[go: up one dir, main page]

WO2023220779A1 - Fertiliser and/or soil improver composition, method of preparation and method of use - Google Patents

Fertiliser and/or soil improver composition, method of preparation and method of use Download PDF

Info

Publication number
WO2023220779A1
WO2023220779A1 PCT/AU2023/050414 AU2023050414W WO2023220779A1 WO 2023220779 A1 WO2023220779 A1 WO 2023220779A1 AU 2023050414 W AU2023050414 W AU 2023050414W WO 2023220779 A1 WO2023220779 A1 WO 2023220779A1
Authority
WO
WIPO (PCT)
Prior art keywords
fertiliser
soil improver
improver composition
urea
eutectic melt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2023/050414
Other languages
French (fr)
Inventor
Ramiz Boulos
Peter Simpson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Veratin Pty Ltd
Original Assignee
Veratin Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from NZ788293A external-priority patent/NZ788293A/en
Application filed by Veratin Pty Ltd filed Critical Veratin Pty Ltd
Publication of WO2023220779A1 publication Critical patent/WO2023220779A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F1/00Fertilisers made from animal corpses, or parts thereof
    • C05F1/005Fertilisers made from animal corpses, or parts thereof from meat-wastes or from other wastes of animal origin, e.g. skins, hair, hoofs, feathers, blood
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05CNITROGENOUS FERTILISERS
    • C05C9/00Fertilisers containing urea or urea compounds
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05CNITROGENOUS FERTILISERS
    • C05C3/00Fertilisers containing other salts of ammonia or ammonia itself, e.g. gas liquor
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F1/00Fertilisers made from animal corpses, or parts thereof
    • C05F1/007Fertilisers made from animal corpses, or parts thereof from derived products of animal origin or their wastes, e.g. leather, dairy products
    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09KMATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K17/00Soil-conditioning materials or soil-stabilising materials
    • C09K17/14Soil-conditioning materials or soil-stabilising materials containing organic compounds only
    • C09K17/18Prepolymers; Macromolecular compounds
    • C09K17/32Prepolymers; Macromolecular compounds of natural origin, e.g. cellulosic materials

Definitions

  • the present invention relates to a fertiliser and/or soil improver composition. More specifically, the present invention relates to one or more organic sources of plant nutrient(s), contained in a fertiliser and/or soil improver composition. The present invention also relates to a method of preparation and method of use of the fertiliser and/or soil improver composition.
  • Fertilisers and soil improvers are commonly used in the agricultural industry and even by home gardeners, and indeed fertilisers and soil improvers have many applications and uses between the two.
  • the growth of plants can be enhanced with the use of fertilisers by providing nutrients for uptake by the plant and/or with the use of soil improvers by enhancing the effectiveness of the soil in which the plant grows.
  • the nutritional aspect of fertilisers and soil improvers can involve the provision of one or more of the three main macronutrients, nitrogen (N), phosphorus (P) and potassium (K), for various growth enhancement activity.
  • a fertiliser and/or soil improver composition for use in enhancing the growth of plants, the composition comprising one or more organic sources of plant nutrient(s), obtained through deconstruction of one or more biological materials by a eutectic melt.
  • a method of preparing a fertiliser and/or soil improver composition comprising one or more organic sources of plant nutrient(s), the method comprising the steps of: a) forming a eutectic melt; and b) deconstructing one or more biological materials using the eutectic melt, to obtain the one or more organic sources of plant nutrient(s).
  • the eutectic melt in the first or second aspect of the present invention comprises at least one hydrogen bond donor (HBD) and at least one hydrogen bond acceptor (HBA).
  • the at least one HBA is a quaternary ammonium salt, such as a choline salt (including a choline halide), and the at least one HBD is a urea, thiourea or a derivative thereof.
  • the eutectic melt comprises a HBA in the form of a choline salt and a HBD in the form of a urea, thiourea, or a derivative of these.
  • the eutectic melt comprises a choline salt and a urea, or urea derivative.
  • the deconstruction of one or more biological materials by a eutectic melt in the first or second aspect of the present invention is at a temperature in a range of approximately: 135 - 170 °C, more preferably 140 - 165°C, or most preferably 145 - 160°C, or even 145 - 155 °C.
  • the temperatures/temperature ranges in the present invention are measured at ambient pressure, or atmospheric pressure, conditions.
  • the molar ratio of the eutectic melt (between the at least one HBD and the at least one HBA) in the first or second aspect of the present invention may be from about 20:1 to about 1:20.
  • the eutectic solvent comprises a choline salt (as a HBA) and a urea, thiourea or a derivative these (as a HBD)
  • the molar ratio of HBD: HBA is in a range of from 10:1 to 1:10; more preferably, from about 8:1 to about 3:1, or most preferably from about 7.5 to about 4:1 (e.g.
  • the HBD being a urea, thiourea or a derivative of these
  • the HBA the choline salt
  • the one or more organic sources of plant nutrient(s) includes, or is in the form of, one or more biomolecules, preferably selected from the group consisting of proteins, peptides, chitins, amino acids, nucleic acids, and combinations thereof.
  • the one or more biomolecules is selected from the group consisting of proteins, peptides, protein or peptide fragments/fractions, amino acids, and combinations thereof, as the, or the predominant, organic source(s) of nitrogen.
  • the one or more biomolecules are in a molecular weight range of from 0 to 40 kDa, from 1 to 30 kDa, or from 1 to 20 kDa.
  • the one or more biomolecules in the first or second aspect of the present invention is obtained through deconstruction of one or more biological materials selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof.
  • the one or more biological materials may originate from a human, or an animal, source (e.g. the biological materials may be human hair, sheep wool and/or chicken feather).
  • the one or more biomolecules obtained preferably comprises at least a protein and/or fragments/fractions thereof, in particular a keratin protein and/or fragments/fractions thereof.
  • the keratin protein and/or fragments/fractions thereof is obtained from wool, through the deconstruction of wool using a eutectic melt.
  • the one or more biomolecules may be fragments/fractions of one or more proteins, obtained from one or more biological materials, through deconstruction of the biological material(s) using a eutectic melt according to the present invention.
  • the one or more biomolecules may be fragment(s)/fraction(s) of a protein (e.g. keratin protein), obtained from wool, through deconstruction of wool (and the protein) using a eutectic melt according to the present invention.
  • step b) provides a eutectic mixture comprising the one or more organic sources of plant nutrient(s), or the one or more biomolecules.
  • the method according to the second aspect of the present invention may comprise one or more further steps of diluting and/or adjusting the pH of the eutectic melt mixture comprising the one or more organic sources of plant nutrient(s) from the deconstructed one or more biological materials.
  • the method may comprise a further step of: c) diluting the eutectic mixture comprising the deconstructed one or more biological materials, or comprising the one or more organic sources of plant nutrient(s).
  • the method comprises a further step of: d) adjusting the pH of the eutectic mixture comprising the deconstructed one or more biological materials, or comprising the one or more organic sources of plant nutrient(s).
  • the order of the steps c) and d) may be alternated, thus step c) may become step d) and step d) may become step c).
  • a method according to the second aspect of the present invention may also comprise a plurality of diluting steps only, a plurality of pH adjusting steps only, or a plurality of diluting and pH adjusting steps (in various orders).
  • a fertiliser and/or soil improver composition prepared according to the method of the second aspect of the present invention, or obtained from the preparation method of the second aspect of the present invention.
  • a method of enhancing the growth of plants comprising a step of applying a fertiliser and/or soil improver composition according to the first, or third, aspect of the present invention to plants and/or soil supporting the plants.
  • a use of a eutectic melt in the preparation of a fertiliser and/or soil improver composition comprising the deconstruction of one or more biological materials using the eutectic melt.
  • the eutectic melt, or the deconstruction of the one or more biological materials is according to that defined in the first or second aspect of the present invention.
  • one or more of the following plant growth parameters may be enhanced: plant height, biomass, normalised difference vegetation index (ND VI), root weight, foliage weight and total plant weight.
  • ND VI normalised difference vegetation index
  • the deconstruction of the biological material(s) using the eutectic melt as in any of the aspects of the present invention may involve a weight to volume ratio of the biological material(s) to the eutectic melt of from about 1 g:l ml to about 1 g:100 ml, to about lg:70ml, lg:50ml, to about lg:25ml, or to about lg:15ml (e.g. a weight to volume ratio of the biological material(s) to the eutectic melt of about 1g: 13.6ml, lg:10ml, lg:8ml, lg:6.1ml, lg:4.5ml, or lg:3ml).
  • a method of enhancing the growth of plants using a fertiliser and/or soil improver composition comprising keratin, keratin fragments/fractions and/or amino acids thereof, which may be obtained through deconstruction of one or more biological materials selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof.
  • the method of enhancing the growth of plants according to the fourth aspect of the present invention may comprise the use of the fertiliser and/or soil improver composition according to the first or third aspect of the present invention as an additive to a known fertiliser and/or soil improver.
  • the method of enhancing the growth of plants may comprise multiple, separate applications of the fertiliser and/or soil improver composition according to the first or third aspect of the present invention to the soil and/or plants.
  • An application of the fertiliser and/or soil improver composition according to the first or third aspect of the present invention may be as a liquid to a surface of soil, proximal to a location of the plant within that soil.
  • the method of enhancing the growth of plants according to the fourth aspect of the present invention may comprise an application of the fertiliser and/or soil improver according to the first or third aspect of the present invention as a concentrate, or in a diluted form, to provide a desired % (w/v) or concentration of one or more nutrient(s), e.g.
  • N, P and/or K 5 - 50% (w/v) of N, P and/or K, or 10 - 40% (w/v) of N, P and/or K, or 5% (w/v), 12% (w/v), 15% (w/v), 20% (w/v), 27% (w/v), 35%, (w/v), 40% (w/v), or 44% (w/v) of N, P and/or K, for example.
  • Figure 1 is a graph displaying the plant height results according to Example I;
  • Figure 2 is a graph displaying the plant biomass results according to Example I;
  • Figure 3 is a graph displaying the ND VI results according to Example I;
  • Figure 4 is a graph displaying the foliage weight results according to Example I.
  • Figure 5 is a graph displaying the root weight results according to Example I.
  • Figure 6 is a graph displaying the total plant weight results according to Example I.
  • a fertiliser and/or soil improver composition comprises one or more organic sources of plant nutrient(s) in a eutectic melt, obtained through deconstruction of one or more biological materials by the eutectic melt.
  • the fertiliser and/or soil improver composition according to the present invention is in a liquid form, which renders the nutrient(s) more accessible/available to plants.
  • the one or more organic sources of plant nutrient(s) are dissolved in a eutectic melt according to the present invention, with the one or more organic sources of plant nutrient(s) being obtained through deconstruction of one or more biological materials by the eutectic melt.
  • the liquid/solvated form enables the fertiliser and/or soil improver composition according to the present invention to be provided as a concentrate, or be diluted to provide a desired level/concentration of the nutrient(s) present, and/or a desired level/concentration of the eutectic solvent components present.
  • additional nutrient element(s), if required, may be conveniently added to a fertiliser and/or soil improver composition according to the present invention.
  • the fertiliser and/or soil improver composition according to the present invention may be applied alone, or together with or subsequent to another composition beneficial or relevant to plant growth.
  • a fertiliser and/or soil improver composition according to the present invention may be applied to soil only to improve or condition the soil before planting, and/or applied at a frequency required after planting.
  • a eutectic melt according to the present invention comprises deep eutectic solvents, with the melting point of the solvent mixture at a temperature significantly lower than the melting point of the individual eutectic solvent components, due to charge delocalisation through hydrogen bond formation.
  • a fertiliser and/or soil improver composition comprising one or more organic sources of plant nutrient(s) (which may be one or more organic sources of nitrogen, or one or more organic nitrogen sources) prepared through deconstruction of one or more biological materials by a eutectic melt, as in any of the aspects of the present invention.
  • a eutectic melt in any of the aspects of the present invention may comprise a quaternary ammonium salt (such as a choline salt) and a urea, thiourea, or a derivative thereof.
  • a quaternary ammonium salt such as a choline salt
  • Other deep eutectic solvents particularly those involving a choline salt, may also be used, for example, a choline salt-polyol eutectic system (such as choline chloride and glycerol), or a choline salt-organic acid eutectic system (such as choline chloride and malonic acid).
  • a choline halide such as choline chloride or choline bromide
  • a urea/urea derivative or thiourea/thiourea derivative based eutectic system is used in the present invention.
  • a choline chloride-urea eutectic melt or system is used in the present invention.
  • a eutectic melt according to the present invention may be used to deconstruct one or more biological materials to provide one or more organic sources of plant nutrient(s).
  • organic source refers to an organic compound or molecule capable of providing/supplying one or more plant nutrients (as opposed to an inorganic nutrient source), or at least providing/supplying one or more of the major plant nutrients N, P and K.
  • the organic source of plant nutrient(s) may be an organic source of nitrogen/organic nitrogen source, including one which provides/supplies predominantly nitrogen but may also provide/supply one or more of other plant nutrients.
  • the one or more organic sources is in the form of one or more biomolecules.
  • biomolecule refers to a molecule present in an organism and capable of contributing to a biological structure or process (e.g. signal transduction, cell division or formation etc.) in the organism.
  • the one or more biomolecules may be selected from the group consisting of proteins, peptides, chitins, amino acids, nucleic acids, and combinations thereof.
  • the one or more biomolecules is selected from the group consisting of proteins, peptides, protein or peptide fragments/fractions, amino acids, and combinations thereof, as the, or the predominant, organic source(s) of nitrogen.
  • a eutectic melt is preferably used to deconstruct one or more biological materials to provide one or more organic sources of plant nutrient(s) in the form of one or more biomolecules.
  • the one or more biological materials is selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof.
  • the one or more organic sources of plant nutrients is one or more organic nitrogen sources (but may also include other plant nutrient sources, e.g. organic phosphorus or potassium sources).
  • the one or more biomolecules is selected from the group consisting of proteins, peptides, protein/peptide fragments/fractions, amino acids, and/or combinations thereof.
  • the one or more biomolecules may be selected from the group consisting of at least one protein, fragments/fractions of the protein(s) including peptide and/or amino acid fragments/fractions of the protein(s), and combinations thereof.
  • a eutectic melt according to the present invention is able to break down such materials, to provide one or more organic plant nutrient sources, such as one or more biomolecules selected from the group consisting of proteins, peptides, amino acids, and/or combinations thereof, which are at least capable of acting as one or more organic nitrogen sources.
  • the one or more biomolecules may comprise (in addition to or in the absence of one or more proteins) peptides and/or amino acids, which may, or may not, be the peptide and/or amino acid fragments of the one or more proteins.
  • the one or more biomolecules may have a molecular weight of less than 50 kilodaltons (kDa), preferably in the range of from 0 to 40 kDa, more preferably from 1 to 30 kDa, most preferably from 1 to 20 kDa.
  • the one or more biomolecules may be or may comprise peptide and/or amino acid fragments of one or more proteins, with the peptide and/or amino acid fragments being in a molecular weight range of from 1 to 20, 1 to 10, 1 to 7, or more preferably from 1 to 5 kDa, e.g. 1, 2, 2.4, 3.5, 4, 5, 5.7, 7, 9.6 kDa.
  • a fertiliser and/or soil improver composition for use in enhancing the growth of plants, the composition comprising one or more biomolecules in a molecular weight range of from 1 to 20 kDa in a eutectic melt, obtained through deconstruction of one or more biological materials by the eutectic melt, wherein the eutectic melt is urea and choline chloride.
  • the one or more biological materials is/are selected from those described herein above, e.g. wool, hair and/or feather.
  • the molecular weight of the one or more biomolecules is/are in a range of from 1 to 10, more preferably from 1 to 5 kDa.
  • the eutectic melt is at a molar ratio in a range as described hereinabove, e.g. at a ratio of urea:choline chloride of 7.5:1.
  • the deconstruction is at a temperature in a range as described hereinabove.
  • a preferred form of protein present in the one or more biological materials according to the present invention is keratin. Keratin is a fibrous structural protein naturally occurring in epithelial cells. There are two varieties of keratin fibres, being a-keratins found in hair and wool etc. and P-keratins, which are harder and are found in such material as nails and beaks. Being a protein, keratin comprises nitrogen from the amino acids which form its structure. The activity of nitrogen on plant growth is well known.
  • compositions comprising the keratin protein and/or its fragments/fractions, including that obtained from wool extracts, possess marked biological stimulant activity and have consequently proved effective as an organic nitrogen source and hence a fertiliser and/or soil improver, or an additive to a fertiliser and/or soil improver, for enhanced plant growth.
  • a eutectic melt as in any of the aspects of the present invention is able to deconstruct one or more biological materials comprising keratin, or a significant portion of keratin, as a structural protein.
  • the one or more biological materials may be selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof. These biological materials may originate from a human and/or non-human animal source. It is known that these biological materials comprise keratin only, or a significant portion of keratin, in the structural makeup of the material.
  • a fertiliser and/or soil improver composition comprising a protein, and/or one or more peptide and/or amino acid fragments thereof, in a eutectic melt.
  • a fertiliser and/or soil improver composition according to the first or third aspect of the present invention comprises protein fragments, e.g.
  • the eutectic melt comprises a HBD:HBA molar ratio in a range of from about 7.5 to about 4:1, e.g. with a HBD:HBA molar ratio of 7.5:1, 6.0:1, 5.2:1, or 4.1:1.
  • the HBD is preferably a urea, thiourea or a derivative of these, more preferably the HBD is urea.
  • the HBA is preferably a choline salt, e.g. choline chloride.
  • the deconstruction of the one or more biological materials using the eutectic melt according to the present invention is in a temperature range of about: 135 - 170°C, preferably 140 - 165°C, more preferably 145 - 160°C, most preferably 145 - 155 °C.
  • wool from sheep is deconstructed to obtain the keratin protein, keratin peptide fragments, and/or amino acids of keratin protein and/or keratin peptides, available for uptake by plants, thereby providing one or more organic nitrogen source to plants.
  • This wool extract has demonstrated fertiliser and/or soil improver activity itself or as an additive to fertiliser and/or soil improver.
  • wool from other animals e.g. Alpaca, Llama, goats or rabbits
  • wool from other animals e.g. Alpaca, Llama, goats or rabbits
  • a method of preparing a fertiliser and/or soil improver composition comprising one or more organic sources of nitrogen, the method comprising the steps of: a) forming a eutectic melt of urea and choline chloride; and b) deconstructing one or more biological materials using the eutectic melt, to provide a eutectic melt or mixture comprising the one or more organic sources of nitrogen.
  • step a) comprises forming a eutectic melt of urea and choline chloride at a molar ratio (of urea:choline chloride) in a range of from about 7.5:1 to 4:1 (e.g. 7:1, 6.5:1, 5.4:1 or 4:1).
  • step b) comprises deconstructing the one more biological materials at a temperature in a range of about: 135 - 170°C, preferably 140 - 165°C, more preferably about 145 - 160°C, most preferably 145 - 155 °C.
  • the one or more biological materials in step b) is wool.
  • step b) provides a eutectic mixture comprising keratin peptides and/or amino acids in the molecular weight range of from 1 to 20 kDa, preferably 1 to 15 kDa, more preferably 1 to 10 kDa, most preferably 1 to 5 kDa.
  • the embodiment of the method according to the second step of the present invention may further comprise one or more steps selected from diluting the eutectic mixture with water, adjusting the pH of the eutectic mixture with an acid or base, and combinations thereof.
  • the eutectic mixture in step b) of the method according to the second aspect of the present invention is a wool extract comprising keratin peptides and/or amino acids in a eutectic melt (or solvent mix) comprising or formed of urea and choline chloride.
  • the wool extract is obtained by the deconstruction of wool using the eutectic melt, and is preferably prepared from the solvent and deconstruction conditions described hereinabove.
  • the keratin peptides and/or amino acids are preferably in a molecular weight range of from 1 to 10 kDa, or from 1 to 5 kDa, e.g. 1, 1.5, 2, 2.4, 3, 4, 4.7 and/or 5 kDa).
  • the wool extract prepared according to the second aspect of the present invention may be stored as a concentrate, or diluted (with or without pH adjustment) and used directly as a fertiliser and/or soil improver composition.
  • a fertiliser and/or soil improver composition in the form of a wool extract comprising one or more organic sources of nitrogen dissolved in a eutectic melt, the one or more organic sources of nitrogen obtained through deconstruction of one or more biological materials by the eutectic melt.
  • the one or more biological materials is preferably wool from one or more animals selected from the group consisting of sheep, Alpaca, Llama, goats, rabbits and combinations thereof.
  • the wool is sheep wool.
  • the eutectic melt is preferably urea and choline chloride.
  • the one or more organic sources of nitrogen, or the one or more biomolecules is/are preferably keratin peptides and/or amino acids in a molecular weight range of from 1 to 10 kDA (e.g. 1, 3, 4.5, 6, 7.8 or 9 kDa), more preferably from 1 to 5 kDA (e.g. 1, 1.5, 2, 2.4, 3,
  • the eutectic melt is preferably at a molar ratio (of urea:choline chloride) in the range of from about 7.5:1 (e.g. 7:1, 6.5:1, 5.4:1 or 4:1).
  • the deconstruction of the one or more biological materials is preferably at a temperature in a range of about: 135 - 170°C, preferably 140 - 165°C, more preferably about 145 - 160°C, most preferably 145 - 155 °C, e.g. 137, 145, 147.2, 150, 152, 155,
  • wool in deconstructing wool, the present inventors found that, with the eutectic melt (or eutectic melt deconstruction) of the present invention, wool may be used as is, i.e. uncleaned and/or with wool fat present.
  • the preparation of an eutectic melt comprises fabrication of keratin through deconstruction of sheep’s wool using a benign choline chloride-urea deep eutectic solvent melt.
  • the eutectic melt may be formulated by mixing choline chloride and urea at a molar ratio of between about 20:1 and 1:20, preferably at a molar ratio of choline chloride to urea of about 4: 1 to about 7.5:1, while heating for a number of minutes, or for hours (depending on the scale of production).
  • Wool is then dissolved in the eutectic melt at a weight to volume ratio of between about 1 g:l ml and about 1 g:100 ml (preferably between about lg:lml and about lg:25ml, more preferably between about lg:lml and about lg:15ml, or even between about lg:lm and about lg:10ml, e.g. about lg:9ml, lg:8ml or lg:6.5ml) using heat.
  • the heating step of the above process would be understood by the skilled person to cause conversion of at least some of the urea content to ammonia.
  • the resultant ammonia gas escapes to the atmosphere, thereby reducing the nitrogen content of the mixture.
  • a pungent smell is also observable, due to the presence of this ammonia.
  • the following examples utilise the ‘urea + choline chloride’ solvent mix and sheep’s wool to illustrate a eutectic melt according to the present invention, its preparation, the deconstruction of a biological material through a eutectic melt and effectiveness of the resultant fertiliser/soil improver composition according to the present invention.
  • Example I Temperature effect on the eutectic melt and/or deconstruction of wool
  • a eutectic melt is prepared by heating urea (20 g, 0.33 mol) and choline chloride (23.2 g, 0.17 mol) at 70 °C until a clear solution was formed. For each temperature study, the eutectic melt is then further heated until 110 °C, 140 °C, 170 °C and 200 °C. When the desired temperature is reached, unwashed/uncleaned wool (1g) was added portion-wise to the eutectic melt over a 2-minute period. The resulting mixture was then continued to be heated at the desired temperature (110 °C, 140 °C, 170 °C, or 200 °C) with stirring, preferably until the wool is fully dissolved.
  • the mixture was then cooled to room temperature, diluted with water (15 ml), stirred for 5 to 30 minutes, then filtered or centrifuged to afford a clear solution comprising the deconstructed/solubilized wool, in the form of keratin protein and fragments/fractions thereof (with the clear solution being a fertiliser and/or soil improver composition in accordance with the first or third aspect of the present invention). Any undissolved wool present is dried and weighted.
  • the filtered solution, or supernatant solution was kept at room temperature for several weeks to observe for storage stability of the solution (e.g. whether there was precipitate formation).
  • the NMR analysis indicates the formation of multiple decomposition products of urea at 200 °C, including biuret, triuret, ammelide and cyanuric acid, some of which may have formed within the temperature range of 170 °C - 200 °C.
  • a temperature range of 140 °C - 170 °C may be used for the eutectic deconstruction of the wool.
  • eutectic deconstruction at 140 °C of 1 g of wool still required a timeframe of approximately 2 hours, which would not be commercially appropriate or economically viable for scaled-up productions.
  • the wool appeared to be fully dissolved after heating at this temperature for 15 minutes; however, the energy input for heating the eutectic melt up to 170 °C would not be economical/viable for commercial scale productions.
  • a temperature for the eutectic deconstruction of a biological material containing keratin protein is preferably in the range of approximately 145 - 165 °C, more preferably approximately 145 - 160 °C, e.g. 145 - 155 °C.
  • a eutectic melt can also be achieved at a HBD:HBA molar ratio higher than 2:1.
  • the present inventors surprisingly found that even at a HBD:HBA molar ratio in a range of from about 7.5:1 to about 4:1, a eutectic melt is still achievable. This was unexpected and would be commercially advantageous, as a significantly less amount of choline chloride, which is considerably more expensive than urea, may be used for the eutectic deconstruction.
  • a significant proportion, or even the bulk of the eutectic solvents remains in a solid form even when heated at approximately 100 °C for 30 minutes, thus a molar ratio range of urea:choline chloride which presents a significant commercial advantage and yet is able to achieve a eutectic melt to ensure proper mixing, wetting and dissolution of the biological material(s) is from about 7.5:1 to about 4:1.
  • Example III Preparation of a preferred embodiment of a fertiliser and/or soil improver composition according to the present invention
  • a fertiliser and/or soil composition according to the present invention is prepared by a method including at least the steps of: a) forming a eutectic melt; and b) deconstructing one or more biological materials selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof using the eutectic melt, to obtain one or more organic nitrogen sources.
  • Step a) may include heating deep eutectic solvents until a eutectic melt is formed.
  • the eutectic solvents preferably comprise a HBA selected from a choline salt (e.g. choline chloride, choline bromide or choline acetate) and a HBD selected urea, a urea derivative, thiourea, a thiourea derivative, and combinations thereof.
  • a HBA selected from a choline salt (e.g. choline chloride, choline bromide or choline acetate) and a HBD selected urea, a urea derivative, thiourea, a thiourea derivative, and combinations thereof.
  • the HBD:HBA molar ratio of the eutectic solvents may be in a ratio of about 10:1 to about 1:10, but preferably in a ratio of from about 8:1 to about 3:1, or from about 7.5:1 to about 4:1.
  • the deconstruction is preferably at a temperature in a range of approximately 145 - 165 °C, or in a range of approximately 145 - 160 °C (e.g. 147 °C, 150 °C, 156°C or 160 °C).
  • the organic nitrogen sources is preferably a keratin protein, or fragments thereof (such as peptide fragments, but there may also be amino acid fragments).
  • the preparation method may comprise a step of diluting a eutectic melt mixture comprising the one or more organic nitrogen sources, e.g. with water, or a liquid comprising one or more plant nutrients (such as a nutrient solution or an existing fertiliser solution in absence of nitrogen, or needing additional nitrogen).
  • a eutectic melt mixture comprising the one or more organic nitrogen sources, e.g. with water, or a liquid comprising one or more plant nutrients (such as a nutrient solution or an existing fertiliser solution in absence of nitrogen, or needing additional nitrogen).
  • the preparation method may also comprise a step of adjusting the pH of the fertiliser and/or soil improver composition prepared to a desired pH range, for example, pH 5.4 - 7.5, or more preferably pH 5.5 - 7 (e.g. pH 5.8, 6.2, 6.5 or 7).
  • a natural organic acid is used for the pH adjustment.
  • Exemplary acids include oxalic, malonic, succinic, tartaric and citric acid.
  • the dilution and pH adjustment steps can either be steps c) and d), or d) and c), i.e. the order of the steps may change, and there may also be additional, or alternating, dilution and/or pH adjustment steps.
  • the present inventors found that, with the fertiliser and/or soil improver composition preparation method of the present invention, a biological material such as wool can be used as is in an uncleaned state, without the need to remove the wool fat (or a fat layer) first. This was both unexpected and has a clear commercial advantage of reducing the preparation of the one or more biological materials before the eutectic deconstruction.
  • Example IV Molecular weight/fragmentation characterization of the one or more biological molecules in a fertiliser and/or soil improver composition according to the present invention
  • Molecular weight/fragmentation analysis is performed on a fertiliser and/or soil improver composition (nV-1) prepared using a method exemplified in Example III, with a sheep wool material being deconstructed using a eutectic melt comprising urea:choline chloride at a ratio in the range of from about 7.5:1 to about 4:1, and with the temperature for the deconstruction of wool being in a range of approximately 145 - 165 °C (or more specifically approximately 145 - 160 °C).
  • nV-1 Sodium dodecyl-sulfate polyacrylamide gel electrophoresis
  • 1 mF of composition nV-1 was desalted by diafiltration using an Amicon Ultra-0.5 mL 3-kDa Centrifugal Filter (Merck- Millipore) following the manufacturer’s instruction.
  • the desalted sample was mixed either with an equal volume of non-reducing SDS-PAGE loading buffer or with an equal volume of reducing buffer (containing 30 mM DTT and 3 mM EDTA).
  • composition nV- 1 contains far less than expected keratin proteins in the ⁇ 10 to 150 kDa range from the deconstruction of wool. This is completely unexpected, given that the temperature of deconstruction was lower than 160 °C.
  • nV-1 a small sample of nV-1 was desalted and analysed by a MALDI mass spectrometer.
  • a fertiliser and/or soil improver composition according to the invention was tested on potted Grosse Lisse tomatoes to evaluate plant growth.
  • Treatments applied were Verigrow-1: choline chloride (2 moles) + urea (1 mole), without heating; Verigrow-2: choline chloride (2 moles) + urea (1 mole), with heating; and Verigrow- 3: choline chloride (2 moles) + urea (1 mole) + wool, with heating (to fully dissolve the wool at a temperature that may be in the range of 170 - 200 °C); each prepared as 1:10 dilutions with water and at 5.4, 10.8 and 21.6 mL/plant (pot); as well as a commercially available seaweed plant treatment (Seasol®) at 141.3 mL/plant.
  • Veasol® seaweed plant treatment
  • Verigrow refers to the 3 test samples (Verigrow -1, Verigrow -2 and Verigrow-3) for this study. Also for this study, Verigrow-3 was at a weight to volume ratio of the wool to eutectic melt in the range of about lg: l ml to about lg:50ml.
  • Table 2 Treatments (Verigrow compositions: 150 mL/1.5 L of water; seaweed treatments: 6.67 mL/2 L of water).
  • Plant height, biomass, normalised difference vegetation index (NDVI) and weights (root and foliage) were measured and all factors were significantly increased by all three Verigrow compositions, with a significant dose response to increasing rates of all Verigrow compositions.
  • Double applications of Verigrow compositions provided greater increases in plant height, biomass, NDVI and weights (root and foliage) than single applications.
  • Single and double applications of the seaweed plant treatment were not significantly different to the untreated control in plant height, biomass, NDVI or weights (root and foliage).
  • a fertiliser and/or soil improver composition (nV-2) is prepared using a method exemplified in Example III, with a sheep wool material (uncleaned and/or containing the fat layer) being deconstructed using a eutectic melt comprising urea:choline chloride at a ratio in the range of from about 7.5:1 to about 4:1 as described herein above, and with the temperature for the deconstruction of wool being in a range of approximately 145 - 165 °C (or more specifically approximately 145 - 160 °C).
  • the composition (nV-2) was also prepared at a weight to volume ratio of the wool to eutectic melt in the range of from about lg:lml to about lg:25 ml.
  • the resultant fertiliser and/or soil improver composition (nV-2) comprises 35% (w/v) of total N (nitrogen) and has pH in the range of pH 5.5 - 6.5.
  • the composition nV-2 was mixed with water (as a carrier) in a ratio of approximately 52 L water to approximately 48 L of nV-2.
  • the field wheat trial was established as a randomised complete block of 5 treatments and 4 replicates in a single bank with each plot 12 meter long by 2 meter wide.
  • the effectiveness of the fertiliser and/or soil improver composition nV-2 according to the present invention on plant growth (wheat in this instance) and/or as an organic nitrogen nutrient source, is compared with a commercial inorganic fertiliser (Flexi-N, which is a commercially available fertiliser distributed by the Australian Company CSBP and contains three forms of N - urea, ammonium and nitrate, with a total N concentration of 42%), and with Urea alone (at 46% total N).
  • Flexi-N which is a commercially available fertiliser distributed by the Australian Company CSBP and contains three forms of N - urea, ammonium and nitrate, with a total N concentration of 42%), and with Urea alone (at 46% total N).
  • Treatments containing 17 units of N were applied either as nV-2 at 35% (w/v) N, Flexi-N at 42% (w/v) N or Urea at 46% (w/v) N.
  • the nV-2 and Flexi-N treatments were liquid banded in-furrow at seeding whilst Urea treatment was top-dressed and incorporated by sowing. All plots were sown to Sceptre wheat at a rate of 91 kg/ha to a depth of 2.5 cm.
  • At 43 days after sowing there was a second application of the Flexi-N and Urea treatments providing a further 42 units of N each, to the plots of these treatments.
  • nV-2 fertiliser and/or soil improver composition is superior in performance compared to the commercial inorganic nitrogen fertiliser Flexi-N and to Urea.
  • 17 units of N applied as nV -2 (35% N) infurrow at seeding was equal to or even better than 59 units of N supplied by Flexi- N (42% N) or Urea (46% N) on the growth, yield and grain quality of Scepter wheat.
  • the results show that per unit of N, the nV-2 fertiliser and/or soil improver composition according to the present invention had a net crop yield that was ⁇ 4.5 times higher than that of Flexi-N and Urea.
  • the composition according to the present invention may still be more effective than, or at least similarly effective to, a commercial, inorganic and/or urea nitrogen fertiliser.
  • a further wheat field trial is conducted using a fertiliser and/or soil improver composition nV-3 in accordance with the present invention, which is prepared in a same or similar manner, and has same/similar chemical properties to nV-1 and nV-2, except with the nv-3 fertiliser being diluted to provide nV-3 (27% N).
  • nv-3 (27% N) fertiliser in accordance with the present invention was administered via liquid banding in furrow at seeding, and spray application via hand boom at 6 weeks after sowing.
  • Example VI comparisons were made with trial plots applied with the commonly used nitrogen fertilisers Flexi-N and Urea.
  • nv-3 (27% N) fertiliser has shown similar or better results to Flexi-N and Urea.
  • nv-3 (27% N) applied at 13 units of N at seeding and 32 units of N 6 weeks after sowing is an optimal product equivalent to urea at 17 units of N at seeding and 42 units of N 6 weeks after sowing.
  • a fertiliser and/or soil improver composition according to the present invention is compatible with chemicals commonly used in agricultural industry, including the herbicides (e.g. Velocity, MPCA 750, Ester 680, Tenet 500 SC etcetera), fungicides (e.g. Uplift, Prosaro, Impact, Oritus 430 and Soprano 500 etcetera), insecticides (e.g. Lemat, Bifenthrin 240, Dimethoate etcetera) and Adjuvants (e.g. SE14 and Adigor etcetera).
  • herbicides e.g. Velocity, MPCA 750, Ester 680, Tenet 500 SC etcetera
  • fungicides e.g. Uplift, Prosaro, Impact, Oritus 430 and Soprano 500 etcetera
  • insecticides e.g. Lemat, Bifenthrin 240, Dimethoate etcetera
  • Adjuvants e.g
  • a fertiliser and/or soil improver composition according to the present invention may be applied simultaneously with, or subsequently to, one or more commonly used agricultural chemicals without the concern of chemical instability or incompatibility leading to precipitation occurring and/or loss of effectiveness of the composition of the present invention and/or the one or more agricultural chemical(s).
  • organic nitrogen is available to plants in the form of protein or as amino acids.
  • the effect of treating wool, as exemplified in the present invention using a eutectic melt according to the present invention means that the resulting keratin protein, and fragments/fractions thereof (such as peptides and amino acids), are bioavailable, or even surprisingly highly bioavailable, as an organic nitrogen source.
  • the nitrogen can be accessed by plants through root uptake in the form of the protein itself via endocytosis, or as simpler amino acids resulting from enzymatic digestion (either by the proteolytic activity of the roots or from microbes present in the soil).
  • the present invention has been found to possess a myriad of advantages over inorganic nitrogen sources, including a longer durability. Further, protein, or fragments/fractions thereof (such as peptides and/or amino acids), as one or more nitrogen sources results in an increase in a plant’s ability to develop a more extensive root network. It is understood that the keratin protein, or fragments/fractions thereof, available from the present composition through the deconstruction of wool, is bioavailable, or even highly bioavailable, as an organic nitrogen source available to plants. The present invention additionally provides the advantages of being an avenue for reducing or eliminating wool waste and also serving as an additional income stream for wool producers. Since other biomaterials such as those mentioned earlier (e.g.
  • feathers, hair, hooves etc. all comprise keratin or predominantly keratin as a structural protein
  • an eutectic melt according to the present invention is at least able to be used to deconstruct such biomaterials, thus turning further waste materials into income streams and environmentally friendly and effective organic sources of plant nutrients.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Environmental & Geological Engineering (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Life Sciences & Earth Sciences (AREA)
  • Soil Sciences (AREA)
  • Materials Engineering (AREA)
  • Fertilizers (AREA)

Abstract

The present invention relates to a fertiliser and/or soil improver composition for use in enhancing the growth of plants, the composition comprising one or more organic sources of plant nutrient(s), obtained through deconstruction of one or more biological materials by a eutectic melt. The present invention also relates to a method of preparing a fertiliser and/or soil improver composition according to the present invention, and a method of enhancing plant growth using a fertiliser and/or soil improver composition according to the present invention.

Description

TITLE
“FERTILISER AND/OR SOIL IMPROVER COMPOSITION, METHOD OF PREPARATION AND METHOD OF USE”
FIELD OF THE INVENTION
[0001] The present invention relates to a fertiliser and/or soil improver composition. More specifically, the present invention relates to one or more organic sources of plant nutrient(s), contained in a fertiliser and/or soil improver composition. The present invention also relates to a method of preparation and method of use of the fertiliser and/or soil improver composition.
BACKGROUND
[0002] Fertilisers and soil improvers are commonly used in the agricultural industry and even by home gardeners, and indeed fertilisers and soil improvers have many applications and uses between the two. The growth of plants can be enhanced with the use of fertilisers by providing nutrients for uptake by the plant and/or with the use of soil improvers by enhancing the effectiveness of the soil in which the plant grows. The nutritional aspect of fertilisers and soil improvers can involve the provision of one or more of the three main macronutrients, nitrogen (N), phosphorus (P) and potassium (K), for various growth enhancement activity.
[0003] Most common fertilisers and soil improvers are synthetic or inorganic. Usually, various chemical treatments and high energy input are required for their manufacture, which can be costly and has direct environmental impacts. Inorganic fertilisers may also acidify the soil with long term use. However, inorganic fertilisers and soil improvers commonly achieve greater results when applied to plants and soil, compared with natural or organic fertilisers and soil improvers. Therefore, whilst organic fertilisers and soil improvers are often favoured, for myriad reasons, not the least of which include environmental concerns, there exists the need to identify new, effective, organic (or organic sources of) fertilisers and soil improvers.
[0004] Traditionally, nitrogen, the most important plant nutrient, although naturally occurs in soil, is commonly supplied through applications of inorganic fertilisers, as proteins (being a possible organic nitrogen source) was not considered directly available to plants. Rather, the view was that plants relied on microbes and soil fauna for the breakdown of the organic material first in order to utilise the nitrogen bound in the organic material. However, it has recently been shown that roots are able to directly access protein, likely through either enzymatic digestion using proteolytic enzymes exuded from the plant’s own roots or through root uptake via endocytosis (see PNAS, 2008, 105:11, 4524).
[0005] There are abundant natural sources of protein in the environment, including in natural waste materials such as wool, feature or other animal components that are not suitable for use or consumption. It is desirable to repurpose such natural waste materials to reduce wastage and environmental impacts. However, it is difficult to break down the protein structures contained in such materials, to render it economically viable to repurpose such materials.
[0006] Conventional methods to dissolve natural waste materials such as wool and feather often include hydrolysing such waste materials with an alkaline solution (e.g. JPH0929080A and CN103145823 A), or with an alkaline -enzymatic process (e.g. Berechet et al., REV. CHIM. (Bucharest), 2018, 69(7): 1649-1654). These methods may involve harmful chemicals, high temperature/pressure requirements and/or arduous preparation steps, thus are undesirable, particularly for large-scale treatment of a waste material. These methods also tend to provide a solid hydrolysate, which is inconvenient or lacks versatility for fertiliser application. The solid hydrolysate may also exhibit poor bioavailability to plants as a nutrient source. [0007] The present invention attempts to overcome at least in part the aforementioned disadvantages and challenges in providing fertilisers and/or soil improvers containing organic sources of plant nutrition element(s).
SUMMARY OF THE INVENTION
[0008] In accordance with a first aspect of the present invention there is provided a fertiliser and/or soil improver composition for use in enhancing the growth of plants, the composition comprising one or more organic sources of plant nutrient(s), obtained through deconstruction of one or more biological materials by a eutectic melt.
[0009] In accordance with a second aspect of the present invention, there is provided a method of preparing a fertiliser and/or soil improver composition comprising one or more organic sources of plant nutrient(s), the method comprising the steps of: a) forming a eutectic melt; and b) deconstructing one or more biological materials using the eutectic melt, to obtain the one or more organic sources of plant nutrient(s).
[0010] The eutectic melt in the first or second aspect of the present invention comprises at least one hydrogen bond donor (HBD) and at least one hydrogen bond acceptor (HBA). Preferably, the at least one HBA is a quaternary ammonium salt, such as a choline salt (including a choline halide), and the at least one HBD is a urea, thiourea or a derivative thereof. In one preferred embodiment, the eutectic melt comprises a HBA in the form of a choline salt and a HBD in the form of a urea, thiourea, or a derivative of these. In a further preferred embodiment, the eutectic melt comprises a choline salt and a urea, or urea derivative.
[0011] In a preferred embodiment, the deconstruction of one or more biological materials by a eutectic melt in the first or second aspect of the present invention is at a temperature in a range of approximately: 135 - 170 °C, more preferably 140 - 165°C, or most preferably 145 - 160°C, or even 145 - 155 °C. The temperatures/temperature ranges in the present invention are measured at ambient pressure, or atmospheric pressure, conditions.
[0012] The molar ratio of the eutectic melt (between the at least one HBD and the at least one HBA) in the first or second aspect of the present invention may be from about 20:1 to about 1:20. In a preferred embodiment in which the eutectic solvent comprises a choline salt (as a HBA) and a urea, thiourea or a derivative these (as a HBD), the molar ratio of HBD: HBA is in a range of from 10:1 to 1:10; more preferably, from about 8:1 to about 3:1, or most preferably from about 7.5 to about 4:1 (e.g. with a HBD:HBA molar ratio of 7.5:1, 7:1, 6.6:1, 6:1, 5.4:1, 5:1, or 4.1:1). Therefore, in this preferred embodiment, the HBD (being a urea, thiourea or a derivative of these) is present at a significantly higher molar ratio than the HBA (the choline salt).
[0013] In a preferred embodiment, the one or more organic sources of plant nutrient(s) according to the first or second aspect of the present invention includes, or is in the form of, one or more biomolecules, preferably selected from the group consisting of proteins, peptides, chitins, amino acids, nucleic acids, and combinations thereof. In a further embodiment, the one or more biomolecules is selected from the group consisting of proteins, peptides, protein or peptide fragments/fractions, amino acids, and combinations thereof, as the, or the predominant, organic source(s) of nitrogen. In one embodiment, the one or more biomolecules are in a molecular weight range of from 0 to 40 kDa, from 1 to 30 kDa, or from 1 to 20 kDa.
[0014] In a preferred embodiment, the one or more biomolecules in the first or second aspect of the present invention is obtained through deconstruction of one or more biological materials selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof. The one or more biological materials may originate from a human, or an animal, source (e.g. the biological materials may be human hair, sheep wool and/or chicken feather). The one or more biomolecules obtained preferably comprises at least a protein and/or fragments/fractions thereof, in particular a keratin protein and/or fragments/fractions thereof. In one embodiment, the keratin protein and/or fragments/fractions thereof is obtained from wool, through the deconstruction of wool using a eutectic melt. In one embodiment, the one or more biomolecules may be fragments/fractions of one or more proteins, obtained from one or more biological materials, through deconstruction of the biological material(s) using a eutectic melt according to the present invention. In one embodiment, the one or more biomolecules may be fragment(s)/fraction(s) of a protein (e.g. keratin protein), obtained from wool, through deconstruction of wool (and the protein) using a eutectic melt according to the present invention.
[0015] In the method according to the second aspect of the present invention, step b) provides a eutectic mixture comprising the one or more organic sources of plant nutrient(s), or the one or more biomolecules. The method according to the second aspect of the present invention may comprise one or more further steps of diluting and/or adjusting the pH of the eutectic melt mixture comprising the one or more organic sources of plant nutrient(s) from the deconstructed one or more biological materials. In one embodiment, the method may comprise a further step of: c) diluting the eutectic mixture comprising the deconstructed one or more biological materials, or comprising the one or more organic sources of plant nutrient(s). In one embodiment, the method comprises a further step of: d) adjusting the pH of the eutectic mixture comprising the deconstructed one or more biological materials, or comprising the one or more organic sources of plant nutrient(s). The order of the steps c) and d) may be alternated, thus step c) may become step d) and step d) may become step c). A method according to the second aspect of the present invention may also comprise a plurality of diluting steps only, a plurality of pH adjusting steps only, or a plurality of diluting and pH adjusting steps (in various orders).
[0016] In accordance with a third aspect of the present invention, there is provided a fertiliser and/or soil improver composition prepared according to the method of the second aspect of the present invention, or obtained from the preparation method of the second aspect of the present invention. [0017] In accordance with a fourth aspect of the present invention, there is provided a method of enhancing the growth of plants comprising a step of applying a fertiliser and/or soil improver composition according to the first, or third, aspect of the present invention to plants and/or soil supporting the plants.
[0018] In accordance with a fifth aspect of the present invention, there is provided a use of a eutectic melt in the preparation of a fertiliser and/or soil improver composition, comprising the deconstruction of one or more biological materials using the eutectic melt. Preferably, the eutectic melt, or the deconstruction of the one or more biological materials, is according to that defined in the first or second aspect of the present invention.
[0019] With the fertiliser and/or soil improver of the present invention, one or more of the following plant growth parameters may be enhanced: plant height, biomass, normalised difference vegetation index (ND VI), root weight, foliage weight and total plant weight.
[0020] The deconstruction of the biological material(s) using the eutectic melt as in any of the aspects of the present invention may involve a weight to volume ratio of the biological material(s) to the eutectic melt of from about 1 g:l ml to about 1 g:100 ml, to about lg:70ml, lg:50ml, to about lg:25ml, or to about lg:15ml (e.g. a weight to volume ratio of the biological material(s) to the eutectic melt of about 1g: 13.6ml, lg:10ml, lg:8ml, lg:6.1ml, lg:4.5ml, or lg:3ml).
[0021] In an embodiment of the method according to the fourth aspect of the present invention there is provided a method of enhancing the growth of plants using a fertiliser and/or soil improver composition comprising keratin, keratin fragments/fractions and/or amino acids thereof, which may be obtained through deconstruction of one or more biological materials selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof. [0022] The method of enhancing the growth of plants according to the fourth aspect of the present invention may comprise the use of the fertiliser and/or soil improver composition according to the first or third aspect of the present invention as an additive to a known fertiliser and/or soil improver.
[0023] The method of enhancing the growth of plants may comprise multiple, separate applications of the fertiliser and/or soil improver composition according to the first or third aspect of the present invention to the soil and/or plants.
[0024] An application of the fertiliser and/or soil improver composition according to the first or third aspect of the present invention may be as a liquid to a surface of soil, proximal to a location of the plant within that soil.
[0025] The method of enhancing the growth of plants according to the fourth aspect of the present invention may comprise an application of the fertiliser and/or soil improver according to the first or third aspect of the present invention as a concentrate, or in a diluted form, to provide a desired % (w/v) or concentration of one or more nutrient(s), e.g. 5 - 50% (w/v) of N, P and/or K, or 10 - 40% (w/v) of N, P and/or K, or 5% (w/v), 12% (w/v), 15% (w/v), 20% (w/v), 27% (w/v), 35%, (w/v), 40% (w/v), or 44% (w/v) of N, P and/or K, for example.
[0026] Throughout the specification, unless the context requires otherwise, the word ‘comprise’ or variations such as ‘comprises’ or ‘comprising’, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
[0027] Furthermore, throughout the specification, unless the context requires otherwise, the word ‘include’ or variations such as ‘includes’ or ‘including’, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. BRIEF DESCRIPTION OF DRAWINGS
[0028] The present invention will now be described, by way of example, with reference to the accompanying drawings, in which:
Figure 1 is a graph displaying the plant height results according to Example I;
Figure 2 is a graph displaying the plant biomass results according to Example I;
Figure 3 is a graph displaying the ND VI results according to Example I;
Figure 4 is a graph displaying the foliage weight results according to Example I;
Figure 5 is a graph displaying the root weight results according to Example I; and
Figure 6 is a graph displaying the total plant weight results according to Example I.
DESCRIPTION OF PREFERRED EMBODIMENT
[0029] In a preferred embodiment of the present invention, a fertiliser and/or soil improver composition comprises one or more organic sources of plant nutrient(s) in a eutectic melt, obtained through deconstruction of one or more biological materials by the eutectic melt.
[0030] Preferably or advantageously, the fertiliser and/or soil improver composition according to the present invention is in a liquid form, which renders the nutrient(s) more accessible/available to plants. The one or more organic sources of plant nutrient(s) are dissolved in a eutectic melt according to the present invention, with the one or more organic sources of plant nutrient(s) being obtained through deconstruction of one or more biological materials by the eutectic melt. The liquid/solvated form enables the fertiliser and/or soil improver composition according to the present invention to be provided as a concentrate, or be diluted to provide a desired level/concentration of the nutrient(s) present, and/or a desired level/concentration of the eutectic solvent components present. Further, being in a liquid form, additional nutrient element(s), if required, may be conveniently added to a fertiliser and/or soil improver composition according to the present invention. Advantageously also, the fertiliser and/or soil improver composition according to the present invention may be applied alone, or together with or subsequent to another composition beneficial or relevant to plant growth.
[0031] A fertiliser and/or soil improver composition according to the present invention may be applied to soil only to improve or condition the soil before planting, and/or applied at a frequency required after planting.
[0032] A eutectic melt according to the present invention comprises deep eutectic solvents, with the melting point of the solvent mixture at a temperature significantly lower than the melting point of the individual eutectic solvent components, due to charge delocalisation through hydrogen bond formation.
[0033] It is not known to date of a fertiliser and/or soil improver composition comprising one or more organic sources of plant nutrient(s) (which may be one or more organic sources of nitrogen, or one or more organic nitrogen sources) prepared through deconstruction of one or more biological materials by a eutectic melt, as in any of the aspects of the present invention.
[0034] A eutectic melt in any of the aspects of the present invention may comprise a quaternary ammonium salt (such as a choline salt) and a urea, thiourea, or a derivative thereof. Other deep eutectic solvents, particularly those involving a choline salt, may also be used, for example, a choline salt-polyol eutectic system (such as choline chloride and glycerol), or a choline salt-organic acid eutectic system (such as choline chloride and malonic acid). More preferably, a choline halide (such as choline chloride or choline bromide) and a urea/urea derivative or thiourea/thiourea derivative based eutectic system is used in the present invention. In a specific embodiment, a choline chloride-urea eutectic melt or system is used in the present invention. [0035] A eutectic melt according to the present invention may be used to deconstruct one or more biological materials to provide one or more organic sources of plant nutrient(s). The term “organic source”, “organic nutrient source”, “organic plant nutrient source”, or variations thereof, as used herein refers to an organic compound or molecule capable of providing/supplying one or more plant nutrients (as opposed to an inorganic nutrient source), or at least providing/supplying one or more of the major plant nutrients N, P and K. In a preferred embodiment, the organic source of plant nutrient(s) may be an organic source of nitrogen/organic nitrogen source, including one which provides/supplies predominantly nitrogen but may also provide/supply one or more of other plant nutrients. In a preferred embodiment, the one or more organic sources is in the form of one or more biomolecules. The term “biomolecule” as used herein refers to a molecule present in an organism and capable of contributing to a biological structure or process (e.g. signal transduction, cell division or formation etc.) in the organism. In a preferred embodiment, the one or more biomolecules may be selected from the group consisting of proteins, peptides, chitins, amino acids, nucleic acids, and combinations thereof. In a further preferred embodiment, the one or more biomolecules is selected from the group consisting of proteins, peptides, protein or peptide fragments/fractions, amino acids, and combinations thereof, as the, or the predominant, organic source(s) of nitrogen.
[0036] In an embodiment according to any of the aspects of the present invention, a eutectic melt is preferably used to deconstruct one or more biological materials to provide one or more organic sources of plant nutrient(s) in the form of one or more biomolecules. The one or more biological materials is selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof. The one or more organic sources of plant nutrients is one or more organic nitrogen sources (but may also include other plant nutrient sources, e.g. organic phosphorus or potassium sources). The one or more biomolecules is selected from the group consisting of proteins, peptides, protein/peptide fragments/fractions, amino acids, and/or combinations thereof. In a further embodiment, the one or more biomolecules may be selected from the group consisting of at least one protein, fragments/fractions of the protein(s) including peptide and/or amino acid fragments/fractions of the protein(s), and combinations thereof.
[0037] Many natural materials from an animal source (including a human animal source) are normally discarded or perceived as waste materials. Advantageously, a eutectic melt according to the present invention is able to break down such materials, to provide one or more organic plant nutrient sources, such as one or more biomolecules selected from the group consisting of proteins, peptides, amino acids, and/or combinations thereof, which are at least capable of acting as one or more organic nitrogen sources. In one embodiment, the one or more biomolecules may comprise (in addition to or in the absence of one or more proteins) peptides and/or amino acids, which may, or may not, be the peptide and/or amino acid fragments of the one or more proteins. In one embodiment, the one or more biomolecules may have a molecular weight of less than 50 kilodaltons (kDa), preferably in the range of from 0 to 40 kDa, more preferably from 1 to 30 kDa, most preferably from 1 to 20 kDa. In a preferred embodiment, the one or more biomolecules may be or may comprise peptide and/or amino acid fragments of one or more proteins, with the peptide and/or amino acid fragments being in a molecular weight range of from 1 to 20, 1 to 10, 1 to 7, or more preferably from 1 to 5 kDa, e.g. 1, 2, 2.4, 3.5, 4, 5, 5.7, 7, 9.6 kDa.
[0038] In one embodiment according to the first or third aspect of the present invention, there is provided a fertiliser and/or soil improver composition for use in enhancing the growth of plants, the composition comprising one or more biomolecules in a molecular weight range of from 1 to 20 kDa in a eutectic melt, obtained through deconstruction of one or more biological materials by the eutectic melt, wherein the eutectic melt is urea and choline chloride. The one or more biological materials is/are selected from those described herein above, e.g. wool, hair and/or feather. Preferably, the molecular weight of the one or more biomolecules is/are in a range of from 1 to 10, more preferably from 1 to 5 kDa. The eutectic melt is at a molar ratio in a range as described hereinabove, e.g. at a ratio of urea:choline chloride of 7.5:1. The deconstruction is at a temperature in a range as described hereinabove. [0039] A preferred form of protein present in the one or more biological materials according to the present invention is keratin. Keratin is a fibrous structural protein naturally occurring in epithelial cells. There are two varieties of keratin fibres, being a-keratins found in hair and wool etc. and P-keratins, which are harder and are found in such material as nails and beaks. Being a protein, keratin comprises nitrogen from the amino acids which form its structure. The activity of nitrogen on plant growth is well known.
[0040] It has presently been found that compositions comprising the keratin protein and/or its fragments/fractions, including that obtained from wool extracts, possess marked biological stimulant activity and have consequently proved effective as an organic nitrogen source and hence a fertiliser and/or soil improver, or an additive to a fertiliser and/or soil improver, for enhanced plant growth.
[0041] Advantageously, a eutectic melt as in any of the aspects of the present invention is able to deconstruct one or more biological materials comprising keratin, or a significant portion of keratin, as a structural protein. In a preferred embodiment, the one or more biological materials may be selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof. These biological materials may originate from a human and/or non-human animal source. It is known that these biological materials comprise keratin only, or a significant portion of keratin, in the structural makeup of the material. With a eutectic melt according to the present invention being capable of deconstructing a hard/rigid protein such as keratin, the present eutectic melt, or deconstruction method, may also be used to deconstruct other proteins, at least those with a similar or less hard/rigid structure compared to keratin. Therefore, in one embodiment, there may simply be a fertiliser and/or soil improver composition comprising a protein, and/or one or more peptide and/or amino acid fragments thereof, in a eutectic melt. [0042] In one embodiment, a fertiliser and/or soil improver composition according to the first or third aspect of the present invention comprises protein fragments, e.g. peptide and/or amino acid fragments, in the molecular weight range of from 1 to 20 kDa, more preferably 1 to 10 kDa, most preferably 1 to 5 kDa, obtained through deconstruction of one or more biological materials by a eutectic melt. The protein fragments may be present in addition to, or in the absence of, one or more proteins which may or may not be present in the fertiliser and/or soil improver composition according to the present invention. In a further embodiment, the eutectic melt comprises a HBD:HBA molar ratio in a range of from about 7.5 to about 4:1, e.g. with a HBD:HBA molar ratio of 7.5:1, 6.0:1, 5.2:1, or 4.1:1. The HBD is preferably a urea, thiourea or a derivative of these, more preferably the HBD is urea. The HBA is preferably a choline salt, e.g. choline chloride. The deconstruction of the one or more biological materials using the eutectic melt according to the present invention is in a temperature range of about: 135 - 170°C, preferably 140 - 165°C, more preferably 145 - 160°C, most preferably 145 - 155 °C.
[0043] In a preferred embodiment of the present invention, wool from sheep is deconstructed to obtain the keratin protein, keratin peptide fragments, and/or amino acids of keratin protein and/or keratin peptides, available for uptake by plants, thereby providing one or more organic nitrogen source to plants. This wool extract has demonstrated fertiliser and/or soil improver activity itself or as an additive to fertiliser and/or soil improver. In other embodiments, wool from other animals (e.g. Alpaca, Llama, goats or rabbits) may also be deconstructed by an eutectic melt according to the present invention, to provide a fertiliser and/or soil improver composition.
[0044] In one embodiment according to the second aspect of the present invention, there is provide a method of preparing a fertiliser and/or soil improver composition comprising one or more organic sources of nitrogen, the method comprising the steps of: a) forming a eutectic melt of urea and choline chloride; and b) deconstructing one or more biological materials using the eutectic melt, to provide a eutectic melt or mixture comprising the one or more organic sources of nitrogen.
[0045] Preferably, in the above embodiment, step a) comprises forming a eutectic melt of urea and choline chloride at a molar ratio (of urea:choline chloride) in a range of from about 7.5:1 to 4:1 (e.g. 7:1, 6.5:1, 5.4:1 or 4:1).
[0046] Preferably, step b) comprises deconstructing the one more biological materials at a temperature in a range of about: 135 - 170°C, preferably 140 - 165°C, more preferably about 145 - 160°C, most preferably 145 - 155 °C.
[0047] In one embodiment, the one or more biological materials in step b) is wool.
[0048] Preferably, step b) provides a eutectic mixture comprising keratin peptides and/or amino acids in the molecular weight range of from 1 to 20 kDa, preferably 1 to 15 kDa, more preferably 1 to 10 kDa, most preferably 1 to 5 kDa.
[0049] The embodiment of the method according to the second step of the present invention may further comprise one or more steps selected from diluting the eutectic mixture with water, adjusting the pH of the eutectic mixture with an acid or base, and combinations thereof.
[0050] In a preferred embodiment, the eutectic mixture in step b) of the method according to the second aspect of the present invention is a wool extract comprising keratin peptides and/or amino acids in a eutectic melt (or solvent mix) comprising or formed of urea and choline chloride. The wool extract is obtained by the deconstruction of wool using the eutectic melt, and is preferably prepared from the solvent and deconstruction conditions described hereinabove. The keratin peptides and/or amino acids are preferably in a molecular weight range of from 1 to 10 kDa, or from 1 to 5 kDa, e.g. 1, 1.5, 2, 2.4, 3, 4, 4.7 and/or 5 kDa). Advantageously, the wool extract prepared according to the second aspect of the present invention may be stored as a concentrate, or diluted (with or without pH adjustment) and used directly as a fertiliser and/or soil improver composition.
[0051 ] In one embodiment according to the first or third aspect of the present invention, there is provided a fertiliser and/or soil improver composition in the form of a wool extract comprising one or more organic sources of nitrogen dissolved in a eutectic melt, the one or more organic sources of nitrogen obtained through deconstruction of one or more biological materials by the eutectic melt. The one or more biological materials is preferably wool from one or more animals selected from the group consisting of sheep, Alpaca, Llama, goats, rabbits and combinations thereof. In one embodiment, the wool is sheep wool. The eutectic melt is preferably urea and choline chloride. The one or more organic sources of nitrogen, or the one or more biomolecules, is/are preferably keratin peptides and/or amino acids in a molecular weight range of from 1 to 10 kDA (e.g. 1, 3, 4.5, 6, 7.8 or 9 kDa), more preferably from 1 to 5 kDA (e.g. 1, 1.5, 2, 2.4, 3,
3.5, 4, 4.7 and/or 5 kDa). The eutectic melt is preferably at a molar ratio (of urea:choline chloride) in the range of from about 7.5:1 (e.g. 7:1, 6.5:1, 5.4:1 or 4:1). The deconstruction of the one or more biological materials is preferably at a temperature in a range of about: 135 - 170°C, preferably 140 - 165°C, more preferably about 145 - 160°C, most preferably 145 - 155 °C, e.g. 137, 145, 147.2, 150, 152, 155,
157.5, 160, 163, 165 and/or 168.2 °C.
[0052] Advantageously, in deconstructing wool, the present inventors found that, with the eutectic melt (or eutectic melt deconstruction) of the present invention, wool may be used as is, i.e. uncleaned and/or with wool fat present. This presents a significant commercial advantage in a commercial production of a fertiliser and/or soil improver composition prepared from sheep wool, which is known to be abundant in lanolin.
[0053] In one embodiment, the preparation of an eutectic melt comprises fabrication of keratin through deconstruction of sheep’s wool using a benign choline chloride-urea deep eutectic solvent melt. The eutectic melt may be formulated by mixing choline chloride and urea at a molar ratio of between about 20:1 and 1:20, preferably at a molar ratio of choline chloride to urea of about 4: 1 to about 7.5:1, while heating for a number of minutes, or for hours (depending on the scale of production). Wool is then dissolved in the eutectic melt at a weight to volume ratio of between about 1 g:l ml and about 1 g:100 ml (preferably between about lg:lml and about lg:25ml, more preferably between about lg:lml and about lg:15ml, or even between about lg:lm and about lg:10ml, e.g. about lg:9ml, lg:8ml or lg:6.5ml) using heat.
[0054] The heating step of the above process would be understood by the skilled person to cause conversion of at least some of the urea content to ammonia. The resultant ammonia gas escapes to the atmosphere, thereby reducing the nitrogen content of the mixture. A pungent smell is also observable, due to the presence of this ammonia.
[0055] The following examples utilise the ‘urea + choline chloride’ solvent mix and sheep’s wool to illustrate a eutectic melt according to the present invention, its preparation, the deconstruction of a biological material through a eutectic melt and effectiveness of the resultant fertiliser/soil improver composition according to the present invention.
Example I - Temperature effect on the eutectic melt and/or deconstruction of wool
[0056] Firstly, a eutectic melt is prepared by heating urea (20 g, 0.33 mol) and choline chloride (23.2 g, 0.17 mol) at 70 °C until a clear solution was formed. For each temperature study, the eutectic melt is then further heated until 110 °C, 140 °C, 170 °C and 200 °C. When the desired temperature is reached, unwashed/uncleaned wool (1g) was added portion-wise to the eutectic melt over a 2-minute period. The resulting mixture was then continued to be heated at the desired temperature (110 °C, 140 °C, 170 °C, or 200 °C) with stirring, preferably until the wool is fully dissolved. The mixture was then cooled to room temperature, diluted with water (15 ml), stirred for 5 to 30 minutes, then filtered or centrifuged to afford a clear solution comprising the deconstructed/solubilized wool, in the form of keratin protein and fragments/fractions thereof (with the clear solution being a fertiliser and/or soil improver composition in accordance with the first or third aspect of the present invention). Any undissolved wool present is dried and weighted. The filtered solution, or supernatant solution, was kept at room temperature for several weeks to observe for storage stability of the solution (e.g. whether there was precipitate formation).
[0057] The results of the temperature study showed that, surprisingly, there is a distinct temperature range within which: i) the wool would reach complete dissolution within a reasonable timeframe; and ii) the deconstructed components of the wool and components of the eutectic melt, would remain in solution without precipitation over time.
[0058] At a temperature of 110 °C, despite continuingly heating the eutectic melt at this temperature for 5 hours with stirring, -75% of the wool remain undissolved. Surprising also, at the elevated temperature of 200 °C, copious amounts of a precipitate also formed after the solution containing the dissolved/deconstructed wool was cooled to room temperature and diluted with water (15 ml). Even when the precipitate was removed by centrifugation, the remaining clear supernatant solution had precipitate forming over the several days after the centrifugation. A subsequent analysis of a sample of the precipitate from the centrifugation by NMR confirmed that the precipitate is a result of urea decomposition, with the urea decomposition pathway not being activated at a lower temperature, but at 200 °C, or more likely within the temperature range of 170 °C - 200 °C. The NMR analysis indicates the formation of multiple decomposition products of urea at 200 °C, including biuret, triuret, ammelide and cyanuric acid, some of which may have formed within the temperature range of 170 °C - 200 °C.
[0059] From this study, a temperature range of 140 °C - 170 °C may be used for the eutectic deconstruction of the wool. However, eutectic deconstruction at 140 °C of 1 g of wool still required a timeframe of approximately 2 hours, which would not be commercially appropriate or economically viable for scaled-up productions. At 170 °C, the wool appeared to be fully dissolved after heating at this temperature for 15 minutes; however, the energy input for heating the eutectic melt up to 170 °C would not be economical/viable for commercial scale productions. Therefore, a temperature for the eutectic deconstruction of a biological material containing keratin protein is preferably in the range of approximately 145 - 165 °C, more preferably approximately 145 - 160 °C, e.g. 145 - 155 °C.
Example II - Effect of the HBD:HBA molar ratio on the eutectic melt
[0060] The above temperature effect study was conducted in a eutectic melt comprising urea (20 g, 0.33 mol) and choline chloride (23.2 g, 0.17 mol), i.e. with a HBD:HBA ratio of 2:1, which is the conventional ratio for a eutectic melt involving urea and choline chloride, in order to achieve a eutectic melt.
[0061] The present inventors found that a eutectic melt can also be achieved at a HBD:HBA molar ratio higher than 2:1. In the instance of urea:choline chloride, the present inventors surprisingly found that even at a HBD:HBA molar ratio in a range of from about 7.5:1 to about 4:1, a eutectic melt is still achievable. This was unexpected and would be commercially advantageous, as a significantly less amount of choline chloride, which is considerably more expensive than urea, may be used for the eutectic deconstruction.
[0062] At a urea:choline chloride molar ratio of 8:1 or higher (e.g. at the molar ratios of 9:1 and 10:1), a significant proportion, or even the bulk of the eutectic solvents, remains in a solid form even when heated at approximately 100 °C for 30 minutes, thus a molar ratio range of urea:choline chloride which presents a significant commercial advantage and yet is able to achieve a eutectic melt to ensure proper mixing, wetting and dissolution of the biological material(s) is from about 7.5:1 to about 4:1.
Example III - Preparation of a preferred embodiment of a fertiliser and/or soil improver composition according to the present invention
[0063] In this Example, a fertiliser and/or soil composition according to the present invention is prepared by a method including at least the steps of: a) forming a eutectic melt; and b) deconstructing one or more biological materials selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof using the eutectic melt, to obtain one or more organic nitrogen sources.
[0064] Step a) may include heating deep eutectic solvents until a eutectic melt is formed.
[0065] The eutectic solvents preferably comprise a HBA selected from a choline salt (e.g. choline chloride, choline bromide or choline acetate) and a HBD selected urea, a urea derivative, thiourea, a thiourea derivative, and combinations thereof.
[0066] The HBD:HBA molar ratio of the eutectic solvents may be in a ratio of about 10:1 to about 1:10, but preferably in a ratio of from about 8:1 to about 3:1, or from about 7.5:1 to about 4:1.
[0067] In step b), the deconstruction is preferably at a temperature in a range of approximately 145 - 165 °C, or in a range of approximately 145 - 160 °C (e.g. 147 °C, 150 °C, 156°C or 160 °C). The organic nitrogen sources is preferably a keratin protein, or fragments thereof (such as peptide fragments, but there may also be amino acid fragments).
[0068] The preparation method may comprise a step of diluting a eutectic melt mixture comprising the one or more organic nitrogen sources, e.g. with water, or a liquid comprising one or more plant nutrients (such as a nutrient solution or an existing fertiliser solution in absence of nitrogen, or needing additional nitrogen).
[0069] The preparation method may also comprise a step of adjusting the pH of the fertiliser and/or soil improver composition prepared to a desired pH range, for example, pH 5.4 - 7.5, or more preferably pH 5.5 - 7 (e.g. pH 5.8, 6.2, 6.5 or 7). If pH adjustment is required, preferably a natural organic acid is used for the pH adjustment. Exemplary acids include oxalic, malonic, succinic, tartaric and citric acid.
[0070] The dilution and pH adjustment steps can either be steps c) and d), or d) and c), i.e. the order of the steps may change, and there may also be additional, or alternating, dilution and/or pH adjustment steps.
[0071] Advantageously, the present inventors found that, with the fertiliser and/or soil improver composition preparation method of the present invention, a biological material such as wool can be used as is in an uncleaned state, without the need to remove the wool fat (or a fat layer) first. This was both unexpected and has a clear commercial advantage of reducing the preparation of the one or more biological materials before the eutectic deconstruction.
Example IV - Molecular weight/fragmentation characterization of the one or more biological molecules in a fertiliser and/or soil improver composition according to the present invention
[0072] Molecular weight/fragmentation analysis is performed on a fertiliser and/or soil improver composition (nV-1) prepared using a method exemplified in Example III, with a sheep wool material being deconstructed using a eutectic melt comprising urea:choline chloride at a ratio in the range of from about 7.5:1 to about 4:1, and with the temperature for the deconstruction of wool being in a range of approximately 145 - 165 °C (or more specifically approximately 145 - 160 °C).
[0073] Firstly, the molecular weight/fragmentation of a sample composition nV-1 according to the present invention is studied by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For SDS-PAGE, 1 mF of composition nV-1 was desalted by diafiltration using an Amicon Ultra-0.5 mL 3-kDa Centrifugal Filter (Merck- Millipore) following the manufacturer’s instruction. The desalted sample was mixed either with an equal volume of non-reducing SDS-PAGE loading buffer or with an equal volume of reducing buffer (containing 30 mM DTT and 3 mM EDTA). Samples were heated for 10 minutes at 90°C, before resolving on a precast gel (SurePAGETM 8-16% Bis-Tris by GenScript) running with MES Buffer at 200 volts for 25 minutes. Gels were stained by a Coomassie Blue R250 solution, before scanning on a GS- 900 calibrated densitometer (Bio-Rad). All Blue Precision Plus Protein standard (Bio-Rad) was used as the molecular weight marker.
[0074] Equivalent to 1, 5, 25 and 75 pL of sample composition nV-1 (expected to contain 20, 100, 500 and 1500 pg of keratin protein) were resolved by SDS-PAGE.
[0075] Surprisingly, the SDS-PAGE results suggest that composition nV- 1 contains far less than expected keratin proteins in the ~10 to 150 kDa range from the deconstruction of wool. This is completely unexpected, given that the temperature of deconstruction was lower than 160 °C.
[0076] Almost all the detected protein bands in the gel appear to accumulate at around the 10 kDa mark, thus suggesting that deconstruction of the wool has mainly resulted in keratin peptide fragments, although there were amino acid fragments detected by SDS-PAGE at < 10 kDa. No apparent difference is detectable between the reduced and non-reduced samples.
[0077] With the above surprising finding, and to shed more light on the size distribution of the peptides in the sample fertiliser and/or soil improver composition nV-1, a small sample of nV-1 was desalted and analysed by a MALDI mass spectrometer. Two different matrices, sinapinic acid (that is more suitable for proteins) and CHCA (more apt for small peptides) were used. Both matrices produced similar profiles (not shown).
[0078] Using the mass spectrum obtained from the CHCA matrix as an example, the spectrum (in the mass range of 500 to 10,000 m/z) showed that nearly all the keratin peptide fragments appear to be in the 1000-4000 Dalton range (i.e. < 5 kDa). It should be noted that all desalting methods readily remove the smaller peptide fragments or amino acids (of <1000 kDa) that are likely to be present in the sample. Example V - Biological activity (pot trial)
[0079] A fertiliser and/or soil improver composition according to the invention was tested on potted Grosse Lisse tomatoes to evaluate plant growth. Treatments applied were Verigrow-1: choline chloride (2 moles) + urea (1 mole), without heating; Verigrow-2: choline chloride (2 moles) + urea (1 mole), with heating; and Verigrow- 3: choline chloride (2 moles) + urea (1 mole) + wool, with heating (to fully dissolve the wool at a temperature that may be in the range of 170 - 200 °C); each prepared as 1:10 dilutions with water and at 5.4, 10.8 and 21.6 mL/plant (pot); as well as a commercially available seaweed plant treatment (Seasol®) at 141.3 mL/plant. Treatments were applied as either a double application, at transplanting (3 -4 leaves on main stem) and 29 days later at 50% inflorescence emergence, or as a single application at 50% inflorescence emergence. The term Verigrow, as used herein, refers to the 3 test samples (Verigrow -1, Verigrow -2 and Verigrow-3) for this study. Also for this study, Verigrow-3 was at a weight to volume ratio of the wool to eutectic melt in the range of about lg: l ml to about lg:50ml.
[0080] The chronology of events and treatments are shown below in Tables 1 and 2 respectively.
Table 1: Chronology of events.
Figure imgf000023_0001
Figure imgf000024_0001
Table 2: Treatments (Verigrow compositions: 150 mL/1.5 L of water; seaweed treatments: 6.67 mL/2 L of water).
Figure imgf000024_0002
Figure imgf000025_0001
[0081] The results are shown in Figures 1 to 6 at various days after applications A or B (DAAA or DAAB).
[0082] Plant height, biomass, normalised difference vegetation index (NDVI) and weights (root and foliage) were measured and all factors were significantly increased by all three Verigrow compositions, with a significant dose response to increasing rates of all Verigrow compositions. Double applications of Verigrow compositions provided greater increases in plant height, biomass, NDVI and weights (root and foliage) than single applications. Single and double applications of the seaweed plant treatment were not significantly different to the untreated control in plant height, biomass, NDVI or weights (root and foliage).
[0083] These results confirm that nitrogen is lost through the heating of the eutectic melt, as evidenced by the superior results achieved by Verigrow -1 when compared with Verigrow-2. However, the results achieved by Verigrow-3, being the composition comprising keratin were similar to those of the unheated mixture, despite the heating process, showing unexpected benefits endowed by a fertiliser and/or soil improver composition according to the present invention. Example VI - Biological activity (field wheat trial)
[0084] A fertiliser and/or soil improver composition (nV-2) is prepared using a method exemplified in Example III, with a sheep wool material (uncleaned and/or containing the fat layer) being deconstructed using a eutectic melt comprising urea:choline chloride at a ratio in the range of from about 7.5:1 to about 4:1 as described herein above, and with the temperature for the deconstruction of wool being in a range of approximately 145 - 165 °C (or more specifically approximately 145 - 160 °C). The composition (nV-2) was also prepared at a weight to volume ratio of the wool to eutectic melt in the range of from about lg:lml to about lg:25 ml.
[0085] The resultant fertiliser and/or soil improver composition (nV-2) comprises 35% (w/v) of total N (nitrogen) and has pH in the range of pH 5.5 - 6.5. For the field application, the composition nV-2 was mixed with water (as a carrier) in a ratio of approximately 52 L water to approximately 48 L of nV-2.
[0086] The field wheat trial was established as a randomised complete block of 5 treatments and 4 replicates in a single bank with each plot 12 meter long by 2 meter wide.
[0087] The effectiveness of the fertiliser and/or soil improver composition nV-2 according to the present invention on plant growth (wheat in this instance) and/or as an organic nitrogen nutrient source, is compared with a commercial inorganic fertiliser (Flexi-N, which is a commercially available fertiliser distributed by the Australian Company CSBP and contains three forms of N - urea, ammonium and nitrate, with a total N concentration of 42%), and with Urea alone (at 46% total N).
[0088] Treatments containing 17 units of N were applied either as nV-2 at 35% (w/v) N, Flexi-N at 42% (w/v) N or Urea at 46% (w/v) N. The nV-2 and Flexi-N treatments were liquid banded in-furrow at seeding whilst Urea treatment was top-dressed and incorporated by sowing. All plots were sown to Sceptre wheat at a rate of 91 kg/ha to a depth of 2.5 cm. [0089] At 43 days after sowing, there was a second application of the Flexi-N and Urea treatments providing a further 42 units of N each, to the plots of these treatments.
[0090] At 41 and 75 days after sowing, the ND VI was recorded for each plot using a hand-held green- seeker.
[0091] At 209 days after sowing, the trial was plot harvested for yield comparisons with grain samples collected from each plot and analysed for quality (protein, moisture, specific weight and screenings) according to industry standards.
[0092] All data collected was statistically analysed with the proprietary agricultural field research program ARM® using an Analysis of Variance at a confidence limit of 95%.
[0093] The results of the field wheat trial is shown below
Table 3: Results of the field wheat trial
Figure imgf000027_0001
[0094] It can be seen that the nV-2 fertiliser and/or soil improver composition is superior in performance compared to the commercial inorganic nitrogen fertiliser Flexi-N and to Urea. In this field trial, 17 units of N applied as nV -2 (35% N) infurrow at seeding was equal to or even better than 59 units of N supplied by Flexi- N (42% N) or Urea (46% N) on the growth, yield and grain quality of Scepter wheat. The results show that per unit of N, the nV-2 fertiliser and/or soil improver composition according to the present invention had a net crop yield that was ~ 4.5 times higher than that of Flexi-N and Urea. With the superior bioavailability of the nitrogen nutrient in a fertiliser and/or soil improver composition according to the present invention, even at a nitrogen concentration significantly less than 35% (w/v) total N (e.g. at a centration in the range of from 10% (w/v) to 35% (w/v), 10% (w/v) to 30% (w/v), or even 10% (w/v) to 25% (w/v) total N), such as 12% (w/v), 15% (w/v), 17.5% (w/v), 20% (w/v), 22.4% (w/v), 25% (w/v), 28%(w/v), or 30% (w/v) total N), the composition according to the present invention may still be more effective than, or at least similarly effective to, a commercial, inorganic and/or urea nitrogen fertiliser.
Example VII - Biological activity (further wheat field trial)
[0095] A further wheat field trial is conducted using a fertiliser and/or soil improver composition nV-3 in accordance with the present invention, which is prepared in a same or similar manner, and has same/similar chemical properties to nV-1 and nV-2, except with the nv-3 fertiliser being diluted to provide nV-3 (27% N).
[0096] Application of the nv-3 (27% N) fertiliser in accordance with the present invention was administered via liquid banding in furrow at seeding, and spray application via hand boom at 6 weeks after sowing. As in Example VI, comparisons were made with trial plots applied with the commonly used nitrogen fertilisers Flexi-N and Urea.
[0097] The present inventors found that even the more diluted nv-3 (27% N) fertiliser has shown similar or better results to Flexi-N and Urea. In particular, the present inventors found that nv-3 (27% N) applied at 13 units of N at seeding and 32 units of N 6 weeks after sowing is an optimal product equivalent to urea at 17 units of N at seeding and 42 units of N 6 weeks after sowing. Example VIII - Chemical compatibility with common agricultural chemicals
[0098] Chemical compatibility testing has shown that a fertiliser and/or soil improver composition according to the present invention (e.g. nv-2) is compatible with chemicals commonly used in agricultural industry, including the herbicides (e.g. Velocity, MPCA 750, Ester 680, Tenet 500 SC etcetera), fungicides (e.g. Uplift, Prosaro, Impact, Oritus 430 and Soprano 500 etcetera), insecticides (e.g. Lemat, Bifenthrin 240, Dimethoate etcetera) and Adjuvants (e.g. SE14 and Adigor etcetera).
[0099] Advantageously, a fertiliser and/or soil improver composition according to the present invention may be applied simultaneously with, or subsequently to, one or more commonly used agricultural chemicals without the concern of chemical instability or incompatibility leading to precipitation occurring and/or loss of effectiveness of the composition of the present invention and/or the one or more agricultural chemical(s).
[00100] As has been previously shown, organic nitrogen is available to plants in the form of protein or as amino acids. The effect of treating wool, as exemplified in the present invention using a eutectic melt according to the present invention means that the resulting keratin protein, and fragments/fractions thereof (such as peptides and amino acids), are bioavailable, or even surprisingly highly bioavailable, as an organic nitrogen source. Even with the one or more biomolecules of the present invention being in a protein form, the nitrogen can be accessed by plants through root uptake in the form of the protein itself via endocytosis, or as simpler amino acids resulting from enzymatic digestion (either by the proteolytic activity of the roots or from microbes present in the soil).
[00101] The present invention has been found to possess a myriad of advantages over inorganic nitrogen sources, including a longer durability. Further, protein, or fragments/fractions thereof (such as peptides and/or amino acids), as one or more nitrogen sources results in an increase in a plant’s ability to develop a more extensive root network. It is understood that the keratin protein, or fragments/fractions thereof, available from the present composition through the deconstruction of wool, is bioavailable, or even highly bioavailable, as an organic nitrogen source available to plants. The present invention additionally provides the advantages of being an avenue for reducing or eliminating wool waste and also serving as an additional income stream for wool producers. Since other biomaterials such as those mentioned earlier (e.g. feathers, hair, hooves etc.) all comprise keratin or predominantly keratin as a structural protein, an eutectic melt according to the present invention is at least able to be used to deconstruct such biomaterials, thus turning further waste materials into income streams and environmentally friendly and effective organic sources of plant nutrients.
[00102] Modifications and variations as would be apparent to a skilled addressee are deemed to be within the scope of the present invention.

Claims

1. A fertiliser and/or soil improver composition for use in enhancing the growth of plants, the composition comprising one or more organic sources of plant nutrient(s), obtained through deconstruction of one or more biological materials by a eutectic melt, wherein the eutectic melt comprises urea and choline chloride.
2. A fertiliser and/or soil improver composition for use in enhancing the growth of plants according to claim 1, wherein the eutectic melt comprises choline chloride and urea.
3. A fertiliser and/or soil improver composition according to claim 2, wherein the molar ratio of urea to choline chloride is from about 8:1 to about 3:1.
4. A fertiliser and/or soil improver composition according to any one of the preceding claims, wherein the one or more organic sources of plant nutrient(s) is obtained from one or more biological materials selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof.
5. A fertiliser and/or soil improver composition according to any one of the preceding claims, wherein the one or more organic sources of plant nutrient(s) is one or more organic sources of nitrogen.
6. A fertiliser and/or soil improver composition according to claim 5, wherein the one or more organic sources of nitrogen is in the form of one or more biomolecules selected from the group consisting of proteins, peptides, amino acids, and combinations thereof.
7. A fertiliser and/or soil improver composition according to claim 5, wherein the one or more biomolecules is selected from the group consisting of keratin, keratin peptide fragments, amino acids, and combinations thereof.
8. A fertiliser and/or soil improver composition according to claim 7, wherein the one or more biomolecules are keratin peptide fragments and/or amino acids.
9. A fertiliser and/or soil improver composition according to claim 7, wherein the keratin peptide fragments and/or amino acids have a molecular weight in the range of from 1 to 10 kDa.
10. A fertiliser and/or soil improver composition according to claim 9, wherein the keratin peptide fragments and/or amino acids have a molecular weight in the range of 1 to 5 kDa.
11. A fertiliser and/or soil improver composition according to any one of the preceding claims, wherein the deconstruction of one or more biological materials by a eutectic melt is at a temperature of approximately 145 - 165°C.
12. A fertiliser and/or soil improver composition according to any one of the preceding claims, wherein the molar ratio of urea to choline chloride is from about 7.5:1 to about 4:1.
13. A fertiliser and/or soil improver according to any one of the preceding claims, wherein a weight to volume ratio of the one or more biological materials to eutectic melt is from about 1 g:l ml to about 1 g:25 ml.
14. A fertiliser and/or soil improver composition according to claim 1, wherein one or more of the following plant growth parameters is enhanced: plant height, biomass, normalised difference vegetation index (ND VI), root weight, foliage weight and total plant weight.
15. A fertiliser and/or soil improver composition for use in enhancing the growth of plants, the composition comprising one or more biomolecules in a molecular weight range of from 1 to 20 kDa in a eutectic melt, obtained through deconstruction of one or more biological materials by the eutectic melt, wherein the eutectic melt is urea and choline chloride.
16. A fertiliser and/or soil improver composition according to claim 15, wherein a molar ratio of urea to choline chloride is in a range of from about 7.5:1 to 4:1.
17. A fertiliser and/or soil improver composition according to claim 15 or 16, wherein the biological material is wool.
18. A fertiliser and/or soil improver composition in the form of a wool extract comprising keratin peptides and/or amino acids dissolved in a eutectic melt comprising urea and choline chloride.
19. A fertiliser and/or soil improver composition according to claim 18, wherein the keratin peptides and/or amino acids are in a molecular weight range of from 1 to 20 kDa.
20. A fertiliser and/or soil improver composition according to claim 18 or 19, wherein the molar ratio of urea to choline chloride is in a range of from about 7.5:1 to 4:1.
21. A fertiliser and/or soil improver composition according to any one of claims 18 to 20, wherein the wool is selected from the group consist of sheep, alpaca, Llama, goats, rabbits and combinations thereof.
22. A method of preparing a fertiliser and/or soil improver composition comprising one or more organic sources of plant nutrient(s), the method comprising the steps of: a) forming a eutectic melt; and b) deconstructing one or more biological materials using the eutectic melt, to obtain the one or more organic sources of plant nutrient(s).
23. A method according to claim 22, the one or more organic sources of plant nutrient(s) is obtained from one or more biological materials selected from the group consisting of wool, hair, feathers, horns, hooves, nails, skins, claws, beaks, and combinations thereof.
24. A method according to claim 22 or 23, wherein the one or more organic sources of plant nutrient(s) is one or more organic sources of nitrogen.
25. A method according to claim 24, wherein the one or more organic sources of nitrogen sources in the form of one or more biomolecules selected from the group consisting of proteins, peptides, amino acids, and combinations thereof.
26. A method according to claim 25, wherein the one or more biomolecules is selected from the group consisting of keratin, keratin peptide fragments, amino acids, and combinations thereof.
27. A method according to claim 26, wherein the one or more biomolecules have a molecular weight in the range of from 1 to 20 kDa.
28. A method according to any one of claims 22 to 27, wherein the eutectic melt comprises choline chloride and urea.
29. A method according to claim 28, wherein the choline chloride and urea mixture is heated.
30. A method according to any one of claims 22 to 29, wherein the deconstruction of one or more biological materials by a eutectic melt is at a temperature of approximately 145 - 165 °C.
31. A method according to any one of claims 22 to 28, wherein a molar ratio of urea to choline chloride is from about 8:1 to about 3:1.
32. A method according to claim 29, wherein the molar ratio of urea to choline chloride is from about 7.5:1 to about 4:1.
33. A method of enhancing the growth of plants comprising a step of applying a fertiliser and/or soil improver composition according to any one of claims 1 to 21 to plants and/or soil supporting the plants.
34. A method according to claim 33, wherein one or more of the following plant growth parameters is enhanced: plant height, biomass, normalised difference vegetation index (ND VI), root weight, foliage weight and total plant weight.
35. A method according to claim 33 or 34, comprising the use of the fertiliser and/or soil improver composition as an additive to a known fertiliser and/or soil improver.
36. A method according to any one of claims 33 to 35 comprising multiple, separate applications of the fertiliser and/or soil improver composition.
37. A method according to any one of claims 33 to 36, comprising an application of the fertiliser and/or soil improver composition as a liquid to a surface of soil, proximal to a location of the plant within that soil.
PCT/AU2023/050414 2022-05-16 2023-05-16 Fertiliser and/or soil improver composition, method of preparation and method of use Ceased WO2023220779A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ788293 2022-05-16
NZ788293A NZ788293A (en) 2022-05-16 Fertiliser and/or soil improver composition, method of preparation and method of use

Publications (1)

Publication Number Publication Date
WO2023220779A1 true WO2023220779A1 (en) 2023-11-23

Family

ID=88834212

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2023/050414 Ceased WO2023220779A1 (en) 2022-05-16 2023-05-16 Fertiliser and/or soil improver composition, method of preparation and method of use

Country Status (1)

Country Link
WO (1) WO2023220779A1 (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013078517A1 (en) * 2011-12-02 2013-06-06 The University Of Western Australia Methods for isolating constituents from nacre-coated shell or pearl
CN108623709A (en) * 2018-06-08 2018-10-09 中国海洋大学 A kind of extracting method of chitin
US20200255354A1 (en) * 2016-09-01 2020-08-13 Veratin Pty Ltd Organic Fertiliser and Soil Improver Comprising Keratin
CN112812323A (en) * 2021-01-18 2021-05-18 沈阳农业大学 Method for extracting humic acid from mineral source
WO2021180996A1 (en) * 2020-03-12 2021-09-16 Consejo Superior De Investigaciones Científicas (Csic) Method for obtaining a phenolic extract from alpeorujo (semi-solid olive oil byproduct)
WO2022036010A1 (en) * 2020-08-11 2022-02-17 Cellacure Llc Green closed loop bio-waste refining process for producing smart active extracts and delivery systems for their application
US20220281779A1 (en) * 2016-09-01 2022-09-08 Veratin Pty Ltd Fertiliser and/or Soil Improver Composition, Method of Preparation and Method of Use

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013078517A1 (en) * 2011-12-02 2013-06-06 The University Of Western Australia Methods for isolating constituents from nacre-coated shell or pearl
US20200255354A1 (en) * 2016-09-01 2020-08-13 Veratin Pty Ltd Organic Fertiliser and Soil Improver Comprising Keratin
US20220281779A1 (en) * 2016-09-01 2022-09-08 Veratin Pty Ltd Fertiliser and/or Soil Improver Composition, Method of Preparation and Method of Use
CN108623709A (en) * 2018-06-08 2018-10-09 中国海洋大学 A kind of extracting method of chitin
WO2021180996A1 (en) * 2020-03-12 2021-09-16 Consejo Superior De Investigaciones Científicas (Csic) Method for obtaining a phenolic extract from alpeorujo (semi-solid olive oil byproduct)
WO2022036010A1 (en) * 2020-08-11 2022-02-17 Cellacure Llc Green closed loop bio-waste refining process for producing smart active extracts and delivery systems for their application
CN112812323A (en) * 2021-01-18 2021-05-18 沈阳农业大学 Method for extracting humic acid from mineral source

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRADIC BOJANA, UROS NOVAK, BLAZ LIKOZAR: "Crustacean shell bio-refining to chitin by natural deep eutectic solvents", GREEN PROCESS SYNTH, vol. 9, 30 October 2019 (2019-10-30), pages 13 - 25, XP093113287, DOI: 10.1515/gps-2020-0002 *
JIANG ZHE, YUAN JIUGANG, WANG PING, FAN XUERONG, XU JIN, WANG QIANG, ZHANG LIANBING: "Dissolution and regeneration of wool keratin in the deep eutectic solvent of choline chloride-urea", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, ELSEVIER BV, NL, vol. 119, 1 November 2018 (2018-11-01), NL , pages 423 - 430, XP093113282, ISSN: 0141-8130, DOI: 10.1016/j.ijbiomac.2018.07.161 *
KATHERINE E. MOORE, ET AL.: "Wool deconstruction using a benign eutectic melt", RSC ADVANCES, vol. 6, no. 24, 1 January 2016 (2016-01-01), pages 20095 - 20101, XP055471486, DOI: 10.1039/C5RA26516A *

Similar Documents

Publication Publication Date Title
AU2024204220A1 (en) Fertiliser and/or soil improver composition, method of preparation and method of use
AU2014206502B2 (en) Melanoidins and their use for improving properties of plants
EP0969722B1 (en) Method for treating cotyledonous plants
CN111662121A (en) Slow-release type bulk blending fertilizer
JP2017525688A (en) Plant inoculation method
TW202339624A (en) Use of a keratin hydrolysate with high contents of free amino acids to stimulate emergence and early growth of plants
US20220281779A1 (en) Fertiliser and/or Soil Improver Composition, Method of Preparation and Method of Use
CN113149766A (en) Organic-inorganic compound fertilizer and preparation method thereof
US20250176560A1 (en) Method of using/applying a keratin hydrolysis peptide solution to promote growth of corn under low light condition
JP2000309502A (en) Plant growth promoter and fertilizer using the plant growth promoter
Szentpétery et al. Effect of nitrogen top-dressing on winter wheat
WO2023220779A1 (en) Fertiliser and/or soil improver composition, method of preparation and method of use
EP4279474A1 (en) Fertiliser and/or soil improver composition, method of preparation and method of use
US20250214906A1 (en) Method For Promoting The Growth Of Corn Under Low Temperature Stress
US10472294B2 (en) Semi-humic composition and methods of use thereof
US20250324978A1 (en) Method of using/applying a keratin hydrolysis peptide solution to promote growth of cotton under low light condition
CA3158923A1 (en) Fertiliser and/or soil improver composition, method of preparation and method of use
Marinova et al. Application of total care and aminobest foliar fertilizers and their influence on the main quality traits in alfalfa (Medicago sativa L.).
US20250206677A1 (en) Method of using/applying a keratin hydrolysis peptide solution to promote the growth of wheat under low temperature condition
US20250081965A1 (en) Method of using/applying a keratin hydrolysis peptide solution upon coffee plants to increase fruiting yield and weight
US20250160336A1 (en) Method of using/applying a keratin hydrolysis peptide solution to promote soybean growth under low light condition
EA028406B1 (en) Fertilizer including highly dispersed sulphur as primary material
US20250204522A1 (en) Method for mitigating herbicide damage to cotton
US20250169507A1 (en) Method Of Using/Applying A Keratin Hydrolysis Peptide Solution To Promote Growth Of Wheat Under Low Light Condition
CN111792964A (en) Microbial fertilizer capable of remarkably increasing soybean yield

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23806447

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23806447

Country of ref document: EP

Kind code of ref document: A1