A method of enhancing metabolite production through stress

Abstract

A method for processing of algal biomass, comprising the step of subjecting the algal biomass to a stress chosen from: physical stress; osmotic stress; temperature stress; and / or preharvest light change stress, to produce the processed algal biomass, wherein the anti-methanogenic metabolite content of the processed algal biomass is higher after exposure to the stress than the anti-methanogenic metabolite content of the algal biomass before exposure to the stress.

Description

A Method of Enhancing Metabolite Production Through StressTECHNICAL FIELD

[0001] The present invention relates to methods for increasing the anti-methanogenic metabolite content of a process algal biomass by subjecting an algal biomass to a stress.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 compliment the previously known algal biomass culture and processing strategies. The present invention seeks to provide an improved or alternative method for processing algal biomass to result inbiomass 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 ofthe application.SUMMARY OF INVENTION

[0008] The present invention provides a method for processing of algal biomass, comprising the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress;Hi. temperature stress; and / or iv. preharvest light change stress to produce a processed algal biomass, wherein the anti-methanogenic metabolite content of the processed algal biomass is higher after exposure to the stress than the anti-methanogenic metabolite content ofthe algal biomass before exposure to the stress.

[0009] The stress may be one stress, or two or three or four stresses applied to the biomass. If more than one stress is applied, the stresses may be applied simultaneously or sequentially.

[0010] The stress may be followed by a period of rest before further processing to extract the bromoform. The period of rest may be provided in a dark environment.

[0011] The algae in the algal biomass may be Asparagopsis taxiformis (A. taxiformis) and / or Asparagopsis armata (A. armata). The anti-methanogenic metabolite may be bromoform.

[0012] If the stress is physical stress, optionally the physical stress to which the algal biomass is subjected may be chosen from: compression, sonication, agitation, and solid and liquid shearing.

[0013] Optionally, the physical stress is administered for up to 120 minutes. The degree of force is preferably sufficient to lead to an increase in the concentration of the anti-methanogenic metabolite content ofthe processed algal biomass without to disrupting (for example by rupturing) the structure of the majority of the cells of the algal biomass.

[0014] If the stress is osmotic stress, optionally the osmotic stress to which the algal biomass is subjected may be provided by exposing the algal biomass to brackish or fresh water, i.e. water that is less salty than sea water.

[0015] Optionally, the osmotic stress is administered for up to 120 minutes. The degree of osmotic stress is preferably sufficient to lead to an increase in the concentration of the anti- methanogenic metabolite content of the processed algal biomass without to disrupting (for example by rupturing) the structure of the majority of the cells of the algal biomass.

[0016] If the stress is temperature stress, optionally the temperature stress to which the algal biomass is subjected may be provided by exposing the algal biomass to a change in the temperature of the algal biomass after harvest, that is, the harvested biomass is exposed to a different temperature to the temperature at which the alga biomass was cultured. The temperature change may be either an increase in temperature of the algal biomass or a decrease in the temperature of the algal biomass.

[0017] Optionally, the temperature stress is administered for up to 120 minutes. The degree of temperature stress is preferably sufficient to lead to an increase in the concentration of the anti- methanogenic metabolite content of the processed algal biomass without to disrupting (for example by freezing or cooking) the structure of the majority of the cells of the algal biomass.

[0018] If the stress is preharvest light change, optionally the preharvest light change stress to which the algal biomass is subjected is a change in the light intensity and / or colour temperature the alga in culture is exposed to 24 h or less before harvesting of the algal biomass.

[0019] The changed light intensity and / or colour temperature stress may be applied to the algal biomass in culture for 24h or less before harvest. The change in light intensity stress may be an increase in light intensity or a decrease in light intensity. The change in colour temperature stress may be a shift in colourtemperature to the warmer end or the cooler end of the colour temperature spectrum.

[0020] If the stress is UV stress, optionally the UV light is applied to the algal biomass to produce a processed algal biomass in the presence of significant amounts of radiation in one or more of the UV wavelengths (wavelength range 100-400 nm; UVA (315-400 nm), UVB (280-315 nm), UVC (100-280 nm)).

[0021] Optionally, the UV light is administered for up to 120 minutes. For example, the UV light is administered for 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, or 120 minutes. Optionally, the intensity of the UV light is not sufficient to disrupt (for example by killing) the cells of the algal biomass. Therefore, the majority of the cells of the algal biomass are still alive after the UV light exposure.

[0022] The processed algal biomass may be stored for period of from 5 min, 10 min, 30 minutes, 1 h 1.5 h, 2h, 2.5 h, 3 h or more than 3 h after being subjected to the stress, to allow the anti- methanogenic metabolite content to increase.

[0023] In one aspect, the present invention provides processed 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 the algal biomass before processing. For example, the present disclosure provides processed 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 the algal biomass before processing.

[0024] In one aspect, the present invention provides processed algal biomass comprising anti- methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g mg / g dry weight. For example, the present disclosure provides processed algal biomass comprising bromoform at from 15, 20, 40, 50, 60 or 70 mg / g dry weight.

[0025] The present disclosure further provides for the use of a stress to produce a processed algal biomass that has a higher anti-methanogenic metabolite content than the anti-methanogenic metabolite content of an algal biomass before processing, comprising the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress;Hi. temperature stress; and / or iv. preharvest light change stress to produce the processed algal biomass.

[0026] The present disclosure further provides a processed algal biomass that has a higher anti- methanogenic metabolite content than the anti-methanogenic metabolite content of an algal biomass before processing, wherein the processing comprises the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress;Hi. temperature stress; and / or iv. preharvest light change stressto produce the processed algal biomass.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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 Figures in which:Figure 1 provides photographs of an Asparagopsis thallus prior to dewatering (desiccation) and UV treatment (top left) and then 60 mins after dewatering and UV treatment (top right); an Asparagopsis thallus prior to treatment with UV light (middle left), then after 60 mins treatment with UV light (middle right); and an Asparagopsis thallus (controls) with no UV light treatment (bottom left), then after 60 mins after no UV light (bottom right).Figure 2 provides photographs of bromoform-rich gland cells in Asparagopsis fragments. At 40x magnification the UV irradiated (left) and non-UV irradiated Asparagopsis gland cells are clearly visible. Note the pinkish hue and high concentration of gland cells in the UV irradiated cells compared to non-irradiated cells.DESCRIPTION OF INVENTIONDetailed Description of the Invention

[0028] Studies show that plants often ramp up their chemical defences in reaction to stresses. In some cases, this defence strategy can divert resources from growth or reproduction, and can provide a crucial advantage by better protecting the plants from damage or consumption. However, unlike constant defences that deplete resources continuously, inducible defences act only when a threat is detected, making for a more resource-efficient defence mechanism.

[0029] Marine algae, too, can enhance their defences in response to a stress. The stress includes a physical stress, for example herbivory or other physical damage. This can involve the rapid transformation of stored, less harmful compounds into more potent deterrents when damage is caused. Seaweeds potential defence mechanism against physical damage involves a well- orchestrated and rapid cellular response. For example, when physical damage occurs, sensor molecules within the seaweed detect the disruption and trigger a signalling cascade. This cascade involves a series of signalling molecules that relay the information throughout the cell. Crucially, these signals lead to changes in gene expression, potentially causing a boost in the production of enzymes responsible for synthesis of chemical deterrents. Additionally, the type and severity of the damage may influence the intensity of this defensive response. Physical stress prompts seaweeds to develop stronger chemical defences. For example, seaweeds in areas facing highherbivory levels are observed to enhance their chemical defence mechanisms more significantly than those in safer habitats.

[0030] Marine algae face stress if exposed to water with a lower dissolved salt concentration than sea water. Fresh water contains less than 1 % salt, for example less than 0.05% salt, and sea water (also known as saltwater) contains more than 3% (on average a salinity of about 3.5%). When algae grown in seawater is exposed to fresh water, the algal cells experience osmotic stress as the cells are hyperosmotic (higher salt concentration) compared to the fresh water, leading to water rushing into their cells, potentially causing cell swelling and disrupting vital functions. This stress induces the cells to increase production of anti-methanogenic metabolites, such as bromoform.

[0031] Marine algae can also enhance their defences in response to a change in the quantity and / or quality of the light immediately prior to harvest of the algae. The change may be a change in the light intensity and / or colour temperature the alga in culture is exposed to. The purpose of the change is to stress the algal cells by changing the conditions they are exposed to immediately prior to harvest of the algae, triggering an increase in anti-methanogenic metabolite production.

[0032] Marine algae can also enhance their defences in response to ultraviolet (UV) light damage from increased solar exposure as a stress. This can involve the rapid transformation of stored, less harmful compounds into more potent deterrents when damage is caused. Seaweeds potential defence mechanism against UV damage involve a well-orchestrated and rapid cellular response. For example, when UV light damage occurs, sensor molecules within the seaweed detect the disruption and trigger a signalling cascade. This cascade involves a series of signalling molecules that relay the information throughout the cell. Crucially, these signals lead to changes in gene expression, potentially causing a boost in the production of enzymes responsible for synthesis of chemical defences. Additionally, the type and severity of the damage may influence the intensity of this defensive response. UV light prompts seaweeds to develop stronger chemical defences.

[0033] Asparagopsis, which produces high concentrations of anti-methanogenic metabolites (including bromoform, a halogenated chemical defence), can change its anti-methanogenic metabolite production rapidly due the abundance of essential precursor compounds. This seaweed naturally generates excess fatty acid molecules, which serve as the foundation for anti- methanogenic metabolites, while the surrounding seawater supplies ample bromide ions for the bromine in the molecule. This abundance allows Asparagopsis to quickly increase anti- methanogenic metabolite production in response to stresses and threats such as grazing, osmotic stress, change in light conditions and / or damage caused by UV light.

[0034] Without being held to any theory, the present inventors believe that, when faced with artificial challenges and stresses, Asparagopsis may utilize its available precursors and specific enzymes to boost bromoform production in a similar manner, thus producing an algal biomass with increased concentration of useful anti-methanogenic metabolites.Method for Post-Harvest Processing

[0035] The present invention provides a method for processing of algal biomass, comprising the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress;Hi. temperature stress; and / or iv. preharvest light change stress to produce the processed algal biomass, wherein the anti-methanogenic metabolite content of the processed algal biomass is higher after exposure to the stress than the anti-methanogenic metabolite content of the algal biomass before exposure to the stress.

[0036] The stress may be one, two, three or four of the above discussed stresses. If more than one stress is applied to the algal biomass, the stresses may be applied simultaneously or sequentially.

[0037] The stress may be followed by a period of rest before further processing to extract the bromoform. The period of rest allows the processed algal biomass to engage the biological pathways that lead to increased bromoform in response to the stress.

[0038] The present invention therefore provides a method for processing of algal biomass, comprising the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress; iii.temperature stress; and / or iv.preharvest light change stress to produce the processed algal biomass,b) resting the processed algal biomass wherein the resting step is carried out for sufficient time for the anti-methanogenic metabolite content wherein the anti-methanogenic metabolite content of the processed algal biomass is higher after exposure to the stress than the anti-methanogenic metabolite content of the algal biomass before exposure to the stress.

[0039] For example, the processed algal biomass may take 5 min, 10 min, 30 minutes, 1 h 1.5 h, 2h, 2.5 h, 3 h, or more than 3 h after being subjected to the stress before the anti-methanogenic metabolite content increases. The processed algal biomass may therefore be rested for period of from 5 min, 10 min, 30 minutes, 1 h 1 .5 h, 2h, 2.5 h, 3 h or more than 3 h after being subjected to the stress, to allow the anti-methanogenic metabolite content to increase.

[0040] The resting period may be carried out in a low light or dark environment.

[0041] After this resting time to increase the anti-methanogenic metabolite content (for example bromoform content), the processed algal biomass may then be subject to further processing steps, such as freeze drying, to produce a product that is suitable for long-term storage and commercial sale.

[0042] Optionally, the algal biomass subject to the stress is algal biomass that has been harvested, cleaned and / or partially dewatered to remove seawater.Cleaning Algal Biomass

[0043] The algal biomass may be cleaned before being subjected to a stress or 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. 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 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.

[0044] 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 algal biomass may therefore be carried out 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.

[0045] A suitable de-watering medium for the Asparagopsis may be provided in the form of a flexible, porous textile or mesh material having apertures of a size sufficient to allow the passage of liquid while retaining the fibrous or particulate matter of the Asparagopsis. In some embodiments, this textile may be woven or nonwoven cotton and constructed from fibres that impart durability, fluid permeability, and resistance to tearing. The open structure of the textile material enables liquid — such as water or seawater / brine — to efficiently drain or be pressed out of the Asparagopsis, while the solid matter remains contained along with certain metabolites including bromoform and dibromoacetic acid. A suitable example material is cheesecloth.

[0046] During operation, the Asparagopsis may be placed in contact with the porous textile. Mechanical pressure, either by compression or manual wringing, is subsequently applied. Under these conditions, the textile’s interstices permit excess liquid to escape, effectively concentrating the solid components. The textile may also be selected or treated to minimise adhesion of plant matter to its surface, facilitating easy removal or further processing of the marine plant. As a result, the textile or mesh material enhances the overall efficiency of de-watering while maintaining the structural integrity of the seaweed throughout the process and enhancing bromoform concentration and avoiding crystallisation of salt which may inhibit bromoform detection.Physical Stress

[0047] The physical stress to which the algal biomass is subjected may be chosen from: compression, sonication, agitation, and solid and liquid shearing. The physical stress in its gross form may be provided by different disrupting methods such as compression, sonication, agitation, and solid and liquid shearing, but on the cellular level, the physical stresses are all performing to deform the algal cells. Without being held to any theory, the inventors believe that this deformation is the trigger for the cells to increase their anti-methanogenic metabolite content. Preferably the deformation is transient, optionally the cells returning to a native conformation, similar to the cell’s conformation before imposition of the physical stress. The type of gross physical stress may vary, but the overall effect of each type of stress is to deform the cells and trigger an increase in anti- methanogenic metabolite content.

[0048] The physical stress may be carried out for a time period of 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min or 120 min. For example, the compression may be carried out for a time period of from 1 min to 120 min, from 1 min to 60 min, from 1 min to 45 min, from 1 min to 30 min, from 1 min to 20 min, from 1 min to 10 min, from 1 min to 5 min, or from 1 min to 2 min.

[0049] The physical stress may be applied to the algal biomass whilst the algal biomass is contained in a flexible, porous textile or mesh material having apertures of a size sufficient toallow the passage of liquid while retaining the fibrous or particulate matter of the Asparagopsis, as described above with regard to dewatering.

[0050] If the physical stress is compression, the compressive force may be administered by manual wringing, squeezing or compression, centrifugation, a hydraulic press, screw press, or a weighted roller or press.

[0051] The degree of force of the compression may be measured in Newtons or by pressure (compressive force over area) in Pascals (Pa). The degree of force of the compression may be from 1 Newton to 100 Newtons. For example, the compression may be carried out at from 1 Newton to 100 Newtons, or from 2 Newtons to 70 Newtons. The pressure applied to the algal biomass may be from 1 kPa to 100 kPa, for example from 2 kPa to 70 kPa.

[0052] The degree of force of the compression may be measured in weight applied. For example, the weight applied to the algal mass may be less than 10 kg, 8kg, 6kg, 5kg, 4kg, 3kg, 2kg or 1 kg. For example, the weight applied to the algal mass may be about 1 kg, 2kg, 2.5kg, 3kg, 3.5kg, 4kg 4.5kg, 5kg, 5.5kg, 6kg, 6.5kg, 7kg, 7.5kg, 8kg, 8.5 kg, 9kg, 9.5kg or 10kg.

[0053] The degree of feree of the compression may be measured in percentage of water removed from the algal mass. For example, the percentage of water removed from the algal mass may be at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the water in the algal mass.

[0054] The compression may be carried out for a time period of 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min or 120 min. For example, the compression may be carried out for a time period of from 1 min to 120 min, from 1 min to 60 min, from 1 min to 45 min, from 1 min to 30 min, from 1 min to 20 min, from 1 min to 10 min, from 1 min to 5 min, or from 1 min to 2 min.

[0055] If the physical stress is solid or liquid shearing, the shearing may be administered by bead milling, high-speed homogenizing, or high-pressure homogenizing. In bead milling, algal biomass is subject to physical stress by applying force through the collisions between the algal biomass and beads. High-speed homogenization (HSH) subjects the algal biomass to physical stress by dynamic cavitation. High-pressure homogenization (HPH) subjects the algal biomass to physical stress via the application of highly pressurized liquid through a thin nozzle.

[0056] The degree of feree of the solid or liquid shearing may be measured in revolutions per minute (rpm). For example, the solid or liquid shearing may be carried out at from 20 rpm to 10,000 rpm, from 20 to 5,000 rpm, from 20 to 2,000 rpm, from 20 to 1000 rpm, from 20 to 500 rpm or from 20 to 200 rpm. In one example, the shearing may be carried out at 200 rpm.

[0057] The solid or liquid shearing may be carried out for a time period of 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min or 120 min. For example, the solid or liquid shearing may becarried out for a time period of from 1 min to 120 min, from 1 min to 60 min, from 1 min to 45 min, from 1 min to 30 min, from 1 min to 20 min, from 1 min to 10 min, from 1 min to 5 min, or from 1 min to 2 min.

[0058] If the physical stress is sonication, the sonication may be administered to the algal biomass by a bath or probe sonicator, or an ultrasonicator.

[0059] The degree of force of the sonication may be measured in Hertz (Hz). For example, the sonication may be carried out at from 15 kHz to 400 kHz, for example from 20 kHz to 40 kHz. In one example, the sonication may be carried out at 20 kHz.

[0060] The sonication may be carried out for a time period of 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min or 120 min. For example, the sonication may be carried out for a time period of from 1 min to 120 min, from 1 min to 60 min, from 1 min to 45 min, from 1 min to 30 min, from 1 min to 20 min, from 1 min to 10 min, from 1 min to 5 min, or from 1 min to 2 min.

[0061] If the physical stress is by agitation, the agitation may be administered to the algal biomass by shakers, jet agitators, paddle agitators, anchor agitators, helical ribbon agitators, propeller agitators, turbine agitators, agitators with screw impellers, retreat curve impellers, hydrofoil impellers, dispersion blade impellers, and coil impellers.

[0062] The degree of force of the agitation may be measured in revolutions per minute (rpm). For example, the agitation may be carried out at from 1 rpm to 5000 rpm, for example from 200 rpm to 2000 rpm. In one example, the agitation may be carried out at from 300 rpm to l OOOprm.

[0063] The agitation may be carried out for a time period of 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min or 120 min. For example, the agitation may be carried out for a time period of from 1 min to 120 min, from 1 min to 60 min, from 1 min to 45 min, from 1 min to 30 min, from 1 min to 20 min, from 1 min to 10 min, from 1 min to 5 min, or from 1 min to 2 min.

[0064] The different sorts of physical stresses all preferably cause transient deformation of the algal cells. Optionally, the degree of force of the physical stress is not sufficient to disrupt (for example by rupturing) the structure of the cells of the algal biomass between the time of imposition of the physical stress that causes deformation and optionally the cells returning to their native conformation (similar to that before imposition of the physical stress). Therefore, the majority of the cells of the algal biomass retain their native conformation or physical structure, with cell walls and / or cell membranes remaining intact. For example, optionally the cell walls and / or cell membranes of at least 90%, at least 95% or at least 99% of cells of the algal biomass are not disrupted by the physical stress.

[0065] After subjecting the algal biomass to the physical stress, at least the anti-methanogenic metabolite content, for example the bromoform content of the processed algal biomass ispreferably increased. This increase may take time after the physical stress has stopped. For example, the processed algal biomass may take 5 min, 10 min, 30 minutes, 1 h 1.5 h, 2h, 2.5 h, 3 h, or more than 3 h after being subjected to the physical stress before the anti-methanogenic metabolite content increases. The processed algal biomass is therefore stored for period of from 5 min, 10 min, 30 minutes, 1 h 1 .5 h, 2h, 2.5 h, 3 h or more than 3 h after being subjected to the physical stress, to allow the anti-methanogenic metabolite content to increase.Osmotic Stress

[0066] Marine algae face stress if exposed to water with a lower dissolved salt concentration (e.g. fresh water or brackish water) than sea water, as the algal cells have the same or similar concentration of salts as the seawater and thus a higher concentration of internal salt than the fresh or brackish water. The osmotic difference leads to an influx of water into the algal cells, potentially causing cell swelling and disrupting vital functions. This stress induces the increased production of anti-methanogenic metabolites, such as bromoform.

[0067] Fresh water is generally defined as having a dissolved salt concentration of less than 0.05%; brackish water generally has a dissolved salt concentration of between about 0.05% and 3.5%, and salt water has a dissolved salt concentration of about 3.5% to 5%. Optionally, the water with a lower dissolved salt concentration than sea water has a dissolved salt concentration of less than 5%; for example, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, 0.01 % or 0.05% dissolved salt concentration.

[0068] The osmotic stress may be carried out for a time period of 30 sec, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min or 120 min. For example, the osmotic stress may be carried out for a time period of from 30 sec min to 120 min, from 30 sec to 60 min, from 30 sec to 45 min, from 30 sec to 30 min, from 30 sec to 20 min, from 30 sec to 10 min, from 30 sec to 5 min, or from 30 sec to 2 min.

[0069] After exposure to the water with a lower dissolved salt concentration than sea water to induce osmotic stress, the algal biomass may be washed in salt water to restore the osmotic balance of any water surrounding the algal biomass, or may simply be dewatered to remove most of the water with a lower dissolved salt concentration than sea water.

[0070] The osmotic stress optionally causes transient swelling of the algal cells due to an influx of water. Optionally, the degree of force of osmotic stress is not sufficient to disrupt (for example by rupturing) the structure of the cells of the algal biomass between the time of imposition of the osmotic stress that causes swelling and optionally the cells returning to their native conformation (similar to that before imposition of the osmotic stress). Therefore, the majority of the cells of the algal biomass retain their native conformation or physical structure, with cell walls and / or cell membranes remaining intact. For example, optionally the cell walls and / or cell membranes of atleast 90%, at least 95% or at least 99% of cells of the algal biomass are not disrupted by the osmotic stress.

[0071] After subjecting the algal biomass to the osmotic stress, at least the anti-methanogenic metabolite content, for example the bromoform content of the processed algal biomass is preferably increased. This increase may take time after the osmotic stress has stopped. For example, the processed algal biomass may take 5 min, 10 min, 30 minutes, 1 h 1.5 h, 2h, 2.5 h, 3 h, or more than 3 h after being subjected to the osmotic stress before the anti-methanogenic metabolite content increases. The processed algal biomass is therefore rested for period of from 5 min, 10 min, 30 minutes, 1 h 1 .5 h, 2h, 2.5 h, 3 h or more than 3 h after being subjected to the osmotic stress, to allow the anti-methanogenic metabolite content to increase.Temperature Stress

[0072] Marine algae can also enhance their defences in response to a change in the temperature of the harvested algal biomass. The temperature change leads to stress in the algal biomass, and the increased production of anti-methanogenic metabolites, such as bromoform. The temperature stress is a stress induced by a change in temperature. The change may be an increase or a decrease, the principle is that the algae is shocked and stressed by the temperature after harvest being different to the temperature the algae experienced during culture. Generally, the temperature algae is exposed to during culture is between about 12 °C and 28 °C.

[0073] The change in temperature stress may be an increase in temperature of 40% or less from the temperature the algal was exposed to during culture (before harvest). The increase in temperature may be a change of 35%, 30%, 25%, 20%, 25%, 10% or 5% from the temperature the algae was exposed to during culture (before harvest). The change in temperature may be a decrease in temperature of 40% or less. The decrease in temperature may be a change of 35%, 30%, 25%, 20%, 25%, 10% or 5% from the temperature the algae was exposed to during culture (before harvest).

[0074] The change in temperature stress may be carried out for a time period of 30 sec, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min or 120 min. For example, the change in temperature stress may be carried out for a time period of from 30 sec min to 120 min, from 30 sec to 60 min, from 30 sec to 45 min, from 30 sec to 30 min, from 30 sec to 20 min, from 30 sec to 10 min, from 30 sec to 5 min, or from 30 sec to 2 min.

[0075] The change in temperature is preferably not sufficient to disrupt (for example by freezing or cooking) the structure of the cells of the algal biomass between the time of imposition of the temperature stress and optionally the cells returning to a temperature similar to that of the algae during culture. Therefore, the majority of the cells of the algal biomass remain alive, with cell walls and / or cell membranes remaining intact. For example, at least 90%, at least 95% or at least 99%of cells of the algal biomass are still alive at the end of the temperature stress. Optionally, the cell walls and / or cell membranes of at least 90%, at least 95% or at least 99% of cells of the algal biomass are not disrupted by the temperature stress.

[0076] After subjecting the algal biomass to the change in temperature, at least the anti- methanogenic metabolite content, for example the bromoform content of the processed algal biomass is preferably increased. This increase may take time after the change in temperature has stopped. For example, the processed algal biomass may take 5 min, 10 min, 30 minutes, 1 h 1 .5 h, 2h, 2.5 h, 3 h, or more than 3 h after being subjected to the change in temperature before the anti-methanogenic metabolite content increases. The processed algal biomass is therefore rested for period of from 5 min, 10 min, 30 minutes, 1 h 1 .5 h, 2h, 2.5 h, 3 h or more than 3 h after being subjected to the change in temperature, to allow the anti-methanogenic metabolite content to increase.Preharvest Light Change Stress

[0077] Marine algae can also enhance their defences in response to a change in the guantity and / or quality of the light immediately prior to harvest of the algae. The change may be a change in the light intensity and / or colour temperature the algae in culture is exposed to. The purpose of the change is to stress the algal cells by changing the conditions they are exposed to immediately prior to harvest of the algae, triggering an increase in anti-methanogenic metabolite production. If the change is carried out too long before harvest, or the change is carried out for too long, the algal cells will adapt to the new conditions and return their anti-methanogenic metabolite production to pre-stress levels.

[0078] If the stress is preharvest light change, optionally the preharvest light change stress to which the algal biomass is subjected is a change in the light intensity and / or colour temperature the algae in culture are exposed to 24 h or less before harvesting of the algal biomass.

[0079] The changed light intensity and / or colour temperature may be applied to the algal biomass in culture for 24h before harvest, or for a shorter period of time (eg applied to the algal biomass in culture for 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 1 1 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, or 23 h), so long as the changed light intensity and / or colour temperature occurs in the period 24 h or less before harvest. The change in light intensity and / or colour temperature may occur 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, or 23 h before harvest.

[0080] Generally, the light intensity algae is exposed to during normal culture is between about 40 pmols m-2s-1and 1 ,500 pmols m-2s-1(for example, in a PBR the light intensity is about 40 pmols m-2s-1to 200 pmols m-2s-1, in an outdoor pond or raceway the light intensity is about 100pmols m-2s-1to 1 ,500 pmols m-2s-1). Generally, the light colour temperature algae is exposed to during normal culture is between about 2,000 and >6,500 Kelvins.

[0081] The change in light intensity may be an increase in light intensity or a decrease in light intensity from the light intensity that the algal biomass has been cultured at for the rest of the culture period. Optionally, the change in light intensity is a 40% change in light intensity or less. The change in light intensity (increase or decrease) may be a change of 35%, 30%, 25%, 20%, 25%, 10% or 5%.

[0082] The change in light colour temperature may be a shift in colour temperature to the warmer end of the colour temperature spectrum (i.e. towards warm white at about 2,000K to 3,500K) or a shift in colour temperature to the cooler end of the colour temperature spectrum (i.e. towards cool white at >6,500K) from the colour temperature that the algal biomass has been cultured at for the rest of the culture period.

[0083] The change in light colour temperature may be a shift towards the warmer end of the colour temperature spectrum of 40% or less. The shift towards the warmer end of the colour temperature spectrum may be a change of 35%, 30%, 25%, 20%, 25%, 10% or 5%. The change in light colour temperature may be shift towards the cooler end of the colourtemperature spectrum of 40% or less. The shift towards the cooler end of the colour temperature spectrum may be a change of 35%, 30%, 25%, 20%, 25%, 10% or 5%.UV light

[0084] The atmosphere blocks about 77% of the Sun's UV, when the Sun is highest in the sky (at zenith), with absorption increasing at shorter UV wavelengths. At ground level with the sun at zenith, sunlight is 44% visible light, 3% ultraviolet, and the remainder infrared. Of the UV radiation that reaches the Earth's surface, about 95% is the longer wavelengths of UV-A, with about 5% UV-B. Almost no UV-C reaches the Earth's surface.

[0085] The UV light to which the algal biomass is exposed may be a significant amount of radiation in one or more of the UV wavelengths (wavelength range 100-400 nm; UV-A (315-400 nm), UV-B (280-315 nm), UV-C (100-280 nm)). The UV light to which the algal biomass is exposed may be one of UV-A, UV-B or UV-C, or a combination of any two or more of these UV wavelengths. In one aspect of the invention, the UV light used is UV-C, as little of the Sun’s UV- C wavelengths reach the Earth’s surface. As a result, many organisms have little ability to protect themselves from damage from such wavelengths and UV-C is most able to induce ROS production in organisms. However, as is well known, UV-A and UV-B can also cause ROS production and DNA, protein and lipid damage.

[0086] Optionally, the intensity of the UV to which the algal biomass is exposed is significantly more intense than the amount of UV in sunlight. For example, the UV to which the algal biomassis exposed may have 1.5 times, 2 times, 2.5 times, 3 times, 10 times, 20 times, 50 times 100 times as much energy in the UV range of wavelengths as sunlight. However, the intensity of the UV to which the algal biomass is exposed may be similarto, or less than sunlight. Exposure under low intensity UV light may require a longer period of exposure to trigger the increase in production of anti-methanogenic metabolites such as bromoform.Table 1 : Typical relative UV levels from different light sources

[0087] Most interior lights (LED, fluorescent, quartz halogen, tungsten filament incandescent), produce significantly less UV radiation than light from the Sun. Therefore, the source of the UV light to which the algal biomass is exposed may be a specialised source of UV light wavelengths. The specialised source of UV light wavelengths may be chosen from: black light lamps (long wave UV radiation); short-wave UV lamps (UV with two peaks in the UV-C band at 253.7 nm and 185 nm); UV gas-discharge lamps; excimer lamps; xenon arc lamps; deuterium arc lamps; mercuryxenon arc lamps; metal-halide arc lamps; UV-LEDs; and gas lasers, laser diodes and solid-state lasers manufactured to emit in the UV range.

[0088] The UV light exposure may be carried out for a time period of 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min or 120 min. For example, the UV light exposure may be carried out for a time period of from 1 min to 120 min, from 1 min to 60 min, from 1 min to 45 min, from 1 min to 30 min, from 1 min to 20 min, from 1 min to 10 min, from 1 min to 5 min, or from 1 min to 2 min.

[0089] Optionally, the intensity of the UV light is not sufficient to disrupt (for example by killing) the cells of the algal biomass. Therefore, the majority of the cells of the algal biomass are still alive after the UV light exposure. For example, optionally at least 90%, at least 95% or at least 99% of cells of the algal biomass are still alive after the UV light exposure.

[0090] After exposing the algal biomass to UV light, at least the anti-methanogenic metabolite content, for example the bromoform content, of the processed algal biomass is preferably increased. This increase may take time after the UV light exposure has stopped. For example, the processed algal biomass may take 5 min, 10 min, 30 minutes, 1 h 1.5 h, 2h, 2.5 h, 3 h, or more than 3 h after being exposed to the UV light exposure before the anti-methanogenic metabolite content increases. The processed algal biomass is therefore stored for period of from 5 min, 10 min, 30 minutes, 1 h 1.5 h, 2h, 2.5 h, 3 h or more than 3 h after being exposed to the UV light exposure, to allow the anti-methanogenic metabolite content to increase.Measuring Anti-methanogenic Metabolite Content

[0091] The anti-methanogenic metabolite content of the algal biomass and the processed algal biomass may be measured by gas chromatography-mass spectrometry (GC-MS), for example by the method of Romanazzi et al. (2021) ACS Agric Sci Technol 1 :436-442 or Paul et al. (2006) Mar Ecol Prog Ser 306:87-101 . For example, the bromoform content of the algal biomass and the processed algal biomass may be measured.

[0092] The inventors have determined that the amount of anti-methanogenic metabolites, such as bromoform, that may be produced and stored in an Asparagopsis cell is determined by the size of the specialized gland cells (also known as vesicle cells) stored within the algal cell. They have found that gland cells could occupy nearly 50% of the total volume of an Asparagopsis cell. Therefore, the amount of anti-methanogenic metabolites that can be produced and stored with an Asparagopsis cell may be very high.

[0093] In one aspect, the present invention provides processed 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 the algal biomass before processing. For example, the present disclosure provides processed 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 the algal biomass before processing.

[0094] In one aspect, the present invention provides processed 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 processed algal biomass comprising bromoform at from 15, 20, 40, 50, 60 or 70 mg / g dry weight.

[0095] Alternatively, the colour of the processed algal biomass may be monitored, for example visually. As the bromoform content of the processed algal biomass increases, the colour of the processed algal biomass may change. For example, for A. taxiformis and A. armata, the colour of the colour of the processed algal biomass may change from red to purple. Such a colour changecan be used as a simple and convenient method to rapidly assess a change in bromoform content without the need for laboratory equipment. For example, after exposure of the algal biomass to a stress to provide a processed algal biomass, the processed algal biomass may be stored for a time to allow the bromoform content of the processed algal biomass to increase, and the increase may be monitored visually by the colour change from red to purple.Algae

[0096] In the present invention, the terms “seaweed” and “algae” are used interchangeably. Algae are simple, non-flowering, and typically eukaryotic photosynthetic aquatic organisms. Algae contain chlorophyll but lack true stems, roots, leaves, and vascular tissue. The algae may be a macroalgae or a microalgae.

[0097] 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 aqueous culture medium, the algae of the present invention may be referred to herein as “algal cells”. When not in association with an aqueous culture medium, the algae of the present invention may be referred to herein as “algal biomass”.

[0098] Algae suitable for use in the present invention are preferably macroalgae. These macroalgae include green, brown and red algae. Brown and red algae are preferred because they typically require weaker light intensity than green algae to grow, which may reduce the electrical cost for onshore farming using artificial lighting. Preferably, 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.

[0099] Algal cells in aqueous 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.

[0100] Optionally, the alga is of the class Florideophyceae. Florideophyceae are multicellular red algae which form biomass fragments. Preferably, the alga is of the order Bonnemaisoniales in the class 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

[0101] 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.

[0102] 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.

[0103] 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.

[0104] The present invention 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 stress as described herein. The invention further provides access to organically produced metabolites, such as cell-wall polysaccharides, which may result in an improved ruminant feed.Other factors

[0105] Optionally, the stress is applied to the algal biomass to produce a processed algal biomass at a temperature of 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 algal biomass is subject to the stress at from 20 °C to 8 °C, or from 18 °C to 10 °C, for example about 18 °C, 15 °C or 10 °C.

[0106] Optionally, the stress is applied to the algal biomass to produce a processed algal biomass without significantly drying the processed algal biomass. For example, the processed algal biomass may retain at least 90%, at least 95% or at least 99% of the water within the agal cells that was present in the algal biomass before processing. This water retained in the algal cells is not the seawater or fresh water that surrounds the algal biomass during growth or the water used for cleaning the algal biomass, but rather is the internal water present within a healthy algal cell.

[0107] Optionally, the algal biomass, such as an 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. Bromoform and dibromoacetic acid have been identified as dominant compounds in A. armata and A. taxiformis.

[0108] Optionally, the processing of the algal biomass by exposure to a stress results in a processed algal biomass that retains most or all of one or more of the metabolites or bioactive compounds present in the algal biomass before processing. Thus, the processed algal biomass may contain the same amount or nearly the same amount of one or more of the metabolites or bioactive compounds that were present in the algal biomass, and / or may contain the same or similar profile of metabolites or bioactive compounds as the algal biomass before processing even if the amounts of each compound or metabolite are reduced. For example, the processed 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 algal biomass, and / or may contain the same or similar profile of lipophilic metabolites or bioactive compounds as the algal biomass before processing even if the amounts of each lipophilic compound or metabolite are reduced.

[0109] The processing of the algal biomass may result in a processed algal biomass that retains most or all of the dibromoacetic acid and / or bromochloroacetic acid compounds present in the algal biomass before processing. Thus, the processed algal biomass may contain the same amount or nearly the same amount of dibromoacetic acid and / or bromochloroacetic acid that was present in the algal biomass, and / or may contain the same profile of dibromoacetic acid and / or bromochloroacetic acid as the algal biomass before processing even if the amounts of each compound are reduced.

[0110] Optionally, the processing of the algal biomass results in a processed algal biomass that retains most or all of the bromide-containing compounds, for example bromoform, present in the algal biomass before processing. Thus, the processed algal biomass may contain the same amount or more bromide-containing compounds, for example bromoform, than was present in the algal biomass. Without being held to any theory, it is believed that the process ofsubjecting the algal biomass to a stress results in some or all of the bromide-containing compounds in the algal biomass releasing their bromide or converting the other bromide- containing compounds in the agal cell to bromoform. Over 100 halogenated compounds may be found in the Asparagopsis cells, including haloforms, haloacids and haloketones, and some or all of these halo-compounds that are bromide compounds may be converted to bromoform.Culture Conditions

[0111] The 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. Alternatively, the algal biomass may be harvested, either from natural sources or from oceanic seaweed farms.

[0112] Optionally, the algal biomass is grown in aqueous culture media at a temperature of from 12 °C to 28 °C; for example, aquaculture growth of algal biomass is often carried out at between about 15 °C and 28 °C.

[0113] 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.

[0114] 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).

[0115] 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).

[0116] Optionally, the pH of the PBR system in which the 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.

[0117] During growth in culture, the algal biomass of the present disclosure is optionally exposed to a light intensity of from 100 pmols m-2.s_1to 1500 pmols m-2.s_1.Uses and Products

[0118] The present disclosure further provides for the use of a stress to produce a processed algal biomass that has a higher anti-methanogenic metabolite content than the anti- methanogenic metabolite content of an algal biomass before processing, comprising the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress; iii.temperature stress; and / or iv.preharvest light change stress to produce the processed algal biomass.

[0119] The present disclosure further provides a processed algal biomass that has a higher anti-methanogenic metabolite content than the anti-methanogenic metabolite content of an algal biomass before processing, wherein the processing comprises the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress; iii.temperature stress; and / or iv.preharvest light change stress to produce the processed algal biomass.

[0120] The algae in the algal biomass may be Asparagopsis taxiformis (A taxiformis) and / or Asparagopsis armata (A. armata). The anti-methanogenic metabolite may be bromoform.

[0121] If the stress is physical stress, optionally the physical stress to which the algal biomass is subjected may be chosen from: compression, sonication, agitation, and solid and liquid shearing.

[0122] Optionally, the physical stress is administered for up to 120 minutes. The degree of force of the physical stress is determined by the type of physical stress chosen. However, the degree of force is preferably sufficient to lead to an increase in the concentration of the anti- methanogenic metabolites of the processed algal biomass without disrupting (for example by rupturing) the structure of the majority of the cells of the algal biomass.

[0123] If the stress is osmotic stress, optionally the osmotic stress to which the algal biomass is subjected may be provided by exposing the algal biomass to brackish or fresh water, i.e. water that is less salty than sea water.

[0124] Optionally, the osmotic stress is administered for up to 120 minutes. The degree of osmotic stress is preferably sufficient to lead to an increase in the concentration of the anti- methanogenic metabolite content of the processed algal biomass without to disrupting (for example by rupturing) the structure of the majority of the cells of the algal biomass.

[0125] If the stress is temperature stress, the change in temperature may be an increase or a decrease in temperature from the temperature the algae was exposed to during culture (before harvest) - the stress resides in the change not the direction of change.

[0126] If the stress is preharvest light change, optionally the preharvest light change stress to which the algal biomass is subjected is a change in the light intensity and / or colour temperature the alga in culture is exposed to 24 h or less before harvesting of the algal biomass. The stress resides in the change not the direction of change, so the light intensity may be an increase or decrease and the change in colour temperature may be to the warmer or cooler end of the spectrum.

[0127] Optionally, the changed light intensity and / or colour temperature is applied to the algal biomass in culture before harvest for 24h or less. Optionally, the changed light intensity and / or colour temperature is applied to the algal biomass in culture 24h or less before harvest.

[0128] If the stress is UV light, optionally the UV light to which the algal biomass is exposed may UV-A, UV-B, UV-C or a combination of any two or more of these UV wavelengths.

[0129] Optionally, the UV light is administered for up to 120 minutes. The intensity and timing of UV light exposure may be varied, for example depending on the wavelength chosen. However, the UV light exposure is preferably sufficient to lead to an increase in the concentration of the anti-methanogenic metabolite content of the processed algal biomass without killing the majority of the cells of the algal biomass.

[0130] The processed algal biomass may be stored for period of from 5 min, 10 min, 30 minutes, 1 h 1 .5 h, 2h, 2.5 h, 3 h or more than 3 h after being subjected to the stress, to allow the anti-methanogenic metabolite content to increase.

[0131] In one aspect, the present invention provides processed 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 the algal biomass before processing.

[0132] In one aspect, the present invention provides processed algal biomass comprising anti-methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g mg / g dry weight. For example, the present disclosure provides processed algal biomass comprising bromoform at from 15, 20, 40, 50, 60 or 70 mg / g dry weight.General

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] The following examples serve to more fully describe the manner of using the above-described 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

[0141] Further features of the present invention are more fully described in the following non-limiting 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 1Effect of Physical Stress on Bromoform ProductionMethodsSample collection

[0142] Asparagopsis taxiformis gametophytes were collected from Point Peron, Western Australia (32°16’31” S, 115°41 ’29” E). Samples were kept immersed in seawater to avoid temperature shock, osmotic shock, and over-exposure to sunlight, and transported in a cooler box filled with seawater from the collection site (~20 °C) for transportation to the Seaweed Aquaculture Research and Hatchery (SARaH) based at the Indian Ocean Marine Research Centre in Watermans Bay, Western Australia. The samples in seawater were stored in the 19 °C room.

[0143] At time 10 minutes, and then at each successive 60 minute interval for up to 3 h, 2g sub-samples of the seaweed were harvested for processing. Initial processing for all treatments (compression, agitation and sonication) involved dip rinsing the seaweed in chilled seawater (to prevent any further loss of bromoform), and then de-watered via centrifugation at 7 x g.

[0144] The seaweed samples were then subject to physical stresses as described below.Sample Processing

[0145] Control algal samples and algal samples exposed to stress to form processed algal biomasses were frozen at -81 °C. The frozen processed algal biomasses were freeze dried at - 55 °C and 200 pbar for 48 hours using a VirTis Benchtop Pro (SP Scientific, USA) (Magnusson et al., 2020). After freeze drying, the dried samples were ground with Spex2010 Geno / Grinder at 1200 rpm for 60 seconds and stored in -80 °C freezer before bromoform extraction.Bromoform Extraction

[0146] 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 withvortex mixer for 5 seconds. Sonication was performed to help break the cell walls within an ultrasonic bath (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

[0147] 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.

[0148] The method for bromoform analysis was based on Paul et al. (2006) 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-1and 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.

[0149] 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).Compression

[0150] To stimulate the Asparagopsis's defensive mechanism, leading to increased bromoform production, this study tested the mechanical force and percentage compression required for bromoform induction. The experimental set-up required a transparent tube open at both ends and with graduated markings every 1 cm interval from 1 -10cm. The graduated tube was used to determine the percentage of compression of the seaweed material (1 cm = 10% compression). The graduated tube has a plunger that fits neatly inside. The graduated tube isplaced on a sieve (1 mm mesh, 200mm diameter) and the entire set-up (graduated tube with plunger and sieve) is placed on top of digital scales to measure the amount of force in kg.

[0151] The graduated tube is filled with Asparagopsis seaweed to the top of the 10cm graduation. The seaweed, graduated tube, plunger and sieve are placed on top of the digital scale and the scale is re-zeroed. The plunger is inserted and pushed down on the seaweed at an initial increment of 1 cm (10cm to 9cm), the weight (in kg) is then recorded and seaweed removed for processing. This is repeated for each successive 10% increment. For example, the graduated tube is reloaded with Asparagopsis seaweed, the plunger is inserted and pushed down until there is 20% compression (ie 10cm to 8cm), and the weight (in kg) is recorded (Table 2). This process is repeated until 90% compression is achieved. After compression, the compressed seaweed is then removed from the graduated tube and placed in a 19 °C room for up to 3 hours.Table 2: Compressive forceResultsCompression

[0152] Physical stress by compression via centrifugation with an equivalent downward force of between 10-20% (2-7 Newtons) versus no compression resulted in a 44.7% increase in bromoform concentration (from 17.0 mg / g to 24.6 mg / g).Table 3: Effect of physical stress by compression on bromoform content

[0153] Given such a percentage increase in bromoform content by the applying of physical stress, if a starting algal biomass contained 20, 30, 40, 50, 60 or 70mg / g bromoform atharvest, the processed algal biomass may contain about 29, 44, 58, 72, 87 or 101 mg / g bromoform after applying of physical stress.Example 2Effect of UV Exposure on Bromoform ProductionMethodsSample collection

[0154] Asparagopsis taxiformis gametophytes were collected from Point Peron, Western Australia (32°16’31” S, 115°41 ’29” E). Samples were kept immersed in seawater to avoid temperature shock and over-exposure to sunlight and transported in a cooler box filled with seawater from the collection site (~20 °C) to keep cool for transportation to the Seaweed Aquaculture Research and Hatchery (SARaH) based at the Indian Ocean Marine Research Centre in Watermans Bay, Western Australia.

[0155] At time 10 minutes, and then at each successive 60 minute interval for up to three hours, 2g sub-samples of the seaweed, that have been stored in the 19 °C room, were harvested for processing. Initial processing involved dip rinsing the seaweed in chilled seawater to prevent any further loss of bromoform.

[0156] Asparagopsis seaweed samples were either dewatered (7 x g centrifugation), or retained in a moist state by being submerged in seawater, before exposure to UV light. Three intact thalli (sub-samples) were placed in each petri-dish. Images were taken of each sub-sample for later comparison after UV exposure.

[0157] Petri-dishes with Asparagopsis seaweed samples were uniformly exposed to UV- C light across several treatment durations (30, 60 and 180 minutes), alongside a control group shielded from UV exposure. This duration of UV treatments aimed to simulate varying levels of exposure to photophysiological stress. UV exposure duration was systematically altered, and the resulting processed algal biomass analysed to determine the resultant changes in seaweed colour, appearance and bromoform concentration.

[0158] Processed algal biomass was then frozen at -81 °C. Frozen processed algal biomass was freeze dried at -55 °C and 200 pbar for 48 hours using a VirTis Benchtop Pro (SP Scientific, USA) (Magnusson et al., 2020). After freeze drying, the dried sample was ground with Spex 2010 Geno / Grinder at 1200 rpm for 60 seconds and stored in -80 °C freezer before bromoform extraction.Sample processing, bromoform extraction and bromoform analysis

[0159] Sample preparation, bromoform extraction, and bromoform analysis to measure bromoform content was done as described in Example 1 .Results

[0160] Dewatered Asparagopsis fragments and seawater submerged Asparagopsis fragments were irradiated with UV light. A change in colour from light brown / white colouration to a pink / red hue was observed within 60 minutes after treatment (Figure 1). Asparagopsis fragments that were not exposed to UV light did not undergo a change of colour.

[0161] From previous observations (not provided), cells that are light brown to white in colour have a bromoform concentration that is typically less than the industry minimum of 6.5 mg / g. Cells that are pink to red colouration have a higher bromoform concentration.

[0162] In the present experiment, the Asparagopsis fragments changed from light brown / white to pink / red, indicating an increase in bromoform concentration. Based on previous measurements of bromoform content in different colours of algal biomass, it appears that the bromoform of the processed algal biomass of the present experiment results in a three-fold (15 mg / g) to seven-fold (40 mg / g) increase in bromoform concentration over Asparagopsis fragments not exposed to UV light.

[0163] Clear changes in bromoform-rich gland cell were also observed after Asparagopsis fragments were irradiated with UV light to produce processed algal biomass, compared to Asparagopsis fragments not irradiated with UV light (Figure 2). Bromoform-rich gland cells in Asparagopsis appear differently under magnification due to their greater density than water, and look like oil droplets.Example 3Effect of UV and Pressure on Bromoform ProductionMethods

[0164] All biomass was harvested from photobioreactors (PBRs) cultivated in sterile seawater enriched with L1 nutrient media. The PBRs were maintained under controlled conditions with continuous aeration at 6 L / min, a temperature of 19°C, and illumination at 50 pmol m-2s-1(white light) under a 12:12 light-dark photoperiod.

[0165] Immediately after harvesting, the biomass was placed onto a 200-micron metal sieve and dewatered by gently compressing the material against the sieve to determine the wet weight of each sample. A control sample (~12 grams) was immediately placed on dry ice for preservation.

[0166] The remaining biomass was divided into two equal portions to assess the effects of different environmental conditions. The first portion was transferred to a 20L bucket, and a photograph of the biomass was taken. A mercury thermometer was inserted into the sample, and the bucket was placed in direct sunlight under ambient conditions in Portarlington, Victoria, for two hours. The second portion was transferred to a separate 20L bucket, which was placed in a dark environment within a temperature-controlled room set to 24°C for the same duration.

[0167] Following the two-hour treatment period, both buckets were removed from their respective environments, and samples were dewatered using one of two methods: a gentle squeeze applied using a sieve (force in kg recorded) or a moderate squeeze using cotton cloth (force in kg recorded). Dewatered samples from each treatment were immediately transferred to well plates and placed on dry ice to preserve their biochemical integrity.

[0168] Sample processing, bromoform extraction and bromoform analysis

[0169] Sample preparation, bromoform extraction, and bromoform analysis to measure bromoform content was done as described in Example 1 .Results

[0170] Bromoform concentrations varied across treatments, with notable differences observed between light and dark conditions, as well as the applied pressure during dewatering (see Table 4). The control sample, which remained untreated, contained 6.09 mg / g of bromoform. In the outdoor treatments exposed to sunlight, bromoform concentrations were slightly affected. The sample dewatered using a sieve under 1 kg of pressure (Outside 1) showed a modest decrease of 5%, resulting in a bromoform concentration of 5.8 mg / g. In contrast, the sample dewatered using cheesecloth under 2.5 kg of pressure (Outside 2) exhibited a 10% increase, with a bromoform concentration of 6.7 mg / g.

[0171] In the indoor treatments conducted under dark, temperature-controlled conditions, bromoform concentrations increased substantially. The samples subjected to cheesecloth dewatering at 2.5 kg of pressure (Inside 1) showed the most significant increase. Replicate 1 contained 12.4 mg / g of bromoform (+103% relative to the control), while replicate 2 contained 12.7 mg / g (+109%). Similarly, the samples dewatered using a sieve at 1 kg of pressure (Inside 2) also displayed elevated bromoform concentrations, with both replicates measuring 8.8 mg / g, reflecting a 44% increase compared to the control.

[0172] These results indicate that dark, temperature-controlled conditions combined with increased pressure during dewatering substantially enhance bromoform retention, while exposure to sunlight and lower compression result in minimal changes.Table 4: Effect of physical stress (UV and pressure) on bromoform contentExample 4Effect of Osmotic Stress on Bromoform ProductionMethods

[0173] All biomass was harvested from photobioreactors (PBRs) cultivated in sterile seawater enriched with L2 nutrient media. The PBRs were maintained under controlled conditions with continuous aeration at 6 L / min, a temperature of 19°C, and illumination at 50 pmol m-2s-1(white light) under a 12:12 light-dark photoperiod.

[0174] Immediately after harvesting, about 10g the biomass by wet weight was placed onto a 200-micron metal sieve and dewatered to remove the seawater of the culture media. The dewatered biomass was immediately placed into 350 ml deionized water for 30 seconds at 20°C. The biomass from the deionized water was then removed to a 200-micron metal sieve and dewatered again to remove the deionized water. The dewatered sample was immediately frozen at -80 °C.Sample processing, bromoform extraction and bromoform analysis

[0175] Sample preparation, bromoform extraction, and bromoform analysis to measure bromoform content was done as described in Example 1 .Results

[0176] The bromoform content of the sample exposed to osmotic stress for 30 sec was 22 mg / g dry weight. This compares to between about 6 mg / g and 13 mg / g bromoform present incontrol samples of Asparagopsis grown in similar conditions but not exposed to osmotic stress. Osmotic stress treatment of the algal biomass resulted in a higher concentration of bromoform.Example 5Compression and DarknessMethodsBiomass Harvesting and Preparation

[0177] Asparagopsis taxiformis tetrasporophyte biomass was maintained with a 13 litre 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, and the total wet weight was recorded. Approximately 70 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 subsequent freeze-drying and further analysis.Experiment A: Compression

[0178] After the standard dewatering over a 200mm sieve, three 10 g samples of fresh Asparagopsis biomass were allocated for immediate manual compression where biomass was placed inside cheese cloth, wrapped up then manually squeezed by hand using one of three levels of compressive force (light [2.2 - 3 kg], medium [3.4 - 4.5kg], and hard [>6kg]) for a minimum of 3 seconds or until water no longer drained from the cheese cloth under that compressive force. The precise compressive force was measured using a dynamometer (Handexer). The wet weight of each sample was recorded before and after compression to quantify water loss. The % change in weight from water removal denotes the percentage of initial biomass weight lost as seawater expelled due to the compressive force.

[0179] The compressed biomass was then snap-frozen on dry ice, and stored in a six- well plate for subsequent freeze-drying and further analysis.Experiment B: Darkness and Compression

[0180] Three additional 10 g samples of Asparagopsis biomass were placed in 1 L glass bottles containing 800 mL of sterile seawater. These bottles were sealed and incubated in a lightproof cardboard box within a temperature control room. Light exclusion was verified using a LiCor PAR meter (LI250Q), with five measurements averaging 0.00 pmol m-2s-1Samples were maintained in complete darkness at 20°C for 2 hours.

[0181] After incubation in darkness, each sample was immediately manually compressed using a gloved fist and the dynamometer to measure the compressive force (medium pressure of between 3.4kg and 4.5kg). The wet weight of each sample was recorded before and after compression to quantify water loss. The de-watered biomass was then snap-frozen on dry ice, and stored for subsequent freeze-drying and further analysis.Freeze Drying samples

[0182] Following treatment and de-watering, all biomass samples were systematically placed in 6-well plates for preservation and subsequent analysis. Each experimental condition was assigned to a specific well, ensuring clear identification and tracking throughout the freeze- drying process.

[0183] 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 gradual sublimation 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 5: Freeze Dry Program for Lanphan HFD-6 Freeze drier

[0184] Bromoform concentration (mg / g) was quantified post-treatment using the Bromoform Extraction and Bromoform Analysis methods of Example 1 , with the percentage increase calculated relative to the untreated control.ResultsExperiment A: Compression

[0185] Fresh biomass samples (10 g) subjected to varying levels of compressive force (light: 3 kg, medium: 3.4 kg, and hard: 6.6 kg) exhibited progressive increases in bromoform concentration. Bromoform content showed a direct correlation with compression force, increasing from 8.57 mg / g in the control sample to 19.5 mg / g under the highest compression level, a 127.3% increase over the control (Table 6).Experiment B: Darkness and Compression

[0186] Incubation of biomass in complete darkness (0.00 pmol m-2s-1PAR, 20°C for 2 hours) after compression further enhanced bromoform concentration. The highest combined effect was observed in the hard compression-dark incubation treatment, where bromoform levels reached 20.3 mg / g, representing a 136.8% increase compared to the control (Table 5).Comparison Across Treatments

[0187] The effect of compressive force and / or post-harvest dark incubation on bromoform concentration in Asparagopsis taxiformis biomass was tested (Table 6).

[0188] Both compression and dark incubation significantly influenced biomass dehydration and bromoform accumulation. Medium and hard compression treatments resulted in higher bromoform concentrations, with the most substantial increases occurring when compression was combined with dark incubation. These findings highlight the potential for optimising post-harvest processing techniques to enhance bromoform yield in Asparagopsis taxiformis.Table 6: Effect of compressive force and post-harvest dark incubation on bromoform concentration

Claims

CLAIMS1 . A method for processing of algal biomass, comprising the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress; rHi. temperature stress; and / or iv. preharvest light change stress to produce the processed algal biomass, wherein the anti-methanogenic metabolite content of the processed algal biomass is higher after exposure to the stress than the anti-methanogenic metabolite content of the algal biomass before exposure to the stress.

2. The method of claim 1 , wherein the algal biomass and processed algal biomass are Asparagopsis taxiformis (A. taxiformis) and / or Asparagopsis armata (A. armata).

3. The method of claim 1 , wherein the anti-methanogenic metabolite is bromoform.

4. The method of claim 1 , wherein the physical stress to which the algal biomass is subjected is chosen from: compression, sonication, agitation, and solid and liquid shearing.

5. The method of claim 4, wherein the degree of force of the physical stress is sufficient to lead to an increase in the concentration of the anti-methanogenic metabolite content of the processed algal biomass without to disrupting the structure of the majority of the cells of the algal biomass.

6. The method of claim 1 , wherein the osmotic stress to which the algal biomass is exposed is exposure to water with a lower dissolved salt concentration than sea water.

7. The method of claim 6, wherein the exposure to the osmotic stress is sufficient to lead to an increase in the concentration of the anti-methanogenic metabolite content of the processed algal biomass without killing the majority of the cells of the algal biomass.

8. The method of claim 1 , wherein the preharvest light change stress is a change in the light intensity and / or colour temperature the algae in culture are exposed to 24 h or less before harvesting of the algal biomass.

9. The method of claim 8, wherein the exposure to the preharvest light change stress is applied to the algal biomass in culture for 24h before harvest.

10. The method of claim 1 , wherein the stress is administered for up to 120 minutes.1 1. The method of claim 1 , wherein the processed algal biomass is stored for period of from 5 min, 10 min, 30 minutes, 1 h 1.5 h, 2h, 2.5 h, 3 h or more than 3 h after being subjected to the stress.

12. The method of claim 1 , wherein the processed 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 the algal biomass before processing.

13. The method of claim 1 , wherein the processed algal biomass comprises anti-methanogenic metabolites at from 15, 20, 40, 50, 60 or 70 mg / g mg / g dry weight.

14. The method of claim 1 , wherein the algal biomass is exposed to two, three or four of the stresses to produce a processed algal biomass.

15. The method of claim 1 , wherein the stresses are carried out simultaneously or sequentially.

16. Use of a stress to produce a processed algal biomass that has a higher anti-methanogenic metabolite content than the anti-methanogenic metabolite content of an algal biomass before processing, comprising the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress;Hi. temperature stress; and / or iv. preharvest light change stress to produce the processed algal biomass.

17. A processed algal biomass that has a higher anti-methanogenic metabolite content than the anti-methanogenic metabolite content of an algal biomass before processing, wherein the processing comprises the step of: a) subjecting the algal biomass to a stress chosen from: i. physical stress; ii. osmotic stress;Hi. temperature stress; and / or iv. preharvest light change stress to produce the processed algal biomass.

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