AU2018454451B2

AU2018454451B2 - Bioavailable nitrogen composition and methods

Info

Publication number: AU2018454451B2
Application number: AU2018454451A
Authority: AU
Inventors: Brian Horsley; Conor O'brien
Original assignee: Multikraft Probiotics Australia Pty Ltd
Current assignee: Multikraft Probiotics Australia Pty Ltd
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2021-04-29
Anticipated expiration: 2038-12-18
Also published as: NZ773610A; CN113454050A; WO2020124119A1; AU2018454451A1; AU2018454451C1

Abstract

Compositions suitable for plant fertilisation are provided. The compositions comprise a nitrogen-containing compound, such as urea, and one or more additional components adapted to increase bioavailability of nitrogen from the nitrogen-containing compound. Associated production methods and methods of increasing nitrogen bioavailability are also provided. Further provided are methods of plant fertilisation and composting using the compositions. Suitable additional components include a microbial culture, an organic acid and a sugar.

Description

TITLE

BIOAVAILABLE NITROGEN COMPOSITION AND METHODS

TECHNICAL FIELD

THIS invention relates to a fertiliser composition containing nitrogen. More particularly, the invention relates, although is not limited, to a composition containing urea and additional components adapted to increase bioavailability of nitrogen from urea.

BACKGROUND

Urea (also referred to as carbamide), is an organic compound of chemical formula CO(NH₂)₂. Urea is widely used as a nitrogen fertiliser for plants, including crop plants. However, urea fertiliser can be associated with relatively low nitrogen use efficiency (NUE).

It is known that urea can enter plants directly, or in derivative forms such as ammonium, e.g. after metabolism by soil microbes. However, nitrogen from urea- based fertiliser can become unavailable to plants through mechanisms such as immobilisation, denitrification, nitrous oxide emission, ammonia volatilisation, and leaching.

Limited effective options exist with respect to fertiliser compositions and associated methods for improving nitrogen bioavailability from urea-based fertilisers. Accordingly, new such compositions, and associated methods, are desirable.

SUMMARY

In one broad form this invention provides a composition comprising a nitrogen-containing compound, such as urea, and one or more additional components adapted to increase bioavailability of nitrogen from the nitrogen-containing compound.

In a first aspect, there is provided a composition comprising a nitrogen- containing compound; and a microbial culture.

Suitably, the composition is a fertiliser composition. In embodiments, the composition is a slow-release fertiliser composition.

In embodiments, the composition of the first aspect is a liquid or semi-liquid composition. The composition may be an aqueous composition. In embodiments, the nitrogen-containing compound of the composition is or includes urea.

In embodiments, the composition of this aspect comprises a sugar. The sugar may be, or be of, a sugar syrup. In an embodiment, the sugar syrup is molasses. The molasses may be or include a sugarcane molasses.

In embodiments, the composition of this aspect further comprises an organic acid. The organic acid may be or include a fulvic acid and/or a humic acid.

In embodiments, the composition of this aspect comprises a plant by-product. In embodiments, the plant by-product is or includes vinasse.

In embodiments, the microbial culture of the composition of this aspect is or includes one or more microbes selected from the group consisting of a lactic acid bacteria; a phototrophic bacteria; a diazotroph; a Nitrobacter species; a Bacillus species; and a yeast. In embodiments, the microbial culture comprises a lactic acid bacteria; a phototrophic bacteria; a diazotroph; a Bacillus species; and a yeast.

The composition of the first aspect will comprise nitrogen. The composition may comprise one or more additional plant nutrients, inclusive of micronutrients and/or macronutrients.

In embodiments, the composition comprises one or more macronutrients other than nitrogen, wherein the macronutrients other than nitrogen are selected from the group consisting of phosphorus; potassium, sulfur, calcium, magnesium, and sodium.

In embodiments, the composition comprises one or more micronutrients selected from the group consisting of copper; zinc; manganese; iron; boron; molybdenum; cobalt; and silicon.

In embodiments, the nitrogen-containing compound is present in the composition at a concentration equivalent to between about 1 and about 60 weight per weight percent (w/w%) nitrogen, including 3 to about 55, 5 to 50 and inclusive of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 55 w/w% nitrogen.

In embodiments, the microbial culture is present in the composition at a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%. In embodiments wherein the composition of the first aspect comprises an organic acid, the organic acid may be present at a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%.

In embodiments wherein the composition of the first aspect comprises a sugar, the sugar may be present at a concentration of between about 0.5 and about 7.5 % w/w%, including about 1, 2, 3, 4, 5, 6, and 7 w/w%.

In embodiments wherein the composition of the first aspect comprises a sugar syrup, the sugar syrup may be present at a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%.

In embodiments wherein the composition of the first aspect comprises a plant by-product, the plant by-product may be present at a concentration of between about 50 and about 95 volume per volume percent (v/v%), including about 55, 60, 65, 70, 75, 80, 85, 90, and 95 v/v%.

In embodiments wherein the composition of the first aspect comprises one or more macronutrients other than nitrogen, the macronutrients other than nitrogen may be present at a total concentration of between about 0.1 and about 3 w/w%, including about 0.5, 1, 1.5, 2, 2.5, and 3 w/w%.

In embodiments wherein the composition of the first aspect comprises one or more micronutrients, the micronutrients may be present at a total concentration of between about 0.01 and about 0.2 w/w%, including about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, and 0.15 w/w%.

In a second aspect, the invention provides a method of producing a composition, including the step of combining a plurality of components wherein the components include a nitrogen-containing compound; and a microbial culture, to thereby produce the composition.

A third aspect of the invention provides a method of increasing bioavailability of nitrogen from a nitrogen-containing compound, including the step of combining a plurality of components including the nitrogen-containing compound; and a microbial culture, to thereby increase the bioavailability of nitrogen from the nitrogen- containing compound.

In embodiments of the second or third aspect, the plurality of components include an organic acid. The organic acid may be a humic acid and/or a fulvic acid. In embodiments of the second or third aspect, the plurality of components include a sugar, such as a sugar syrup. The sugar syrup may be molasses.

In embodiments of the second or third aspect, the plurality of components include a plant by-product. The plant by-product may be vinasse.

In embodiments of the second or third aspect, the method includes the step of fermenting the combined components.

In embodiments of the second or third aspect wherein the method includes the step of fermenting the combined components, the fermenting may be for a duration of about 1 day to at least about 14 days, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 days.

In embodiments of the second or third aspect wherein the method includes the step of fermenting the combined components, the fermenting may be performed at ambient temperature. In embodiments, the fermenting may be performed at elevated temperature. In embodiments, the elevated temperature is between about 30°C and about 60°C, including about 35, 40, 45, 50, and 55°C.

In embodiments of the second or third aspect, the method includes the step of cooling or resting the combined components.

In embodiments of the second or third aspect wherein the method includes the step of cooling or resting the combined components, the cooling or resting may be for a duration of about 1 day to at least about 14 days, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 days.

In embodiments of the second or third aspect wherein the method includes the step of cooling or resting the combined components, the cooling or resting may be at ambient temperature, such as a temperature of about 10°C to about 30°C, including about 15, 20, and 25 °C.

A fourth aspect of the invention provides a fertiliser composition produced according to the method of the second aspect. In embodiments, the composition of the fourth aspect is a composition of the first aspect.

In a fifth aspect, the invention provides a method of fertilising a plant, including the step of applying a composition comprising a nitrogen-containing compound; and a microbial culture, to the plant.

In an embodiment of the fifth aspect, the plant is a crop plant. In a sixth aspect, the invention provides a method of composting organic matter, including the step of applying a composition comprising a nitrogen-containing compound; and a microbial culture, to the organic matter.

In an embodiment of the sixth aspect, the organic matter is plant material.

In a seventh aspect, the invention provides a method of increasing nitrogen bioavailability in a growth medium, including the step of applying a composition comprising a nitrogen-containing compound; and a microbial culture, to the growth medium.

In an embodiment of the seventh aspect, the growth medium is soil.

In embodiments of the fifth to seventh aspects, the applied composition comprises an organic acid. The organic acid may be a humic acid and/or a fulvic acid.

In embodiments of the fifth to seventh aspects, the applied composition comprises a sugar, such as a sugar syrup. The sugar syrup may be molasses.

In embodiments of the fifth to seventh aspects, the applied composition comprises a plant by-product. The plant by-product may be vinasse.

In embodiments of the fifth to seventh aspects, the applied composition is a composition of the first or fourth aspects.

It will be appreciated that the indefinite articles“a” and“an” are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, “a” nitrogen- containing compound includes one nitrogen-containing compound, one or more nitrogen-containing compounds or a plurality of nitrogen-containing compounds. Similarly,“a” microbial culture includes one microbial culture, one or more microbial cultures, or a plurality of microbial cultures.

As used herein, unless the context requires otherwise, the words“comprise”, “ comprises” and“ comprising” will be understood to mean 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 THE FIGURES

Figure 1 sets forth a schematic illustration of a typical soil nitrogen cycle, showing relative mobility of various nitrogen forms. Figure 2 sets forth a schematic illustration of the experimental timeline used for Example 4.

Figure 3 sets forth soil microbial biomass nitrogen at day 30 in all treatments in three soils, as per Example 4. Composition 2 is labelled‘Multikraft Dunder’ in Figure 3.

Figure 4 sets forth soil soluble organic and inorganic nitrogen concentrations (mg nitrogen per kilogram of dry soil) in incubated soils as per Example 4. Soluble organic nitrogen (protein, peptides, amino acids) is shown in the left column. Ammonium concentrations are shown in the centre column. Nitrate values are shown in the right column. Top row is the Bundaberg grey sandy soil, middle row is the Bundaberg red loam soil, bottom row is the Kalbar alluvial vertisol. Control treatments are shown in black with downward triangles. Urea treatments are shown in red with upwards triangles. Dunder treatments are shown in blue with squares. Multikraft Probiotic Dunder (Composition 2) is shown in green with circles.

DETAILED DESCRIPTION

The present invention is at least partly predicated on the identification of components suitable for combining with a nitrogen-containing compound to increase the bioavailability of nitrogen from the nitrogen-containing compound for plant fertilisation.

As used herein, “ bioavailability”, “bioavailable” , and the like, will be understood to refer to a state or form in which a plant nutrient, particularly nitrogen, is accessible by a plant from the environment. It will be appreciated that a state of being more bioavailable, or having greater bioavailability, refers to a state in which the plant nutrient is comparatively more accessible by a plant from the environment and may include an increased level of stability of certain bioavailable forms. It may be that increased bioavailability results from reduced conversion of the nitrogen source into less usable or more mobile forms.

By way of non-limiting example, as the skilled person will appreciate, plants may obtain nitrogen from the environment as ammonium (NH4⁺), and also as nitrate (NO3 ) and nitrite (NO2 ). However, ammonium has substantially less mobility or greater stability in soil as compared to nitrate and nitrite. Furthermore, substantial loss of nitrate and nitrite occurs to the atmosphere in the form of nitrous oxide and the like. Accordingly, for the purposes of this invention (unless the context requires otherwise) nitrogen in ammonium form will be considered to have greater bioavailability, or to be more bioavailable, than nitrogen in nitrate and/or nitrite form and may be considered a stabilised form of nitrogen which is available to plants.

It will be appreciated that the scope of“bioavailability” .“ bioavailable and the like, may further refer to a state or form in which a plant nutrient, particularly nitrogen, is stored, for later access by a plant. This may include a greater % of the nitrogen being present in a more stable form than would otherwise be the case without intervention by compositions of the present invention. By way of non-limiting example, as the skilled person will appreciate, nitrogen incorporated within microbial biomass, and/or in proteins, peptides, and amino acids, can represent effective storage of nitrogen for later access by a plant.

It will be understood that, in the context of the invention, the bioavailability of nitrogen from the nitrogen-containing compound can be measured under various circumstances.

In at least some embodiments, increased bioavailability of nitrogen may be measured within a composition as described herein comprising the nitrogen- containing compound along with one or more additional components.

In at least some embodiments, increased bioavailability of nitrogen may be measured after application of a composition as described herein to a substrate or growth medium, such as soil or compost, although without limitation thereto. By way of non-limiting example, there may be an increase in bioavailable nitrogen from the nitrogen-containing compound at one or more time points after application of a composition as described herein to a substrate, as compared to application of the nitrogen-containing compound itself, or the nitrogen-containing compound in an alternative composition, to the substrate. This may include maintenance of a bioavailable nitrogen source in that form, such as a reduced conversion of that form into a less bioavailable or more mobile form, for a greater period of time.

In some embodiments, an increase in bioavailable nitrogen from a nitrogen- containing compound in the context of the invention will be associated with a relative increase in ammonium derived from the nitrogen-containing compound. In some embodiments, an increase in bioavailable nitrogen from a nitrogen- containing compound will be associated with a relative decrease in nitrate and/or nitrite derived from the nitrogen-containing compound.

In some embodiments an increase in bioavailable nitrogen from a nitrogen- containing compound will be associated with a relative increase in nitrogen as microbial biomass, and/or protein, peptides, or amino acids, from the nitrogen- containing compound.

In the description that follows, various concentrations are given using weight and/or volume -based percentages. It will be readily appreciated by the skilled person that such percentages can be interconverted where density properties (e.g. specific gravity) of applicable solution(s) are known. By way of example, in Table 1 herein, concentration values for some measured components of a typical fertiliser composition are provided in both w/w% and w/v%, in view of the specific gravity of the composition. More generally, w/w%, w/v%, and v/v% will be readily interconvertible by the skilled person, as desired, based on the disclosure of this specification as a whole.

Compositions

One aspect of the invention is directed to a composition comprising a nitrogen-containing compound; and a microbial culture.

The composition of this aspect is typically a fertiliser composition. As used herein, a“fertiliser composition” refers to a composition for application to a plant to provide the plant with one or more nutrients that typically assist with growth and/or development of the plant. The composition may be a “slow release” fertiliser composition, adapted to release one or more nutrients (typically including nitrogen) gradually (typically over a period of days to months), for uptake by a plant.

It will be understood that, included within the scope of “fertiliser composition” as used herein is a“ precursor composition” , which will be understood to be a composition that is for application to a plant after one or more processing steps. In a typical embodiment, the fertiliser composition is a precursor composition that is for application to a plant after a step of fermentation of the composition by or involving a microbial culture. Typically, the microbial culture is the microbial culture of the composition. The nitrogen-containing compound according to the composition of this aspect typically also contains carbon. More typically, the nitrogen-containing compound contains carbon, hydrogen, oxygen, and nitrogen.

Typically, the nitrogen-containing compound contains at least about 20% nitrogen by weight (i.e. 20 w/w% nitrogen), including at least 25, 30, 35, 40, and 45 w/w% nitrogen.

Typically, the nitrogen-containing compound is crystalline.

Typically, the nitrogen-containing compound has a high solubility in water.

Typically, the nitrogen-containing compound is urea, or an analogue or derivative thereof.

As used herein, an“ analogue” refers to a compound that is structurally similar to an original or ‘parent’ compound (such as urea), but differs somewhat in composition (e.g., an atom or component thereof, or functional group is different, added, or removed).

Analogues may or may not have different chemical or physical properties than the parent compound and may or may not have altered biological and/or chemical activity. By way of non-limiting example, the analogue may be more hydrophilic or it may have altered reactivity as compared to the parent compound. The analogue may mimic the chemical and/or biological activity of the parent compound (i.e., it may have similar or identical activity), or, in some cases, may have increased or decreased activity. An analogue may be a naturally or non-naturally occurring (e.g. synthetic) variant of a parent compound.

As used herein, a“ derivative” refers to a chemically or biologically modified version of a parent compound (such as urea) that is structurally similar to an original or parent compound and (actually or theoretically) derivable from that parent compound. A“ derivative” differs from an“ analogue” in that a parent compound may be the starting material to generate a derivative, whereas the parent compound need not necessarily be used as the starting material to generate an analogue.

The microbial culture according to the composition of this aspect typically includes a plurality of microbes.

In typical embodiments, the microbial culture includes at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 120, 140, or 150 microbial isolates. In one typical embodiment the microbial culture includes greater than about 80 isolates.

In typical embodiments, the microbial culture includes at least: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 microbial species. In one typical embodiment the microbial culture includes greater than about 45 microbial species.

Typically, the microbial culture will be adapted to perform fermentation, wherein the nitrogen-containing compound of the composition is capable of being used a nitrogen source for the fermentation.

In a typical embodiment, the microbial culture includes a lactic acid bacteria, such as one or more bacteria of the genera Abiotrophi ; Aerococcus; Carnobacterium ; Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella. Typically, the lactic acid bacteria includes a bacteria of the genera Lactobacillus. In one typical embodiment, the lactic acid bacteria includes Lactobacillus casei and/or Lactobacillus plantarum.

In certain embodiments, the microbial culture includes a Nitrobacter species, such as one or more of Nitrobacter winogradskyi, Nitrobacter hamburgensis, Nitrobactervulgaris and Nitrobacter alkalicus.

In a typical embodiment, the microbial culture includes a phototrophic bacteria, such as one or more of an aerobic anoxygenic phototrophic bacteria; a bacteria of the Chlorobium genus; a bacteria of the Chloroflexi class; a bacteria of the Chromatium genus; a Cyanobacteria, a bacteria of the Chlorobiaceae family; a bacteria of the Halobacterium genus; a heliobacteria; a bacteria of the Herpetosiphon genus; a purple bacteria; a purple non-sulfur bacteria; a bacteria of the genus Rhodopseudomonas, a bacteria of the genus Rhodobacter, a bacteria of the genus Rhodoferax; a bacteria of the genus Rhodospirillum; and a sulfur-reducing bacteria. In one typical embodiment the phototrophic bacteria includes Rhodopseudomonas palustris.

In a typical embodiment, the microbial culture includes a diazotroph, typically a nitrogen-fixing bacteria, such as one or more bacteria of the genera Rhizobium; Acetobacter, Azotobacter, Azo spirillum, Trichodesmium, Anabaena, Lrankia, Burkholderia, Sinorhizobium, Bradyrhizobium, Herbaspirillum, and Azoarcus. In one typical embodiment the diazotroph includes Acetobacter tropicalis, Acetobacter lovaniensis, Acetobacter syzygib, and/or one or more Rhizobium species.

In a typical embodiment, the microbial culture includes a Bacillus or Lysinibacillus species, such as Bacillus subtilis, Lysinibacillus sphaericus, and/or Bacillus amyloliquefaciens.

In a typical embodiment, the microbial culture includes a yeast, such as one or more yeasts of the genera Aureobasidium, Brettanomyces, Candida ; Cryptococcus, Debaryomyces, Hanseniaspora, Kluyveromyces, Kuraishia, Leucosporidiunv, Malassezia, Metschnikowia, Pachysolew, Pichiw, Rhodotorulw, Saccharomyces, Saccharomycodes, Schizosaccharomyces, Torulaspora ; Trichosporon and Zygosaccharomyces. In one typical embodiment the yeast includes Saccharomyces cerevisiae.

In one typical embodiment of the composition of this aspect, the microbial culture includes a Lactobacillus species; typically Lactobacillus casei and/or Lactobacillus plantarum, a Rhodopseudomonas species, typically Rhodopseudomonas palustris, an Acetobacter species, typically Acetobacter tropicalis, Acetobacter lovaniensis, and/or Acetobacter syzygii, a Rhizobium species; a Bacillus or Lysinibacillus species, typcially Bacillus subtilis, Lysinibacillus sphaericus, and/or Bacillus amyloliquefaciens, and a Saccharomyces species, typically Saccharomyces cerevisiae.

With reference to the examples, it will be appreciated that one typical composition as described herein comprises the commercial microbial culture mix ‘MicroLife’ (https://www.multikraft.com.au/MicroLife). Although MicroLife is suitable according to compositions of this aspect, it is considered that a range of other microbial mixes are also suitable.

It will be appreciated that MicroLife comprises lactic acid bacteria (. Lactobacillus ); phototrophic bacteria ( Rhodopseudomonas ); nitrogen-fixing bacteria ( Acetobacter and Rhizobium ); Bacillus bacteria; and yeast ( Saccharomyces ). It is considered that such a combination of lactic acid bacteria; phototrophic bacteria, nitrogen-fixing bacteria, Bacillus bacteria, and yeast, may be particularly beneficial for compositions as described herein. A Nitrobacter species may also be included. Typically, the composition of this aspect further comprises a sugar, inclusive of monosaccharide, disaccharide, oligosaccharide, and polysaccharide sugars. Typically, the sugar is a monosaccharide or a disaccharide. In some embodiments, the sugar is selected from the group consisting of glucose (dextrose); sucrose; and fructose.

In certain embodiments wherein the composition comprises a sugar, the sugar is, or is of, a sugar syrup. As used herein,“ sugar syrup” (which may also be referred to more simply as‘syrup’) will be understood to refer to any solution, typically an aqueous solution, containing a high concentration of one or more sugars. Typically, the concentration of sugar in the sugar syrup is greater than 30, 40, 50, 60, 70, or 80 w/w%. More typically the concentration of sugar is greater than about 50 w/w%. The solution will typically have relatively high viscosity, and may be a liquid or a semi liquid.

In embodiments, the sugar syrup of the composition of this aspect is molasses. As the skilled person will understand, molasses is a syrup resulting from extracting and boiling of juice from sources including sugarcane or sugar beet. As the skilled person will appreciate, molasses typically has a specific gravity between about 1.4 and about 1.5, and greater than 50 w/w% sugar. Typically, the molasses is sugarcane molasses.

In certain embodiments, the composition of this aspect comprises an organic acid. Typically, the organic acid includes fulvic acid and/or humic acid.

As the skilled person will appreciate, fulvic acid and humic acid are two classes of acidic organic polymers that can be extracted from humus. Naturally occurring fulvic and humic acids are best described as a family of organic acids, however, formulas of CuHnOs for fulvic acid; and C9H9NO6 for humic acid may be used.

In some embodiments, the composition of this aspect further comprises a plant by-product. As used herein, unless the context requires otherwise, a “plant by product (which may also be referred to as a‘secondary plant product’), will be understood to be a product that results from, or during the course of, processing a plant material to form a different product (which may be referred to as a‘primary plant product’). For the sake of clarity, a “plant by-producf includes within its scope any substance, agent, or component that has the characteristics of a secondary plant product produced during the processing of a plant material to form a primary plant product. That is, the description of a component as a“plant by-producf herein will be understood to impose no inherent limitation with respect to how the component has been produced.

It will be appreciated that the term‘co-product’, may be used in some contexts to describe individual products produced during the course of processing of a plant material. In at least some embodiments, the scope of “ plant by-producf may encompass one or more respective products alternatively described as plant co products.

Typically, the plant by-product according to the composition of this aspect is a by-product of sugar and/or alcohol production. Typically, the plant by-product is vinasse.

As will be understood by the skilled person, vinasse may be alternatively referred to as stillage, dunder or biodunder, and is a remaining product after sugarcane or sugar beet is processed to produce a primary product such as crystalline sugar, molasses, or ethanol, and the primary product is removed. In some embodiments, the vinasse may be defined as a by-product of biomass distillation wherein the biomass may be one or more of sugar crops (including sugarcane and beet), starch crops (including corn, wheat, rice and cassava), and cellulosic materials (including harvesting crop residues, sugarcane bagasse and woods). In one embodiment, and as used in the examples herein, the vinasse may be biodunder from the Biostil fermentation/distillation process and as supplied by Wilmer Bioethanol (https://wilmarbioethanol.com/what-is-biodunder). Typically, the vinasse according to the composition of this aspect is vinasse as results from molasses production, typically sugarcane molasses production. Typically, the vinasse according to the composition of this aspect comprises vegetable matter; yeast biomass; and plant nutrients, such as potassium, sodium, nitrogen, calcium, magnesium, phosphorous and/or sulfur. The use of the vinasse of biodunder can provide significant benefits to the composition in terms of, amongst others, solubilisation of the nitrogen-containing compound and generation of heat. The composition of this aspect will comprise nitrogen. Typically, the composition further comprises one or more additional plant nutrients. The one or more additional plant nutrients may be plant micronutrients and/or plant macronutrients.

Typically, the composition of this aspect comprises one or more macronutrients other than nitrogen selected from the group consisting of phosphorus; potassium; sulfur; calcium; magnesium; and sodium.

Typically, the composition of this aspect comprises one or more micronutrients selected from the group consisting of copper; zinc; manganese; iron; boron; molybdenum; cobalt; and silicon.

In a typical embodiment, the nitrogen-containing compound is present in the composition of this aspect at a concentration equivalent to between about 1 and about 60 w/w% nitrogen, including about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 55 w/w% nitrogen. More typically, the nitrogen-containing compound is present at between about 3 and about 55 w/w% nitrogen or between about 5 and about 50 w/w% nitrogen. In one typical embodiment the nitrogen-containing compound is present in the composition of this aspect at a concentration of about 15 w/w% nitrogen.

In some typical embodiments, total nitrogen content of the composition of this aspect is between about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, and 60 w/w% nitrogen. More typically, total nitrogen content of the composition of this aspect is between about 10 and about 50 w/w% nitrogen. In one typical embodiment, total nitrogen content of the composition is about 15 w/w% nitrogen.

In a typical embodiment, the composition of this aspect comprises ammonium at a concentration equivalent to between about 0.1 and at least about 1 w/w% nitrogen, including about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 w/w% nitrogen. In one typical embodiment, the composition of this aspect comprises ammonium at a concentration equivalent to about 0.5 w/w% nitrogen.

In a typical embodiment, the composition of this aspect comprises total nitrate and nitrite equivalent to less than about 0.005 w/w% nitrogen, including less than about 0.003, 0.001, 0.0008, and 0.0006 w/w% nitrogen. In one typical embodiment, the composition of this aspect comprises total nitrate and nitrate equivalent to less than about 0.0006 w/w% nitrogen.

In a typical embodiment, the composition of this aspect comprises total nitrite equivalent to less than about 0.005 w/w% nitrogen, including less than about 0.003, 0.001, 0.0008, and 0.0006 w/w% nitrogen. In one typical embodiment, the composition of this aspect comprises total nitrate equivalent to less than about 0.006 w/w% nitrogen.

In a typical embodiment, the composition of this aspect comprises total nitrate equivalent to less than about 0.00001 w/w% nitrogen, including less than about 0.000005 w/w% nitrogen, and less than about 0.0000005 w/w% nitrogen.

In some typical embodiments, the composition of this aspect comprises greater than about 0.5% of total nitrogen as ammonium, including greater than about 1, 1.5, 2, 2.5, and 3% of total nitrogen as ammonium.

In some typical embodiments, the composition of this aspect comprises less than about 0.01% total nitrogen as nitrate and nitrite, including less than about 0.009, 0.007, 0.005, and 0.003%.

Without limiting the scope of the term substantially as used herein, it will be understood that typical embodiments as described above may be referred to as ‘substantially free’ of nitrate and/or nitrite, or containing ‘no substantial’ nitrate and/or nitrite, or similar.

Typically, the microbial culture is present in the composition of this aspect at a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%. More typically, the microbial culture is present at a concentration of between about 2 and about 7 v/v%. In one typical embodiment, the microbial culture is present at a concentration of about 5 v/v%.

In embodiments wherein the composition of this aspect comprises an organic acid, the organic acid is present at a total concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%. More typically, the organic acid is present at a total concentration of between about 2 and about 7 v/v%. In one typical embodiment, the organic acid is present at a total concentration of about 5 v/v%. In embodiments, the composition comprises fulvic acid at a concentration of between about 0.5 and about 4 v/v%, including about 1, 2, and 3 v/v%. More typically, the composition comprises fulvic acid at a concentration of about 2 v/v%.

In an embodiment, the composition comprises humic acid at a concentration of between about 1 and about 6 w/v%, including about 2, 3, 4, and 5 v/v%. More typically, the composition comprises humic acid at a concentration of about 3 v/v%.

In certain embodiments wherein the composition of this aspect comprises a sugar, the sugar is at a total concentration of between about 0.5 to about 10 w/w%, including about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9.5 w/w%. In one typical embodiment, the sugar is present at a total concentration of about 3 w/w%.

In embodiments, the composition comprises a sugar syrup at a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%. More typically, the sugar syrup is present at a concentration of between about 2 and about 10 v/v%. In one typical embodiment, the sugar syrup is present at a concentration of about 5 v/v%.

In embodiments wherein the composition of this aspect comprises a plant by product, the plant by-product is present at a concentration of between about 50 and about 95 volume per volume percent (v/v%), including about 55, 60, 65, 70, 75, 80, 85, 90, and 95 v/v%. More typically, the plant by-product is present in the composition of this aspect at a concentration of between about 70 and about 90 v/v%. In one typical embodiment, the plant by-product is present in the composition at a concentration of about 85 v/v%.

In embodiments, the composition of this aspect comprises one or more macronutrients other than nitrogen at a total concentration of between about 0.1 and about 5 w/w%, including about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 w/w%. More typically, the total concentration of the one or more macronutrients other than nitrogen is between about 1 and about 3 w/w%. In one typical embodiment, the total concentration of one or more macronutrients other than nitrogen in the composition of this aspect is about 2 w/v%.

In certain embodiments, the composition of this aspect comprises phosphorus at a concentration of between 0.004 and about 0.04 w/w%, including about 0.005, 0.01, 0.02, and 0.03 w/w%. More typically, the composition of this aspect comprises phosphorus at about 0.015 w/w%.

In embodiments, the composition of this aspect comprises phosphate at concentration equivalent to between about 0.0003 and about 0.003 w/w% phosphorus, including about 0.0006, 0.0008, 0.001, and 0.002 w/w% phosphorus. More typically, the composition of this aspect comprises phosphate at a concentration equivalent to about 0.001 w/v% phosphorus.

In an embodiment, the composition of this aspect comprises potassium at a concentration of between about 0.25 and about 2.5 w/w%, including about 0.5, 1, 1.5 and 2 w/w%. More typically, the composition of this aspect comprises potassium at about 1 w/w%.

In embodiments, the composition of this aspect comprises sulfur at a concentration of between about 0.05 and about 0.5 w/w%, including about 0.1, 0.2, 0.3, and 0.4 w/w%. More typically, the composition of this aspect comprises sulfur at a concentration of about 0.2 w/w%.

In embodiments, the composition of this aspect comprises calcium at a concentration of between about 0.03 and about 0.3 w/w%, including about 0.05, 0.1, and 0.2 w/w%. More typically, the composition of this aspect comprises calcium at a concentration of about 0.15 w/w%.

In an embodiment, the composition of this aspect comprises magnesium at a concentration of between about 0.03 and about 0.3 w/w%, including about 0.05, 0.1, and 0.2 w/w%. More typically, the composition of this aspect comprises magnesium at a concentration of about 0.1 w/w%.

In certain embodiments, the composition of this aspect comprises sodium at a concentration of between about 0.01 and about 0.1 w/w%, including about 0.02, 0.04, 0.06, and 0.08 w/w%. More typically, the composition of this aspect comprises sodium at about 0.05 w/w%.

In embodiments, the composition of this aspect comprises one or more micronutrients at a total concentration of between about 0.01 and about 0.2 w/w%, including about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 w/w%. More typically, the total concentration of micronutrients is about 0.03 and about 0.1 w/w%. In one typical embodiment, the total concentration of micronutrients is about 0.07 w/w%.

In an embodiment, the composition of this aspect comprises copper at a concentration of between about 0.0003 and about 0.003 w/w%, including about 0.0005, 0.001, and 0.002 w/w%. More typically, the composition of this aspect comprises copper at a concentration of about 0.001 w/w%.

In embodiments, the composition of this aspect comprises zinc at a concentration of between about 0.0006 and about 0.006 w/w%, including about 0.001, 0.002, and 0.003, 0.004, and 0.005 w/w%. More typically, the composition of this aspect comprises copper at a concentration of about 0.003 w/w%.

In embodiments, the composition of this aspect comprises manganese at a concentration of between about 0.0006 and about 0.006 w/w%, including about 0.001, 0.002, and 0.003, 0.004, and 0.005 w/w%. More typically, the composition of this aspect comprises manganese at a concentration of about 0.002 w/w%.

In an embodiment, the composition of this aspect comprises iron at a concentration of between about 0.01 and about 0.1 w/w%, including about 0.02, 0.04, 0.06, and 0.08 w/w%. More typically, the composition of this aspect comprises iron at about 0.05 w/w%.

In embodiments, the composition of this aspect comprises boron at a concentration of between about 0.001 and about 0.01 w/w%, including about 0.002, 0.004, and 0.006, and 0.008 w/w%. More typically, the composition of this aspect comprises boron at a concentration of about 0.005 w/w%.

In an embodiment, the composition of this aspect comprises molybdenum at a concentration of between about 0.00004 and about 0.0004 w/w%, including about 0.00005, 0.0001, 0.0002, and 0.0003 w/w%. More typically, the composition of this aspect comprises molybdenum at a concentration of about 0.0001 w/w%.

In embodiments, the composition of this aspect comprises cobalt at a concentration of between about 0.00004 and about 0.0004 w/w%, including about 0.00005, 0.0001, 0.0002, and 0.0003 w/w%. More typically, the composition of this aspect comprises cobalt at a concentration of about 0.00015 w/w%. In an embodiment, the composition of this aspect has a pH of between about 7 and about 9, including about 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, and 8.8. More typically, the composition of this aspect has a pH of about 8.

In certain embodiments, the composition of this aspect has electrical conductivity of about 10 to about 50 dS/m, including about 15, 20, 25, 30, 35, 40, and 45 dS/m. More typically, the composition of this aspect has electrical conductivity of about 30 dS/m.

In an embodiment, the composition of this aspect comprises total dissolved salts at a concentration of between about 0.5 to about 5 w/w%, including about 1, 1.5 2, 2.5, 3, 3.5, 4, and 4.5 w/v%. More typically, the composition of this aspect comprises total dissolved salts at a concentration of about 2 w/w%.

In a typical embodiment, the composition of this aspect has a specific gravity of between about 0.5 to about 2 g/ml, including about 0.75, 1, 1.25, and 1.5 g/ml. More typically, the composition of this aspect has a specific gravity of about 1.2 g/ml.

Values for measured characteristics of one typical embodiment of a composition of this aspect, referred to as Composition 2, are set forth in Table 1.

Typically, compositions of this aspect have values for at least one of the measured characteristics set forth in Table 1 within about 10% and about 1000%; within about 20% and about 500%; within about 40% and about 250%; or within about 80% and about 125% percent of the values set forth in Table 1.

More typically, compositions of this aspect have values for a plurality of the measured characteristics set forth in Table 1 within about 10% and about 1000%; within about 20% and about 500%; within about 40% and about 250%; or within about 80% and about 125% percent of the values set forth in Table 1.

In one typical embodiment, compositions of this aspect have values for each of the measured characteristics set forth in Table 1 within about 10% and about 1000%; within about 20% and about 500%; within about 40% and about 250%; or within about 80% and about 125% percent of the values set forth in Table 1.

Production of compositions

Another aspect of the invention provides a method of producing a composition, including the step of combining a plurality of components wherein the components include a nitrogen-containing compound; and a microbial culture, to thereby produce the composition.

Any one of the plurality of components, for this aspect, may be selected from those components described as forming, or potentially forming, part of the composition of the first aspect.

In embodiments, the plurality of components according to the method of this aspect include an organic acid.

Typically, the plurality of components include a sugar, such as a sugar syrup.

In some typical embodiments, the plurality of components include a plant by product.

In embodiments, the composition produced according to the method of this aspect is a fertilising composition, inclusive of precursor compositions as hereinabove described. In some embodiments, the composition is a slow release fertiliser composition.

In embodiments, the nitrogen-containing compound, the plant by-product, the microbial culture, the organic acid, and the sugar according to the method of this aspect are as described for the composition of the preceding aspect.

In some typical embodiments of the method of this aspect:

(a) the nitrogen-containing compound, the organic acid, and the sugar are combined by dissolving in water;

(b) the microbial culture is combined with the combination as per (a).

In some typical embodiments of the method of this aspect:

(a) the nitrogen-containing compound is combined with a plant by-product;

(b) an organic acid is combined with the combination as per (a);

(c) a sugar syrup is combined with the combination as per (b); and

(d) the microbial culture is combined with the combination as per (c).

Typically, the method of this aspect includes mixing the combined components. Mixing may take any suitable form, as are well known in the art, such as mixing with mechanical stirrers or agitators, or shaking or vibration of a vessel containing the plurality of components.

Mixing of the plurality of components according to the method of this aspect may be performed for any or all of (a)-(b) or (a)-(d), above. In a typical embodiment, mixing of the components occurs until the sugar or sugar syrup is dissolved, or substantially dissolved. Typically, the microbial culture is combined with the other components after mixing until the sugar syrup is dissolved, or substantially dissolved.

According to the method of this aspect, in embodiments, the nitrogen- containing compound is combined with the plurality of components to a concentration equivalent to between about 1 and about 30 w/w% nitrogen, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 w/w% nitrogen. More typically, the nitrogen-containing compound is combined to a concentration of between about 10 and about 20 w/w% nitrogen. In one typical embodiment the nitrogen-containing compound is combined to a concentration of about 15 w/w% nitrogen.

According to the method of this aspect, typically, the microbial culture is combined with the plurality of components to a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%. More typically, the microbial culture is combined to a total concentration of between about 2 and about 7 v/v%. In one typical embodiment, the microbial culture is combined to a total concentration of about 5 v/v%.

According to the method of this aspect, typically, the organic acid is combined with the plurality of components to a total concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%. More typically, the organic acid is combined to a total concentration of between about 2 and about 7 v/v%. In one typical embodiment, the organic acid is combined to a total concentration of about 5 v/v%.

According to the method of this aspect, typically, the sugar syrup is combined with the plurality of components to a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 v/v%. More typically, the sugar syrup is combined to a concentration of between about 2 and about 10 v/v%. In one typical embodiment, the sugar syrup is combined to a concentration of about 5 v/v%.

According to the method of this aspect, typically, the plant by-product is combined with the plurality of components to a concentration of between about 50 and about 95 v/v%, including about 55, 60, 65, 70, 75, 80, 85, 90, and 95 v/v%. More typically, the plant by-product is combined to a concentration of between about 70 and about 90 v/v%. In one typical embodiment, the plant by-product is combined to a concentration of about 85 v/v%.

Typically, the method of this aspect includes the step of fermenting the combined components. Typically, the fermentation step occurs using the microbial culture.

It will be appreciated that, in embodiments of this aspect wherein the method includes the step of fermenting the combined components, the combined components prior to the fermenting step may be considered a precursor composition, as hereinabove described.

In typical embodiments, the fermenting step according to the method of this aspect is for a duration of about 1 day to greater than about 14 days, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 days. More typically, the fermentation step is for a duration of about at least about 7 days.

In typical embodiments, the fermenting step according to the method of this aspect is performed at an elevated temperature. As used herein “ elevated temperature” will be understood to be a temperature above room temperature, typically above about 25 °C. Typically, the fermenting step is performed at a temperature between about 30°C and about 60°C, including about 35, 40, 45, 50, and 55°C. More typically, the fermenting step is performed at a temperature of about 38°C. Alternatively, the fermenting step may be performed at ambient or room temperature, or about ambient or room temperature.

The method of this aspect may include the step of cooling or resting the combined components. Typically, the step of cooling or resting the combined components occurs after the step of fermenting the combined components, although without limitation thereto.

Typically, the step of cooling or resting the combined components is for a duration of about 1 day to greater than about 14 days, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 days. More typically, the step of cooling or resting the combined components is for a duration of at least about 7 days.

Typically, the step of cooling or resting the combined components is performed at a temperature at or below room temperature or ambient temperature. In some typical embodiments the cooling or resting step is performed at a temperature of about 10°C to about 30°C, including about 15, 20, and 25°C.

The invention also provides a composition produced according to the method of this aspect, inclusive of precursor compositions.

Typically, compositions produced according to the method of this aspect will have characteristics in respect of concentration of the one or more macronutrients; concentration of one or more micronutrients; pH; electrical conductivity; specific gravity; total dissolved salts; concentration of nitrate; concentration of nitrite; concentration of ammonium; and/or concentration of phosphate, as described for the preceding aspect.

Increasing bioavailability of nitrogen

Another aspect of the invention provides a method of increasing bioavailability of nitrogen from a nitrogen-containing compound, including the step of combining a plurality of components including the nitrogen-containing compound; and a microbial culture, to thereby increase the bioavailability of nitrogen from the nitrogen-containing compound.

Typically, the plurality of components combined according to the method of this aspect include an organic acid.

Typically, the plurality of components combined according to the method of this aspect include a sugar, such as a sugar syrup.

In some typical embodiments, the plurality of components combined according to the method of this aspect include a plant by-product.

Typically, the nitrogen-containing compound, the microbial culture, the organic acid, the sugar, and the plant by-product according to the method of this aspect are as hereinabove described.

The method of this aspect typically includes fermenting steps as described for the preceding aspect. The method of this aspect may include combining; mixing; and/or cooling steps all as described for the preceding aspect.

According to the method of this aspect, the bioavailability of nitrogen from the nitrogen-containing compound may be increased or enhanced by chemically and/or physically altering the nitrogen-containing compound. Typically, the bioavailability of nitrogen from the nitrogen-containing compound is increased or enhanced by modifying or controlling the derivatisation or conversion of the nitrogen-containing compound into a different chemical form.

In a typical embodiment, bioavailability of nitrogen is increased or enhanced, at least in part, by increasing or enhancing the conversion of the nitrogen-containing compound to ammonium.

In a typical embodiment, the bioavailability of nitrogen is increased or enhanced, at least in part, by decreasing or inhibiting the conversion of nitrogen to nitrite and/or nitrate.

In some typical embodiments, bioavailability of nitrogen from the nitrogen- containing compound is increased or enhanced, at least in part, by attaching the nitrogen to, or otherwise incorporating the nitrogen within, an organic compound, such as a protein, peptide, or amino acid (although without limitation thereto).

In some typical embodiments, bioavailability of nitrogen from the nitrogen- containing compound is increased or enhanced, at least in part, by incorporating the nitrogen within microbial biomass.

According to the method of this aspect, typically, the bioavailability of nitrogen from the nitrogen containing compound is increased by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200%.

Plant fertilisation, composting, and/or growth medium conditioning

Other aspects of the invention provide methods of fertilising a plant, composting organic matter, and/or increasing nitrogen bioavailability in a growth medium, including the step of applying a composition comprising a nitrogen- containing compound; and a microbial culture to the plant, the organic matter, and/or the growth medium.

Typically, the composition applied according to these aspects is a composition as hereinabove described.

In aspects of the invention providing a method of fertilising a plant, typically, the amount or degree of bioavailable nitrogen accessible by the plant is increased or enhanced by fertilisation of the plant with the composition.

In aspects providing a method of fertilising a plant, the step of applying the composition to the plant may take any suitable from. The step of applying the composition to the plant may be or include direct application, such as foliar or root application. Additionally, or alternatively, the step of applying the composition to the plant may be or include indirect application, such as application to a growth medium or compost in, on, or with which the plant is growth.

The plant that is fertilised according to aspects of the invention providing a method of fertilising a plant, may be any suitable plant. Typically, the plant is a monocotyledonous plant or a dicotyledonous plant. The plant may be a grass of the Poaceae family, such as sugar cane; a Gossypium species, such as cotton; a berry, such as a strawberry; a tree species, inclusive of fruit trees such as apple and orange and nut trees such as almond; an ornamental plant, such as an ornamental flowering plant, inclusive of rosaceous plants such as rose; a vine, inclusive of fruit vines such as grapes; a cereal, including sorghum, rice, wheat, barley, oats, and maize; a leguminous species, including beans such as soybean and peanut; a solanaceous species, including tomato and potato; a brassicaceous species, including cabbage and oriental mustard; a cucurbitaceous plants, including pumpkin and zucchini; a rosaceous plants, including rose; an asteraceous plant, including lettuce, chicory, and sunflower, although without limitation thereto. Typically, the plant is a crop, inclusive of cereals, leguminous plants, brassicaceous plants, asteraceous plants, fruit trees, and other fruit and vegetable crops.

According to aspects of the invention providing a method of composting organic matter, the organic matter that is composted typically is or includes plant matter. The organic matter may include leaves, grass clippings, peat moss, wood chips, bark, and/or straw, although without limitation thereto. Typically, composting of the organic matter results in an increase in nitrogen bioavailability in the organic matter.

In embodiments of aspects providing a method of composting organic matter, the step of applying the composition to the organic matter is performed in situ to organic matter that is positioned in, on, or around a plant. It will be appreciated that, in these embodiments, composting can be performed in situ , e.g. as a mulch for a plant, as compared to first performing composting prior to mulching using organic matter. The step of applying the composition to the organic matter can additionally or alternatively be performed in any other suitable manner. By way of non-limiting example, the composition can be applied to organic matter held or stored in a container or compost bin, as are known in the art.

According to aspects of the invention providing a method of increasing nitrogen bioavailability in a growth medium, the growth medium may be any substance or agent suitable for supporting plant growth, inclusive of natural and artificial mediums as are well-known in the art. In typical embodiments, the growth medium is a soil. By way of non-limiting example, the soil may be a sandy soil (such as a grey sandy soil), a loamy soil (such as a red loam soil), or an alluvial and/or vertisol soil (such as a black alluvial vertisol soil).

In some embodiments of aspects providing a method of increasing nitrogen bioavailability in a growth medium, the step of applying the composition to the growth medium, such as a soil, to increase nitrogen bioavailability in the growth medium is performed prior to cultivation of a plant or crop in the growth medium. Additionally, or alternatively, the step of applying the composition to the growth medium may be performed during cultivation of a plant or crop in the growth medium.

In typical embodiments of methods of these aspects nitrogen bioavailability is increased or enhanced, at least in part, by increasing or enhancing the amount or concentration of ammonium accessible by the plant, in the compost, and/or in the growth medium.

In some typical embodiments, nitrogen bioavailability is increased or enhanced, at least in part, by increasing or enhancing the amount or concentration of nitrogen within an organic compound, such as a protein, peptide, or amino acid, accessible by the plant, in the compost, and/or in the growth medium.

In some typical embodiments, nitrogen bioavailability is increased or enhanced by increasing or enhancing the amount or concentration of nitrogen within microbial biomass accessible by the plant, in the compost, and/or in the growth medium.

According to the method of this aspect, typically, the bioavailability of nitrogen from the nitrogen containing compound is increased by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200%. EXAMPLES

Example 1. Composition 1

This example provides a protocol for production of a fertiliser composition of the invention, referred to as Composition 1.

Materials

- Purified water

- Urea

- Sugar (e.g. glucose, fructose, and/or sucrose)

- Organic acid (fulvic acid and/or humic acid)

- Microbial mix containing lactic acid bacteria; phototrophic bacteria; nitrogen-fixing bacteria; Bacillus bacteria; and yeast.

Methods

For 1000L of fertiliser composition, 850L of water containing 33 w/w% urea (~15 w/w% nitrogen), 5 w/w% organic acid, and 5 w/w% sugar is combined with 50L microbial culture, and thoroughly mixed. The mixture is the placed in a fermentation chamber at 38°C, and fermented for at least 7 days.

Example 2. Composition 2

This example provides a protocol for production of a fertiliser composition of the invention, referred to as Composition 2.

Materials

- Dunder. A type of vinasse that is a semi-liquid (approximately 30-40% solids) by-product of sugarcane molasses fermentation for ethanol production, and contains vegetable matter, yeast biomass, potassium, sodium, nitrogen, calcium, magnesium, phosphorous and sulphur. A suitable vinasse/biodunder may be sourced from Wilmar BioEthanol (https://wilmarbioethanol.com/what-is-biodunder).

- Urea. Urea is pre-added to the dunder at approximately 15 w/w% nitrogen (~33 w/w% total urea).

- Sugarcane molasses.

- Humic acid.

- Fulvic acid.

- Microbial cultures. The commercial ‘MicroLife’ mix manufactured by Multikraft Probiotics Australia is suitable, however other microbial mixes may be used. The manufacturer brochure for MicroLife can be found at https://www.multikraft.com.au/image /catalog/Brochure%20PDF/microlife.pdf, and is incorporated herein by reference. MicroLife includes a combination of over 80 isolates from over 45 microbial species, including lactic acid bacteria, such as Lactobacillus casei and Lactobacillus plantarunv, photosynthetic bacteria, such as Rhodopseudomonas palustris nitrogen-fixing bacteria, such as Acetobacter tropicalis, Acetobacter lovaniensis, Acetobacter syzygii, and Rhizobium species; Bacillus species, such as Bacillus subtilis, Lysinibacillus sphaericus, and Bacillus amyloliquefaciens, and yeasts, such as Saccharomyces cervisiae.

Methods

For 1000L of fertiliser composition, 850L of dunder (containing urea) is combined with 50L molasses, 30L humic acid, 20L fulvic acid, and 50L MicroLife. Subsequently, fermentation, then cooling is performed.

Steps are as follows:

(1) Place 500L dunder (containing 15 w/w% nitrogen from urea) into mixing bowl;

(2) Turn on mixing agitators;

(3) Add 20L fulvic acid;

(4) Add 30L humic acid;

(5) Add 50L molasses;

(6) Mix until molasses is thoroughly dissolved;

(7) Add 50L MicroLife;

(8) Pump out mixing bowl contents into storage container;

(9) Add an additional 350L dunder + urea to storage container;

(10) Place storage container in fermentation chamber at 38°C;

(11) Ferment precursor composition in storage container for 7 days;

(12) Remove storage container from fermentation chamber, and cool 7 days at room temperature (approximately 25 °C).

Example 3. Analysis of Composition 2

A sample of Composition 2 as described in Example 2 has been analysed for plant macronutrient content, plant micronutrient content, pH, electrical conductivity, total dissolved salts, specific gravity, nitrate content, nitrite content, ammonium content, and phosphate content. Prior to analysis, samples were digested on a hotblock digestor with nitric acid and hydrochloric acid.

Nutrient element analyses were performed using inductively coupled plasma mass spectrometry (ICP-MS), or inductively coupled plasma optical emission spectrometry (ICP-OES) for sulfur. For nitrogen, a LECO CNS2000 Analyser was used. pH was assessed using a standard pH meter. Electrical conductivity was assessed using a standard EC meter. Total dissolved salt was calculated using electrical conductivity.

Results are presented in Table 1.

Example 4. Assessment of nitrogen characteristics in soil after application of fertiliser composition

Introduction

Most conventional nitrogen (N) fertiliser applied to agricultural fields is quickly converted from ammonium (NH4+) into nitrate (NO3-) (Subbarao et ah, 2006). This can be unfavourable for a number of reasons, as nitrate is easily lost from the cropping system via leaching (Di and Cameron, 2002; Rasiah et ah, 2003) and as greenhouse gasses (Allen et ah, 2010). Nitrate is approximately 10 times more mobile in soil than ammonium or low molecular weight organic nitrogen (Figure 1), and there is substantial interest in devising efficient crop systems which retain nitrogen in organic or ammonium forms for longer (Ruser and Schulz, 2015; Soares et ah, 2015; Subbarao et ah, 2012; Subbarao et ah, 2013; Zhang et ah, 2015).

Dunder is a by-product (or‘co-product’) of ethanol production from sugarcane molasses (Bieske, 1979), and can be used as a liquid fertiliser after amendment with nutrients such as nitrogen and phosphorous. Nitrogen from urea-amended dunder is also rapidly converted to nitrate after application to soil (Brackin, 2016). Accordingly, compositions comprising dunder in combination with other components suitable for stabilising the nitrogen forms, and/or slowing down the in-soil transformation of nitrogen into, e.g., nitrate, may facilitate reduced nitrogen losses, and increase bioavailable nitrogen.

This study aimed to assess the rate of transformation of nitrogen, and the forms of nitrogen present, after application of a dunder-based fertiliser composition to soil. Methods

This experiment investigated the efficacy of Composition 2, as set forth in Example 2, for enhancing bioavailable nitrogen in soil. The experiment ran for 60 days. The experiment was conducted in a microcosm system as described in Inselsbacher et al (2009), incorporated herein by reference. The experiment was conducted at ambient temperature for southeast Queensland, Australia, in a glasshouse, during September and October. Three soil types were included, as nitrogen stabiliser products can have varied effectiveness depending on soil properties (Brackin, 2016). Two soils were collected from the Bundaberg region (a grey sandy soil and a red loam), and one from the Scenic Rim region (black alluvial vertisol).

Details of experimental design are provided in Table 2 and Figure 2. Three soils; four treatments (three nitrogen treatments and a control); five replicates; and five harvest time points were used for a total of 300 microcosms.

For each of the nitrogen treatments, nitrogen was applied at the same rate. Dunder was applied at a rate of 80F per hectare, equivalent to about 16.2 kg nitrogen per hectare. Composition 2 and plain urea were applied at the same rate of nitrogen (i.e. -16.2 kg per hectare), with urea dissolved in distilled water to achieve accurate application rates.

Microcosms (5 replicates per treatment per soil) were removed from the experiment at each harvest point, and destructively sampled to measure soil nitrogen pools. At each time point, ongoing profiling of the nitrogen forms available was conducted. Organic nitrogen (protein, peptide and amino acid) was quantified using the ninhydrin assay and subtracting ammonium (Amato and Fadd, 1988; Joergensen and Brookes, 1990), ammonium (Kandeler and Gerber, 1988) and nitrate (Miranda et al., 2001). Nitrogen immobilised in the soil microbial biomass was quantified using the fumigation - extraction method (Joergensen and Brookes, 1990).

Results and discussion

Soil microbial biomass

At day 30, soil microbial biomass was significantly higher for the Composition 2 treatment than for any other treatment, in all soils (Figure 3). Baseline microbial biomass in the untreated control treatment varied substantially between soils - from approximately 2 mg N kg ¹ in the Bundaberg grey sand, up to approximately 20 mg N kg ¹ in the Kalbar soil. Addition of Composition 2 had the greatest effect in the Bundaberg red loam - and the least effect in the Kalbar soil (seemingly due to the high existing microbial biomass in this soil). Microbial biomass was increased by 6, 18 and 8 mg N kg ¹ in the grey, red, and Kalbar soils respectively compared to the control treatments. In the grey sand and red loam soils, the non- amended (or‘raw’) dunder treatment also showed a trend towards increased microbial biomass.

Addition of organic matter such as compost, leaf litter and other organic amendments typically leads to increased soil microbial biomass, as local populations increase in response to increased resource availability (Brackin et ah, 2013, 2014; Brackin et ah, 2017b; Buckley et ah, 2016), however this tends to be relatively short lived, and microbial populations return to the previous size as resources are consumed (Brackin et ah, 2013).

In the current scenario, the higher levels in the Composition 2 treatment compared to the unamended dunder suggest that the probiotic amendment of the dunder plays a major role in the observed increased microbial biomass - either through surviving probiotic organisms introduced in the composition, or a subsequent boom of microbes which consume or decompose those introduced with the composition.

- Soluble organic nitrogen

Soluble organic nitrogen was significantly increased in the dunder and Composition 2 treatments over the 60 days of the experiment, but not in the urea treatment (Figure 4).

In all three soils, the soluble organic nitrogen pool peaked at day 7 in the dunder treatment, and at day 14 in the Composition 2 treatment. This strongly suggests that nitrogen which has been previously bound in the microbial biomass is being released as microbes die and are broken down by enzymes (Brackin et ah, 2014), and the slight delay in peak organic N in the Composition 2 treatment is associated with the higher microbial biomass in this treatment (Figure 3).

Organic nitrogen remained higher in the Composition 2 treatment throughout the remainder of the experiment. Organic nitrogen decreased in the dunder and Composition 2 treatments at a reasonably consistent rate, and appeared to be mineralised into ammonium by microbes. Decomposition of protein to amino acids (rather than the subsequent mineralisation of amino acids into ammonium) is thought to be the main bottleneck in soil nitrogen cycling (Jan et ah, 2009), and soil protein breakdown rates correlate with plant productivity (Simpson et ah, 2017). While protein can be used by plants as a nitrogen source (Paungfoo-Lonhienne et ah, 2008), it becomes substantially more accessible when broken down to peptides or amino acids which are both used by plants (Brackin et ah, 2015; Falkengren-Grerup et ah, 2000; Gioseffi et ah, 2012; Jamtgard et ah, 2007; Soper et ah, 2011; Svennerstam et ah, 2011; Thornton and Robinson, 2005).

- Soil inorganic nitrogen

Ammonium and nitrate levels in all fertilised soils were significantly higher than unfertilised control treatments, demonstrating that all formulations are successful in delivering nitrogen in useable forms to the soil (Figure 4). In all three soils, conventional urea fertiliser is rapidly converted to ammonium, and subsequently to nitrate. Composition 2 has a markedly delayed release curve of ammonium - and subsequently delayed nitrification. This indicates that release of ammonium from Composition 2 was effectively slowed down, achieving a key aim of efficient fertilisation. Slightly different patterns of nitrogen releases occurred in each of the three soils, indicating that soil-specific interactions, or the differing soil microbial communities in the three soils influenced nitrogen release and transformation rates.

In the grey sandy soil from Bundaberg, ammonium concentrations in the urea and dunder treatments peaked at day 14 and decreased across the remainder of the experiment. By contrast, Composition 2 treatment had had peak ammonium concentrations at day 30. Composition 2 treatment had lower nitrate concentrations than the other two fertilisers on day 7 and 14, but had higher concentrations by day 30, which were maintained until day 60.

In the red loam soil from Bundaberg, ammonium concentrations in the urea and dunder treatments peaked at day 7 and decreased mildly across the remainder of the experiment. By contrast, the Composition 2 had the highest ammonium concentrations by day 14, and peak ammonium concentrations at day 60. Nitrate concentrations increased steadily in all treatments across the experiment. Composition 2 treatment had lower nitrate concentrations than the other two fertilised treatments from day 7 to 30, all three treatments had similar nitrate concentrations by day 60.

In the alluvial vertisol from Kalbar, ammonium concentrations were highest in the Composition 2 treatment throughout the experiment until day 30. Concentrations were negligible in all treatments by day 60. Nitrate concentrations were lower in the Composition 2 treatment than the other two fertilised treatments from day 7 through to day 60. In all three soils, control treatments (receiving no N fertiliser) had negligible ammonium concentrations throughout, but nitrate concentrations increased during the 60 day experiment.

The Composition 2 treatment generally had higher levels of ammonium, and the lowest levels of nitrate across the three soils. An examination of the ratios of ammonium to nitrate show higher ammonium relative to nitrate in soils treated with dunder and Composition 2 (Figure 5). In the Kalbar soil, the Composition 2 treatment showed a higher ratio of ammonium to nitrate than all other treatments across the entire experiment. The Bundaberg red soil shows similar patterns from day 14 onwards. By contrast this pattern only emerged by day 30 in the Bundaberg grey soil. This metric is a desirable trait from an efficiency perspective, as ammonium is much less prone to loss than nitrate (Abaas et al., 2012). This is especially important in crops such as sugarcane, which have a strong preference for ammonium rather than nitrate under conditions of high supply (Robinson et al., 2011).

Conventional fertilisers usually result in initial nitrogen concentrations which are well above plant uptake capacity; resulting in large pools of soluble nitrogen in the soil which cannot yet be taken up by the plant, and are highly vulnerable to loss in rainfall or irrigation events (Brackin et al., 2017a; Brackin et al., 2015). Approaches to improve nitrogen use efficiency include the use of‘enhanced efficiency fertilisers’ which act to delay the release of nitrogen, or to slow the transition of ammonium into loss prone nitrate. This is achieved through nitrification inhibitors such as DMPP, DCD and nitrapyrin (Abalos et al., 2014; Brackin and Schmidt, 2016; Ruser and Schulz, 2015; Yang et al., 2016), and encapsulated fertilisers (Soares et al., 2015; Timilsena et al., 2015; Verburg et al., 2014). Both of these approaches have seen some reductions in nitrogen losses via the gas nitrous oxide, however improvements in crop yield have largely been elusive (Abalos et al., 2014). The effectiveness of these‘enhanced efficiency fertilisers’ is also highly patchy - for example high clay or organic matter contents substantially reduced the effectiveness of DMPP (Barth et al., 2001; Brackin and Schmidt, 2016); DCD is less effective in neutral than in acidic soils, and has been the cause of a contamination scare in New Zealand milk products (Yang et al., 2016). Similarly, encapsulated slow release fertilisers can burst simultaneously under some conditions, causing soil nitrogen‘spikes’ which promote higher loss than conventional fertilisers (Wang et al., 2016a; Wang et al., 2016b).

This study strongly indicates that Composition 2 is the least loss-prone of the assessed nitrogen treatments. It has inherently slower‘release rates’ of nitrogen from organic forms into the soluble inorganic forms ammonium and nitrate. It also results in higher soil nitrogen held in the microbial biomass, which will also be released gradually over time as the microbial population returns to the typical size.

Despite the slow nitrogen release pattern of Composition 2, it still resulted in increased soil ammonium concentrations by day 7 in all soils, indicating that there is no major penalty in initial plant-available nitrogen (which tends to be unnecessarily high in conventionally fertilised treatments), and this suggests that there is therefore no need to apply this product at a higher rate of nitrogen than other fertilisers to achieve similar results.

Conclusions

- Nitrogen in Composition 2 is split between a number of pools - it is contained in the microbial biomass, dissolved organic nitrogen (protein, peptide and amino acid), and ammonium, but relatively little in the form of nitrate.

- Due to this nitrogen composition, Composition 2 effectively acts as a slow release fertiliser as the nitrogen is mineralised over time. This maintains bioavailability of nitrogen in soil treated with Composition 2.

- Composition 2 also resulted in significantly increased soil microbial biomass compared to controls and other treatments

- Release of nitrogen in Composition 2 treated soil continued for 60 days, and kinetics suggest it may continue until ~ 90 days after application.

Throughout the specification, the aim has been to describe typical embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

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TABLES

Table 1. Results of analysis of a typical fertiliser composition of the invention.

Table 2. Details of experimental design for Example 4.

Claims

1. A fertiliser composition comprising a nitrogen-containing compound; a microbial culture; an organic acid; and a sugar.

2. The composition of claim 1, wherein the composition is liquid or semi-liquid.

3. The composition of claim 1 or claim 2, wherein the nitrogen-containing compound is urea.

4. The composition of any preceding claim, wherein the microbial culture includes one or more microbes selected from the group consisting of a lactic acid bacteria; a phototrophic bacteria; a diazotroph; a Nitrobacter species; a Bacillus species; and a yeast.

5. The composition of claim 4, wherein the microbial culture includes a lactic acid bacteria; a phototrophic bacteria; a diazotroph; a Bacillus species; and a yeast.

6. The composition of any preceding claim, wherein the organic acid is fulvic acid and/or humic acid.

7. The composition of any preceding claim, wherein the sugar is of a molasses.

8. The fertiliser composition of any preceding claim, comprising a plant by product.

9. The composition of claim 8, wherein the plant by-product is vinasse.

10. The composition of any preceding claim, wherein the nitrogen-containing compound is present in the composition at a concentration equivalent to between about 1 and about 60 w/w% nitrogen, including about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 55 w/w% nitrogen. 11. The composition of any preceding claim, wherein the organic acid is present at a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, and 14 v/v%.

12. The composition of any preceding claim, wherein the sugar is present at a concentration of between about 0.5 and about 7.5 w/w%, including about 1, 2, 3, 4, 5, and 6 w/w%.

13. The composition of claim 7, wherein the molasses is present at a concentration of between about 1 and about 15 v/v%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, and 14 v/v%.

14. The composition of claim 8 or claim 9, wherein the plant by-product is present at a concentration of between about 50 and about 95 v/v%, including about 55, 60, 65, 70, 75, 80, 85, and 90 v/v%.

15. The composition of any preceding claim, comprising one or more macronutrients other than nitrogen.

16. The composition of claim 15, wherein the macronutrients other than nitrogen are selected from the group consisting of phosphorus; potassium, sulfur, calcium, magnesium, and sodium.

17. The composition of any preceding claim, comprising one or more micronutrients.

18. The composition of claim 17, wherein the micronutrients are selected from the group consisting of copper; zinc; manganese; iron; boron; molybdenum; cobalt; and silicon.

19. The composition of claim 15 or claim 16, wherein the total concentration of the macronutrients other than nitrogen is between about 0.1 and about 3 w/w%, including about 0.5, 1, 1.5, 2, 2.5, and 3 w/w%.

20. The composition of claim 17 or claim 18, wherein the total concentration of the micronutrients is between about 0.01 and about 0.2 w/w%, including about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 w/w%.

21. A method of producing a composition, including the step of combining a plurality of components wherein the components include a nitrogen -containing compound; a microbial culture; an organic acid; and a sugar, to thereby produce the composition.

22. A method of increasing bioavailability of nitrogen from a nitrogen-containing compound, including the steps of combining a plurality of components including the nitrogen-containing compound; a microbial culture; an organic acid; and a sugar, to thereby increase the bioavailability of nitrogen from the nitrogen-containing compound.

23. The method of claim 21 or claim 22, wherein the plurality of components include a plant by-product.

24. The method of any one of claims 21-23, including the step of fermenting the combined components.

25. The method of claim 24, wherein the fermenting is for a duration of about 1 day to at least about 14 days, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 days.

26. The method of claim 24 or claim 25, wherein the fermenting is performed at a temperature of between about 30°C and about 60°C, including about 35, 40, 45, 50, and 55°C.

27. The method of claim 24 or claim 25, wherein the fermenting is performed at ambient or room temperature.

28. The method of any one of claims 21-27, including the step of cooling or resting the combined components.

29. A fertiliser composition produced according to the method of claim 21 or any one of claims 23-28.

30. A method of fertilising a plant, including the step of applying the composition of any one of claims 1-20 or claim 29 to the plant, to thereby fertilise the plant.

31. The method of claim 30, wherein the plant is a crop plant.

32. A method of composting organic matter, including the step of applying the composition of any one of claims 1-20 or claim 29 to the organic matter, to thereby compost the organic matter.

33. The method of claim 32, wherein the organic matter is plant material.

34. A method of increasing nitrogen bioavailability in a growth medium, including the step of applying the composition of any one of claims 1-20 or claim 29 to the growth medium, to thereby increase nitrogen bioavailability in the growth medium.

35. The method of claim 34, wherein the growth medium is soil.