WO2024201362A1 - Process for the extraction of biostimulants and amino acids from kelp - Google Patents

Abstract

The present invention relates to processes for the extraction of biostimulants and amino acids from brown macroalgae including dry kelp hinge pieces and to products from such an extraction process, as well as to methods for stimulating plant growth with the use of the biostimulants and amino acids extracted by the processes of the invention.

Description

PROCESS FOR THE EXTRACTION OF BIOSTIMULANTS AND AMINO ACIDS FROM KELP FIELD OF THE INVENTION The current invention relates to processes for the extraction of biostimulants and amino acids from brown macroalgae including dry kelp hinge pieces and to products from such an extraction process, as well as to methods for stimulating plant growth with the use of the biostimulants and amino acids extracted by the processes of the invention. BACKGROUND OF THE INVENTION The ever-increasing demand for food, a result of the exponential population increase, exhort a greater demand on the agricultural sector to ensure sustainable food production. This results in an increase in the frequency at which arable land needs to be exploited, which eventually leads to a decrease in food productivity due to a decrease in soil fertility. In addition, climate change and the increase in urbanization also threatens the availability of arable land. Resultantly, more fruits and vegetables needs to be produced on a smaller area of land. In order to keep up with the high demand and overcoming the various disadvantages, whilst appropriately managing arable lands, fertilizers fulfilling multiple roles must be developed. These fertilizers must provide adequate nutrients and nutrient uptake regulators, growth regulators, plant protectants and stress regulators, and the like. Therefore, the classically used fertilizers needs to be, in most cases, supplemented with organic biostimulants obtained from appropriate sources. An example of one such source of organic biostimulants is seaweed, which has been used by humans for millennia. The use of seaweed dates backs to 600 BC, first utilized in China (Waalaand, 1981). However, seaweed utilization has only recently received considerable interest, especially in the agricultural area. Seaweeds were predominantly used as a meal (McHugh, 2003). The seaweeds would generally be dried, milled and subsequently used as a fertilizer and a soil conditioner. Although the powdered seaweed has a low phosphorus content, it has an appropriate amount of nitrogen and potassium, rendering it a suitable fertilizer. At the same time, the large amount of insoluble carbohydrates improves the moisture retention properties of soil. In addition, some of the carbohydrates act as a gel that can hold the soil together, decreasing the extent of soil erosion, especially in areas prone to soil loss whilst at the same time retaining water. Certain seaweed meals have also been used to raise the pH of acidic soil, acting as an organic replacement for lime. However, seaweed meals are becoming less popular compared to the liquid suspension or concentrated extracts obtained through the processing of seaweeds (McHugh, 2003). The mode of action of the liquid concentrate is much quicker compared to the seaweed meal, in addition to being more dilute and easier to handle and transport. Various seaweed extracts and/or suspensions are sold commercially under the following brand names: Maxicrop from the United Kingdom, Goëmill from France, Algifert produced in Norway, Kelpak 66 from South Africa and Seasol which is produced in Australia. These seaweed extracts are all obtained from brown seaweeds, while the species of brown seaweed used varies between countries. The processing of the seaweeds is also different when compared against each other. Freeze-thaw cycles are used during the production of the Goëmill seaweed extract, from the brown seaweed Ascophyllum. The freezing of the wet seaweed, crushing and homogenization, results in cellular lysis expelling the cellular content into a creamy suspension with particle size varying between 6-10 micrometers. A cell- burst method is used during the preparation of Kelpak (McHugh, 2003). The wet seaweed, Ecklonia maxima, is harvested and then milled in order to decrease the size of the seaweed particles. The smaller seaweed particles are then subjected to extremely high pressures, followed by being passed into a low-pressure chamber where the cells burst open, releasing the cellular content to give the liquid concentrate containing very fine particles. Other products such as Maxicrop and Seasol employs an alkaline extraction method, followed by filtration, to give a clear liquid concentrate (McHugh, 2003). The extract obtained from seaweeds has been applied to numerous fruits, vegetables and various other crops which has shown beneficial effects on the plants, especially in terms of crop performance and yield, early seed germination and establishment, increased resistance to biotic and abiotic stress and also an improved nutrient uptake (McHugh, 2003; Khan, 2009). The advantageous effects that the seaweed extracts have on plants is in all probability a result of a synergistic effect between the various chemical components such as macro- and micronutrients, vitamins, cytokinins and auxins, amino acids and betaines, obtained from the seaweed extract (McHugh, 2003; Khan, 2009). One of the main attributes of seaweed extracts ascribed to significantly stimulate growth-response in plants, is the growth-regulatory substances such as cytokinins and auxins (Khan, 2009; Tiwari, 2015; Misurcova, 2010). Of the cytokinins, trans- zeatin and trans-zeatin riboside, and their dihydro analogues, are the more commonly encountered cytokinins. Indole-3-acetic acid is one of the more common auxins found in seaweeds. The indole-3-acetic acid has shown noteworthy root- promoting activity, which is characteristic of auxin activity (Khan, 2009). Betaines have been reported to relieve osmotic stress brought on by soils with a high salt concentration, especially in times of drought. It has also been reported that betaines can enhance leaf chlorophyll content through decreasing chlorophyll degradation, while further acting as a source of nitrogen and a osmolyte (Khan, 2009; Tiwari, 2015; Misurcova, 2010). Unusual and complex polysaccharides such as alginates, fucoidan and laminaran are only found in seaweeds and not in land plants. They have various biological activities, such as inducing natural defence responses in plants, plant gene regulation and retention of water, amongst others. As was mentioned earlier, seaweed extraction in order to obtain the abovementioned biostimulants, can be accomplished through several routes, most of which involves the use of freshly harvested seaweed with a high-water content. However, the use of dry seaweed as the kelp starting material for extraction, is far less common. Extraction procedures using dry seaweed generally employ an alkaline extraction method with either a sodium hydroxide, sodium carbonate or potassium carbonate as base. The effect of temperature and pH on the extract and the extraction procedure was recently investigated. The authors had used the giant kelp Macrocystis pyrifera, dried it and milled to a fine powder (Dominguez-Briceno, 2014). The powdered kelp was subjected to alkaline extraction conditions where the pH and temperature of the reactions where varied. The plant growth promoting activity of the extracts obtained where also evaluated in the mung bean bioassay, and as a function of the growth of tomato plants. The authors noted that extraction under alkaline conditions and at elevated temperature resulted in mixtures with very high viscosities, in some instances forming a thick paste. However, a decrease in the viscosity of the final solution was noted when the pH and temperature were increased. This was attributed to the depolymerization of the alginates, yielding mono- and oligomeric fragments through glycosidic bond cleavage (Dominguez- Briceno, 2014). Under the alkaline conditions, the degradation occurs mainly via β- elimination processes. It was determined that the viscosity of the mixture has a significant influence on the extract, not only during the extraction procedure, but also during the mung bean assays. Extracts with low viscosities showed significant improved rooting activity compared to mixtures with high viscosities. The extract, obtained after the extraction of the nutrients at pH 11 and at a temperature of 80 °C, had the highest rooting activity compared to the other extraction procedure with different variables. In addition, the rooting activity of the extract obtained at pH 11 and at 80 °C was only an order of magnitude lower compared to the rooting activity induced by Kelpak. Finally, the tomato seedlings also exhibited significant growth when treated with extract obtained at pH 11 and at 80 °C. The study indicated that chemical extraction of the various growth regulators and of the nutrients in the seaweed, even under harsh temperature and pH conditions, had a significant beneficial effect on plant growth and defence (Dominguez-Briceno, 2014). There is however a need for an improved, simplified and/or more cost effective procedure where little or no waste is generated for the efficient extraction of the biostimulants, amino acids and macro- and micro-nutrients particularly from a dry brown macroalgae product including dried kelp hinge pieces to produce a liquid product, particularly where the liquid product is versatile and can be simply adapted for use in either a neutral / alkaline environment or an acid environment. Furthermore, the costs and efficacy of such processes have a significant impact on the economic feasibility of the such a process and therefore need to be carefully controlled in order to be economically feasible. SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided a method for extraction of an extract comprising biostimulants and amino acids from dried brown macroalgae, in particular kelp, including Ecklonia maxima or Durvillaea potatorum, preferably Durvillaea potatorum, the method consisting of the following steps: a) adding water at a ratio of about 7-19 water to about 1 dry kelp (w/w), heated by a heating source to between about 55 °C to about 77 °C, or about 62 °C to about 75 °C, or about 68 °C to about 72 °C to a first reaction vessel; b) adding the dried brown macroalgae pieces at a ratio of about 7-19 water to about 1 dry kelp (w/w), to the hot water and agitating the dried brown macroalgae in the water with an agitator for at least about 50 minutes, or at least about 55 minutes, or at least about 60 minutes, or at least about 65 minutes, or at least about 70 minutes, or longer as desired; c) adding an organic acid, including citric acid or acetic acid, but preferably citric acid, and more preferably a citric acid monohydrate crystal at, at least a ratio of 0.06 w/w to 1 dry kelp to the first reaction vessel, whilst agitating for at least about 20 minutes, or at least about 30 minutes, or at least about 40 minutes, or at least about 50 minutes, or at least about 60 minutes, or at least about 90 minutes, or at least about 120 minutes, or longer as desired, wherein the citric acid may be added as crystals or as a solution, preferably as a citric acid solution; d) adding an alkali selected from the group consisting of sodium or potassium hydroxide or a mixture thereof, either dry or as a solution, at an alkali to kelp ratio of 0.13-0.36 (w/w), wherein preferably the solution is a mixture of sodium hydroxide and potassium hydroxide containing at least 50 % w/w each, or more preferably a potassium hydroxide solution, to the first reaction vessel while agitating for at least about 30 minutes, or at least about 60 minutes, or at least about 120 minutes, or at least about 3 hours, or at least about 4 hours, or at least about 5 hours, or at least about 6 hours, or longer as desired to yield a brown macroalgae extract having a pH of at least 6 but preferably at least above 6.5, and optionally including subjecting the brown macroalgae extract solution of this step d) to high shear mixing for at least about 60 minutes, or at least about 90 minutes, or at least about 120 minutes, or longer as desired; e) optionally adding an ammonium phosphate solution, such that a ratio of solid mono ammonium phosphate to dry kelp of at least 0.05 w/w is obtained, to the first reaction vessel while agitating for at least about 30 minutes, or at least about 40 minutes, or at least about 50 minutes, or at least about 60 minutes, or at least about 70 minutes, or longer as desired; f) optionally, while still agitating, adding either a ferrous sulphate heptahydrate or copper sulphate heptahydrate solution, such that the ratio of metal sulphate to dry kelp is at least 0.03 (w/w), to the first reaction vessel, and followed immediately thereafter adding a hydrogen peroxide solution such that the ratio of hydrogen peroxide to dry kelp pieces is at least 0.03 (w/w), to the reaction vessel, and followed immediately thereafter adding an ascorbic acid solution such that the ascorbic acid to dry kelp (w/w) ratio is at least 0.006 and continuing to agitate for at least about 5 minutes, or at least about 10 minutes, or at least about 15 minutes, or at least about 20 minutes, or longer if desired, to yield a brown macroalgae extract in the form of a suspension comprising biostimulants and amino acids; and g) wherein where the pH is below about 13, or below about 12, or below about 11 or below about 10, adding a preservative including a sodium benzoate, potassium sorbate or boric acid solution such that ratio of the dry preservative to dry kelp is at least 0.1 (w/w), to the brown macroalgae extract in the first reaction vessel while agitating for at least about 30 minutes, or at least about 40 minutes, or at least about 50 minutes, or at least about 60 minutes, or at least about 70 minutes, or longer as desired, wherein where the pH is above about 13, or above about 12, or above about 11 or above about 10, this step is optional; and h) optionally filtering the brown macroalgae extract before collecting it. The dried brown macroalgae may comprise dried kelp hinge pieces. The method for extraction of a brown macroalgae extract may be a method for extraction of a clear extract comprising biostimulants and amino acids in which calcium alginate or alginic acids have been precipitated and removed, wherein the method further comprises additional steps after step d), or after any one of the optional steps e) to h) above of: AI) where the pH range is greater than about 10 or about 11 or about 12 or about 13, adding a calcium nitrate (Ca(NO₃)₂) or a calcium chloride (CaCl2) solution such that the ratio of dry calcium nitrate or chloride product to dry kelp is at least 0.49 (w/w), to the brown macroalgae extract in the first reaction vessel while agitating for at least about 10 minutes, or at least about 20 minutes, or at least about 30 minutes, or longer if desired, thereby to precipitate out calcium alginate in the brown macroalgae extract; AII) filtering the brown macroalgae extract of step AI) thereby to separate a clear brown macroalgae liquid comprising biostimulants and amino acids from the precipitated calcium alginate, including by pressure plate filtration and/or membrane squeezing; and AIII) collecting the clear brown macroalgae liquid having a pH of greater than about 10 or about 11 or about 12 or about 13 comprising biostimulants and amino acids; or BI) alternatively, adding an acid solution, including acetic acid, citric acid, or phosphoric acid, preferably a 85 wt% phosphoric acid solution to the brown macroalgae extract in the first reaction vessel until a pH of less than about 4, less than about 3, less than about 2, or less than about 1 (i.e. an acidic pH) is reached, at about room temperature or about 25°C, while agitating, for at least about for at least about 30 minutes, or at least about 60 minutes, or at least about 90 minutes, or at least about 100 minutes, or longer as desired, thereby to precipitate out the alginates in the brown macroalgae extract as alginic acid; BII) filtering the brown macroalgae extract of step BII), thereby to separate a clear brown macroalgae liquid having a pH of less than about 4, less than about 3, less than about 2, or less than about 1 comprising biostimulants and amino acids from the precipitated alginates, including by pressure plate filtration, a filter press and/or membrane squeezing; and BIII) collecting the clear brown macroalgae liquid comprising biostimulants and amino acids. In one embodiment, the temperature in first reaction vessel is maintained throughout the method at about 55 °C to about 77 °C, or about 62 °C to about 75 °C, or about 68 °C to about 72 °C. In a second embodiment, the initial temperature of the water and brown macroalgae pieces in the first reaction vessel is heated to about 55 °C to about 77 °C, or about 62 °C to about 75 °C, or about 68 °C to about 72 °C, after which the heating source is switched off for the remainder of the method. According to a second aspect of the invention, there is provided a method of formulating the brown macroalgae extract of the invention into a plant biostimulant composition or fertiliser, including a liquid or granular plant biostimulant composition or fertiliser, wherein the plant biostimulant composition or fertiliser may be neutral having a pH of between about 4 to about 8, alkaline having a pH of greater than about 8 or acidic having a pH of less than about 4, wherein where the liquid plant biostimulant composition or fertiliser is neutral or alkaline, the brown macroalgae extract is a brown macroalgae suspension according to any one of steps d) to g) above including calcium alginate and/or alginic acids, or wherein where the liquid plant biostimulant composition or fertiliser is acidic, the brown macroalgae extract is a clear liquid where calcium alginate or alginic acids have been precipitated out according to steps AI) to AIII) or BI) to BIII) above; and wherein where the granular plant biostimulant composition or fertiliser is neutral, alkaline or acidic, the brown macroalgae extract is derived from the brown macroalgae suspension according to any one of steps d) to g) above including calcium alginate and alginic acids, or from the clear liquid where calcium alginate or alginic acids have been precipitated out according to steps AI) to AIII) or BI) to BIII) above. According to a fourth aspect of the invention, there is provided a method of stimulating plant growth with the use of the brown macroalgae extract comprising biostimulants and amino acids produced according to the method of the invention or with the use of the plant biostimulant composition or fertiliser of the invention. DETAILED DESCRIPTION OF DRAWINGS FIGURE 1: shows selected growth regulators in a commercial kelp product “K- kelp”, and kelp extracted from Australian bull kelp (n-hepa = n- hydroxyethyl phthalimide; iaa = indole-3-acetic acid; i3ca = indole-3- carboxylic acid; ica = indole-3-carboxaldehyde; sa = salicylic acid; acc = 1-aminocyclopropane-1-carboxylic acid); FIGURE 2: shows selected amino acids in K-kelp and kelp extracted from Australian Bull kelp; FIGURE 3: shows changes in root number at varying IAA concentrations; FIGURE 4: shows changes in root number at varying kelp concentrations for the different kelp sources; FIGURE 5: shows changes in SPAD number (chlorophyll concentration) with various treatments. Acronyms: G-Kelp is the test kelp of the process of the invention; K-Kelp is the commercially available kelp; + A is with alginates; - A is without alginates; NS is no drought stress; WS is with drought stress. Different letters above each bar indicates statistical significant differences. Letters that are the same indicate no significant differences between those treatments; FIGURE 6: shows changes in root, shoot and total dry biomass with various treatments. Acronyms: G-Kelp is the test kelp of the process of the invention; K-Kelp is the commercially available kelp; + A is with alginates; - A is without alginates; NS is no drought stress; WS is with drought stress. Different letters above each bar of the same color indicates statistical significant differences. Letters that are the same indicate no significant differences between those treatments; FIGURE 7: shows changes in H2O2 concentration with varying treatments. Acronyms: G-Kelp is the test kelp of the process of the invention; K- Kelp is the commercially available kelp; + A is with alginates; - A is without alginates; NS is no drought stress; WS is with drought stress. Different letters above each bar of the same colour indicates statistical significant differences. Letters that are the same indicate no significant differences between those treatments; FIGURE 8: shows SOD activity with various treatments; and FIGURE 9: shows APX activity with various treatments. DETAILED DESCRIPTION OF THE INVENTION The current invention relates to processes for the extraction of biostimulants and amino acids from brown macroalgae, including dry kelp hinge pieces and to products from such an extraction process, as well as to methods for stimulating plant growth with the use of the biostimulants and amino acids extracted by the processes of the invention. The following description of the invention is provided as an enabling teaching of the invention, is illustrative of the principles of the invention and is not intended to limit the scope of the invention. It will be understood that changes can be made to the embodiment/s depicted and described, while still attaining beneficial results of the present invention. Furthermore, it will be understood that some benefits of the present invention can be attained by selecting some of the features of the present invention without utilising other features. Accordingly, those skilled in the art will recognise that modifications and adaptations to the present invention are possible and can even be desirable in certain circumstances, and are a part of the present invention. The applicant has developed a novel method for the extraction of various biostimulants and amino acids from dried brown macroalgae comprising alginates, in particular Ecklonia maxima or Australian Bull kelp (Durvillaea potatorum) hinge pieces. It is however to be expected that any dried brown macroalgae comprising alginates may be successfully processed to produce the biostimulants and amino acids according to the method developed by the applicant. In an exemplary method this entails the step-wise addition of various chemical reagents to dried kelp hinge pieces after a hydration step. The particular steps of the invention have been shown by the applicant to ensure both sufficient cellular lysis and therefore efficient extraction of the targeted compounds, in addition to keeping the viscosity of the mixture relatively low throughout the extraction procedure. The extraction process is completed in a reasonably short amount of time, especially when compared to other brown macroalgae extraction methods. Using an extraction method to produce a kelp suspension (including alginates present), 14.7-ton product/ton dry chips can be produced with 0.025-ton waste/ton product. When using an extraction method for a clear kelp liquid where alginates are removed, 10.9-ton product/ton dry chips can be produced with 0.33-ton alginate cake/ton product. Furthermore, the extraction conditions are mild, with a high extraction efficiency resulting to little or no waste generated after the extraction procedure. The brown macroalgae products can be used as prepared, in neutral, alkaline or acidic aqueous fertilisers or plant biostimulants or granular fertilisers or plant biostimulants. The alginates can be removed from the brown macroalgae suspension, yielding a clear brown macroalgae liquid and the respective alginate. It was determined, through LC-QqQ-MS analysis, that the brown macroalgae extracts contained various auxins, cytokinins and amino acids at concentrations comparable to commercially available kelp products. Moreover, microbiological analysis of the brown macroalgae extract confirmed that the preservatives used in the neutral and acidic extracts, either potassium sorbate or boric acid, effectively inhibited both bacterial and fungal growth, ensuring product stability and shelf-life. EXAMPLE 1 Example 1 is based on an adapted method provided in Qin (CN101538172) which constitutes the closest prior art relating to the process of the invention. The process is performed as follows: • Weigh specified amount of water and dry kelp according to batch card shown in Table 1. • Add water in glass beaker, place on hot plate and insert thermocouple probe into the water. • Insert agitator from overhead stirrer into the water and start agitator. • Insert temperature set point on hot plate and start. • While water is agitating and heating, add the dry kelp. Leave for 1 hour. • Add specified amount of EDTA (ethylenediamine tetraacetic acid) as well as solid KOH (potassium hydroxide). • After 1 hour, the viscosity will be very high, add ferrous sulphate heptahydrate, hydrogen peroxide as well as ascorbic acid in specified amounts. • The viscosity will decrease; ensure that everything is in suspension again. • The mixture can be placed in a kitchen blender to break large chunks into smaller pieces. • Use a Buchner filter with vacuum filtration to separate the solids larger than 300 μm from filtrate. • Add specified amount of boric acid to the filtrate. Table 1: Batch card for test procedure

It was observed by the applicant that the solution produced according to this method becomes extremely viscous after 1 hour when EDTA and KOH have been added to the mixture. After the addition of ferrous sulphate heptahydrate, hydrogen peroxide and ascorbic acid the viscosity decreased. However, the technoeconomic feasibility of the process does not result in a profitable process, particularly due to the high cost of EDTA. Furthermore, the process yields a kelp extract that is only suitable for addition to fertiliser formulations that have a weakly acidic or neutral pH. Furthermore, the product of the process is a kelp suspension including alginates. It is necessary to improve extraction efficiency, especially in terms of a reduction in waste generated and to provide a higher solid content in the extract, whilst at the same time, improving the ease of extraction through decreasing the viscosity of the mixture throughout the extraction procedure. The applicant therefore went on to modify the process further to achieve the objectives above. EXAMPLE 2 The following batch card was used on plant scale to produce an alkaline kelp suspension. Table 2: Batch card for plant scale kelp extraction using sodium based extractant Unit Value Water L 10528 Kelp Kg 890 Citric Acid Kg 50 Sodium Hydroxide 46% solution kg 444 Total kg 11912 • Add water into the reactor and turn agitator on (See Table 2) • Start circulating the water through an external heater overnight, until the desired temperature of 70^oC is reached • Transfer the required mass of dried kelp pieces into the reactor and allow residence time of 1 hour to hydrate • After the 1 hour residence time, add citric acid into the reactor • Allow further 2 hours residence time • Add the sodium hydroxide solution into the reactor and allow residence time of 6 hours • Use a high shearing pump for at least 2 hours to aid in homogenizing the suspension to produce a final product EXAMPLE 3 The following batch card was evaluated as an alternative process on a plant scale to produce an alkaline kelp suspension. Table 3: Batch card for plant scale kelp extraction using potassium based extractant Unit Value Water L 9170 Kelp Kg 510 Citric Acid Kg 30 Potassium Hydroxide 50% solution Kg 370 Total kg 10080 • Add water into the reactor and turn agitator on (See Table 3) • Start circulating the water through an external heater overnight, until the desired temperature of 70^oC is reached • Transfer the required mass of dried kelp pieces into the reactor and allow residence time of 1 hour to hydrate • After the 1 hour residence time, add citric acid into the reactor • Allow 2 hours residence time • Add the potassium hydroxide solution into the reactor and allow residence time of 6 hours • Use a high shearing pump for at least 2 hours to aid in homogenizing the suspension to produce a final product EXAMPLE 4 The following batch card was used on plant scale to produce a clear alkaline kelp liquid. Table 4: Batch card for plant scale alginate free alkaline kelp extraction using potassium based extractant Unit Value Water L 11660 Kelp Kg 650 Citric Acid Kg 40 Potassium Hydroxide 50 wt.% solution kg 460 Calcium Chloride Solution 40 wt.% kg 800 Total kg 13610 • Add water into the reactor and turn agitator on (See Table 4) • Start circulating the water through an external heater overnight, until the desired temperature of 70^oC is reached • Transfer the required mass of dried kelp pieces into the reactor and provide residence time of 1 hour to hydrate • After the 1 hour residence time, add citric acid into the reactor • Provide 2 hours residence time • Add the potassium hydroxide solution into the reactor and allow residence time of 6 hours • Use a high shearing pump for at least 2 hours to aid in homogenizing the suspension • Add the calcium chloride solution slowly into the reactor. Calcium alginate will precipitate • Allow 30 minutes residence time • Transfer the suspension through a filter press to obtain at least 65wt% of total mass as clear kelp liquid EXAMPLE 5 The following batch card was used to produce a clear acid kelp liquid. Table 5: Batch card for plant scale alginate free acidic kelp extraction using potassium based extractant Unit Value Water L 12650 Kelp kg 700 Citric Acid Kg 40 Potassium Hydroxide 50% solution Kg 510 Phosphoric acid 85% Kg 830 Total Kg 14730 • Add water into the reactor and turn agitator on (See Table 5) • Start circulating the water through an external heater overnight, until the desried temperature of 70^oC is reached • Transfer the required mass of dried kelp pieces into the reactor and provide residence time of 1 hour to hydrate • After the 1 hour residence time, add citric acid into the reactor • Provide 2 hours residence time • Add the potassium hydroxide solution into the reactor and allow residence time of 6 hours • Use a high shearing pump at least 2 hours to aid in homogenizing the suspension • Add the phosphoric acid liquid slowly into the reactor to reach a pH of at least 3. Alginic acid will precipitate • Allow 30 minutes residence time • Transfer the suspension through a filter press to obtain at least 65wt% of total mass as clear kelp liquid. EXAMPLE 6 The first adaptation of the method involves the addition of mono-ammonium phosphate. Table 6: Batch card for test procedure • Add specified amount of water and dry kelp (see Table 6 above) to glass beaker. • Insert agitator, from overhead stirrer, and thermocouple probe into the water. Set temperature set point on hot plate to 70 °C. • Initiate stirring, at 100 RPM (as determined using a tachometer), and heating to 70 °C. • Stir mixture for 60 minutes, maintaining stirring speed at 100 RPM. • Add specified amount of EDTA and KOH in one batch, whilst stirring. • Stir mixture for 30 minutes at 70 °C. Mixture becomes very dark and very viscous, with the stirring speed dropping down from 100 RPM to 40 RPM. • Add specified amount of mono-ammonium phosphate (“MAP”) whilst stirring. • Stir mixture for 30 minutes at 70 °C. • Add ferrous sulphate heptahydrate followed by hydrogen peroxide. • Stir mixture for 2 minutes. • Add ascorbic acid and stir mixture for an additional 5 minutes at 70 °C. • Add boric acid whilst stirring at 70 °C. • Stir mixture for 20 minutes. • Filter mixture through a filter cloth with an aperture size of < 300 μm using a pressure filtration apparatus. The applicant determined that addition of mono-ammonium phosphate had a significant influence on the viscosity of the reaction mixture, as well as the extraction efficiency and allowed for an improved mixing which directly results to more kelp hinge pieces being degraded and dissolved in the solution. This is clearly evident in the large decrease of waste residue remaining after filtration of the mixture, compared to the standardized extraction procedure. The above data and observations substantiates the addition of mono-ammonium phosphate to the reaction mixture, and the gains obtained from the addition of the phosphate salt outweighs the cost of adding the chemical. EXAMPLE 7 Citric acid was explored as both a pre-treatment agent as well as a chelating agent. Table 7: Batch card for test procedure • Add specified amount of water and dry kelp (see Table 7) to glass beaker. • Insert agitator, from overhead stirrer, and thermocouple probe into the water. Set temperature set point on hot plate to 70 °C. • Initiate stirring, at 100 RPM (as determined using a tachometer), and heating to 70 °C. • Stir mixture for 60 minutes, maintaining stirring speed at 100 RPM. • Add specified amount of citric acid whilst stirring. • Stir mixture for 30 minutes at 70 °C. Mixture’s viscosity decreases slightly, with the stirring speed increasing from 100 RPM to 105 RPM. • Add KOH pellets whilst stirring at 70 °C. • Stir mixture for 30 minutes at 70 °C. Mixture’s viscosity increased, with the stirring speed decreasing from 105 RPM to 60 RPM. • Add specified amount of mono-ammonium phosphate whilst stirring. • Stir mixture for 30 minutes at 70 °C. • Add ferrous sulphate heptahydrate followed by hydrogen peroxide. • Stir mixture for 2 minutes. • Add ascorbic acid and stir mixture for an additional 5 minutes at 70 °C. • Add sodium benzoate whilst stirring at 70 °C. • Stir mixture for 20 minutes. • Filter mixture through a filter cloth with an aperture size of < 300 μm using a pressure filtration apparatus. In addition, a second extract where alginates are removed by precipitation to yield a clear kelp liquid from the kelp suspension can be obtained from this process. Where desired, the process includes the following additional steps: • Dissolve 50 g of calcium nitrate (Ca(NO₃)₂) in 100 g H₂O. • Stir solution slowly with an anchor impeller. • Add the kelp suspension drop-wise to the Ca(NO₃)₂ water solution, whilst stirring slowly. • Filter off the clear kelp liquid from the brown calcium alginate via vacuum filtration using a Whatmann number 4 filter paper. The applicant was able to demonstrate that the use of citric acid as both a proton source and a chelator, instead of EDTA, significantly improved the ease of extraction through decreasing the viscosity of the reaction mixture throughout the extraction procedure. The viscosity of the final mixture was also found to be lower compared to previous extraction procedures. Table 8 below summarizes the results obtained from the elemental analysis of the bull kelp suspension obtained via extraction with citric acid as a proton donor and as a chelator. Table 8: Elemental analysis of kelp extract obtained by using citric acid as a proton source and chelator The final pH of the mixture is more neutral at pH 6.8, due to the use of sodium benzoate as preservative instead of boric acid. In addition, the lower viscosity ultimately leads to a greater ease of extraction, and a decrease in the energy required during the mixing of the solution/suspension. The lower viscosity throughout the entire extraction procedure indicated that citric acid can sufficiently protonate calcium alginate to yield the more soluble alginic acid, which directly affects the mixture’s viscosity. Furthermore, potassium alginate formation is much more favoured through the deprotonation of alginic acid compared to a calcium- potassium cation exchange reaction. Finally, the excess calcium in solution is chelated by the citrate anion, resulting to the viscosity of the final mixture being low, and more importantly, remaining low. Additional processing of the kelp suspension obtained after filtration also allowed for the separation of the alginates from the mixture to yield the kelp solution as a clear brown liquid. This was readily accomplished through the drop-wise addition of the kelp suspension to a concentrated calcium nitrate water solution. The alginates in the kelp suspension, mainly in the form of potassium alginate, immediately precipitates out as calcium alginate when it is added to the calcium nitrate solution. This allows for the isolation of the clear kelp liquid after filtration. The removal of the alginates from the suspension resulted to a slight decrease in both the solid content (decreased from 9% to 7.5%) and pH of the solution, with the final pH being 5.6. However, the viscosity of the solution after removal of the alginates decreased significantly, from 545 cP to 25.5 cP. The clear kelp liquid also has a higher nitrogen concentration, as determined through elemental analysis (Table 9). The higher nitrogen content is a result of using Ca(NO₃)₂, which fulfils a two-fold role. It readily dissolves in water yielding free calcium ions that can coordinate to the anionic oxygens on the alginate backbone. Secondly, the nitrate ions dissolved in the solution improve the fertilizer properties of the product. Table 9: Elemental analysis of clear kelp liquid containing no alginates The current method involving the use of citric acid as a proton source and a chelator, was determined to be the optimized method for the standardized reaction. From this method, various alterations to the procedure were further explored. One such alteration is the replacement of the iron catalyst with copper. The addition of copper instead of iron might be useful in soils saturated with iron or with a shortage of copper. EXAMPLE 8 A variation of the citric acid method involves the replacement of iron with copper. • Add specified amount of water and dry kelp (see Table 10) to glass beaker. • Insert agitator, from overhead stirrer, and thermocouple probe into the water. Set temperature set point on hot plate to 70 °C. • Initiate stirring, at 100 RPM (as determined using a tachometer), and heating to 70 °C. • Stir mixture for 60 minutes, maintaining stirring speed at 100 RPM. • Add specified amount of citric acid whilst stirring. • Stir mixture for 30 minutes at 70 °C. Mixture’s viscosity decreases slightly, with the stirring speed increasing from 100 RPM to 105 RPM. • Add KOH pellets whilst stirring at 70 °C. • Stir mixture for 30 minutes at 70 °C. Mixture’s viscosity increased, with the stirring speed decreasing from 105 RPM to 63 RPM. • Add specified amount of mono-ammonium phosphate whilst stirring. • Stir mixture for 30 minutes at 70 °C. • Add copper sulphate pentahydrate followed by hydrogen peroxide. • Stir mixture for 2 minutes. • Add ascorbic acid and stir mixture for an additional 5 minutes at 70 °C. • Add sodium benzoate whilst stirring at 70 °C. • Stir mixture for 20 minutes. • Filter mixture through a filter cloth with an aperture size of < 300 μm using a pressure filtration apparatus. For precipitation of alginates to yield a clear kelp liquid from the kelp suspension obtained: • Dissolve 50 g of Ca(NO₃)₂ in 100 g H₂O. • Stir mixture with an anchor impeller. • Add the kelp suspension drop-wise to the Ca(NO₃)₂ water solution, whilst stirring slowly. • Filter off the clear kelp liquid from the brown calcium alginate via vacuum filtration using a Whatmann number 4 filter paper. Table 10: Batch card for test procedure The applicant determined that the replacement of iron with copper had significant effects on the reaction mixture. The viscosity of the mixture throughout the extraction, as well as the final mixture’s viscosity, was determined to be lower compared to the standard citric acid method. This significantly improved operational handling, especially in terms of mixing and filtering. The lower viscosity will also improve the handling efficiency during the infield application of the final product. The extraction efficiency is similar to the standard citric acid method, with the differences being a slight decrease in the solid content of the mixture compared to the standard method, but with a significant decrease of the viscosity of the mixture of more than double. This is a direct result of the use of copper instead of iron for the generation of the reactive oxygen species for the oxidative depolymerization reaction. The use of copper, in addition with ascorbic acid, could also lead to the in- situ generation of singlet oxygen, which will react differently compared to the hydroxyl radicals. Singlet oxygen also has a lower relative oxidative strength compared to the hydroxyl radical. This results to a ‘softer’ reactive species in solution, which could be beneficial especially if the hydroxyl radical proves to be too harsh, resulting to degradation of the desired biostimulants and amino acids compounds. The alginates and the kelp liquid were also individually isolated from the suspension, again through precipitation of the alginates as calcium alginate with calcium nitrate. Both solid content and pH were found to be similar to the clear kelp extract obtained through the standard citric acid method. Elemental analysis (See table 11) of the clear kelp liquid indicated a slightly higher concentration of nitrogen compared to the extract obtained in Example 7. As can be expected, the concentration of copper is significantly higher at 42.4 ppm, compared to the extract obtained through the standard citric acid method. Table 11: Elemental analysis of clear kelp liquid containing no alginates EXAMPLE 9 The applicant then conducted a pilot plant trial. • Add specified amount of H2O (see Table 12) to reactor. • Heat H2O to 65 °C in reactor fitted with an anchor impeller. • Add Kelp hinge pieces to water at 65 °C whilst stirring at 75 RPM. • Stir mixture for 60 minutes, allowing for sufficient hydration of dried kelp hinge pieces. • Add citric acid whilst stirring at 75 RPM at 65 °C. • Stir mixture at 75 RPM and at 65 °C for 30 minutes. • Add KOH flakes to reaction mixture whilst stirring at 75 RPM at 65 °C. • Stir mixture at 75 RPM and at 65 °C for 30 minutes. Mixture’s viscosity increases significantly. • Add mono-ammonium phosphate whilst stirring at 75 RPM, at 65 °C. • Stir mixture for 30 minutes at 65 °C. Mixture becomes dark and viscous, with the stirring speed remaining at 75 RPM. • Add ferrous sulphate heptahydrate. • Add 50% hydrogen peroxide via a peristaltic pump, over a period of 5 minutes. Mixture’s viscosity decreased significantly. • Add ascorbic acid, directly after hydrogen peroxide addition. • Mixture was stirred at 65 °C with a stirring speed of 75 RPM, for 5 minutes. • Add sodium benzoate whilst stirring at 75 RPM, at 65 °C, and stirred mixture for 20 minutes. • The mixture was filtered, whilst hot, through a sieve with a 2 mm aperture size. Table 12: Batch card for pilot plant procedure

Use of a high shear mixer for extraction of the targeted compounds from the kelp hinge pieces, had significantly improved the ease of extraction through efficient mixing of the viscous mixture during the extraction procedure. EXAMPLE 10 Even though the extraction procedure is very efficient with the addition of the reagents as a solid, there is difficulty with the handling of solids, especially with the large quantities typically used at plant scale procedures. Resultantly, the addition of the reagents as aqueous solutions was included to determine the effects of adding the reagents as an aqueous solution. The addition of solutions would simplify the extraction procedure, as the reagent solutions can easily be pumped into the reactor, compared to the addition of the solids via a hopper and/or a screw feeder. The applicant therefore investigated optimization of the pilot plant procedure involving the addition of the reagents as aqueous solutions, as follows: • Add specified amount of H2O (see Table 13, below) to reactor. • Heat up H2O to 65 °C in reactor fitted with an anchor impeller. • Add kelp hinge pieces to water at 65 °C whilst stirring at 75 RPM. • Stir mixture for 60 minutes, allowing for sufficient hydration of dried kelp hinge pieces. • Add citric acid dissolved in H2O whilst stirring at 75 RPM at 65 °C. • Stir mixture at 75 RPM and at 65 °C for 30 minutes. • Add KOH flakes dissolved in H2O to reaction mixture whilst stirring. • Stir mixture at 75 RPM and at 65 °C for 60 minutes. • Mixture’s viscosity increases significantly. • Add a solution of mono-ammonium phosphate in H2O whilst stirring at 75 RPM, at 65 °C. • Stir mixture for 60 minutes at 65 °C. Mixture becomes dark and viscous, with the stirring speed remaining at 75 RPM. • Add ferrous sulphate heptahydrate dissolved in H2O. • Add 50% hydrogen peroxide via a peristaltic pump, over a period of 5 minutes. • During the addition of hydrogen peroxide, the viscosity visually decreased. • Add a solution of ascorbic acid in H2O, directly after hydrogen peroxide addition. • Mixture was stirred at 65 °C with a stirring speed of 75 RPM, for 10 minutes. • Add a solution of sodium benzoate in H2O whilst stirring at 75 RPM, at 65 °C, and stirred mixture for 40 minutes. • The mixture was filtered, whilst hot, through a sieve with a 2 mm aperture size. Table 13: Batch card for pilot plant reaction optimization with addition of the reagents as aqueous solutions Adding the reagents as aqueous solutions did not have a negative effect on the extraction procedure. On the contrary, it was noted that the more diluted reaction mixture allowed for improved ease of mixing. Table 14: Results for optimization of the standard pilot plant extraction procedure The extraction efficiency was found to be satisfactory, with the waste residue remaining after the extraction determined to be 4%. Both the pH and density were determined to be similar to previous results. The lower solid content and viscosity is a direct result of the more diluted mixture. The addition of aqueous solutions greatly improved the ease of extraction, while the extraction efficiency was not negatively affected. In order to obtain a solid content similar to that obtained in previous extractions, the amount of water initially added must be reduced in order to compensate for the water added, that was required to prepare the respective solutions. EXAMPLE 11 Two different kelp products can be obtained, as described below, by employing the same extraction methodology, with only minor alterations needed to be made to the batch card, to obtain the desired kelp product. This will further differentiate the kelp extraction procedure, as the two different kelp products can be used under different conditions and for different end-uses. The type of kelp product to be prepared will also dictate which preservative is to be used, in order to sufficiently inhibit fungal and bacterial growth and ensure product stability and shelf-life. The two kelp products to be prepared are a kelp suspension or alginate-free liquid to be used in neutral or alkaline environments, and an acidic kelp liquid to be used in products with a low pH. Optimization pilot plant extraction procedure for the isolation of a kelp suspension that could be treated with base, or added to a neutral or alkaline environment, involved the use of potassium sorbate as a preservative. In addition, the alginates can be removed as calcium alginate. The extraction was completed as follows: • Add specified amount of H2O (see Table 15) to reactor. • Heat up H2O to 68 °C in reactor fitted with an anchor impeller. • Switch off heating source. • Add kelp hinge pieces to water at 68 °C whilst stirring at 75 RPM. • Stir mixture for 60 minutes, allowing for sufficient hydration of dried kelp hinge pieces. • Add citric acid dissolved in H2O whilst stirring. • Stir mixture at 75 RPM for 30 minutes. • Add KOH dissolved in H2O to reaction mixture whilst stirring at 75 RPM. • Stir mixture at 75 RPM for 60 minutes. • Mixture’s viscosity increases significantly. • Add a solution of mono-ammonium phosphate in H2O whilst stirring. • Stir mixture for 60 minutes. • Add copper sulphate pentahydrate dissolved in H2O. • Add 50% hydrogen peroxide via a peristaltic pump, over a period of 5 minutes. • During the addition of hydrogen peroxide, the viscosity visually decreased. • Add a solution of ascorbic acid in H2O, directly after hydrogen peroxide addition. • Mixture was stirred at 75 RPM, for 10 minutes. • Add a solution of potassium sorbate in H2O whilst stirring. Stirred mixture for 60 minutes. • The mixture was filtered, whilst hot, through a sieve with a 2 mm aperture size. For precipitation of calcium alginate (optional) to yield a clear kelp liquid from the kelp suspension mixture above: • Prepare concentrated solution of Ca(NO₃)₂ in H₂O. • Re-circulate concentrated Ca(NO₃)₂ solution via a centrifugal pump. • Dose kelp suspension into the solution of Ca(NO₃)₂ via a peristaltic pump. • Filter the kelp liquid/calcium alginate slurry through pressure plate filtration followed by membrane squeezing. Table 15: Batch card for optimized extraction of bull kelp on pilot plant scale Processing of the kelp hinge pieces was determined to be sufficient, as could be expected from the optimized extraction procedure. The substitution of both the iron catalyst for copper, as well as the sodium benzoate preservative with potassium sorbate, improved the processing of the kelp hinge pieces, as compared to previous attempts. The viscosity of the mixture remained relatively low throughout the extraction procedure, decreasing the energy required for mixing. Table 16 summarizes the results obtain for the extraction procedure. Table 16: Results for optimized extraction procedure A high extraction efficiency was noted for the procedure, while the physical properties of the mixture was determined to be similar to previously prepared kelp suspensions. The copper catalyst, a sufficient replacement for the iron catalyst, will not form an unstable mixture in an aqueous environment over a given period. It was determined that the potassium sorbate is an excellent preservative for the kelp suspension, as it inhibited almost all bacterial growth, in addition to completely inhibiting fungal growth. Further processing of the kelp suspension involved the isolation of the alginates, yielding calcium alginate and a clear kelp liquid. This was accomplished by subjecting the kelp suspension to an aqueous calcium nitrate solution, whereby calcium alginate precipitates out of the alginate/kelp liquid slurry. Calcium alginate is then readily isolated via pressure plate filtration followed by membrane squeezing. The calcium alginate has excellent moisture retaining properties, which resulted to the isolation of the alginates with a high kelp liquid content (split of 76% to 24%), even after membrane squeezing. The solid content of the alginate filter cake was determined to be only 31%, indicating that more than 65% of the wet alginate filter cake still consisted of kelp liquid. A longer squeezing time could improve the filtration of the kelp liquid from the filter cake, isolating an alginate cake with a lower liquid content. The physical properties of the clear kelp liquid were found to slightly different from the kelp suspension. The pH was found to be lower at 5.4, while the sold content also decreased to 7.8%. EXAMPLE 12 By applying minor adjustment to the optimised method, a clear kelp liquid could also be prepared on the same method as the kelp suspension was prepared. The change made to the process is the substitution of the preservative potassium sorbate for boric acid. This allows for the kelp liquid to be added to mixtures with a low pH. Potassium sorbate precipitates out of aqueous acidic mixtures as sorbic acid. This would result to microbial contamination in the kelp liquid, as there is no preservative to inhibit bacterial and fungal growth. Boric acid is stable under acidic conditions, and is sufficient to inhibit microbial growth, as was determined during microbiological analysis of the kelp liquid (see below). The optimised method below was used for the preparation of the kelp suspension containing boric acid as preservative, followed by the precipitation of alginates as alginic acid through the addition of phosphoric acid: • Add specified amount of H2O (see Table 17) to reactor. • Heat up H2O to 68 °C in reactor fitted with an anchor impeller. • Switch off heating source. • Add Kelp hinge pieces to water at 68 °C whilst stirring at 75 RPM. • Stir mixture for 60 minutes, allowing for sufficient hydration of dried kelp hinge pieces. • Add citric acid dissolved in H2O whilst stirring. • Stir mixture at 75 RPM for 30 minutes. • Add KOH dissolved in H2O to reaction mixture whilst stirring at 75 RPM. • Stir mixture at 75 RPM for 60 minutes. • Mixture’s viscosity increases significantly. • Add a solution of mono-ammonium phosphate in H2O whilst stirring at 75 RPM. • Stir mixture for 60 minutes. • Add copper sulphate pentahydrate dissolved in H2O. • Add 50% hydrogen peroxide via a peristaltic pump, over a period of 5 minutes. • During the addition of hydrogen peroxide, the viscosity visually decreased. • Add a solution of ascorbic acid in H2O, directly after hydrogen peroxide addition. • Mixture was stirred at 75 RPM, for 10 minutes. • Add a solution of boric acid in H2O whilst stirring at 75 RPM. Stirred mixture for 60 minutes. • The mixture was filtered, whilst hot, through a sieve with a 2 mm aperture size. • Slowly add 85% H₃PO₄ to kelp suspension whilst stirring at 75 RPM, in a turbulator fitted with an anchor impeller at room temperature. • Stir mixture for 60 minutes at room temperature • Filter the kelp liquid/alginic acid slurry via pressure plate filtration followed by membrane squeezing. Table 17: Batch card for optimized extraction of bull kelp on pilot plant scale for use in acid environments The extraction procedure was the similar as for the preparation of the kelp suspension containing potassium sorbate as preservative. The only difference is the low solubility of boric acid in water, as compared to potassium sorbate. In order to compensate for the low solubility of boric acid, the amount of water that was initially added to the kelp hinge pieces was decreased. Table 18 below summarizes the results obtain for the extraction procedure Table 18: Results for optimized extraction procedure The extraction efficiency was similar to that of the extraction procedure employed for the preparation of the potassium sorbate containing kelp suspension. The solid content is slightly lower, at 8.2% compared against the potassium sorbate containing kelp suspension at 8.4%. The pH is also lower at 6.46, as was expected due to the use of boric acid as preservative. Alginic acid is more soluble in water when compared against calcium alginate, and is generally completely soluble in very diluted solutions. However, in a concentrated alginate mixture, the formation of alginic acid from potassium alginate results to the precipitation of the alginate out of the solution. Consequently, the kelp suspension was acidified through the addition of phosphoric acid, which allowed for the precipitation of alginic acid with subsequent filtration, yielding the clear kelp liquid at low pH as well as the alginic acid filter cake. The moisture containing properties of alginic acid did not allow for isolation of a filter cake with a low water content, as was the case for the calcium alginate filter cake. The filtration efficiency was determined to be similar to the filtration efficiency of the calcium alginate filtration. As could be expected, the physical properties of the clear kelp liquid were very different when compared against the kelp suspension, or the kelp liquid obtained after removal of the calcium alginates. The solid content was high, at 10.3%, a result of the addition of phosphoric acid to precipitate alginic acid. The pH was low, at 2.05, while the density was slightly higher at 1.06 g/ml. The optimized extraction procedure allows for easy isolation of the kelp suspension from the starting dried kelp hinge pieces. The method has been significantly optimized, obtaining a kelp suspension in a relatively short time with minimal energy input and maximum extraction efficiency, whilst being stable towards microbial degradation. In addition, by slightly adjusting the batch card, the same extraction methodology allows for the preparation of different kelp products, to be used in neutral, alkaline or acidic environments. This further differentiates the extraction procedure, as the different kelp products can be used in almost any fertilizer product, including with a neutral or high pH, or with a low pH. EXAMPLE 13 The optimized batch card for kelp suspension production, containing alginates, to be used in a neutral or alkaline environment, is illustrated in Table 19 below. The changes to the previously optimized process, includes an increase in the amount of water added for extraction, as well as the use of diluted hydrogen peroxide. Table 19: optimized batch card for use in a neutral or alkaline environment The fine-tuned batch card allows for the preparation of the alginate containing kelp suspension, to be used in a neutral or alkaline environment, with agitator torque requirements remaining low enough. Additionally, the slight alterations to the batch card did not have a negative impact on the extraction efficiency. The physical properties of the kelp suspension, did not differ markedly from the kelp product obtained, during the finalized pilot plant trials. The solid content of the kelp liquid, also remained relatively unchanged when compared against the pilot plant kelp extract. Furthermore, the kelp suspension/kelp waste split after filtration, was likewise comparable to the above-mentioned pilot plant kelp extraction procedure. The kelp waste residue was dried, specifically to compare against the starting amount of dried kelp hinge pieces. It was determined that, the extraction efficiency was 97.7% on a dried kelp basis (kelp hinge pieces compared against dried kelp waste). This implies that, more than 97% of the mass of the solid kelp hinge pieces, is extracted. Resultantly, the waste obtained after extraction of the kelp hinge pieces is, on a dry basis, less than 3%. EXAMPLE 14 As was mentioned above, the batch card for the production of boric acid containing kelp suspension, was also modified and the batch size reduced for adaptation of the process for use in an acid environment. The changes to the batch card made includes 10% dilution of the batch size. Table 20 below illustrates the optimized batch card for kelp production containing boric acid as preservative, to be used in acidic mixtures (Note: boric acid experiment was done with 50% hydrogen peroxide, and not the diluted 30% hydrogen peroxide). Table 20: optimized batch card for use in an acid environment The physical properties, as well as the extraction efficiency, was found to be similar to the previously discussed extraction procedure and batch card for boric acid containing kelp suspension production. It was determined that, the extraction efficiency was also more than 97%, on a dried kelp basis, for the production of boric acid containing kelp suspension. Again, this implies that the waste remaining after extraction is less than 3%, on dry waste basis. EXAMPLE 15 It was noted above that; a small amount of dried waste is obtained after the finalized extraction procedure had been completed. In order to further decrease the amount of waste, a high-shearing step was introduced as above, which eliminated the waste almost completely. However, an alternative method towards high-shear mixing was investigated, which involves the re-extraction of the kelp waste. The re-extraction of the dried kelp waste residue further decreases the amount of waste accumulated, and also decrease the cost of waste disposal. The method entailed the use of dried kelp waste, of which was obtained after the extraction of the kelp hinge pieces on a pilot plant scale. The dried kelp residue was grinded to a finer powder, before the extraction procedure was initiated. o Dry kelp waste residue, at 65 °C until completely dry. o Crush dried kelp waste residue to a fine powder. o Add specified amount of H2O to reactor. o Heat up H2O to 65 °C in reactor fitted with an anchor impeller. o Add kelp waste powder to water at 65 °C whilst stirring at 200 RPM. o Stir mixture for 60 minutes. o Add citric acid dissolved in H2O whilst stirring at 200 RPM. o Stir mixture at 200 RPM for 30 minutes. o Add KOH flakes dissolved in H2O to reaction mixture whilst stirring at 200 RPM. o Stir mixture at 200 RPM for 60 minutes. o Add a solution of mono-ammonium phosphate in H2O whilst stirring. o Stir mixture for 60 minutes. o Add CuSO₄∙5H₂O dissolved in H2O. o Add hydrogen peroxide whilst stirring at 200 RPM. o Add a solution of ascorbic acid in H2O, directly after hydrogen peroxide addition. o Mixture was stirred at 200 RPM, for 10 minutes. o Add a solution of potassium sorbate in H2O whilst stirring at 200 RPM. Stir mixture for 40 minutes. o Filter mixture, through a sieve with a 1 mm aperture size. Table 21: Batch card used for extraction of kelp waste residue The viscosity of the mixture throughout the extraction procedure was very low, especially when compared against the extraction procedure involving the kelp hinge pieces. This could be determined by recording the agitator torque requirements during the experiment. This resulted to extraction of the kelp waste residue with relative ease. Furthermore, it was determined that the kelp extract obtained after extraction, had slightly different physical properties, as compared against the kelp extract obtained from the extraction of kelp hinge pieces. The viscosity was lower, at 41.9 cP. The pH was also higher at 7.71, but still well within the neutral range. The solid content was also comparable to the optimized kelp extraction procedure; determined to be 8.6%. The extraction efficiency, was however, lower in comparison with the optimised procedure. The kelp filtrate/kelp residue split after filtration, was 80.2% filtrate and 19.8% waste residue. Additionally, the extraction efficiency based on a dried kelp basis, was determined to be 67.7%, which is significantly lower when compared against the 97% extraction efficiency for extraction of the kelp from the starting hinge pieces. However, additional extraction of the waste residue obtained after extraction of the kelp liquid from the hinge pieces, significantly reduces the accumulated waste. The presence and concentration of the targeted growth regulators and amino acids in the kelp extract obtained after extraction of the waste residue are predicted to be less to zero, when compared against the extract obtained from the kelp hinge pieces. Nevertheless, various organic compounds would still be present in the mixture, especially sugar derivative and various polysaccharides. Additionally, the kelp extract could be added to a liquid kelp batch obtained after extraction of the kelp hinge pieces. The utilization of the kelp waste obtained after extraction of the kelp liquid from the kelp hinge pieces, decreases the amount of kelp waste accumulated. Product Quality The composition of the kelp suspension containing alginates and the acidic kelp liquid with alginates removed, was analysed to obtain a full elemental composition. Furthermore, the amino acid and biostimulant composition in the kelp samples was analysed. Table 22 below depicts the elemental composition of the alginate containing kelp suspension. Table 22: Elemental composition of kelp suspension containing alginates Table 23 below depicts the elemental composition of the acidic kelp liquid, with alginates removed as alginic acid. Table 23: Elemental composition of kelp suspension without alginates Biostimulant concentration was also measured, and is used during quality control checks. Table 24 below depicts the biostimulant concentration of the kelp suspension containing alginates, acidic kelp liquid with alginates removed, as well as the commercially available K-Kelp for comparative purposes. Table 24: Biostimulant concentration of kelp suspensions

Furthermore, the amino acid composition also plays a major role in the kelp liquids. As such, the amino acid composition of the alginate containing kelp suspension, the acidic kelp liquid without alginates, as well as the K-Kelp commercial product for comparative purposes, was determined and depicted in Table 25 below. Table 25: Amino acid concentration of kelp suspensions EXAMPLE 17 Characterisation of products Analysis of growth regulators with LC-QqQ-MS The extracts obtained were analysed with LC-QqQ-MS in order to determine the presence and evaluate the concentration of selected auxins, cytokinins and betaines. The selected auxins, cytokinins and betaines include: • Auxins o Indole-3-acetic acid o Indole-3-carboxylic acid o Indole-3-carboxaldehyde o N-(2-hydroxyethyl)phthalimide • Cytokinins o Zeatin • Betaines o Trimethylglycine Sample preparation for analysis was as followed: 50 mL of the kelp sample was freeze dried, and the resulting extract was dissolved in 10 mL of a methanol:water (50:50 v/v) solution. The methanol:water solution was prepared with mass spectrometry grade methanol and MiliQ water. The samples were filtered through a nylon filter cloth with an aperture size of 0.22 μm. The filtered samples were injected in the LC-QqQ-MS: 3 replicates were prepared for each sample, and each replicate was injected three times. The quantification was done using the multiple reaction monitoring (MRM) method. The obtained values are only “estimates”, since no internal standards were used (from the start of the sample preparation) to account for any loss of the analyte; and, no other selective/targeted sample preparation was applied (to selectively pre-concentrate each analyte). Matrix effect could have interfered in MS analyses, affecting subsequently the detected signals. However, comparison against commercially available kelp products will provide valuable information with regards to growth regulator concentration in the kelp extract samples. The kelp extracts analysed were compared against a commercially available kelp product. It was found that, for both biostimulants and amino acids, that the kelp extracts were comparable to the commercial product. The growth regulator content in the (a) commercial product, (b) kelp suspension containing alginates and (c) the acidic kelp liquid with alginates removed, is graphically represented in Figure 1. The commercial product has a slightly higher concentration of the growth regulators N-hydroxyethyl phthalimide and indole-3- carboxylic acid. However, the kelp samples extracted from Australian Bull kelp has a higher concentration of 1-aminocyclopropane-1-carboxylic acid, when compared against the commercial product. All three kelp samples analysed, showed similar levels of the growth regulators zeatin, indole-3-carboxyaldehyde and indole-3-acetic acid. Furthermore, salicylic acid was not detected in the commercial product, while high concentrations of salicylic acid was analysed for in the Australian Bull kelp extracted samples. Finally, betaine was only detected in the kelp suspension sample containing alginate. It is worth noting that the level of betaine in the alginate containing kelp suspension sample, was significantly high. Figure 2 graphically illustrates the various amino acids, and their relative concentrations, in the three kelp samples analysed. It could be deduced from the figure that the commercial kelp product has on average, a higher concentration of various amino acids when compared against kelp extracted from Australian bull kelp. However, glycine was not detected in the commercial kelp product sample, while the bull kelp extracted kelp products contained significantly high levels of glycine. The results indicated that the extraction methodology is sufficient to extract the targeted growth regulators and amino acid from the dried bull kelp hinge pieces. Furthermore, the processing conditions are mild enough, and does not result to significant decomposition of the auxins, cytokinins and amino acids. In addition, the amount of growth regulators and amino acids are comparable to the well-known, commercially available, kelp product. Microbiological Analysis of Kelp Extract The kelp extract, containing various preservatives, were analysed for both bacterial and fungal contamination. It is necessary to ensure that no microbial growth occurs during storage of the product, as this would result to product instability and finally, product decomposition. Kelp samples containing different preservative or a combination of various preservatives, at different pH values, were analysed for microbial contamination. The microbiological analysis of the kelp extract was done according to the following method and through using the following materials: • The following Agar plates were prepared according to manufacturer’s instructions: o Nutrient Agar (NA) o Salmonella Shigella Agar (SSA) o Malt Extract Agar (MEA): Both chloramphenicol and streptomycin were added to the agar solution at a concentration of 100 ppm and 300 ppm, respectively, for selective growth of fungal colonies. • Serial dilutions of up to 10-3 were prepared for each sample and three replicates (100 μl) of each dilution factor were plated. Three replicates (100 μl) of a neat sample were also plated. Thereafter, nutrient agar and salmonella shigella agar plates were incubated for 24 hours at 32 °C. Malt extract agar plates were incubated for 5 days at 28 °C. Colony counts from the inoculated and incubated plates were then recorded and averaged. The amount of bacterial and fungal growth is given as colony forming units per millilitre (CFU/ml). Table 26: Bacterial and Fungal growth of kelp samples containing different preservatives at various pH values

Table 26 summarises the bacterial and fungal growth. The preservative that showed the highest inhibition of bacterial growth was potassium sorbate, especially when the concentration of potassium sorbate was doubled. Furthermore, fungal growth was completely inhibited by potassium sorbate. Additionally, it was determined that boric acid was also an efficient preservative, as it significantly inhibited bacterial growth and completely inhibited fungal growth. Boric acid, however, did not inhibit bacterial growth to the same extent as did potassium sorbate. As was experimentally noted, sodium benzoate was insufficient to inhibit both bacterial and fungal growth. Mung Bean Essay Procedure A study was undertaken to evaluate the efficiency of extracted kelp from the proposed processes according to Example 9 (“G-kelp”) above compared to commercially available kelp (“K-kelp”). The main emphasis of this study was to prove that the Kelp extracted in this study, performs equally or better than the market competitor. The following sections provides results obtained from both the Mung Bean Bio Assay as well as a tomato pot trials where extracted kelp from the proposed processes is compared against the competitor K-Kelp. Table 27: Mung Bean Bio Essay protocol Table 27 provides a complete description of the Mung Bean Bio Assay as used to test the Kelp products. Figure 3 shows the calibration curve for lateral root formation (number) using different concentrations of IAA (auxin). The curve shows a linear correlation between auxin concentration and lateral root formation. Figure 4 shows the kelp treated Mung bean data obtained. A clear distinction is noticed between G- Kelp treated Mung beans and K-Kelp treated Mung beans. The G-Kelp treated Mung beans had improved root formation, compared against K-Kelp treated Mung beans. The G-kelp treated mung beans performed significantly better in terms of root stimulation, which points to a higher auxin activity. Although the total auxin analysis (concentration) in the G-Kelp and K-Kelp products were very similar, the activity of the G-Kelp seemed to be superior. Greenhouse Pot Trials Table 28 shows the protocol to evaluate extracted Kelp on tomato plants under stress conditions. Table 28: Pot trial protocol used to evaluate extracted Kelp product From the results in Figure 5, it can be determined that all kelp treatments had a significant effect on the Soil Plant Analysis Development (“SPAD”) readings (chlorophyll concentration) of the tomato plants. Under stress conditions (such as drought), plants produce oxidative components such as reactive oxygen species as well as hydrogen peroxide (H2O2). These species cause damage to plant materials such as chlorophyll, which is noted in the drought stress treatments in Figure 5. Soil Plant Analysis Development The unstressed plants had significantly higher chlorophyll concentration, compared to the stressed plants. In the kelp treated stressed plants, this damage was somewhat mitigated, resulting in statistically higher chlorophyll concentration in the kelp treated stressed plants, when compared to stressed plants that were not treated with the kelp products. No significant difference could be seen between the different kelp treatments. Dry Biomas The stress related damage manifested in lower biomass for the stressed plants (Figure 6). Root, shoot and total dry biomass was significantly lower in the stressed plants, relative to the control (unstressed) plants. The kelp treated stressed plants resulted in higher biomass compared to stressed untreated control plants. Although the total biomass between the kelp treatments did not differ significantly, the G-Kelp treatment had significantly higher root mass compared to the K-Kelp treatment. Stress Biomarkers Plants respond to environmental stress by producing large amounts of oxidative chemical species. Plants have evolved to reduce these species by making use of several plant enzymes and free radical scavengers such as amino acids. One of the first steps in reducing the oxidative species such as oxygen radicals (O2-), is it subsequent conversion to H2O2. This is done by the activity of a very specific family of enzymes called superoxide dismutase (“SOD”). H2O2 is less damaging than the radical species, but a build-up of H2O2 still causes damage to plant material. Therefore, H2O2 has to be converted into inert, non-toxic compounds by other plant enzymes. One of the enzymes responsible for this is ascorbate peroxidase (APX), which converts H2O2 to water (H2O). The activity of SOD and APX is thus crucial in protecting plants against stress. Unstressed plants will naturally have lower SOD and APX activity than stressed plants, because less radicals are produced. Kelp has been shown to further increase the activity of these enzyme, although the mechanism for upregulation remains unclear. Figure 7 depicts the leaf hydrogen peroxide concentration. The activity of the superoxide dismutase was also measured, and is graphically depicted in Figure 8. Additionally, the ascorbate peroxidase enzyme activity was also determined and is graphically depicted in Figure 9. It can be seen that in both types of kelp treatments, the SOD and APX activity of kelp treated stressed plants was increased, compared to the stressed control plants. Correspondingly, the kelp treatments also had lower H2O2 concentration compared to the untreated stressed plants. The unstressed plants had the lowest H2O2 concentration as well as the lowest SOD and APX activities. The SOD activity of the different kelp treatments was almost identical, but the G-Kelp had a significantly lower H2O2 content, compared to the K-Kelp. This is likely due to the fact that the APX activity in the G-Kelp treated plants were significantly higher than the K-Kelp treated plants. Because the SOD of the different kelp treatments did not differ significantly, it means that all the different kelp treated plants experienced an equivalent amount of stress (equal oxidative species forming). On the other hand, the lower H2O2 concentration in the G-Kelp plants, is indicative of the fact that the G-Kelp plants were coping better with the same amount of stress. REFERENCES 1. Waalaand, J., 1981. Commercial Utilization. In: The Biology of Seaweeds. Berkeley: University: California Press, pp.726-741. 2. McHugh, D., 2003. A guide to seaweed industry. FAO Fisheries Technical Paper, Volume 441, pp.1-105. 3. Khan, W., Menon, U., Subramanian, S., Jithesh, M., Rayorath, P., Hodges, D., Critchley, A., Craigie, J., Norrie, J., Prithiviraj, B., 2009. Seaweed Extracts as Biostimulants of Plant Growth and Development. Journal of Plant Growth Regulation, 28, pp.386-399. 4. Tiwari, B. T. D., 2015. Seaweed sustainability: Food and non-food applications. London: Elsevier. Misurcova, L., 2010. Chemical composition of seaweeds. In: Handbook of Marine Macroalgae: Biotechnology and Applied Phycology. s.l.:John Wiley and Sons, pp.171-192. 5. Dominguez-Briceno, D., Hernández-Carmona G., Moyo, M., Stirk, W., van Staden, J., 2014. Plant growth promoting activity of seaweed liquid extracts produced from Macrocystis pyrefera under different pH and temperature conditions. Journal of Applied Phycology, Volume 26, pp.2203-2210.

Claims

CLAIMS 1. A method for extraction of an extract comprising biostimulants and amino acids from dried brown macroalgae, the method comprising the following steps: a) adding water at a ratio of between 7 - 19 water to about 1 dry kelp (w/w), heated by a heating source to a temperature between 55 °C and 77 °C, to a first reaction vessel; b) adding the dried brown macroalgae pieces at a ratio of between 7 - 19 water to about 1 dry kelp (w/w), to the hot water and agitating the dried brown macroalgae in the water with an agitator for between 50 and 70 minutes; c) adding an organic acid at a ratio of at least 0.06 w/w to 1 dry kelp, to the first reaction vessel, whilst agitating for a period of time between 20 and 120 minutes, wherein the organic acid is added as crystals or as a solution; d) adding an alkali selected from the group consisting of sodium hydroxide or potassium hydroxide, or a mixture thereof, at an alkali to kelp ratio of 0.13- 0.36 (w/w), wherein the solution is a mixture of sodium hydroxide and potassium hydroxide containing at least 50 % w/w each, to the first reaction vessel while agitating between 30 minutes and 6 hours, until it yields a brown macroalgae extract having a pH of at least 6; and e) adding a preservative including a sodium benzoate, potassium sorbate or boric acid solution such that the ratio of the dry preservative to dry kelp is at least 0.1 (w/w) when the pH is below 13, to the brown macroalgae extract in the first reaction vessel while agitating for between 30 and 70 minutes, wherein where the pH is above 10. 2. The method according to claim 1, wherein the brown macroalgae is selected from the group consisting of Ecklonia maxima, Durvillaea potatorum, and combinations thereof. 3. The method according to claim 1 wherein the organic acid is selected from the group consisting of citric acid, acetic acid, and combinations thereof. 4. The method according to claim 4 wherein the citric acid or acetic acid, or citric acid and acetic acid in combination is a monohydrate crystal or a solution. 5. The method according to claim 1 wherein the alkali is added dry or as a solution. 6. The method according to claim 1 wherein the solution in step d) is agitated until yielding a brown macroalgae extract having a pH of at least 6. 7. The method according to claim 6 wherein the pH is at least 6.5. 8. The method according to claim 1 further comprising a step after step d), the step comprising subjecting the brown macroalgae extract solution to high shear mixing for between 60 and 120 minutes. 9. The method according to claim 1 further comprising a step after step d), the step comprising adding an ammonium phosphate solution, such that a ratio of solid mono ammonium phosphate to dry kelp of at least 0.05 w/w is obtained, to the first reaction vessel while agitating for between 30 and 70 minutes. 10. The method according to claim 1 further comprising a step after step d), the step comprising adding either a ferrous sulphate heptahydrate or copper sulphate heptahydrate solution while agitating, such that the ratio of metal sulphate to dry kelp is at least 0.03 (w/w), to the first reaction vessel. 11. The method according to claim 10 comprising a further step of adding a hydrogen peroxide solution to the reaction vessel such that the ratio of hydrogen peroxide to dry kelp pieces is at least 0.03 (w/w). 12. The method according to claim 11 comprising a further step of adding an ascorbic acid solution such that the ascorbic acid to dry kelp (w/w) ratio is at least 0.006 and continuing to agitate for between 5 and 20 minutes, to yield a brown macroalgae extract in the form of a suspension comprising biostimulants and amino acids. 13. The method according to claim 1 wherein the pH is below a value selected from 12, 11, or 10. 14. The method according to claim 1 wherein the pH is above a value selected from 12, 12, or 13. 15. The method according to claim 1 comprising a further step of filtering the brown macroalgae extract before collecting it. 16. The method according to claim 1 wherein the dried brown macroalgae comprises dried kelp hinge pieces. 17. The method according to claim 1 being a method for extraction of a clear extract comprising biostimulants and amino acids in which calcium alginate or alginic acids have been precipitated and removed by comprising a further step after step d) of adding a calcium nitrate (Ca(NO₃)₂) or a calcium chloride (CaCl2) solution where the pH is greater than 10, such that the ratio of dry calcium nitrate or chloride product to dry kelp is at least 0.49 (w/w), to the brown macroalgae extract in the first reaction vessel while agitating for between 10 and 30 minutes to precipitate out calcium alginate in the brown macroalgae extract. 18. The method according to claim 17 comprising a further step of filtering the brown macroalgae extract to separate a clear brown macroalgae liquid comprising biostimulants and amino acids from the precipitated calcium alginate. 19. The method according to claim 18 wherein the step of filtering comprises a pressure plate filtration or membrane squeezing, or pressure plate filtration and membrane squeezing in combination. 20. The method according to claim 17 comprising a further step of collecting the clear brown macroalgae liquid having a pH of greater than about 10 comprising biostimulants and amino acids. 21. The method according to claim 1 being a method for extraction of a clear extract comprising biostimulants and amino acids in which calcium alginate or alginic acids have been precipitated and removed by comprising a further step after step d) of adding an acid solution selected from the group consisting of acetic acid, citric acid, phosphoric acid, and combinations thereof, to the brown macroalgae extract in the first reaction vessel until an acidic pH is reached, at 25°C, while agitating, for between 30 and 100 minutes to precipitate out the alginates in the brown macroalgae extract as alginic acid. 22. The method according to claim 21 wherein the acid solution is a 85 wt% phosphoric acid solution. 23. The method according to claim 21 wherein the acidic pH is a value below 4, 3, 2 or 1. 24. The method according to claim 21 comprising a further step of filtering the brown macroalgae extract to separate a clear brown macroalgae liquid having an acid pH comprising biostimulants and amino acids from the precipitated alginates. 25. The method according to claim 24 wherein the acidic pH is a value below 4, 3, 2 or 1. 26. The method according to claim 18 wherein the filtering comprises a pressure plate filtration or membrane squeezing, or pressure plate filtration and membrane squeezing in combination. 27. The method according to claim 21 comprising a further step of collecting the clear brown macroalgae liquid comprising biostimulants and amino acids. 28. The method according to claim 1 wherein the temperature in first reaction vessel is maintained throughout the method at between 55 °C to about 77 °C. 29. The method according to claim 1 wherein the initial temperature of the water and brown macroalgae pieces in the first reaction vessel is heated to a temperature between 55 °C and 77 °C, after which the heating source is switched off for the remainder of the method. 30. A method according to any of the preceding claims whereby the brown macroalgae extract is formulated into a plant biostimulant composition or fertiliser. 31. The method according to claim 30 wherein the plant biostimulant composition is a liquid plant biostimulant composition or a granular plant biostimulant composition. 32. The method according to claim 30 wherein the fertiliser is a liquid plant fertiliser or a granular plant fertiliser. 33. The method according to claim 30 having a neutral pH value between 4 and 8. 34. The method according to claim 30 having an alkaline pH value of greater than 8. 35. The method according to claim 30 having an acidic pH value of below 4. 36. The method according to claims 33 or 34 wherein the brown macroalgae extract is a brown macroalgae suspension comprising calcium alginate or alginic acids. 37. The method according to claim 35 wherein the brown macroalgae extract is a clear liquid where calcium alginate or alginic acids have been precipitated out. 38. The method according to any one of claims 33, 34 or 35, wherein the brown macroalgae extract is derived from the brown macroalgae suspension comprising calcium alginate and alginic acids. 39. The method according to any one of claims 33, 34 or 35, wherein the brown macroalgae extract is a clear liquid where calcium alginate or alginic acids have been precipitated out. 40. A method according to any of the preceding steps for stimulating plant growth with the use of the brown macroalgae extract comprising biostimulants and amino acids.