A method of enhancing metabolite production

Abstract

A method for producing a cultured algal biomass comprising an antimethanogenic compound, wherein the method comprises the step of culturing the algal biomass: at a light intensity of from 1 µmols m-2.s-1 to 40 µmols m-2.s-1; in a culture media supplemented with bromide; and / or in a culture media supplemented with vanadium.

Description

A Method of Enhancing Metabolite ProductionTECHNICAL FIELD

[0001] The present disclosure relates to methods for increasing the anti-methanogenic metabolite content of cultured algal biomass using growth media additives and / or control of light exposure, and cultured algal biomass with an increased anti-methanogenic metabolite content produced by such methods.BACKGROUND ART

[0002] Livestock production, particularly ruminants, contributes to anthropogenic greenhouse gas (GHG) emissions globally. The majority of GHG emissions from livestock production are in the form of methane (CH4), which is produced largely through enteric fermentation, and to a lesser extent manure decomposition. Enteric CH4emissions not only contribute to total agricultural GHG emissions but also represent an energy loss amounting to 1 1% of dietary energy consumption. Therefore, reducing enteric CH4emissions decreases the total agricultural contribution to climate change and can improve productivity through conservation of feed energy.

[0003] Organic feed supplements derived from algae can be used modify the rumen environment in ruminant livestock and directly inhibit methanogenesis, resulting in lower enteric CH4production. For example, the red seaweeds Asparagopsis taxiformis (A. taxiformis) and Asparagopsis armata (A. armata) can be used modify the rumen environment, directly inhibiting methanogenesis and lowering enteric CH4production by up to 80%. As a natural defence mechanism, Asparagopsis spp. synthesize and store halogenated CH4analogues such as bromoform and dibromochloromethane within specialized gland cells.

[0004] Asparagopsis spp. have been found to reduce CH4more effectively compared to similar inclusions of pure bromoform in vitro, likely due to both the presence of multiple anti- methanogenic CH4analogues such as bromo- and iodo-methanes and -ethanes that work synergistically, and that the various methanogen species are sensitive to different CH4inhibitor metabolites.

[0005] However, the amount of anti-methanogenic metabolites in algae such as Asparagopsis spp., may be low and therefore large amounts of algae are required to generate the desired amounts of metabolites. This results in difficulties in algal growth (use of resources, waste disposal etc) and in animal administration (ruminants are known to reject feed containing high amounts of algae, possibly due to taste).

[0006] There is therefore a need to provide new methods to increase the anti-methanogenic metabolite content of algal biomass; or at least the provision of alternative methods to complimentthe previously known algal biomass culture and processing strategies. The present invention seeks to provide an improved or alternative method for culturing algal biomass to result in biomass with an anti-methanogenic metabolite content that is higher than that of wild-harvested algal biomass.

[0007] The previous discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.SUMMARY OF INVENTION

[0008] The present disclosure provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass at a light intensity of from 1 pmols nr2.s-1to 40 pmols nr2.s’1; b) culturing the algal biomass in culture media supplemented with bromide; and / or c) culturing the algal biomass in culture media supplemented with vanadium.

[0009] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising: a) culturing the algal biomass at a light intensity of from 1 pmols nr2.s-1to 40 pmols nr2.s’1; b) culturing the algal biomass in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media; and / or c) culturing the algal biomass in culture media supplemented with vanadium, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0010] The culturing in the presence of a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1, bromide and / or vanadium may occur for all of the culturing of the algal biomass, or may occur for some of the culturing of the algal biomass. For example, if the algal biomass is cultured for several days to allow the biomass concentration to rise to a level that allows commercially relevant amounts of biomass to be harvested, the culturing in the presence of a light intensity of from 1 pmols m-2.s_1to 40 pmols nr2.s-1, bromide and / or vanadium may occur for only a few hours of that total culturing period.

[0011] In one embodiment, the method for culturing an algal biomass or use of growing conditions to produce a cultured algal biomass results in a cultured algal biomass comprising more antimethanogenic metabolites than wild harvested algal biomass.

[0012] The bromide may be in the form of NaBr and / or KBr, and / or the vanadium may be in the form of sodium orthovanadate (Na3VO4). The bromide may be at a concentration in the culture media of from 0.1 mg / L to 80 g / L bromide. Optionally, the bromide may be provided in the form of NaBr and / or KBr, for example at from 0.01 g / L to 100 g / L NaBr and / or KBr in the culture media. The vanadium may be at a concentration in the culture media of from 0.05 pg / L to 70 mg / L vanadium. Optionally, the vanadium may be provided in the form of sodium orthovanadate, for example from 0.01 pg / L to 200 mg / L sodium orthovanadate in the culture media.

[0013] The present disclosure provides a cultured algal biomass comprising anti-methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g dry weight. The present disclosure provides cultured algal biomass comprising bromoform at from 15, 20, 40, 50, 60 or 70 mg / g dry weight The cultured algal biomass may be produced by the methods or using the culture conditions provided above.

[0014] The present disclosure provides cultured algal biomass comprising anti-methanogenic metabolites at from 1 .5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti-methanogenic metabolites as wild harvested algal biomass. The present disclosure provides cultured algal biomass comprising bromoform at from 1 .5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti-methanogenic metabolites as wild harvested algal biomass. The cultured algal biomass may be produced by the methods or using the culture conditions provided above.

[0015] Optionally, the cultured algal biomass is in the form of cultured algal biomass fragments (aggregates of cells) with an average diameter of from 1 mm to 20 mm. The cultured algal biomass may be produced by the methods or using the culture conditions provided above.

[0016] Optionally, the cultured algal biomass is a cultured Asparagopsis biomass.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:Figure 1 is a diagram of an example photobioreactor (PBR) cell that may be used to grow a cultured algal biomass in accordance with the present disclosure.Figure 2 is a diagram of an example PBR cell (left) and PBR cells connected in series (right) that may be used to grow a cultured algal biomass in accordance with the present disclosure.DESCRIPTION OF INVENTIONDetailed Description of the Invention

[0018] It is desirable to have a high content of anti-methanogenic metabolites in algae biomass, to reduce the amount of algal biomass that need be provided to ruminants to have an anti- methanogenic effect, and to reduce the cost and environmental footprint of culturing algal biomass to provide suitable quantities of anti-methanogenic metabolites.

[0019] Exemplary algal biomasses with measured anti-methanogenic metabolites in the form of bromoform include wild-harvested Asparagopsis biomass having 7.7 mg / g dry weight bromoform (disclosed in WO2023 / 150832), and cultured Asparagopsis biomass having 13.56 mg / g dry weight bromoform (disclosed in WO2023 / 215946).High Anti-methanogenic Metabolite Cultured Algal Biomass

[0020] The present disclosure provides cultured algal biomass comprising anti-methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g dry weight. For example, the present disclosure provides cultured algal biomass comprising bromoform at from 15, 20, 40, 50, 60 or 70 mg / g dry weight.

[0021] In one aspect, the present disclosure provides cultured algal biomass comprising anti- methanogenic metabolites at from 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti-methanogenic metabolites as wild harvested algal biomass. For example, the present invention provides cultured algal biomass comprising bromoform at from 1 .5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much bromoform as wild harvested algal biomass.

[0022] Optionally, the algal biomass is cultured algal biomass that has been harvested, cleaned and / or partially dewatered to remove seawater.Algal Biomass

[0023] The cultured algal biomass may be in the form of tetrasporophytes or gametophytes.

[0024] Optionally, the cultured algal biomass is in the form of cultured algal biomass fragments (aggregates of cells) with an average diameter of from 1 mm to 20 mm. For example, the average diameter of the cultured algal biomass fragments may be from 5 mm to 10 mm. Cultured algal biomass fragments may be grown from tetrasporophytes which have been macerated into small fragments and then allowed to regrow into a suitable size in a photobioreactor (PBR) system. Alternatively, the cultured algal biomass fragments may be gametophytes of a suitable size grown in a PBR system.Measuring Anti-methanogenic Metabolite Content

[0025] The anti-methanogenic metabolite content of the cultured algal biomass may be measured by gas chromatography-mass spectrometry (GC-MS) or by high performance liquid chromatography (HPLC), for example by the methods of Romanazzi et al. (2021) ACS Agric Sci Technol 1 :436-442 or Paul et al. (2006) Mar Ecol Prog Ser 306:87-101. In one aspect, the anti- methanogenic metabolite content in the form of the bromoform content of the cultured algal biomass may be measured.Algae

[0026] In the present disclosure, the terms “seaweed” and “algae” are used interchangeably. Algae are simple, non-flowering, and typically eukaryotic photosynthetic aguatic organisms. Algae contain chlorophyll but lack true stems, roots, leaves, and vascular tissue. The algae may be a macroalgae or a microalgae.

[0027] A microalgae is unicellular algae throughout its lifecycle while a macroalgae has at least one multicellular stage during its life cycle When in association with an agueous culture medium, the algae of the present disclosure may be referred to herein as “algal cells”. When not in association with an agueous culture medium, the algae of the present disclosure may be referred to herein as “algal biomass”.

[0028] Algae suitable for use in the present disclosure are preferably macroalgae. These macroalgae include green, brown and red algae. Brown and red algae are preferred because they typically reguire weaker light intensity than green algae to grow, which may reduce the electrical cost for onshore farming using artificial lighting. Optionally, the macroalgae is red algae. Red algae is preferred because it tends to produce extracellular material, including cell-wall polysaccharides, which may result in an improved ruminant feed.

[0029] Algal cells in agueous culture medium may form multi-cellular clusters, especially under growth conditions. These clusters may be referred to as “biomass fragments” or simply “fragments”. Fragments may range in size from a diameter of less than 2 mm to greater than 8 mm. A biomass fragment having a diameter of less than 2 mm may be referred to as “very small”, a biomass fragment having a diameter of from 2 mm to 4 mm may be referred to as “small”, a biomass fragment having a diameter of from 4 mm to 6 mm may be referred to as “medium”, while a biomass fragment having a diameter of greater than 6 mm may be referred to as “large”. The term “diameter” in the context of biomass fragments does not limit the shape of a fragment and refers to the greatest axial dimension. Biomass fragments may form during the gametophyte or sporophyte phase. The sporophyte of an algae may be, and preferably is, a tetrasporophyte.

[0030] Optionally, the alga is of the class Florideophyceae. Florideophyceae are multicellular red algae which form biomass fragments. Optionally, the alga is of the order Bonnemaisoniales in theclass Florideophyceae. Bonnemaisonialea form biomass fragments in the sporophyte phase including as tetrasporophytes. Optionally, the alga is an Asparagopsis spp. as described below. The Asparagopsis spp. may be, and is preferably, Asparagopsis taxiformis (A. taxiformis) and / or Asparagopsis armata (A. armata). Asparagopsis spp. are macroalgae, although the algae may be in the form of very small (microscopic) fragments, for example after maceration prior to inoculation or during early growth. The macroalgal Asparagopsis fragments may initially be as small as one or a few cells and may be microscopic; however, the cells will undergo substantial cell divisional and form clearly visible macroalgal fragments after a day or more growth.Asparagopsis

[0031] A. taxiformis and A. armata have a gametophyte and tetrasporophyte life-stages when maintained in culture. Tetrasporophytes can produce tetraspores via asexual reproduction (meiosis). Tetraspores released into the water column can settle on substratum and develop into gametophytes.

[0032] Both Asparagopsis species’ gametophyte and tetrasporophyte life-stages are sources of halogenated compounds, with important antifungal and antibiotic activity. The tetrasporophyte stage tends to have more halogenated compounds per unit biomass than the gametophyte stage due to less structural biomass.

[0033] The commercial demand for these two species is due not only to their inherent ability to produce biologically active metabolites (e.g. bromoform as well as small quantities of other bromine, chlorine and iodine-containing methanes, ethanes, ethanols, acetaldehydes, acetones, 2-acetoxypropanes, propens, epoxypropanes, acroleins and butenones), but also to partition and store these compounds in specialized storage or gland cells to prevent autotoxicity. In addition to producing powerful anti-methanogenic uses, Asparagopsis may represent a significant source of other bioactive compounds responsible for antioxidant and cytotoxic activity in pharmaceutical and veterinary settings.

[0034] The present disclosure provides the ability to commercially increase the anti- methanogenic metabolite content of algae, such as A. taxiformis and / or A. armata, by subjecting the algal biomass to suitable culture conditions as described herein. The disclosure further provides access to organically produced metabolites, such as cell-wall polysaccharides, which may result in an improved ruminant feed.Culture Conditions

[0035] The cultured algal biomass of the present disclosure may be grown or cultured in a photobioreactor (PBR) system. As example of a suitable PBR is provided in PCT / AU2023 / 051035.

[0036] Optionally, the PBR system may have H-connectors covered with a lid equipped with a filter to prevent or reduce contamination during gas exchange and / or an inlet for supplying sterile medium by pipetting to prevent or reduce contamination during medium supplementation. A cultured biomass without contamination or with reduced contamination may contribute to increased production and / or accumulation of bromoform.

[0037] Optionally, the cultured algal biomass is grown in aqueous culture media at a temperature of from 12 °C to 28 °C.

[0038] Generally, the water flow rate or velocity is determined and controlled by the aeration flow rate - the movement of the air bubbles coincidentally moves the surrounding water. However, if desired a separate water pump may be attached to an algal culture PBR system to assist in water flow control. Optionally, the water velocity is from 0.1 m / s to 0.7 m / s.

[0039] Algae can be grown on different forms of nitrogen (including NO" and NH+as well as organic forms of nitrogen) and phosphate (including inorganic and organic forms of phosphorous), trace metals (including copper, zinc, manganese, molybdenum, iron, and cobalt), and vitamins (including vitamin B12, biotin, and thiamin).

[0040] Suitable nutrient media are known to the skilled reader for algal growth. These include commercially available nutrient media (including Cell-Hi™ algal nutrient medium); nutrient media developed from seawater nutrient media recipes (including F / 2 media, L1 media, Provasoli Enriched Seawater, Enriched Seawater, Erdschreiber's Medium); or organic forms of nutrient media (fish farm effluent, sewerage effluent, animal effluent).

[0041] Optionally, the pH of the PBR system in which the cultured algal biomass is grown is maintained at from pH 7 to pH 9. For example, the pH may be maintained at from pH 7.5 to pH 9.0, or at about pH 8.1 to 8.8, being the average pH of seawater.Light Source

[0042] Without being held to any theory, a low light intensity during algal biomass growth may increase the anti-methanogenic metabolite content, for example the bromoform content, of the cultured algal biomass by promoting, accelerating or enhancing anti-methanogenic metabolite biosynthesis; by increasing accumulation of anti-methanogenic metabolites; and / or by stopping, suspending or reducing degradation or excretion of anti-methanogenic metabolites. Furthermore, the low light intensity assists in preventing or reducing the growth of other microorganisms, both algae other than the desired species of algae of the present disclosure, or bacterial and fungal contaminants.

[0043] Generally, the light intensity algae is exposed to during normal culture is between about 40 and 1 ,500 pmols of photons nr2s-1(for example, in a PBR the light intensity is about 40 pmolsm-2s-1to 200 pmols m-2s-1, in an outdoor pond or raceway the light intensity is about 100 pmols m-2s-1to 1 ,500 pmols m-2s-1).

[0044] During growth in culture, the cultured algal biomass of the present disclosure is optionally exposed to a light intensity of from 1 pmols m-2.s_1to 40 pmols nr2.s-1, or 50 pmols m-2.s_1to 20 pmols nr2.s’1. The cultured algal biomass of the present disclosure may be grown under a light intensity of from 1 pmols m-2.s_1to 40 pmols nr2.s-1for at least 1 day of algal growth. For example, the algal biomass may be cultured for at least 2 day, 3 days, 4 days, 5 days, 5 days, 6 days, 7 days or more under a light intensity of from 1 pmols m-2.s_1to 40 pmols nr2.s’1.

[0045] The decreased light intensity may be provided for only some of the days of cultivation of the algal biomass. For example, the algal biomass may be cultured under normal light intensity (generally from 40 pmols m-2.s_1to 1 ,500 pmols m-2.s_1) for some days of the culture period, and may be grown under the decreased light intensity for other days of the culture period. The decreased light intensity may be administered if contamination is detected in the culture, and maintained until the contamination is reduced. Alternatively, the decreased light intensity may be administered nearthe end of the culture period, for a few days (eg from 1 to 7 days) before harvest to increase the anti-methanogenic metabolite content of the algal biomass prior to harvest.

[0046] The light source may be natural light or may be provided by man-made electrical lighting. If is used, the electrical lighting may provide wavelengths of from 400 nm to 700 nm, to maximise algal growth.

[0047] The length of the day (photoperiod) is a robust seasonal signal that eukaryotic algae can use to initiate or complete different developmental programs. Multicellular algae such as the macroalgal red algae (Rhodophyta) have a triphasic (gametophyte, carposporophyte, and tetrasporophyte) life cycle.

[0048] During growth in culture, the cultured algal biomass of the present disclosure may be exposed to a photoperiod of from 8:16 to 14:10 light:dark cycle, more preferable a 10:14 light:dark cycle. This photoperiod maximises the tetrasporophyte stage of the algal life cycle, more for example maximising the tetrasporophyte stage of Asparagopsis’ life cycle.

[0049] The efficient growth of cultured biomass under controlled conditions may contribute to increased production and / or accumulation of anti-methanogenic metabolites, including bromoform.Media Supplementation

[0050] Nutrient media, such as commercially available nutrient media; nutrient media developed from seawater nutrient media recipes; or organic forms of nutrient media may be supplemented to promote bromoform production. For example, nutrient media may besupplemented with bromide and / or vanadium to promote anti-methanogenic metabolite, for example bromoform, production.

[0051] During growth in culture, the cultured algal biomass of the present disclosure is optionally exposed to bromide and / or vanadium. The cultured algal biomass of the present disclosure may be grown in the presence of bromide and / or vanadium for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 hours, or during 1 day of algal growth. For example, the algal biomass may be cultured for at least 2 day, 3 days, 4 days, 5 days, 5 days, 6 days, 7 days or more in the presence of bromide and / or vanadium.Bromide

[0052] Culture media supplemented with bromide may contribute to an increase in the anti- methanogenic metabolite content in the algal biomass by promoting, accelerating or enhancing anti-methanogenic metabolite biosynthesis; by increasing accumulation of anti-methanogenic metabolites; and / or by stopping, suspending or reducing degradation or excretion of anti- methanogenic metabolites. In one aspect, culture media supplemented with bromide may contribute to an increase in the bromoform content in the algal biomass by promoting, accelerating or enhancing bromoform biosynthesis; by increasing accumulation of bromoform; and / or by stopping, suspending or reducing degradation or excretion of bromoform.

[0053] The culture media used in the PBR system growing the cultured algal biomass of the present disclosure may optionally be supplemented with bromide, optionally in the form of bromide salts and / or halogenated organic compounds. If the bromide is in the form of a bromide salt, the bromide salts may be chosen from NaBr, KBr, LiBr, NH4Br, MgBr2, FeBr2, CaBr2, or MnBr2. Alternatively, if the bromide is in the form of a halogenated organic compound, the halogenated organic compound may be chosen from dibromoacetic acid; bromochloroacetic acid; or bromine-containing methanes, ethanes, ethanols, acetaldehydes, acetones, 2- acetoxypropanes, propens, epoxypropanes, acroleins and butenones. In one aspect, the bromide used to supplement the culture media is in the form of NaBr and / or KBr.

[0054] Exemplary supplementation ranges include bromide, in the form of a salt or a halogenated organic compound, at from 0.01 g / L to 100 g / L bromide in the culture media. Optionally, the bromide is provided in the culture media at from 0.01 g / L to 100 g / L, 0.05 g / L to 50 g / L, 0.1 g / L to 10 g / L, 0.1 g / L to 80 g / L. Optionally, the bromide is at from 0.1 g / L to 80 g / L in the culture media.

[0055] If the bromide is in the form of NaBr and / or KBr, exemplary supplementation ranges include NaBr and / or KBr at from 0.01 g / L to 100 g / L, 0.05 g / L to 50 g / L, 0.1 g / L to 10 g / L, 0.01 g / L to 80 g / L, in the culture media. Optionally, the NaBr and / or KBr is at from 0.1 g / L to 80 g / L in the culture media.Vanadium

[0056] Culture media supplemented with vanadium may contribute to an increase in the anti- methanogenic metabolite content in the algal biomass by promoting, accelerating or enhancing anti-methanogenic metabolite biosynthesis; by increasing accumulation of anti-methanogenic metabolites; and / or by stopping, suspending or reducing degradation or excretion of anti- methanogenic metabolites. In one aspect, culture media supplemented with vanadium may contribute to an increase in the bromoform content in the algal biomass by promoting, accelerating or enhancing bromoform biosynthesis; by increasing accumulation of bromoform; and / or by stopping, suspending or reducing degradation or excretion of bromoform. Vanadium plays a role in enhancing anti-methanogenic metabolite production, including bromoform production, through multiple biochemical mechanisms.

[0057] One of vanadium’s primary functions is to boost the activity of vanadium-dependent bromoperoxidase (V-BPO) enzymes, which are essential in catalysing the oxidation of bromide ions (Br) by hydrogen peroxide (H2O2) to produce bromoform and other halogenated organic compounds. This enzymatic pathway is central to Asparagopsis' ability to generate bromoform, a compound known for its potential in reducing methane emissions when used as a cattle feed supplement. Providing a bioavailable source of vanadium may enhance the efficiency and stability of V-BPO enzymes, allowing for more consistent and increased production of bromoform under optimal conditions.

[0058] Beyond its role as a cofactor supplier, vanadium functions as a potent phosphatase inhibitor, preventing the dephosphorylation of key regulatory proteins involved in cellular signalling. This inhibition maintains the activation of metabolic pathways responsible for halogenated compound biosynthesis, including halogenated compounds that function as anti- methanogenic metabolites. Phosphatases typically regulate cellular processes by removing phosphate groups from proteins, which can deactivate enzymes or signalling molecules. By blocking these enzymes, vanadium prolongs the active state of pathways associated with secondary metabolite production, thereby promoting sustained anti-methanogenic metabolite synthesis. This regulatory effect may also reduce cellular feedback inhibition, ensuring continuous anti-methanogenic metabolite biosynthesis even under variable environmental conditions. In one aspect of the invention, the phosphatase inhibition is not complete phosphatase inhibition. It is preferred that the phosphatase inhibition is partial inhibition, sufficient to cause stress to the algae and thus promote anti-methanogenic metabolite production.

[0059] Another effect of vanadium is its ability to induce mild oxidative stress within Asparagopsis cells. This stress response can trigger the production of secondary metabolites, including anti-methanogenic metabolites, as a part of the alga’s natural defence mechanism against environmental stressors. Oxidative stress results from an imbalance between reactiveoxygen species (ROS) generation and the cell’s antioxidant defences. By increasing ROS levels in a controlled manner, vanadium can stimulate the production of halogenated compounds, including anti-methanogenic metabolites, without causing cytotoxic damage. This stress-induced metabolic shift aligns with the organism’s evolutionary adaptation to defend against herbivory and microbial invasion.

[0060] The concentration and duration of vanadium exposure are parameters that can influence the trade-off between biomass accumulation and anti-methanogenic metabolite yield. At lower concentrations, vanadium may stimulate enzyme activity without severely affecting growth rates, whereas higher concentrations could prioritise anti-methanogenic metabolite production at the expense of biomass expansion, due to the metabolic burden imposed by prolonged oxidative stress and phosphatase inhibition. This dose-dependent effect can be integrated into cultivation systems to enhance the yield of bromoform-rich Asparagopsis.

[0061] Vanadium exists in multiple oxidation states. Optionally, the vanadium of the present invention is provided in the V5+oxidation state. Without being held to this theory, it is thought that the V5+oxidation state is the most biologically active in phosphatase inhibition. However, vanadium compounds with other vanadium oxidation states may be used to supplement the nutrient media the algae is cultured in.

[0062] The vanadium my be in the form of sodium orthovanadate (Na3VO4; V5+), sodium metavanadate (NaVO3; V5+), ammonium metavanadate (NH4VO3; V5+), vanadyl sulfate (VOSO4; V4+), vanadium pentoxide (V2OS; V5+); vanadium complexes (e.g., Schiff base vanadium complexes, peroxovanadates). Optionally, the vanadium of the present invention is provided in the form of sodium orthovanadate, sodium metavanadate, vanadium pentoxide, and / or ammonium metavanadate. For example, the vanadium of the present invention may be provided in the form of sodium orthovanadate.

[0063] Exemplary supplementation ranges include vanadium at from 0.01 p / L to 200 mg / L vanadium in the culture media. Optionally, the vanadium is provided in the culture media at from 0.01 pg / L to 70 mg / L, 0.01 pg / L to 10 mg / L, ,0.01 p / L to 1 mg / L, ,1 p / L to 1 mg / L.

[0064] If the vanadium is in the form of sodium orthovanadate, supplementation ranges include sodium orthovanadate at from 0.01 p / L to 200 mg / L, 0.01 pg / L to 70 mg / L, 0.01 pg / L to 10 mg / L, ,0.01 p / L to 1 mg / L,1 p / L to 1 mg / L.

[0065] In one aspect of the invention, the algal biomass is grown in a culture media supplemented with bromide salts, optionally in the form of NaBr and / or KBr, optionally at from 0.01 g / L to 100 g / L in the culture media, and vanadium salts, optionally in the form of sodium orthovanadate (Na3VO4), optionally at from 0.01 p / L to 200 mg / L in the culture media.

[0066] In one aspect of the invention, the algal biomass is grown under a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1for at least one day, and in a culture media supplemented with bromide salts, optionally in the form of NaBr and / or KBr, optionally at from 0.01 g / L to 100 g / L in the culture media. For example, the algal biomass may be grown under a light intensity of from 5 pmols m-2.s_1to 20 pmols m-2.s_1for at least one day, and in a culture media supplemented with bromide salts, optionally in the form of NaBr and / or KBr, optionally at from 0.1 g / L to 10 g / L bromide in the culture media.

[0067] In one aspect of the invention, the algal biomass is grown under a light intensity of from 1 pmols rrr2. s-1to 40 pmols m-2.s_1for at least one day, and in a culture media supplemented with vanadium salts, optionally in the form of sodium orthovanadate (Na3VO4), optionally at from from 0.01 p / L to 200 mg / L vanadium in the culture media. For example, the algal biomass may be grown under a light intensity of from 5 pmols m-2.s_1to 20 pmols m-2.s_1for at least one day, and in a culture media supplemented with vanadium salts, optionally in the form of sodium orthovanadate (Na3VO4), optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0068] In one aspect of the invention, the algal biomass is grown under a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1for at least one day, and in a culture media supplemented with bromide salts, optionally in the form of NaBr and / or KBr, optionally at from 0.01 g / L to 100 g / L bromide in the culture media, and vanadium salts, optionally in the form of sodium orthovanadate (Na3VO4), optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media. For example, the algal biomass may be grown under a light intensity of from 5 pmols m-2.s_1to 20 pmols m-2.s_1for at least one day, and in a culture media supplemented with bromide salts, optionally in the form of NaBr and / or KBr, optionally at from 0.1 g / L to 10 g / L bromide in the culture media and vanadium salts, optionally in the form of sodium orthovanadate (Na3VO4), optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.Metabolites or bioactive compounds

[0069] Optionally, the cultured algal biomass, such as cultured Asparagopsis biomass, contains one or more metabolites or bioactive compounds chosen from the following: phytosterols; tocopherols; carotenoids; bromoform; dibromoacetic acid; bromochloroacetic acid; bromine-, chlorine- and iodine-containing methanes, ethanes, ethanols, acetaldehydes, acetones, 2- acetoxypropanes, propens, epoxypropanes, acroleins and butenones. The metabolites or bioactive compounds includes anti-methanogenic metabolites. The anti-methanogenic metabolites bromoform and dibromoacetic acid have been identified as dominant compounds in A. armata and A. taxiformis.

[0070] Optionally, the cultured algal biomass retains most or all of one or more of the metabolites or bioactive compounds present in wild-harvested algal biomass. Thus, the cultured algal biomassmay contain the same amount or nearly the same amount of one or more of the metabolites or bioactive compounds that are present in wild-harvested algal biomass, and / or may contain the same or similar profile of metabolites or bioactive compounds as wild-harvested algal biomass even if the amounts of each compound or metabolite are reduced. For example, the cultured algal biomass may contain the same amount or nearly the same amount of one or more lipophilic metabolites or bioactive compounds that were present in the wild-harvested algal biomass, and / or may contain the same or similar profile of lipophilic metabolites or bioactive compounds as the wild-harvested algal biomass even if the amounts of each lipophilic compound or metabolite are reduced.

[0071] Optionally, the cultured algal biomass comprises more anti-methanogenic metabolites, including bromoform, than wild harvested algal biomass. The present disclosure provides a cultured algal biomass comprising anti-methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g dry weight. The present disclosure provides cultured algal biomass comprising anti- methanogenic metabolites at from 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti-methanogenic metabolites as wild harvested algal biomass. The present disclosure further provides cultured algal biomass comprising bromoform at from 15, 20, 40, 50, 60 or 70 mg / g dry weight. The present disclosure provides cultured algal biomass comprising bromoform at from 1 .5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti- methanogenic metabolites as wild harvested algal biomass.

[0072] Optionally, the cultured algal biomass is in the form of cultured algal biomass fragments (aggregates of cells) with an average diameter of from 1 mm to 20 mm.Cleaning Algal Biomass

[0073] The cultured algal biomass may be cleaned after harvesting from the PBR, but before other processing steps are carried out. The algal biomass may be washed in seawater and / or fresh water (for example deionised water) to remove contaminants such as salt, solid grit and sand, and biomass from other, undesired species of seaweed and / or bacteria and fungi. The cleaning may be carried out in steps, with some washes being conducted using seawater and some washes being conducted using fresh water. For example, the cultured algal biomass may be cleaned by washing in seawater at the point of collection, the washed one or more times in a separate processing location using fresh water.

[0074] It is known that exposure to temperatures above 20 °C elevates production of reactive oxygen species in algal cells, increasing the release of bromoform. Bromoform itself is also a volatile compound (vapor pressure of 5.40 mm Hg at 25°C) and is not found in liguid form outside the cell at room temperature. The cleaning of the cultured algal biomass may therefore be carriedout at room temperate, or a temperature lower than room temperature. For example, the cleaning may be carried out at 25 °C, 22 °C, 20 °C, 18 °C, 16 °C, 14 °C, 12 °C, 10 °C, 8 °C, 7 °C, 6 °C, 5 °C, 4 °C, 3 °C, 2 °C, or 1 °C. In one aspect, the cleaning is carried out at between 20 °C and 8 °C, or between 18 °C and 10 °C, for example about 18 °C, 15 °C and 10 °C.Methods

[0075] The present disclosure provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass at a light intensity of from 1 pmols nr2.s-1to 40 pmols nr2.s’1.

[0076] The present disclosure further provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media.

[0077] The present disclosure further provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass in culture media supplemented with vanadium salts, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0078] The present disclosure provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass at a light intensity of from 1 pmols nr2.s-1to 40 pmols nr2.s’1; b) culturing the algal biomass in culture media supplemented with bromide; and / or c) culturing the algal biomass in culture media supplemented with vanadium.

[0079] The present disclosure provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass at a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1and in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media.

[0080] The present disclosure provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass at a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1and in culture media supplemented with vanadium, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0081] The present disclosure further provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media, and vanadium, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0082] The present disclosure provides a method for culturing an algal biomass to produce a cultured algal biomass, wherein the method comprises the step of: a) culturing the algal biomass at a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1and in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L in the culture media, and vanadium, optionally at from 0.01 p / L to 200 mg / L in the culture media.

[0083] In the methods above, if the bromide is in the form of NaBr and / or KBr, the NaBr and / or KBr may be at a concentration in the culture media of from 0.01 g / L to 100 g / L NaBr and / or KBr in the culture media.

[0084] In the methods above, if the vanadium is in the form of sodium orthovanadate, the sodium orthovanadate may be at a concentration in the culture media of from 0.01 p / L to 200 mg / L sodium orthovanadate in the culture media.

[0085] The methods of the present disclosure may produce a cultured algal biomass comprising anti-methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g dry weight. The methods of the present disclosure may produce a cultured algal biomass comprising anti-methanogenic metabolites at from 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti-methanogenic metabolites as wild harvested algal biomass.

[0086] The methods of the present disclosure may produce a cultured algal biomass comprising bromoform at from 15, 20, 40, 50, 60 or 70 mg / g dry weight. The methods of the present disclosure may produce a cultured algal biomass comprising bromoform at from 1 .5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti-methanogenic metabolites as wild harvested algal biomass.

[0087] The methods of the present disclosure may produce a cultured algal biomass in the form of cultured algal biomass fragments (aggregates of cells) with an average diameter of from 1 mm to 20 mm.Use of Growing Conditions

[0088] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising: a) culturing the algal biomass at a light intensity of from 1 pmols nr2.s-1to 40 pmols nr2.s’1.

[0089] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising: a) culturing the algal biomass in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media.

[0090] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising: a) culturing the algal biomass in culture media supplemented with vanadium, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0091] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising: a) culturing the algal biomass at a light intensity of from 1 pmols rrr2.s-1to 40 pmols m-2.s_1; b) culturing the algal biomass in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media; and / or c) culturing the algal biomass in culture media supplemented with vanadium, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0092] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising: a) culturing the algal biomass at a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1and in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media.

[0093] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising: a) culturing the algal biomass at a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1and in culture media supplemented with vanadium, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0094] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising:a) culturing the algal biomass in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media, and vanadium, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0095] The present disclosure provides for the use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising: a) culturing the algal biomass at a light intensity of from 1 pmols m-2.s_1to 40 pmols m-2.s_1and in culture media supplemented with bromide, optionally at from 0.01 g / L to 100 g / L bromide in the culture media, and vanadium, optionally at from 0.01 p / L to 200 mg / L vanadium in the culture media.

[0096] In the growing conditions above, if the bromide is in the form of NaBr and / or KBr, the NaBr and / or KBr may be at a concentration in the culture media of from 0.01 g / L to 100 g / L NaBr and / or KBr in the culture media.

[0097] In the growing conditions above, if the vanadium is in the form of sodium orthovanadate, the sodium orthovanadate may be at a concentration in the culture media of from 0.01 p / L to 200 mg / L sodium orthovanadate in the culture media.

[0098] The growing conditions of the present disclosure may produce a cultured algal biomass comprising anti-methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g dry weight.

[0099] The growing conditions of the present disclosure may produce a cultured algal biomass comprising anti-methanogenic metabolites at from 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much antimethanogenic metabolites as wild harvested algal biomass.

[0100] The growing conditions of the present disclosure may produce a cultured algal biomass comprising bromoform at from 15, 20, 40, 50, 60 or 70 mg / g dry weight.

[0101] The growing conditions of the present disclosure may produce a cultured algal biomass comprising bromoform at from 1 .5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti-methanogenic metabolites as wild harvested algal biomass.

[0102] The growing conditions of the present disclosure may produce a cultured algal biomass in the form of cultured algal biomass fragments (aggregates of cells) with an average diameter of from 1 mm to 20 mm.General

[0103] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0104] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0105] Any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0106] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0107] The invention described herein may include one or more range of values (eg. Size, displacement and field strength etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Hence “about 80 %” means “about 80 %” and also “80 %”. At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0108] Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited,but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0109] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. The term “active agent” may mean one active agent, or may encompass two or more active agents.

[0110] The following examples serve to more fully describe the manner of using the abovedescribed invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these methods in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.EXAMPLES

[0111] Further features of the present invention are more fully described in the following nonlimiting Examples. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad description of the invention as set out above.Example 1Development of PBR for Algal GrowthMaterials and methodsSeaweed fragmentation and PBR inoculation

[0112] Asparagopsis taxiformis seaweed juveniles used for inoculating the PBR were clonally produced from the fragmentation of mature tetrasporophytes. Fragmentation was achieved by macerating tetrasporophytes within a 50ml beaker half-filled with seawater. Maceration was achieved by using a device with a fast-rotating cutter which chopped the tetrasporophytes into thousands of fragments within 20-30 seconds. The fragments range in size from 0.3-1 ,5mm in length, with each fragment having the potential to grow into new large tetrasporophytes. The PBR was inoculated with fragments on two separate but linked trials:Inoculation 1: Fragmentation of sixty mature tetrasporophytes, between 6-8 mm in diameter and weighing between 8-10 mg each, with a total biomass of 0.5 g fresh weight. Production time for this inoculation biomass was 19 days PBR operation without a water change.Inoculation 2: Tetrasporophyte biomass, produced after 19 days of operation from ‘Inoculation T, was then re-fragmented. The fragments were then used to inoculate the PBR for 23 days of continuous PBR operation without a water change.Photobioreactor design, set-up and operational parameters

[0113] PBR conditions were set up as outlined in Table 1 and described below.1 . The Series 160L multi-tubular airlift photobioreactor was set up on appropriate racking indoors to control environmental conditions and prevent contamination (Figure 1 and 2).2. Twenty 90 mm acrylic tubes of 1000 mm length were connected in series using PVC fittings (90 mm T-sections and 90-degree elbows) to form a closed-loop recirculating system. The PBR is made up of 10 tubes on the front and 10 tubes on the rear, connected at the base by two 90-degree elbows (to form a U-bend) and connected at the top with two T-sections to form a H-section where the tops were open to allow gas exchange.Every second acrylic tube was aerated at its base using compressed air fed through an air stone (upwelling tube) whilst every alternate acrylic tube acted as a downwelling tube. The PBR system was filled with filtered seawater (160 litres) that spanned 20 metres of acrylic tubes. Four 400-Watt metal halide lights (cool white light) were mounted on the racking system to illuminate the PBR. Without shading, the lights provide 400 pmolsof photons m-2.s_1of light intensity measured as photosynthetically active radiation (PAR) to the front tubes, and 350 pmols of photons nr2.s-1of light intensity measured as PAR to the rear tubes. For the experimental trials, we applied shading in the form of 70% shade cloth to achieve 80 pmols of photons m-2.s_1of light intensity measured as PAR to the front tubes, and 70 pmols of photons m-2.s'1of light intensity measured as PAR to the rear tubes. There was a spread of light intensity across the tubes (from top to bottom). The highest light intensity was achieved in the middle of the tubes (80 pmols of photons m-2s-1) and this decreased by 50% in the top and bottom 20cm of tube to supply 50 pmols of photons nr2.s’1. During the first two days after Inoculation 1 and Inoculation 2, low light levels (50% of production level) were maintained to reduce stress on freshly fragmented tetrasporophytes. After two days, light intensity was increased to production levels at 70-80 pmols of photons nr2.s-1. The photoperiod was set to a 10:14 (light:dark) photoperiod. The temperature of the system was maintained at 19 °C in a climate-controlled room. Nutrient media was supplied as a 10% dose of Provassoli's Enriched Seawater (PES) nutrient media (full dose is 20ml / L) at 2ml / L. A low dose was used to reduce contamination spread (if contamination present), but the nutrient dose was sufficient for Asparagopsis growth. A 10% or 2ml / L dose was added every three days to maintain adequate nutrient supply to support growth. Aeration was set at 2 L / min to provide sufficient lift for the fragments and to circulate water around the PBR system.Table 1 : PBR growth conditionsGrowth Assessment

[0114] Biomass (fresh weight, g) was weighed at the beginning of each inoculation and then weighed after 19 days for Inoculation 1 and after 23 days for Inoculation 2.

[0115] Tetrasporophyte diameter was measured on 20 randomly selected fragments after fragmentation, and then 20 randomly selected mature tetrasporophytes after 19 days for Inoculation 1 and after 23 days for Inoculation 2.

[0116] Bromoform analysis was only performed at the end of the production cycle for inoculation 2. Harvested tetrasporophyte biomass was dip-rinsed in 10 °C deionized water for 1 second, repeating three times to remove salts. Dewatering was applied by using a centrifuge (Eppendorf 5430 R, Eppendorf South Pacific Pty. Ltd., NSW) at a spinning rate of 56 x g for a duration of 5 minutes.Sample Processing

[0117] Control and test algal samples were frozen at -81 °C. The frozen biomasses were freeze dried at -55 °C and 200 pbarfor48 hours using a VirTis Benchtop Pro (SP Scientific, USA) (Magnusson et al., 2020). After freeze drying, the dried samples were ground with Spex 2010 Geno / Grinder at 1200 rpm for 60 seconds and stored at -80 °C before bromoform extraction.Bromoform Extraction

[0118] To extract the bromoform, 10 mg of fine ground sample was extracted with HPLC grade methanol (CH3OH aka MeOH; Sigma-Aldrich, Australia), with 10 pg mL’1naphthalene (CI0H8; Sigma-Aldrich, Australia) as an internal standard. The methanol sample was mixed with vortex mixer for 5 seconds. Sonication in an ultrasonic bath was performed to help break the cell walls (Codyson CD-4821 , China) for 30 minutes. The sonicated sample was placed in a vortex for another 5 seconds then centrifuged (Eppendorf 5430 R, Eppendorf South Pacific Pty. Ltd., NSW) for 3000 rpm at 4 °C for 10 minutes (Romanazzi et al., 2021). The clear supernatant from the centrifuged sample was transferred to a vial. This process was repeated, and the second supernatant was combined with the first extract. For analysis, 20 pL of supernatant was diluted with MeOH to 1000 pL in a mass spectrometry vial to create solution with 50 dilution factors.Bromoform Analysis

[0119] Bromoform analysis involved preparing and running the standard curve samples at the beginning of the process. Standard curve was prepared at 2.5, 10, 25, 50, 100, 150, 200 pg mL-1bromoform (Sigma-Aldrich, Australia) in MeOH with 10 pg mL-1naphthalene as the internal standard, then diluted the same as the sample.

[0120] The method for bromoform analysis was based on Paul et al. (2006) Mar Ecol Prog Ser 306:87-101 , using QP2010 gas chromatograph-mass spectrometer (Shimadzu Corp., Japan) in splitless mode fitted with a ZebronTM ZB-WAXplusTM column, 30 m x 0.25 mm ID with 0.25 pm film thickness (Phenomonex, Torrance, USA). Settings for the GC-MS condition were 40 °C (1 min) for column temperature, then to 250 °C at 16°C min-1 and held for 2 min; carrier gas with helium controlled at 36.3 cm.s-1linear velocity (50.5 kPa starting pressure); injection port setting of 250 °C in splitless mode with 1 .5 min sampling time; GC-MS interface temperature of 250 °C; ion source temperature of 200 °C and solvent cut-off time of 4.5 min. 1 pL was injected into the GC with Single Taper inlet liner, 3.4 mm internal diameter, 95 mm length.

[0121] Data acquisition and processing were processed using GC Solutions (Shimadzu) in selected ion monitoring (SIM) mode. Qualifier ion peaks at m / z 249.7, 251 .8, 253.8, 255.8 [1 :2:2:1 ] and quantified at ion peak m / z 172.8 were selected to detect the concentration of bromoform. Internal standard (naphthalene) was quantified by detecting the qualifier ion peaks at m / z 127 and 129 [1 :1] and quantified at ion peak m / z 128 (Paul et al., 2006). Calculated concentration of bromoform was stated in milligrams of CHBr3per gram of dry seaweed biomass (mg.g-1).ResultsPBR operation

[0122] The operational parameters of the PBR were consistent during both production periods. Temperature (19°C), light intensity (70-80 micromoles of photons m-2s-1), and salinity (35-36ppt) were stable throughout the production periods. There was no significant contamination in the tetrasporophyte inoculation culture. The nutrient dosing regime, which was administered at 10% (2 ml / L) of full Provassoli's Enriched Seawater (PES) nutrient media doses every three days, provided sufficient nutrients to support positive, continuous daily growth, without significant contamination.Growth and size of tetrasporophytes

[0123] Inoculation 1: After 0.5 g (0.4 to 1.5mm diameter) of fragments were added to the photobioreactor, the operational parameters of the PBR achieved a growth rate of 13% per day after 19 days, achieving 5g of biomass and reaching a diameter of 5-7mm.

[0124] Inoculation 2: The 5 g produced from inoculation 1 was re-fragmented (similar size range 0.3 to 1 ,4mm) and added to the photobioreactor after a 100% change of seawater and new nutrient media. During fragmentation of this quantity of tetrasporophytes there was a loss of some biomass (~0.5g). The PBR achieved a growth rate of 8% after 23 days with 6-8mm diameter tetrasporophytes.Bromoform content

[0125] In this study, the bromoform analysis was conducted at the conclusion of the Inoculation 2 growth phase. The decision to analyze the bromoform concentration at this specific time point was to capture the cumulative effects of the growth conditions and environmental factors encountered throughout the entire production cycle. The measured bromoform concentration of 21.37 mg / g within the freeze-dried product serves as an important reference point for evaluating the success of the growth conditions. The relatively high concentration of bromoform compared to the control concentration of 13.12 mg / g suggests that the conditions provided during this phase were conducive to bromoform production and / or accumulation and highlights Asparagopsis’ potential for biogenic bromoform synthesis and / or maintenance under controlled conditions.Table 2: Asparagopsis growth and bromoform contentExample 2Effect of Supplementation with Bromide Salts on Bromoform ProductionMaterials and methodsSeaweed collection and preparation

[0126] Asparagopsis taxiformis gametophytes were collected from Point Peron, Western Australia. Gametophytes are found at this site year-round in temperatures 16°C to 24.5°C (Integrated Marine Observing System, 2022). Gametophytes were hand-collected by harvesting whole thalli from the reef, placed in a catch bag, then transported back to the Indian Ocean Marine Research Centre - Watermans Bay lab in a cooler box filled with sea water from the site. At the lab, gametophytes were transferred to a holding tank within a constant environment room at 19°C, low intensity white light (~50 pmol photons m-2 s-1) and a 12:12 photoperiod. The holding tank was filled with clean (1 pm), UV sterilized sea water and aerated to maintain water movement.

[0127] Asparagopsis taxiformis gametophyte grows clonally along its rhizomatous stolons. Fragments were made up of the new apical shoots emerging from the stolon. Fragments of stolons were used, rather than the thallus, to standardize the samples. 10-20mm long fragments were excised with only one cut, thereby reducing stress and / or infection points. Cleaned fragments were used for trials. To clean fragments, epiphytes and epifauna were removed using forceps, then the seaweed fragments were rinsed once for 3 seconds in autoclaved seawater. Five fragments were assigned to each culture vessel. Fragments were then assigned to a culture flask with Cell-Hi® nutrient media.Culture system

[0128] 5g of Asparagopsis taxiformis gametophyte fragments were cultured in 750 mL culture media in 1 L culture vessels within a constant environment room at 19 °C. The culture racks were illuminated by a LED light (Ledzeal® Mirage X200) mounted above, at an intensity of 50 pmols m-2.s_1and a 10:14 light:dark photoperiod. Air lines were supplied to individual culture vessels on the culture rack. Culture vessels were dosed with Cell-Hi™ F2P media [0.1 g L-1, recommendeddosage for seaweed] (Varicon Aqua, UK) diluted with filtered sea water. Cell-Hi™ is a commercial aquaculture mixture based on Guillard’s F / 2 recipe (Guillard, 1975).Bromide salt supplementation

[0129] To test the effect of bromide salt supplementation in seawater on Asparagopsis bromoform production, seawater was supplemented with either no bromide salts or with KBr and NaBr at 0.1 , or 1 g / L. Seaweed was then cultivated for one week.Sample processing, bromoform extraction and bromoform analysis

[0130] Sample preparation, bromoform extraction, and bromoform analysis to measure bromoform content was done as described in Example 1 . The results are shown in Table 3.Results

[0131] Supplementation with 0.1 g / L NaBr and KBr increased the bromoform concentration above the controls where no supplementation was applied. KBr showed a greater increase compared to NaBr. The maximum concentration achieved was 20.9 mg / g bromoform dry weight, which was nearly three times the concentration without bromide salt supplementation. Cell-Hi™, based on F2 media solution, is a commercially available nutrient solution that can sustain Asparagopsis growth; however, bromide in seawater can become depleted or be insufficient in a closed culture vessel to sustain increased bromoform production.Table 3: Analysis of bromoform content

[0132] The results demonstrate a significant increase in bromoform concentration in Asparagopsis when supplemented with sodium bromide (NaBr) and potassium bromide (KBr) at a concentration of 0.1 g / L compared to controls without bromide salt supplementation.

[0133] The maximum concentration of bromoform achieved with supplementation reached 20.9 mg / g dry weight, which is nearly three times higher than the concentration observed in controls without supplementation. This substantial increase underscores the effectiveness of KBr above NaBr supplementation in promoting, accelerating or enhancing bromoform biosynthesis; in increasing accumulation of bromoform; and / or in stopping, suspending or reducing degradationor excretion of bromoform by Asparagopsis. The addition of NaBr may have caused osmotic stress since sodium is highly available in seawater compared to potassium, which is a limiting macronutrient.

[0134] This study highlights the importance of bromide availability in sustaining increased bromoform production and / or accumulation in Asparagopsis closed circuit cultures.Example 3Enhancing Bromoform Production - Supplementation with Sodium Orthovanadate (Na-jVOMethods

[0135] To investigate the effects of sodium orthovanadate on Asparagopsis biomass, a controlled incubation experiment was conducted.Media Preparation

[0136] Under sterile conditions in a laminar flow hood, a 1000 mL glass flask was filled with 1 L of sterile seawater. Using a micropipette, 1 pL of sodium orthovanadate (1.84 g / L) stock solution was added to the flask. The solution was gently swirled to ensure thorough mixing and left to stand for 5 minutes as per standard laboratory protocols.Introduction of Asparagopsis Biomass

[0137] A 10 g portion of Asparagopsis was placed into the prepared sodium orthovanadate solution. Immediately after introduction, a photograph was taken to document the initial condition ofthe algae.Dark Incubation

[0138] The flask containing the Asparagopsis and sodium orthovanadate solution was transferred to a temperature-controlled room maintained at 24 °C. The flask was placed in a dark environment by enclosing it within a cardboard box and incubated for 2 hours. A second photograph was taken immediately before the flask was removed from the dark environment.Post-Treatment Handling

[0139] After the 2-hour incubation period, the flask was removed from the dark environment. The contents were poured over a metal sieve, and light pressure was applied using the back of a gloved hand to remove excess water. The entire 10 g sample was immediately placed on dry ice for preservation.Sample processing, bromoform extraction and bromoform analysis

[0140] Sample preparation, bromoform extraction, and bromoform analysis to measure bromoform content was done as described in Example 1 .Results

[0141] After incubation with culture media supplemented with sodium orthovanadate, bromoform production was enhanced in Asparagopsis compared to the control. In untreated samples, bromoform concentration was 6.09 mg / g, whereas samples exposed to sodium orthovanadate exhibited a bromoform concentration of 11.1 mg / g, representing an 82% increase (Table 4). This substantial enhancement suggests that vanadium plays a regulatory role in halogenated metabolite biosynthesis, likely through its function as a phosphatase inhibitor, sustaining the activation of pathways involved in bromoform synthesis.

[0142] The observed increase in bromoform production may be attributed to the prolonged phosphorylation state of key regulatory enzymes, leading to sustained metabolic flux toward halogenation reactions. Additionally, vanadium may contribute to higher intracellular hydrogen peroxide (H2O2) levels, further promoting the activity of vanadium-dependent haloperoxidases (V-HPOs), the enzymes responsible for catalysing bromoform synthesis.

[0143] These results highlight the potential of vanadium as a metabolic modulator in optimising bromoform yield.Table 4: Effect of (Na3VO4) on bromoform productionExample 4Effect of Supplementation with Potassium Bromide on Bromoform ProductionMethods

[0144] To enhance bromoform concentration in cultured Asparagopsis taxiformis tetrasporophytes, the culture medium was supplemented with potassium bromide.

[0145] A total of 10 grams of Asparagopsis was used to inoculate two 1 L flasks, with approximately 5 grams allocated to each flask. The flasks were filled with sterile seawater enriched with L2 nutrient media, and the seawater was supplemented with potassium bromide at a concentration of 0.1 g / L. Cultures were maintained under controlled conditions with continuousaeration at 2 L / min, a temperature of 19°C, light intensity of 50 pmol m2s1(white light), and a 12:12 light-dark photoperiod.

[0146] The first flask (5 g of Asparagopsis) was harvested after 24 hours - Trial A. The biomass was dewatered by placing it in cheesecloth and applying medium pressure (3 kg) to remove excess water. The dewatered sample was then transferred immediately into a 6-well plate and placed on dry ice for preservation.

[0147] The second flask (5 g of Asparagopsis) was cultivated for an additional seven days to assess bromoform accumulation overtime - Trial B. Afterthe seven-day period, the biomass was removed from the flask, dewatered using cheesecloth as described for Trial A, and transferred into a 6-well plate before being immediately placed on dry ice.Sample processing, bromoform extraction and bromoform analysis

[0148] Sample preparation, bromoform extraction and bromoform_analysis to measure bromoform content was done as described in Example 1 .Results

[0149] After 24 hours of exposure to 0.1 g / L potassium bromide in Trial A, Asparagopsis cultures showed a substantial increase in bromoform concentration compared to the control. The bromoform concentration in the potassium bromide-treated culture reached 16.8 mg / g, representing a 93% increase relative to the control sample, which contained 8.7 mg / g. This result indicates that potassium bromide supplementation significantly enhances bromoform production within a short incubation period.

[0150] After 7 days exposure to 0.1 g / L potassium bromide in Trial B, the bromoform concentration increase by ~60%, from 8.57 to 13.7 mg / g, also indicating a significant increase during longer term cultivation.Table 5: Effect of potassium bromide on bromoform productionExample 5Effect of Supplementation During Culturing with KBr and Darkness on Bromoform ProductionMethodBiomass Harvesting and Preparation

[0151] Asparagopsis taxiformis tetrasporophyte biomass was maintained in a 13 L photobioreactor and maintained at 20°C, 50 pmol m-2s-1PAR on 12:12 (L:D) photoperiod, L1 nutrient media, and 5 litre min air flow rate. All biomass from within the photobioreactor was harvested and then strained over a 200 mm metal sieve to remove excess seawater. Approximately 40 g of Asparagopsis biomass was separated for experimental treatments. To establish a control, a 10 g sample of freshly harvested and strained biomass was immediately collected and snap-frozen on dry ice, then stored for subseguent freeze-drying and further analysis.Extended Darkness and Potassium Bromide (KBr) Treatments

[0152] Three additional 10 g samples were subjected to extended darkness culturing treatments of 4 hours, 12 hours, and 20 hours in the presence of potassium bromide (KBr). Each sample was placed in a 1 L flask containing 1 L of filtered seawater, L1 nutrient media, and 0.1 g / L KBr for further culturing. For all treatments (4-hour, 12-hour, and 20-hour), KBr was preweighed onto a petri dish, dissolved in a known volume of seawater, and then added to the culturing flasks. The 20-hour culturing treatment commenced at 3:30 pm, the 4-hour treatment at 4:30 pm, and the 12-hour treatment at 8:30 pm. Flasks for the 4-hour and 12-hour culturing treatments were maintained in a temperature control room at 19°C under 40 pmol m-2s-1fullspectrum white light at the flask midpoint.

[0153] At the end of each time point, biomass was recovered and de-watered. The dewatered biomasses were then snap-frozen on dry ice, then stored for subseguent freeze-drying and further analysis.Freeze Drying samples

[0154] Following treatment and de-watering, all biomass samples were systematically placed in 6-well plates for preservation and subseguent analysis. Each experimental condition was assigned to a specific well, ensuring clear identification and tracking throughout the freeze-drying process.

[0155] All samples were snap-frozen on dry ice immediately following placement in the well plates and stored at -80°C prior to freeze-drying. The freeze-drying process was conducted using a Lanphan HFD-6 freeze dryer, following a controlled six-stage program to ensure gradualsublimation and sample integrity. This freeze-drying protocol was selected to minimize thermal degradation and preserve the biochemical composition of the biomass for downstream analysis. The freeze-dry program included the following conditions:1 . Stage 1 : -30°C for 0 minutes (initial stabilization) with no vacuum applied (0.0 Pa).2. Stage 2: -5°C for 480 minutes under vacuum pressure between 45.0-50.0 Pa.3. Stage 3: 0°C for 480 minutes under vacuum pressure between 45.0-50.0 Pa.4. Stage 4: 5°C for 480 minutes under vacuum pressure between 45.0-50.0 Pa.5. Stage 5: 10°C for 480 minutes under vacuum pressure between 80.0-90.0 Pa.6. Stage 6: 15°C for 641 minutes under vacuum pressure between 80.0-90.0 Pa.Table 6: Freeze Dry Program for Lanphan HFD-6 Freeze drierResults

[0156] Culturing of Asparagopsis taxiformis biomass in darkness for 4, 12, and 20 hours in the presence of KBr (0.1 g / L) influenced bromoform concentration.

[0157] All KBr treatments resulted in a significant increase in bromoform content relative to the control (8.57 mg / g). The highest bromoform concentration (17.6 mg / g) was observed in the 4-hour KBr treatment, representing a 105.4% increase over the control. The 12-hour and 20-hour KBr treatments resulted in bromoform concentrations of 14.1 mg / g (64.5% increase) and 14.9 mg / g (73.9% increase), respectively. These results demonstrate that short-term (4-hour) culturing with KBr yields the highest bromoform concentrations.Table 7: Effect of potassium bromide (KBr) incubation on bromoform production

Claims

CLAIMS1 . A method for producing a cultured algal biomass comprising an antimethanogenic compound, wherein the method comprises the step of culturing the algal biomass: a) at a light intensity of from 1 pmols m-2.s_1to 40 pmols nr2.s-1; b) in culture media supplemented with bromide; and / or c) in culture media supplemented with vanadium.

2. Use of growing conditions to produce a cultured algal biomass comprising an antimethanogenic compound, the growing conditions comprising culturing the algal biomass: a) at a light intensity of from 1 pmols m-2.s_1to 40 pmols nr2.s-1; b) in culture media supplemented with bromide; and / or c) in culture media supplemented with vanadium.

3. The method of claim 1 or 2, wherein: a) the bromide is in the form of NaBr and / or KBr; and / or b) the vanadium is in the form of sodium orthovanadate (Na3VO4).

4. The method of any one of claims 1 to 3, wherein: a) the bromide is at a concentration at from 0.01 g / L to 100 g / L in the culture media; and / or b) the vanadium is a concentration at from 0.01 p / L to 200 mg / L in the culture media.

5. The method of any of claims 1 to 4, wherein the cultured algal biomass is a cultured Asparagopsis biomass.

6. The method of any of claims 1 to 5, wherein the cultured algal biomass comprises more anti-methanogenic metabolites than wild harvested algal biomass.

7. The method of any of claims 1 to 6, wherein the cultured algal biomass comprises antimethanogenic metabolites at from 1 .5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times as much anti-methanogenic metabolites as wild harvested algal biomass.

8. The method of any of claims 1 to 7, wherein the method produces a cultured algal biomass comprising anti-methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g dry weight.

9. The method of any of claims 1 to 8, wherein the cultured algal biomass is in the form of cultured algal biomass fragments with an average diameter of from 1 mm to 20 mm.

10. The method of any of claims 1 to 9, wherein the anti-methanogenic metabolite is bromoform.

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