodiments, the microbial consortium in the composition comprises at least about 60% whole cells of Gammaproteobacteria methanotrophs.

[0056] In some embodiments, the microbial consortium in the composition comprises at least about 70% whole cells of Gammaproteobacteria methanotrophs.

[0057] In some embodiments, the microbial consortium in the composition comprises at least about 80% whole cells of Gammaproteobacteria methanotrophs.

[0058] In some embodiments, the microbial consortium in the composition comprises at least about 90% whole cells of Gammaproteobacteria methanotrophs.

[0059] In some embodiments, the microbial consortium in the composition comprises at least about 99% whole cells of Gammaproteobacteria methanotrophs.

[0060] Thus, in some embodiments, the microbial consortium in the composition consists essentially of whole cells of Gammaproteobacteria methanotrophs.

[0061] In some embodiments, the microbial consortium in the composition consists of whole cells of a Gammaproteobacterial methanotroph.

[0062] In some embodiments, the gammaproteobacterial methanotrophs used in the biostimulant compositions are selected from the group consisting of Methylococcus, Methylomonas, Methylobacter, Methyloglobulus, Methylovulum, Methylomicrobium, Methylosarcina, Methylosphaera, Methyloprofundus, Methylosoma, Methyloccumis, and the like. The present invention relates to a type I or type X methanotroph belonging to a genus selected from the group consisting of Methylobacterium ylocucumis, Methylocaldum, Methyloparacoccus, Methylogaea, Methylomagnum, Methyloterricola, Methylothermus, Methylohalobius, Methylomarinovum, Methylomarinum and Crenothrix, or any combination thereof.

[0063] In some embodiments, the gammaproteobacterial methanotroph used in the biostimulant composition is a Type I or Type X methanotroph selected from the group including Methylococcus spp., Methylomonas spp., Methylobacter spp., Methyloglobulus spp., Methyloblum spp., Methylomicrobium spp., Methylsarcinia spp., Methylosphaera spp., Methyloprophundus spp., Methylosoma spp., Methyloccumis spp., Methylocardum spp., Methyloparacox spp., Methylogaea spp., Methylomagnum spp., Methylotelicola spp., Methylothermus spp., Methylohalobius spp., Methylomarinum spp., Methylomarinobum spp., and Crenothrix spp., or any combination thereof.

[0064] In some embodiments, the gammaproteobacterial methanotroph is selected from the group consisting of Methylococcus capsulatus, Methylococcus mobilis, Methylomicrobium kenyense, Methylomicrobium alcaliphilum, Methylomicrobium alcaliphilum 20Z, Methylomicrobium buryatense 5G, Methylomicrobium buryatense 4G, Halomonas pantelleriensis, Methylomicrobium album, Methylomonas methanica, MB 126, Methylobacter tundripaludum, Methyloblum miyakonense, Methylomonas rubra, Methylomonas koyamae, Methylomonas methancia, Methylomonas denitrificans, Methylomonas paludis, Methylomonas lenta, Methylomarinum vadi, Methylococcus thermophilus, Methylobacter whittenburyi, Crenothrix polyspora, Clonothrix fusca fusca, Methylobacter bovis, Methylomonas aurantiaca, Methylomonas fodinarum, Methylobacter vinelandii, Methylomicrobium japanense, Methylococcus bacterium, Methylocystis methanolicus, Methyloccumis oryzae, Methylogaea oryzae, Methylsarcina lacus, Methylosoma difficile, and combinations thereof.

[0065] In some embodiments of the composition, the gammaproteobacterial methanotroph is Methylococcus capsulatus, Methyloccumis oryzae, Methylogaea oryzae, Methylomicrobium alkaliphilum, Methylomicrobium alkaliphilum 20Z, Methylomicrobium briatense 5G, Methylomicrobium briatense 4G, Halomonas pantelleriensis, Methylobacter tundripaludum, Methylobacter uchtenbrii, Methylobacter marinus, Methylobacter luteus, Methylsarsina lacus, Methylsarsina fibrata, Methylotericola oryzae, Methylosoma difficile, Methylomonas methanica, Methylomonas denitrificans, Methyl The fungus is selected from the group consisting of Chiromonas koyamae, Methylomicrobium album, Methylomicrobium agile, Methylobum miyakonense, Methylobum psychorotolerans, Methylomagnum isuhizawai, Methylohalobius crimeensis, Crenothrix polyspora, Methyloprofondus sedimenthi, and combinations thereof.

[0066] In some embodiments, the microbial consortium totals about 1 x 10 per gram or milliliter of the composition. 3 Total cells ~ approx. 5 x 10 10 The total number of cells includes all values and ranges therein. As stated above, of these total cells, at least 50% of the total cells are Gammaproteobacterial methanotrophs.

[0067] In some embodiments, the gammaproteobacterial methanotroph used in the biostimulant composition is Methylococcus capsulatus.

[0068] Thus, in some embodiments, the microbial consortium within the biostimulant composition comprises at least about 60% to about 100% whole cells of Methylococcus capsulatus, including all values and ranges therein.

[0069] In some embodiments, the microbial consortium within the composition comprises at least about 60% whole cells of Methylococcus capsulatus.

[0070] In some embodiments, the microbial consortium within the composition comprises at least about 70% whole cells of Methylococcus capsulatus.

[0071] In some embodiments, the microbial consortium within the composition comprises at least about 80% whole cells of Methylococcus capsulatus.

[0072] In some embodiments, the microbial consortium within the composition comprises at least about 90% whole cells of Methylococcus capsulatus.

[0073] In some embodiments, the microbial consortium in the composition comprises at least about 99% whole cells of Methylococcus capsulatus.

[0074] In some embodiments, the microbial consortium within the composition consists essentially of whole cells of Methylococcus capsulatus.

[0075] In some embodiments, the microbial consortium in the composition consists of whole cells of Methylococcus capsulatus.

[0076] In some embodiments, the microbial consortium totals about 1 x 10 per gram or milliliter of the composition. 3 Total cells ~ approx. 5 x 10 10 Contains all Methylococcus capsulatus cells and includes all values and ranges therein.

[0077] In some embodiments, the microbial consortium comprises a total of about 5 x 10 per gram or milliliter of the composition. 3 Total cells ~ approx. 5 x 10 10 Contains whole cells of Methylococcus capsulatus.

[0078] In some embodiments, the microbial consortium totals about 1 x 10 per gram or milliliter of the composition. 3 Total cells ~ approx. 1 x 10 10 Contains whole cells of Methylococcus capsulatus.

[0079] In some embodiments, the microbial consortium is at least about 0.5 x 10 per gram or per milliliter of the composition. 3 Total cells ~ approx. 2.5 x 10 10 Contains whole cells of Methylococcus capsulatus.

[0080] In some embodiments, the microbial consortium is at least about 1 x 10 per gram or per milliliter of the composition. 5 Total cells ~ approx. 1 x 10 8 Contains whole cells of Methylococcus capsulatus.

[0081] In some embodiments, apart from the Gammaproteobacterial methanotrophs, the microbial consortium comprises other plant growth-promoting microorganisms / microorganisms (PGPM) selected from the group including, but not limited to, nitrogen-fixing microorganisms, phosphorus-solubilizing microorganisms, mineral-solubilizing microorganisms, plant hormone-secreting microorganisms, organic acid-secreting bacteria, other plant beneficial microorganisms, and combinations thereof. Thus, the PGPM comprise from about 1% to about 50% of the consortium.

[0082] Thus, in some embodiments, the microbial consortium comprises at least 50% whole cells of Gammaproteobacteria methanotrophs along with other plant growth-promoting microorganisms selected from the group including, but not limited to, nitrogen-fixing microorganisms, phosphorus-solubilizing microorganisms, mineral-solubilizing microorganisms, plant hormone-secreting microorganisms, organic acid-secreting bacteria, other plant beneficial microorganisms, and combinations thereof.

[0083] Thus, in some embodiments, the microbial consortium comprises Methylococcus capsulatus together with other plant growth-promoting microorganisms selected from the group including, but not limited to, nitrogen-fixing microorganisms, phosphorus-solubilizing microorganisms, mineral-solubilizing microorganisms, plant hormone-secreting microorganisms, organic acid-secreting bacteria, other plant beneficial microorganisms, and combinations thereof.

[0084] Thus, in some embodiments, the microbial consortium comprises at least 50% whole cells of Methylococcus capsulatus together with other plant growth-promoting microorganisms selected from the group including, but not limited to, nitrogen-fixing microorganisms, phosphorus-solubilizing microorganisms, mineral-solubilizing microorganisms, plant hormone-secreting microorganisms, organic acid-secreting bacteria, other plant beneficial microorganisms, and combinations thereof.

[0085] In some embodiments, the plant growth-promoting microorganism is a plant growth-promoting bacterium (PGPB), a plant endophytic bacterium, a plant endophytic fungus, an epiphytic bacterium, an epiphytic fungus, a mycorrhizal fungus, a vesicular-arbuscular mycorrhiza (VAM), or any combination thereof.

[0086] In some embodiments, the plant growth-promoting bacteria (PGPB) are plant growth-promoting rhizobacteria (PGPR).

[0087] Thus, in some embodiments, the microbial consortium of the present disclosure is composed primarily of Gammaproteobacterial methanotrophs, such as M. capsulatus.

[0088] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 90:10.

[0089] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 80:20.

[0090] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 70:30.

[0091] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 60:40.

[0092] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 50:50.

[0093] Apart from the microbial consortium comprising whole cells of a gammaproteobacterial methanotroph, such as M. capsulatus, the biostimulant composition also comprises at least one metabolite, at least one medium-derived nutrient, and optionally at least one agriculturally acceptable excipient.

[0094] Thus, the present disclosure provides a biostimulant composition comprising a microbial consortium having a gammaproteobacterial methanotroph, such as M. capsulatus, at least one metabolite, at least one medium-derived nutrient, and optionally at least one agriculturally acceptable excipient.

[0095] In some embodiments, the biostimulant composition comprises a microbial consortium having Methylococcus capsulatus, at least one metabolite, at least one medium-derived nutrient, and optionally at least one agriculturally acceptable excipient.

[0096] In some embodiments, the biostimulant composition comprises a microbial consortium having Methylococcus capsulatus, at least one metabolite, at least one medium-derived nutrient, and at least one agriculturally acceptable excipient.

[0097] In some embodiments, the biostimulant composition comprises a microbial consortium having at least one methanotroph and at least one plant growth-promoting microorganism (PGPM), at least one metabolite, at least one medium-derived nutrient, and optionally at least one agriculturally acceptable excipient.

[0098] In all embodiments of the biostimulant compositions of the present disclosure, the microbial consortium of whole cells totals about 1 x 10 per gram or per milliliter of the composition. 3 cells ~ approx. 5 x 10 10 It contains cells and all values and ranges within them.

[0099] In some embodiments, a total of 1×10 per gram or per milliliter of the composition 3 cells ~ approx. 5 x 10 10 The consortium of cells comprises about 0.1% to about 80% of the biostimulant composition of the present disclosure. The remainder of the composition comprises about 0.1% to about 10% of at least one metabolite, about 0.1% to about 10% of at least one medium-derived nutrient, and optionally about 0.01% to about 90% of at least one agriculturally acceptable excipient. The above ranges expressly encompass all values and ranges therein.

[0100] In some embodiments, the metabolites include components derived from a culture broth. In some embodiments, the metabolites include components derived from a culture broth, wherein the culture broth is obtained by culturing a methanotroph. In particular, the metabolites include components produced by Gammaproteobacterial methanotrophic cells as a result of culturing those cells in a culture medium.

[0101] In some embodiments, metabolites include components selected from the group including, but not limited to, carbohydrates, lipids, sugars, fatty acids, proteins, peptides, nucleic acids, nucleotides, amino acids, vitamins, organic acids, salts, minerals, extracellular enzymes, osmolytes, bacterially derived components, minerals, and combinations thereof.

[0102] In some embodiments, the metabolite comprises a component selected from the group comprising a peptide or mixture of peptides, free amino acids, nucleic acids, nucleotides, vitamins, carbohydrates, lipids, sugars, fatty acids, salts, minerals, osmolytes, extracellular enzymes, bacterial components, ash, and combinations thereof.

[0103] In some embodiments, the medium-derived nutrients include components derived from the culture medium.

[0104] In some embodiments, medium-derived nutrients include components derived from the culture medium used to culture methanotrophs, more specifically Gammaproteobacterial methanotrophs. Thus, medium-derived nutrients include components that are not produced by the methanotrophic cells, but rather are added as part of the culture medium during cultivation of the methanotrophic cells. In specific embodiments, medium-derived nutrients are components in the culture medium used to culture the methanotrophs. In some embodiments, the terms "medium-derived nutrients" and "non-cellular nutrients" are used interchangeably and have the same meaning.

[0105] In some embodiments, the nutrients from the medium include inorganic nutrients.

[0106] In some embodiments, nutrients from the medium include minerals.

[0107] In some embodiments, nutrients from the medium include ions, salts, or combinations thereof.

[0108] In some embodiments, the ions are cations, anions, or a combination thereof.

[0109] In some embodiments, the cation is selected from the group including sodium, potassium, magnesium, manganese, cobalt, zinc, copper, iron, calcium, boron, nickel, molybdenum, calcium, and combinations thereof.

[0110] In some embodiments, the anion is selected from the group including sulfate, chloride, nitrate, phosphate, borate, and combinations thereof.

[0111] In some embodiments, nutrients from the medium comprise salts selected from the group including sodium salts, potassium salts, magnesium salts, manganese salts, cobalt salts, zinc salts, copper salts, iron salts, calcium salts, boron salts, nickel salts, and combinations thereof.

[0112] In some embodiments, nutrients from the medium include salts selected from the group including sodium chloride, potassium nitrate, magnesium sulfate, calcium chloride, sodium molybdate, ferrous sulfate, zinc sulfate, cobalt chloride, borate, zinc chloride, manganese chloride, nickel chloride, copper sulfate, sodium / potassium phosphates, and combinations thereof.

[0113] In some embodiments, the nutrients from the medium include chelated salts, in which the salts are bound to chelating agents.

[0114] In some embodiments, the chelating agent is selected from the group including ethylenediaminetetraacetic acid (EDTA), citric acid, hydroxyaminopolycarboxylic acids, diethylenetriaminepentaacetic acid, hydroxyethylenediaminetriacetic acid, tetrakishydroxymethylphosphonium sulfate, nitrilotriacetic acid, and glutamic acid-diacetic acid, and combinations thereof.

[0115] In some embodiments, nutrients from the medium include components selected from the group including sodium, potassium, magnesium, manganese, cobalt, zinc, copper, iron, calcium, molybdenum, boron, nickel, sulfate, chloride, nitrate, phosphate, borate, salts, chelating salts, and combinations thereof.

[0116] In some embodiments, the agriculturally acceptable excipient comprises a component selected from the group comprising a carrier, a cytoprotectant, an adjuvant, a surfactant, a stabilizer, a preservative, a diluent, a suspending agent, a dispersing agent, a co-solvent, and combinations thereof.

[0117] In some embodiments, the carrier is selected from the group including, but not limited to, lignite, bentonite, peat, vermiculite, charcoal, soil mixtures, compost, and combinations thereof.

[0118] In some embodiments, the cytoprotective agent is selected from the group including, but not limited to, polyethylene glycol (PEG), polyvinyl alcohol, sodium alginate, gelatin, gellan, welan, and combinations thereof.

[0119] In some embodiments, the adjuvant is selected from the group including, but not limited to, xanthan gum, carboxymethylcellulose (CMC), gum arabic, polyvinylpyrrolidone (PVP), and combinations thereof.

[0120] In some embodiments, the surfactant is selected from the group including, but not limited to, cationic surfactants, anionic surfactants, nonionic surfactants, silicon-based surfactants, and combinations thereof.

[0121] In some embodiments, the surfactant is selected from the group including, but not limited to, natural surfactants, semi-synthetic surfactants, synthetic surfactants, and combinations thereof.

[0122] In some embodiments, the surfactant is selected from the group including, but not limited to, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), gum arabic, sodium alginate, Silwet L-77, Tween 20, Tween 80, Triton X100, and combinations thereof.

[0123] In some embodiments, the stabilizer or preservative is selected from the group including, but not limited to, potassium sorbate, sorbic acid, trehalose, citric acid, polyglutamic acid, and combinations thereof.

[0124] In some embodiments, the diluent is selected from the group comprising an ionic buffer-based diluent solution, saline, and combinations thereof.

[0125] Thus, in some embodiments, the biostimulant composition comprises a microbial consortium having at least 50% whole cells of gammaproteobacteria methanotrophs, one or more components: metabolites including carbohydrates, sugars, lipids, fatty acids, proteins, amino acids, peptides, organic acids, nucleic acids, nucleotides, vitamins, other cellular metabolites, minerals and osmolytes, and one or more components: non-cellular nutrients including minerals, salts, ions, and optionally one or more agriculturally acceptable excipients.

[0126] In some embodiments, the biostimulant composition comprises a microbial consortium having at least 50% whole cells of gammaproteobacteria methanotrophs, one or more components: metabolites including carbohydrates, sugars, lipids, fatty acids, proteins, amino acids, peptides, organic acids, nucleic acids, nucleotides, vitamins, other cellular metabolites, minerals and osmolytes, and one or more components: non-cellular nutrients including sulfate, phosphate, chloride, nitrate, sodium, potassium, magnesium, manganese, calcium, copper, cobalt, molybdenum, zinc, nickel and iron, and optionally one or more agriculturally acceptable excipients.

[0127] In some embodiments, the biostimulant composition comprises a microbial consortium having at least 50% Methylococcus capsulatus whole cells, one or more components: metabolites including carbohydrates, sugars, fatty acids, lipids, proteins, peptides, amino acids, vitamins, organic acids, and osmolytes, one or more components: media nutrients including minerals, salts, and ions, and optionally one or more agriculturally acceptable excipients.

[0128] In some embodiments, the biostimulant composition comprises a microbial consortium having at least 50% Methylococcus capsulatus whole cells, one or more components: metabolites including carbohydrates, sugars, fatty acids, lipids, proteins, peptides, amino acids, vitamins, organic acids, and osmolytes, nutrients from a medium including one or more components: sulfate, phosphate, chloride, nitrate, sodium, potassium, magnesium, calcium, copper, cobalt, molybdenum, zinc, nickel, iron, and optionally one or more agriculturally acceptable excipients.

[0129] In some embodiments, the biostimulant composition is in liquid or solid form.

[0130] In some embodiments, the biostimulant composition is in a liquid or solid form, including but not limited to, liquid spray, dust, granular, bead, soluble powder, wettable powder, pellet, microencapsulated, emulsifiable concentrate, encapsulated suspension, dry flowable form, liquid flowable form, and the like. These forms provide examples of various ways in which the biostimulant composition of the present disclosure can be formulated, but the activity of the composition does not depend on or change depending on the form. Therefore, those skilled in the art can use the composition of the present disclosure in the form that is most suitable for their purpose.

[0131] Thus, in some embodiments, the microbial consortium totals about 1 x 10 per gram of solid composition or per milliliter of liquid composition. 3 Total cells ~ approx. 5 x 10 10 Contains all cells.

[0132] In some embodiments, the microbial consortium totals about 1 x 10 per gram of solid composition or per milliliter of liquid composition. 3 Total cells ~ approx. 5 x 10 10 It contains 100 whole cells, of which at least 50% are gammaproteobacterial methanotrophs.

[0133] In some embodiments, the microbial consortium totals about 1 x 10 per gram of solid composition or per milliliter of liquid composition. 3 Total cells ~ approx. 5 x 10 10 This includes whole cells of gammaproteobacterial methanotrophs such as M. capsulatus, including all values and ranges therein.

[0134] In some embodiments, the biostimulant composition comprises whole cells in a concentration of 1×10 for a solid form composition. 3 cells / g ~5×10 10 cells / g or 1 x 10 for liquid form compositions 3 cells / ml ~ 5 x 1010 The composition contains a concentration of about 0.1% to about 10% metabolites and about 0.1% to about 10% nutrients from the medium at a concentration ranging from about 0.1% to about 10% cells / ml, in which case at least 50% of the total cells in the microbial consortium in the composition are gammaproteobacterial methanotrophs, such as M. capsulatus.

[0135] In some embodiments, the biostimulant composition comprises whole cells in a concentration of 1×10 for a solid form composition. 3 cells / g ~5×10 10 cells / g or 1 x 10 for liquid form compositions 3 cells / ml ~ 5 x 10 10 The composition contains a concentration of about 0.1% to about 10% metabolites, a concentration of about 0.1% to about 10% nutrients from the medium, and an agriculturally acceptable excipient at a concentration of about 0.01% to about 90% at a concentration of about 10% to about 10% cells / ml, wherein at least 50% of the total cells in the microbial consortium in the composition are gammaproteobacterial methanotrophs, such as M. capsulatus.

[0136] In an exemplary embodiment, the metabolites in the composition, in solid form, comprise about 70% protein, about 15% carbohydrate, about 7% lipid, and about 8% minerals.

[0137] In a preferred embodiment, the metabolites in the composition, in liquid form, comprise about 15% protein, 5% carbohydrates, 3% lipids, and about 3% minerals.

[0138] As discussed above, in some embodiments, apart from the Gammaproteobacterial methanotroph, such as M. capsulatus, the biostimulant composition further comprises a plant growth-promoting microorganism. Thus, in some embodiments, the consortium comprises between about 1% and about 50% of at least one plant growth-promoting microorganism.

[0139] Thus, in some embodiments, the composition comprises: a microbial consortium comprising one or more gammaproteobacterial methanotrophic bacteria and one or more plant growth-promoting microorganisms (PGPMs); at least one metabolite, at least one medium-derived nutrient; and Optionally, at least one agriculturally acceptable excipient Includes:

[0140] In some embodiments, the methanotrophic bacteria, metabolites, non-cellular nutrients, agriculturally acceptable excipients, and plant growth-promoting microorganisms (PGPMs) are as defined above.

[0141] In some embodiments, the composition comprises: a microbial consortium comprising at least 50% whole cells of a gammaproteobacterial methanotroph, such as M. capsulatus, and one or more plant growth-promoting microorganisms (PGPM), in a concentration of about 1 x 10 for the solid composition; 3 cells / g ~5×10 10 cells / g or 1 x 10 for liquid compositions 3 cells / ml ~ 5 x 10 10 At total whole cell concentrations in the range of cells / ml, Metabolite(s) at a concentration ranging from about 0.1% to 10% a nutrient(s) from the medium at a concentration ranging from about 0.1% to 10%, and Optionally, agriculturally acceptable excipients at concentrations ranging from about 0.01% to 90%. include.

[0142] In some embodiments, the composition comprises: a microbial consortium comprising at least 50% whole cells of a gammaproteobacterial methanotroph, such as M. capsulatus, and one or more plant growth-promoting microorganisms (PGPM), in a concentration of about 1 x 10 for the solid composition; 3 cells / g ~5×10 10cells / g or 1 x 10 for liquid compositions 3 cells / ml ~ 5 x 10 10 At total whole cell concentrations in the range of cells / ml, Metabolite(s) at a concentration ranging from about 0.1% to 10% a nutrient(s) from the medium at a concentration ranging from about 0.1% to 10%, and Agriculturally acceptable excipients at concentrations ranging from approximately 0.01% to 90% include.

[0143] In some embodiments, the biostimulant composition defined above comprises total solids at a concentration ranging from about 0.5% to 50%, crude protein at a concentration ranging from about 0.1% to 70%, and minerals, carbohydrates, and lipids at concentrations ranging from about 0.1% to 30%.

[0144] In some embodiments, the above-defined biostimulant composition has a pH ranging between 4-10.

[0145] In some embodiments, the biostimulant compositions of the present disclosure, including a consortium having at least 50% Gammaproteobacteria methanotrophs, metabolites, nutrients from the medium, and optionally an agriculturally acceptable excipient, can be combined with methanotroph-derived hydrolysate composition(s), such as those obtained by lysis / processing of methanotroph cells or other microbial cell-based biostimulants known in the art.

[0146] In all embodiments of the present disclosure, a biostimulant composition as described above comprising a microbial consortium of whole cells, wherein said consortium comprises at least 50% Gammaproteobacteria methanotroph whole cells, provides at least one of the following benefits: Improve or enhance plant performance; Increase the availability or efficient utilization by plants of at least one nutrient selected from, but not limited to, nitrogen, phosphorus, and potassium; or Reducing the need for external addition of at least one nutrient selected from nitrogen, phosphorus and potassium, either individually or as part of a fertilizer.

[0147] Application of biostimulant compositions As multiple benefits are associated with the use of biostimulant compositions, the present disclosure therefore also relates to the application of such biostimulant compositions to plants.

[0148] In some embodiments, application of the biostimulant composition to the plant is by a method comprising contacting or applying the plant biostimulant composition described above to the plant or part thereof.

[0149] In some embodiments, application of the biostimulant composition or associated method promotes plant growth and / or performance.

[0150] In some embodiments, application of the biostimulant composition or associated method improves or enhances plant performance.

[0151] In some embodiments, application of the biostimulant composition or related methods increases the availability or efficient utilization by plants of at least one nutrient selected from, but not limited to, nitrogen, phosphorus, and potassium.

[0152] In some embodiments, application of the biostimulant composition or associated method reduces the need for exogenous addition of at least one nutrient selected from nitrogen, phosphorus, and potassium, either individually or as part of a fertilizer.

[0153] In some embodiments, the method comprises: obtaining a plant biostimulant composition comprising a gammaproteobacterial methanotroph as described above, and contacting a plant or part thereof with the plant biostimulant composition, wherein the method promotes plant growth and / or performance.

[0154] In some embodiments of the methods of treating plants to promote plant growth, yields are increased by about 1% to 500% compared to methods that do not use the plant biostimulant compositions described herein.

[0155] In some embodiments of the methods of treating plants to promote plant growth, yields are increased by about 1% to 250% compared to methods that do not use the plant biostimulant compositions described herein.

[0156] In some embodiments of the methods of treating plants to promote plant growth, yields are improved by about 1% to 100% compared to methods that do not use the plant biostimulant compositions described herein.

[0157] In some embodiments of the methods of treating plants to promote plant growth, yields are improved by about 1% to 50% compared to methods that do not use the plant biostimulant compositions described herein.

[0158] In some embodiments of the methods of treating plants to promote plant growth, yields are improved by about 1% to 10% compared to methods that do not use the plant biostimulant compositions described herein.

[0159] In some embodiments of the methods of treating plants to promote plant growth, yields are improved by about 1.5- to 10-fold compared to methods that do not use the plant biostimulant compositions described herein.

[0160] In some embodiments, the biostimulant composition is contacted or applied in an amount ranging from about 0.1 L / acre to 10 L / acre.

[0161] In some embodiments, the biostimulant composition is contacted or applied in an amount ranging from about 0.5 L / acre to 5 L / acre.

[0162] In some embodiments, the biostimulant composition is in solid or liquid form and is contacted with or applied to the plant at a concentration ranging from about 1 ml per liter to about 50 ml per liter for liquid forms or from 1 gm per kilogram to about 50 gm per kilogram for solid forms.

[0163] In some embodiments, the biostimulant composition is in solid or liquid form and is contacted with or applied to the plant at a concentration ranging from about 1× to 100,000× dilution of the solid or liquid form of the composition, including all values and ranges therein.

[0164] In some embodiments, the biostimulant composition is in a liquid or solid form, including but not limited to, liquid spray, dust, granules, beads, soluble powder, wettable powder, pellet, microencapsulated, emulsifiable concentrate, encapsulated suspension, dry flowable form, liquid flowable form, and the like. These forms provide examples of various ways in which the biostimulant composition of the present disclosure can be formulated, but the activity of the composition does not depend on or change depending on the form. Therefore, those skilled in the art can use the composition of the present disclosure in the form that is most suitable for their purpose.

[0165] In some embodiments, the biostimulant compositions as described above are contacted with or applied to the above-ground parts of plants, including leaves, as a foliar spray.

[0166] In some embodiments, the biostimulant composition as described above is contacted or applied to the plant through its soil.

[0167] In some embodiments, the biostimulant composition is contacted with or applied to the plant through its soil or through a foliar spray, as described above, in a single dose or multiple doses.

[0168] In some embodiments, the biostimulant composition as described above is contacted or applied to the plant through its seed.

[0169] In some embodiments, the biostimulant composition is contacted with or applied to the plant through its soil, seeds, or via foliar application, as described above, in a single dose or multiple doses, with each subsequent dose being administered 1 to 90 days apart per crop cycle.

[0170] In some embodiments, the application of the biostimulant composition is not affected or altered by seeding rate, planting date, harvest time, and other standard / conventional agricultural management practices, and therefore, those skilled in the art can freely adjust the application practices of the composition depending on the plant or crop of interest.

[0171] In some embodiments, the amount of biostimulant composition to be applied to plants is known to those skilled in the art.Therefore, said amount does not form a limiting feature of the present disclosure.What is important is the components of the composition, and the most important are the microbial consortium, the types of cells therein, and the total number and ratio of cells therein.Depending on the plant, those skilled in the art will not have difficulty adjusting the dosage of the composition that needs to be applied to plants, as long as it meets the above criteria.

[0172] Improving plant performance As previously mentioned, when the biostimulant composition of the present disclosure is applied to or comes into contact with a plant, it improves or enhances its performance.

[0173] Thus, the present disclosure provides for the use of the whole cell biostimulant compositions described above to enhance or improve agricultural / plant productivity or performance.

[0174] In some embodiments, plant performance is enhanced or improved by applying or contacting the plant or its seeds with a whole cell-based biostimulant composition as described above.

[0175] In some embodiments, the present disclosure provides whole cell-based biostimulant compositions comprising gammaproteobacterial methanotrophs, metabolites, and nutrients from the medium to improve or enhance plant performance.

[0176] In all embodiments for improving or enhancing plant performance, the characteristics of the methanotroph (whole cell) based biostimulant composition are as described in one or more of the preceding embodiments.

[0177] In some embodiments, enhanced or improved agricultural productivity is measured as the difference in increase in agricultural productivity (such as crop yield, productivity, or other beneficial parameter) when agricultural production is carried out with and without the use / application of the biostimulant compositions described herein.

[0178] In some embodiments, the plants to which the biostimulant compositions of the present disclosure are applied are selected from, but not limited to, agricultural crops, horticultural crops, plantation crops, or any combination thereof.

[0179] In some embodiments, the crop is selected from the group including, but not limited to, cereals, millets, pulses / legumes, cash crops, oil crops, and combinations thereof.

[0180] In some embodiments, the horticultural crop is selected from the group including, but not limited to, vegetable crops, medicinal crops, aromatic crops, floriculture crops, fruit crops, spices and plantation crops, and combinations thereof.

[0181] In some embodiments, the plant is selected from the group including, but not limited to, radish, spinach, coriander, chili pepper, guar gram, potato, green bean, tomato, lettuce, corn, rice, marigold, broccoli, soybean, chili pepper, grape, English cucumber, pomegranate, wheat, carrot, maize, broad bean, sunflower, pea, canola, barley, mint, corn, saffron, and combinations thereof.

[0182] In some embodiments, enhancing or improving plant performance includes, but is not limited to, having a stimulating / promoting effect on plant growth, yield, nutrient utilization efficiency, or any combination thereof. In one embodiment, enhancing or improving plant performance includes having a stimulating / promoting effect on plants as measured by increased production or number of below-ground or above-ground biomass such as roots, shoots, leaves, flowers, stamens, stigmas, anthers, fruits, seeds, etc., increased photosynthetic activity during controlled or adverse conditions, increased efficiency of nutrient / mineral availability, absorption and utilization, reduced use of chemical fertilizers, increased accumulation of metabolites, or any combination thereof.

[0183] In some embodiments, improving or enhancing plant performance comprises stimulating or promoting a quantitative or qualitative plant attribute selected from the group including biomass production, yield, photosynthetic activity, nutritional value, secondary metabolites and nutrient utilization efficiency, or any combination thereof.

[0184] In some embodiments, the effect of improved or enhanced plant performance is: an increase in the number, size or quality of below-ground or above-ground biomass selected from the group including roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits and seeds or any combination thereof; Increased photosynthetic activity or chlorophyll content Increased protein, dietary fiber, beta-carotene or essential oil content, plant-specific metabolites, or any combination thereof; Efficient absorption or utilization of available or exogenously supplied nutrients or minerals The measurement is made through one or more of the following:

[0185] In some embodiments, those skilled in the art will understand that the attributes set forth above and the methods by which they are measured do not constitute an exhaustive list and are provided for illustrative purposes only. Enhancement or improvement of other plant performance attributes or parameters not explicitly captured herein also falls within the scope of the present disclosure. What is important is the fact that plants can grow or survive better when a whole cell biostimulant of the present disclosure is applied.

[0186] In some embodiments, the biostimulant compositions as described above are used to improve yields and other parameters in cultivation practices selected from the group including, but not limited to, hydroponics, aeroponics, vertical farming, indoor horticulture, turfgrass, and combinations thereof.

[0187] The present disclosure further relates to a method of improving or enhancing the performance of a plant comprising contacting the plant with a biostimulant composition as described above.

[0188] In some embodiments, a method of improving or enhancing plant performance comprises contacting a plant with a biostimulant composition comprising a gammaproteobacterial methanotroph, metabolites, nutrients from the medium, and an agriculturally acceptable excipient, as described above.

[0189] In some embodiments, a method for improving or enhancing plant performance comprises contacting a plant with a biostimulant composition comprising Methylococcus capsulatus, metabolites, nutrients from a medium, and an agriculturally acceptable excipient, as described above.

[0190] In some embodiments of the method for improving or enhancing plant performance, the plant biostimulant composition as described above is applied as a foliar spray in contact with or to above-ground parts of the plant, including leaves, to improve plant performance.

[0191] In some embodiments of the method of improving or enhancing plant performance, a plant biostimulant composition as described above is contacted with or applied to soil to improve plant performance.

[0192] In some embodiments of the method of improving or enhancing plant performance, a plant biostimulant composition as described above is contacted with or applied to the seeds of a plant to improve plant performance.

[0193] In some embodiments of the method for improving or enhancing plant performance, the plant biostimulant composition as described above is contacted with or applied to above-ground parts of the plant, including shoots, flowers, fruits, or any combination thereof, to improve plant performance.

[0194] In some embodiments of the method for improving or enhancing plant performance, the plant biostimulant composition as described above is contacted with or applied to seeds to improve plant performance, in some embodiments, the plant biostimulant composition is applied as a seed coating, seed dressing, or seed treatment.

[0195] In some embodiments of the method for improving or enhancing plant performance, the plant biostimulant composition as described above is contacted with or applied to the whole plant to improve plant performance.

[0196] In some embodiments of the method for improving or enhancing plant performance, a plant biostimulant composition as described above is contacted with or applied to a plant or part thereof through any known application mode to improve plant performance.

[0197] In some embodiments of the methods for improving or enhancing plant performance, yield is improved by about 1% to 500% compared to methods without the use of the plant biostimulant compositions described herein.

[0198] In some embodiments of the methods for improving or enhancing plant performance, yield is improved by about 1% to 250% compared to methods without the use of the plant biostimulant compositions described herein.

[0199] In some embodiments of the methods for improving or enhancing plant performance, yield is improved by about 1% to 100% compared to methods without the use of the plant biostimulant compositions described herein.

[0200] In some embodiments of the methods for improving or enhancing plant performance, yield is improved by about 1% to 50% compared to methods without the use of the plant biostimulant compositions described herein.

[0201] In some embodiments of the methods for improving or enhancing plant performance, yield is improved by about 1% to 10% compared to methods without the use of the plant biostimulant compositions described herein.

[0202] In some embodiments of the methods for improving or enhancing plant performance, yields are improved by about 1.5- to 10-fold compared to methods without the use of the plant biostimulant compositions described herein.

[0203] In some embodiments, application of the biostimulant composition of the present disclosure to a plant results in an increase in biomass production of at least about 23% to about 43% compared to a plant to which the composition is not applied.

[0204] In some embodiments, application of the biostimulant composition of the present disclosure to plants results in an increase in biomass production of at least about 11% to about 15% compared to plants to which the composition is not applied and instead a commercially available biostimulant comprising nitrogen-fixing, phosphorus-solubilizing, and zinc-solubilizing bacteria is applied.

[0205] In some embodiments, application of a biostimulant composition of the present disclosure to a plant results in an increase in its pod yield of at least about 22% compared to a plant to which the composition is not applied.

[0206] In some embodiments, application of the biostimulant composition of the present disclosure to a plant results in an increase in its pod yield of at least about 8% to about 15% compared to a plant to which the composition is not applied and instead a commercially available biostimulant comprising nitrogen-fixing, phosphorus-solubilizing, and zinc-solubilizing bacteria is applied.

[0207] In some embodiments, application of the biostimulant composition of the present disclosure to a plant results in an increase in plant yield of at least about 27% to about 48% compared to a plant to which the composition is not applied.

[0208] In some embodiments, application of the biostimulant composition of the present disclosure to a plant results in an increase in the number of tap roots of at least about 7% to about 10% compared to a plant to which the composition is not applied.

[0209] In some embodiments, application of the biostimulant composition of the present disclosure to plants results in at least about a 32% increase in photosynthetic efficiency as measured by SPAD readings compared to plants to which the composition is not applied.

[0210] In some embodiments, application of the biostimulant composition of the present disclosure to plants results in an increase in dietary fiber content of at least about 20% compared to plants to which the composition is not applied.

[0211] In some embodiments, application of a biostimulant composition of the present disclosure to a plant results in an increase in protein content of at least about 44% compared to a plant to which the composition is not applied.

[0212] nitrogen fixation As mentioned above, one of the ways that the biostimulant composition of the present disclosure improves or enhances plant performance is by improving the plant's nutrient utilization efficiency. Nitrogen is one of the most important nutrients required for proper plant growth, development, performance, and / or survival. Therefore, if plants can efficiently utilize atmospheric nitrogen, this will result in better growth, development, performance, and / or survival.

[0213] Thus, in some embodiments, plant performance is enhanced or improved as a direct result of the increased nitrogen fixation promoted by the biostimulant compositions of the present disclosure. Thus, when the biostimulant compositions of the present disclosure are applied to plants, nitrogen fixation is promoted and nitrogen and related compounds become available to the plant, which then utilizes them for efficient plant growth and survival.

[0214] In some embodiments, the biostimulant compositions of the present disclosure promote increased nitrogen fixation, resulting in better plant uptake or utilization of nitrogen / nitrogen-derived compounds or metabolites, which in turn results in enhanced or improved plant growth as indicated by quantitative or qualitative plant attributes selected from the group including biomass production, yield, photosynthetic activity, nutritional value, secondary metabolites and nutrient utilization efficiency, or any combination thereof.

[0215] Thus, the present disclosure provides methods for promoting better nitrogen fixation in plants, resulting in either increased availability of nitrogen to plants or efficient utilization of nitrogen by plants, or both.

[0216] Thus, the present disclosure provides methods for increasing nitrogen fixation in plants, resulting in either increased availability of nitrogen to plants or efficient utilization of nitrogen by plants, or both.

[0217] In some embodiments, the biostimulant compositions of the present disclosure, when contacted with or applied to plants, promote better or increased nitrogen fixation, resulting in increased availability of nitrogen to plants, or efficient utilization of nitrogen by plants, or both.

[0218] In all embodiments of the method for promoting better nitrogen fixation, the biostimulant composition used is as described in any of the above embodiments. The method by which the biostimulant is contacted with or applied to the plant is also as described in any of the above embodiments. For the sake of brevity and to avoid repetition, each of these embodiments will not be described again herein. However, each of the above embodiments is fully within the scope of the method for promoting nitrogen fixation in plants of the present invention.

[0219] In some embodiments, the biostimulant composition increases, enhances, improves or makes better nitrogen fixation in a plant compared to nitrogen fixation by the same plant in the absence of the biostimulant composition of the present disclosure.

[0220] In some embodiments of the method for improving nitrogen fixation, the method comprises: Developing a biostimulant composition of the present disclosure comprising a microbial culture having at least 50% whole cells of Gammaproteobacteria methanotrophs, optionally together with other PGPMs, metabolite(s), nutrient(s) from the medium, and agriculturally acceptable excipient(s); and applying the composition to a plant, wherein the methanotrophs in the composition fix atmospheric nitrogen and increase its availability to the plant.

[0221] In some embodiments of the method for improving nitrogen fixation, the method comprises: Supplying methane to grow gammaproteobacterial methanotrophs such as M. capsulatus; Developing a biostimulant composition of the present disclosure comprising a microbial culture having at least 50% whole cells of Gammaproteobacteria methanotrophs, optionally together with other PGPMs, metabolite(s), nutrient(s) from the medium, and agriculturally acceptable excipient(s); and applying the composition to a plant, wherein the methanotrophs in the composition fix atmospheric nitrogen and increase its availability to the plant, the amount of fixed nitrogen being greater than the nitrogen fixed by the same plant without the composition.

[0222] Nitrogen availability or efficient utilization of nitrogen is increased through better nitrogen fixation promoted by the biostimulant compositions of the present disclosure, which in turn is a direct result of increased expression of nitrogenase genes selected from the group including nifA, nifD, nifH and nifK, or any combination thereof, in the microbial whole cells present in the biostimulant.

[0223] Thus, in some embodiments, the improved nitrogen fixation promoted by the biostimulant compositions of the present disclosure is the result of increased expression of nitrogenase family genes selected from the group including nifA, nifD, nifH, and nifK, or any combination thereof, in the gammaproteobacterial methanotrophs present in the biostimulant.

[0224] In some embodiments, the improved nitrogen fixation promoted by the biostimulant compositions of the present disclosure is the result of increased expression of nitrogenase family genes selected from the group including nifA, nifD, nifH, and nifK, or any combination thereof, in M. capsulatus present in the biostimulant.

[0225] In some embodiments, application of the biostimulant compositions of the present disclosure to plants allows gammaproteobacterial methanotrophs to grow in the absence of an external nitrogen source by regulating the expression of Nif genes, thus activating the nitrogenase machinery for fixing nitrogen in the environment.

[0226] In some embodiments, the method reduces the need for exogenous nitrogen or nitrogen-containing fertilizer in a plant by at least about 10% to about 100% compared to the need for exogenous nitrogen or nitrogen-containing fertilizer in a plant not contacted with the biostimulant composition of the present disclosure.

[0227] In a similar manner as above, the biostimulant compositions of the present disclosure also promote better plant availability and / or utilization of other nutrients, including phosphorus and potassium.

[0228] Thus, the present disclosure also provides a method of increasing the availability of phosphorus, potassium, or any combination thereof to a plant, comprising contacting or applying to the plant a biostimulant composition as described in any of the above embodiments.

[0229] Similarly, the present disclosure also provides a method for increasing the availability of nitrogen, phosphorus, and potassium to a plant, comprising contacting or applying to the plant a biostimulant composition as described in any of the above embodiments. The method by which the biostimulant is contacted with or applied to the plant is also as described in any of the above embodiments.

[0230] In some embodiments, the methods increase the availability of nitrogen, phosphorus, and potassium, or any combination thereof, in the soil for uptake by plants.

[0231] In some embodiments, the method increases the availability of nitrogen, phosphorus, and potassium, or any combination thereof, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a method that does not use a composition defined herein.

[0232] In some embodiments, application of a biostimulant composition of the present disclosure to a plant results in about a 36% increase in nitrogen uptake compared to a plant to which the composition is not applied.

[0233] In some embodiments, application of a biostimulant composition of the present disclosure to a plant results in about a 53% increase in phosphorus uptake compared to a plant to which the composition is not applied.

[0234] In some embodiments, application of a biostimulant composition of the present disclosure to a plant results in about a 39% increase in potassium uptake compared to a plant to which the composition is not applied.

[0235] As previously mentioned, one of the key objectives of the present disclosure is to utilize methane generated from agricultural activities and other sources in a manner that is also beneficial to reducing carbon footprints. Thus, the use of the biostimulant compositions of the present disclosure achieves this objective.

[0236] Use of methane and reduction of its atmospheric content Thus, the present disclosure also provides a method for increasing atmospheric methane utilization, comprising contacting or applying to a plant a biostimulant composition as described above.

[0237] The biostimulant composition of the present disclosure contains a microbial consortium with at least 50% whole cells of gammaproteobacteria methanotrophs, and therefore functions as an efficient tool used for methane utilization. In other words, simply applying this composition to a plant allows the gammaproteobacteria methanotrophs to utilize methane generated by the same plant, or present in the atmosphere as a result of agricultural activities, or from any other source, and use it for their growth and survival. As a result, methane levels in the vicinity of this composition are reduced.

[0238] In some embodiments, the gammaproteobacterial methanotrophs of the present disclosure utilize methane through carbon capture, which in turn is utilized for their growth and survival.

[0239] In some embodiments, methane utilization by a consortium in a biostimulant composition of the present disclosure comprising at least 50% whole cells of Gammaproteobacteria methanotrophs recycles at least about 0.1 kg of methane per kg of biostimulant used.

[0240] In some embodiments, the methane utilized by gammaproteobacterial methanotrophs can be separated into two buckets. Atmospheric methane, which is normally present in the dry air of the atmosphere at about 1-2 ppm, or Methane emissions concentrated in specific areas as a result of high agricultural or industrial activity

[0241] While the gammaproteobacteria methanotrophs present in the consortium in the biostimulant compositions herein also utilize atmospheric methane, the focus of this disclosure is to enable the methanotrophs to utilize methane that has become concentrated in certain areas, particularly due to agricultural activities. In other words, plants that emit high levels of methane serve as important methane sources for these methanotrophs. Because methane is a harmful greenhouse gas, its atmospheric concentration must be continually reduced. The use of the biostimulant compositions of the present disclosure facilitates exactly this. Because the compositions of the present invention contain gammaproteobacteria methanotrophs, when applied to plants that result in high levels of methane production, the organisms utilize it, helping to improve plant nitrogen fixation and performance.

[0242] In all embodiments relating to the method of utilizing methane by a consortium present in a biostimulant composition, the biostimulant composition itself and the method to be applied for the utilization of methane by the consortium therein are as described in any of the composition or method embodiments set forth above. For the sake of brevity and to avoid repetition, each of those embodiments will not be described again herein. However, each of said embodiments falls fully within the scope of the method of enhancing methane utilization.

[0243] As previously mentioned, since the composition provides multiple benefits in terms of enhancing plant performance and / or increasing plant nitrogen fixation, the utilization of methane by the same composition serves as an added benefit. The combination of these attributes makes the biostimulant composition a highly efficient and environmentally friendly product.

[0244] Concurrent methane utilization and nitrogen fixation in plants. Therefore, the present disclosure also provides a method for simultaneously promoting methane utilization and nitrogen fixation in a plant, comprising contacting or applying to the plant a biostimulant composition as described above.

[0245] In some embodiments, the present disclosure provides a method for simultaneously promoting methane utilization and nitrogen fixation in a plant, comprising contacting or applying a biostimulant composition comprising a microbial consortium of whole cells, wherein the consortium comprises at least 50% Gammaproteobacterial methanotroph whole cells.

[0246] In some embodiments, the microbial consortium comprises at least about 60% to about 100% whole cells of Gammaproteobacteria methanotrophs.

[0247] In some embodiments, the gammaproteobacterial methanotroph is a Type I or Type X methanotroph belonging to a genus selected from the group including Methylococcus, Methylomonas, Methylobacter, Methyloglobulus, Methyloblum, Methylomicrobium, Methylsarcina, Methylosphaera, Methyloprophundus, Methylosoma, Methyloccumis, Methylocardum, Methyloparacox, Methylogaea, Methylomagnum, Methylotelicola, Methylothermus, Methylohalobius, Methylomarinobum, Methylomarinum, and Crenothrix, or any combination thereof.

[0248] In some embodiments, the gammaproteobacterial methanotroph is Methylococcus capsulatus.

[0249] In some embodiments, the composition is in solid or liquid form and comprises at least one metabolite, at least one nutrient derived from the medium, and optionally at least one agriculturally acceptable excipient.

[0250] In some embodiments, the microbial consortium of whole cells is about 1 x 10 per gram or per milliliter of the composition. 3 cells ~ approx. 5 x 10 10 cells, constituting from about 0.1% to about 80% of the composition, with the remainder of the composition being from about 0.1% to about 10% of at least one metabolite, from about 0.1% to about 10% of at least one medium-derived nutrient, and optionally from about 0.01% to about 90% of at least one agriculturally acceptable excipient, including all values and ranges therein.

[0251] In some embodiments, the metabolites are selected from the group comprising carbohydrates, lipids, sugars, fatty acids, proteins, peptides, amino acids, nucleic acids, nucleotides, vitamins, organic acids, salts, minerals, osmolytes, extracellular enzymes, bacterially derived components and minerals, or any combination thereof; the media-derived nutrients are selected from the group comprising ions and salts, or combinations thereof; and the agriculturally acceptable excipients are selected from the group comprising carriers, cytoprotectants, adjuvants, surfactants, stabilizers, preservatives, diluents, suspending agents, dispersing agents and co-solvents, or any combination thereof.

[0252] In some embodiments, in addition to the Gammaproteobacterial methanotrophs, the consortium comprises from about 1% to about 50% of at least one plant growth-promoting microorganism selected from the group including nitrogen-fixing microorganisms, phosphorus-solubilizing microorganisms, mineral-solubilizing microorganisms, plant hormone-secreting microorganisms, organic acid-secreting bacteria, and plant beneficial microorganisms, or any combination thereof.

[0253] Thus, in some embodiments, the microbial consortium of the present disclosure is composed primarily of Gammaproteobacterial methanotrophs, such as M. capsulatus.

[0254] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 90:10.

[0255] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 80:20.

[0256] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 70:30.

[0257] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 60:40.

[0258] In some embodiments, the microbial consortium of the present disclosure comprises a Gammaproteobacterial methanotroph, such as M. capsulatus, and one or more PGPMs in a ratio of about 50:50.

[0259] In some embodiments, the consortium in the composition utilizes methane and promotes nitrogen fixation; and Improve or enhance plant performance; reducing the need for external addition of at least one nutrient selected from nitrogen, phosphorus and potassium, either individually or as part of a fertilizer; or · It's both.

[0260] In all embodiments of the method for simultaneously promoting methane utilization and nitrogen fixation in plants, the biostimulant composition used is as described in any of the above-mentioned embodiments. Similarly, the method for applying the biostimulant composition to plants is also as described in any of the above-mentioned embodiments. For the sake of brevity and to avoid repetition, each of these embodiments will not be described again herein. However, each of the above embodiments fully falls within the scope of the method for simultaneously promoting methane utilization and nitrogen fixation in plants.

[0261] More specifically, the present disclosure provides a method for simultaneously enhancing methane utilization and nitrogen fixation / availability in a plant, comprising contacting or applying to the plant a biostimulant composition comprising a consortium of whole cells comprising at least 50% gammaproteobacterial methanotroph whole cells, optionally together with metabolite(s), media-derived nutrient(s), and agriculturally acceptable excipient(s).

[0262] In some embodiments, the gammaproteobacteria methanotrophs in the biostimulant composition utilize atmospheric methane to facilitate / enable nitrogen fixation in plants.

[0263] In some embodiments, the methanotrophic organisms in the compositions of the methods utilize atmospheric methane to increase nitrogen fixation, thereby increasing nitrogen availability to plants.

[0264] In some embodiments, the Gammaproteobacteria methanotrophs in the consortium of the composition are at least about 2 x 10 per gram of plant part, particularly root, weight. 8 It has an average colonization capacity of 100 bacterial cells per unit of plant part.

[0265] In some embodiments, the plant part is selected from the group comprising roots, rhizomes, seeds, stems, flowers, stigmas, stamens, anthers, fruits, leaves, shoots, and combinations thereof.

[0266] In some embodiments, the Gammaproteobacteria methanotrophs in the consortium of the above-defined compositions are capable of increasing nitrogen availability to plants by fixing atmospheric nitrogen in the soil.

[0267] In some embodiments, when a biostimulant composition defined herein is contacted or applied to a plant / crop, the gammaproteobacteria methanotrophs in the composition can promote the utilization of atmospheric methane, concomitantly increasing nitrogen availability to the plant by fixing atmospheric nitrogen in the soil, compared to methods without using a composition defined herein.

[0268] In some embodiments, the gammaproteobacterial methanotrophs in the above-defined compositions can utilize atmospheric methane for their growth and metabolism and fix atmospheric nitrogen in the soil as ammonia, nitrite, nitrate or other nitrogen-containing compounds for enhanced nitrogen uptake and assimilation by plants.

[0269] In some embodiments, the plant is a legume, a non-legume, or a combination thereof.

[0270] In some embodiments, the plant is an agricultural crop, a horticultural crop, a plantation crop, or any combination thereof.

[0271] In some embodiments, the plant is an agricultural crop selected from the group including, but not limited to, cereals, millet, pulses / legumes, cash crops, oil crops, and combinations thereof.

[0272] In some embodiments, the plant is a horticultural crop selected from the group including, but not limited to, vegetable crops, medicinal crops, aromatic crops, floriculture crops, fruit crops, spice and plantation crops, and combinations thereof.

[0273] Nitrogen availability or efficient utilization of nitrogen is increased through better nitrogen fixation promoted by the biostimulant compositions of the present disclosure, which in turn is a direct result of increased expression of nitrogenase genes selected from the group including nifA, nifD, nifH and nifK, or any combination thereof, in the microbial whole cells present in the biostimulant.

[0274] Thus, in some embodiments, the improved nitrogen fixation with concomitant methane utilization promoted by the biostimulant compositions of the present disclosure is the result of increased expression of nitrogenase family genes selected from the group including nifA, nifD, nifH, and nifK, or any combination thereof, in the gammaproteobacterial methanotrophs present in the biostimulant.

[0275] In some embodiments, the improved nitrogen fixation with concomitant methane utilization promoted by the biostimulant compositions of the present disclosure is the result of increased expression of nitrogenase family genes selected from the group including nifA, nifD, nifH, and nifK, or any combination thereof, in M. capsulatus present in the biostimulant.

[0276] In some embodiments, a consortium of biostimulant compositions of the present disclosure comprising at least 50% whole cells of gammaproteobacteria methanotrophs promotes the nitrogen fixation referred to above while recycling at least about 0.1 kg of methane per kg of biostimulant used.

[0277] In some embodiments, the gammaproteobacterial methanotrophs of the present disclosure utilize methane through carbon capture, which in turn is utilized for their growth and survival, as well as to facilitate nitrogen fixation.

[0278] As mentioned above, the biostimulant composition of the present disclosure promotes better availability of at least one nutrient selected from, but not limited to, nitrogen, phosphorus, and potassium to plants or increases the utilization of said nutrients by plants, automatically reducing the need for externally added artificial / chemical / synthetic fertilizers containing these nutrients.

[0279] Reduced need for external nutrients and fertilizers Thus, the present disclosure provides a method for reducing the need for exogenous addition of at least one nutrient or nutrient-containing fertilizer for plant growth, development, performance, and / or survival, comprising contacting or applying to a plant a biostimulant composition as described above.

[0280] In some embodiments, the nutrients are selected from the group including, but not limited to, nitrogen, phosphorus, and potassium, or any combination thereof.

[0281] In some embodiments, the fertilizer is a chemical fertilizer.

[0282] In some embodiments, the method comprises reducing chemical fertilizer inputs required for plant growth and productivity, the method comprising contacting or applying to the plant a biostimulant composition of the present disclosure.

[0283] In some embodiments, plant contact or application of the biostimulant composition reduces the typically conventional amount of nitrogen-containing fertilizer, phosphorus-containing fertilizer, potassium-containing fertilizer, or any combination thereof, required to produce improved plant yield.

[0284] In some embodiments, those skilled in the art will readily know and understand the amounts of such fertilizers conventionally used in the normal course of agriculture, and the biostimulant compositions of the present disclosure reduce the need for external addition of such fertilizers.

[0285] In some embodiments, contacting or applying the biostimulant composition to a plant reduces the amount of nitrogen-containing fertilizer, including glutamine, ammonia, ammonium, urea, sulfur-coated urea, methylene urea, polymer-coated urea, isobutylidene diurea, nitrate, nitrite, ammonium-containing molecules, nitrate-containing molecules, or nitrite-containing molecules, or any combination thereof, required to result in improved plant yield.

[0286] In some embodiments, contacting or applying the biostimulant composition to a plant reduces the amount of phosphorus-containing fertilizers, including, but not limited to, diammonium phosphate, monoammonium phosphate, simple superphosphate, ammonium dihydrogen phosphate, ammonium phosphate, superphosphate, tricalcium phosphate, or any combination thereof, as a source of phosphate.

[0287] In some embodiments, contacting or applying the composition to a plant reduces the amount of potassium-containing fertilizer, including, but not limited to, potassium chloride, potassium sulfate, potassium nitrate, potassium magnesium sulfate, kainite, or combinations thereof, as a source of potassium.

[0288] In some embodiments, the method reduces the need for exogenous addition of at least one of nitrogen, phosphorus, and potassium for plant growth, development, performance, and / or survival by at least about 10% to about 100% compared to the need for added nitrogen, phosphorus, and potassium, respectively, in plants not contacted with the biostimulant composition of the present disclosure.

[0289] In some embodiments, the method reduces the amount of chemical fertilizer required for plant growth by at least about 10% compared to a method that does not use a biostimulant composition defined herein.

[0290] In some embodiments, the method reduces the amount of chemical fertilizer required for plant growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a method that does not use a biostimulant composition defined herein.

[0291] In some embodiments, applying a biostimulant composition of the present disclosure to a plant in an amount 50% less than conventional NPK fertilizer results in an increase in biomass production of at least about 50% compared to a plant grown under the same conditions with 50% less NPK fertilizer and no application of the composition.

[0292] In some embodiments, applying the biostimulant composition of the present disclosure to a plant at 50% less than conventional NPK results in an increase in biomass production of at least about 42% compared to a plant grown at 100% normal NPK and not receiving the composition.

[0293] In some embodiments, the present disclosure provides a method for reducing the need for exogenous addition of at least one nutrient or nutrient-containing fertilizer for plant growth, development, performance, and / or survival, comprising contacting or applying a biostimulant composition comprising a microbial consortium of whole cells, wherein the consortium comprises at least 50% Gammaproteobacterial methanotroph whole cells.

[0294] In some embodiments, the microbial consortium comprises at least about 60% to about 100% Gammaproteobacteria methanotroph whole cells, including all values and ranges therein.

[0295] In some embodiments, the gammaproteobacterial methanotroph is a Type I or Type X methanotroph belonging to a genus selected from the group including Methylococcus, Methylomonas, Methylobacter, Methyloglobulus, Methyloblum, Methylomicrobium, Methylsarcina, Methylosphaera, Methyloprophundus, Methylosoma, Methyloccumis, Methylocardum, Methyloparacox, Methylogaea, Methylomagnum, Methylotelicola, Methylothermus, Methylohalobius, Methylomarinobum, Methylomarinum, and Crenothrix, or any combination thereof.

[0296] In some embodiments, the gammaproteobacterial methanotroph is Methylococcus capsulatus.

[0297] In some embodiments, the composition is in solid or liquid form and comprises at least one metabolite, at least one nutrient derived from the medium, and optionally at least one agriculturally acceptable excipient.

[0298] In some embodiments, the microbial consortium of whole cells is about 1 x 10 per gram or per milliliter of the composition. 3 cells ~ approx. 5 x 10 10 cells, constituting from about 0.1% to about 80% of the composition, with the remainder of the composition being from about 0.1% to about 10% of at least one metabolite, from about 0.1% to about 10% of at least one medium-derived nutrient, and optionally from about 0.01% to about 90% of at least one agriculturally acceptable excipient, including all values and ranges therein.

[0299] In some embodiments, the metabolites are selected from the group comprising carbohydrates, lipids, sugars, fatty acids, proteins, peptides, amino acids, nucleic acids, nucleotides, vitamins, organic acids, salts, minerals, osmolytes, extracellular enzymes, bacterially derived components and minerals, or any combination thereof; the media-derived nutrients are selected from the group comprising ions and salts, or combinations thereof; and the agriculturally acceptable excipients are selected from the group comprising carriers, cytoprotectants, adjuvants, surfactants, stabilizers, preservatives, diluents, suspending agents, dispersing agents and co-solvents, or any combination thereof.

[0300] In some embodiments, in addition to the Gammaproteobacterial methanotrophs, the consortium comprises from about 1% to about 50% of at least one plant growth-promoting microorganism selected from the group including nitrogen-fixing microorganisms, phosphorus-solubilizing microorganisms, mineral-solubilizing microorganisms, plant hormone-secreting microorganisms, organic acid-secreting bacteria, and plant beneficial microorganisms, or any combination thereof.

[0301] In some embodiments, the consortium in the composition reduces the need for external addition of at least one nutrient selected from nitrogen, phosphorus, and potassium, either individually or as part of a fertilizer; and Improve or enhance plant performance; Utilize methane and simultaneously promote nitrogen fixation, or · It's both.

[0302] In all embodiments of the method for reducing the need for external addition of at least one nutrient or nutrient-containing fertilizer for plant growth, development, performance, and / or survival, the biostimulant composition used is as described in any of the above-mentioned embodiments. Similarly, the method for applying the biostimulant composition to the plant is also as described in any of the above-mentioned embodiments. For the sake of brevity and to avoid repetition, each of these embodiments will not be described again herein. However, each of the above embodiments fully falls within the scope of the method for reducing the need for external addition of at least one nutrient or nutrient-containing fertilizer.

[0303] In some embodiments of the above methods for reducing the amount of fertilizer required to grow a plant, the plant is a legume, a non-legume, or a combination thereof.

[0304] In some embodiments of the above methods for reducing the amount of fertilizer required for plant growth, the plant is an agricultural crop, a horticultural crop, a plantation crop, or any combination thereof.

[0305] In some embodiments of the above methods of reducing the amount of fertilizer required to grow a plant, the plant is an agricultural crop selected from the group including cereals, millet, pulses / legumes, cash crops, oil crops, and combinations thereof.

[0306] In some embodiments of the above methods of reducing the amount of fertilizer required for plant growth, the plant is a horticultural crop selected from the group including vegetable crops, medicinal crops, aromatic crops, floriculture crops, fruit crops, spice and plantation crops, and combinations thereof.

[0307] The present disclosure further relates to a method of maintaining soil fertility comprising planting a plant or part thereof that has been contacted with or applied to a biostimulant composition of the present disclosure.

[0308] In some embodiments of the method of maintaining soil fertility, the method further comprises harvesting the plant.

[0309] In some embodiments according to the method of maintaining soil fertility, the method comprises sowing seeds that have been contacted with or applied with a biostimulant composition of the present disclosure.

[0310] In some embodiments of the method for maintaining soil fertility, the method maintains nitrogen levels in the soil, reducing the need for fertilizer, preferably nitrogen-containing fertilizer, for plant growth.

[0311] In some embodiments of the method for maintaining soil fertility, the method reduces the need for nitrogen-containing fertilizers, phosphorus-containing fertilizers, potassium-containing fertilizers, or any combination thereof.

[0312] While the previous embodiments highlighted the importance of the biostimulant composition of the present disclosure and how it affects plant performance, the following embodiments provide methods for preparing said composition comprising a microbial consortium having at least 50% whole cells of Gammaproteobacteria methanotrophs, optionally together with at least one metabolite, at least one medium-derived nutrient, and at least one agriculturally acceptable excipient.

[0313] Preparation of Biostimulant Compositions Therefore, the present disclosure also provides a method for preparing a biostimulant composition comprising a gammaproteobacterial methanotroph, at least one metabolite or medium-derived nutrient, and optionally at least one agriculturally acceptable excipient as defined above, comprising: - obtaining a mixture comprising a gammaproteobacterial methanotroph, at least one metabolite and nutrients from a culture medium, and optionally adding an agriculturally acceptable excipient to the mixture to prepare a composition; or - mixing the gammaproteobacterial methanotroph, at least one metabolite, and nutrients from the culture medium, optionally together with an agriculturally acceptable excipient, to prepare a composition. The present invention relates to a method comprising:

[0314] In some embodiments, the method comprises: - obtaining a mixture comprising a gammaproteobacterial methanotroph, at least one metabolite, and at least one nutrient from a culture medium, and optionally adding an agriculturally acceptable excipient to the mixture to prepare a composition; or - mixing the gammaproteobacterial methanotroph, at least one metabolite, and at least one nutrient from the medium, optionally together with an agriculturally acceptable excipient, to prepare a composition. Includes:

[0315] In some embodiments of the above method, the Gammaproteobacteria methanotrophs are present in an amount of about 1 x 10 per gram or ml of the solid composition. 3 cells ~ approx. 5 x 10 10 A range of cells is present in the consortium with a total concentration of all cells in the range of cells, including all values and ranges therein.

[0316] Therefore, the present disclosure also provides a method for preparing a biostimulant composition comprising a Gammaproteobacterial methanotroph, optionally together with at least one metabolite, at least one nutrient from the medium and at least one agriculturally acceptable excipient as defined above, comprising: Fermentation in the presence of a methane source to obtain Gammaproteobacterial methanotrophs, treating the fermentation broth containing the cells; and mixing the cells with at least one metabolite, a nutrient from the culture medium, and / or an agriculturally acceptable excipient to prepare a biostimulant composition of the present disclosure; The present invention relates to a method comprising:

[0317] In some embodiments, the method comprises: Cultivating Gammaproteobacteria methanotrophs in a nutrient mixture (culture medium) in the presence of methane as the sole carbon and energy source; harvesting the gammaproteobacterial methanotrophs together with the metabolite(s) and nutrient(s) from the medium to obtain the biostimulant composition of the present disclosure; Includes:

[0318] In some embodiments, the method comprises: Cultivating Gammaproteobacteria methanotrophs in a nutrient mixture (culture medium) in the presence of methane as the sole carbon and energy source; harvesting the gammaproteobacterial methanotroph together with the metabolite(s) and nutrient(s) from the medium to obtain a mixture; and Optionally, adding an agriculturally acceptable excipient(s) to the mixture to obtain the biostimulant composition of the present disclosure. Includes:

[0319] In some embodiments, the gammaproteobacterial methanotroph is Methylococcus capsulatus.

[0320] Thus, in some embodiments, the method comprises culturing Methylococcus capsulatus in a culture medium under suitable culture conditions, followed by harvesting the Methylococcus capsulatus to prepare the biostimulant composition of the present disclosure.

[0321] In some embodiments, the method comprises: Fermenting or culturing Methylococcus capsulatus in a culture medium in the presence of methane under suitable temperature and pressure conditions; harvesting Methylococcus capsulatus together with metabolite(s) and nutrient(s) from the medium to obtain the biostimulant composition of the present disclosure; Cultivating Methylococcus capsulatus in a nutrient mixture (culture medium) in the presence of methane under suitable temperature and pressure conditions; Harvesting Methylococcus capsulatus together with metabolite(s) and nutrient(s) from the medium to obtain a mixture containing Methylococcus capsulatus, metabolites, and non-cellular nutrients. and Optionally in some embodiments, the method further comprises: Cultivating Methylococcus capsulatus to obtain a mixture containing Methylococcus capsulatus cells, metabolite(s) and nutrient(s) from the medium; and Optionally, adding an agriculturally acceptable excipient to the mixture to prepare the biostimulant composition of the present disclosure. Includes:

[0322] In some embodiments, the method comprises: adding agriculturally acceptable excipient(s) to the mixture to obtain the biostimulant composition of the present disclosure; Includes:

[0323] In a preferred embodiment of the present disclosure, the method for cultivating methanotrophs is carried out in accordance with the descriptions and examples of International Application No. PCT / IB2017 / 052688 and / or International Application No. PCT / IB2019 / 059664, the descriptions of which are incorporated herein by reference in their entirety.

[0324] In a preferred embodiment of the present disclosure, the method for culturing Methylococcus capsulatus is carried out according to International Application No. PCT / IB2017 / 052688 and / or International Application No. PCT / IB2019 / 059664, the disclosures of which are incorporated herein by reference in their entirety. In another preferred embodiment of the present disclosure, the method for culturing Methylococcus capsulatus in a culture medium in the presence of methane under suitable culture conditions is described in International Application No. PCT / IB2017 / 052688 and / or International Application No. PCT / IB2019 / 059664, the disclosures of which are incorporated herein by reference in their entirety.

[0325] In some embodiments, after culturing, the cells along with components from the medium are mixed with at least one agriculturally acceptable excipient.

[0326] In some embodiments, the excipients used are selected from known adjuvants and cytoprotective agents and are used at 0.01%, 0.5% or 1% (weight / volume) in the composition.

[0327] In some embodiments, after the fermentation process, the methanotrophic cells, along with components from the medium, are mixed with at least one bacterial group, the cells being from a group including nitrogen-fixing, mineral-dissolving, plant hormone-producing, or plant growth-promoting bacteria.

[0328] In some embodiments of the above methods, the prepared composition comprises Methylococcus capsulatus, at least one metabolite selected from, but not limited to, carbohydrates, sugars, proteins, amino acids, nucleic acids, nucleotides, peptides, fatty acids, lipids, vitamins, organic acids, osmolytes, and salts, and at least one medium-derived nutrient selected from, but not limited to, salts, minerals, and ions.

[0329] As previously mentioned, the biostimulant compositions of the present disclosure may contain a total of about 1 x 10 3 Total cells ~ approx. 5 x 10 10In some embodiments, the microbial consortium comprises a total of about 5 x 10 total cells. 3 Total cells ~ approx. 5 x 10 10 In other embodiments, the microbial consortium comprises a total of about 1 x 10 total cells. 3 Total cells ~ approx. 1 x 10 10 Contains all cells.

[0330] Importantly, the consortium must contain at least 50% Gammaproteobacteria methanotroph whole cells, hi some embodiments, the microbial consortium within the biostimulant composition contains at least about 60% to about 100% Gammaproteobacteria methanotroph whole cells, all values and ranges therein.

[0331] Therefore, while following the method steps for preparing the biostimulant composition of the present disclosure, including a gammaproteobacterial methanotroph, such as M. capsulatus, is paramount. If the consortium within the composition is composed solely of gammaproteobacterial methanotrophs, such as M. capsulatus, the method steps are as set forth above, whereby cell culturing, harvesting, and mixing with metabolite(s), medium-derived nutrient(s), and / or excipient(s) are applicable to each gammaproteobacterial methanotroph cell. However, if other PGPMs are present as part of the consortium apart from the gammaproteobacterial methanotrophs, such PGPMs may be cultured and / or harvested together with the gammaproteobacterial methanotrophs, or may be cultured and / or harvested separately and then included in the composition as an additional step in the method described in the preceding embodiment.

[0332] Those skilled in the art will understand that the sequence of steps and methods for culturing and harvesting cells and mixing them with their metabolite(s), nutrient(s) from the medium and / or excipient(s) is routine in the art and can therefore be carried out by any known technique. What is important is the fact that the final biostimulant composition so prepared must satisfy the following: - a whole-cell microbial consortium, the consortium comprising at least 50% whole cells of gammaproteobacteria methanotrophs; Total whole cells: Approximately 1 x 10 per gram or milliliter of composition 3 cells ~ approx. 5 x 10 10 at concentrations ranging from 0.1 to 0.2 cells, and optionally, at least one metabolite at a concentration ranging from about 0.1% to about 10%; optionally, at least one medium-derived nutrient at a concentration ranging from about 0.1% to about 10%; and Optionally, at least one agriculturally acceptable excipient at a concentration ranging from about 0.01% to about 90%.

[0333] In some embodiments, the gammaproteobacterial methanotroph used in the biostimulant composition is a Type I or Type X methanotroph belonging to a genus selected from the group including Methylococcus, Methylomonas, Methylobacter, Methyloglobulus, Methyloblum, Methylomicrobium, Methylsarcina, Methylosphaera, Methyloprophundus, Methylosoma, Methyloccumis, Methylocardum, Methyloparacox, Methylogaea, Methylomagnum, Methylotelicola, Methylothermus, Methylohalobius, Methylomarinobum, Methylomarinum, and Crenothrix, or any combination thereof.

[0334] In some embodiments, the gammaproteobacterial methanotrophs used in the biostimulant compositions include type I or type X methanotrophs, such as Methylococcus spp., Methylomonas spp., Methylobacter spp., Methyloglobulus spp., Methyloblum spp., Methylomicrobium spp., Methylsarcina spp., Methylosphaera spp., Methyloprofen ... In some embodiments, the species is selected from the group comprising: Saccharomyces spp., Methylosoma spp., Methyloccumis spp., Methylocardum spp., Methyloparacox spp., Methylogaea spp., Methylomagnum spp., Methylotelicola spp., Methylothermus spp., Methylohalobius spp., Methylomarinobum spp., Methylomarinum spp., and Crenothrix spp., or any combination thereof.

[0335] In some embodiments, the gammaproteobacterial methanotroph is selected from the group consisting of Methylococcus capsulatus, Methylococcus mobilis, Methylomicrobium kenyense, Methylomicrobium alkaliphilum, Methylomicrobium alkaliphilum 20Z, Methylomicrobium briatense 5G, Methylomicrobium briatense 4G, Halomonas pantelleriensis, Methylomicrobium albumen, Methylomonas methanica, MB 126, Methylobacter tundripaldurum, Methyloblum miyakonense, Methylomonas rubra, Methylomonas koyamae, Methylomonas metanchia, Methylomonas denitrificans, Methylomonas pardis, Methylomonas lenta, Methylomarinum vidii, Methylococcus thermophilus, Methylobacter uchttenbrii, Crenothrix polyspora, Chronothrix fusca, Methylobacter bovis, Methylomonas aurantiaca, Methylomonas fodinarum, Methylobacter vinelandii, Methylomicrobium japanense, Methylococcus bacterium, Methylocystis methanolux, Methyloccumis oryzae, Methylogaea oryzae, Methylsarcina lacus, Methylosoma difficile and combinations thereof.

[0336] In the above method of preparing a composition, the methanotroph, metabolites, nutrients from the medium, and agriculturally acceptable excipients are as described in the previous embodiment.

[0337] In some embodiments, the present disclosure also provides: a microbial consortium comprising one or more Gammaproteobacterial methanotrophs and one or more plant growth-promoting microorganisms (PGPMs); at least one metabolite, at least one medium-derived nutrient; and Optionally, agriculturally acceptable excipients 1. A method for preparing a composition comprising: - Obtaining a mixture comprising a microbial consortium having a gammaproteobacterial methanotroph and at least one PGPM, at least one metabolite and at least one nutrient from a culture medium, and optionally adding an agriculturally acceptable excipient to prepare a composition; or - obtaining a mixture comprising a gammaproteobacterial methanotroph, at least one metabolite and at least one nutrient from a culture medium, and adding at least one plant growth-promoting microorganism (PGPM) and, optionally, an agriculturally acceptable excipient to prepare a composition; or - preparing a composition by mixing the microbial consortium, at least one metabolite, and at least one nutrient from the culture medium, optionally together with an agriculturally acceptable excipient. The present invention provides a method comprising:

[0338] In some embodiments of the above method, obtaining a mixture comprising a microbial consortium, metabolites, and nutrients from a culture medium comprises culturing a methanotroph and a plant growth-promoting microorganism (PGPM) in the same or different culture medium under suitable culture conditions to obtain the mixture.

[0339] In some embodiments, to prepare the compositions of the present disclosure, Methylococcus capsulatus was cultured in a culture medium in the presence of methane under suitable culture conditions. Because the cultivation and growth requirements of Methylococcus capsulatus are well known in the art, conventional methods for this purpose were employed. These culture conditions were performed according to the descriptions and examples in International Application No. PCT / IB2017 / 052688 and / or International Application No. PCT / IB2019 / 059664, the descriptions of which are incorporated herein by reference in their entirety. After cultivation, the cells, along with components from the medium, were mixed with at least one agriculturally acceptable excipient and used to analyze plant performance. In another example, after the fermentation process, the methanotrophic cells, along with components from the medium, were mixed with at least one bacterial group, the cells being derived from a group including nitrogen-fixing, mineral-dissolving, plant hormone-producing, or plant growth-promoting bacteria. Various ratios of methanotroph cells were mixed with the bacteria, and accordingly the cell to bacteria ratio was varied as follows: 90:10, 80:20, 70:30, 60:40 or 50:50.

[0340] Thus, in some embodiments, the microbial consortium of the present disclosure is composed primarily of M. capsulatus.

[0341] In some embodiments, the microbial consortium of the present disclosure comprises M. capsulatus and one or more PGPMs in a ratio of about 90:10.

[0342] In some embodiments, the microbial consortium of the present disclosure comprises M. capsulatus and one or more PGPMs in a ratio of about 80:20.

[0343] In some embodiments, the microbial consortium of the present disclosure comprises M. capsulatus and one or more PGPMs in a ratio of about 70:30.

[0344] In some embodiments, the microbial consortium of the present disclosure comprises M. capsulatus and one or more PGPMs in a ratio of about 60:40.

[0345] In some embodiments, the microbial consortium of the present disclosure comprises M. capsulatus and one or more PGPM in a ratio of about 50:50.

[0346] In some embodiments, the concentration of methanotrophic cells, such as M. capsulatus, in the composition is typically targeted to be greater than 90%. In some embodiments, the concentration of methanotrophic cells, such as M. capsulatus, in the composition is targeted to be greater than 80%. In some embodiments, the concentration of methanotrophic cells, such as M. capsulatus, in the composition is targeted to be greater than 70%. In some embodiments, the concentration of methanotrophic cells, such as M. capsulatus, in the composition is targeted to be greater than 60%.

[0347] In some embodiments, at least 50% methanotrophic cells, such as M. capsulatus cells, are mixed with at least 50% cells from one group of nitrogen-fixing, mineral-dissolving, plant hormone-producing bacteria, or plant growth-promoting bacteria.

[0348] In some embodiments, the composition is mixed with at least one agriculturally acceptable excipient. In some embodiments, the excipient used is selected from known adjuvants and cytoprotective agents. In some embodiments, the excipient is mixed in the composition at 0.01%, 0.1%, 0.5%, or 1% (weight / volume).

[0349] In some embodiments, the composition comprises 1 x 10 sucrose, ... 5 ~1×10 8The composition contained M. capsulatus cells at 0.5% cells / ml. The composition contains 2-3% protein and 1-2% total salts (micronutrients). The composition is formulated with an agriculturally acceptable excipient at 0.5%. In some embodiments, the excipient used is 0.5% DMSO. In other embodiments, the excipient used is 0.5% DMSO and 2% xanthan gum. Thus, this composition forms a biostimulant composition of the present disclosure in which the microbial consortium is primarily composed of M. capsulatus.

[0350] In some embodiments, total protein was analyzed using established methods with HPLC and Kjeldahl, and micronutrients were analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and ion chromatography (IC).

[0351] In some embodiments, the cells from the M. capsulatus fermentation broth are present in a composition having a total cell count of 1 x 10 5 ~1×10 8 The composition is mixed with phosphate-solubilizing bacteria at a ratio of 90:10, at 100 cells / ml. The composition contains 2-3% protein and 1-2% total salts (micronutrients). This composition is formulated with an agriculturally acceptable excipient at 0.5%. In some embodiments, the excipient used is 0.5% DMSO. In other embodiments, the excipient used is 0.5% DMSO and 2% xanthan gum. Thus, in some embodiments, the ratio of cells from M. capsulatus fermentation to PGPM, such as phosphate-solubilizing bacteria, is 90:10, 80:20, 70:30, 60:40, or 50:50. These compositions thus form biostimulant compositions of the present disclosure in which the microbial consortium is comprised of M. capsulatus along with other plant growth-promoting microorganisms.

[0352] In some embodiments, for use of the compositions on plants, the compositions are in liquid form and a sufficient quantity (qs) of water based on the end use application is added to each of the compositions prepared herein.

[0353] In some embodiments, the composition is applied to plants as a solid formulation. To prepare a solid formulation, the composition is mixed with a carrier material and dried until the total moisture content of the formulation is less than 10%. The composition is dried by air drying, spray drying, drum drying, or vacuum tray drying.

[0354] In some embodiments of the above methods, the microbial consortium, methanotrophs, plant growth-promoting microorganisms (PGPMs), metabolites, nutrients from the medium, agriculturally acceptable excipients, and concentrations / amounts of said components in the composition are as defined in the previous embodiments.

[0355] As mentioned above, the biostimulant composition of the present disclosure prepared in this way is important from an agricultural and environmental point of view, as it meets multiple attributes and overcomes at once the challenges related to the high content of methane emitted by agricultural activities, better agricultural productivity and reduced use of external fertilizers (something not provided by any of the currently available biostimulants).

[0356] Uses of Biostimulant Compositions Thus, the present disclosure also provides uses of the biostimulant compositions of the present disclosure for: Improving or enhancing the performance of plants; or Simultaneously promoting methane utilization and nitrogen fixation in plants, or reducing the need for external addition of at least one nutrient or nutrient-containing fertilizer for plant growth, development, performance, and / or survival; or Increase nitrogen fixation in plants, or Any combination of them.

[0357] In some embodiments, improving or enhancing plant performance is characterized by at least one of the following: Stimulation or promotion of quantitative or qualitative plant attributes selected from the group including biomass production, yield, photosynthetic activity, nutritional value, secondary metabolites and nutrient utilization efficiency, or any combination thereof; an increase in the number, size or quality of below-ground or above-ground biomass selected from the group including roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits and seeds, or any combination thereof; Increased photosynthetic activity or chlorophyll content Increased protein, dietary fiber, beta-carotene or essential oil content, plant-specific metabolites, or any combination thereof; or · Efficient absorption or use of available or externally supplied nutrients selected from the group including nitrogen, phosphorus and potassium, or any combination thereof.

[0358] In some embodiments, the biostimulant composition is in solid or liquid form and is contacted or applied to the plant through the soil of the plant or through an aerial or non-aerial part of the plant selected from the group including roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits and seeds, or any combination thereof, at a concentration ranging from about 1 ml per liter to about 50 ml per liter for solid forms or from 1 gm per kilogram to about 50 gm per kilogram for solid forms.

[0359] In some embodiments, the biostimulant composition is in solid or liquid form and is contacted or applied to the plant through the soil of the plant or through an above-ground or non-aerial part of the plant selected from the group including roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits and seeds, or any combination thereof, at a concentration ranging from 1× to 100,000× dilution of the solid or liquid form of the composition, including all values and ranges therein.

[0360] The present disclosure also provides a) contacting or applying a biostimulant composition of the present disclosure to a crop; and b) Harvesting crops to obtain agricultural or horticultural products The present invention provides the use of a biostimulant composition of the present disclosure in a method for making an agricultural or horticultural product comprising:

[0361] In some embodiments, the agricultural or horticultural product is selected from the group including, but not limited to, food grains, vegetables, fruits, tubers, nuts, cereals, grains, millet, pulses, oil crops, floriculture crops, medicinal plants, aromatic plants, spices and plantation crops, grasses, and combinations thereof.

[0362] The present disclosure also provides a) a biostimulant composition of the present disclosure, and b) a hydrolysate-based biostimulant composition containing a protein-derived component in an amount of about 30% or less by weight of the composition; Including, The protein-derived component is obtained from a methanotrophic bacterium. The Company offers biostimulant products.

[0363] In all embodiments of the method of use provided herein, the biostimulant composition used is as described in any of the above embodiments. Similarly, the method of applying the biostimulant composition to plants is also as described in any of the above embodiments. For the sake of brevity and to avoid repetition, each of these embodiments will not be described again herein. However, each of these embodiments is fully within the scope of the use.

[0364] Therefore, the present disclosure generally aims to provide a unique and alternative approach / method for simultaneously achieving the reduction of methane emissions in agriculture (through the efficient utilization of said methane) and the availability / fixation of nitrogen in plants. To achieve this, a biostimulant composition based on gammaproteobacteria methanotrophs as described above is provided. Furthermore, methods using said composition are related to environmentally friendly approaches for reducing atmospheric methane, fixing atmospheric nitrogen in plants, and / or reducing the use of chemical fertilizers, among other benefits, while said composition is also used in agricultural applications to improve plant / crop performance.

[0365] It is important to understand that fermentation is an economical and scalable process that has been utilized for decades to bring products to market in various sectors, including food, feed, health, and consumer products. The compositions and methods of the present disclosure utilize the use of methane, a harmful greenhouse gas, to enable unique biostimulant compositions. This allows the biostimulant compositions to be economical / cost-effective while achieving sustainability and consistency in production. Improved plant performance provides attractive returns to users, primarily agricultural communities and farmers, enabling wider use of the biostimulant compositions.

[0366] Additional embodiments and features of the present disclosure will be apparent to those skilled in the art based on the description provided herein. The embodiments of the present disclosure illustrate various features and advantageous details thereof in the description. Descriptions of well-known / conventional methods and techniques are omitted so as not to unnecessarily obscure the embodiments of the present disclosure. Furthermore, the disclosure of the present disclosure provides examples illustrating the above-described embodiments, and certain specific aspects are used to illustrate the embodiments of the present disclosure. The examples used herein for such illustration are intended merely to facilitate understanding of how the embodiments of the present disclosure can be implemented and to further enable those skilled in the art to implement the embodiments of the present disclosure. Therefore, the following examples should not be construed as limiting the scope of the examples of the present disclosure. [Example]

[0367] Materials used The Methylococcus capsulatus strain used in this disclosure has been deposited with the Microbial Type Culture Collection (MTCC) and Gene Bank (MTCC25398) in accordance with the Budapest Treaty. The geographic origin and source of this strain is the United Kingdom, and after procurement, it was managed by String Bio Private Limited. Furthermore, all plants / crops were used in the following experiments / examples only to verify the technical effects of the product (methanotroph-derived biostimulant composition) of the present disclosure. None of these plants / crops were used in the preparation / development of the biostimulant product of the present disclosure.

[0368] Example A Preparation of methanotroph-based whole cell compositions To prepare the compositions of the present disclosure, Methylococcus capsulatus was cultured in the presence of methane under suitable culture conditions. Because the cultivation of Methylococcus capsulatus and its growth requirements are well known in the art, conventional methods for this purpose were employed. These culture conditions were carried out according to the descriptions and examples in International Application No. PCT / IB2017 / 052688 and / or International Application No. PCT / IB2019 / 059664, the specifications of which are incorporated herein by reference in their entirety.

[0369] After cultivation, the components from the medium and the cells were mixed with at least one agriculturally acceptable excipient and used for plant performance analysis. In another example, during the post-fermentation process, the components from the medium and the methanotrophic cells were mixed with at least one group of bacteria. The bacteria were from groups including nitrogen-fixing, mineral-dissolving, plant hormone-producing, or plant growth-promoting bacteria. Various ratios of methanogenic cells were mixed with the bacteria. Accordingly, the cell to bacteria ratio was varied as follows: 90:10, 80:20, 70:30, 60:40, or 50:50.

[0370] Typically, the concentration of methanotrophic cells in the composition was targeted to be greater than 90%. In some instances, the concentration of methanotrophic cells in the composition was targeted to be greater than 80%. In some instances, the concentration of methanotrophic cells in the composition was targeted to be greater than 70%. In some instances, the concentration of methanotrophic cells in the composition was targeted to be greater than 60%. In other instances, 50% methanotrophic cells were mixed with 50% cells from a group of nitrogen-fixing, mineral-dissolving, plant hormone-producing, or plant growth-promoting bacteria. This composition was then mixed with at least one agriculturally acceptable excipient. The excipients used were selected from known adjuvants and cytoprotectants. The excipients were mixed in the composition at 0.01%, 0.1%, 0.5%, or 1% (weight / volume).

[0371] In one example of preparing the composition, the composition is prepared by adding 1 x 10 sucrose to 100 ml of sucrose harvested after fermentation in the presence of methane. 5 ~1×10 8The composition contained M. capsulatus cells at 0.5% cell / ml. The composition was analyzed for protein and contained 2-3% protein and 1-2% total salts (micronutrients). Total protein was analyzed using established methods using HPLC and Kjeldahl. Micronutrients were analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and ion chromatography (IC). The composition was formulated with an agriculturally acceptable excipient at 0.5%. In some instances, the excipient used was 0.5% DMSO. In other instances, the excipient was 0.5% DMSO and 2% xanthan gum. Thus, this composition is an example of a biostimulant composition of the present disclosure in which the microbial consortium is primarily composed of M. capsulatus.

[0372] In another example of preparing the composition, cells from M. capsulatus fermentation are mixed with phosphate-dissolving bacteria in a 90:10 ratio, resulting in a total cell count of 1 x 10 5 ~1×10 8 The concentration was determined to be 100 cells / ml. The composition was analyzed for protein and contained 2-3% protein and 1-2% total salts (micronutrients). This composition was formulated with an agriculturally acceptable excipient at 0.5%. In some instances, the excipient used was 0.5% DMSO. In other instances, the excipients were 0.5% DMSO and 2% xanthan gum. The ratio of cells derived from the fermentation of M. capsulatus to phosphate-dissolving bacteria was varied as appropriate: 90:10, 80:20, 70:30, 60:40, or 50:50. Thus, these compositions are examples of biostimulant compositions of the present disclosure in which the microbial consortium is comprised of M. capsulatus along with other plant growth-promoting microorganisms.

[0373] To use the compositions on plants, in some cases the compositions are in liquid form, and a sufficient quantity (qs) of water based on the end use application was added to each of the compositions prepared herein.

[0374] In some cases, the compositions were applied to plants as solid formulations. To prepare solid formulations, the compositions were mixed with a carrier material and dried until the total moisture content in the formulation was <10%. The compositions were dried by air drying, spray drying, drum drying, or vacuum tray drying. Table 1 provides examples of typical methanotroph-based whole cell compositions.

[0375] [Table 1]

[0376] Example 1 Application of methanotroph-based whole cell compositions improved spinach biomass yield To understand the effect of methanotroph-based whole-cell compositions on improving the yield of spinach (Spinacia oleracea), a field trial experiment was designed following a Randomized Complete Block Design (RCBD). Seed rate, fertilization, planting date, harvest time, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole-cell composition. Seeds were sown in the field, and the first treatment of spinach plants with the methanotroph-based whole-cell composition was carried out 15 days after sowing, followed by a second application at an interval of 10 days. Treatments were carried out either as soil application or foliar spray. The final whole-cell composition used in this experiment was prepared as listed in Example A. The primary treatment consisted of 1 x 10 M. capsulatus cells. 5 ~1×10 8 The composition was contained at a cell count of cells / ml. Control plants received both foliar and soil applications of the composition. Plants were harvested 40-45 days after sowing. Above-ground biomass was used to determine yield improvement. All observations were obtained from a uniform sampling of plants under identical conditions, unless otherwise noted.

[0377] The results of the experiment are shown in Figure 1. As observed, both foliar and soil applications of the methanotroph-based whole cell composition in spinach showed a significant improvement in product biomass of ~23-36% compared to control plants that received water spray. The described results regarding improved yield in spinach further support the ability of methanotroph-based whole cell compositions to produce agriculturally relevant results such as improved yield in open-field conditions.

[0378] Example 2 Application of methanotroph-based whole cell composition in guar bean improved pod yield To understand the effect of a methanotroph-based whole-cell composition on pod yield of guar bean (Cyamopsis tetragonoloba), a plot study was designed following a completely randomized block design (RCBD). Plant population, fertilization, planting date, harvest time, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole-cell composition. Seeds were sown in the field, and the first foliar application of the methanotroph-based whole-cell formulation was applied 30 days after sowing (DAS). The second and third foliar applications were made at 45 and 60 DAS, respectively. The final whole-cell composition used in this experiment was prepared as listed in Example A. 2 x 10 5 ~1×10 8 The total cell count contained cells / ml, with >90% of the cells being M. capsulatus. The compositions were applied to plants as foliar sprays or soil treatments. Pods harvested from multiple pickings were measured to determine overall yield improvement. Unless otherwise noted, all observations were made from a uniform sampling of plants under identical conditions.

[0379] The results of the experiment are shown in Figure 2. As observed, plants treated with the methanotroph-based whole cell composition showed a significant improvement in pod yield of -22% compared to the negative control. The described results on yield improvement in cluster bean further support the ability of methanotroph-based whole cell compositions to produce agriculturally relevant results such as improved pod yield in open-field conditions.

[0380] Example 3 Application of methanotroph-based whole cell compositions improved yields of cereal and horticultural crops The effect of the methanotroph-based whole cell composition on improving yield in other agriculturally important crops such as sweet corn, chili pepper, coriander, broad bean, and marigold is shown in Table 2. The plant population, planting date, harvest time, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole cell composition. The experiments were conducted in different locations in the farmer's field but under identical conditions. For testing and validation, the final whole cell composition used in this experiment was 1 x 10 5 ~1×10 8 The compositions were composed of a total cell count of 1000 cells / ml, with >80% of the cells being M. capsulatus, and were prepared as listed in Example A. The compositions were applied either through the soil or by foliar spray. The yield results obtained show that the methanotroph-based whole cell compositions of the present disclosure demonstrated significant yield improvements in various crops compared to their respective controls. The results / yield improvements further demonstrate the ability of the methanotroph-based whole cell compositions to enhance / promote plant growth or performance in a diverse group of crops.

[0381] [Table 2]

[0382] Example 4 Application of a methanotroph-based whole-cell composition improved nutrient utilization efficiency in spinach. To understand the effect of methanotroph-based whole-cell compositions on improving nutrient uptake in spinach (Spinachia oleracea), a field trial experiment was designed following a completely randomized block design (RCBD). Plant populations, fertilization, planting dates, harvest times, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole-cell composition. Seeds were sown in the field, and the first soil application of the methanotroph-based whole-cell composition was made 15 days after sowing (DAS). The second soil application was made 25 days after sowing. For testing and validation, the final whole-cell composition used in this experiment was 1 x 10 5 ~1×10 8 The compositions were composed of a total cell count of 0.01 cells / ml, with >80% of the cells being M. capsulatus, and were prepared as listed in Example A. Water containing the appropriate adjuvant was used for soil application to control plants. To understand the effect of the methanotroph-based whole cell compositions on plant nutrient uptake, nitrogen, potassium, and phosphorus levels were analyzed. Samples taken from uniform sampling of plants were pooled. After drying, the samples were analyzed for nitrogen, potassium, and phosphorus levels according to standard protocols.

[0383] The results of the experiment are shown in Figure 3. As observed, plants treated with the methanotroph-based whole cell composition showed significantly improved uptake of N (nitrogen -36%), P (phosphorus -53%), and K (potassium -39%). The results / improved nutrient uptake further demonstrate the ability of the methanotroph-based whole cell composition to affect plant nutrient utilization efficiency under field conditions, leading to reduced fertilizer use.

[0384] Example 5 Application of methanotroph-based whole-cell compositions improved nutrient utilization efficiency in coriander To understand the effect of a methanotroph-based whole-cell composition on improving fertilizer utilization and biomass in coriander (Coriandrum sativum), a plot-based experiment using a completely randomized block design (RCBD) was designed. This experiment also served to understand whether the whole-cell composition reduced the need for external fertilizer addition. Plant populations, planting dates, harvest times, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole-cell composition. Fields were seeded with two different levels of nitrogen, phosphorus, and potassium. One set of the experiment applied the recommended amounts of nitrogen (18.5 kg / acre urea), phosphorus (30.4 kg / acre diammonium phosphate), and potassium (23.3 kg / acre potassium chloride) (100% NPK), while the second set applied only half the recommended amounts of nitrogen, phosphorus, and potassium (50% NPK). Two foliar applications were made to coriander at 20 and 30 days after sowing (DAS). The final total cell composition used in this experiment was 5 x 10 6 ~5×10 7 The composition contained a total cell count of cells / ml, with >70% of the cells being M. capsulatus, and was prepared as listed in Example A. Cells were diluted in water at a 500x dilution and sprayed on control plants. Plant biomass data was recorded at harvest. All observations were obtained from a uniform sampling of plants unless otherwise noted.

[0385] The results of the experiment are shown in Figure 4. As observed, plants treated with the methanotroph-based whole-cell composition showed a significant improvement in coriander biomass. Furthermore, the biomass increase observed in plants grown on 50% NPK was not only superior to the corresponding control (>50%), but also significantly higher than the 100% NPK control (42%). The described results further support the ability of methanotroph-based whole-cell compositions to deliver agriculturally relevant results by simultaneously improving yield and nutrient use efficiency (reduced fertilizer use). Use of the disclosed whole-cell composition shows significant improvements over both 50% and 100% NPK use. Indeed, importantly, the results demonstrate that including only 50% NPK results in better results with the whole-cell composition compared to the 100% NPK and whole-cell composition and the 100% NPK-only control plants. Therefore, these results are somewhat surprising, demonstrating that whole-cell compositions can not only increase plant yield but also reduce the need for NPK fertilizer. Furthermore, since this gas is used as the sole carbon source for producing the methanotroph-based whole cell composition, producing the methanotroph-based whole cell composition can also reduce methane levels or recover carbon.

[0386] Example 6 Effect of different proportions of whole cell components of gammaproteobacterial methanotrophs on improving rice seed germination To determine the optimal level of gammaproteobacterial methanotrophs in the whole cell composition of the present disclosure that can result in efficient seed germination, the following experiment was performed. The final whole cell composition used in this experiment contained a total of 1 x 10 5 ~1×10 8The optimal level of the gammaproteobacterial methanotroph-based whole-cell composition that could improve / increase seed germination efficiency was determined by varying the ratio of Methylococcus capsulatus to other bacteria (Table 3). Rice seeds were soaked overnight in the methanotroph-based whole-cell composition. Reference seeds were soaked in water. The ability of the optimal methanotroph-based whole-cell composition to improve germination efficiency was recorded. Fifteen seedlings from each of three different replicates were randomly sampled and used for data recording.

[0387] [Table 3]

[0388] Results from these experiments are shown in Figure 20. As observed, there was a direct correlation between the percentage of methanotrophic cell population and germination efficiency. Furthermore, the results demonstrate the importance of methanotrophic cells in whole cell-based biostimulant compositions in improving early seed germination.

[0389] Example 7 Application of a methanotroph-based whole cell composition improved chlorophyll levels in spinach A field trial experiment was designed to understand the effect of a methanotroph-based whole cell composition on chlorophyll levels in spinach (Spinachia oleracea). Growth conditions and application of the methanotroph-based whole cell composition were similar to those in Examples 1 and 4. Leaf chlorophyll was measured using a Soil Plant Analysis Development (SPAD) meter.

[0390] The results of the experiment are shown in Figure 5. As observed, plants treated with the methanotroph-based whole cell composition showed a significant improvement in the SPAD index (~32% increase compared to the control) and therefore better photosynthetic efficiency.

[0391] Example 8 Regulation of Nif gene expression in methanotrophs. To understand the ability of methanotrophs to regulate Nif gene (nitrogenase group) expression and thereby activate nitrogenase activity, cells were cultured in mineral salt medium lacking any nitrogen source and supplied with methane as a carbon source. Cells were harvested at 0 h, 30 min, 1 h, and 48 h and stored in a -80°C freezer until further analysis. Total RNA was extracted, and cDNA was synthesized from all samples. Quantitative real-time PCR (qPCR) was performed to check the expression of NifA, NifD, NifK, and NifH. The expression levels of all genes at 0 h were set as 1, and the relative expression levels at other time points were determined. Expression of the transcriptional regulator NifA showed biphasic expression, while there was a steady increase in the expression of NifD, NifK, and NifH.

[0392] Results from these experiments are shown in Figure 6. As observed, methanotrophs were able to grow in the absence of an external nitrogen source by regulating the expression of Nif genes and thus activating the nitrogenase machinery for fixing environmental nitrogen. This data supports the results seen in the field, where methanotroph-based whole-cell compositions can reduce the need for added external nitrogen. Nitrogen requirements may be reduced through a level of nitrogen fixation caused by Nif gene expression in methanotroph cells.

[0393] Example 9 Application of a methanotroph-based whole-cell composition improved yields of multiple plants compared to a commercial control containing nitrogen-fixing, phosphorus-solubilizing, and zinc-solubilizing bacteria. A plot-based experiment following a completely randomized block design (RCBD) was designed to understand the effect of a methanotroph-based whole-cell composition on pod yield of broad bean (Vicia faba) and biomass of coriander (Coriandrum sativum) compared with commercially available controls. Similar effects were also investigated for guar bean and spinach. Plant populations, planting dates, harvest times, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole-cell composition. For broad bean, three foliar applications were performed at 20, 40, and 55 days after sowing (DAS). The treatments for guar bean were the same as those mentioned in Example 2. For coriander, two foliar or soil applications were performed at 20 and 30 DAS. The treatment for spinach was the same as mentioned in Example 1 / 4. The final whole cell composition used in these experiments was prepared as in Example 1. >90% Methylococcus capsulatus cells were added at 1 × 10 5 ~1×10 8 The total cell count is expressed as cells / ml. The water vehicle was sprayed on the control plants. The leading commercial product was used as the commercial control. It is a carrier-based microbial consortium containing nitrogen-fixing, phosphate- and zinc-solubilizing bacteria along with plant growth-promoting microorganisms. The manufacturer's recommended amount was applied at each time point. Pods harvested at multiple pickings were measured to understand the overall yield improvement of broad beans. In the case of coriander, plant biomass data was recorded. Unless otherwise noted, all observations were obtained from uniform sampling of plants.

[0394] The results of the experiments are shown in Figures 7-10 and Table 4. As observed, plants treated with the methanotroph-based whole cell composition showed significant improvements in pod yield for faba bean and guar bean (Figures 7-8), and biomass for coriander and spinach (Figures 9, 10) compared to both the commercial product and the control. The described results regarding yield improvements further support the ability of the methanotroph-based whole cell composition to provide agriculturally relevant results compared to commercial products available in the market.

[0395] [Table 4]

[0396] Example 10 Application of the methanotroph-based whole cell composition improved yields compared to a commercial control containing methylotrophic bacteria. To understand the effect of a methanotroph-based whole-cell composition on the fruit yield of chili pepper (Capsicum annuum) and spinach (Spinachia oleracea) compared with commercially available controls, a plot trial experiment following a completely randomized block design (RCBD) was designed. Plant populations, fertilization, planting date, harvest time, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole-cell composition. For chili pepper, seedlings were transplanted into the field, and three foliar sprays were administered at 25, 40, and 60 days after transplanting (DAT). For spinach, the experimental design mentioned in Examples 1 and 4 was followed. The whole-cell composition in the final formulation containing adjuvants was prepared as listed in Example A. >90% Methylococcus capsulatus cells 5 × 10 6 ~5×10 7The total cell count was expressed as cells / ml. Control plants were sprayed with water containing the appropriate vehicle. A Methylobacterium-based microbial product (Pink pigmented facultative methylotroph (PPFM)) was used as a commercial control. The manufacturer's recommended dose was applied as a foliar spray at each time point. Fruit harvested at multiple pickings was measured to determine overall chili pepper yield improvement. For spinach, plant biomass data was recorded. All observations were obtained from uniform sampling of plants unless otherwise noted.

[0397] The results of the experiments are shown in Figures 11 and 12. As observed, plants treated with the methanotroph-based whole cell composition showed a significant improvement in chili pepper fruit yield (Figure 11) and spinach biomass (Figure 12) compared to both the commercial product and the reference. The described results regarding yield improvement further support the ability of the methanotroph-based whole cell composition to provide agriculturally relevant results compared to commercial products available on the market.

[0398] Example 11 Application of a methanotroph-based whole-cell composition improved early seedling establishment of rice. To understand the ability of methanotroph-based whole cell compositions to improve seedling establishment, the following experiment was performed. The whole cell compositions in the final formulation were prepared as listed in Example A and contained ∼2 × 10 5 ~2×10 8The concentration of methanotroph-based whole-cell compositions was 1000 cells / ml, with >70% of the cells being M. capsulatus. Rice seeds were soaked overnight in the methanotroph-based whole-cell composition, and then transferred to a Petri dish containing a germination sheet or moistened filter paper. Reference seeds were soaked in water. A Methylobacterium-based microbial product (pink-pigmented facultative methylotroph (PPFM)) was used as a commercial control. The manufacturer's recommended dose was used for seed treatment. The following experiment was conducted to examine the effect of the methanotroph-based whole-cell composition on improving seedling establishment. The concentration of methanotroph on early seedling establishment by improving root and shoot growth was recorded. Thirty seeds from three different replicates were randomly sampled and used to record data.

[0399] Results from these experiments are shown in Figure 13. As observed, seeds treated with the methanotroph-based whole cell composition showed a significant increase in root (-18%) and shoot length (-10%) compared to seeds treated with the commercial control. The results further demonstrate the ability of the methanotroph-based whole cell composition to aid in early seedling establishment by regulating root and shoot growth.

[0400] Example 12 Application of methanotroph-based whole cell compositions improved carrot yield To understand the effect of a methanotroph-based whole cell composition on the yield of carrot (Daucus carota) taproot, a plot trial experiment following a completely randomized block design (RCBD) was designed. Plant populations, fertilization, planting dates, harvest times, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole cell composition. A single foliar spray was administered to carrots 30 DAS later. The final whole cell composition used in this experiment was prepared as listed in Example A and contained approximately 1 x 10 5 ~1×10 8The concentration of M. capsulatus was determined by the concentration of M. capsulatus cells / ml, with >90% of the cells being M. capsulatus. Control plants were sprayed with water containing the appropriate vehicle. Taproot weights were recorded to determine overall yield improvement. Unless otherwise noted, all observations were obtained from a uniform sampling of plants under identical conditions.

[0401] The results of the experiment are shown in Figure 14. As observed, the carrot yield improvement was -7-10%. The described results on carrot yield improvement further support the ability of methanotroph-based whole cell compositions to produce agriculturally relevant results such as improved taproot growth in field conditions.

[0402] Example 13 Application of methanotroph-based whole-cell compositions improved spinach biomass yield under hydroponic conditions. A hydroponic-based experiment was designed to understand the effect of a methanotroph-based whole-cell composition on improving the yield of spinach (Spinachia oleracea). Seed rate, nutrient application, planting date, harvest time, and other standard management practices were consistent with local agricultural practices, except for the application of the methanotroph-based whole-cell composition. The first foliar application of the methanotroph-based whole-cell composition to spinach plants was performed 15 days after sowing, followed by a second application at an interval of 10 days. The final whole-cell composition used in this experiment was prepared as listed in Example A and contained ~5 x 10 7 ~5×10 8 The cells / ml contained >90% Methylococcus capsulatus cells. Water containing the appropriate adjuvant was used for both foliar spray and soil application to control plants. Plants were harvested 40-45 days after sowing. Aboveground biomass was used to determine yield improvement. Unless otherwise noted, all observations were obtained from a uniform sampling of plants under identical conditions.

[0403] The results of the experiment are shown in Figure 15. As observed, foliar application of the methanotroph-based whole cell composition on spinach showed a significant improvement in crop biomass of -43% compared to control plants that received water spray. The described results on improved yield in spinach further support the ability of methanotroph-based whole cell compositions to produce agriculturally relevant results such as improved yield even under hydroponic conditions.

[0404] Example 14 Application of methanotroph-based whole-cell compositions improved dietary fiber and protein A field trial experiment was designed to understand the effect of a methanotroph-based whole-cell composition on dietary fiber and protein in spinach (Spinachia oleracea). Growth conditions and application of the methanotroph-based whole-cell composition were similar to those in Examples 1 and 4. A commercial control containing the carrier-based microbial consortium used in the previous examples at the recommended dose was applied at the appropriate time. Leaf dietary fiber and total protein were measured according to standard protocols previously reported.

[0405] The results of the experiment are shown in Figure 16. As observed, plants treated with the methanotroph-based whole cell composition showed a significant increase in dietary fiber (-20%) and protein (-44%), thus indicating better crop quality.

[0406] Example 15 Application of methanotroph-based whole cell compositions in spinach Spinachia oleracea plantlets were grown in coco peat and supplemented with the required amounts of nitrogen, phosphorus, and potassium. One week after transplanting (i.e., 17 days after sowing), in a separate experiment, the first foliar spray or soil application of a methanotroph-based whole-cell composition with an agronomically acceptable excipient (adjuvant) was performed. Thereafter, foliar sprays or soil applications were performed every seven days. No additional methane was supplemented for the growth of methanotrophs in the composition. The whole-cell composition in the final formulation containing adjuvant contained approximately 1 × 10 5~1×10 8 The compositions consisted of 1000 cells / ml, with >80% of the cells being M. capsulatus, and were prepared as listed in Example A. Water containing the appropriate adjuvant was used as a control for both foliar and soil applications. Plants were harvested 45 days after sowing. Plants were measured for biomass yield and other morphological characteristics. Total shoot biomass was collected and recorded 45 days after sowing. Results shown are from 5-10 biological replicates, and the effects in plants treated with the compositions were compared to the respective controls. Student's t-test: * P<0.05. Error bars indicate mean ± SE.

[0407] The results of the experiment are shown in Figure 17. As observed, plants treated with the methanotroph-based whole cell composition showed a significant increase in shoot biomass compared to the control. The results / improvement in shoot biomass further demonstrate the ability of the methanotroph-based whole cell composition of the present invention to fix atmospheric nitrogen for uptake by the plant and utilize atmospheric methane as an energy and metabolic source to enhance / promote plant growth or performance.

[0408] Example 16 Application of methanotroph-based whole cell compositions in radish Raphanus sativus var. Longipinnatus (radish) seeds were grown in coco peat and supplemented with required amounts of nitrogen, phosphorus, and potassium. 20 days after germination, in separate experiments, methanotroph-based whole-cell compositions were applied as foliar sprays or soil applications with agriculturally acceptable excipients (adjuvants). The whole-cell compositions in the final formulations containing adjuvants were prepared as listed in Example A and contained approximately 1 x 10 5 ~1×10 8The concentration of the composition was composed of cells / ml, with >80% of the cells being M. capsulatus. Water containing the appropriate adjuvant was used as a control for both foliar spray and soil application. Plants were harvested 50 days after sowing. Plants were measured for biomass yield and other morphological characteristics. Fresh root and root-shoot biomass were collected and recorded 50 days after sowing. Results shown are from five biological replicates, and the effects in plants treated with the composition were compared to the respective controls. Student's t-test: ** P < 0.01; *** P<0.001. Error bars indicate mean ± SE.

[0409] The results of the experiment are shown in Figure 18. As observed, plants treated with the methanotroph-based whole cell composition showed a significant increase in root and shoot biomass compared to the control. The results / improvement in shoot biomass further demonstrate the ability of the methanotroph-based whole cell composition of the present invention to fix atmospheric nitrogen for uptake by the plant and utilize atmospheric methane as an energy and metabolic source to enhance / promote plant growth or performance.

[0410] Example 17 Application of methanotroph-based whole cell compositions in tomatoes Ten-day-old tomato seedlings were transferred to coco peat and supplemented with required amounts of nitrogen, phosphorus, and potassium. Ten days after transfer, the methanotroph-based whole cell composition with an agriculturally acceptable excipient (adjuvant) was applied as a first foliar spray or soil application, followed by two more applications. The whole cell composition in the final formulation containing the adjuvant was prepared as listed in Example A and contained approximately 1 x 10 5 ~1×10 8The concentration of the composition was comprised of cells / ml, with >90% of the cells being M. capsulatus. Water containing the appropriate adjuvant was used as a control for both foliar and soil applications. Sixty days after transplanting, plants were measured for fruit number and other morphological characteristics. Two months after transplanting, the number of mature fruits was recorded. Results shown are from 5-10 biological replicates, and the effects in plants treated with the composition were compared to the respective controls. Student's t-test: *** P<0.001. Error bars indicate mean ± SE.

[0411] The results of the experiment are shown in Figure 19. As observed, plants treated with the methanotroph-based whole cell composition exhibited a significantly greater number of mature fruits compared to the control. The results / improvement in shoot biomass further demonstrate the ability of the methanotroph-based whole cell composition of the present invention to fix atmospheric nitrogen for uptake by the plant and utilize atmospheric methane as an energy and metabolic source, enhancing / promoting plant growth / performance and fruit yield.

[0412] The foregoing description of specific embodiments reveals the general nature of the embodiments herein, such that others, by applying their current knowledge, can readily modify and / or adapt such specific embodiments for various uses without departing from the general concept; therefore, such adaptations and modifications should, and are intended to, be understood within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the phraseology and terminology used herein are for purposes of description and not of limitation. Thus, while the embodiments of the present disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments of the present disclosure can be practiced with modification within the spirit and scope of the embodiments described herein.

[0413] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising" or "including", wherever used, will be understood to mean the inclusion of the stated element, integer or step, or group of elements, integers or steps, and not the exclusion of other elements, integers or steps, or groups of elements, integers or steps.

[0414] Throughout this specification, the terms "combinations thereof" or "any combination thereof" or "any combinations thereof" are used interchangeably and are intended to have the same meaning, as commonly known in the art of patent disclosure.

[0415] As used herein, the term "comprising," when placed before a description of steps in a method, means that the method encompasses one or more steps in addition to the explicitly recited steps, and that the additional step or steps may occur before, between, and / or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c; a method of steps a, b, c, and x; and a method of steps x, a, b, and c. Furthermore, the term "comprising," when placed before a description of method steps, does not (and may) require sequential performance of the recited steps, unless the context clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses methods of performing the steps in the order of a, c, and b; c, b, and a; and c, a, and b, etc.

[0416] As used herein and in the appended claims, the singular forms "a," "an," and "the" include both singular and plural references unless the context clearly dictates otherwise. For example, the term "inserted at a position," as used herein with respect to a polypeptide sequence, refers to insertion at one or more (1, 2, 3, etc.) amino acid positions in the polypeptide sequence. The use of the phrase "at least" or "at least one" suggests the use of one or more elements or components or amounts, which may be for use in embodiments of the disclosure that achieve one or more desired purposes or results. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0417] With respect to the use of virtually any plural and / or singular term herein, one of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be explicitly stated herein for clarity. The suffix "(s)" at the end of any term in this disclosure contemplates both the singular and plural forms of said term within its scope.

[0418] Numerical ranges stated in the format "from x to y" include the stated values and values within the respective measurement accuracy known to those skilled in the art. When several preferred numerical ranges are stated in this format, it goes without saying that all ranges formed by combining the different endpoints are also included.

[0419] The terms "about" or "approximately," as used herein in reference to measurable values such as parameters, amounts, temporal durations, and the like, are meant to encompass variations of the specified value and variations from the specified value, such as variations of no more than + / - 10%, no more than + / - 5%, no more than + / - 1%, and no more than + / - 0.1% of the specified value, to the extent that such variations are appropriate for practice in the disclosed invention. It should be understood that the value to which the modifier "about" or "about" refers is itself specifically, and preferably, disclosed.

[0420] As used herein, "include" (any form of "include", such as "include"), "have" (and "have"), any form of "having", such as "comprise", "including" (and any form of "including", such as "including"), "containing", "comprising" or "comprises" will be understood to be inclusive and mean the inclusion of a specified element, integer or step or group of elements, integers or steps, but not the exclusion of other elements, integers or steps or groups of elements, integers or steps.

[0421] With regard to the embodiments characterized herein, each embodiment is intended to be read not only independently but also in combination with other embodiments. For example, if embodiment 1 describes three options A, B, and C, embodiment 2 describes three options D, E, and F, and embodiment 3 describes three options G, H, and I, it should be understood that the specification unambiguously discloses embodiments corresponding to the combinations of A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I;

[0422] Any discussion of documents, acts, materials, devices, articles and the like which has been included in the present specification is for the purpose of providing a context for the present disclosure only and is not to be construed as an admission that any or all of these matters form part of the prior art body or were common general knowledge in the art relevant to the present disclosure as existing anywhere prior to the priority date of this application.

Claims

1. A biostimulant composition comprising a microbial consortium of whole cells, said consortium comprising at least 50% whole cells of Gammaproteobacteria methanotrophs.

2. 2. The biostimulant composition of claim 1, wherein the microbial consortium comprises at least about 60% to about 100% whole cells of Gammaproteobacteria methanotrophs.

3. 2. The biostimulant composition of claim 1, wherein the gammaproteobacterial methanotroph is a type I or type X methanotroph belonging to a genus selected from the group consisting of Methylococcus, Methylomonas, Methylobacter, Methyloglobulus, Methyloblum, Methylomicrobium, Methylsarcinia, Methylosphaera, Methyloprophus, Methylosoma, Methyloccumis, Methylocardum, Methyloparacox, Methylogaea, Methylomagnum, Methylotelicola, Methylothermus, Methylohalobius, Methylomarinobum, Methylomarinum and Crenothrix, or any combination thereof.

4. 2. The biostimulant composition of claim 1, wherein the gammaproteobacterial methanotroph is Methylococcus capsulatus.

5. 2. The biostimulant composition of claim 1, wherein the composition is in solid or liquid form and comprises at least one metabolite, at least one nutrient derived from the culture medium, and optionally at least one agriculturally acceptable excipient.

6. The microbial consortium of whole cells is about 1 x 10 per gram or per milliliter of the composition. ３ cells to approximately 5 x 10 １０ 6. The biostimulant composition of claim 5, comprising about 0.1% to about 80% of said composition, with the remainder of said composition consisting of about 0.1% to about 10% of at least one metabolite, about 0.1% to about 10% of at least one medium-derived nutrient, and optionally about 0.01% to about 90% of at least one agriculturally acceptable excipient.

7. the metabolites are selected from the group comprising carbohydrates, lipids, sugars, fatty acids, proteins, peptides, amino acids, nucleic acids, nucleotides, vitamins, organic acids, salts, minerals, osmolytes, extracellular enzymes, bacterial-derived components and minerals, or any combination thereof; the nutrients from the medium are selected from the group comprising ions and salts, or a combination thereof; 6. The biostimulant composition of claim 5, wherein the agriculturally acceptable excipient is selected from the group comprising carriers, cell protectants, adjuvants, surfactants, stabilizers, preservatives, diluents, suspending agents, dispersing agents and co-solvents, or any combination thereof.

8. 2. The biostimulant composition of claim 1, wherein in addition to the Gammaproteobacteria methanotrophs, the consortium comprises from about 1% to about 50% of at least one plant growth-promoting microorganism selected from the group consisting of nitrogen-fixing microorganisms, phosphorus-dissolving microorganisms, mineral-dissolving microorganisms, plant hormone-secreting microorganisms, organic acid-secreting bacteria, and plant beneficial microorganisms, or any combination thereof.

9. wherein the consortium in the composition utilizes methane, and the composition comprises: a. improving or enhancing the performance of the plant; b. increasing the availability or efficient use of at least one of nitrogen, phosphorus, and potassium by the plant; c. reducing the need for external addition of at least one nutrient selected from nitrogen, phosphorus and potassium, either individually or as part of a fertilizer; or d. Any combination of a. to c. The biostimulant composition of claim 1.

10. a) Improve or enhance the performance of plants b) simultaneously promoting methane utilization and nitrogen fixation in plants; or c) reducing the need for externally added nutrients and / or nutrient-containing fertilizers for plant growth, development, performance, or survival; 1. A method, comprising: growing a plant using a nutrient selected from the group consisting of nitrogen, phosphorus, potassium, and any combination thereof; 10. The method, comprising contacting or applying to the plant the biostimulant composition of claim 1.

11. Improving or enhancing the performance of the plant comprises stimulating or promoting a quantitative or qualitative plant attribute selected from the group including biomass production, yield, photosynthetic activity, nutritional value, secondary metabolites and nutrient utilization efficiency, or any combination thereof; or The effect of the improved or enhanced plant performance is an increase in the number, size or quality of below-ground or above-ground biomass selected from the group including roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits and seeds or any combination thereof; b. Increased photosynthetic activity or chlorophyll content; c. Increased protein, dietary fiber, beta-carotene or essential oil content, or any combination thereof; d. Efficient absorption or utilization of available or exogenously supplied nutrients or minerals; or Any combination of e. a. to d. The method of claim 10, wherein the measurement is performed by

12. the performance of the plant is improved or enhanced through increased nitrogen fixation in the plant resulting in at least one of increased availability of nitrogen to the plant or efficient utilization of nitrogen by the plant; or The plant performance is improved or enhanced through increased expression of nitrogenase genes selected from the group comprising nifA, nifD, nifH and nifK, or any combination thereof, in the whole cells of the microorganism present in the biostimulant composition; or 11. The method of claim 10, wherein the plant performance is simultaneously improved or enhanced with methane utilization by the consortium comprising at least 50% whole cells of Gammaproteobacteria methanotrophs.

13. The biostimulant composition is in solid or liquid form and is contacted with or applied to the plant at a concentration ranging from about 1× to 100,000× the dilution of the solid or liquid form of the biostimulant composition; or 11. The method of claim 10, wherein the biostimulant composition contacts or is applied to the plant through its soil or through above-ground or non-aerial parts of the plant selected from the group including roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits and seeds, or any combination thereof.

14. 11. The method of claim 10, wherein the biostimulant composition improves or enhances the performance of the plant by about 1% to about 500% or about 1.5 times to about 10 times compared to the respective performance of a plant not contacted with the biostimulant composition of claim 1.

15. Increase nitrogen fixation in the plant resulting in either increased availability of nitrogen to the plant or more efficient utilization of nitrogen by the plant, or both; or 11. The method of claim 10, wherein the nitrogen fixation is promoted by increasing the expression of nitrogenase genes selected from the group consisting of nifA, nifD, nifH and nifK, or any combination thereof, in the whole cells of the microorganisms present in the biostimulant composition.

16. 11. The method of claim 10, wherein the methane utilization by the consortium comprising at least 50% whole cells of gammaproteobacteria methanotrophs recycles at least about 0.1 kg of methane per kg of biostimulant composition used.

17. 11. The method of claim 10, wherein the need for exogenous addition of at least one of nitrogen, nitrogen-containing fertilizer, phosphorus, phosphorus-containing fertilizer, potassium, and potassium-containing fertilizer for the growth, development, performance, or survival of the plant is reduced by at least about 10% to about 100% compared to the need for addition of nitrogen or nitrogen-containing fertilizer in a plant not contacted with the biostimulant composition of claim 1.

18. 11. The method of claim 10, wherein the composition improves or enhances the performance of the plant by either increasing the availability or efficient utilization of at least one of nitrogen, phosphorus, and potassium by the plant, or both.

19. A method for preparing the biostimulant composition of claim 1, comprising combining a consortium comprising at least 50% whole cells of gammaproteobacteria methanotrophs with at least one metabolite and nutrient from the medium, and optionally at least one agriculturally acceptable excipient.

20. 1. A biostimulant product comprising: a. the biostimulant composition of claim 1, and b. A hydrolysate-based biostimulant composition comprising a protein-derived component in an amount of about 30% or less by weight of said composition. Includes A biostimulant product, wherein the protein-derived component is obtained from a methanotrophic bacterium.

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