Microbial inoculant composite material

Abstract

A microbial inoculant composite material for use in agriculture, forestry or horticulture, comprising one or more microbial inoculants and a moisture-sorption component The moisture-sorption component is in the form of a powder, granules, capsules, or pellets, and is comprised of a microbial cell extract and a moisture-sorption particulate material comprising at least 5 wt.% smectite clay mineral. The clay in the moisture-sorption component forms layered stacks and the microbial cell extract is intercalated between individual clay layers in the stacks.

Description

[0001] MICROBIAL INOCULANT COMPOSITE MATERIAL

[0002] TECHNICAL FIELD

[0003] The present disclosure is related to a microbial inoculant composite material for use in agriculture, forestry or horticulture.

[0004] BACKGROUND ART

[0005] Plant growth-promoting and crop-protecting microbial inoculants, commonly referred to as biopesticides and biofertilizers, or collectively as "biologicals", offer a sustainable alternative to synthetic chemical pesticides and fertilizers. These inoculants enhance fertilizer-use efficiency, leading to higher crop yields. For instance, strains of Bacillus aryabhattai have demonstrated the ability to increase phosphate bioavailability by exuding organic acids that liberate phosphate from recalcitrant phosphate salts. Other microbes may produce metabolites that antagonize fungi or deter insects.

[0006] Microbial inoculant products are typically applied as dormant spores directly to seeds, soil, or as foliar sprays, depending on the intended function and required management intervention. Regardless of the mode of action— whether through the exudation of secondary metabolites, exoenzymes, or peptides— the spores must first enter active growth and maintain a viable population to be effective.

[0007] However, the field performance of microbial inoculants is often inconsistent. This inconsistency is not well understood, leading to a mismatch between farmer expectations and actual results. Consequently, adoption rates are lower than potential, limiting the positive environmental impact of this technology.

[0008] The soil microbiome is a complex and dynamic ecosystem influenced by factors such as soil moisture, organic content, temperature, pH, and plant hosts. Applied microbial inoculants are also sensitive to these conditions. Under desiccating conditions or in soils with low organic content, the microbes may not achieve the viable population needed for effectiveness.

[0009] Therefore, techniques are needed to enhance the performance of microbial inoculants, ensuring more consistent and effective results across varying environmental conditions.SUMMARY OF THE INVENTION

[0010] It is an object of the present disclosure to provide a microbial inoculant composite material for use in agriculture, forestry or horticulture and that result in a more consistent and effective performance of the microbial inoculant under varying environmental conditions.

[0011] The invention is defined by the appended independent patent claims. Non-limiting embodiments emerge from independent claims, the appended drawings and the following description.

[0012] According to a first aspect, there is provided a microbial inoculant composite material for use in agriculture, forestry or horticulture, comprising one or more microbial inoculants and a moisture-sorption component, wherein the moisture-sorption component is in the form of a powder, granules, capsules, or pellets, and wherein the moisture-sorption component is comprised of a microbial cell extract and a moisture-sorption particulate material comprising at least 5 wt.% smectite clay mineral, wherein the clay in the moisture-sorption component forms layered stacks and the microbial cell extract is intercalated between individual clay layers in the stacks.

[0013] The moisture-sorption component improves the efficacy of the microbial inoculant. The moisture-sorption component functions as a source of nutrition (from the microbial cell extract) and a moisture reservoir (the moisture-sorption component particulate material comprising clay) to the microbial inoculant. With this composite material it is possible to increase the time window for the microbe to experience optimal temperatures in the soil. This may increase the robustness and reliability of the microbial inoculant(s) leading to a more cost-effective and sustainable management tool for us in agriculture, forestry or horticulture. The composition offers a pH-optimal microsite for the microbial inoculant to enter log phase and establish a viable population. In the event of low, limiting soil temperature, as can be the case when seed is sown in spring, the enduring moisture reservoir and organic nutrition increase the probability that the microbe will maintain its potential for growth until concomitant soil temperature permits a viable population to be established.At least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, or at least 90 wt.% of the microbial inoculant composition, based on dry weight, may consist of the microbial inoculant and the moisture-sorption component.

[0014] A weight ratio of moisture-sorption component to microbial inoculant component in the microbial inoculant composition may be 10,000:1 to 1:1.

[0015] Apart from the microbial inoculant and moisture-sorption component, the microbial inoculant composition may comprise e.g. binders or other substances such as benign inactive ballast.

[0016] The moisture-sorption component may consist to at least 95 wt.%, based on dry weight, of microbial cell extract and moisture-sorption particulate material.

[0017] The moisture-sorption component may consist to at least 95 wt.%, based on dry weight, of microbial cell extract and clay.

[0018] 1 to 90 wt.%, based on dry weight, of the moisture-sorption component in the microbial inoculant composite material may consist of clay.

[0019] The clay comprises at least 5 wt.%, based on dry weight, smectite clay mineral.

[0020] The smectite clay mineral may be montmorillonite, nontronite, saponite, hectorite or any combination thereof. Bentonite clay, which is dominated by montmorillonite, may be used.

[0021] The clay in the moisture-sorption component forms layered stacks and the microbial cell extract is intercalated between individual clay layers in the stacks. The clay may have an average basal spacing (dOOl) between 1.2 nm and 4.1 nm.

[0022] In one embodiment, the moisture-sorption particulate material may comprise clay and a superabsorbent hydrogel.The moisture-sorption component may consist to at least 95 wt.% of the microbial cell extract, the clay and the superabsorbent hydrogel.

[0023] 1-90 wt.%, based on dry weight, of the moisture-sorption component may consist of clay and superabsorbent hydrogel.

[0024] A weight ratio of clay to superabsorbent hydrogel in the moisture-sorption component may be 10:1 to 1:10.

[0025] In the moisture-sorption component, the superabsorbent hydrogel may be mixed with the clay.

[0026] The superabsorbent hydrogel may be selected from one or more of cellulose-based hydrogels, starch-based hydrogels, chitosan-based hydrogels, alginate-based hydrogels, gelatine-based hydrogels, poly(acrylamide-co-acrylate) and polyvinyl alcohol-based hydrogels.

[0027] The microbial cell extract may be yeast extract.

[0028] The microbial inoculant may be selected from biofertilizers, biopesticides, microbial agents, beneficial microbes, bioinoculants, microbial consortia and soil probiotics or any combination thereof.

[0029] The microbial inoculant may be selected from nitrogen-fixing bacteria, phosphate-solubilizing microorganisms, mycorrhizal fungi, plant growth-promoting rhizobacteria, biocontrol agents, organic matter decomposers, protozoa and nematodes or any combination thereof.

[0030] The microbial inoculant composite material may be in the form of a powder, granules, capsules, liquid, suspension, slurry, pellets or any combination thereof.

[0031] According to a second aspect there is provided a method of producing a microbial inoculant composite material for use in agriculture, forestry or horticulture, comprising:

[0032] providing one or more microbial inoculant,providing a moisture-sorption component, wherein the moisture-sorption component is in the form of a powder, granules, capsules, or pellets and is comprised of a microbial cell extract and a moisture-sorption particulate material comprising a clay which comprises at least 5 wt.% smectite clay mineral or a mixture of said clay with a superabsorbent hydrogel, wherein providing said moisture-sorption component comprises the steps of: pre-swelling said clay to exfoliate its layers, subsequently mixing the pre-swelled clay, optionally also comprising the superabsorbent hydrogel, with the microbial cell extract to intercalate the cell extract between the exfoliated clay layers, and drying the mixture; and

[0033] mixing the moisture-sorption component to microbial inoculant in a ratio of 10,000:1 to 1:1.

[0034] According to a third aspect there is provided a use of the microbial inoculant composite material described above as a soil fertility enhancer, a plant growth promoter, a disease resistance agent, a stress tolerance improver, in agriculture, forestry or horticulture.

[0035] In addition the microbial inoculant composite material may be used to reduce the use of different chemicals in agriculture, forestry or horticulture

[0036] According to a fourth aspect there is provided a method of applying a microbial inoculant composite material to soil, plant or seed, comprising:

[0037] providing one or more microbial inoculants,

[0038] providing a moisture-sorption component according to the first aspect, ,

[0039] applying the moisture-sorption component and the microbial inoculant(s) to said soil, plant or seed in a weight ratio of 10,000:1 to 1:1.

[0040] The microbial inoculant and the moisture-sorption component may be mixed prior to application to the soil, plant or seed; mixed simultaneously as applied to the soil, plant or seed, or may be applied in sequence to the soil, plant or seed.

[0041] The microbial inoculant composite material may be coated on at least a portion of a seed.

[0042] The microbial inoculant composite material may be applied in an amount of 1-100 kg / 10000 m2soil.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Fig. 1 schematically illustrates how the microbial cell extract is intercalated between layers of clay in a moisture-sorption component.

[0044] Fig. 2 shows a graph of sorption-desorption isotherms for a moisture-sorption component comprising intercalated yeast extract between montmorillonite clay layers compared to a sandy clay loam agricultural soil.

[0045] Fig. 3 shows a graph of sorption-desorption isotherms for a moisture-sorption component comprising intercalated yeast extract between montmorillonite clay layers compared to a montmorillonite-rich clay sample.

[0046] Fig. 4 schematically illustrates the major steps of a method for producing a microbial inoculant composite material for use in agriculture, forestry or horticulture.

[0047] Fig. 5 schematically illustrates the major steps of a method of applying a microbial inoculant composite material to soil, plant or seed.

[0048] Fig. 6 shows powder X-Ray Diffraction (XRD) data comparing a moisture-sorption component prepared according to an embodiment of the present disclosure (top curve) with a comparative macroscopic hydrogel composition prepared according to a comparative method (bottom curve).

[0049] DETAILED DESCRIPTION

[0050] Below is described in more detail a microbial inoculant composite material for use in agriculture, forestry or horticulture and its method of production and use.

[0051] The microbial inoculant composite material is comprised of one or more microbial inoculants and a moisture-sorption component, wherein the moisture-sorption component is comprised of a microbial cell extract, such as yeast extract, and a moisture-sorption particulate material comprising clay. The microbial inoculant composite material may be in the form of a powder, granules, capsules, liquid, suspension, slurry, pellets or any combination thereof. Themoisture-sorption component may for example be in the form of a powder, granules, capsules, or pellets. The microbial inoculant may be in be in the form of a powder, granules, capsules, pellets or in liquid form.

[0052] That the moisture-sorption component is comprised of a microbial cell extract and a moisturesorption particulate material comprising clay means that the microbial cell extract may be uniformly distributed throughout / blended with the moisture-sorption component particulate material comprising clay. The microbial cell extract may be mixed with the moisture-sorption component particulate material comprising clay in a non-uniform manner, creating regions with higher or lower concentrations of microbial cell extract. The microbial cell extract may be encapsulated within cavities formed in the moisture-sorption component particulate material comprising clay. Alternatively, the microbial cell extract may be layered between sheets of moisture-sorption component particulate material comprising clay, creating a stratified / sandwiched structure.

[0053] The microbial inoculant may be biofertilizers, biopesticides, microbial agents, beneficial microbes, bioinoculants, microbial consortia and soil probiotics or any combination thereof and may be selected from nitrogen-fixing bacteria, phosphate-solubilizing microorganisms, mycorrhizal fungi, plant growth-promoting rhizobacteria, biocontrol agents, organic matter decomposers, protozoa and nematodes or any combination thereof.

[0054] The moisture-sorption component can improve the efficacy of the microbial inoculant when applied to soil, plant or seed. The moisture-sorption component functions as a source of nutrition (from the microbial cell extract) and a moisture reservoir (the moisture-sorption component particulate material comprising clay) to the microbial inoculant. Using the composite material it is possible to increase the time window for the microbe to experience optimal temperatures in the soil. This may increase the robustness and reliability of the microbial inoculant(s) leading to a more cost-effective and sustainable management tool for us in agriculture, forestry or horticulture. The composition offers a pH-optimal microsite for the microbial inoculant to enter log phase and establish a viable population. In the event of low, limiting soil temperature, as can be the case when crops are sown in spring, the enduring moisture reservoir and organic nutrition increase the probability that the microbe willmaintain its potential for growth until concomitant soil temperature permits a viable population to be established.

[0055] A weight ratio of moisture-sorption component to microbial inoculant may be 10,000:1 to 1:1, or 5,000:1 to 1:1 or 4,000:1 to 1:1, or 3,000:1 to 1:1, or 2,000:1 to 1:1, or 1,000:1 to 1:1, or 900:1 to 1:1, or 800:1 to 1:1, or 700:1 to 1:1, or 600:1 to 1:1, or 500:1 to 1:1, or 400:1 to 1:1, or 300:1 to 1:1, or 200:1 to 1:1, or 100:1 to 1:1, or 50:1 to 1:1, or 10:1 to 1:1, or 1,000:1 to 10:1, or 1,000:1 to 50:1, or 1,000:1 to 100:1, or 1,000:1 to 200:1, or 1,000:1 to 300:1, or 1,000:1 to 400:1, or 1,000:1 to 500:1, or 1,000:1 to 600:1, or 1,000:1 to 700:1, or 1,000:1 to 800:1, or 1,000:1 to 900:1, or 200:1 to 800:1, or 300:1 to 700:1, or 400:1 to 600:1.

[0056] In one example, for a granule application rate of 1-30 kg / 10,000 m2of granules, the soil may be inoculated with a beneficial microbe by spraying a liquid preparation as described by the manufacturer (1.2xl012CFU / L) at rate in the order of 1-5 L / 10,000 m2.

[0057] The moisture-sorption component may be comprised to at least 95 wt.%, based on dry weight, of microbial cell extract and clay, or at least 96 wt.%, at least 97 wt.%, at least 98 wt.% or at least 99 wt.%. The rest may be e.g. binders or other substances such as benign inactive ballast.

[0058] The microbial cell extract may comprise constituents that are more or less ionic and may comprise constituents that are ionic enough to interact with clay minerals. Microbial cell extract, such as yeast extract, is a complex mixture of the cell contents of microbial cells, such as yeast cells, including proteins, peptides, amino acids, metabolites, ions, carbohydrates and lipids. It comprises a complex mixture of cations, anions and neutral molecules in both inorganic and organic form. The large majority of the nitrogen is in the form of free amino acids and peptides, which carry both positive and negative charge depending on the different pKa values of the different organic molecules, as well as pH. In turn, the profile of release of the constituent components of yeast extract from the particles will depend on charge and size.

[0059] 1 to 90 wt.%, 1 to 80 wt.%, or 1-70 wt.%, or 1-60 wt.%, or 1-50 wt.%, or 1-40 wt.%, or 1-30 wt.%, or 10 to 90 wt.%, or 20 to 90 wt.%, or 30 to 90 wt.%, or 40 to 90 wt.%, or 50 to 90 wt.%, or 60 to 90 wt.%, or 70 to 90 wt.%, or 80 to 90 wt.%, or 20 to 80 wt.%, or 30 to 70 wt.%, or 30-50 wt.%, or 40-50 wt.% or 30-40 wt.% of the moisture-sorption component in the microbial inoculant composite material may consist of clay, based on dry weight. A ratio of microbial cell extract to clay in the moisture-sorption component may be 1:10-1:1, 1:10-1:2, 1:10-1:3, 1:10-1:4, 1:10-1:5, 1:10-1:6, 1:10-1:7, 1:10-1:8, 1:10-1:9, 1:9-1:2, 1:8-1:2, 1:7-1:2, 1:6-1:2, 1:5-1:2, 1:4-1:2, or 1:3-1:2. In one embodiment, the moisture-sorption component is comprised to at least 95 wt.%, based on dry weight, of microbial cell extract and clay, and 20-50 wt.% of the moisture-sorption component is clay.

[0060] Clay is a naturally occurring and desirable component of agricultural soil due to its water and nutrient holding capacity. The clay may comprise at least 5 wt.%, or at least 10 wt.%, 20 wt.%, 30 wt.%, 40 wt.%, 50 wt.%, 60 wt.%, 70 wt.%, 80 wt.% or 90 wt.%, or 5-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100%, based on dry weight, smectite clay mineral. The rest of the clay component may be constituted by nonsmectite accessory minerals commonly found in clays, such as silicates, magnesium oxides, aluminium oxides, manganese oxides, iron-oxides, sand and / or silt. The smectite clay mineral may be montmorillonite, nontronite, saponite, hectorite or any combination thereof.

[0061] Bentonite clay, which is dominated by montmorillonite, may be used.

[0062] Smectite-type clays have a layered structure, the clay component forms layered stacks in the moisture-sorption component and at least a portion of the microbial cell extract is intercalated or interposed between individual clay layers in the stacks. The microbial cell extract may also be arranged on the edge of clay layers, on the outer surface of the stacked clay layers etc. The degree of stacking of the clay layers, i.e. the number of stacks and number of layers per stack, can be determined by means of transmission electron microscopy and / or XRD (X-Ray Diffraction Analysis).

[0063] The stacking of clay layers may be caused by electrostatic forces along with Van der Waals forces.

[0064] Clay layers may be stacked into primary particles; primary particles may be stacked together to form larger particles. This may be caused by ionic forces along with van der Waals forces. The particle may have any shape, such as spherical-, platelet- or cylinder-shape, and any size such as being on the nanometre, micrometre, millimetre or centimetre scale, and is suitablefor being provided at or close to a plant. In one example, the moisture-sorption component may have an average diameter of 0.01-4 mm. The moisture-sorption component may be comprised solely of clay and microbial cell extract or 80-100 wt.%, based on dry weight, of the component may be comprised of clay and microbial cell extract. The rest may for example be a coating layer, a binder, etc.

[0065] In the moisture-sorption component a majority, or at least a portion, of the clay layers may not be arranged in stacks, i.e. non-aggregated (dispersed) clay layers, and present as single entities or with edge to face ionic association (house of cards structure) or as no more than two clay layers with face to face contact where the inter-layer space is absent of microbial cell extract. This house of cards structure may, however, mostly be present in wet / hydrated particles, while this structure might collapse when the particle is dried.

[0066] The number of stacks in the moisture-sorption component is dependent on e.g. the amount of clay and the size of the particle. There may be a varying number of individual clay layers (also called tactoid / aggregate) in a stack, such as at least 2 up to 100 clay layers or more. The stacks of clay layers in the moisture-sorption component may be disorganized, bent, and vary in size. The degree of stacking of the clay layers / platelets, i.e. the number of layers per stack, can be determined by means of transmission electron microscopy and / or XRD.

[0067] Each individual clay layer may have a thickness of about 1 nm and the other two dimensions being about 30-1000 nm. An average distance between the layers of the clay in a stack in the composition may be of at least 1.0 nm, or at least 3 nm. These thicknesses and distances may, however, deviate from the above and depend on the microbial cell extract used, and on the amounts and ratios of clay and microbial cell extract in the moisture-sorption component.

[0068] Fig. 1 schematically illustrates how the microbial cell extract is intercalated between layers of clay in the moisture-sorption component. The average basal spacing (dooi) being in a range between 1.2 nm and 4.1 nm. The space between the internal faces of adjacent clay platelets, layers, is the interlayer, which may be occupied by materials, such as the microbial cell extract. The sum of the distance and platelet thickness is the "dOOl" basal spacing (platelet spacing), which can be measured by X-ray diffraction. The specific surface area of the moisture-sorption component may be greater than 700 m2 / g and there may be a large aspect ratio, such as greater than 50.The clay particles may be formed though pre-swelling of a clay component consisting of at least 50% smectite clay mineral in an aqueous solvent. Before pre-swelling, the clay component may be pre-treated in different ways, such as e.g. to enrich sodium in the clay component. Pre-swelling may for example take place in water, distilled water, a buffer or water with a salt content of at most 0.3 M. In the pre-swelling step 100, the clay component, usually added as a dry powder, is mixed with the aqueous solvent at a ratio of 5% to 50% (w / v). Mixing of clay component and solvent may for example take place by stirring or by mixing during incubation or followed by incubation for at least 4 hours or at least 8 hours or up to 48 hours or more to ensure complete swelling and exfoliation of the clay layers.

[0069] To measure if the pre-swelling method used results in a clay component that has reached a completely swollen or sufficiently swollen state, a simple approach would be to measure the volume of the clay component before and after swelling. If the wet volume of the swollen clay is four times larger or more than the volume of the dry clay (before being mixed with the solvent), the pre-swelling step has resulted in a sufficiently swollen clay. Another way to measure the degree of clay swelling could be to spin down the solids as the clay is undergoing swelling and measure the volume. Generally, the clay should swell to an even consistency and have an average basal distance between layers of at least 2 nm. The pre-swelling step 100 may take place at room temperature or at a higher temperature, such as 20-50°C.

[0070] The pre-swelled clay component is thereafter mixed with the microbial cell extract using e.g. kneading or stirring. The mixing step may take place at room temperature or at a higher temperature such as 20-80°C. The mixing step may be followed by an incubation step of up to 24 hours or more to ensure adequate diffusion of the yeast extract amongst and between the exfoliated montmorillonite layers. This can be measured using XRD. Adequate diffusion does not necessarily mean complete, homogenous, infiltration of the microbial cell extract into the inter-clay-layer spaces. While complete diffusion is ideal, adequate mixing means the active microbial cell extract sufficiently resides in the interlayer space such that the release rate of microbial cell extract from the moisture-sorption component is reduced. In a heterogeneous structure some (at least 5% of the total volume of the material of the particle) of the microbial cell extract is intercalated and the remaining fraction remains as unincorporated microbial cell extract in the moisture-sorption component.Thereafter the mixture is possibly extruded, dried and possibly crushed into smaller particles.

[0071] XRD measurements were used to compare the structure of clay particles with yeast extract and pure clay. The XRD data for montmorillonite clay intercalated with yeast extract did not display a (001) diffraction peak due to unlimited and unordered swelling. The native montmorillonite clay, displayed a (001) diffraction peak at 7.4° 20, and a corresponding dooi of approx. 12.4 A due to water uptake at ambient relative humidity. For the clay-yeast extract composite particles the XRD results showed that swelling of the montmorillonite layer was not ordered or limited, since the (001) peak of clay peak was lost in the presence of yeast extract, meaning that massive intercalation of yeast extract is not limited, only determined by the yeast extract to clay ratio.

[0072] The release patterns of yeast cell extract from such formed layered moisture-sorption component were compared to moisture-sorption component comprising a non-layered mixture of the same clay and yeast extract. The release of yeast extract from a non-layered component with yeast extract was immediate and near complete. In contrast, the release from a component with intercalated yeast extract was delayed.

[0073] This demonstrates that an intercalated microbial cell extract is retained over time and has multiple elution cycles as compared to the same amounts of microbial cell extract and clay as a mere admixture where clay layers are stacked in aggregate with an average basal distance of less than 2 nm. The release of microbial cell extract held between each single clay layer is delayed because it requires the material to first swell, and then for ions to be released from the cationic and anionic exchange sites on the face and edge of the clay layer respectively.

[0074] The release rate can be modified to make microbial cell extract available at optimal levels over time, depending on the intended outcome of application of the microbial cell extract. Release can be further delayed or peak concentrations reduced, thus extending the efficacy of a treatment, by increasing the ratio of clay to the microbial cell extract. The montmorillonite clay then has an ongoing function serving as an exchange surface for charge ions.

[0075] The described moisture-sorption component retains its size and shape during handling and application to a desired area. Upon contact with water or moisture, the clay layers of the clay component of the particle re-swell and exfoliate as water enters the inter-layer space. Thisallows the microbial cell extract to dissolve and diffuse out of the component, such that the microbial cell extract can reach the intended area or plant. A particle comprising a non-layered mixture of clay and microbial cell extract has an immediate and near complete release of the microbial cell extract when contacted with water / moisture. The above described particle with microbial cell extract intercalated between clay layers in the stack exhibits on the other hand a more delayed, controlled and buffered release of the microbial cell extract from the particle when contacted with water / moisture / soil, offering an alternative to non-biodegradable polymers. By adjusting the amount of clay component, the amount of microbial cell extract and the ratio between clay and microbial cell extract in the moisture-sorption component, the component may be adapted to different requirements and conditions, such as time for full release of the microbial cell extract.

[0076] The microbial cell extract may be fully mixed with the clay in a homogenous phase. Hence, the amount of the microbial cell extract intercalated in a clay particle is mainly determined by the ratio of the microbial cell extract to clay. The amount of intercalated microbial cell extract in the particles is unlimited and may be at least 40, or at least 50 wt.% of the total dry weight of the moisture-sorption component (as measured byXRD).

[0077] The above-described moisture-sorption component shows a combined effect of delayed and staggered / multi-phased release rate. The nitrogen source in microbial cell extract is largely in organic form as 20 different proteinogenic amino acids. Each amino acid has a unique isoelectric point and therefore will carry a unique charge depending on pH. The 20 different amino acids will each be released according to idiosyncratic rates depending on charge and size. A delayed and staggered / multi-phased controlled release rate is beneficial as it helps to match available nutrition with the nutrient demand needed for healthy and stress tolerant growth, reducing losses and improving efficiency. The delayed and staggered / multi-phased release is compatible with biologically sensitive process such as seed germination where small changes in osmotic potential surrounding the seed can arrest germination, allowing the granule to be precisely placed in close proximity to the seed in the seeding furrow at rates that are reduced compared to broadcast applications.

[0078] If similar clay components instead were formed from clay and urea, this would result in a limited amount of intercalated urea of at most about 35 wt.% of the total dry weight of theparticle (as measured with XRD). Urea is a small charge neutral molecule with a molecular weight of 60.06 g / mol with a nitrogen content of 46%. Urea does not contain phosphate, potassium or any other plant nutrient and the density of urea is no more than 1.3 g / cm3. Excess urea not intercalated will re-crystallize outside the clay particles.

[0079] Using urea as a fertilizer, most of the nitrogen in urea is made available for plant uptake upon hydrolysis and the formation of ammonia by free urease enzymes in the soil. It is well established that the ammonia derived from urea can damage root tips, show characteristic symptoms of leaf tip burn, or arrest seed germination if applied in too close proximity.

[0080] Therefore, broadcast fertilizer application, i.e. the fertilizer is spread evenly across the soil surface, is practiced to reduce the effective concentration at the seed. Further, nonintercalated and recrystallized urea outside the clay particle is available for immediate and highly concentrated release to the plant, which may cause harm to the plant as mentioned above rather than improving the growth and germination thereof. The release rate of urea is not influenced by ionic associations, and soil concentration of urea will increase according to a single, acute, release rate function. The release rate of intercalated and non-intercalated urea in this way is not compatible with sensitive biological process such as seed germination if granules are placed in close proximity to the seed, which means such granules cannot be used for precision placement in the seeding furrow and must be broadcast or banded away from the seeding furrow at high overall rates.

[0081] Fig. 2 shows a graph of an adsorption-desorption isotherm for a moisture-sorption component of bentonite clay intercalated with yeast extract and an adsorption-desorption hysteresis of a sandy clay loam agricultural soil. Moisture retention of the moisture-sorption component is far greater than that of the soil, demonstrating the moisture-sorption component has the potential to improve and extend moisture retention under desiccating conditions.

[0082] Fig. 3 shows a graph of an adsorption-desorption isotherm for a moisture-sorption component of bentonite clay intercalated with yeast extract and an adsorption-desorption isotherm of a bentonite clay alone. The nature of moisture adsorption-desorption of the moisture-sorption component differs in multiple ways from clay alone. The moisture-sorption component showshigher sorption levels at higher relative humidity and lower sorption levels at lower relative humidity, albeit, well above the soil sorption level seen in Fig. 2 at any given relative humidity.

[0083] From these graphs it can be seen that the moisture-sorption potential of the moisturesorption component comprising clay intercalated with yeast extract creates an enduring moisture reservoir relative to the soil and, as such, a hydrating microenvironment suitable for the proliferation of a microbial inoculant sustained by the prolonged and even release of organic nutrition. It is likely that at this range of relative humidity; one, two, or three hydration layers are sorbed and assembled in an orderly way between the clay layers. In the moisturesorption component, the yeast extract compliments the clay sorption potential, but the orderly hydration layers are disrupted by interactions between water and ions in the yeast extract, or by interactions between ions in the yeast extract and the clay. The yeast extract-driven increase in sorption capacity at higher relative humidities (>80%) is greater than the yeast extract driven reduction in sorption at lower relative humidities (<80%).

[0084] In one embodiment, the moisture-sorption particulate material may comprise clay (as described above) and in addition a superabsorbent hydrogel. The superabsorbent hydrogel may be mixed with the clay. If the clay is mixed with the superabsorbent hydrogel it may prevent / counteract collapse of the superabsorbent hydrogel. The superabsorbent hydrogel may be selected from one or more of cellulose-based hydrogels, starch-based hydrogels, chitosan-based hydrogels, alginate-based hydrogels, gelatine-based hydrogels, poly(acrylamide-co-acrylate) and polyvinyl alcohol-based hydrogels. Superabsorbent hydrogels are well-known for their water-absorbing and water-retaining properties. Superabsorbent hydrogels generally experience a decrease in their swelling capacity and reduced performance in the presence of salt, which may be present in the soil. By mixing clay with the superabsorbent hydrogel in the moisture-sorption component, the clay may prevent / counteract collapse of the superabsorbent hydrogel when applied to soil. Such microbial inoculant composite material may also improve the efficacy of the microbial inoculant. The moisture-sorption component functioning as a source of nutrition (from the microbial cell extract) and a moisture reservoir (the moisture-sorption component particulate material comprising clay and superabsorbent hydrogel) to the microbial inoculant. If the superabsorbent hydrogel is coated on the clay it may reduce dust shedding of the clay particle.The moisture-sorption component may be comprised to at least 95 wt.%, or at least 96 wt.%, at least 97 wt.%, at least 98 wt.% or at least 99 wt.% of the microbial cell extract, the clay and the superabsorbent hydrogel. The rest may be e.g. binders or other substances such as benign inactive ballast.

[0085] 1 to 90 wt.%, 1 to 80 wt.%, or 1-70 wt.%, or 1-60 wt.%, or 1-50 wt.%, or 1-40 wt.%, or 1-30 wt.%, or 10 to 90 wt.%, or 20 to 90 wt.%, or 30 to 90 wt.%, or 40 to 90 wt.%, or 50 to 90 wt.%, or 60 to 90 wt.%, or 70 to 90 wt.%, or 80 to 90 wt.%, or 20 to 80 wt.%, or 30 to 70 wt.%, or 30-50 wt.%, or 40-50 wt.% or 30-40 wt.%, based on dry weight, of the moisture-sorption component may consist of clay and superabsorbent hydrogel.

[0086] A ratio of microbial cell extract to clay and superabsorbent hydrogel in the moisture-sorption component may be 1:10-1:1, 1:10-1:2, 1:10-1:3, 1:10-1:4, 1:10-1:5, 1:10-1:6, 1:10-1:7, 1:10-1:8, 1:10-1:9, 1:9-1:2, 1:8-1:2, 1:7-1:2, 1:6-1:2, 1:5-1:2, 1:4-1:2, or 1:3-1:2. In one embodiment, the moisture-sorption component is comprised to at least 95 wt.%, based on dry weight, of microbial cell extract, clay and superabsorbent hydrogel, and 20-50 wt.% of the moisturesorption component is clay.

[0087] A weight ratio of clay to superabsorbent hydrogel in the moisture-sorption component may be 1:10 to 10:1, or 1:8 to 10:1, 1:6 to 10:1, 1:4 to 10:1, 1:2 to 10:1, 1:10 to 8:1, 1:10 to 6:1, 1:10 to 4:1, 1:10 to 2:1, 1:3 to 2:1, or 1:2 to 2:1, or 1:1 to 2:1, or 1:4 to 1:1, or 1:4 to 1:2, or 1:4 to 1:3.

[0088] The clay and microbial cell extract may dominate the moisture-sorption component and the superabsorbent hydrogel may comprise up to 10% on a dry weight basis.

[0089] In Fig. 4 is illustrated a method of producing a microbial inoculant composite material for use in agriculture, forestry or horticulture, comprising:

[0090] providing 1 one or more microbial inoculants,

[0091] providing 2 a moisture-sorption component, wherein the moisture-sorption component is comprised of a microbial cell extract and a moisture-sorption particulate material comprising clay or a mixture of clay and superabsorbent hydrogel,mixing 3 the microbial inoculants and the moisture-sorption component in a ratio of 10,000:1 to 1:1.

[0092] In Fig. 5 is illustrated a method of applying a microbial inoculant composite material to soil, plant or seed, comprising:

[0093] providing 10 one or more microbial inoculants,

[0094] providing 20 a moisture-sorption component, wherein the moisture-sorption component is comprised of a microbial cell extract and particulate material comprising clay or a mixture of clay and superabsorbent hydrogel,

[0095] applying 30 the microbial inoculant and the moisture-sorption component in a ratio of 10,000:1 to 1:1 to said soil, plant or seed.

[0096] The clay or the clay and superabsorbent hydrogel based moisture-sorption component may be mixed with the microbial inoculant prior to application or simultaneously as applied to soil or applied in sequence to the soil, seed or plant.

[0097] The microbial inoculant composite material can be applied to the soil, incorporated into the soil, incorporated into potting substrate, placed in close proximity of a seed at sowing or placed in close proximity of roots at planting of a tree seedling for delayed release of the active ingredient and retaining of moisture. It composite material could be applied as a powder to seeds. The moisture-sorption component could be sprayed as a suspended powder onto leaves. The composite material may comprise the moisture-sorption component as aggregates, granules or pellets. The composite material could be in powder form.

[0098] Alternatively, the composite material could be a suspension such as a sprayable suspension.

[0099] The microbial inoculant composite material described above may be used as a soil fertility enhancer, a plant growth promoter, a disease resistance agent, a stress tolerance improver, in agriculture, forestry or horticulture.

[0100] In addition the microbial inoculant composite material may be used to reduce the use of different chemicals in agriculture, forestry or horticultureThe microbial inoculant composite material may be coated on at least a portion of a seed.

[0101] The microbial inoculant composite material may be applied in an amount of 1-100 kg / 10000 m2soil.

[0102] EXPERIMENTAL

[0103] Preparation of moisture-sorption component comprising clay

[0104] A 10 % (w / v) montmorillonite-rich bentonite clay, consisting of at least 50% smectite clay mineral, was pre-swelled by gently mixing the clay with tap water and allowed to stand for >48 hours at room temperature to ensure complete exfoliation of the montmorillonite layers. A pre-swelled clay component, a viscous gel resulted. Dry yeast extract powder was added, bringing the mixture to 20% (w / v) with a dry content ratio of 1:1 (clay component:yeast extract powder). The gel was intermittently manually kneaded in a plastic bag and allowed to incubate in a water bath at 65 degrees Celsius for 24 hours to ensure adequate diffusion of the yeast extract amongst and between the exfoliated montmorillonite layers. The gel was then pressed through a hole, 7 mm in diameter, to form a sausage-like shape and dried at 50 degrees Celsius. The dried material was then crushed into smaller particles with a particle size distribution from 1-4 mm.

[0105] Preparation of moisture-sorption component comprising clay and superabsorbent hydrogel

[0106] A 10 % (w / v) montmorillonite-rich bentonite clay, consisting of at least 50% smectite clay mineral, was pre-swelled by gently mixing the clay with tap water and allowed to stand for >48 hours at room temperature to ensure complete exfoliation of the montmorillonite layers. A pre-swelled clay component, a viscous gel resulted. In a separate vessel, a 10% (w / v) carboxymethyl cellulose gel was prepared by adding tap water and stirring for 24 hours. The resulting gels were then mixed by kneading at a ratio of 10 parts pre-swelled clay to 1 part carboxymethyl cellulose gel giving a final gel with 10 wt.% dry matter. Dry yeast extract powder was added, bringing the mixture to 20% (w / v) with a dry content ratio of 1:1 (clay component:yeast extract powder). The gel was intermittently manually kneaded in a plastic bag and allowed to incubate in a water bath at 65 degrees Celsius for 24 hours to ensure adequate diffusion of the yeast extract amongst and between the exfoliated montmorillonitelayers. The gel was then pressed through a hole, 7 mm in diameter, to form a sausage-like shape and dried at 50 degrees Celsius. The dried material was then crushed into smaller particles with a particle size distribution from 1-4 mm.

[0107] Application of microbial inoculant composite material to soil

[0108] Example 1

[0109] At the time of seeding a crop, moisture-sorption component is applied in close proximity to the seed, for example by drilling it directly into the seeding furrow at a rate of 1-30 kg / 10,000 m2. In the same operation, the soil is inoculated with a beneficial microbial inoculant by spraying a liquid preparation as described by the manufacturer (1.2xl012CFU / L) at rate in the order of 1-5 L / 10,000 m2.

[0110] Example 2

[0111] The moisture-sorption component is applied at seedling as in Example 1. The microbe inoculant is provided directly onto the seed.

[0112] Example 3

[0113] A powdered form of the moisture-sorption component is applied to the seed together with a microbial inoculant prior to sowing.

[0114] Example 4

[0115] The moisture-sorption component is applied as a side dressing once the crop is established. Microbe inoculated directly together as a side dressing.

[0116] Comparative Example 1: Structural Analysis via XRD

[0117] A comparative experiment was conducted to assess the structural differences between a dry, particulate moisture-sorption component prepared according to the present disclosure and a comparative highly water-saturated hydrogel composition.

[0118] Powder XRD measurements were performed in reflection mode with CuKa radiation (0.15418 nm) in the 20 range of 4-40 degrees.Preparation of the Example of the present disclosure:

[0119] A moisture-sorption component was prepared using yeast extract and bentonite clay. 1 part (by weight) bentonite clay was pre-swelled in 5 parts of water, allowing for complete delamination (exfoliation) of the clay layers. Subsequently, the pre-swelled clay was mixed with 1 part yeast extract. The resulting suspension was dried and pelletized to form a final dry particulate product. To simulate fully water-saturated conditions during field application, the particulate product was mixed thoroughly with 5 parts water prior to XRD measurement.

[0120] Preparation of the Comparative Example:

[0121] A comparative macroscopic hydrogel was prepared containing 1 part yeast extract, 1 part hectorite clay, and 1.6 parts polyacrylate-based polymer, which was water-saturated with 100 parts of water. Crucially, in this comparative method, the polyacrylate polymer and hectorite clay were mixed with water first, forming a polymer-clay matrix. Subsequently, the yeast extract was added to this matrix. The composition inherently forms a highly water-saturated hydrogel and cannot be obtained or tested as a dry particulate; therefore, it was tested in its inherent hydrogel state.

[0122] Results:

[0123] Fig. 6 shows the XRD data for the inventive product (top) and the comparative product (bottom).

[0124] The product prepared according to the present disclosure shows distinct clay diffraction peaks, including peaks from accessory minerals present in the native bentonite. The most pronounced clay peak is centered around 6.2 degrees 20, which is indicative of a 14 A (1.4 nm) basal spacing. This basal spacing is consistent with the presence of organic material (yeast extract) intercalated between the individual clay layers. The data demonstrates that the yeast extract interacts favorably with the exfoliated clay surfaces, and this ordered, intercalated structure is retained even underwater-saturated conditions.

[0125] Conversely, the comparative product shows no ordered clay structure. It exhibits only two amorphous peaks centered around 10 and 28-29 degrees 20, which are assigned to the amorphous polymer and amorphous water structures, respectively. Because the clay wassimultaneously mixed with the cross-linking hydrogel polymers prior to the addition of the yeast extract, the polymers encapsulated the clay, preventing intercalation. Consequently, no structural order or yeast extract-clay intercalation is observed.

[0126] This demonstrates that the specific sequence of pre-swelling and mixing is required to achieve the claimed intercalated structure, and that merely mixing clay, yeast extract, and hydrogel polymers does not inherently result in intercalation.

Claims

CLAIMS1. A microbial inoculant composite material for use in agriculture, forestry or horticulture, comprising:one or more microbial inoculants, anda moisture-sorption component, wherein the moisture-sorption component is in the form of a powder, granules, capsules, or pellets, and wherein the moisture-sorption component is comprised of a microbial cell extract and a moisture-sorption particulate material comprising at least 5 wt.% smectite clay mineral,wherein the clay in the moisture-sorption component forms layered stacks and the microbial cell extract is intercalated between individual clay layers in the stacks.

2. The microbial inoculant composite material of claim 1, wherein a weight ratio of moisture-sorption component to microbial inoculant is 10,000:1 to 1:1.

3. The microbial inoculant composite material of claim 1 or 2, wherein the moisturesorption component consists to at least 95 wt.% of microbial cell extract and moisturesorption particulate material.

4. The microbial inoculant composite material of any of claims 1-3, wherein the moisturesorption component consists to at least 95 wt.% of microbial cell extract and clay.

5. The microbial inoculant composite material of any of claims 1-4, wherein 1-90 wt.% of the moisture-sorption component consists of clay.

6. The microbial inoculant composite material of any of claims 1-3, wherein the moisturesorption particulate material comprises clay and a superabsorbent hydrogel.

7. The microbial inoculant composite material of claim 6, wherein the moisture-sorption component consists to at least 95 wt.% of microbial cell extract, clay and superabsorbent hydrogel.

8. The microbial inoculant composite material of claim 6 or 7, wherein 1-90 wt.% of the moisture-sorption component consists of clay and superabsorbent hydrogel.

9. The microbial inoculant composite material of any of claims 6 to 8, wherein a weight ratio of clay to superabsorbent hydrogel in the moisture-sorption component is 10:1 to 1:10.

10. The microbial inoculant composite material of any of claims 6 to 9, wherein the superabsorbent hydrogel is mixed with the clay.

11. The microbial inoculant composite material of any of claims 6-10, wherein the superabsorbent hydrogel is selected from one or more of cellulose-based hydrogels, starch-based hydrogels, chitosan-based hydrogels, alginate-based hydrogels, gelatine-based hydrogels, poly(acrylamide-co-acrylate) and polyvinyl alcohol-based hydrogels.

12. The microbial inoculant composite material of any of the preceding claims, wherein the microbial cell extract is yeast extract.

13. The microbial inoculant composite material of any of the preceding claims, wherein the microbial inoculant is selected from biofertilizers, biopesticides, microbial agents, beneficial microbes, bioinoculants, microbial consortia and soil probiotics or any combination thereof.

14. The microbial inoculant composite material of claim 13, wherein the microbial inoculant is selected from nitrogen-fixing bacteria, phosphate-solubilizing microorganisms, mycorrhizal fungi, plant growth-promoting rhizobacteria, biocontrol agents, organic matter decomposers, protozoa and nematodes or any combination thereof.

15. The microbial inoculant composite material of any of the preceding claims, wherein the microbial inoculant composite material is in the form of a powder, granules, capsules, liquid, suspension, slurry, pellets or any combination thereof.

16. The microbial inoculant composite material of any of the preceding claims, wherein the clay has an average basal spacing (dooi) between 1.2 nm and 4.1 nm.

17. Method of producing a microbial inoculant composite material for use in agriculture, forestry or horticulture, comprising:providing (1) one or more microbial inoculant(s),providing (2) a moisture-sorption component, wherein the moisture-sorption component is in the form of a powder, granules, capsules, or pellets and is comprised of a microbial cell extract and a moisture-sorption particulate material comprising a clay which comprises at least 5 wt.% smectite clay mineral or a mixture of said clay with a superabsorbent hydrogel, wherein providing said moisture-sorption component comprises the steps of: pre-swelling said clay to exfoliate its layers, subsequently mixing the pre-swelled clay, optionally also comprising the superabsorbent hydrogel, with the microbial cell extract to intercalate the cell extract between the exfoliated clay layers, and drying the mixture; andmixing (3) the moisture-sorption component to microbial inoculant in a ratio of 10,000:1 to 1:1.

18. Use of the microbial inoculant composite material of any of the claims 1-16 as a soil fertility enhancer, a plant growth promoter, a disease resistance agent, a stress tolerance improver, in agriculture, forestry or horticulture.

19. Method of applying a microbial inoculant composite material to soil, plant or seed, comprising:providing (10) one or more microbial inoculants,providing (20) a moisture-sorption component of any of claims 1-16,applying (30) the moisture-sorption component and the microbial inoculant(s) to said soil, plant or seed in a weight ratio of 10,000:1 to 1:1.

20. The method of claim 19, wherein the microbial inoculant and the moisture-sorption component are mixed prior to application to said soil, plant or seed; mixed simultaneously as applied to said soil, plant or seed, or are applied in sequence to said soil, plant or seed.

21. The method of claim 19, wherein the microbial inoculant composite material is coated on at least a portion of a seed.

22. The method of any of claims 19-21, wherein the microbial inoculant composite material is applied in an amount of 1-100 kg / 10,000 m2soil.

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