Enzyme-containing coating for fertilizer products

Abstract

The present invention relates to a coated fertilizer product comprising a fertilizer particle having an outer surface and containing an inorganic phosphorus source, and a coating on at least part of the outer surface of the fertilizer particle, wherein the coating comprises at least one enzyme being a phytase. The invention further relates to a process for the preparation of a corresponding fertilizer product and to its use in crop cultivation.

Description

New International patent application Supersol Biyoteknoloji A.§.SUPBI.0003 WOENZYME-CONTAINING COATING FOR FERTILIZER PRODUCTS

[0001] The present invention relates to an enzyme-containing coating for a fertilizer product, a corresponding fertilizer product, a process for the preparation of a fertilizer product, and to corresponding uses in crop cultivation.Background of the invention

[0002] Conventional phosphorus-based fertilizers often suffer from issues such as low bioavailability or uptake of inorganic phosphorus, residual phosphate accumulation in soil and increased water footprint. Additionally, the use of such conventional fertilizers contributes to increased transport costs and carbon footprint due to their bulk compared to more effective fertilizers.

[0003] In the prior art, there have been some attempts to improve or control the release of inorganic phosphorus from conventional fertilizers, such as diammonium phosphate (DAP) or triple super phosphate (TSP), and to increase the bioavailability or uptake of phosphorus by plants.

[0004] For example, the efficacy of DAP coated with bacteria has been investigated by Murad et al. in Sei. Rep. 2024, 14, 11389-11398. In turn, the effect of coating TSP with different biopolymers, such as lignin or carrageenan, has been investigated by Fertahi et al. in ACS Sustainable Chem. Eng. 2019, 7, 10371-10382.

[0005] Nevertheless, there is still a continuous need for improvements, especially with regard to efficiency and environmental performance. The present invention aims to address these challenges by providing a fertilizer product that allows for improved bioavailability and increased total uptake of phosphorus by plants, reduced residual phosphate in the soil, optimized water and carbon footprints.Summary of the invention

[0006] According to one aspect of the present invention, a fertilizer product is provided which comprises:- a fertilizer particle having an outer surface, and containing an inorganic phosphorus source, and- a coating on at least part of the outer surface of the fertilizer particle characterized in that the coating comprises at least one enzyme being a phytase.

[0007] Another aspect of the present invention relates to a process for the preparation of a fertilizer product, preferably a fertilizer product as defined hereinabove, the process comprising the following steps:(a) providing a fertilizer particle having an outer surface, and comprising an inorganic phosphorus source;(b) providing a coating composition, the coating composition comprising at least one enzyme being a phytase;(c) coating the outer surface of the fertilizer particle provided in step (a) at least partially with the coating composition provided in step (b) to obtain a coated fertilizer particle; and(d) optionally drying the coated fertilizer particle obtained in step (c).

[0008] Another aspect of the present invention relates to a fertilizer product obtainable by the process disclosed herein.

[0009] Stiller another aspect of the present invention relates to the use of the fertilizer product disclosed herein for crop cultivation, in particular com (Zea mays).

[0010] Advantageous embodiments of the present invention are defined in the dependent claims.

[0011] According to one embodiment, the inorganic phosphorus source of the fertilizer particle is selected from one or more of the following:- superphosphates, preferably single superphosphate, double superphosphate and triple superphosphate, more preferably double superphosphate and triple superphosphate- ammonium phosphates, preferably monoammonium phosphate and diammonium phosphate- any combination thereof.

[0012] According to another embodiment, the fertilizer particle further comprises an inorganic nitrogen source or an inorganic potassium source.

[0013] According to an alternative embodiment, the fertilizer particle further comprises an inorganic nitrogen source and an inorganic potassium source.

[0014] According to another embodiment, the fertilizer particle further comprises at least one element selected from the group consisting of calcium, magnesium, sulfur and combinations thereof and / or at least one trace element selected from the group consisting of boron, copper, iron, manganese, molybdenum, zinc and combinations thereof.

[0015] According to another embodiment, the coating further comprises at least one polymer, preferably a biopolymer, more preferably a biopolymer selected from the group consisting of starch, alginate, lignin, carrageenan, chitosan and combinations thereof, still more preferably alginate, most preferably sodium alginate.

[0016] According to another embodiment, the coating further comprises at least one bacterial species capable of converting organic phosphorus into inorganic phosphorus and / or increasing solubility of inorganic phosphorus, preferably the bacterial species is Bacillus spp., more preferably the bacterial species is selected from the group consisting of Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus subtilis and any combination thereof, most preferably the bacterial species is Bacillus amyloliquefaciens and / or Bacillus megaterium.

[0017] According to another embodiment, the coating further comprises at least one microbial enzyme, preferably selected from the group consisting of a-amylase, cellulase and chitinase.

[0018] According to another embodiment, the coating further comprises at least one preservative, preferably the preservative is a non-reducing sugar, most preferably trehalose; and / or the coating further comprises at least one amino acid.

[0019] According to still another embodiment, the coating includes a first coating layer and a second coating layer different from the first coating layer and provided above the first coating layer, wherein the phytase is present in the first coating layer.

[0020] According to still another embodiment, the fertilizer product has an average coating weight ranging from 0.01 to 50 wt.%, preferably from 0.05 to 35 wt.%, most preferably from 0.1 to 10 wt.%, for example 1 to 5 wt.%, based on the total weight of the fertilizer product.

[0021] Where the term “comprising” is used herein, it does not exclude other elements. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0022] Terms like “obtainable” or “definable” and “obtained” or “defined” are used interchangeably. This e.g. means that, unless the context clearly dictates otherwise, the term “obtained” does not mean to indicate that e.g. an embodiment must be obtained by e.g. the sequence of steps following the term “obtained”, though such a limited understanding is always included by the terms “obtained” or “defined” as a preferred embodiment.Detailed description

[0023] In the following, advantageous embodiments will be discussed in more detail. It is to be understood that these details apply to all aspects of the present invention which includes the fertilizer product, its preparation, and corresponding uses.

[0024] The fertilizer product according to the present invention comprises:- a fertilizer particle having an outer surface, and containing an inorganic phosphorus source, and- a coating on at least part of the outer surface of the fertilizer particle characterized in that the coating comprises at least one enzyme being a phytase.

[0025] The inventors have found that applying a phytase-containing layer on a conventional phosphorus-based fertilizer provides sufficient phytase activity and thereby allows for an increased total uptake of phosphorus by plants. As a result, this leads to a faster and more productive build-up of biomass.

[0026] In principle, the fertilizer particle of the fertilizer product according to the present invention may be any kind of fertilizer known in the art, and being in the form of particles as long as it contains an inorganic phosphorus source. Usually, inorganic phosphorus is provided in the form of phosphate. Therefore, in one embodiment, the inorganic phosphorus source comprises phosphate (PCM3-, HPCU2-, H2PO4- and any combination thereof), preferably dihydrogen phosphate (H2PO4-), more preferably monocalcium phosphate (Ca(H2PO4)2).

[0027] For example, according to one embodiment, the inorganic phosphorus source of the fertilizer particle is selected from one or more of the following, including any combination thereof:- superphosphates, preferably single superphosphate, double superphosphate and triple superphosphate, more preferably double superphosphate and triple superphosphate- ammonium phosphates, preferably monoammonium phosphate and diammonium phosphate.

[0028] The term “superphosphate” refers to a group of phosphorus-based fertilizers obtainable by breaking down naturally occurring, water-insoluble calcium phosphate with mineral acids. This term covers single superphosphate (SSP), double superphosphate (DSP) and triple superphosphate (TSP). All these fertilizers are granular, solid products that contain phosphate that can be washed out with water, i.e. water-soluble.

[0029] Specifically, single superphosphate (SSP) is obtainable by extraction of phosphate rock with sulfuric acid. Single superphosphate is approximately a 1 :1 mixture of Ca(H2PO4)2 and CaSCU. Triple superphosphate (TSP) is obtainable by treating phosphate rock with phosphoric acid. It primarily consists of monocalcium phosphate, Ca(H2PO4)2. Double superphosphate (DSP) is obtainable by extraction of phosphate rock with a mixture of phosphoric and sulfuric acids. Its chemical composition thus may be seen to correspond to a mixture of single and triple superphosphate.

[0030] Typically, fertilizer-grade triple superphosphate contains 40 to 50 wt.%, preferably approx. 45 wt.%, P2O5 equivalents based on its total composition. In turn, single superphosphate contains 15 to 25 wt.%, preferably approx. 20 wt.%, P2O5 equivalents based on its total composition. Accordingly, double superphosphate contains 30 to 40 wt.%, preferably approx. 35 wt.%, P2O5 equivalents based on its total composition.

[0031] Additionally or alternatively to superphosphates, the inorganic phosphorus source of the fertilizer particle is selected from ammonium phosphates. In a preferred embodiment, the inorganic phosphorus source is monoammonium phosphate (MAP), diammonium phosphate (DAP) or a combination thereof.

[0032] In some embodiments, the fertilizer particle essentially consists of or (exclusively) consists of one or more of the inorganic phosphorus sources disclosed herein. For example, the fertilizer particle essentially consists of or (exclusively) consists of diammonium phosphate (DAP) or triple superphosphate (TSP).

[0033] In a further embodiment, the fertilizer particle further comprises an inorganic nitrogen source or an inorganic potassium source. Accordingly, the fertilizer particle of the fertilizer product may include one of the following: an inorganic phosphorus source and an inorganic nitrogen source, or an inorganic phosphorus source and an inorganic potassium source. Alternatively, the fertilizer particle further comprises an inorganic nitrogen source and an inorganic potassium source. In this embodiment, the fertilizer particle of the fertilizer product thus may comprise acombination of an inorganic phosphorus source, an inorganic nitrogen source and an inorganic potassium source (NPK fertilizer).

[0034] In each of the foregoing embodiments, the fertilizer particle may further comprise at least one element selected from the group consisting of calcium, magnesium, sulfur and combinations thereof and / or at least one trace element selected from the group consisting of boron, copper, iron, manganese, molybdenum, zinc and combinations thereof.

[0035] The fertilizer product of the present invention is characterized in that it contains at least one enzyme being a phytase which is provided in the form of a coating on at least part of the surface of the fertilizer particle. In turn, the coating may further comprise at least one polymer which may be any polymer that is suitable for application on granular fertilizers (fertilizer particles), preferably by spray-coating.

[0036] According to one embodiment, the at least one polymer is a biopolymer (as opposed to non-biopolymers). As used herein, the term “biopolymer” refers to natural polymers produced by the cells of living organisms and corresponding structurally identical synthetic polymers.

[0037] Regardless of the structure and origin of the polymer, it is advantageous if the polymer is biodegradable with regard to the intended use. Therefore, in one embodiment, the polymer is biodegradable. In a particularly preferred embodiment, the polymer is a biodegradable biopolymer.

[0038] In cases where the at least one polymer is a biopolymer, said biopolymer may be selected from the group consisting of starch, alginate, lignin, carrageenan, chitosan and combinations thereof. A particularly preferred biopolymer is alginate, more preferably an alkali metal or alkaline earth metal alginate, optionally in combination with com starch. Particularly suitable alkali metals or alkaline earth metals in alginates include sodium, potassium and magnesium. Most preferably, the biopolymer is sodium alginate, optionally in combination with com starch.

[0039] The enzyme of the fertilizer product according to the present invention is a phytase. Phytases are phosphatase enzymes that catalyze the hydrolysis of phyticacid (myo-inositol hexakisphosphate) - an indigestible, organic form of phosphorus that is found in many plant tissues, especially in grains and oil seeds - and releases a usable form of inorganic phosphorus.

[0040] In principle, there are no limitations regarding the phytase suitable for the purposes of the present invention. However, in a preferred embodiment, the phytase is obtained from microorganisms (microbial phytase), more preferably from bacteria (bacterial phytase).

[0041] Microbes are the most preferred source of phytases owing to the fact that they are economically feasible and higher yields can be achieved. Furthermore, the product can be easily modified, conditions can be easily optimized, and production is independent of seasonal fluctuations. Also, many microbial phytases are known to be more stable and show greater catalytic activity compared to plant-based phytase. Specifically, such phytases are reported to be stable at a wide range of temperatures and pH. Corresponding phytases have been described, for example, by Balwani et al. in BiocataL Agric. Biotechnol. 2017, 12, 23-29 and by Gurung et al. in BioMed Res. Int. 2013, Article ID 329 121.

[0042] A suitable commercially available phytase is Fitazim which is a liquid formulation having a phytase activity of approx. 0.27 U / ml. This product was registered by Supersol Biotechnology Inc. (License number 1249) with the Ministry of Agriculture and Forestry of Turkiye under registration number 2022TO2086.

[0043] The coating of the fertilizer product according to the present invention may comprise additional components.

[0044] For example, in one embodiment, the coating further comprises at least one bacterial species capable of converting organic phosphorus into inorganic phosphorus and / or increase solubility of inorganic phosphorus. It has been found that the presence of these bacterial species further contributes to the total uptake of phosphorus, in particular in combination with phytase.

[0045] In principle, it is possible to use any bacterial species that is capable of converting organic phosphorus into inorganic phosphorus and / or increasing solubilityof inorganic phosphorus. This includes both Gram-negative and Gram-positive bacteria. In a preferred embodiment, the bacterial species belongs to the group of so- called plant growth-promoting rhizobacteria (also known as PGPRs).

[0046] Examples of suitable Gram-negative bacteria include Pseudomonas spp. , in particular Pseudomonas fluorescens, and Pantoea spp., in particular Pantoea agglomerans.

[0047] Preferably, in one embodiment of the present invention, the bacterial species is Gram-positive. More preferably, the bacterial species is Bacillus spp., still more preferably the bacterial species is selected from the group consisting of Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus subtilis and any combination thereof. Most preferably, the bacterial species is Bacillus amyloliquefaciens and / or Bacillus megaterium.

[0048] Among the foregoing preferred bacterial species, particularly useful bacterial strains are Bacillus amyloliquefaciens TV 17-C, and Bacillus megaterium TV-3D. A preferred strain of Bacillus amyloliquefaciens TV 17-C is registered under GenBank accession no. MK478843.1.

[0049] Accordingly, in an exemplary embodiment of the present invention, the inorganic phosphorus source comprises phosphate, preferably any of the superphosphates or ammonium phosphates disclosed herein (e.g., TSP or DAP), and (if present) the at least one polymer is a biopolymer, preferably selected from the group consisting of starch, alginate, lignin, carrageenan, chitosan and combinations thereof, more preferably any of the alginates disclosed herein (e.g., sodium alginate).

[0050] In another exemplary embodiment, the inorganic phosphorus source comprises phosphate, preferably any of the superphosphates or ammonium phosphates disclosed herein (e.g., TSP or DAP), and the bacterial species is Bacillus spp., preferably Bacillus amyloliquefaciens and / or Bacillus megaterium (e.g., Bacillus amyloliquefaciens TV 17-C or Bacillus megaterium TV-3D).

[0051] In another exemplary embodiment, the at least one polymer (if present) is a biopolymer, preferably selected from the group consisting of starch, alginate, lignin,carrageenan, chitosan and combinations thereof, more preferably any of the alginates disclosed herein (e.g., sodium alginate), and the bacterial species is Bacillus spp., preferably Bacillus amyloliquefaciens and / or Bacillus megaterium (e.g., Bacillus amyloliquefaciens TV 17-C or Bacillus megaterium TV-3D).

[0052] In still another exemplary embodiment, the inorganic phosphorus source comprises phosphate, preferably any of the superphosphates or ammonium phosphates disclosed herein (e.g., TSP or DAP), the at least one polymer (if present) is a biopolymer, preferably selected from the group consisting of starch, alginate, lignin, carrageenan, chitosan and combinations thereof, more preferably any of the alginates disclosed herein (e.g., sodium alginate), and the bacterial species is Bacillus spp., preferably Bacillus amyloliquefaciens and / or Bacillus megaterium (e.g., Bacillus amyloliquefaciens TV 17-C or Bacillus megaterium TV-3D).

[0053] According to another embodiment, the coating further comprises at least one microbial enzyme, preferably selected from the group consisting of a-amylase, cellulase and chitinase. These additional enzymes may be particularly useful when a corresponding biopolymer is used as the at least one polymer present in the coating. For example, the use of a-amylase may be particularly advantageous when the coating contains starch.

[0054] According to another embodiment, the coating further comprises at least one preservative, preferably the preservative is a non-reducing sugar, most preferably trehalose. Trehalose and other non-reducing sugars are important carbon sources for many bacteria and can support bacterial growth. Additionally or alternatively, the coating may further comprise at least one amino acid, preferably at least one plantbased amino acid.

[0055] The coating which comprises at least one phytase enzyme may be applied in one or more coating layers.

[0056] According to a first embodiment, the fertilizer product includes a coating layer (“all-in-one coating”) comprising the at least one phytase enzyme and, optionally, one or more of the other components disclosed herein (e.g., at least one polymer, an inorganic nitrogen source, an inorganic potassium source, other elements, at least onebacterial species, at least one enzyme other than phytase, at least one preservative and / or at least one amino acid). In a preferred variant of this embodiment, the fertilizer product includes a coating layer which comprises at least one enzyme being a phytase, at least one polymer and, optionally, one or more of the other components disclosed herein (e.g., an inorganic nitrogen source, an inorganic potassium source, other elements, at least one bacterial species, at least one enzyme other than phytase, at least one preservative and / or at least one amino acid). In another preferred variant of this embodiment, the fertilizer product includes a coating layer which comprises at least one enzyme being a phytase, at least one bacterial species and, optionally, one or more of the other components disclosed herein (e.g., at least one polymer, an inorganic nitrogen source, an inorganic potassium source, other elements, at least one enzyme other than phytase, at least one preservative and / or at least one amino acid). In still another preferred variant of this embodiment, the fertilizer product includes a coating layer which comprises at least one enzyme being a phytase, at least one polymer, at least one bacterial species and, optionally, one or more of the other components disclosed herein (e.g., an inorganic nitrogen source, an inorganic potassium source, other elements, at least one enzyme other than phytase, at least one preservative and / or at least one amino acid).

[0057] In some embodiments, the same coating composition is provided in one coating layer (“single coating”) or in several coating layers (e.g., 2, 3, 4 or 5 layers), together referred to as “the coating”, whereby particularly high coating weights can be achieved. In this case, each coating layer may have a composition as disclosed herein for the first embodiment.

[0058] In a second embodiment, different coating compositions are provided as separate coating layers, together also referred to as “the coating”. This design has proven to be particularly advantageous. Apart from achieving high coating weights, it is possible to shield pH sensitive components, such as bacteria, from the acidic environment that is typically found in direct proximity to the fertilizer particle or a fertilizer product when in contact with soil. Therefore, the coating may comprise a first coating layer and a second coating layer different from the first coating layer wherein the second coating layer is provided above the first coating layer, and wherein the atleast one enzyme being a phytase is present in the first coating layer. Preferably, the at least one bacterial species as disclosed herein is present in the second coating layer.

[0059] In a particularly preferred variant of the second embodiment, the coating comprises a first coating layer comprising the at least one enzyme being a phytase, and a second coating layer being different from the first coating layer and comprising at least one bacterial species as described hereinabove, wherein the second coating layer is provided above the first coating layer, and wherein the first and, optionally, the second coating layer independently comprises a polymer, preferably a biopolymer, more preferably selected from the group consisting of starch, alginate, lignin, carrageenan, chitosan, and combinations thereof, and most preferably any of the alginates disclosed herein (e.g., sodium alginate). Preferably, the bacterial species is Bacillus spp., more preferably Bacillus amyloliquefaciens and / or Bacillus megaterium (e.g., Bacillus amyloliquefaciens TV 17-C or Bacillus megaterium TV-3D).

[0060] Unless specified otherwise, the coating of the fertilizer product according to the present invention may be provided either directly or indirectly on at least part of the outer surface of the fertilizer particle. Accordingly, in one embodiment, the coating of the present invention is provided directly on the outer surface of the fertilizer particle or, in an alternative embodiment, a polymer base layer is provided directly on the outer surface of the fertilizer particle so that the coating of the present invention is provided above the polymer base layer. The polymer base layer may be characterized by the presence of any of the polymers disclosed herein while being essentially free or free of enzymes and / or bacterial species. Optionally, an additional polymer protective layer may be provided as the outermost layer (e.g., in order to increase the shelf life of the fertilizer product).

[0061] In a preferred embodiment, the coating of the fertilizer product according to the present invention is provided directly on at least part of the outer surface of the fertilizer particle which means that the coating layer (in cases where the coating consist of one coating layer) or the first coating layer (in cases where there are several coating layers) is in direct contact with the fertilizer particle.

[0062] According to still another embodiment, the fertilizer product has an average coating weight ranging from 0.01 to 50 wt.%, preferably from 0.05 to 35 wt.%, most preferably from 0.1 to 10 wt.%, for example 1 to 5 wt.%, based on the total weight of the fertilizer product.

[0063] According to another aspect of the present invention, a process is provided for the preparation of a fertilizer, wherein said process comprises the following steps:(a) providing a fertilizer particle having an outer surface, and comprising an inorganic phosphorus source as disclosed herein;(b) providing a coating composition, the coating composition comprising at least one enzyme being a phytase as disclosed herein;(c) coating the outer surface of the fertilizer particle provided in step (a) at least partially with the coating composition provided in step (b) to obtain coated fertilizer particles; and(d) optionally drying the coated fertilizer particle obtained in step (c).

[0064] In a preferred embodiment, the process is a process for the preparation of a fertilizer product as described above.

[0065] A suitable commercially available phytase is Fitazim which is a liquid formulation. Alternatively, the phytase can be provided as a wettable powder formulation which is obtainable by adding, e.g., 1 wt.% trehalose and 2 wt.% glycerol, to a liquid phytase-containing formulation, and drying the obtained composition by any method known to the skilled person, for example by spray-drying.

[0066] For example, the coating composition provided in step (b) may further comprise other components as disclosed herein, including at least one polymer, an inorganic nitrogen source, an inorganic potassium source, other elements, at least one bacterial species, at least one enzyme other than phytase, at least one preservative and / or at least one amino acid.

[0067] Where used, the bacterial species can be provided as a liquid formulation in a suitable medium. An exemplary medium may contain one or more (preferably all) of the following: glucose (e.g., 10 g / l), yeast extract (e.g., 5 g / l), ZnSO4-7H2O (e.g.,1 mg / l), MgSO4'7H2O (e.g., 1 mg / l), MnCl2 (e.g., 1 mg / l), polyethylene glycol (e.g., 1 g / l). Alternatively, the bacterial species can be provided as a wettable powder which can be obtained from the liquid formulation by using a centrifuge to separate the bacteria from the liquid growth medium. The pellet obtained after centrifugation is dried using spray drying. The total number of viable cells can be tested using the spread plate method.

[0068] It is possible to repeat coating step (c) several times (e.g. , 2, 3 or 4 times) so that the coating of the fertilizer product is constituted by several coating layers (e.g., 2, 3, 4 or 5) having the same composition.

[0069] In an alternative embodiment, the coating of the fertilizer product is constituted by two or more different coating layers. In this case, the process of the present invention comprises the provision of one or more further coating compositions different from the coating composition provided in step (b) and one or more additional coating steps different from coating step (c).

[0070] As explained above, the coating of this embodiment may comprise a first coating layer and a second coating layer different from the first coating layer wherein the second coating layer is provided above the first coating layer, and wherein the at least one enzyme being a phytase is present in the first coating layer. Preferably, at least one bacterial species as described above is present in the second coating layer. Accordingly, in one embodiment, the process of the present invention comprises the following steps:(a) providing a fertilizer particle having an outer surface, and comprising an inorganic phosphorus source as disclosed herein;(b1) providing a first coating composition, the coating composition comprising at least one enzyme being a phytase as disclosed herein;(b2) providing a second coating composition, the second coating composition comprising at least one bacterial species as disclosed herein;(c1) coating the outer surface of the fertilizer particle provided in step (a) at least partially with the coating composition provided in step (b1);(c2) coating the product obtained in step (c1 ) at least partially with the coating composition provided in step (b2); and(d) optionally drying the product obtained in step (c2).

[0071] The first coating composition provided in step (b1) and / or the second coating composition provided in step (b2) may further comprise at least one polymer. In a preferred embodiment of this process, the polymers are biopolymers, more preferably the polymers are independently selected from the group consisting of starch, alginate, lignin, carrageenan, chitosan and combinations thereof, and most preferably any of the alginates disclosed herein (e.g., sodium alginate). Preferably, the bacterial species is Bacillus spp., more preferably Bacillus amyloliquefaciens and / or Bacillus megaterium (e.g. , Bacillus amyloliquefaciens TV 17-C or Bacillus megaterium TV-3D).

[0072] As explained above, a polymer base layer may be provided directly on the outer surface of the fertilizer particle so that the coating of the present invention is provided above said polymer base layer. The polymer base layer may be characterized by the presence of any of the polymers disclosed herein while being essentially free or free of enzymes and / or bacterial species. Optionally, a polymer protective layer may be provided as the outermost layer.

[0073] In a preferred embodiment, the coating of the fertilizer product according to the present invention is provided directly on at least part of the outer surface of the fertilizer particle. This means that the coating layer (in cases where the coating consists of one coating layer) or the first coating layer (in cases where there are several different or identical coating layers) is in direct contact with the fertilizer particle.

[0074] Another aspect of the present invention relates to a fertilizer product obtainable by the process disclosed herein.

[0075] Still another aspect of the present invention relates to the use of at least one enzyme, preferably phytase, as additive in a fertilizer product containing an inorganic phosphorus source. In a preferred embodiment, the enzyme is used as additive for increasing the total uptake of phosphorus by plants in fertilization. The inorganic phosphorus source may be any inorganic phosphorus source disclosed hereinabove. Similarly, the at least one enzyme may be any enzyme, preferably aphytase, as disclosed herein. In this aspect, the fertilizer product may be provided in the form of particles wherein the at least one enzyme may be associated with the fertilizer particles, for example as additive incorporated into the fertilizer particles or, preferably, as a coating provided on at least part of the outer surface of the fertilizer particles. In other embodiments, the fertilizer product according to this aspect may include one or more further components disclosed herein, including an inorganic nitrogen source, an inorganic potassium source, other elements, at least one bacterial species, at least one enzyme other than phytase, at least one preservative and / or at least one amino acid.

[0076] Still another aspect of the present invention relates to the use of the fertilizer product disclosed herein for crop cultivation, in particular com (Zea mays). Further examples cover cereal cultivation in general (in particular wheat, emmer wheat, triticale, spelt, rye, barley, oats, millet, quinoa and rice), legumes (in particular soy), all kind of vegetable, and other crops (e.g., cotton and rapeseed). It has been found that the use of the fertilizer product according to the present invention leads to an accelerated or stronger growth, and higher yield compared to, for example, conventional TSP or DAP.

[0077] In one embodiment, the fertilizer product is used in crop cultivation, wherein the dosage is less than 100 g / m2, preferably less than 50 g / m2, and more preferably less than 35 g / m2, for example 25 g / m2or less. In a preferred embodiment, the fertilizer product is used in com cultivation (Zea mays), wherein the dosage is less than 100 g / m2, preferably less than 50 g / m2, and more preferably less than 35 g / m2, for example 25 g / m2or less. Suitable ranges for these uses include 1 to 100 g / m2, preferably 5 to 50 g / m2, and more preferably 10 to 35 g / m2, for example approx. 25 g / m2.

[0078] The scope and interest of the invention may be better understood on the basis of the following examples which are intended to illustrate embodiments of the present invention. However, they are not to be construed to limit the scope of the claims in any manner whatsoever.Examples

[0079] In the following examples, the performance of different fertilizer products according to the present invention was tested under field conditions using different coating designs and different dosages.Preparation of fertilizer productsEnzyme component

[0080] A commercially available microbial phytase enzyme (Fitazim) was used as the enzyme component (> 0.27 U / ml microbial phytase enzyme activity).Bacteria component

[0081] Production of liquid medium containing bacteria (> 1 x io8cfu / ml) was carried out in a growth medium (glucose 10 g / l, yeast extract 5 g / l, ZnSGU FhO 1 mg / l, MgSO4-7H2O 1 mg / l, MnCl2 1 mg / l, polyethylene glycol 1 g / l). After adjusting the pH of the medium to 7.00, it is sterilized in an autoclave at 121 °C for 15 minutes. Then, bacterial inoculation is performed into this medium (the inoculum ratio varies between 2 % and 5 %). After inoculating the bacteria into the medium, it is incubated in a shaking incubator at 180 rpm and 30°C for 48 hours. After incubation, the total number of viable cells is tested using the spread plate method, and the bacterial content is determined.

[0082] This way, two different liquid formulations were prepared, one containing Bacillus amyloliquefaciens (TV 17-C) and the other containing Bacillus megaterium (TV-3D).Enzyme coating (E)

[0083] Sodium alginate (1 wt.%), com starch (1 wt.%), amino acids (1 wt.%), and preservative (trehalose, 1 wt.%) were mixed with Fitazim.Bacteria coating with one bacterial species (B1)

[0084] The bacteria component prepared as a liquid formulation described above was directly used as coating formulation.

[0085] Bacterial species used: Bacillus amyloliquefaciens (TV 17-C)Bacteria coating with one bacterial species (B2)

[0086] The bacteria component prepared as a liquid formulation described above was directly used as coating formulation.

[0087] Bacterial species used: Bacillus megaterium (TV-3D)Bacteria coating with two bacterial species (B 1 / B2)

[0088] The bacteria components prepared as a liquid formulation described above were mixed in a 1 :1 ratio and directly used as coating formulation.

[0089] Bacterial species used: Bacillus amyloliquefaciens (TV 17-C) + Bacillus megaterium (TV-3D)Coating of fertilizer particles

[0090] Different fertilizers were provided according to the following scheme:

[0091] In all cases, commercially available triple super phosphate from Gubreta§ Inc. was used as the fertilizer particle and coated by spraying. The coating process was carried out by use of a lab scale rotary drum equipped with a spray nozzle. For this purpose, the granular fertilizer was placed in the drum and rotated while thecorresponding coating composition was applied by spraying. The same rotary drum was used for each coating step. It was cleaned after each coating step and the spray nozzle was replaced. The coating weight was between 1 and 5 wt.%, based on the total weight of the fertilizer product.Experimental design of field trials and application details

[0092] All experiments were conducted in a greenhouse in Kumluca, Antalya (Turkiye).

[0093] The setup included two blocks referred to as Block A and Block B, each having 14 m2in size. Each of these blocks was separated into seven plots of 2 m2size and tested with a respective one of the fertilizer products according to Examples 1 to 7.

[0094] Garden cress (Lepidium sativum) was planted in each plot with 7 g of seeds per plot. The respective fertilizer product was used both above and below the seeds, mixed, and 20 liters of water were added per plot:Block A: 50 g / m2fertilizer productBlock B: 25 g / m2fertilizer productObservation periodsDay 0

[0095] Start of trials, sowingDay 3

[0096] It was observed that the seeds started to germinate. The formation of hypocotyls (the stem part below the seed leaves) and cotyledons (the first leaves from the seed) began.Day 10

[0097] The development of hypocotyls and cotyledons continued. The first signs of true leaf primordia were noted.Day 14

[0098] True leaf primordia began to form in plots 1 , 2, 4, 5, and 6. Development in the other plots was slightly behind.From day 25

[0099] The growth of the plants was closely monitored and photographed regularly.Results - Block A

[0100] Day 25 (see Fig. 1a)

[0101] Day 47 (see Fig. 1 b)

[0102] Day 83 (see Fig. 1c)Results - Block B

[0103] Day 25 (see Fig. 2a)

[0104] Day 47 (see Fig. 2b)

[0105] Day 83 (see Fig. 2c)Comparison at half dose

[0106] Example B1 (left) vs. Example A7 (right); see Fig. 3a

[0107] Example B2 (left) vs. Example A7 (right); see Fig. 3b

[0108] Example B3 (left) vs. Example A7 (right); see Fig. 3c

[0109] Example B4 (left) vs. Example A7 (right); see Fig. 3d

[0110] Example B5 (left) vs. Example A7 (right); see Fig. 3e

[0111] Example B6 (left) vs. Example A7 (right); see Fig. 3fCorn cultivation (Zea mays)All-in-one coating with two bacterial species (E+B 1 / B2)Sodium alginate (1 wt.%), com starch (1 wt.%), amino acids (1 wt.%), and preservative (trehalose, 1 wt.%) were mixed with commercial Fitazim (> 0.27 U / ml). Subsequently, Bacillus amyloliquefaciens (TV 17-C) and Bacillus megaterium (TV-3D) as described above were added.Coating of fertilizer particles

[0112] Different fertilizer products were provided according to the following scheme, and using the coating process described above:

[0113] The above fertilizer products were used in the following field trials and compared against TSP and DAP, respectively:

[0114] The field trials with Zea mays showed better performance with the fertilizer product of the present invention even at half dose compared to both conventional TSP and DAP.Conclusion

[0115] The above trials show that the fertilizer product of the present invention performs better than conventional fertilizers, such as TSP or DAP, for both Lepidium sativum and Zea mays.

[0116] Specifically, better performance means accelerated or stronger growth, and higher yield compared to, for example, conventional TSP or DAP. This observation is made even when the dosage of the fertilizer product according to the present invention is lower (25 g / m2) than the dosage of TSP or DAP (50 g / m2).

Claims

Claims1. A fertilizer product comprising:- a fertilizer particle having an outer surface, and containing an inorganic phosphorus source, and- a coating on at least part of the outer surface of the fertilizer particle characterized in that the coating comprises at least one enzyme being a phytase.

2. The fertilizer product according to claim 1 , wherein the inorganic phosphorus source is selected from one or more of the following:- superphosphates, preferably single superphosphate, double superphosphate and triple superphosphate, more preferably double superphosphate and triple superphosphate- ammonium phosphates, preferably monoammonium phosphate and diammonium phosphate- any combination thereof.

3. The fertilizer product according to any of claims 1 or 2, wherein the fertilizer particle further comprises an inorganic nitrogen source or an inorganic potassium source.

4. The fertilizer product according to any of claims 1 or 2, wherein the fertilizer particle further comprises an inorganic nitrogen source and an inorganic potassium source.

5. The fertilizer product according to any of claims 1 to 4, wherein the fertilizer particle further comprises at least one element selected from the group consisting of calcium, magnesium, sulfur and combinations thereof and / or at least one trace element selected from the group consisting of boron, copper, iron, manganese, molybdenum, zinc and combinations thereof.

6. The fertilizer product according to any of claims 1 to 5, wherein the coating further comprises at least one polymer, preferably a biopolymer, more preferably a biopolymer selected from the group consisting of starch, alginate, lignin, carrageenan, chitosan and combinations thereof, still more preferably alginate, most preferably sodium alginate.

7. The fertilizer product according to any of claims 1 to 6, wherein the coating further comprises at least one bacterial species capable of converting organic phosphorus into inorganic phosphorus and / or increasing solubility of inorganic phosphorus, preferably the bacterial species is Bacillus spp., more preferably the bacterial species is selected from the group consisting of Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus subtilis and any combination thereof, most preferably the bacterial species is Bacillus amyloliquefaciens and / or Bacillus megaterium.

8. The fertilizer product according to any of claims 1 to 7, wherein the coating further comprises at least one microbial enzyme, preferably selected from the group consisting of a-amylase, cellulase and chitinase.

9. The fertilizer product according to any of claims 1 to 8, wherein the coating further comprises:- at least one preservative, preferably the preservative is a non-reducing sugar, most preferably trehalose; and / or- at least one amino acid.

10. The fertilizer product according to any of claims 1 to 9, wherein the coating includes a first coating layer and a second coating layer different from the first coating layer and provided above the first coating layer, wherein the phytase is present in the first coating layer.

11. The fertilizer product according to any of claims 1 to 10, wherein the fertilizer product has average coating weight ranging from 0.01 to 50 wt.%, preferably from 0.05to 35 wt.%, most preferably from 0.1 to 10 wt.%, based on the total weight of the fertilizer product.

12. A process for the preparation of a fertilizer product, the process comprising the following steps:(a) providing a fertilizer particle having an outer surface, and comprising an inorganic phosphorus source;(b) providing a coating composition, the coating composition comprising at least one enzyme being a phytase;(c) coating the outer surface of the fertilizer particle provided in step (a) at least partially with the coating composition provided in step (b) to obtain a coated fertilizer particle; and(d) optionally drying the coated fertilizer particle obtained in step (c).

13. The process according to claim 12, wherein the fertilizer product is a fertilizer product according to any of claims 1 to 11.

14. A fertilizer product obtainable by the process according to claim 12.

15. Use of the fertilizer product according to any of claims 1 to 11 or claim 14 for crop cultivation, in particular com (Zea mays).

16. Use of phytase as additive in a fertilizer product containing an inorganic phosphorus source.

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