Urea-based granular fertilizers with enhanced nitrogen use efficiency

Abstract

New type urea-based granular fertilizers provided with a multiple-layer coating. The coating comprises, independently distributed among the layers: an ammonium converting microorganism, an ureolytic microorganism, an oil-binder mixture and an anticaking agent. The present granules are meant for administration to the soil for purpose of fertilization. They may be administered a such or as part of a composition comprising them. The granules of the invention provide a prolonged fertilizing effect combined with an enhanced conversion of urea in plant-absorbable nitrogen, mainly in form of nitrites and nitrates, advantageously accompanied by a reduced loss on nitrogen in form of gaseous ammonia and a prolonged duration of action.

Description

[0001] TITLE

[0002] UREA-BASED GRANULAR FERTILIZERS WITH ENHANCED NITROGEN USE

[0003] EFFICIENCY

[0004] STATE OF THE ART

[0005] Since the beginning of 20th century, the fertilizer demand has continually increased as a function of the population growth. The first essential step for the fertilizer production is the nitrogen fixation which is mainly made by the long-known Haber-Bosh process (HB). The HB process is based on the nitrogen reduction by hydrogen. Although the HB process has greatly increased the efficiency during the last decades, it is still a highly energyconsuming process.

[0006] Urea is the most used nitrogen fertilizer worldwide, and it is produced by the conversion of NH3 derived from HB process. In 2018 / 2019 over 107 106 tons of nitrogen fertilizers was produced by industry and approximately 55% of the them were based on urea as nitrogen source. Its diffusion is particularly due to its high nitrogen content (46%), relatively low cost, availability in most markets, high water solubility, low corrosion capacity and compatibility to most fertilizers as well as high foliar uptake. Nevertheless, the nitrogen use efficiency in soil for urea is fairly low, often averaging <50%.

[0007] Many factors are responsible for the low nitrogen use efficiency, such as: leaching of fertilizer from the site of application due to environmental factors (rain, wind, sun); slow or insufficient conversion of the fertilizer in plant-absorbable nitrogen (in particular nitrates or nitrites); loss of available nitrogen by conversion in gaseous ammonia, etc.

[0008] Agricultural systems should ensure food security for the growing global population in the context of climate change and the improvement of fertilizers efficiency while reducing their environmental impact is one of the biggest challenges. For this reason, efforts have been made to increase fertilizer nitrogen use efficiency, maximize profit and reduce negative impacts on the environment, guaranteeing that the nitrogen nutrient supply matches with plant demand during the growing season.

[0009] Different strategies are studied to overcome the limits of low nitrogen use efficiency of urea. One of the most challenging is directed to the use of slow and controlled release fertilizers, and biofertilizers. Slow and controlled release fertilizers are considered those containing a plant nutrient in a form, which either (i) delays its availability for plant uptake and use after application, or (ii) remain available to the plant significantly longer than in the traditional fertilizer. These can be achieved by two principal types of strategies as: (i) the use of semipermeable coatings for controlled solubility of the fertilizer in water, protein materials, occlusion, chemicals, slow hydrolysis of water-soluble compounds of lower molecular weights, and some other unknown means; (ii) the utilization of semipermeable materials and sensors of chemical or biological origin within the fertilizer. These are materials able to change in response to an external stimulus.

[0010] Slow and controlled release arrangements have however the limitation that they do not increase the overall amount of nitrogen available for absorption provided by the fertilizer. In particular, they are largely ineffective on the causes of low nitrogen use efficiency, i.e. slow conversion of the fertilizer in plant-absorbable nitrogen and loss of ammonia. Therefore, the overall improvement in nitrogen use efficiency provided by these systems remains low.

[0011] Bioproducts are known, which can slowly release in situ the nutrients that plant need using the sun as source of energy; the nutrients can be produced by microorganisms present in the bioproduct. Different microorganisms are considered, such as bacteria, enzymes and algae, each one with advantages and drawbacks. Microorganisms may have a high capability to generate nitrates and nitrites and thus, in principle to efficiently degrade the original nitrogen source: however, this also increases the incidence of the side reactions leading ammonia formation and nitrogen loss, thus resulting in modest improvements in nitrogen use efficiency.

[0012] The patent publication CN104557312A discloses a granular fertilizer comprising various nutrients, including e.g. 0-400 parts of urea powder, where the granule is coated with a double-walled encapsulated mixture of microorganisms of the Bacillus species; there are no examples of combinations of species, nor there is described a separate layering of microorganisms.

[0013] The patent publication CN101885646A describes a composition based on a biological bacteria (nitrogen-fixing Bacillus) being adsorbed and completely integrated into a mixture of humic acid, urea and bentonite; the resulting mixture is coated with one adhesive layers including carboxymethylcellulose, guar gum and vegetable oil. The patent publication CN107032914A discloses a urea-humic acid granule, first sprayed with a mixture of palm oil and tartaric acid, and then with a mixture of microorganisms of the Bacillus species.

[0014] The patent publication US2020255355A1 discloses core particles of fertilizer, e.g. urea granules, coated by a shell including at least one polymer layer and one or more species of microbes.

[0015] The patent publication WO2024064213A1 discloses core granules containing a mixture of organic-inorganic materials coated with one or more spore-forming microorganisms, e.g. Penicillum bilabiae, then mixed with e.g. urea to obtain a fertilizers composition.

[0016] The article Microb. Cell Fact. (2020), [Online] vol.19 no.1 , 23.1.2020, discloses the ureolytic ectivity of Sporosarcina pasteurii.

[0017] The need remains thus still unmet for improved nitrogen fertilizers, in particular those with a high nitrogen use efficiency; such products would ideally allow a reduction of the amount of administered product per soil surface unit and, consequently, the environmental impact and costs.

[0018] SUMMARY OF THE INVENTION

[0019] A new type of urea-based granular fertilizers is now provided by the present Applicants, which improves nitrogen use efficiency and plant growth. They are characterized by a combination of product structure, excipients, and microorganisms, which result in enhanced conversion in plant-absorbable products, reduced nitrogen loss and longstanding efficacy in terms of plant growth. The fertilizer is based on urea granules provided with a multiple-layer coating; the coating comprises, independently distributed among the layers:

[0020] (a) an ammonium converting microorganism,

[0021] (b) an ureolytic microorganism,

[0022] (c) an oil-binder mixture and

[0023] (d) an anticaking agent.

[0024] In accordance with the invention, the present granules are administered to the soil for purpose of fertilization. They may be administered a such (granular product), or as part of a composition comprising them, both included in the present invention. After administration to the soil, in consequence of environmental factors, in particular humidity and rain, the granules structure slowly degrades and, consequently a contact of urea and microorganisms in a wet environment is established. In these conditions, in a first reaction, led by the component (b), urea is degraded in a targeted manner to carbonate and ammonium / ammonia; in a second reaction, the component (a) oxidizes ammonium / ammonia into nitrate and nitrite, thus efficiently converting it into bio- assimilable compounds by plants. The produced carbonate precipitates with calcium in the environment in the form of calcium carbonate. Differently from a simple administration of pure urea to the soil, the present method and product reduces the emission of CO2 generally associated with the degradation of urea.

[0025] The granules of the invention provide a prolonged fertilizing effect combined with an enhanced conversion of urea in plant-absorbable nitrogen, mainly in form of nitrites and nitrates, advantageously accompanied by a reduced loss on nitrogen in form of gaseous ammonia and a prolonged duration of action. The nitrogen use efficiency is thereby significantly increased.

[0026] DESCRIPTION OF THE DRAWING

[0027] Figures 1A-1 B: both these figures show embodiments of the invention wherein the urea granule is coated as follows:

[0028] (i) the innermost layer is the component (c) - oil / binder mixture

[0029] (ii) the external part of the coating is formed by a two-layers system, the internal one being component (a) - ammonium converting microorganism and the outermost one being the component (d) - anticaking agent.

[0030] Between the substructures (i) and (ii) there can be present an intermediate layer of: component (b) - ureolytic microorganism (cf. Figure 1A), or component (b+d) - ureolytic microorganism + anticaking agent (cf. Figure 1 B).

[0031] Figure 2A: shows embodiments of the invention wherein the components are coated on the urea granule in the sequence, from innermost to outermost layer: (b)-(c)-(b)-(a)-(d). Figure 2B: shows the same arrangement of Figure 2A, with the difference that the two layers (b) are replaced by layers (b+d).

[0032] Figure 2C: shows embodiments of the invention wherein the components are coated on the urea granule in the sequence, from innermost to outermost layer: (d)-(c)-(b)-(a)-(d). Figure 2D: shows the same arrangement of Figure 2B, with the difference that the two layers (d) are replaced by layers (b+d). Figure 3A: shows embodiments of the invention wherein the components are coated on the urea granule in the sequence, from innermost to outermost layer: (d)-(a+c)-(b)-(d). Figure 3B: shows the same arrangement of Figure 3A, with the difference that the innermost layer (b) is replaced by layer (d).

[0033] Figure 4A: shows embodiments of the invention wherein the components are coated on the urea granule in the sequence, from innermost to outermost layer: (a+c)-(b)-(d).

[0034] Figure 4B: shows the same arrangement of Figure 4A, with the difference that the layers and (d) are combined in a single layer.

[0035] Figures 5: shows embodiments of the invention wherein the components are coated on the urea granule in the sequence, from innermost to outermost layer: (c)-(d)-(a)-(b)-(d).

[0036] Figure 6A-C: shows the results of the study of generation of plant-absorbable nitrogen described in Example 3, for the granules of the invention; the amount of urea, ammonia and nitrate in soil were extracted by solid-liquid extraction and quantified by colorimetric method with UV-Vis spectrophotometer.

[0037] Figure 7A and 7B show the results of the study of long-term fertilizer effect described in Example 3, for the granules of the invention.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] The terms “granule” or “granules” are used here indifferently to encompass both the singular and the plural. These terms, including the adjective “granular” are herein meant broadly to include all micronized solid forms obtained by drying a corresponding liquid phase: the term thus includes e.g. pellets, prilled forms, etc.

[0040] The terms “microorganism” used herein is not limited to specific classes, but includes microorganisms in general, such as yeasts, fungi, spores thereof, algae, etc. as long as they have ammonium-converting or ureolytic properties as defined herein.

[0041] The preparation of the urea granules “as such”, i.e. prior to coating, can be performed according to conventional techniques, for example, dry granulation, wet granulation, extrusion, etc. The average size of the uncoated granules can be comprised between about 0.5 and 5.0 mm, e.g. 0.7 to 4.0 mm, or 1 .0 to 3.0 mm, etc.

[0042] Throughout this application, the term “about” indicates a tolerance of ± 20% of the given value. The urea component is present only in the granule, not in the coatings. The term “multiple layer coating” used herein indicates the result of applying two or more coating layers to the urea granule. Although there is no strict upper limit for the number of layers, best results in terms of the invention aims are obtained with a total number of 3 to 5 layers; more than 5 layers are not preferred because of complexity of manufacturing and excessive embedding of the core urea granule, which may excessively reduce the fertilizer availability to the soil. The overall coating, i.e. consisting of all the layers applied and dried upon the granule, provides a coating thickness preferably comprised between about 0.02 and 0.8 mm, e.g. 0.05 to 0.4 mm, or 0.2 to 0.4 mm, etc..

[0043] The characteristic components of the present invention, i.e.: (a) the ammonium converting microorganism, (b) the ureolytic microorganism, (c) the oil-binder mixture and (d) the anticaking agent, are variously present within the layers. Preferably, the microorganisms (a) and (b) are not present in the composition as homogeneous mixture, but are separated from each other, i.e. they are present in different layers; even more preferably, the layer with the ureolytic microorganism is closer to the urea core than the layer with the ammonium-converting microorganism. The separate layering of the two microorganisms also allows to optimize the respective layer compositions, with the aims of increasing adhesion and viability of the respective microbial raw materials, while avoiding possible interference between the respective metabolic activities.

[0044] For example, each of the characterising components can be present as an own layer; alternatively, it is possible to combine two or more of characterising components into a single layer; it is also possible to have two or more layers containing the same characteristic component(s), separated from each other by layer(s) with different composition.

[0045] The ammonium converting microorganism (component (a)) is preferably of the Bacillus species, more preferably Bacillus subtilis. A particularly preferred microorganism is the Bacillus subtilis strain deposited by Casale at CCOS with Deposit number 2113. The ammonium converting property is defined herein as the capacity to convert ammonia and ammonium salt / ion into nitrites and / or nitrates and / or proteins. Further microorganisms that convert ammonium to nitrites chosen, without limitation, among the genus Bacillius, Pseudomonas, Nitrosomonas, Nitrosospira, Nitrosococcus, Ammonia-oxidizing archaea and NitrosoIobus and microorganisms that convert nitrites to nitrates are chosen among the genus Bacillus, Pseudomonas, Rhodococcus, Nitrobacter, Nitrospina, and Nitrococcus, all of which are included in the present invention. The ureolytic microorganism (component (b)) is preferably of the Sporosracina species, more preferably Sporosracina pasteurii. A particularly preferred microorganism is the Sporosracina pasteurii strain deposited by Casale at CCOS with Deposit number 2114. The ureolytic property is defined herein as the capacity to convert urea into ammonium salt / ion, ammonia and carbonates. Further ureolytic microorganisms can be chosen, without limitation, among the genus Bacillus, Rhodococcus, Mycoplasma, Pseudomonas, Stutzeri monas, Micrococcus, Pseudoalteromonas, etc.

[0046] Preferably, the microorganisms (a) and (b) contained in the coated granule are present, independently from each other, in a ratio with urea comprised between 200 000 and 20 000 000 CFU / g urea.

[0047] The oil-binder mixture (component (c)) is conveniently made by mixing a vegetable oil with a formulation binder. Vegetable oils can be chosen, without limitation, in the group consisting of castor oil, coconut oil, com oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, safflower oil, sesame oil, soybean oil, sunflower oil, nut oil, almond oil, hazelnut oil, macadamia oil, pecan oil, pine nut oil, pistachio oil, walnut oil, pumpkin seed oil, etc. and mixtures thereof. The binder can be selected among those conventionally used for this purpose. Examples thereof are molasses, starch, dextran, gum arabic, guar gum, etc. and mixtures thereof.

[0048] The weight ratio of binder to oil in the component (c) is widely variable and can be adapted to the type of coating material and degree of adherence desired therefor; particularly effective ratios are found in the range between 2:1 to 10:1 , more preferably 4:1 to 8:1 , most preferably of about 6:1 .

[0049] The anticaking agent (component (d)) is conveniently selected among those excipients know for this property. Examples thereof are sodium lignosulfate, potassium lignosulfate, calcium lignosulfate, biuret, maltodextrin, carboxymethyl starch, tricalcium phosphate, powdered cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, calcium phosphate, sodium silicate, silicon dioxide, calcium silicate, magnesium trisilicate, talcum powder, sodium aluminosilicate, potassium aluminium silicate, calcium aluminosilicate, bentonite, zeolite, aluminium silicate, stearic acid, polydimethylsiloxane.

[0050] Optionally, in addition to the characteristic components (a)-(d) of the invention, further formulative excipients can be incorporated into the coating layers, used in function of their known properties. Examples thereof are protective and preservative agents such as sucrose, trehalose, glycerol, bovine serum protein (BSA), mannitol, lactose etc. lactose. Minor amounts of water, e.g. less than 1 wt% can also be present, in particular in the component (c).

[0051] A further object of the invention is process to obtain the above-described coated granules, comprising the steps of applying, to urea granules, a sequence of layers comprising the said components (a)-(d), independently distributed among the layers.

[0052] These components, inclusive of any optional formulative excipients, can be applied to the urea granule by any available coating technique, for example by spray drying, polar drying, lyophilization, dipping in liquid and sieving, etc. Polar drying is a high-throughput electrostatic drying technique ideal for heat-sensitive and oxygen-sensitive material including living microorganisms, probiotics, and proteins; polar dryers and operating methods are provided e.g. by Fluid Air Europe, Montrouge (FR). In all these techniques, the coating material is generally applied in excess to the granules and, at the end of the process, the residual excess material is recovered e.g. by sieving.

[0053] In particular, the microorganisms (a) and (b) can be applied, independently from each other, in form of a powder, by spray drying, polar drying, lyophilization, and similar techniques. The application in powder form for is preferably used for component (a).

[0054] Alternatively, the microorganisms are provided as a solution or suspension of microorganism or spores thereof in a liquid medium, directly applied to the granule, e.g. via dipping in liquid and sieving, etc. The liquid medium is variably chosen among e.g. water, a buffered saline or, in case of spores, a standard sporulation medium. An example of sporulation medium is a water solution composed of meat extract 0.2-3 g / L, peptone 0.5-5 g / L, KCI 0.05-2 g / L, MgSO4*7H2O 0.01 -1 g / L, NaOH 1-10M in water at 5-100 pL / L, Ca(NO3)2*4H2O 0.01 to 1 g / L, MnCI2*4H2O 0.0001 to 0.01 g / L, FeSO4*7H2O 0.001 - 0.01 g / L. The application in liquid form is preferably used for component (b).

[0055] Subject to the above definitions, various combinations of coatings are contemplated by the present invention.

[0056] In particular, each component may be applied as independent coating. Alternatively, combinations of components can be applied, in particular according to the following embodiments. According to a first embodiment, the components (a) and (c) are combined in at least one layer. This embodiment is particularly useful when (a) is provided as a powder, in which case the “wet” nature of component (c) aids in fixing of the powder onto the granular substrate. In operation, the combination of (a) and (c) can be formed prior to coating, by forming a liquid mixture of components (a) and (c) which is subsequently applied to the granule; alternatively, it can be formed in situ by applying one of the two components immediately after the other, such as the liquid component incorporates in situ the solid one.

[0057] According to a second embodiment, the components (b) and (d) are combined in at least one layer. This embodiment is particularly useful when (b) is provided as a liquid medium, in which case the “wet” nature of the latter aids in fixing the anticaking agent onto the granular substrate. In operation, the combination of (b) and (d) can be formed prior to coating, by forming a liquid mixture of components (b) and (d) which is subsequently applied to the granule; alternatively, it can be formed in situ by applying one of the two components immediately after the other, so that the liquid component incorporates in situ the solid one.

[0058] According to a third embodiment, the sequence of layers (a) and (b) (or vice versa) is applied to the urea granule previously coated with component (c). Also in this embodiment, the fixing of the solid microorganism layer(s) is aided by the “wet” nature of the component (c).

[0059] In addition, in all forms of the invention, the component (d) is preferably present at least in the outermost coating layer, i.e. the last deposited coating of the final granule.

[0060] Particularly preferred sequences of coatings, numbered I to XI starting from the innermost to the outermost are those in accordance with the following table:

[0061] (-) layer not present

[0062] Particularly preferred within the above table are the configurations from I, II, III, IV, V, VI, and VII, wherein the ureolytic microorganism (b) is layered more proximally to the urea granular core, than the ammonium-converting microorganism. This configuration maximizes the conversion of urea into plant-useful nutrients by promoting, after administration and contact with environmental humidity, an initial reaction of the urea with the first-to-contact (ureolytic) microorganism; as the humidity increases within the product, the high amounts of generated ammonium are made more mobile and come into contact with the ammonium converting microorganism, which is already available in wetted state.

[0063] In accordance with the invention, the present granules are administered to the soil for purpose of fertilization. The granules may be administered a such, or as part of a formulation comprising them. Accordingly, the present invention includes the use of the present granules as fertilizers, as well as for the manufacturing of a fertilizer formulation, the latter being also meant for use as fertilizer.

[0064] After administration of the present granules to the soil, in consequence of environmental factors, in particular humidity and rain, the granule structure slowly degrades and a contact of urea and microorganisms in a wet environment is formed. In these conditions, in a first reaction, led by the component (b), urea is degraded in a targeted manner into carbonate and ammonium / ammonia; in a second reaction, the component (a) oxidizes ammonium / ammonia into nitrate and nitrite, thus converting the ammonia into bio- assimilable compounds by plants. The produced carbonate precipitates with calcium in the environment in the form of calcium carbonate. This course of reactions is most advantageously implemented by a preferred configuration of the present invention, whereby the layer containing the ureolytic microorganism is more proximate to the urea granule than the layer containing the ammonium converting microorganism: i.e. in the final coated granule, the ureolytic microorganism is layered more internally than the ammonium converting microorganism. In this configuration, once administered to the ground, in presence of environmental humidity the urea degradation reaction is the first in time to occur, and the whole microbial load for this reaction is in strict contact with the urea substrate to be degraded; subsequently, a high-efficiency conversion of ammonium to plant-useful nitrates is driven by the high amount of generated ammonium substrate; meanwhile, the wetting of the layer with the ammonium converting microorganism, even if not immediately in contact with the corresponding ammonium substrate, is also useful since it enhances the viability of the microorganism, to the benefit of an efficient microbial reaction. Therefore, any risk of lack of contact between microorganisms and or substrates, which would reduce the overall generation of plant-useful nitrates, is minimized.

[0065] Accordingly, a preferred embodiment of the invention is an urea granule provided with a multiple-layer coating, said coating comprising the following components, independently distributed among the layers: (a) an ammonium converting microorganism; (b) an ureolytic microorganism; (c) an oil-binder mixture; (d) an anticaking agent, wherein the layer containing the ureolytic microorganism is more proximate to the urea granule than the layer containing the ammonium converting microorganism. Granular products comprising the granules in accordance with this preferred embodiment, and compositions comprising said granular products are also part of the invention.

[0066] A correlated preferred object of the invention is process to obtain urea granules provided with a multiple-layer coating, comprising the steps of applying, to urea granules, a sequence of layers comprising the following components, independently distributed among the layers: an ammonium converting microorganism, an ureolytic microorganism, an oil-binder mixture, an anticaking agent, with the proviso that the ureolytic microorganism is applied to the granule before the ammonium converting microorganism.

[0067] The granules of the invention provide a prolonged fertilizing effect combined with an enhanced conversion of urea in plant-absorbable nitrogen, mainly in form of nitrites and nitrates, advantageously accompanied by a reduced loss on nitrogen in form of gaseous ammonia. The nitrogen use efficiency is thereby significantly increased.

[0068] The invention is now described by the following non-limitative experimental examples.

[0069] EXPERIMENTALS

[0070] Example 1 - Preparation of granules

[0071] A granular product in accordance with the present invention (Fig. 1 B) has been prepared using the following components:

[0072] Urea 100 g

[0073] (a) Bacillus subtilis spores 1 .6E+09 CFU / mL 0.6 mL (9.6E+06 CFU / g urea)

[0074] (c) “wet” layer sunflower oil 0.6 mL, molasses 3.6 mL, water 0.6 mL

[0075] ( b +d) Sporosarcina [powder obtained by polar drying, mixes 1 :10 with maltodextrin, final content 1.8E+06 CFU / g urea] 10 g; biuret pasteurii 5g, calcium lignosulfonate 5g

[0076] (d) Anticaking agent maltodextrin 5g, biuret 5g and calcium lignosulfonate 10g

[0077] Component (c) is heated to 40°C in a water bath, then added to the 100g of urea, then stirred vigorously by Vortex Shaker for 30 seconds.

[0078] Component (b+d) is added to the resulting granules, and the granules are stirred vigorously by Vortex Agitator for 30 seconds. The excess of component (b+d) is recovered by mechanical sieve (stirring for 30 seconds).

[0079] The granules are mixed with 0.6 mL of component (a) and stirred vigorously by Vortex shaker for 30 seconds.

[0080] Component (d) is added to the resulting granules, and the granules are stirred vigorously by Vortex Agitator for 30 seconds. The excess of component is recovered by mechanical sieve (stirring for 30 seconds).

[0081] The resulting granules are stored in a dark plastic container and are ready for use.

[0082] Example 2 - Preparation of granules

[0083] A granular product in accordance with the present invention (Fig. 2b) has been prepared using the following components: Urea 50 g

[0084] (b) Bacillus subtilis spores 1 .2E+09 CFU / mL 0.3 mL (7.2+06 CFU / g)

[0085] (c) “wet” layer sunflower oil 0.3 mL, molasses 1 .8 mL, water 0.3 mL

[0086] ( b +d) Sporosarcina [powder obtained by polar drying, mixes 1 :10 with maltodextrin, final content 5E+06 CFU / kg urea] 5 g; biuret pasteurii 2.5g, calcium lignosulfonate 2.5g

[0087] (e) Anticaking agent maltodextrin 2.5g, biuret 2.5g and calcium lignosulfonate 5g

[0088] Component (c) is heated to 40°C in a water bath, then added to the 50g of urea, then stirred vigorously by Vortex Shaker for 30 seconds.

[0089] Component (b+d) is added to the resulting granules, and the granules are stirred vigorously by Vortex Agitator for 30 seconds. The excess of component (b+d) is recovered by mechanical sieve (stirring for 30 seconds).

[0090] The granules are mixed with 0.6 mL of component (a) and stirred vigorously by Vortex shaker for 30 seconds.

[0091] Component (d) is added to the resulting granules, and the granules are stirred vigorously by Vortex Agitator for 30 seconds. The excess of component is recovered by mechanical sieve (stirring for 30 seconds).

[0092] The resulting granules are stored in a dark plastic container and are ready for use.

[0093] Example 3 - Testing of granules

[0094] 3. 1 Generation of plant-absorbable nitrogen

[0095] Urea granules coated with microorganisms were tested with different crop to validate their performance in comparison with traditional granular urea.

[0096] Soil tests were conducted on 2 different conditions: soil treated with urea granules (reference - state of the art) and soil treated with urea coated granules coated according to Example 1

[0097] The experiments for each condition were repeated 11 times. For each condition the same procedure was followed:

[0098] A mass of 10 g soil was added to a small holed pot with a layer of filter paper inside, then, 10 mg urea reference or coated were added in the samples. After one hour, 100 mL of water was added to each pot in order to simulate the watering and promote the leaching of urea and related compounds. Then, leaching with 100 mL of water was further performed after 4 and 7 days of incubation at room temperature.

[0099] At the end of the experiment, the amount of urea, ammonia and nitrate in soil were extracted by solid-liquid extraction and quantified by colorimetric method with UV-Vis spectrophotometer.

[0100] The final data are represented in Figures 6A, 6B and 6C. The results are presented as the median of the 11 replicates for each condition and the error bar is the MAD i.e. the median absolute deviation.

[0101] 3.2 Evaluation of ammonia generation

[0102] A test for release of gaseous ammonia from soil was carried out by adding 10 mg of urea reference or coated in 10 g of soil, kept in an airtight container. The release of gaseous ammonia from soil was qualitatively assessed for 11 days of treatment by inserting a special sensor with a colorimetric indicator into the bottle cap. The tests were conducted in independent triplicates. The results are presented in the following Table.

[0103] Qualitative data: nd: gaseous ammonia non-detected; + / ++ / +++: qualitatively increasing concentrations of gaseous ammonia.

[0104] The results of Tests in 3.1 and 3.2 show that samples treated with coated urea, which is the subject of this invention, maintain a higher soil content of urea, ammonium and nitrate. This shows that the invention makes it possible to reduce leaching losses of urea and ammonia into the soil, while also increasing the nitrate content, derived from urea conversion.

[0105] 3.3 Evaluation of long-term fertilizer effect

[0106] The granules of Example 1 were administered as a fertilizer to greenhouse tomato plant crops at the reference concentration of 150 kg / hectare. Reference granular urea was used as a control. The plants were grown for 129 days. The experiments for each condition were repeated 4 times. For each condition the same procedure was followed: Pot of 4 L were filled up to 3 / 4 with soil then, 0.6 g of urea were place in the soil and the tomato plant were planted and covered by a layer of soil. 1 .3 g of urea were then placed above the soil. 200 mL of water was added to each pot in order to simulate the watering and promote the leaching of urea and related compounds.

[0107] Soil moisture, humidity and temperature of the greenhouse were monitored every 2 days. Parameters of main stem’s height, number of leaves, number of flowers, and number of fruits were measured twice a week. At the end of the campaign, all the fruits were harvested and the dry mass of the plant with and without roots was measured.

[0108] As a comparison between the reference and the invention, the number of fruits per plant was counted as an indicator of productivity and leaf length as an indicator of plant development.

[0109] The graph related to the number of fruits is presented in Figures 7A and 7B.

[0110] In both cases, the invention positively impacts on the measured parameters and allows for a significantly higher number of fruits than the reference treatment and an average leaf length.

[0111] Overall, these results allow us to state that the invention:

[0112] (i) Control the release of urea into the soil, reducing losses of gaseous nitrogen and promoting the accumulation of readily bio-accumulative nitrogen compounds such as nitrate

[0113] (ii) Due to the effects at (i) and the action of microorganisms, the invention enables higher productivity and better development of treated plants.

Claims

CLAIMS1. Urea granule provided with a multiple-layer coating, said coating comprising the following components, independently distributed among the layers:(a) an ammonium converting microorganism(b) an ureolytic microorganism(c) an oil-binder mixture(d) an anticaking agent2. The granule of claim 1 , wherein the ammonium converting microorganism is Bacillus subtilis.

3. The granule of claims 1 -2, wherein the ureolytic microorganism is Sporosarcina pasteurii.

4. The granule of claims 1-3, including layers, preferably one or two layers, which contain the components (a) and (c) in combination.

5. The granule of claims 1-4, including layers, preferably one or two layers, which contain the components (b) and (d) in combination.

6. The granule of claims 1-5, wherein the component (d) is present at least in the outermost coating layer.

7. The granule of claims 1-6, wherein the coating comprises 3, 4 or 5 layers.

8. The granule of claims 1-3, wherein the coating contains a layers sequence selected from sequences (l)-(XI):(-) layer not present9. The granule according to any of the preceding claims, wherein the microorganism (a) is applied as solid powder, preferably by spray drying, polar drying and / or lyophilization, and the microorganism (b) is applied in form of spores suspended in a liquid medium.

10. The granule according to any of the preceding claims, wherein the components (a) and (b) are present, independently from each other, in a ratio with urea comprised between 200 000 and 20 000 000 CFU / g urea.

11. The granule according to any of the preceding claims, wherein the oil is selected from castor oil, coconut oil, com oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, safflower oil, sesame oil, soybean oil, sunflower oil, nut oil, almond oil, hazelnut oil, macadamia oil, pecan oil, pine nut oil, pistachio oil, walnut oil, pumpkin seed oil, and mixtures thereof.

12. The granule according to any of the preceding claims, wherein the binder is selected among molasses, starch, dextran, gum arabic, guar gum, and mixtures thereof.

13. The granule according to any of the preceding claims, wherein the anticaking agent is selected from sodium lignosulfate, potassium lignosulfate, calcium lignosulfate, biuret, maltodextrin, carboxymethyl starch, tricalcium phosphate, powdered cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, calciumphosphate, sodium silicate, silicon dioxide, calcium silicate, magnesium trisilicate, talcum powder, sodium aluminosilicate, potassium aluminium silicate, calcium aluminosilicate, bentonite, zeolite, aluminium silicate, stearic acid, polydimethylsiloxane, and mixtures thereof.

14. A process to obtain the granule in accordance with claims 1 -13, comprising the steps of applying, to urea granules, a sequence of layers comprising the following components, independently distributed among the layers:(a) an ammonium converting microorganism(b) an ureolytic microorganism (c) an oil-binder mixture(d) an anticaking agent15. A granular product comprising granules in accordance with claims 1-13.

16. A composition comprising the granular product in accordance with claim 15.

17. Use of the product or composition in accordance with claims 15-16 as agricultural fertilizer.

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