RELATED APPLICATIONS

[0001] This PCT application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 62/196,670, filed July 24, 2015. This application is incorporated herein by reference in its entirety for all purposes.

FIELD

[0002] Embodiments of the present disclosure generally relate to compositions, methods and systems for the isolation, separation and/or purification of undesirable and/or contaminating agents from desirable products in biomass extracts. In certain embodiments, compositions, methods and systems are provided for separation and/or isolation of pheophorbide and arsenic from astaxanthin in algal extracts using novel extraction methods. In some embodiments, the novel extraction methods can include supercritical fluid extraction (SFE).

BACKGROUND

[0003] Processes for extracting products from various biological materials such as, but not limited to, microorganism biomass have been developed for a variety of commercial industries, including the dietary supplement, the nutraceutical, and the personal care industries. A significant challenge in working with biological materials is the ability to remove and isolate harmful contaminants and undesirable substances from a desired product during processing of the biological materials in a cost-efficient manner. These harmful contaminants and undesirable substances may be present in the biological material itself (endogenous contaminants), and/or may be introduced during processing, for example, due to contact with contaminated industrial air, water and equipment (exogenous contaminants).

[0004] In contrast to exogenous contaminants, which can often be reduced by implementing additional steps to decontaminate processing equipment, certain endogenous substances naturally present in the biological materials may be altered or transformed into harmful or undesirable substances during the processing of the biological materials. Reducing endogenous contaminants and harmful substances from biological material is generally more challenging, often requiring costly alterations to the isolation and purification processes, many of which can also adversely impact the quality and quantity of the final product(s). However, given the commercial importance and potential health benefits of various sought-after products isolated from biological materials such as microorganism biomass, improved extraction methods and systems are needed.

SUMMARY

[0005] Embodiments of the present disclosure include systems for extracting one or more target products from biomass material. In accordance with these embodiments, the system can include one or more biomass material solubilization compartments. These biomass material solubilization compartments can contain a solution including biomass material and one or more supercritical fluids. Further, these systems can include one or more biomass material purification compartments. These biomass material purification compartments can contain an essentially insoluble purification matrix and, in some embodiments, at least one filter. The one or more biomass material purification compartments can be fluidly coupled to the one or more biomass material solubilization compartments so that the solution of the biomass material and the one or more supercritical fluids flows from the one or more biomass material solubilization to the one or more biomass material purification compartments to produce a substantially purified biomass product. In some embodiments, the solution comprising the biomass material and one or more supercritical fluids contacts the essentially insoluble purification matrix in the one or more biomass material purification compartments to facilitate isolation and/or separation of harmful and/or undesirable agents from desirable products contained in the biomass material.

[0006] In some embodiments, the one or more biomass material solubilization compartments and the one or more biomass material purification compartments are fluidly coupled within a single vessel. In other embodiments, the one or more biomass material solubilization compartments and the one or more biomass material purification compartments can include separate vessels fluidly coupled using one or more enclosed channels. Systems can also include one or more supercritical fluid inlets and one or more product extract outlets. In some embodiments, the system further includes one or more biomass material entry and removal ports, one or more purification matrix entry and removal ports, and one or more supercritical fluid entry and removal ports. In still other embodiments, the biomass material can further include a flow aid to improve flow distribution and uniformity across the biomass bed and to prevent flow channeling during processing of the biomass material.

[0007] In some embodiments, biomass material disclosed herein includes biomass from single-celled organisms. In some embodiments, the biomass material comprises an algal biomass or algal biomass extract. In accordance with these embodiments, the biomass material is algal biomass or algal biomass extract from Haematococcus pluvialis. In certain embodiments, the one or more supercritical fluids includes carbon dioxide and/or one or more co-solvents. In some embodiments, one or more functional groups can include one or more sulfonic acid functional groups or one or more carboxylic acid functional groups. In other embodiments, one or more functional groups can include one or more trimethylammonium sulfonate functional groups or one or more polyethylene amine functional groups. In other embodiments, the biomass material can include biomass extract first subjected to one or more biomass extraction processes, wherein the systems and methods of the present disclosure further purifies the biomass extract. In still other embodiments, the essentially insoluble purification matrix includes an ion-exchange resin with a macroporous polystyrene resin having one or more sulfonic acid functional groups that is capable of binding one or more of pheophorbide, chlorophyll, pheophytin, other chlorophyll metabolites, arsenic, and other heavy metals. Systems encompassed by these embodiments can be used to produce a substantially purified biomass product such as astaxanthin substantially free from pheophorbide and arsenic.

[0008] Embodiments herein can also include methods for extracting a target product from biomass material, such as biomass from single-celled organisms, which include combining the biomass material with one or more supercritical fluids, such as supercritical carbon dioxide, in one or more biomass material solubilization compartments to form a solution of solubilizable biomass material. These methods also include causing the solution of solubilizable biomass material to flow from the one or more biomass material solubilization compartments to one or more biomass material purification compartments, which contain an essentially insoluble purification matrix, such as an ion-exchange resin, and in some cases, at least one filter. In accordance with these embodiments, the essentially insoluble purification matrix can be used to isolate and separate any undesirable and/or harmful agents (e.g., pheophorbide, arsenic, and other undesirable agents) from the solubilizable biomass material, in order to allow for the desirable products (e.g., astaxanthin) to be collected in a substantially purified form, essentially free of the undesirable agents, or wherein the undesirable agents are below a specific allowable threshold. In some embodiments, methods can also include further purifying biomass material or biomass material extract that has been subjected to one or more biomass extraction processes.

[0009] Embodiments of the present disclosure also include a composition for obtaining a substantially purified biomass product. In accordance with these embodiments, a composition can include a source of biomass or biomass extract, such as biomass from single-celled organisms, one or more supercritical fluids to solubilize the biomass or biomass extract, and an essentially insoluble purification matrix. The essentially insoluble purification matrix can facilitate removal of one or more undesired products from the biomass or biomass extract to produce a substantially purified biomass product. In certain embodiments, compositions disclosed herein can include an algal biomass or algal extract, carbon dioxide and/or one or more co-solvents, one or more flow aids to prevent channeling, and an ion exchange resin having one or more sulfonic acid functional groups.

[0010] As used herein, the terms "biomass," and "biomass material" can generally refer to biomass obtained from any plant-, microorganism- and/or animal-based biological material, including but not limited to, single-celled organisms, microorganisms, fungi, yeast, algae, microalgae, cyanobacteria, diatoms, bacteria, krill, fish, and the like.

[001 1] As used herein, the terms "extract" and "biomass extract" generally refer to biomaterials from non-animal sources (described above) that have been subjected to at least one extraction process.

[0012] The terms "determine," "calculate," and "compute," and variations thereof, as used herein, are used interchangeably and can include any type of methodology, process, mathematical operation or technique. [0013] It is to be noted that the term "a" or "an" entity refers to one or more of that entity. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0014] As used herein, "at least one," "one or more," and "and/or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and/or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together. When each one of A, B, and C in the above expressions refers to an element, such as X, Y, and Z, or class of elements, such as Xi-X _n , Yi-Y _m , and Zi-Z ₀ , the phrase is intended to refer to a single element selected from X, Y, and Z, a combination of elements selected from the same class (e.g., X ₁ and X ₂ ) as well as a combination of elements selected from two or more classes (e.g., Y ₁ and Zo).

[0015] The term "means" as used herein shall be given its broadest possible interpretation in accordance with 35 U.S.C. § 1 12(f). Accordingly, a claim incorporating the term "means" shall cover all structures, materials, or acts set forth herein, and all of the equivalents thereof. Further, the structures, materials or acts and the equivalents thereof shall include all those described in the summary, brief description of the drawings, detailed description, abstract, and claims themselves.

[0016] It should be understood that every maximum numerical limitation given throughout this disclosure is deemed to include each and every lower numerical limitation as an alternative, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this disclosure is deemed to include each and every higher numerical limitation as an alternative, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this disclosure is deemed to include each and every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0017] The preceding is a simplified summary of the disclosure to provide an understanding of some aspects of the disclosure. This summary is neither an extensive nor exhaustive overview of the disclosure and its various aspects, embodiments, and configurations. It is intended neither to identify key or critical elements of the disclosure nor to delineate the scope of the disclosure but to present selected concepts of the disclosure in a simplified form as an introduction to the more detailed description presented below. As will be appreciated, other aspects, embodiments, and configurations of the disclosure are possible utilizing, alone or in combination, one or more of the features set forth above or described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are incorporated into and form a part of the specification to illustrate several examples of the present disclosure. These drawings, together with the description, explain the principles of the disclosure. The drawings simply illustrate examples of how the disclosure can be made and used and are not to be construed as limiting the disclosure to only the illustrated and described examples. Further features and advantages will become apparent from the following, more detailed, description of the various aspects, embodiments, and configurations of the disclosure, as illustrated by the drawings referenced below.

[0019] FIG. 1 is a representative diagram of a system for extracting one or more products from biomass having a biomass material solubilization compartment and a biomass material purification compartment fluidly coupled within a single vessel, according to one embodiment of the present disclosure.

[0020] FIG. 2 is a representative diagram of a system for extracting one or more products from biomass having a biomass material solubilization compartment and a biomass material purification compartment fluidly coupled in separate vessels using enclosed channels, according to one embodiment of the present disclosure.

[0021] FIG. 3 is a representative diagram of a system for extracting one or more products from biomass having a biomass material solubilization compartment and a biomass material purification compartment fluidly coupled in separate vessels using enclosed channels, according to one embodiment of the present disclosure. DETAILED DESCRIPTION

[0022] In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details may be modified through routine experimentation. In some cases, well-known methods or components have not been included in the description.

[0023] Embodiments of the present disclosure generally relate to compositions, methods and systems for the isolation and separation of undesirable agents from desirable products in biomass material and biomass extracts. In certain embodiments, the present disclosure provides compositions, methods and systems for separation and isolation of pheophorbide and arsenic from the target product, astaxanthin, in algal extracts using supercritical fluid extraction (SFE).

[0024] Embodiments of the present disclosure provide improved systems and methods for enhancing the quality of extracted or purified compounds from biological materials. Biomass material used in accordance with these methods and systems can include any plant- and/or animal-based biological material, including but not limited to, single-celled organisms, microorganisms, fungi, yeast, algae, microalgae, cyanobacteria, diatoms (e.g., Phaeodactylum tricornutum), bacteria, krill, and fish {e.g., tuna, menhaden, and the like). In some aspects, compositions, methods and systems disclosed herein can be used to extract one or more target products from, for example, biomass of the microalga Haematococcus pluvialis, and to provide a substantially purified target product by removing one or more harmful and/or undesirable agents using ion-exchange resins having affinity for the harmful and/or undesirable agents present in the algal biomass. In other aspects, compositions, methods and systems disclosed herein can also be used to extract and/or purify astaxanthin from Haematococcus pluvialis, to extract and/or purify fucoxanthin and/or EPA from Phaeodactylum tricornutum, as well as to extract and/or purify various components of oil from fish such as tuna and menhaden.

[0025] As illustrated in FIG. 1, embodiments of the present disclosure can include a system 100 for extracting one or more products from biomass materials using supercritical fluid and ion-exchange resins. In one embodiment, the system 100 can include a single vessel 115 containing both a biomass material solubilization compartment 105 and a biomass material purification compartment 110. As illustrated, the biomass material solubilization compartment 105 can be fluidly coupled to the biomass material purification compartment 110 that lies downstream. Generally, the biomass material solubilization compartment 105 contains a solvent, such as supercritical carbon dioxide, and/or a co-solvent, while the biomass material purification compartment 110 contains an ion-exchange resin. Co-solvents can include, but are not limited to, methanol, ethanol, acetone and water.

[0026] As illustrated in FIG. 1, a mixture containing biomass material, such as biomass from single-celled organisms, can be solubilized and purified within a single vessel 115. As would be recognized by one of ordinary skill in the art based on the present disclosure, not all components of biomass are soluble. Solubilized biomass generally refers to the biomass components that are capable of being solubilized (e.g., solubilizable). In some embodiments, the biomass material can contain biomass from single-celled organisms and can have an optional flow aid to prevent channeling during the solubilization and purification processes. For example, the vessel 115 can be used to purify microorganism biomass, such as biomass from algae or in certain aspects, biomass from Haematococcus pluvialis, using one or more supercritical fluids introduced through a supercritical fluid inlet 120 that opens up into a head space 125 for storing the supercritical fluid. When one or more supercritical fluids flowing from the supercritical fluid inlet 120 through the supercritical fluid head space 125 are brought into contact with the biomass material, the supercritical fluids solubilize certain components of the biomass material, such as lipids, carotenoids, and other soluble byproducts. In some embodiments, a filter or separator 130 can be positioned between the supercritical fluid storage head space 125 and the biomass material solubilization compartment 105, and/or a filter or separator 135 can be positioned between the biomass material solubilization compartment 105 and the downstream biomass material purification compartment 110. In some embodiments, a filter or separator 140 can be located between the biomass material purification compartment 110 and tail space 145. In accordance with these embodiments illustrated in FIG. 1, the above described components are contained within a single vessel 115. [0027] Generally, a supercritical fluid (also referred to as SCF) refers to a substance that is at a temperature (Tc) and pressure (Pc) above its critical point where distinct liquid and gas phases do not exist. This critical point represents the highest temperature and pressure at which the substance can exist as a vapor and liquid in equilibrium. In some embodiments, the supercritical fluid can be carbon dioxide (C0 ₂ ). Carbon dioxide usually behaves as a gas in air at standard temperature and pressure (STP), or as a solid called dry ice when frozen. If the temperature and pressure of carbon dioxide are both increased from STP to be at or above the critical point for carbon dioxide, it exhibits properties between a gas and a liquid. For carbon dioxide, the point where it exists as a supercritical fluid occurs above its critical temperature (approximately 304.25 K) and its critical pressure (approximately 72.9 atm or 7.39 MPa). Supercritical C0 ₂ is an effective solvent due to its solubilization efficiency, its low toxicity, and its relatively low T _c and Pc of C0 ₂ , which allow most compounds to be extracted from biological materials with little damage or denaturing using supercritical C0 ₂ . However, as one of ordinary skill in the art would readily understand based on the present disclosure, other supercritical fluids can be used with the various embodiments of the present, including but not limited to, water, methane, ethane, propane, ethylene, propylene, methanol, ethanol, acetone, nitrous oxide, dimethyl ether and other similar supercritical fluids.

[0028] In some embodiments, as the one or more supercritical fluids flowing through the biomass material solubilization compartment 105 have effectively solubilized the components of the biomass capable of being solubilized (e.g., solubilizable biomass), the solution including biomass material and one or more supercritical fluids flows from the biomass material solubilization compartment 105, through a filter or separator 135, and into the biomass material purification compartment 1 10. The biomass material purification compartment 1 10 contains an essentially insoluble purification matrix to facilitate the removal and isolation of various harmful and/or undesirable products from the biomass solute. In some aspects, the essentially insoluble purification matrix is an ion-exchange resin.

[0029] Generally, ion exchange resins (also referred to as ion-exchange polymers or polymeric catalyst resin) are a type of insoluble matrix that can range in size from 40-1000 μπι in diameter. The resin can be designed with varying degrees of porosity (micro or macro- porous) in order to optimize contact with material of interest, and the resin can have varying degrees of solubility, as would be recognized by one of ordinary skill in the art based on the present disclosure. The resin can include a catalyst (e.g., functional groups), the arrangement or layout of which within the resin can have important effects on the efficiency and/or utility of the resin for a given extraction process. Optimizing extraction conditions pertaining to a given resin often involves determining the optimal size, porosity, and catalyst construction of a given resin as a starting point. In some embodiments, substantially similar resins from different sources (e.g. , vendors) may produce vastly different reaction rates because of different proprietary manufacturing processes which affect performance of the resin. Examples of this may include polymeric differences, disparities in size uniformity throughout resins, and/or the thoroughness of the cleaning or washing of unwanted monomers or contaminants between substantially similar resins. Differences in these areas, for example, would cause substantially similar resins to perform differently, including producing differences in yield and product quality/purity. In some embodiments, a macro-porous resin having sulfonic acid functional groups (e.g. , catalysts) with a particle size distribution of approximately 250-750μπι produced favorable results. Suitable resins that can be used with the methods and systems of the present disclosure include, but are not limited to, DIAION RCP 160M resin (Mitsubishi Chemical Corp.), AMBERLYST 15, hydrogen form (Sigma Aldrich), AMBERLITE FPC22 H (Dow Chemical Company). Additionally, resins that did not perform as effectively include AMBERLITE IR120 H (Dow Chemical Company) and ALUMINA ACID, Act. 1 (Sorbtech).

[0030] Often provided in the form of beads or other matrices, ion-exchange resins are generally porous with a large amount of surface area to facilitate a high degree of capture of a target or sought-after compound or substance and the concomitant release of other compounds or substances from the resin. Ion-exchange resins can be based on cross-linked polystyrene. Ion exchanging sites are generally introduced after polymerization of the matrices. Additionally, in the case of polystyrene, cross-linking can be introduced via co- polymerization of styrene and, for example, a low percentage of divinylbenzene. Typically, cross-linking decreases ion-exchange capacity of the resin and prolongs the time needed to accomplish the ion exchange processes, but improves the robustness of the resin. Particle size also influences resin parameters. Ion-exchange resins can be bead-shaped and/or constructed as membranes.

[0031] Additionally, ion-exchange resins can contain various functional groups that bind certain compounds and substances and facilitate their removal from biological materials such as biomass. For example, ion-exchange resins can have functional groups that are strongly acidic, including but not limited to, sulfonic groups (e.g., sodium polystyrene sulfonate or polyAMPS). Ion-exchange resins can have functional groups that are also weakly acidic, including but not limited to, carboxylic acid groups. Ion-exchange resins can have functional groups that are strongly basic, including but not limited to, quaternary groups (e.g., trimethylammonium sulfonate or polyAPTAC). Ion-exchange resins can also have functional groups that are also weakly basic, including but not limited to, primary, secondary, and/or tertiary amino groups (e.g., polyethylene amine). In some embodiments of the present disclosure, macroporous polystyrene-based ion-exchange resins comprising sulfonic acid functional groups (e.g., strong acid functional groups) can be used to isolate and remove various harmful and/or undesirable agents from biomass and extracts thereof, including but not limited to, one or more of pheophorbide, chlorophyll, pheophytin, other chlorophyll metabolites, arsenic, and other heavy metals. Other resins with different matrix materials, porosity, or functional groups may be used for the removal of other undesirable agents.

[0032] When passing through the biomass material purification compartment 1 10, various harmful and/or undesirable agents can be captured in a selected ion-exchange resin, thereby separating these agents from the biomass extract. The biomass extract can pass through a filter or separator 140. The substantially purified biomass extract product having reduced contamination of the undesirable agent can flow into a tail space 145, before exiting the system 100 through a solute in solution extract outlet 150. The substantially purified biomass extract product can then be collected and stored for additional processing, sent to another vessel for additional processing, and/or put into a separator at subcritical fluid conditions in order to precipitate out the solute from the subcritical gas.

[0033] The ratio of biomass to ion-exchange resin can be determined empirically based on, for example, the absorption/adsorption characteristics of the ion-exchange resin, the concentration of the undesirable substances to be removed from the biomass, supercritical fluid flow rate, and granularity of the biomass to be extracted, and the uniformity of supercritical fluid flow distribution across the cross section of the extraction vessel, as would be readily understood by one of ordinary skill in the art based on the present disclosure. In some embodiments, biomass to resin ratios can range from about 50:50 biomass to resin, to about 95:5. In some embodiments, biomass to resin ratios can be about 85: 15, about 80:20 about 75:25 about 70:30, about 65:35 about 60:40, and about 55:45 or any suitable ratio depending on the biomass and the resin used.

[0034] In some embodiments, cultures of microorganisms can be used as a source of biomass. Certain exemplary sources for biomass include algae such as Chlorophyta species. Certain Chlorophyta species of use in embodiments disclosed herein include, but are not limited to, Haematococcus pluvialis. Haematococcus pluvialis can be used as a source of biomass due to the ability of Haematococcus pluvialis to produce a high concentration and high quality astaxanthin. In accordance with these embodiments, an algal culture can be dewatered, and algal cells can be fractured and dried prior to supercritical C0 ₂ solubilization, as described above. System 100 can be used to extract astaxanthin and other valuable lipids from the algal biomass, while concomitantly isolating and removing harmful and/or undesirable agents such as pheophorbide and arsenic from the algal biomass extract, producing substantially purified astaxanthin. Although modifications to standard cultivation and post-harvest processes for Haematococcus pluvialis can reduce the content of chlorophyll and chlorophyll-breakdown products in the algal biomass, these measures can be insufficient to comply with regulatory requirements and standards. Additionally, various other post- harvest treatments to reduce pheophorbide and arsenic can also lead to a marked decrease in astaxanthin quantity/yield as well as product degradation, significantly increasing the cost of the process and potentially introducing residual solvents into the final product {e.g., ethanol and acetone in when using conventional extraction means). Embodiments of the present disclosure directly address these limitations and overcome these issues by providing a substantially purified, higher quality product with significantly reduced costs and labor.

[0035] As illustrated in FIG. 2, embodiments disclosed herein can include a system 200 for extracting one or more products from biomass materials using supercritical fluid and ion- exchange resins. System 200 is substantially similar to system 100, except that the one or more biomass material solubilization compartments 115 and the one or more biomass material purification compartments 110 are housed in separate extraction vessels and fluidly coupled using one or more enclosed channels 155. The enclosed channels 155 facilitate the transfer or flow of the solution including the biomass material and one or more supercritical fluids from the vessel containing the biomass material solubilization compartment 105 and to the vessel containing biomass material purification compartment 110. The closed channels 155 can be constructed of various high strength corrosion resistant materials (e.g. metals) to withstand supercritical fluid conditions, and can contain valves or other mechanisms to ensure proper flow, as would be readily recognized by one of skill in the art based on the present disclosure.

[0036] As illustrated in FIG. 3, embodiments disclosed herein can include a system 300 for extracting one or more products from biomass extracted materials using supercritical fluid and ion-exchange resins. System 300 is substantially similar to system 100, except that the one or more biomass material solubilization compartments 105 and the one or more biomass material purification compartments 110 include separate extraction vessels fluidly coupled using one or more channels 155 (e.g. enclosed), and the biomass material is biomass extract 160. Supercritical fluid can be introduced into one or more biomass material solubilization compartments 105, where it contacts the biomass extract 160.

[0037] In some embodiments, a flow aid can be used as illustrated in FIGS. 1 and 2, where biomass material is being extracted. One or more flow aids can be mixed with the biomass material so it does not compact and form flow channels, where the supercritical fluid contact becomes very concentrated in the channeled area. This channeling effect reduces contact in other areas and reduces extraction yield and efficiency. Any suitable flow aids can be used, as would be recognized by one of ordinary skill based on the present disclosure, including, for example, diatomaceous earth. In contrast, as illustrated in FIG. 3, biomass material of the present disclosure also includes biomass that has been subjected to an extraction process and is therefore less dense and/or has less volume. As such, biomass extract material does not necessarily require the addition of a flow aid to prevent channeling. As one of skill in the art would readily recognize based on the present disclosure, the purification methods and systems described herein can be used to further purify biomass materials that has been subjected to an extraction process, but for various reasons, have not reached a degree of quality required, for example, for food grade products (see, e.g., Table 1 below).

[0038] The systems 100, 200 and 300 of FIGS. 1, 2 and 3, respectively, can include a plurality of biomass material solubilization compartments 105 arranged in parallel or in series, as well as a plurality of biomass material purification compartments 110, arranged in parallel or in series. The various arrangements of the biomass material solubilization compartments 105 and the biomass material purification compartments 110 in a particular system may depend on a variety of factors, including but not limited to, the type of product being extracted, the source of the biomass, the type of supercritical fluid and/or the type of insoluble purification matrix being used (and its degree of solubility), and the like. In some embodiments, vessels containing the biomass material solubilization compartments 105 and the biomass material purification compartments 110 in a particular system may be arranged to improve further operation queuing and/or to reduce the time required to remove and/or replace biomass, supercritical fluid, and/or the insoluble purification matrix. The systems of the present disclosure may also include at least one biomass material entry and removal port, at least one essentially insoluble purification matrix entry and removal port, and at least one supercritical fluid entry and removal port, in order to facilitate the recharging of the system for further rounds of extraction and purification.

[0039] Embodiments disclosed herein can also include methods for extracting or isolating a product from biomass material such as biomass from single-celled organisms. In accordance with these embodiments, the methods can include combining the biomass material with one or more supercritical fluids, such as supercritical carbon dioxide, in one or more biomass material solubilization compartments to form a solution. The method also includes causing the solution to flow from the one or more biomass material solubilization compartments to one or more biomass material purification compartments, which contain an essentially insoluble purification matrix, such as an ion-exchange resin, and in some cases, at least one filter. The essentially insoluble purification matrix can be used to isolate and separate any undesirable and/or harmful agents (e.g., pheophorbide, arsenic, and the like) from the solubilized solution, so that the desirable products can then be collected in a substantially purified form. EXAMPLES

[0040] The following examples are included to illustrate various embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practice of the claimed methods, compositions and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0041] As described in exemplary Table 1, extraction methods of the present disclosure were performed using biomass from H. pluvialis (also referred to as oleoresin). The U.S. Pharmacopeia (USP) dictates standards for both the quality and purity of various food ingredients, which are listed in the Food Chemicals Codex (FCC). For example, according to USP-FCC astaxanthin monographs, the allowable threshold for pheophorbide contamination is 0.02%, while the allowable threshold for arsenic contamination is 2.0 ppm (0.0002%).

[0042] In one example, unprocessed biomass from H. pluvialis ("Solix-11") was used as the starting biomass material, and it contained approximately 0.41% pheophorbide and 1.7 ppm arsenic. Using conventional acetone extraction methods, biomass from H. pluvialis was processed in three 16 g loads, for a total of 48 g of oleoresin processed per 8.7 g of resin. As described in Table 1, pheophorbide contamination remained above the allowable threshold, while arsenic contamination was not detected.

[0043] Using supercritical carbon dioxide extraction methods, as disclosed herein, 126 kg of biomass from H. pluvialis ("1503-01BM") was subjected to four rounds of extraction using 22 kg of resin. As described in Table 1, the quality of the extract produced using supercritical carbon dioxide extraction methods improved compared to conventional acetone extraction. Pheophorbide contamination was below the allowable threshold, while arsenic contamination was not detected. Additionally, as would be appreciated by one of ordinary skill in the art, the use of acetone or ethanol in microorganism biomass extraction generally introduces acetone or ethanol into the extract product, requiring additional steps to remove and/or purify the extract, which often do not completely eliminate the acetone or ethanol. When using supercritical carbon dioxide extraction methods, as disclosed herein, additional steps to remove residual ethanol and/or acetone are not required.

[0044] Table 1: Extraction of pheophorbide and arsenic from microorganism biomass

[0045] Supercritical carbon dioxide extraction methods, as described herein, were also effective when commercial amounts of biomass from H. pluvialis was used as the starting material. As shown in Table 2, three different lots of commercially scaled amounts of H. pluvialis were subject to supercritical carbon dioxide extraction using ion exchange resin to remove undesired pheophorbide during extraction. In each of the lots shown in Table 2, the percentage of pheophorbide before treatment with the ion exchange resin {i.e., pre-resin treatment) was significantly reduced, as compared to after treatment {i.e., post-resin treatment). The percent pheophorbide reduction for Lot# AYPS150902P was 77.5%; the percent pheophorbide reduction for Lot# AYPS150903P was 66.7%; and the percent pheophorbide reduction for Lot# 201602-02F was 95%. These data clearly demonstrate the efficacy of supercritical carbon dioxide extraction methods described herein for removing undesired components from biomass material, even when applied on a commercial scale.

[0046] Table 2: Commercial extraction of pheophorbide from H. pluvialis biomass

H. pluvialis | 201602-02F | 0Λ | 0.005 |

[0047] In various aspects, embodiments, and configurations, can include components, methods, processes, systems and/or apparatus substantially as depicted and described herein, including various aspects, embodiments, configurations, sub combinations, and subsets thereof. Those of skill in the art will understand how to make and use the various aspects, embodiments, and configurations, after reading the present disclosure. The present disclosure, in various aspects, embodiments, and configurations, includes providing compositions and processes in the absence of items not depicted and/or described herein or in various aspects, embodiments, and configurations hereof, including in the absence of such items as may have been used in previous compositions or processes, e.g. , for improving performance, achieving ease and\or reducing cost of implementation.

[0048] The foregoing discussion of the disclosure has been presented for purposes of illustration and description. The foregoing is not intended to limit the disclosure to the form or forms disclosed herein. In the foregoing Detailed Description for example, various features of the disclosure are grouped together in one or more, aspects, embodiments, and configurations for the purpose of streamlining the disclosure. The features of the aspects, embodiments, and configurations of the disclosure may be combined in alternate aspects, embodiments, and configurations other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention that the claimed disclosure requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed aspects, embodiments, and configurations. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate preferred embodiment of the disclosure.

[0049] Moreover, though the description of the disclosure has included description of one or more aspects, embodiments, or configurations and certain variations and modifications, other variations, combinations, and modifications are within the scope of the disclosure, e.g. , as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative aspects, embodiments, and configurations to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.