Dual extraction method

Abstract

A dual extraction method is provided for isolating halogenated carbon compounds and pigments from biomass containing both. The method involves forming a slurry of the biomass in an aqueous solvent, incubating under conditions that disrupt cells and extract target compounds, and purging volatile halogenated carbon compounds using gas. The volatile extract is contacted with an organic solvent or oil to yield a halogenated carbon compound-rich extract and a depleted volatile phase. Pigments remain in the aqueous phase, which may be re-extracted. The method accommodates various biomass sources, including Asparagopsis species. Devices for performing the method are also disclosed.

Description

DUAL EXTRACTION METHODPRIORITY DOCUMENTS

[0001] The present application claims priority from Australian Provisional Patent Application No. 2024902461 titled “DUAL EXTRACTION METHOD” and filed on 8 August 2024, the content of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to a dual extraction method for extracting products having different physicochemical properties from a biomass. In one form, present disclosure relates to a dual extraction method for extracting bromoform and R-phycoerythrin from a biomass.BACKGROUND

[0003] Methane is a greenhouse gas with significant potency. Enteric fermentation in ruminant animals within the agricultural sector constitutes a primary anthropogenic source of methane. Due to growing anxieties regarding climate change as a result of global warming, various mitigation strategies are being actively implemented to achieve reductions in methane production1. Synthetic solutions have been explored; however, concerns regarding their long-term cost and safety have spurred a drive towards the development of natural solutions derived from biological sources2.

[0004] Certain biomass, such as marine biomass, contain aqueous and organic solvent or oil soluble compounds with commercial applications. An example is Asparcigopsis taxiformis, which is a subtropical marine seaweed classified within the phylum Rhodophyta (red algae). This seaweed flourishes in warm temperate waters and can be found along the coastlines of Western Australia, the Hawaiian Islands, Baja California (Mexico), and various locations off Southern California (including San Diego and Catalina Island)3,4. A. taxiformis has garnered recent attention for its potential to inhibit ruminant methanogenesis by exceeding 80%4. This remarkable capability is attributed to its ability to produce and accumulate elevated quantities of halogenated compounds, such as bromoform, dibromochloromethane, etc4,5. Consequently, various techniques have been developed to incorporate A. taxiformis into cattle feed. Common methods involve the inclusion of freeze-dried A. taxiformis biomass or the addition of A. taxiformis crude oil extract, with the latter method demonstrating superior stabilising effects on bioactive compounds6.

[0005] Beyond its antimethanogenic properties, Asparagopsis serves as a rich source of red photosynthetic pigment proteins, including R-Phycoerythrin (R-PE) and other phycobiliproteins7’8. R-PE is gaining significant traction as a viable alternative to synthetic dyes within the food, dairy, and cosmetic industries. Additionally, R-PE of analytical grade purity finds applications as fluorescent molecular probes, pharmaceuticals, and therapeutics9. Established methods exist for the extraction and purification of R-PE and other phycobiliproteins from seaweed. Physical tissue disruption techniques, such as maceration, homogenization, sonication and microwave-assisted extraction in appropriate buffers, have been demonstrated to be the most effective methods10.

[0006] It would be helpful if both bromoform and R-PE, could be extracted simultaneously from the same Asparagopsis biomass without affecting the extraction efficiency and purity of the products. This is challenging as bromoform is volatile in nature and needs to be extracted in an organic solvent or oil, while R-PE is a water-soluble pigment that must be extracted in an aqueous solvent. Similarly, it would be helpful if commercially useful compounds having different physicochemical properties could be extracted simultaneously from the same biomass without affecting the extraction efficiency and purity of the products.

[0007] There is thus a need to provide a method for the dual extraction of commercially useful compounds having different physicochemical properties, such as halogenated carbon compounds and / or pigments, that desirably improves upon existing methods.SUMMARY

[0008] Through research, the inventors have developed a dual extraction method suitable for extracting commercially useful compounds having different physicochemical properties from a biomass. In one application of the method, the inventors have exemplified a dual extraction method suitable for extracting one or more halogenated carbon compound and one or more pigment from a biomass of red seaweed of the genus Asparagopsis . Typically, extraction methods are specific for a single compound or a single class of compounds, e.g., a halogenated carbon compound or a pigment. Halogenated carbon compounds and pigments from Asparagopsis species are soluble in different solvents and so require different extraction techniques. The extraction of halogenated carbon compounds from Asparagopsis species through, e.g., submersion in oil, may indeed extract the halogenated carbon compounds, but it leaves an oil -rich biomass that is unsuitable for downstream uses. Other compounds extracted by the oil cause the oil to develop a gellike consistency, which upon further downstream processing can result in loss of the extract18. Extraction of halogenated carbon compounds using methanol leaves a toxic biomass that must be disposed of. The inventors have developed a process in view of these drawbacks.

[0009] Accordingly, in a first aspect, there is provided a method of extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, the method comprising the steps of: contacting the biomass with an aqueous solvent to provide a slurry; incubating the slurry under conditions and for a period of time sufficient to extract the one or more halogenated carbon compound and one or more pigment from the biomass to produce a halogenated carbon compound-rich volatile extract, an extracted biomass residue and a first extract comprising a pigment-rich aqueous solvent comprising the one or more pigment; contacting an organic solvent or oil with the halogenated carbon compound-rich volatile extract to provide a halogenated carbon compound-depleted volatile extract and a second extract comprising a halogenated carbon compound-rich organic solvent or oil comprising the one or more halogenated carbon compound; and optionally separately recovering the first and second extracts.

[0010] In certain embodiments, the first extract is recovered. In certain embodiments, the second extract is recovered.

[0011] In certain embodiments, the method further comprises the step of introducing a gas to the pigmentrich aqueous solvent within the slurry to purge the halogenated carbon compound -rich volatile extract from the slurry. In certain embodiments, the introduced gas is the halogenated carbon compound-depleted volatile extract. As such, the method further comprises the step of recirculating the halogenated carbon compound-depleted extract to the slurry to purge the halogenated carbon compound-rich volatile extract from the slurry.

[0012] In certain embodiments, the conditions under which the slurry is incubated disrupt cells of the biomass and comprise a disrupting process selected from the group consisting of: homogenising, macerating, sonicating, microwaving, enzyme treatment, freeze-thawing, heating and combinations thereof.

[0013] In certain embodiments, the method further comprises replacing the extracted biomass residue with a new biomass comprising both the one or more halogenated carbon compound and the one or more pigment.

[0014] In certain embodiments, the method further comprises re-extracting the extracted biomass residue by replacing the first extract with aqueous solvent to provide a slurry and incubating the slurry under conditions and for a period of time sufficient to extract more of the one or more pigment from the biomass to produce a re-extracted biomass residue and an additional first extract comprising a pigment-rich aqueous solvent comprising the one or more pigment.

[0015] In certain embodiments, the method comprises a pretreatment step, comprising incubating the biomass at a temperature below 0 degrees Celsius prior to forming the slurry.

[0016] In certain embodiments, the conditions under which the slurry is incubated comprise incubating the slurry at a temperature in the range of about 0 to about 60 degrees Celsius. In certain embodiments, the slurry is incubated at room temperature.

[0017] In certain embodiments, the aqueous solvent comprises a chelating agent. In certain embodiments, the chelating agent comprises one or more chelating agent selected from the group consisting of citric acid, acetic acid, carbonic acid, Tris-HCl, phosphoric acid, sodium phosphate and ethylenediaminetetraacetic acid (EDTA). In certain embodiments, the chelating agent is EDTA.

[0018] In certain embodiments, a pH of the aqueous solvent is between 4 and 9. In certain embodiments, the pH of the aqueous solvent is about 7.

[0019] In certain embodiments, a volume-to-weight ratio of organic solvent or oil to biomass is in the range of about 1 : 1 to 1 :200

[0020] In certain embodiments, the oil is an edible oil. In certain embodiments, the edible oil is a seed or vegetable oil.

[0021] In certain embodiments, a weight-to-weight ratio of the aqueous solvent to the biomass is in the range of 10: 1 to 1:2.

[0022] In certain embodiments, the biomass is from an Asparcigopsis species, Laminaria species, Rhodymenia species, Ulva species, Dictyosyphon species, Porosira species, Nitzschia species, Thalassiosira species, Synechococcus species, or Ditylum species.

[0023] In certain embodiments, the aqueous solvent causes cells of the biomass to lyse by osmotic shock to release the one or more halogenated carbon compound and the one or more pigment.

[0024] In certain embodiments, the one or more pigment comprises R-phycoerythrin, phycocyanin, allophycocyanin or combinations thereof. In certain embodiments, an absorbance ratio of the R- phycoerythrin in the pigment-rich aqueous solvent measured using A565 / A280 is in the range of 0.1 to 2.0.

[0025] In certain embodiments, yield of the R-phycoerythrin (DW) in the pigment-rich aqueous solvent is at least 1 mg / g biomass wet weight. In certain embodiments, the method further comprises recovering the first extract. In certain embodiments, the method further comprises isolating the one or more pigment from the recovered first extract. In certain embodiments, the method further comprises isolating the R- phycoerythrin from the recovered first extract.

[0026] In certain embodiments, a yield of the one or more halogenated carbon compound in the halogenated carbon compound-rich organic solvent or oil is at least 4 mg / g biomass dry weight. In certain embodiments, the method further comprises recovering the second extract.

[0027] In certain embodiments, the one or more halogenated carbon compound comprises bromoform, dibromochloromethane, dibromomethane, 1,2-dibromoethylene, tribromoethylene, bromochloroacetic acid or combinations thereof.

[0028] In a second aspect, there is provided a pigment extracted by the method of the first aspect.

[0029] In a third aspect, there is provided a halogenated carbon compound extracted by the method of the first aspect.

[0030] In a fourth aspect, there is provided an animal feed supplement comprising the recovered second extract with the one or more halogenated carbon compound or the one or more extracted halogenated carbon compound of the third aspect.

[0031] In a fifth aspect, there is provided an animal feed comprising the recovered second extract with the one or more halogenated carbon compound, the one or more extracted halogenated carbon compound of the third aspect or the supplement of the fourth aspect.

[0032] In a sixth aspect, there is provided a device for extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, comprising: a first vessel for containing a slurry comprising an aqueous solvent and the biomass; a second vessel for containing an organic solvent or oil; an outlet from the headspace of the first vessel; an inlet in the second vessel at a position below a surface level of the organic solvent or oil when contained in the second vessel; a first conduit connecting the first vessel outlet and the second vessel inlet.

[0033] In certain embodiments, the device further comprises an outlet from a headspace of the second vessel; an inlet in the first vessel at a position below a surface level of the aqueous solvent when contained in the first vessel; and a second conduit connecting the second vessel outlet and the first vessel inlet.

[0034] In certain embodiments, the device further comprises a pump to move a volatile extract through the first conduit from the first vessel to the second vessel.

[0035] In certain embodiments, the device further comprises an agitator in the first vessel.

[0036] In certain embodiments, the device further comprises a sparger in the second vessel and connected to the second conduit.

[0037] In certain embodiments, the biomass is from an Asparcigopsis species, Laminaria species, Rhodymenia species, Ulva species, Dictyosyphon species, Porosira species, Nitzschia species, Thalassiosira species, Synechococcus species, or Ditylum species.

[0038] In a seventh aspect, there is provided a method of extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, comprising performing the method of the first aspect using the device of the sixth aspect.

[0039] In an eighth aspect, there is provided a method of extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both the one or more organic solvent or oil soluble volatile compound and the one or more aqueous solvent soluble compound, the method comprising the steps of: contacting the biomass with an aqueous solvent to provide a slurry; incubating the slurry under conditions and for a period of time sufficient to extract the one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from the biomass to produce an organic solvent or oil soluble volatile compound -rich volatile extract, an extracted biomass residue and a first extract comprising an aqueous solvent soluble compoundrich aqueous solvent comprising the one or more aqueous solvent soluble compound; contacting an organic solvent or oil with the organic solvent or oil soluble volatile compound-rich volatile extract to provide an organic solvent or oil soluble volatile compound-depleted volatile extract and a second extract comprising an organic solvent or oil soluble volatile compound-rich organic solvent or oil comprising the one or more organic solvent or oil soluble volatile compound; and optionally separately recovering the first and second extracts.

[0040] In a ninth aspect, there is provided a device for extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound, comprising: a first vessel for containing a slurry comprising an aqueous solvent and the biomass; a second vessel for containing an organic solvent or oil; an outlet from the headspace of the first vessel; an inlet in the second vessel at a position below a surface level of the organic solvent or oil when contained in the second vessel; a first conduit connecting the first vessel outlet and the second vessel inlet.

[0041] In a tenth aspect, there is provided a method of extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both the one or more organic solvent or oil soluble volatile compound and the one or more aqueous solventsoluble compound, comprising performing the method of the eighth aspect using the device of the ninth aspect.BRIEF DESCRIPTION OF DRAWINGS

[0042] Embodiments of the present disclosure will be discussed with reference to the accompanying drawings wherein:

[0043] Figure 1 is a flow diagram of an embodiment of the method of the disclosure;

[0044] Figure 2 is a schematic diagram of an embodiment of the device of the disclosure;

[0045] Figure 3 is a schematic diagram of an embodiment of the device of the disclosure;

[0046] Figure 4 is a schematic diagram of an embodiment of the device of the disclosure;

[0047] Figure 5 is a schematic diagram of an embodiment of the device of the disclosure;

[0048] Figure 6 is a graph showing bromoform concentration (mg) in oil (ml) obtained for the baseline testing, intermediate level testing and upper limit testing extraction tests; and

[0049] Figure ? (a) is a photograph of the crude R-PE extracts after first round (Extract 1, El) of extraction, second round (Extract 2, E2), third round (Extract 3, E3) and fourth round (Extract 4, E4) of re -extraction, (b) is a graph showing the absorption spectra of the four R-PE extracts - Extract 1 (8-fold dilution), Extract 2 (1.3-fold dilution), Extract 3 and Extract 4. The insert shows an enlarged spectrum over 400nm to 600nm for Extract 1 which displays the characteristic R-PE absorption peaks.DESCRIPTION OF EMBODIMENTS

[0050] As mentioned above, through research, the inventors have developed a dual extraction method for extracting one or more halogenated carbon compound and one or more pigment from a biomass. The inventors have developed the method in view of the drawbacks of known methods.

[0051] Accordingly, in a first aspect, and as shown in Figure 1, there is provided a method of extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, the method comprising the steps of: contacting the biomass with an aqueous solvent to provide a slurry; incubating the slurry under conditions and for a period of time sufficient to extract the one or more halogenated carbon compound and one or more pigment from the biomass to produce a halogenated carbon compound-rich volatile extract, anextracted biomass residue and a first extract comprising a pigment-rich aqueous solvent comprising the one or more pigment; contacting an organic solvent or oil with the halogenated carbon compound-rich volatile extract to provide a halogenated carbon compound-depleted volatile extract and a second extract comprising a halogenated carbon compound-rich organic solvent or oil comprising the one or more halogenated carbon compound; and optionally separately recovering the first and second extracts.

[0052] Additionally, the method may allow for further utilisation of the spent biomass for other applications.

[0053] In certain embodiments, the method further comprises separately recovering the first and second extracts. In certain embodiments, the first extract is recovered. In certain embodiments, the second extract is recovered.

[0054] In certain embodiments, the method further comprises the step of introducing a gas to the pigmentrich aqueous solvent within the slurry to purge the halogenated carbon compound -rich volatile extract from the slurry. In certain embodiments, the introduced gas is air or an inert gas. In certain embodiments, the introduced gas is the halogenated carbon compound-depleted volatile extract. As such, in certain embodiments, the method further comprises the step of recirculating the halogenated carbon compound- depleted volatile extract to the slurry to purge the halogenated carbon compound-rich volatile extract from the slurry (e.g. the volatile extracts may move in a cyclical manner). This means that the halogenated carbon compound-rich volatile extract moves from the slurry to contact the organic solvent or oil, then the halogenated carbon compound-depleted volatile extract returns to the slurry to purge more of the halogenated carbon compound from the slurry. As would be appreciated by the person skilled in the art, a single passage of the halogenated carbon compound-rich volatile extract through the organic solvent or oil may remove only a portion of the one or more halogenated carbon compound. As such, the halogenated carbon compound-depleted volatile extract may contain residual or undissolved halogenated carbon compound(s). Further recirculation through the organic solvent or oil will remove more of the one or more halogenated carbon compound. This process may be repeated until, e.g., the organic solvent or oil is saturated with the one or more halogenated carbon compound, until a concentration of the one or more halogenated carbon compound in the organic solvent or oil stops increasing (e.g. remains substantially constant), or until a concentration of the one or more halogenated carbon compound in the halogenated carbon compound-rich volatile extract stops decreasing (e.g. remains substantially constant). In certain or alternative embodiments, the halogenated carbon compound-depleted volatile extract is purged to the atmosphere.

[0055] In certain embodiments, the organic solvent or oil is contacted with the halogenated carbon compound-rich volatile extract in a manner to increase contact between the solvent and the volatile extract. As the person skilled in the art would understand, an increase in the surface area of volatile extractcontacting the solvent enhances mass transfer of the one or more halogenated carbon compound from the volatile extract to the solvent or oil. In certain embodiments, mass transfer is increased by decreasing the size of volatile extract bubbles in the solvent or oil (e.g. by forming microbubbles). Bubble size may be decreased using a sparger, e.g., a porous material with small holes that allow the escape of the volatile extract into the solvent or oil, or by modulating the flow rate of volatile extract, e.g., using an oscillatory pattern. Typical sparger pore sizes are in the range of about 1 pm to about 200 pm, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or 60 pm. The person skilled in the art could readily modulate bubble size to increase mass transfer.

[0056] In certain embodiments, the aqueous solvent is prepared, and then the biomass is contacted with the aqueous solvent. In certain embodiments, the biomass is added to the aqueous solvent. In alternative embodiments, the aqueous solvent is added to the biomass. The present inventors have found that the aqueous solvent is particularly effective at extracting one or more halogenated carbon compound and one or more pigment as the aqueous phase causes an osmotic shock to the biomass and the cells of the biomass are disrupted. In certain embodiments, the aqueous solvent causes cells of the biomass to lyse by osmotic shock to release the one or more halogenated carbon compound and the one or more pigment. In certain embodiments, the temperature difference of the biomass and the aqueous solvent causes cell lysis by thermal shock to release the one or more halogenated carbon compound and the one or more pigment.

[0057] As above, the biomass comprises both the one or more halogenated carbon compound and the one or more pigment. In certain embodiments, the biomass is from a marine species. In certain embodiments, the biomass is from a marine microalga, marine macroalga or marine cyanobacterium. In certain embodiments, the biomass is from an Asparcigopsis species, Laminaria species, Rhodymenia species, Ulva species, Dictyosyphon species, Porosira species, Nitzschia species, Thalassiosira species, Synechococcus species, or Ditylum species. In certain embodiments, the biomass is from an Asparagopsis species. In certain embodiments, the Asparagopsis species is Asparagopsis taxiformis, A. armata, or A. svedelii. The Asparagopsis species may be at any of the different life stages. In certain embodiments, the biomass is from an A. taxiformis tetrasporophyte. In certain embodiments, the biomass is from an A. taxiformis gametophyte. The person skilled in the art would understand that the method can be applied to different biomass, such as Asparagopsis species from any life stage, and that the parameters of the method can be adjusted as appropriate for the particular biomass or life stage, e.g., the conditions and period of time could be adjusted to enhance extraction for a biomass or life stage with thicker and tougher cell walls.

[0058] In certain embodiments, the step of contacting the biomass is commenced within 12 hours of removal of the biomass from culture. In certain embodiments, the step of contacting the biomass is commenced within 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour or 1 minute of removal of the biomass from culture. The culture environment may include, e.g., the ocean where the biomass grows in the wild, or a controlled environment (e.g., aquarium, or laboratory). Commencing the step of contacting the biomass within 12hours of removal from culture may reduce the loss of bromoform. In other embodiments, the biomass is removed from culture and stored below 0 degrees Celsius until used in the contacting step. For example, between minus 20 and minus 80 degrees Celsius. As would be appreciated by the person skilled in the art, storage below the freezing temperature of water minimises bromoform loss and enables long-term storage of excess biomass until further use. In certain embodiments, the biomass is removed from culture and stored less than or equal to 4 degrees Celsius until used in the contacting step. As would be appreciated by the person skilled in the art, storage at a temperature less than or equal to 4 degrees Celsius minimises bromoform loss and enables long-term storage of excess biomass until further use.

[0059] In certain embodiments, the conditions under which the slurry is incubated disrupt cells of the biomass and comprise a disrupting process selected from the group consisting of: homogenising, macerating, sonicating, microwaving, enzyme treatment, freeze-thawing, heating and combinations thereof. As would be appreciated by the person skilled in the art, the conditions for the process of disrupting the cells would be determined based on the volume or weight of the biomass and the particular process. For example, sonicating may take 5-30 minutes to disrupt the cells, whereas homogenising may take 30 seconds to 30 minutes. The person skilled in the art could readily determine whether the cells were disrupted by visually examining the disrupted cells by, e.g., the naked eye or using a microscope. Suitable enzymes may include cell wall degrading enzymes, such as glycosyl-hydrolases, oxidoreductases, lyases, and esterases. Freeze-thawing may include adjusting the temperature below and above 0 degrees Celsius for one or more times to disrupt the cells. Heating may include increasing the temperature as described elsewhere herein with reference to the incubation.

[0060] In certain embodiments, the disrupting process is performed one or more time. In certain embodiments, the disrupting process is performed a plurality of times. In particular embodiments, the disrupting process is performed two or more times, such as two, three, four, five or six times. In certain embodiments, the disrupting process is performed for at least 30 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, the disrupting process is once, about every 1, 2, 5, 10, 20, 30, 40, or 50 minutes, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 11, 12 hours, daily, every second day, every third day, every fourth day, every fifth day, every sixth day, or weekly. In particular embodiments, the disruption process is performed continuously. In particular embodiments, the disruption process is performed continuously for 15 days. In particular embodiments, the disruption process is followed by agitation. In certain embodiments, the disruption process is followed by agitation with the agitation duration ranging from 30 minutes to continuous agitation for 15 days. In certain embodiments, the disruption is replaced by agitation.

[0061] Disrupting the cells releases the cell contents, including the one or more halogenated carbon compound and one or more pigment. As such, a disrupting process will cause the release of one or morehalogenated carbon compound and one or more pigment, which will be solubilised by the organic solvent or oil and aqueous solvent, respectively, over a period of time. Therefore, following a disrupting process, the concentration of one or more halogenated carbon compound in the organic solvent or oil will increase, until all of the released halogenated carbon compound(s) is solubilised (ie the concentration stops increasing). Then, the disrupting process may be performed again. In certain embodiments, the disrupting process is performed when a concentration of the one or more halogenated carbon compound in the organic solvent or oil stops increasing. The concentration of the one or more halogenated carbon compound in the organic solvent or oil may be determined using routine tests, such as methanol extraction followed by GC- MS. Alternatively, suitable methods include high-performance liquid chromatography (HPLC) with UV or MS detection, headspace gas chromatography with electron capture detection (GC-ECD), or nuclear magnetic resonance (NMR) spectroscopy. Where appropriate, the concentration may also be inferred from total halogen content determined by argentometric titration or elemental analysis.

[0062] In certain embodiments, the method further comprises replacing the extracted biomass residue with a new biomass comprising both the one or more halogenated carbon compound and the one or more pigment. The extracted biomass residue may be replaced with or without replacing the second extract (ie the halogenated carbon compound-rich organic solvent or oil comprising the one or more halogenated carbon compound). In certain embodiments, the extracted biomass residue is replaced with new biomass and the second extract is replaced with new organic solvent or oil. In alternative embodiments, the extracted biomass residue is replaced with new biomass and the second extract is not replaced with new organic solvent or oil. In certain embodiments, the extracted biomass residue is replaced with the new biomass between 1 and 10 times with or without replacing the second extract. The relative amounts of the new biomass and aqueous solvent may be as described elsewhere herein with reference to the biomass and aqueous solvent.

[0063] In certain embodiments, the method further comprises re-extracting the extracted biomass residue by replacing the first extract with aqueous solvent to provide a slurry and incubating the slurry under conditions and for a period of time sufficient to extract more of the one or more pigment from the biomass to produce a re-extracted biomass residue and an additional first extract comprising a pigment-rich aqueous solvent comprising the one or more pigment. In certain embodiments, the step of re-extracting the biomass is performed two, three, four or more times or until no more of one or more pigment is detected in the extract (e.g. additional first extract). The steps of re-extracting the biomass are performed according to the extracting step described elsewhere herein.

[0064] In certain embodiments, the method comprises a pretreatment step, comprising incubating the biomass at a temperature less than or equal to 4 degrees Celsius prior to forming the slurry. In certain embodiments, the pretreatment step comprises incubating the biomass at a temperature below -80, -70, -60, -50, -40, -30, -25-, -20, -15, -10 , -5, 0 or 4 degrees Celsius prior to forming the slurry. Lowering thetemperature below 0 degrees C may cause the biomass to freeze, which may facilitate disruption of the cells of the biomass as a result of thermal shock when thawed to room temperature . Lowering the temperature below 4 degrees C but above 0 degrees Celsius may facilitate disruption of the cells of the biomass as a result of thermal shock brought to room temperature. In certain embodiments, when the conditions under which the slurry is incubated do not comprise one of the above disruption processes (e.g., homogenising, sonicating, microwaving or enzyme treatment), the pretreatment step comprises incubating the biomass at a temperature below -80, -70, -60, -50, -40, -30, -25-, -20, -15, -10, -5 or 0 degrees Celsius. In certain embodiments, both pretreatment and disruption processes of the biomass are carried out. In any of the above embodiments when the biomass is incubated at a temperature below 0 degrees Celsius, a time period of the incubation may comprise at least 1 minute and up to, e.g., 5 years. In certain embodiments, the time period of the incubation may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 36, 27, 28, 29, 30 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. Alternatively, the above embodiments where the biomass is incubated at a temperature below 0 degrees Celsius may be optionally excluded.

[0065] In certain embodiments, the conditions under which the slurry is incubated comprise incubating the slurry at a temperature below 4 degrees Celsius prior to disrupting the cells. In certain embodiments, the conditions under which the slurry is incubated comprise incubating the slurry at a temperature below - 80, -70, -60, -50, -40, -30, -25-, -20, -15, -10, -5, 0 or 4 degrees Celsius prior to disrupting the cells. In certain embodiments, the slurry is incubated at the temperature below 4 degrees Celsius to lower the temperature of the slurry to a temperature above 4 degrees C. In other embodiments, the slurry is incubated at the temperature below 4 degrees Celsius to lower the temperature of the slurry to a temperature below 4 degrees C. Lowering the temperature below 0 degrees C may cause the aqueous solvent to freeze, which may facilitate disruption of the cells of the biomass. In alternative embodiments, when the conditions under which the slurry is incubated do not comprise one of the above disruption processes (e.g., homogenising, sonicating, microwaving or enzyme treatment), the conditions under which the slurry is incubated may comprise incubating the slurry at a temperature below -80, -70, -60, -50, -40, -30, -25-, -20, -15, -10, -5, 0 or 4 degrees Celsius. In any of the above embodiments when the slurry is incubated at a temperature below 4 degrees Celsius, a time period of the incubation may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 36, 27, 28, 29, 30 minutes, or 1, 2, 3, 5, 6, 8, 10, or 12 hours. The slurry may then be incubated at a higher temperature, as detailed below. Alternatively, the above embodiments when the slurry is incubated at a temperature below 4 degrees Celsius may be optionally excluded.

[0066] In certain embodiments, the conditions under which the slurry is incubated comprise incubating the slurry at a temperature in the range of about 0 to about 60 degrees Celsius. However, the person skilled in the art would appreciate the halogenated carbon compound(s) may be volatile and release of the one ormore halogenated carbon compound from the biomass may increase at temperatures above 30 degrees Celsius. In certain embodiments, the slurry is incubated at a temperature between 1 and 38 degrees Celsius, such as between 4 and 30 degrees Celsius, or between 10 and 30 degrees Celsius, or at room temperature, such as about 18 to about 25 degrees Celsius. In particular embodiments, the slurry is incubated at room temperature. In certain embodiments, room temperature is 20-25 degrees Celsius. In particular embodiments, the slurry is incubated at a temperature appropriate for the maximum activity of the enzyme used for disrupting the cells, e.g., between about 20 and about 38 degrees Celsius. In particular embodiments, the slurry is incubated at a temperature between about 50 and about 60 degrees Celsius to liberate the one or more halogenated carbon compound from the biomass, which can then be trapped and dissolved in the organic solvent without compromising the one or more pigment. At such an elevated temperature, one or more halogenated carbon compound may be liberated from the biomass as a volatile compound which might otherwise be non-volatile, e.g., at room temperature.

[0067] The slurry is incubated for a period of time to extract the one or more halogenated carbon compound and one or more pigment. In certain embodiments, the slurry is incubated until complete extraction of the one or more halogenated carbon compound and one or more pigment. In certain embodiments, “complete extraction” refers to a state in which at least 90%, 95%, 97%, 98%, 99% or substantially all of the extractable halogenated carbon compound and / or pigment originally present in the biomass is transferred into the respective solvent phases. As mentioned above, the period of time could readily be determined by the person skilled in the art based on the volume or weight of the biomass and any process of cell disruption. The person skilled in the art could determine whether the one or more pigment had been extracted into the aqueous solvent by, e.g., visually examining the biomass for the characteristic colour of the one or more pigment (e.g. the pink colour of R-phycoerythrin) or absence thereof, in the biomass or spectroscopically based on their characteristic peaks at 499, 565 nm and a 545 shoulder or purity index A565 / A280. In certain embodiments, complete extraction of the pigment may be inferred when the biomass lacks visible pigment and the absorbance spectrum or A565 / A280 value remains substantially unchanged in subsequent extractions, or when less than 5%, 2%, or 1% of the initial yield is obtained upon re -extraction. The person skilled in the art could determine whether one or more halogenated carbon compound had been extracted into the organic solvent or oil using routine tests, e.g., performing a methanol extraction of the halogenated carbon compound-rich organic solvent or oil and the extracted biomass residue to test for the one or more halogenated carbon, as described elsewhere in this disclosure. In certain embodiments, complete extraction of the halogenated carbon compound may be inferred when the concentration in the organic solvent or oil ceases to increase over time or additional extractions yield less than 5%, 2%, or 1% of the initial quantity.

[0068] In certain embodiments, the period of time of the incubation is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 18, about 24, about 30, about 36, about 42 or about 48 hours, about 3, about 4, about 5, about 6 or about 7 days, or about 2, about3, or about 4 weeks. In certain embodiments, the period of time of the incubation is between 1 minute and 2 months, for example, between 1 minute and 2 weeks, 10 minute and 1 week, 30 minutes and 3 days, 1 hour and 2 days, 2 hours and 1 day, 5 hours and 12 hours, or about 8 hours, 1 day and 15 days, 1 day and 30 days, 1 day and 60 days. In particular embodiments, the period of time of the incubation is between 6 hours and 1 month. In certain embodiments, the period of time of the incubation is between 1 day and 1 month. If sonicating is used for cell disruption, the period of time of the incubation may, in certain embodiments, be at least 1 minute, e.g. between 6 hours and 1 month, 1 day and 1 month, 1 minute and 1 week, 10 minutes and 2 days, 30 minutes and 1 day, 1 minute and 1 hour, 2 minutes and 30 minutes, or about 10, 15, 20, 25 or 30 minutes. If homogenising is used for cell disruption the period of time of the incubation may be at least 1 minute, e.g. between 1 minute and 1 week, 10 minutes and 2 days, 30 minutes and 1 day, 1 minute and 1 hour, 2 minutes and 30 minutes, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes. If microwaving is used for cell disruption, the period of time of the incubation may be at least 1 minute, e.g., between 6 hours and 1 month, 1 day and 1 month, 30 seconds to 30, 1 minute and 20 minutes, 2 minutes and 15 minutes, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes. If an enzyme is used for cell disruption, the period of time of the incubation may be at least 1 minute, e.g., between 6 hours and 1 month, 1 day and 1 month, 1 minute and 12 hours, 2 minutes and 6 hours, 5 minutes and 4 hours, 10 minutes and 2 hours, or about 20, 40 or 60 minutes, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. For any of the cell disruption processes, the person skilled in the art could readily adjust the conditions to ensure that the cells were ruptured. Alternatively, when the conditions under which the slurry is incubated do not comprise one of the above disruption processes (e.g., homogenising, sonicating, microwaving or enzyme treatment), the incubation time to extract the one or more halogenated carbon compound and one or more pigment may be considerably longer, such as between 1 day and 2 months. The slurry may be periodically agitated, e.g., by shaking or stirring, to expose the surfaces of the biomass to the aqueous solvents. In certain embodiments, the slurry is agitated for a period of time of at least 1 minute, e.g. between 1 minute and 1 hour, 2 minutes and 30 minutes, or about 10, 15, 20, 25, 30 minutes or continuously agitated. The slurry may be agitated as above and as appropriate, e.g., every minute, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 60 minutes, 2 hours, 4 hours, 8 hours, 12 hours, every 24 hours or continuously stirred or agitated.

[0069] In certain embodiments, the aqueous solvent comprises a chelating agent. In certain embodiments, the chelating agent comprises one or more chelating agent selected from the group consisting of citric acid (e.g. citrate buffer), acetic acid (e.g. acetate buffer), carbonic acid (e.g. carbonate buffer), Tris-HCl (e.g. Tris-HCl buffer), phosphoric acid (e.g. phosphate buffer), sodium phosphate or EDTA. In particular embodiments, the chelating agent is EDTA. In certain embodiments, the EDTA is at least one of disodium EDTA (EDTA 2Na), sodium calcium EDTA (EDTA Na Ca), or tetrasodium EDTA (EDTA 4Na). In particular embodiments, the EDTA is disodium EDTA (EDTA 2Na). In certain embodiments, the chelating agent is provided in the aqueous solvent at a concentration of at least 0.0 ImM, for example, between 0.01and 200mM, 0.01 and lOOmM, 0.05 and 50mM, 0.05 and 20mM, 0.1 and 5mM, 0.1 and 2mM, 0.1 and ImM. In certain embodiments, the chelating agent is provided in the aqueous solvent at a concentration of 0.01 to 50 mM. In certain embodiments, the chelating agent is provided at a concentration of about 5 mM. By way of non-limiting example only, particular concentrations may be preferable for different chelating agents, e.g., citrate buffer (about lOOmM), acetate buffer (about lOOmM), carbonate buffer (about lOOmM), Tris-HCl buffer (about 50mM), phosphate buffer (about 50 mM), and EDTA (about ImM). The concentration of the chelating agent may be readily determined by the person skilled in the art, who would understand that the concentration could be optimised based on the composition of the extract , then adjusting the concentration of chelating agent as appropriate. In particular embodiments in which the chelating agent is EDTA, the concentration is about ImM, or between about 0.05mM and 5mM.

[0070] In certain embodiments, the aqueous solvent is at a pH between about 4 and about 9. The person skilled in the art would understand that the one or more pigment should be relatively stable at a pH between 4 and 9. However, the present inventors have found that when the pigment is R-phycoerythrin, the pigment is sensitive to pH changes. As such, the pH of the aqueous solvent should preferably be adjusted to between 4 and 9 before forming the slurry. In certain embodiments, the pH is 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9. Preferably, the pH is close to neutral or between about 6 and 8. In particular embodiments, the pH is about 7, e.g., 6.7, 6.8. 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8. By way of non-limiting example only, particular pH ranges may be preferable for different chelating agents, e.g., citrate buffer (pH 5.0), acetate buffer (pH 6.0), carbonate buffer (pH 9.6), Tris-HCl buffer (pH 7.2), phosphate buffer (pH 6.7), and EDTA (pH 7.3). The person skilled in the art would understand that the pH could be optimised by starting with a given biomass and a given chelating agent at one of the above pH ranges, and then adjusting the pH as appropriate.

[0071] In certain embodiments, the organic solvent is any suitable solvent that can solubilise the one or more halogenated carbon compound. In certain embodiments, the organic solvent or oil is substantially non-volatile. The person skilled in the art would appreciate that a solvent or oil with a boiling point below 100 degrees Celsius is considered to be volatile, whereas that with a boiling point of 100 degrees Celsius or higher is considered to be non-volatile. In certain embodiments, the organic solvent is one or more of acetone, benzene, chloroform, dichloromethane, diethyl ether, ethanol, hexane, methanol, petroleum ether, pyridine, toluene, xylene. As would be appreciated by the person skilled in the art, some organic solvents are highly volatile or toxic, and so may be unsuitable, depending upon the downstream application of the produced second extract comprising the halogenated carbon compound-rich organic solvent. As such, in certain embodiments, an oil may be preferable. In certain embodiments, the oil is an edible oil. In certain embodiments, the edible oil is a seed or vegetable oil. In certain embodiments, the edible oil may be selected from the group consisting of almond oil, apricot oil, argan oil, avocado oil, Brazil nut oil, canola oil, cashew oil, coconut oil, colza oil, com oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seedoil, grapeseed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, pecan oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil, and vegetable oil or any combination thereof. In particular embodiments, the edible oil is selected from the group consisting of soy, safflower, cotton, linseed, peanut, olive, sunflower, canola, rape seed, com, palm oil and combinations thereof. In particular embodiments, the edible oil is soy, sunflower, canola, com, rapeseed oil, or combinations thereof.

[0072] The method may be advantageously conducted to minimise the use of edible oil. In certain embodiments, a volume-to-weight ratio of organic solvent to biomass is in the range of about 1 : 1 to 1 :200. For example, a 1: 1 ratio would mean 1 litre of organic solvent and 1 kilogram (kg) of biomass. In certain embodiments, a volume-to-weight ratio of organic solvent or oil to biomass is about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10, about 1: 15, about 1:20: about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1: 100, about 1: 150 or about 1:200. In particular embodiments, a volume-to-weight ratio of organic solvent or oil to biomass in the range of about 1:5 to 1:25, about 1: 10 to about 1: 15, or about 1 : 20 to about 1 : 50. As would be appreciated by the person skilled in the art, the ratio of organic solvent to biomass could be calculated on a weight-to-weight ratio. If we use canola oil as an example, the person skilled in the art would understand that the density of canola oil is approximately 0.914-0.917 kg / 1 at 20°C, therefore, they could convert a quantity of oil at 20°C from kg to litres by dividing by, e.g., 0.917, or could convert a quantity of oil at 20°C from litres to kg by multiplying by, e.g., 0.917.

[0073] In certain embodiments, a weight-to-weight ratio of the aqueous solvent to the biomass is in the range of 10: 1 to 1:2. In certain embodiments, a weight-to-weight ratio of the aqueous solvent to the biomass is 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.5: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, 0.5: 1 or 1:2. In particular embodiments, a weight-to-weight ratio of the aqueous solvent to the biomass is in the range of about 4: 1 to about 1: 1, such as about 3: 1 to about 1.5: 1, about 2: 1 or about 1:2. The person skilled in the art would appreciate that efficient extraction of the one or more pigment would occur when all of the biomass was exposed to the aqueous solvent. As such, in certain embodiments, a sufficient volume of aqueous solvent is used to submerge the biomass.

[0074] As mentioned above, the method involves separately recovering the first and second extracts. As such, there may be a first extract, a second extract and the extracted biomass residue. The first extract may be separated from the extracted biomass residue by filtration using, e.g., a membrane or sieve. The membrane or sieve may have an appropriate pore size to retain the spent biomass residue, e.g., at least 0.2 pm, such as between about 100 pm and about 0.5pm, or about 5 and about 0.4pm, or about 10 to about 0.22pm.

[0075] In certain embodiments, the method further comprises isolating the one or more pigment from the pigment-rich aqueous solvent. The person skilled in the art would appreciate that suitable methods of isolating the one or more pigment include, e.g., concentrating and then precipitating the pigment by, e.g., saturating the solution with ammonium sulphate or with magnesium sulphate . The isolated pigment most commonly finds application as a natural pigment or molecular label and as an active ingredient in cosmeceuticals, nutraceuticals, and biomedicine. In certain embodiments, the one or more pigment comprises R-phycoerythrin, phycocyanin, allophycocyanin or combinations thereof. In certain embodiments, the method further comprises isolating the one or more pigment from the recovered first extract. In particular embodiments, the pigment comprises R-phycoerythrin. In certain embodiments, the method further comprises isolating the R-phycoerythrin from the recovered first extract.

[0076] The disclosed method provides a pigment-rich aqueous solvent with the one or more pigment. A purity of the pigment may be determined by measuring absorbance using a spectrophotometer and calculating an absorbance ratio using A565 / A280. In certain embodiments, an absorbance ratio of the pigmentrich aqueous solvent measured using A565 / A280 is greater than 0. 1. In certain embodiments, an absorbance ratio of the pigment-rich aqueous solvent measured using A565 / A280 is greater than 0.6. In certain embodiments, the absorbance ratio of the pigment-rich aqueous solvent measured using A565 / A280 is greater at least 1. In certain embodiments, the absorbance ratio of the pigment-rich aqueous solvent measured using A565 / A28o is greater than 0.1, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1.0, 1,1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, or 3.0. In certain embodiments, an absorbance ratio of the R-phycoerythrin in the pigment-rich aqueous solvent measured using A565 / A280 is in the range of 0. 1 to 2.0. The person skilled in the art would understand that the absorbance ratio of A565 / A280 is a commonly used measure of purity and that other methods could also be used. As the person skilled in the art would appreciate, the pigment-rich aqueous solvent is a crude extract. The one or more pigment may be separated from the other molecules in the pigment -rich aqueous solvent using various methods, for example, ultrafiltration or (NH^SC precipitation followed by gel filtration and ion-exchange chromatography. Following such a separation technique, the absorbance ratio measured using A565 / A280 will increase, e.g., to be greater than 3 or 4.

[0077] As above, in certain embodiments, the one or more pigment comprises R-phycoerythrin. The method extracts the R-phycoerythrin from the biomass into the aqueous solvent to provide a pigment-rich (e.g. an R-phycoerythrin-rich) aqueous solvent. The concentration of R-phycoerythrin in the pigment-rich (e.g. R-phycoerythrin-rich) aqueous solvent may vary, depending upon the amount of biomass and the amount of aqueous solvent and the type of biomass. As would be appreciated by the person skilled in the art, a lower volume of the aqueous solvent containing all of the extractable R-phycoerythrin from the biomass will have a higher R-phycoerythrin concentration than a higher volume of the aqueous solvent containing the same amount of R-phycoerythrin. In certain embodiments, a concentration of the R- phycoerythrin (DW) in the pigment-rich (e.g. R-phycoerythrin-rich) aqueous solvent is at least 0.5 mg / g A.taxiformis wet weight. In certain embodiments, a concentration of the R-phycoerythrin (DW) in the pigment-rich (e.g. R-phycoerythrin-rich) aqueous solvent is at least 1 mg / g A. taxiformis wet weight. As the wet weight was found to be approximately six times the dry weight, the concentration of R- phycoerythrin on a dry weight basis is 6-times higher. As such, in certain embodiments, a concentration of the R-phycoerythrin (DW) in the pigment-rich (e.g. R-phycoerythrin-rich) aqueous solvent is at least 3 mg / g A. taxiformis dry weight. In certain embodiments, a concentration R-phycoerythrin (DW) in the pigment-rich (e.g. R-phycoerythrin-rich) aqueous solvent is 0.5 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 36, 27, 28, 29, 30, 35, 40, 45, 50, 55 or 60 mg / g A. taxiformis wet weight, which equates to 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 216, 162, 168, 174, 180, 210, 240, 270, 300, 330 or 360 mg / g A. taxiformis dry weight.

[0078] The method extracts the one or more halogenated carbon compound from the biomass into the organic solvent or oil to provide the halogenated carbon compound-rich organic solvent or oil (ie the second extract). The concentration of the one or more halogenated carbon compound in the halogenated carbon compound-rich organic solvent or oil may vary, depending upon the amount of biomass and the amount of organic solvent or oil. As would be appreciated by the person skilled in the art, a lower volume of organic solvent or oil containing all of the extractable halogenated carbon compound from the biomass will have a higher halogenated carbon compound(s) concentration than a higher volume of organic solvent or oil containing the same amount of halogenated carbon compound. As would be apparent to the person skilled in the art, an A. taxiformis tetrasporophyte may contain a lower amount of halogenated carbon compound(s) (such as bromoform) in a given quantity of biomass, compared to the A. taxiformis gametophyte. In certain embodiments, a concentration of the one or more halogenated carbon compound in the halogenated carbon compound-rich organic solvent or oil is at least 5 mg / mL. In particular embodiments, a concentration of the one or more halogenated carbon compound in the halogenated carbon compound-rich organic solvent or oil is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg / mL. In particular embodiments, a concentration of the one or more halogenated carbon compound in the halogenated carbon compound-rich organic solvent or oil is in the range of about 5 to about 60mg / mL, about 8 to about 30 mg / mL or about 8 to about 17 mg / mL. In certain embodiments, a yield of the one or more halogenated carbon compound (mg) extracted per gram of dry weight (dw) of A. taxiformis is at least 4 mg. In certain embodiments, a mass of the one or more halogenated carbon compound (mg) extracted per gram of dry weight (dw) of A. taxiformis is at least 4, 5, 6, 7, 8, 9 or 10 mg. In certain embodiments, a mass of the one or more halogenated carbon compound (mg) extracted per gram of dry weight (dw) of A. taxiformis is in the range of about 4 to about 8 mg, or about 4 to about 6 mg.

[0079] As above, the method further comprises recovering the second extract. In certain embodiments, the method further comprises isolating the one or more halogenated carbon compound from the recoveredsecond extract. As such, in certain embodiments, the method further comprises isolating the one or more halogenated carbon compound from the one or more halogenated carbon compound-rich organic solvent or oil. In alternative embodiments, the one or more halogenated carbon compound is not isolated from the halogenated carbon compound-rich organic solvent or oil. Rather, the halogenated carbon compound-rich organic solvent or oil is the final product or may be used as an ingredient for formulated products. Formulated products might include, e.g., animal feed or supplements. In certain embodiments, the one or more halogenated carbon compound comprises bromoform, dibromochloromethane, dibromomethane, 1,2- dibromoethylene, tribromoethylene, bromochloroacetic acid or combinations thereof.

[0080] In a second aspect, there is provided a pigment extracted by the method of the first aspect. As mentioned, in certain embodiments, the one or more pigment comprises R-phycoerythrin, phycocyanin, allophycocyanin or combinations thereof. As would be appreciated by the person skilled in the art, R- phycoerythrin in soluble form (e.g., in an aqueous solution) may take the form of a high -concentration liquid. The high-concentration liquid may contain dissolved solutes, such as a chelating agent as found in the aqueous solvent and include other features of the aqueous solvent, such as the disclosed pH. The person skilled in the art would also understand that the solution could contain dissolved or suspended phycoerythrin in ultrapure water. The extracted R-phycoerythrin finds application as a fluorescent probe used in food, cosmetics, immunodiagnostics, analytical reagents and electrophoretic procedures.

[0081] In a third aspect, there is provided a halogenated carbon compound extracted by the method of the first aspect. As above, the halogenated carbon compound may comprise bromoform, dibromochloromethane, dibromomethane, 1,2-dibromoethylene, tribromoethylene or bromochloroacetic acid. As would be appreciated by the person skilled in the art, a purified halogenated carbon compound, such as bromoform, may take the form of a colourless liquid at room temperature. However, it may also be stored in alcohol, benzene, chloroform, ether, petroleum ether, acetone and oils. As such, the halogenated carbon compound, such as bromoform, extracted by the method of disclosure may take the form of a bromoform -rich organic solvent or oil (e.g. a halogenated carbon compound -rich organic solvent or oil). The extracted bromoform or bromoform-rich organic solvent or oil (or halogenated carbon compound-rich organic solvent or oil) may find application in animal feed supplements or animal feeds to reduce methane emissions.

[0082] In a fourth aspect, there is provided an animal feed supplement comprising an effective amount of the second extract (ie the halogenated carbon compound-rich organic solvent or oil ) of the method of the disclosure or the extracted halogenated carbon compound of the disclosure. The animal feed supplement may be in any form that is suitable for feeding to an animal, particularly a ruminant. For example, the supplement may be a liquid (e.g., dissolved or suspended in a carrier, such as water or oil), or a solid. Suitable solid forms include powders (e.g., dried, lyophilised, ground, milled), granulates, coated carriers (e.g., spray-dried granules), and the like.

[0083] In a fifth aspect, there is provided an animal feed comprising an effective amount of the second extract (ie the halogenated carbon compound-rich organic solvent or oil) of the method of the disclosure, the extracted halogenated carbon compound of the disclosure or the supplement of the disclosure. The animal feed may be in any form that is suitable for feeding to an animal, particularly a ruminant. The animal feed may be in the form of a block (e.g., a lick -block or a fat block), a blend (e.g., with a meal, such as rapeseed meal, flaxseed meal, cottonseed meal, soybean meal or cornmeal), or a diet supplement (such as a milk supplement), or added to the animal's normal drinking water (either dissolving or remaining suspended in the water).

[0084] In certain embodiments, the animal is ruminant. In certain embodiments, the ruminant is a domesticated ruminant, for example, in particular embodiments, the ruminant is a cow, goat, sheep, llama, alpaca, bison, elk, reindeer, yak, deer, buffalo, or camel.

[0085] In certain embodiments, the effective amount is 0.01% to 5% dry weight of the ruminant's diet. In particular embodiments, the effective amount is 0.02%, 0.05%, 0.1%, 0.5%, 1%, 1.5, 2%, 2.5% or 3% dry weight of the ruminant's diet. For example, if the ruminant consumes 10 kg of feed per day, then the effective amount is between about 2 g and 300 g. The person skilled in the art would be able to formulate an appropriate dosage form and regime guided by the published literature, e.g., WO2015109362A2. For example, in certain embodiments, the administering is daily, every second day, every third day or every fourth day. In particular embodiments, the administering occurs every time the animal is fed. In embodiments, administering the supplement or feed according to the above regimes occurs for a period of at least 5, 10, 25, 50, 100, 250 or 350 days.

[0086] Figures 2, 3, 4 and 5 illustrate embodiments of a device for extracting one or more halogenated carbon compound and one or more pigment from a biomass of an Asparagopsis species. Advantageously, the device may be used to practice the method of the first aspect. As such, references to features in the Figures 2, 3, 4 and 5 are to be understood in the context of the method of the first aspect or as described elsewhere herein, unless otherwise indicated.

[0087] In a sixth aspect, and as illustrated in Figures 2, there is provided a device (10) for extracting one or more halogenated carbon compound and one or more pigment from a biomass (14) comprising both the one or more halogenated carbon compound and the one or more pigment, comprising: a first vessel (12) for containing a slurry comprising an aqueous solvent (16) and the biomass (14); a second vessel (26) for containing an organic solvent or oil (32); an outlet (23) from the headspace (13) of the first vessel, an inlet (28) in the second vessel (26) at a position below a surface level of the organic solvent or oil (32) when contained in the second vessel (26), and a first conduit (22) connecting the first vessel outlet (23) and the second vessel inlet (28).

[0088] As would be appreciated from the method of the first aspect, a biomass (14) and an aqueous solvent (16) are contained in the first vessel (12) and provide a slurry. The slurry may be incubated under conditions and for a period of time sufficient to extract the one or more halogenated carbon compound and the one or more pigment from the biomass to produce a halogenated carbon compound-rich volatile extract, an extracted biomass residue and a first extract comprising a pigment-rich aqueous solvent comprising the one or more pigment. The conditions may comprise, for example, homogenising, macerating, sonicating, microwaving, enzyme treatment, freeze-thawing, heating and combinations thereof, as described elsewhere herein. The halogenated carbon compound-rich volatile extract from the headspace (13) of the first vessel (12) moves through the first conduit (22) to the organic solvent or oil (32) when contained in the second vessel (26). The organic solvent or oil is contacted with the halogenated carbon compound-rich volatile extract to provide a halogenated carbon compound-depleted volatile extract and a second extract comprising a halogenated carbon compound-rich organic solvent or oil comprising the one or more halogenated carbon compound. At the end of the extraction, the first and second extracts may be separately recovered from the first and second vessels, respectively.

[0089] The first vessel (12) may be any suitable size, shape or material for containing the aqueous solvent and the biomass (14). Suitable materials include non-reactive materials, e.g., glass, stainless steel, plastic or aluminium. In certain embodiments, other than the first conduit (22) connected to the headspace (13) of the first vessel (12), the vessel is hermetically sealed.

[0090] As shown in Figures 2 to 5, the first conduit is sized and shaped to allow the movement of the halogenated carbon compound-rich volatile extract (34) from the headspace of the first vessel (12) to the second vessel (26). In embodiments, the outlet (23) is positioned at a side wall or top of the vessel (12). As shown in Figures 2 to 5, the first conduit is positioned so that the halogenated carbon compound-rich volatile extract exits the first conduit beneath a surface of the organic solvent or oil (32). The inlet (28) is at a position below a surface level of the organic solvent or oil (32) when contained in the second vessel (26). The first conduit may be connected to the second vessel (26) at a position below (Figures 2, 3, and 5) or above (Figure 4) a surface level of the organic solvent or oil (32) when contained in the second vessel (26). As such, the first conduit may enter the second vessel at any position, e.g., through a top, side wall or bottom of the vessel.

[0091] In certain embodiments and as shown in Figures 3, 4 and 5, the device further comprises an outlet (37) from a headspace (27) of the second vessel (26); an inlet (38) in the first vessel (12) at a position below a surface level of the aqueous solvent (16) when contained in the first vessel (12); and a second conduit (36) connecting outlet (37) and the inlet (38). The second conduit is for the purpose of introducing a gas to the pigment-rich aqueous solvent (ie the first extract) in the first vessel within the slurry to purge the halogenated carbon compound-rich volatile extract from the slurry. In certain embodiments, other than the second conduit (36) connected to the headspace (27) of the second vessel (26) and the first conduit (22)connected to the first vessel (12), the second vessel is hermetically sealed. In certain embodiments, other than the first conduit (22) connected to the headspace (13) of the first vessel (12) and the second conduit (36) connected to the second vessel (26), the first vessel is hermetically sealed. The second conduit is sized and shaped to allow the movement of the halogenated carbon compound -depleted volatile extract (40) from the headspace of the second vessel (20) to the first vessel (12). In embodiments, the second conduit is connected to a side wall or top of the second vessel (26). As shown in Figures 3 to 5, the second conduit (36) is positioned so that the halogenated carbon compound -depleted volatile extract (40) exits the conduit beneath a surface of the aqueous solvent (16). In embodiments, the outlet (37) is positioned at a side wall or top of the vessel (26). The inlet (38) is at a position below a surface level of the aqueous solvent (16) when contained in the first vessel (12). The second conduit may be connected to the first vessel (12) at a position below (Figures 2 to 5) or above (not illustrated) a surface level of the aqueous solvent (16) when contained in the first vessel ( 12). As such, the second conduit may enter the first vessel at any position, e.g., through atop, side wall or bottom of the vessel.

[0092] In non-illustrated embodiments, a third conduit is connected to a source of gas and to the first vessel. The third conduit is for the purpose of introducing a gas to the pigment-rich aqueous solvent (ie the first extract) in the first vessel within the slurry to purge the halogenated carbon compound-rich volatile extract from the slurry. The third conduit is sized and shaped to allow the movement of the gas from the source to the first vessel (12). The third conduit may be positioned so that the gas exits the conduit beneath the surface of the aqueous solvent (16). The third conduit may enter the first vessel at any position, e.g., through a top, side wall or bottom of the vessel. The gas may be, e.g., air or an inert gas. In certain embodiments, the outlet (37) of the second vessel (26) vents to the atmosphere. As such, the second conduit (36) may be absent.

[0093] In certain embodiments and as illustrated in Figures 3, 4 and 5, the device further comprises a pump (24) to move the halogenated carbon compound-rich volatile extract through the first conduit (22) from the first vessel (12) to the second vessel (26). The pump may also modulate the flow rate of gas and / or modulate the pattern of gas flow, e.g., using an oscillatory pattern, which may modulate bubble size to increase absorption of the halogenated carbon compound in the organic solvent or oil. The pump may be any suitable pump for moving a gas, e.g., a peristaltic pump, gear pump, diaphragm pump or centrifugal pump.

[0094] In certain embodiments and as illustrated in Figure 5, the device (10) further comprises an agitator (20) in the first vessel (12). The agitator (20) may be a stirrer (e.g. magnetic stirrer, paddle stirrer, impeller, sparger, or fluid jet) and serve to mix the slurry of aqueous solvent and biomass. Alternatively, the agitator may be, e.g., a macerator and physically disrupt cells in the biomass as described elsewhere herein. In certain embodiments, the device (10) is configured to perform one or more disrupting process as described elsewhere herein. For example, one or more disrupting process selected from the group consisting of: homogenising, macerating, sonicating, microwaving, enzyme treatment, freeze-thawing, heating andcombinations thereof. The person skilled in the art would readily be able to modify the first vessel (12) to include, e.g., a homogeniser, macerator, sonicator, microwave, temperature regulation system or combinations thereof. In certain non-illustrated embodiments, an outlet of the second conduit (36) may be positioned such that the entering gas performs the function of the agitator. In certain embodiments, the outlet (38) of the second conduit (36) comprises a sparger (not illustrated).

[0095] In certain embodiments and as illustrated in Figure 5, the device further comprises a sparger (30) in the second vessel (26) and connected to the second conduit (36). The sparger may include, e.g., a porous material with small holes that allow the escape of gas into the organic solvent or oil.

[0096] In certain embodiments and as illustrated in Figure 5, the first vessel (12) further comprises a safety valve (18). As the device (10) may operate as a closed system, the safety valve is used to protect against the build-up of excessive pressure. The person skilled in the art could readily determine a safe operating pressure of the device and hence select an appropriate safety valve. In non-illustrated embodiments, the second vessel comprises a safety valve.

[0097] In non-illustrated embodiments, a water trap may be installed between the first vessel (12) and the second vessel (26), e.g., between the first vessel (12) pump (24) to prevent contamination of the pump and / or cross contamination from first extract to the second extract. In non-illustrated embodiments, a water trap may be installed between the second vessel (26) and first vessel (12) to prevent cross contamination from the second extract to the first extract. Alternatively, or in addition, an oil separator may be installed between the second vessel (26) and first vessel (12) to prevent cross contamination from the second extract to the first extract. The device may comprise one or more sample ports for sampling the aqueous solvent (16), the headspace (13) of the first vessel (12), the organic solvent or oil (32), or the headspace (27) of the second vessel (26).

[0098] In a seventh aspect, there is provided a method of extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, comprising performing the method of the first aspect using the device of the sixth aspect. As such, references to any features are to be understood in the context of the first and sixth aspects or as described elsewhere herein, unless otherwise indicated. In certain embodiments, the biomass is of an Asparagopsis species, as described elsewhere herein.

[0099] As would be appreciated by the person skilled in the art, the method can be applied to any biomass from any species from which two products of different physicochemical properties can be isolated simultaneously. Therefore, the method is generally applicable to the extraction of one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass. As such, in an eighth aspect, there is provided a method of extracting one or more organic solventor oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both the one or more organic solvent or oil soluble volatile compound and the one or more aqueous solvent soluble compound, the method comprising the steps of: contacting the biomass with an aqueous solvent to provide a slurry; incubating the slurry under conditions and for a period of time sufficient to extract the one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from the biomass to produce an organic solvent or oil soluble volatile compound-rich volatile extract, an extracted biomass residue and a first extract comprising an aqueous solvent soluble compound-rich aqueous solvent comprising the one or more aqueous solvent soluble compound; contacting an organic solvent or oil with the organic solvent or oil soluble volatile compound-rich volatile extract to provide an organic solvent or oil soluble volatile compound-depleted volatile extract and a second extract comprising an organic solvent or oil soluble volatile compound-rich organic solvent or oil comprising the one or more organic solvent or oil soluble volatile compound; and optionally separately recovering the first and second extracts.

[0100] As would be appreciated by the person skilled in the art, the eighth aspect may be understood in view of the disclosure of the other aspects of this disclosure. The one or more organic solvent or oil soluble volatile compound may be understood with reference to the one or more halogenated carbon compound. The one or more aqueous solvent soluble compound may be understood with reference to the one or more pigment. In particular, the method of the eighth aspect may be performed in accordance with the description of the conditions of the method of the first aspect.

[0101] In certain embodiments, the biomass is from a vertebrate, invertebrate, plant, phytoplankton, seaweed, bacterium, yeast, fungus or microalga. In certain embodiments, the biomass is from a marine species. In certain embodiments, the biomass is from a whale, dolphin, reptile, bony or cartilaginous fish, mollusc, echinoderm such as Echinometra mathaei. crustacean, such as a shrimp or prawn including Litopenaeus vanname i, Macrobrachium rosenbergii, Penaeus monodon, Fenner openaeus chinensis. and Penaeus japonicus, diatom, dinoflagellate, haptophyte, green alga such as those from the genera Codium, Halimeda, Porphyra, Palmaria, Chondrus and Ulva, red alga such as those from the genera Kappaphycus, , brown alga (Phaeophyta) such as those from the genera Alaria, Ascophyllum, Bifurcaria, Carpophyllum, Cladosiphon, Cystophora, Cystoseira, Dictyota, Ecklonia, Ectocarpus, Eisenia, Fucus, Himanthalia, Hizikia, Ishige, Kjellmaniella, Laminaria, Myagropsis, Padina, Pelvetia, Petalonia, Saccharina, Sargassum, Schytosiphon, Sporochnus, Turbinaria and Undaria, cyanobacterium such as those from the genera Arthrospira, marine bacteria such as those from the genera Agrobacterium, Alcaligenes, Actinobacteria, Actinomadura, Alteromonas, Bacillus, Brevibacterium, Brevundimonas, Cellulophaga,, Collimonas, Chromobacterium, Erythrobacter, Hahella, Janthinobacterium, Marinomonas, Serratia, Zooshikella, Streptomyces, Nostoc, Pelagibacter, Paracoccus, Pseudomonas, Pseudoalteromonas, Shewanella and Vibrio, yeast such as those from the genera Rhodosporidium, Sporabolomyces,Rhodotorula. or Phaffia, fungi such as those from the genera Eurotium, Halorosellinia, Microsporum, Aspergillus, Hortaea, Phaeotheca, Trimmatostroma, Penicillium, Talaromyces, Altemaria, Microsphaeropsis, Monodyctis, Nigrospora, Paecilomyces, Phomopsis, Stemphylium, Caloplcicci and Fusarium, microalgae such as those from the genera Dunaliella, Haematococcus, Nannochloropsis, Porphyridium, Tetraselmis, Chlorella, Oscillatoria, Spirulina, EuryhaUnema. Desertifilum, P seudanahaena, Limnothrix, Phormidium, Gracilaria, Lynghya, Halomicronema, Geitlerinema, Porphyridium, Nostoc, Synechocystis, Dunaliella, Spirulina, Porphyridium, Scenedesmus, and Muriellopsis, or seaweed such as those from the genera Asparagopsis, Sargassum. In certain embodiments, the biomass is from Asparagopsis taxiformis, A. armata, or A. svedelii, Echinometra mathaei, openaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, Fenner openaeus chinensis. Penaeus japonicus. cophyllum nodosum, Bifurcaria bifurcata, Fucus spiralis, Himanthalia elongata, Laminaria ochroleuca, Laminaria saccharina, Pelvetia canaliculata, Sargassum muricum, Undaria pinnatifida, Kappaphycus alvarezii Pseudomonas aeruginosa, Serratia marcescens, Janthinobacterium species strain UV13, Rhodotorula species (Ambyl09), Arthrospira maxima, Aspergillus chevalieri TM2-S6, Euryhalinema species, Desertifilum species, Haematococcus pluvialis, Dunaliella salina, or Oscillatoria species. In certain embodiments, the biomass is from an Asparagopsis species, Laminaria species, Rhodymenia species, Ulva species, Dictyosyphon species, Porosira species, Nitzschia species, Thalassiosira species, Synechococcus species, or Ditylum species. In certain embodiments, the biomass is from an Asparagopsis species. The person skilled in the art could readily select an appropriate biomass with products having different physicochemical properties, e.g., a one or more organic solvent or oil soluble volatile compound and a one or more aqueous solvent soluble compound.

[0102] In certain embodiments, the one or more organic solvent or oil soluble volatile compound comprises a terpenoid (e.g., limonene, fucosterol, phytol, a-humulene, squalene), a furan (e.g., 2- acetylfuran, 5 -methylfurfural), a sulfur-containing compound (e.g., dimethyl sulfide, dimethyl disulfide, methyl mercaptan), a halogenated compound (e.g., bromoform, dibromomethane, tribromomethane, bromophenols, chloromethane), a hydrocarbon such as an alkane, alkene, or alkyne (e.g., heptadecane, pentadecene, 1-nonene, tetradecane), an alcohol (e.g., l-octen-3-ol, farnesol), an aldehyde or ketone (e.g., hexanal, octanal, benzaldehyde, 2-heptanone, 6-methyl-5-hepten-2-one), an ester (e.g., ethyl acetate, butyl butyrate, isoamyl acetate), heterocyclic compound (e.g., indole, pyrrole, benzofuran derivative), , phenylpropanoid or polyphenol (e.g., eugenol, cinnamaldehyde, phloroglucinol), or a component of an essential oil or hydro distillate, or combinations thereof. The person skilled in the art could select an appropriate organic solvent or oil based on the solubility and volatility characteristics of the one or more organic solvent or oil soluble volatile compound and the disclosure herein.

[0103] In certain embodiments, the one or more aqueous solvent soluble compound comprises a chlorophyll, phycobilin, or carotenoid or combinations thereof. In certain embodiments, the one or moreaqueous solvent soluble compound comprises a R-phycoerythrin, phycocyanin, allophycocyanin, astaxanthin, carrageenan, canthaxanthin, lutein, polyhydroxylated naphthoquinone, zeaxanthin, a-carotene, P-carotene, tunaxanthin, taraxanthin, doradexanthins, phenazine -1 -carboxylic acid, chlorophylls, xanthophylls, prodigiosin, violacein, tetrahydroauroglaucin, polysaccharide, carotenoid, vitamin, alkaloid, saponin, polyphenol, glysoside, monosaccharide, disaccharide, glycan, chitin, chitosan, agar, alginate, gelatin, pectin, xanthone, coumarin, phenolic acid, lignan, enzyme, hormone, recombinant product, amino acid, fatty acid, lipid, peptide, antibiotic, antibody, sterol, or flavoglaucin or combinations thereof. The person skilled in the art could select an appropriate aqueous solvent based on the characteristics of the one or more aqueous solvent soluble compound and the disclosure herein.

[0104] In a ninth aspect, there is provided a device for extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound, comprising: a first vessel (12) for containing a slurry comprising an aqueous solvent (16) and the biomass (14); a second vessel (26) for containing an organic solvent or oil (32); an outlet (23) from the headspace (13) of the first vessel, an inlet (28) in the second vessel (26) at a position below a surface level of the organic solvent or oil (32) when contained in the second vessel (26), and a first conduit (22) connecting the first vessel outlet (23) and the second vessel inlet (28).

[0105] Advantageously, the device may be used to practice the method of the eighth aspect. The device is illustrated in Figures 2, 3, 4 and 5. The working of the device may be understood with reference to the structural description in the sixth aspect. The one or more organic solvent or oil soluble volatile compound may be understood with reference to the one or more halogenated carbon compound. The one or more aqueous solvent soluble compound may be understood with reference to the one or more pigment.

[0106] In a tenth aspect, there is provided a method of extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound, comprising performing the method of the eighth aspect using the device of the ninth aspect.

[0107] In an eleventh aspect, there is provided an aqueous solvent soluble compound extracted by the method of the eighth or tenth aspects.

[0108] In a twelfth aspect, there is provided an organic solvent or oil soluble volatile compound extracted by the method of the eighth or tenth aspects.

[0109] In thirteenth aspect, there is provided a system for extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment. The system comprises interconnected vessels and conduits configured to allow volatile transfer of the halogenated carbon compound from an aqueous slurry to an organic solvent or oil phase, while retaining the pigment in the aqueous solvent. The system may optionally be operated as a closed-loop circuit.

[0110] In particular embodiments, the system comprises: a first container (e.g., vessel) configured to receive and contain a slurry comprising an aqueous solvent and a biomass, the biomass comprising both the one or more halogenated carbon compound and the one or more pigment; e configured to receive and contain an organic solvent or oil capable of solubilising the one or more halogenated carbon compound; a first conduit configured to connect an outlet from a headspace of the first container to an inlet positioned below a surface level of the organic solvent or oil in the second container; and optionally, a second conduit configured to connect an outlet from a headspace of the second container to an inlet positioned below a surface level of the aqueous solvent in the first container.

[0111] In use, the system facilitates movement of a halogenated carbon compound -rich volatile extract from the first container to the second container, where the volatile compound dissolves into the organic solvent or oil. The volatile stream may be moved passively (e.g., by pressure differential) or actively (e.g., by a pump). In certain embodiments, the second conduit allows recirculation of a halogenated carbon compound-depleted volatile extract back into the first container to assist with further purging of the halogenated carbon compound from the slurry.

[0112] In certain embodiments, the first container further comprises an agitator, such as a stirrer, paddle, or magnetic impeller, to maintain mixing of the slurry. In some embodiments, the first container includes a macerator or cell disruption unit (e.g., sonicator or homogeniser) to disrupt cells of the biomass and thereby enhance release of the halogenated carbon compound and pigment. The system may also include temperature control means (e.g., a heating jacket or chilled bath) for incubating the slurry at a desired temperature.

[0113] In particular embodiments, the second container comprises a sparger, diffuser, or porous medium at the inlet to increase contact between the incoming volatile stream and the organic solvent or oil. In some embodiments, the second container includes a gas outlet for venting residual gases or for directing a portion of the depleted volatile extract back to the first container.

[0114] The system may be constructed from materials compatible with aqueous buffers and organic solvents or oils, such as stainless steel, glass, PTFE, or chemically resistant polymers. Components may be scaled for bench-scale, pilot-scale, or industrial-scale operation.

[0115] The system may be provided as a kit including: (i) the first and second containers; (ii) the conduits; (iii) optionally, a pump or flow regulator; (iv) optionally, an agitator or disruption module; (v) optionally, a sparger; and (vi) instructions for use or assembly.

[0116] In some embodiments, the system or kit is pre -configured for use with Asparcigopsis taxiformis biomass or other marine algae known to contain halogenated carbon compounds and pigments such as R-phycoerythrin.

[0117] The system or kit may comprise the device as described elsewhere herein. The system or kit may be used to perform a method as described elsewhere herein.

[0118] The meaning of terms in any aspect may be understood with reference to any other aspect unless indicated otherwise by context. The features of various aspects may also be combined or substituted unless indicated otherwise by context.EXAMPLES

[0119] Example 1: Extraction of bromoform and R-phycoerythrin from Asparagopsis taxiformis

[0120] Materials and Methods

[0121] The following materials and chemicals were used, with sources shown in brackets: disodium ethylenediaminetetraacetate dihydrate (EDTA-2Na) (Rowe Scientific, WA), canola oil (Woolworths, WA), deionised water, Marine pH tester (Hanna Instruments), peristaltic pump (FlexFlo), plastic tubing, drill press (Full Boar, FBDDP-750) and sodium hydroxide (Rowe Scientific, WA).

[0122] Extraction buffer was prepared by adding EDTA-2Na (1 mM) in deionised water to required volumes and the pH was adjusted to 7 using 0.5 M sodium hydroxide solution.

[0123] Collection of A. taxiformis biomass

[0124] A. taxiformis gametophytes (ATG) were collected from Abrolhos Island, WA (- 28.717164179124083, 113.82108354232697) during August-September 2023. The ATG were transported to Fremantle, WA within 9 hrs by air transport in a sterile esky with seawater of the same temperature as that of the collection site. The ATG were then separated from the seawater by filtration and stored at -20 °C until further use.

[0125] A. taxiformis gametophytes (ATG) were also collected near Rottnest Island (- 32.02489398214646, 115.53078935581746). The ATG were transported to Fremantle in a sterile esky with seawater of the same temperature as that of the collection site. The ATG were then separated from the seawater by fdtration and stored at -20 °C until further use. The period of time between collection and storage at -20 °C was 4-6 hours. While not used in this experiment, A. taxiformis tetrasporophytes may be cultivated in the lab, from lab germinated carpospores, and then used in the extraction process. The ATG are referred to as biomass and were used in the extraction as detailed below.

[0126] Construction and working of the device

[0127] The device is represented in a schematic diagram (Figure 4). The device (10) comprised an extractor vessel (12), which was hermetically sealed. A. taxiformis biomass (14) was added to the vessel with 1 mM EDTA-2Na (pH 7) aqueous buffer (16). The top cover of the vessel was fitted with a safety valve ( 18), a provision for motorised agitation / maceration system (20) and a tube (22) exiting the top cover and connected to peristaltic pump (24).

[0128] The device also comprised a hermetically sealed cylindrical column (26) which was connected to the output from the pump (24) at the inlet point (28). A diffuser stone (30) was placed inside the column at the inlet point (28). The column (26) contained canola oil (32). The bromoform-containing volatile extract (34) was bubbled through the column (26). The outlet (37) at the top of the column (26) was connected via tube (36) to the extractor vessel ( 12) at an inlet (38) below the surface of the buffer (16).

[0129] The R-Phycoerythrin (R-PE) was extracted into the aqueous buffer, and the bromoform was released into the headspace above. The extraction of R-PE and bromoform was enhanced by macerating and agitating the biomass in the extractor (12). This was achieved by a motorised agitation / maceration system (20), which comprised an automatic drill press connected to a rod penetrating the top cover of the extraction unit. Cutting blades were fixed to the base of the rod to enable maceration and blending.

[0130] A peristaltic pump (24) sucked gas from the headspace of the extractor vessel (12) and into the column (26) containing canola oil. The absorption of bromoform in the oil was accelerated by increasing the contact surface area using a diffusion stone (30) at the bottom of the column (26). The bromoformcontaining gas was bubbled through the column and dissolved in the canola oil. The gas containing residual or undissolved bromoform (40) was circulated back to the extractor (12) at the inlet (38), where it passed through the aqueous buffer and back into the column (26). While not pictured, a water trap can be installed between the extractor vessel (12) and peristaltic pump (24) to prevent cross-contamination. The extraction process was continued until complete extraction of bromoform was achieved. For R-PE, subsequent reextractions of the same biomass (14) with buffer (16) were carried out to achieve complete extraction. A sample collection port was connected to a wall of the column (26), from which sample was collected usinga syringe. A sample collection port was connected adjacent the inlet (38) of the extractor vessel (12) to filter and collect R-PE extract.

[0131] Extraction of bromoform

[0132] Three bromoform extractions were conducted using the above-described device. The specific experimental conditions are outlined in Table 1 (below).TABLE 1: EXPERIMENTAL CONDITIONS FOR TESTS FOR THE BROMOFORM EXTRACTION

[0133] For these extractions, frozen biomass was weighed and added to the extractor vessel (12) with 1 mM EDTA-2Na (pH 7) buffer in the amounts indicated. The amount of biomass added can vary depending on the capacity of the vessel. The extractor vessel was sealed with a lid. The biomass was allowed to thaw for 1 hour before performing maceration or agitation. As the biomass started to thaw, the algae cells lysed due to thermal shock and osmotic pressure and released bromoform-containing volatileextract and R-Phycoerythrin (R-PE). The extractions were carried out at room temperature (21-22 °C), as bromoform and R-PE are stable at those temperatures11 12.

[0134] For the baseline testing, one year old frozen ATG biomass was used, which had impurities including green seaweeds and corals. In the column (26), 100 ml of canola oil was added. The peristaltic pump was set to a flow rate of 400 ml / min. Oil samples (2.5 mL) were collected at different time intervals during the extractions as mentioned in the Table 1. The oil samples were covered in aluminium foil and stored at 4 °C until the end of experiment and outsourced for testing . The testing parameters are detailed below. For the intermediate and upper limit testing, the agitation frequency and duration were varied to get a broader range of extraction efficiency of the device.

[0135] The bromoform was tested using Purge-&-Trap (P & T) GC-MS by Analytical Services Tasmania (AST). The testing parameters were as follows: 0.1 g oil was extracted in 10 mL methanol, followed by 1 min vortexing and 30 min in a tumbling -wheel. This methanol extract was then centrifuged and diluted with deionised water, where the dilution factor depended on the concentration of bromoform in the oil. P & T analysis was conducted using a Tekmar Aquatek autosampler (5mL sample) and Tekmar Lumin P & T unit with Vocarb 3000 trap, coupled to an Agilent 8860 gas chromatograph with 5977B mass spectrometer. A Restek Rxi-624SIL MS column (20m x 0.18mm x 1.0mm) column was used, with Helium carrier gas at 0.8mL / min constant flow, and 80: 1 split injection, and an injector temperature of 150 °C. The GC oven was temperature programmed as follows: 60 °C for 1 min, then 20 °C / min ramp to 200 °C. The mass spectrometer was operated in selected-ion-monitoring (SIM) mode, with dwell times optimised in each segment to give a total cycle time of -250 ms (Table 2). The bromoform was detected based on the MS peaks and Retention time (RT) (Table 2).TABLE 2: RETENTION TIMES AND M / Z FOR BROMOFORM AND INTERNAL STANDARDSUSED

[0136] Note: HS: Headspace; RT: Retention time; P&T: Purge and Trap

[0137] Calibration standards were prepared from certified reference materials over the range 10- 400mg / L (in vial).

[0138] The concentration of bromoform in the sample was calculated:

[0140] For bromoform extraction with dual extraction, the efficiency was determined as bromoform concentration in oil (mg / mL) (eq 2), bromoform concentration (mg) per gram dry weight of A. thaliana (eq 3) and bromoform concentration (mg) per gram of dry weight (dw) of A. thaliana (eq 4). The wet weight of biomass was found to be 6 times the dry weight, when the wet biomass (filtered and pat dried) was dried to completely remove any moisture.

[0141] Bromoform cone, in oil (— ) = bromoform (— ) x density of canola oil —) / 1000 (2)

[0144] Extraction of R-Phycoerythrin

[0145] The R-PE extraction yield and purity was determined for the fresh biomass extract under intermediate level testing conditions. The aqueous buffer containing the crude extract was collected and the biomass was separated from the extract (Extract 1) using filters of 100 pm and 5 pm pore size. The biomass was then re-extracted by adding fresh aqueous buffer and incubated at 4 °C for 24 hrs. The biomass was then separated from the extract (Extract 2) using a mesh filter. The process was repeated to obtain Extract 3. For Extract 4, the biomass and aqueous buffer were incubated for 18 days under the same conditions. A known volume of the extracts (i.e., extract fraction) was collected and dried using a hot air oven. The yield of the R-PE extract was calculated using the below equation (eq 5). The density of the buffer was determined as 0.94 g / mL.

[0147] The extracts were analysed using UV-Vis spectrophotometer (Peak instruments, model C- 7100) to characterize the R-PE and to determine the purity of the extracts. Absorption spectra of Vitamin B 12 were taken as standard for instrument validation. The extracts were fdtered through 0.22-micron fdters prior to taking absorption measurements. Absorption spectra for the extracts after appropriate dilutions were collected in the range of 220-800 nm. The purity of the extracts was determined by calculating the ratio of absorbances at 565 nm and 280 nm.

[0148] Results and Discussion

[0149] The major factors influencing the extraction efficiency were identified as agitation frequency and duration, based on the results of the three tests. These included baseline testing (one blend / maceration for 1 min per day over 12 days of extraction), intermediate level testing (one blend / maceration for 10 min at the beginning of extraction) and upper limit testing (one blend / maceration for 10 min followed by continuous mild agitation for 5 days). Figure 6 depicts the concentration of bromoform in the oil extract over time for the three different tests performed. Table 3 depicts the final results obtained for the three different tests performed.TABLE 3: BROMOFORM EXTRACTION EFFICIENCY WITH VARIOUS PRELIMINARY TESTS

[0150] The baseline test with agitation frequency of one blend per day with agitation duration of 1 min was carried out for 12 days and oil samples were collected at regular intervals. A saturation point was observed after the 7thday which could indicate complete extraction of the biomass (Figure 6). With complete extraction, bromoform concentration of 14.35 mg / ml of oil was achieved in 7 days. A slight loss in bromoform after 7thday was noted, which could be attributed to frequent opening and closing of the column (H) for sample collection, as bromoform losses have been reported when the bromoform is kept in an open container11. This issue was solved by adding a sample port in the column for sample collection using a syringe.

[0151] For establishing the intermediate level of extraction efficiency, the biomass was macerated for 10 minutes once per extraction. The bromoform concentration of 14.35 mg / ml oil was achieved in 5 days. However, the aim was to obtain complete extraction in 1 -2 days. It was speculated that biomass slurry was not sufficiently agitated to liberate bromoform entrapped in the slurry. Thus, another test was carried out where the biomass slurry in the extractor vessel was continuously agitated following the 10 min intense maceration ('upper limit testing'). The continuous agitation improved the bromoform extraction efficiency (65 mg / mL per kg dw A. taxiformis). Further experimentation and optimization will be carried out to obtain complete extraction within 1-2 days.

[0152] The R-PE extraction yield and purity was determined for the fresh biomass extract under intermediate level testing conditions. The crude extract after the first round of extraction had an intense reddish pink colour, which started to fade with subsequent two rounds of re-extraction (Figure 7a). However, as the fourth round of extraction was carried out with 18 days of incubation, further extraction of R-PE was achieved and the purity increased (Table 4).

[0153] Characterization of R-PE in the crude extracts was carried out using UV-Vis absorption spectroscopy. Vitamin B 12 in deionized water was used as a control and maximum absorption peaks (Amax) were obtained at 550 nm and 362 nm which matched the literature13, thus validating instrument parameters. Absorption spectra of R-PE crude extracts revealed higher absorption in the range of 290-350 nm (Figure 7b), which could be attributed to polysaccharides, phenolic acids, polyphenols, vitamins, etc14,15. The peakat 280 nm corresponds to proteins and the peaks in the range of 280-580 nm corresponds to phycobilliproteins16. R-Phycoerythrin has characteristic absorption peaks at 499, 565 nm and a 545 shoulder9. Similar absorption peaks were observed in the R-PE crude extract (Figure 7b insert).

[0154] The yield of Extract 1 was 42.9 mg / g ATG, which decreased gradually with subsequent reextractions (Table 4). The purity of R-PE was determined by taking the ratio of absorbances at 565 nm and 280 nm (Purity index (PI) = Ases nm / A28o nm). The purity determines the use for various applications i.e., food-grade (PI = 0.7), reactive grade (PI=3.9) and analytical grade (PI >4.0)10. The first three extracts had a lower PI, which resulting from higher absorbance at 280 nm due to the presence of other impurities, such as proteins. In the fourth round of re -extraction, the concentration of undesirable proteins decreased resulting in increase in PI. Further downstream processing may obtain higher purity R-PE.

[0155] Further optimization of the dual extraction process may increase the extraction efficiency of bromoform and R-PE.TABLE 4: YIELD AND PURITY OF R-PE IN THE CRUDE EXTRACT

[0156] The disclosed dual extraction device enables the simultaneous extraction of bromoform and R-PE from Asparcigopsis taxiformis.

[0157] The disclosed method has various advantages compared to existing literature, in terms of quality and quantity of the products. The commonly used technique for extracting bromoform in oil from Asparcigopsis taxiformis is an oil immersion method. While a higher biomass to oil ratio has been reported to increase bromoform content in oil17 18, a higher biomass to oil ratio leads to gel formation, which hinders the extraction process and downstream use requires further treatment (e.g. heat treatment) with resulting product losses18. In contrast, in the disclosed method the biomass does not contact the oil. Therefore, the bromoform can be extracted into a small amount of oil to the point of saturation by, e.g., using multiple batches of biomass with the same oil. The resulting highly concentrated bromoform oil facilitates transportation and storage. With the oil immersion technique, where the biomass is homogenised in the oil, the oil will have impurities of residual biomass debris17, thus compromising the purity of oil and the spentbiomass. In the disclosed method, the oil is not in contact with the biomass and so the oil can be easily separated without biomass cross contamination. Moreover, the R-PE crude extract does not contact with the oil, so an aqueous product is obtained that is not contaminated by oil. The spent biomass is also available for further uses and is not contaminated by oil. As such, the disclosed method may provide a high extraction efficiency of both products (e.g. can be near 100 %), the bromoform -containing oil may be highly concentrated and substantially pure, and the clean spent biomass with no oil residues may be available for further applications.

[0158] References:(1) Camer-Pesci, B.; Laird, D. W.; van Keulen, M.; Vadiveloo, A.; Chalmers, M.; Moheimani, N. R. Opportunities of Asparagopsis Sp. Cultivation to Reduce Methanogenesis in Ruminants: A Critical Review. Algal Research 2023, 76. 103308. https: / / doi.Org / 10.1016 / j.algal.2023.103308.(2) Tseten, T.; Sanjoijo, R. A.; Kwon, M.; Kim, S.-W. Strategies to Mitigate Enteric Methane Emissions from Ruminant Animals. J Microbiol Biotechnol 2022, 32 (3), 269-277. https: / / doi.org / 10.4014 / jmb.2202.02019.(3) Seaweed aquaculture. https: / / www.fish.wa.gov.au / Fishing-and-Aquaculture / Aquaculture / Aquaculture-Regions / Pages / seaweed-aquaculture.aspx (accessed 2024-03-19).(4) Wasson, D. E.; Yarish, C.; Hristov, A. N. Enteric Methane Mitigation through Asparagopsis Taxiformis Supplementation and Potential Algal Alternatives. Front. Anim. Sci. 2022, 3. https: / / doi.org / 10.3389 / fanim.2022.999338.(5) Machado, L.; Magnusson, M.; Paul, N.; Kinley, R.; de Nys, R.; Tomkins, N. Identification of Bioactives from the Red Seaweed Asparagopsis Taxiformis That Promote Antimethanogenic Activity in Vitro. Journal of Applied Phycology 2016, 28. https: / / doi.org / 10.1007 / sl0811-016-0830-7.(6) Kinley, R. D.; Tan, S.; Turnbull, J.; Askew, S.; Harris, J.; Roque, B. M. Exploration of Methane Mitigation Efficacy Using AsparagopsisMcmcA Bioactives Stabilized in Edible Oil Compared to Freeze- Dried Asparagopsis in vitro. American Journal of Plant Sciences 2022, 13 (7), 1023-1041. https: / / doi.org / 10.4236 / ajps.2022.137068.(7) Patwary, Z. P.; Zhao, M.; Paul, N. A.; Cummins, S. F. Identification of Reproductive Sex-Biased Gene Expression in Asparagopsis Taxiformis (Lineage 6) Gametophytes. Journal of Phycology n / a (n / a). https: / / doi.org / 10. l l l l / jpy.13419.(8) Zanolla, M.; Romanazzi, D.; Svenson, J.; Sherwood, A.; Stengel, D. B. Bromoform, Mycosporine- like Amino Acids and Phycobiliprotein Content and Stability in Asparagopsis Armata during Long-Term Indoor Cultivation. JApplPhycol 2022, 34 (3), 1635-1647. https: / / doi.org / 10.1007 / sl0811-022-02706-1.(9) George, R.; John, J. A. Phycoerythrin as a Potential Natural Colourant: A Mini Review.International Journal of Food Science & Technology 2023, 58 (2), 513-519. https: / / doi.org / 10. I l l 1 / ijfs.16229.(10) Kovaleski, G.; Kholany, M.; Dias, L. M. S.; Correia, S. F. H.; Ferreira, R. A. S.; Coutinho, J. A. P.; Ventura, S. P. M. Extraction and Purification of Phycobiliproteins from Algae and Their Applications. Front. Chem. 2022, 70. https: / / doi.org / 10.3389 / fchem.2022.1065355.(11) Tan, S.; Harris, J.; Roque, B. M.; Askew, S.; Kinley, R. D. Shelf-Life Stability of Asparcigopsis Bromoform in Oil and Freeze-Dried Powder. J Appl Phycol 2023, 35 (1), 291-299. https: / / doi.org / 10.1007 / sl0811-022-02876-y.(12) Munifah, I.; Musfiroh, S.; Munandar, A.; Surilayani, D. Thermal and pH Stability of the Red Seaweed (Rhodophyceae) Phycoerythrin Pigments from Kupang, Indonesia. IOP Conf. Ser.: Earth Environ. Sci. 2022, 978 (1), 012043. https: / / doi.Org / 10.1088 / 1755-1315 / 978 / l / 012043.(13) Umimoto, K.; Matsuura, M.; Shimamoto, Y.; Tachibana, K.; Kamada, A.; Miyata, M. mp480, Simple measurement of vitamin bl2 by optical spectroscopy. Nephrology Dialysis Transplantation 2016, 31 (suppl_l), i501. https: / / doi.org / 10.1093 / ndt / gfwl94.35.(14) Rajapakse, N.; Kim, S.-K. Nutritional and Digestive Health Benefits of Seaweed. Adv Food Nutr Res 2011, 64, 17-28. https: / / doi.org / 10.1016 / B978-0-12-387669-0.00002-8.(15) Liu, J.; Yong, H.; Yao, X.; Hu, H.; Yun, D.; Xiao, L. Recent Advances in Phenolic-Protein Conjugates: Synthesis, Characterization, Biological Activities and Potential Applications. RSC Advances2019, 9 (61), 35825-35840. https: / / doi.org / 10.1039 / C9RA07808H.(16) Hu, I.-C. Chapter 14 - Production of Potential Coproducts from Microalgae. In Biofuels from Algae (Second Edition),' Pandey, A., Chang, J.-S., Soccol, C. R., Lee, D.-J., Chisti, Y., Eds.; Biomass, Biofuels, Biochemicals; Elsevier, 2019; pp 345-358. https: / / doi.org / 10.1016 / B978-0-444-64192-2.00014-7.(17) Magnusson, M.; Vucko, M. J.; Neoh, T. L.; de Nys, R. Using Oil Immersion to Deliver a Naturally- Derived, Stable Bromoform Product from the Red Seaweed Asparagopsis Taxiformis. Algal Research2020, 51, 102065. https: / / doi.Org / 10.1016 / j.algal.2020.102065.(18) Nys, R. D.; Magnusson, M. E. Novel Composition. AU2021102541A4, July 1, 2021. https: / / patents.google.com / patent / AU2021102541A4 / en (accessed 2024-04-01).

[0159] The present disclosure is industrially applicable as it provides a dual extraction method suitable for extracting commercially useful compounds having different physicochemical properties from a biomass. In one application of the method, the inventors have exemplified a dual extraction method suitable for extracting one or more halogenated carbon compound and one or more pigment from a biomass of red seaweed of the genus Asparagopsis . In the exemplified method, the extracted bromoform finds application in reducing methane emissions from ruminants and R-phycoerythrin finds commercial application as a natural pigment or molecular label and as an active ingredient in cosmeceuticals, nutraceuticals, and biomedicine.

[0160] The present disclosure may provide numerous advantages over prior methods of extracting bromoform and R-phycoerythrin. For example, the method provides a dual extraction method for thesimultaneous extraction of these two valuable products. The exemplified method uses predominantly edible oils and water, which are inexpensive, naturally abundant, safe and easily used in commercial products. The method may be conducted at ambient conditions indoors or outdoors, so may have low energy requirements. However, the use of active processes to disrupt the cells of the biomass may enhance extraction. The extraction may be facilitated by the effects of hypotonic-induced osmotic lysis, in which cell walls are ruptured or disrupted to release the bromoform and R-phycoerythrin. In contrast to conventional oil-only methods of bromoform extraction which require a ratio of oil / biomass at least 1: 1, the ratio of organic phase to biomass used in the disclosed method is flexible and may be lower than 0.05 : 1 (ie 5% of the initial biomass) to reduce usage of the organic phase (e.g. oil).

[0161] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge.

[0162] It will be understood that the terms “comprise” and “include” and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, are to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied.

[0163] In some cases, a single embodiment may, for succinctness and / or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further, a phrase referring to “at least one of’ a list of items refers to any combination of those items, including single members. As an example, “at least one of: a, b, or c” is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c.

[0164] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and / or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.STATEMENTS

[0165] The present disclosure may provide:1. A method of extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, the method comprising the steps of: contacting the biomass with an aqueous solvent to provide a slurry; incubating the slurry under conditions and for a period of time sufficient to extract the one or more halogenated carbon compound and one or more pigment from the biomass to produce a halogenated carbon compound-rich volatile extract, an extracted biomass residue and a first extract comprising a pigment-rich aqueous solvent comprising the one or more pigment; contacting an organic solvent or oil with the halogenated carbon compound -rich volatile extract to provide a halogenated carbon compound-depleted volatile extract and a second extract comprising a halogenated carbon compound-rich organic solvent or oil comprising the one or more halogenated carbon compound; and separately recovering the first and second extracts.2. The method of statement 1, further comprising the step of introducing a gas to the pigment -rich aqueous solvent within the slurry to purge the halogenated carbon compound-rich volatile extract from the slurry.3. The method of statement 2, wherein the introduced gas is the halogenated carbon compound- depleted volatile extract.4. The method of any one of the preceding statements, wherein the conditions under which the slurry is incubated disrupt cells of the biomass and comprise a disrupting process selected from the group consisting of: homogenising, macerating, sonicating, microwaving, enzyme treatment, freeze-thawing, heating and combinations thereof.5. The method of statement 4, wherein the disrupting process is performed when a concentration of the one or more halogenated carbon compound in the organic solvent or oil stops increasing.6. The method of statement 5, wherein the disrupting process is performed two or more times.7. The method of any one of the preceding statements, further comprising replacing the extracted biomass residue with a new biomass comprising both the one or more halogenated carbon compound and the one or more pigment.8. The method of statement 7, wherein the extracted biomass residue is replaced with the new biomass between 1 and 10 times.9. The method of any one of the preceding statements, further comprising re -extracting the extracted biomass residue by replacing the first extract with aqueous solvent to provide a slurry and incubating the slurry under conditions and for a period of time sufficient to extract more of the one or more pigment from the biomass to produce a re-extracted biomass residue and an additional first extract comprising a pigmentrich aqueous solvent comprising the one or more pigment.10. The method of statement 9, wherein the step of re-extracting the biomass is performed two, three, four or more times.11. The method of any one of the preceding statements, further comprising replacing the second extract comprising the one or more halogenated carbon compound with organic solvent or oil.12. The method of any one of the preceding statements, wherein the method comprises a pretreatment step, comprising incubating the biomass at a temperature below 4 degrees Celsius prior to forming the slurry.13. The method of any one of the preceding statements, wherein the conditions under which the slurry is incubated comprise incubating the slurry at a temperature in the range of about 0 to about 60 degreesCelsius.14. The method of statement 13, wherein the slurry is incubated at room temperature.15. The method of any one of the preceding statements, wherein the period of time of the incubation is between 6 hours and 1 month.16. The method of any one of the preceding statements, wherein the aqueous solvent comprises a chelating agent.17. The method of statement 16, wherein the chelating agent comprises one or more chelating agent selected from the group consisting of citric acid, acetic acid, carbonic acid, Tris-HCl, phosphoric acid, sodium phosphate and ethylenediaminetetraacetic acid (EDTA).18. The method of statement 17, wherein the chelating agent is EDTA.19. The method of any one of statements 16 to 18, wherein the chelating agent is provided in the aqueous solvent at a concentration of 0.01 to 200 mM.20. The method of any one of the preceding statements, wherein a pH of the aqueous solvent is between 4 and 9.21. The method of any one of the preceding statements, wherein a volume-to-weight ratio of organic solvent or oil to biomass is in the range of about 1 : 1 to 1 :200.22. The method of any one of the preceding statements, wherein the oil is an edible oil.23. The method of statement 22, wherein the edible oil is a seed or vegetable oil.24. The method of any one of the preceding statements, wherein a weight-to-weight ratio of the aqueous solvent to the biomass is in the range of 10: 1 to 1:2.25. The method of any one of the preceding statements, wherein the biomass is from an Asparcigopsis species, Laminaria species, Rhodymenia species, Ulva species, Dictyosyphon species, Porosira species, Nitzschia species, Thalassiosira species, Synechococcus species, or Ditylum species.26. The method of any one of the preceding statements, wherein the biomass is from an Asparagopsis taxiformis tetrasporophyte.27. The method of any one of statements 1 to 25, wherein the biomass is from an Asparagopsis taxiformis gametophyte.28. The method of any one of the preceding statements, wherein the aqueous solvent causes cells of the biomass to lyse by osmotic shock to release the one or more halogenated carbon compound and the one or more pigment.29. The method of any one of the preceding statements, wherein the step of incubating the biomass is commenced within 12 hours of removal of the biomass from culture.30. The method of any one of the preceding statements, wherein the one or more pigment comprises R-phycoerythrin, phycocyanin, allophycocyanin or combinations thereof.31. The method of statement 30, wherein an absorbance ratio of the R-phycoerythrin in the pigmentrich aqueous solvent measured using A565 / A280 is greater than 0.1.32. The method of any one of the preceding statements, wherein a yield of the R-phycoerythrin (DW) in the pigment-rich aqueous solvent is at least 1 mg / g biomass wet weight.33. The method of any one of the preceding statements, further comprising isolating the one or more pigment from the recovered first extract.34. The method of any one of the preceding statements, wherein a yield of the one or more halogenated carbon compound in the halogenated carbon compound-rich organic solvent or oil is at least 4 mg / g biomass dry weight.35. The method of any one of the preceding statements, further comprising isolating the one or more halogenated carbon compound from the recovered second extract.36. The method of any one of the preceding statements, wherein the one or more halogenated carbon compound comprises bromoform, dibromochloromethane, dibromomethane, 1,2 -dibromoethylene, tribromoethylene, bromochloroacetic acid or combinations thereof.37. A pigment extracted by the method of any one of statements 1 to 36.38. A halogenated carbon compound extracted by the method of any one of statements 1 to 36.39. An animal feed supplement comprising the recovered second extract from the method of any one of statements 1 to 36 or the halogenated carbon compound of statement 38.40. An animal feed comprising the recovered second extract from the method of any one of statements 1 to 36, the halogenated carbon compound of statement 38, or the supplement of statement 39.41. A device for extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, comprising: a first vessel for containing a slurry comprising an aqueous solvent and the biomass; a second vessel for containing an organic solvent or oil; an outlet from the headspace of the first vessel; an inlet in the second vessel at a position below a surface level of the organic solvent or oil when contained in the second vessel; a first conduit connecting the first vessel outlet and the second vessel inlet.42. The device of statement 41, further comprising an outlet from a headspace of the second vessel; an inlet in the first vessel at a position below a surface level of the aqueous solvent when contained in the first vessel; and a second conduit connecting the second vessel outlet and the first vessel inlet.43. The device of statement 41 or 42, further comprising a pump to move a volatile extract through the first conduit from the first vessel to the second vessel.44. The device of any one of statements 41 to 43, further comprising an agitator in the first vessel.45. The device of any one of statements 41 to 44, further comprising a sparger in the second vessel and connected to the second conduit.46. The device of any one of statements 41 to 45, wherein the biomass is from an Asparcigopsis species, Laminaria species, Rhodymenia species, Ulva species, Dictyosyphon species, Porosira species, Nitzschia species, Thalassiosira species, Synechococcus species, or Ditylum species.47. A method of extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, comprising performing the method of any one of statements 1 to 36 using the device of any one of statements 41 to 45.48. A method of extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both the one or more organic solvent or oil soluble volatile compound and the one or more aqueous solvent soluble compound, the method comprising the steps of: contacting the biomass with an aqueous solvent to provide a slurry; incubating the slurry under conditions and for a period of time sufficient to extract the one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from the biomass to produce an organic solvent or oil soluble volatile compound-rich volatile extract, an extracted biomass residue and a first extract comprising an aqueous solvent soluble compound -rich aqueous solvent comprising the one or more aqueous solvent soluble compound; contacting an organic solvent or oil with the organic solvent or oil soluble volatile compound -rich volatile extract to provide an organic solvent or oil soluble volatile compound -depleted volatile extract and a second extract comprising an organic solvent or oil soluble volatile compound-rich organic solvent or oil comprising the one or more organic solvent or oil soluble volatile compound; and optionally separately recovering the first and second extracts.49. A device for extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound, comprising: a first vessel for containing a slurry comprising an aqueous solvent and the biomass; a second vessel for containing an organic solvent or oil;an outlet from the headspace of the first vessel; an inlet in the second vessel at a position below a surface level of the organic solvent or oil when contained in the second vessel; a first conduit connecting the first vessel outlet and the second vessel inlet.50. The device of statement 49, further comprising an outlet from a headspace of the second vessel; an inlet in the first vessel at a position below a surface level of the aqueous solvent when contained in the first vessel; and a second conduit connecting the second vessel outlet and the first vessel inlet.51. The device of statement 49 or 50, further comprising a pump to move a gas or volatile extract through the first conduit from the first vessel to the second vessel.52. The device of any one of statements 49 to 51, further comprising an agitator in the first vessel.53. The device of any one of statements 49 to 52, further comprising a sparger in the second vessel and connected to the second conduit.54. A method of extracting one or more halogenated carbon compound in organic solvent or oil and one or more pigment in an aqueous solvent from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, comprising performing the method of statement 48 using the device of any one of statements 49 to 53.

Claims

CLAIMS1. A method of extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, the method comprising the steps of: contacting the biomass with an aqueous solvent to provide a slurry; incubating the slurry under conditions and for a period of time sufficient to extract the one or more halogenated carbon compound and one or more pigment from the biomass to produce a halogenated carbon compound-rich volatile extract, an extracted biomass residue and a first extract comprising a pigment-rich aqueous solvent comprising the one or more pigment; contacting an organic solvent or oil with the halogenated carbon compound-rich volatile extract to provide a halogenated carbon compound-depleted volatile extract and a second extract comprising a halogenated carbon compound-rich organic solvent or oil comprising the one or more halogenated carbon compound; and separately recovering the first and second extracts.

2. The method of claim 1, further comprising the step of introducing the halogenated carbon compound-depleted volatile extract to the pigment-rich aqueous solvent within the slurry to purge the halogenated carbon compound-rich volatile extract from the slurry.

3. The method of claim 1 or 2, wherein the conditions under which the slurry is incubated disrupt cells of the biomass and comprise a disrupting process selected from the group consisting of: homogenising, macerating, sonicating, microwaving, enzyme treatment, freeze-thawing, heating and combinations thereof.

4. The method of any one of the preceding claims, further comprising replacing the extracted biomass residue with a new biomass comprising both the one or more halogenated carbon compound and the one or more pigment.

5. The method of any one of the preceding claims, further comprising re-extracting the extracted biomass residue by replacing the first extract with aqueous solvent to provide a slurry and incubating the slurry under conditions and for a period of time sufficient to extract more of the one or more pigment from the biomass to produce a re-extracted biomass residue and an additional first extract comprising a pigmentrich aqueous solvent comprising the one or more pigment.

6. The method of any one of the preceding claims, wherein the method comprises a pretreatment step, comprising incubating the biomass at a temperature below 4 degrees Celsius prior to forming the slurry.

7. The method of any one of the preceding claims, wherein the conditions under which the slurry is incubated comprise incubating the slurry at a temperature in the range of about 0 to about 60 degrees Celsius or at room temperature and wherein the period of time of the incubation is between 6 hours and 1 month.

8. The method of any one of the preceding claims, wherein the aqueous solvent comprises one or more chelating agent selected from the group consisting of citric acid, acetic acid, carbonic acid, Tris-HCl, phosphoric acid, sodium phosphate and ethylenediaminetetraacetic acid (EDTA).

9. The method of any one of the preceding claims, wherein a pH of the aqueous solvent is between 4 and 9.

10. The method of any one of the preceding claims, wherein a volume-to-weight ratio of organic solvent or oil to biomass is in the range of about 1 : 1 to 1 :200.

11. The method of any one of the preceding claims, wherein the oil is an edible oil.

12. The method of any one of the preceding claims, wherein a weight -to-weight ratio of the aqueous solvent to the biomass is in the range of 10: 1 to 1:2.

13. The method of any one of the preceding claims, wherein the biomass is from an Asparcigopsis species, Laminaria species, Rhodymenia species, Ulva species, Dictyosyphon species, Porosira species, Nitzschia species, Thalassiosira species, Synechococcus species, or Ditylum species.

14. The method of any one of the preceding claims, wherein the one or more pigment comprises R- phycoerythrin, phycocyanin, allophycocyanin or combinations thereof.

15. The method of any one of the preceding claims, wherein a yield of the R-phycoerythrin (DW) in the pigment-rich aqueous solvent is at least 1 mg / g biomass wet weight.

16. The method of any one of the preceding claims, wherein a yield of the one or more halogenated carbon compound in the halogenated carbon compound-rich organic solvent or oil is at least 4 mg / g biomass dry weight.

17. The method of any one of the preceding claims, wherein the one or more halogenated carbon compound comprises bromoform, dibromochloromethane, dibromomethane, 1,2-dibromoethylene, tribromoethylene, bromochloroacetic acid or combinations thereof.

18. An animal feed supplement comprising the recovered second extract from the method of any one of claims 1 to 17.

19. An animal feed comprising the recovered second extract from the method of any one of claims 1 to 17, or the supplement of claim 18.

20. A device for extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, comprising: a first vessel for containing a slurry comprising an aqueous solvent and the biomass; a second vessel for containing an organic solvent or oil; an outlet from the headspace of the first vessel; an inlet in the second vessel at a position below a surface level of the organic solvent or oil when contained in the second vessel; a first conduit connecting the first vessel outlet and the second vessel inlet.

21. The device of claim 20, further comprising an outlet from a headspace of the second vessel; an inlet in the first vessel at a position below a surface level of the aqueous solvent when contained in the first vessel; and a second conduit connecting the second vessel outlet and the first vessel inlet.

22. A method of extracting one or more halogenated carbon compound and one or more pigment from a biomass comprising both the one or more halogenated carbon compound and the one or more pigment, comprising performing the method of any one of claims 1 to 17 using the device of claim 20 or 21.

23. A method of extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both the one or more organic solvent or oil soluble volatile compound and the one or more aqueous solvent soluble compound, the method comprising the steps of: contacting the biomass with an aqueous solvent to provide a slurry; incubating the slurry under conditions and for a period of time sufficient to extract the one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from the biomass to produce an organic solvent or oil soluble volatile compound-rich volatile extract, an extracted biomass residue and a first extract comprising an aqueous solvent soluble compound -rich aqueous solvent comprising the one or more aqueous solvent soluble compound; contacting an organic solvent or oil with the organic solvent or oil soluble volatile compound -rich volatile extract to provide an organic solvent or oil soluble volatile compound -depleted volatile extract and a second extract comprising an organic solvent or oil soluble volatile compound-rich organic solvent or oil comprising the one or more organic solvent or oil soluble volatile compound; and optionally separately recovering the first and second extracts.

24. A device for extracting one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound from a biomass comprising both one or more organic solvent or oil soluble volatile compound and one or more aqueous solvent soluble compound, comprising: a first vessel for containing a slurry comprising an aqueous solvent and the biomass; a second vessel for containing an organic solvent or oil; an outlet from the headspace of the first vessel; an inlet in the second vessel at a position below a surface level of the organic solvent or oil when contained in the second vessel; a first conduit connecting the first vessel outlet and the second vessel inlet.

25. The device of claim 24, further comprising an outlet from a headspace of the second vessel; an inlet in the first vessel at a position below a surface level of the aqueous solvent when contained in the first vessel; and a second conduit connecting the second vessel outlet and the first vessel inlet.

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