[0001] This PCT application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 62/210,909, filed August 27, 2015. This application is incorporated herein by reference in its entirety for all purposes.

FIELD

[ΘΘ02] 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 extraction methods including, but not limited to, extraction by organic solvents.

BACKGROUND

[ΘΘ03] 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 can 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. Other embodiments disclose biomass material solubilization compartments can contain a solution comprising the biomass material and one or more organic solvents. 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. In other embodiments, 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 organic solvents 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, a solution including biomass material and one or more organic solvents can contact 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.

[0008] 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 to one another using one or more enclosed channels. The systems may also include one or more organic solvent inlets and one or more product extract outlets. In some embodiments, certain systems can further include one or more biomass material entry and removal ports, one or more purification matrix entry and removal ports, and one or more organic solvent entry and removal ports. In still other embodiments, biomass material can further includes 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 includes an algal biomass or algal biomass extract. In accordance with these embodiments, the biomass material can be an algal biomass or algal biomass extract from Haemaiococcus pluvialis. Other algal or single cell organism type biomass can include, but are not limited to, genera; Phaeodactylum, ChloreUa, Nannochloropsis, and Spirulina. In certain embodiments, the one or more organic solvents includes acetone 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 (organic or inorganic species or varieties), and other heavy metals. Systems encompassed by some of these embodiments can be used to produce a substantially purified biomass product such as astaxanthin substantially free from undesirable agents, for example, 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 can include combining the biomass material with one or more organic solvents, such as acetone, ethanol and/or methanol, in one or more biomass material solubilization compartments to form a solution of solubilizable biomass material. These methods can 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 can 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., astaxanthm) 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. For example, when a target product has intended use for human consumption a "food grade" material can be used, such as an approved ion exchange resin meeting FDA 21 CFR 173.25 regulations. In other embodiments, removal of undesirable agents by this method can be combined with other extraction techniques including, but not limited to, leach or percolation, pressing or milling, (e.g. bead milling, counter current, and shallow bed extractions).

[0009] Embodiments of the present disclosure can 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 organic solvents 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, one or more organic solvents 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] In yet other embodiments, methods can include using a combination of ion exchange resin separation technologies disclosed herein with other filtering or filter aid materials. In accordance with these embodiments, these filter materials can include, but not limited to, silica, activated carbon, charcoal, diatomaceous or bleaching earth. These materials may be layered in the form of a cake, or as a mix of comingled materials/products. These combinations can also include using the ion exchange resin or a combination of ion-exchange resin with described material as a "plug or polishing filter" in order to assist with scavenging phenols, heavy metals, organic or other volatile contaminants.

[0011] As used herein, the terms "biomass," and "biomass material" generally refer to biomass obtained from any plant- and/or animal-based biological material, including but not limited to, single-celled organisms, microorganisms, fungi, yeast, algae, microalgae, cya obacteria, diatoms, bacteria, krili, fish, and the like.

[0012] As used herein, the terms "extract" and "biomass extract" generally refer to biomaterials from biological material (described above) that have been subjected to at least one extraction process.

[0013] 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.

[0014] 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.

[0015] 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 Z\~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 _! and X ₂ ) as well as a combination of elements selected from two or more classes (e.g., and

Zo).

[0018] 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.

[0017] 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.

[0018] 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

[ΘΘ19] 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 preferred and alternative 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.

[0020] 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 to one another as two separate vessels, according to some embodiments of the present disclosure.

[0021] FIG. 2 is a representative series diagram of a system for purifying one or more products from biomass having at least three separate vessels, a biomass material solubilization extraction vessel, a second ion-exchange extraction vessel and a filter aid extraction vessel fluidly coupled using enclosed channels, according to some embodiments of the present disclosure.

[0022] FIG. 3 is a representative diagram of a system for purifying one or more products from an extract in a single vessel, according to some embodiments of the present disclosure.

[0023] FIG. 4 is a representative diagram of a system for purifying one or more products from an extract having an ion-exchange resin extraction vessel and a filter aid system extraction vessel as two separate vessels fluidly coupled using enclosed channels, according to some embodiments of the present disclosure.

DETAILED DESCRIPTION

[0024] 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 extraction methods that include various organic solvents.

[0025] 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 material, including but not limited to, single-celled organisms, microorganisms, fungi, yeast, algae, microalgae, cyanobacteria, diatoms (e.g., Phaeodactylum tricormUum), 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 plmialis, 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 Haemaiococcus pluvialis, to extract and/or purify fucoxanthin and/or EPA from Phaeodaciylum iricornutum, as well as to extract and/or purify various components of oil from fish such as tuna and menhaden.

[0026] As illustrated in FIG. 1 , embodiments of the present disclosure can include a system 100 for extracting and/or enriching one or more products from biomass materials using organic solvents and ion-exchange resins. In some embodiments, system 100 can include at least two vessels 1 15 A and 1 15B containing a biomass material solubilization compartment 105 and a solvent and an extraction vessel 115B having a compartment for receiving an organic solvent/extract solute 125 from 115A. As illustrated, the biomass material solubilization compartment 105 can be fluidly coupled to the organic solvent headspace 160. In certain embodiments, the organic solvent headspace 160 can contain an organic solvent, such as acetone and/or ethanol, and/or a co-solvent, while the biomass material solubilization compartment 105 can receive the solvent for generating an organic solvent/extract from the biomass. Solvents contemplated herein can include, but are not limited to, methanol, ethanol, acetone and water. In some embodiments disclosed herein, solvents can also include, but are not limited to, one or more of, acetone, methanol, ethanol, isopropanol, 1-butanol, 2-butanol, 1 -pentanol, acetates (methyl, ethyl, propyl, isobutyl, and isopropyl), hexane, isohexane, heptane, pentane, acetic acid, anisole, 3 -methyl- 1 -butanol, methylethylketone, methylisobutylketone, water and the like.

[ΘΘ27] 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, extraction vessel 1 15B, , 215B, 215C, 315, 415A and 415B can be used to purify target products from microorganism biomass, such as extract from algae or in certain aspects, astaxanthin esters from Haematococcus pluvialis, using one or more organic solvents introduced through an organic solvent inlet 165 that opens up into a head space 160 for dispersing the organic solvent. When one or more organic solvents flowing from the organic solvent inlet 165 through the organic solvent head space 160 are brought into contact with biomass material, the organic solvents solubilize certain components of the biomass material, for example, lipids, carotenoids, and other soluble byproducts of the biomass material. In some embodiments, a filter or separator 135 can be positioned between the organic solvent head space 60 and the biomass compartment 105, and/or a filter or separator 80 can be positioned between the biomass material solubilization compartment 105 and the downstream organic solvent/extract solute tail space 170. In some embodiments, the organic solvent/extract solute can pass through an outlet 175 through a channel 190 to a second extract vessel 115B into an organic solvent/extract solute inlet 120 into headspace 125 and dispersed and exposed to ion-exchange resin 185. In certain embodiments a filter or separator is positioned between the organic solvent/extract solute headspace 125 and the ion exchange resin chamber 185 in order to contain the Ion exchange resin and enhance purification of a target compound. In other embodiments, a filter aid 1 10 is positioned between separator or filter 140 and a purified extract tailspace 145.

[0028] In some embodiments, as the one or more organic solvents flowing through the biomass material solubilization compartment 105 have effectively soiubilized the components of the biomass capable of being soiubilized (e.g., soiubilizable biomass), the solution including biomass material and one or more organic solvents flows from the biomass material containing compartment 105, through a filter or separator 180, and into the organic solvent/extract solute tailspace 170. Extraction vessel 115B can contain an essentially insoluble purification matrix to facilitate removal and isolation of various harmful and/or undesirable agents from the biomass solute. In some aspects, the essentially insoluble purification matrix can be an ion-exchange resm 185.

[0029] It is understood by one skilled in the art that 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- Ι ΟΟΟμηι in diameter. Resins 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. Resins can include a catalyst (e.g., functional groups), the arrangement or layout of which within the resm 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 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 resms, and/or the thoroughness of the cleaning or washing of unwanted monomers or contaminants between substantially similar resins. Differences in these areas, for example, may cause substantially similar resms 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 250-750μηι is capable of performing favorable results. Suitable resins that can be used with certain 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 resms 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 bioraass. 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, carboxyiic acid groups. Ion-exchange resins can have functional groups that are strongly basic, including but not limited to, quaternar groups (e.g., trimethylammonium sulfonate or poly APT AC). 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 185, various harmful and/or undesirable agents can be captured in a selected ion-exchange resin, thereby separating these agents from the remaining biomass extract. The remaining biomass extract can pass through a filter aid 110 through a filter or separator 140. The substantially purified biomass extract product having reduced contamination of the undesirable agent can flow into a tailspace 145, before exiting the system 150 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 to, for example, precipitate out the solute from the organic solvent(s).

[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, organic solvent flow rate, and granularity of the biomass to be extracted, and the uniformity of organic solvent 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: 1 5, 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 Haematococcus pluvialis can be used as a source of algal biomass due to the ability of Haematococcus pluvialis to produce a high concentration and high quality astaxanthm. In accordance with these embodiments, an algal culture can be dewatered, and algal cells can be fractured and dried prior to solubilization, as described above. Systems 100 and 200, 300 or 400 can be used to extract astaxanthm and other valuable lipids from the algal biomass, while Systems 100, 200, 300 or 400 can concomitantly isolate and remove harmful and/or undesirable agents for example, pheophorbide and arsenic from the algal biomass extract, producing substantially purified astaxanthm. 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 and/or not cost effective 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 contaminants into the final product. 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] 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, for example, the organic solvent 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, diatomaeeous earth. In contrast, biomass material of the present disclosure can include 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, purification and separation 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 attained a desired degree of quality, for example, for food grade products (see, e.g.. Table 1 below). In some embodiments, solubilizable biomass materials can be independently solubilized in one or more organic solvents in lieu of being solubilized in an extraction vessel as depicted herein. Pre-solubilized biomass material can instead directly enter a purification pre-determined extraction vessel through, for example, one or more enclosed channels or inlets.

[0038] Systems 100, 200, 300 and 400 of FIGS. 1, 2, 3 and 4, respectively, can include a single or a plurality of extraction vessels 115 arranged in parallel or in series. In certain embodiments, each extraction vessel 115 can include various compartments (e.g., 105, 125, 145) arranged within a single vessel 115. The various arrangements of the extraction vessels 115 and the compartments (e.g., 105, 125, 145) in a particular system 100, 200, 300, 400 can depend on a variety of factors, including but not limited to, types of product being extracted, source of the biomass, type of organic solvent, types of undesirable agents targeted for removal from a biomass and/or type of insoluble matrix being used (and its degree of solubility), and the like.

[ΘΘ37] Referring to FIG. 1 , in this embodiment, system 100 includes a first extraction vessel 115A and a second extraction vessel 115B. In some embodiments, the first extraction vessel 1 15A can contain a compartment 105 for housing biomass and a compartment 160 for holding a solvent (e.g. an organic solvent) in a headspace above the biomass allowing fluid communication between the organic solvent headspace 160 and the biomass compartment 105. Solvents can be introduced to the first extraction vessel 115A via a solvent inlet 165. The solvent inlet 165 can be positioned to permit the introduction of the solvent to the organic solvent headspace 160 of the first extraction vessel 1 1 5 A. As depicted in system 100, the first extraction vessel 115 A can contain one or more filter/separators 135, 180 positioned to filter a solvent and/or filter a solvent/biomass composition prior to exiting the extraction vessel. The first extraction vessel 1 15A can also contain a solvent/extract compartment 170, which holds a solvent/extract composition prior to eluting the composition from the first extraction vessel 1 15A through a solvent/extract solute outlet 175. Solutes (e.g. organic solvent/extract) eluted from the first extraction vessel 115A can then be introduced by an inlet 120 to a second extraction vessel 1 15B in series.

[0038] In other embodiments, the second extraction vessel 1 15B can include a compartment 185 that contains an ion exchange resm. Further, the second extraction vessel 115B can include a compartment 125 for holding a solvent/extract solute in a headspace or in another compartment of the extraction vessel having fluid communication with an ion- exchange resin 185. The at least second extraction vessel 1 1 5B can further include one or more filter separators 130, 135, 140 for removing or separating out solute agents. In addition, the at least second extraction vessel 115B can include a compartment 145 for a purified extract in fluid communication with the compartment 185 containing the ion-exchange resin and/or a compartment 110 with a filter aid. Purified extract from the at least second extraction vessel 115B can exit the extraction vessel 115B through a purified extract outlet 150 for further processing or for use as a purified product

[0039] System 200 represented in Fig. 2 can include three or more extraction vessels 215A, 215B, 215C. Each extraction vessel 215A, 215B, 215C of system 200 performs a predetermined functional feature of further purification and/or separation of a biomass, in parallel vessels or in series. In a first extraction vessel 215 A, biomass 105 can be combined with organic solvent from a compartment 160 optionally thru a separator/filter 135 to form an organic solvent/extract found in an organic solvent/extract solute compartment 170. In a second extraction vessel 215B, organic solvent/extract of the first extraction vessel 215 A can be introduced by fluid connection or enclosed channel 190 or manually to the second extraction vessel 215B having a compartment 125 for receiving the organic solvent/extract and a compartment 185 having an ion exchange resin for purifying out certain undesirable agents from an extract. The at least second extraction vessel 215B can include one or more filter /separators 130, 140 before and/or after the compartment 1 85 containing the ion- exchange resin 185. In at least the second extraction vessel 215B of system 200, an extract solute/organic solvent composition can be produced and held in an organic solvent/extract solute tailspace 245. The extract solute/organic solvent composition can then be introduced either manually or by fluid connection or enclosed channel 290 to at least a third extraction vessel 215C through an inlet 255 and optionally held or transitioned through headspace 225. Then extract solute/organic solvent can then transition through filter aid 110 which can include one or more of a separator/filter 240, 235 in fluid communication with filter aid 1 10. Once an extract solute/organic solvent is transitioned through at least a filter aid of system 200, a purified extract can be held or transitioned through a purified extract tailspace 145 to a purified extract outlet 150 for further purification and/or use.

[0040] As illustrated in FIG. 2, embodiments of the present disclosure can include a system 200 for extracting one or more products from biomass materials using organic solvents and ion-exchange resins. System 200 is substantially similar to system 100, except that the one or more biomass material solubilization compartments 115A, 115B and the one or more biomass material purification compartments 110, included in system 100, are housed in separate extraction vessels 215 A, 215B, and 215C. In embodiments, the separate extraction vessels 215A, 215B, 215C are fluidly coupled using one or more enclosed channels 290. The enclosed channels 190 facilitate the transfer or flow of the solution including the biomass material and one or more organic solvents from the vessel containing the biomass material solubilization compartment 105 and to the vessel containing biomass material purification compartment 110. The closed channels 290 can be constructed of various high strength corrosion resistant materials (e.g., metals) to withstand organic solvents, 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.

[ΘΘ41] Uses of purified extracts of systems 100, 200, 300 and 400 can include pharmaceutical uses, health supplements or other over-the-counter uses, or any other use depending on the product sought and the purified composition produced herein.

[0042] System 300 can include a single extraction vessel 31 5 housing a complete extraction system for use with any extract contemplated herein. Extract can be introduced to extraction vessel 315 by any method (e.g. manually, automated and the like). An extract contemplated of use in system 300 is any crude or prepared extract by any method. Once the extract enters extract headspace 310 it can flow either manually or by automation through separator/filter 330 to ion exchange resin 185. Extraction vessel 315 of system 300 can include a separator/filter 330 positioned between extract headspace 310 and ion exchange resm 185 allowing addition purification and or separation of extract components or agents. Ion-exchange resm 185 can be in fluid communication with second separator/filter 320 as well as a filter aid 1 10. Extracts introduced to extraction vessel 315 of system 300 can continue through to filter aid 1 10 through separator/filter 325 as illustrated in Fig. 3. Once the extract has traversed filter aid 1 10, a purified extract can he generated for storage in a purified extract tailspace 145 or flow out of the extraction vessel 31 5 through purified extract outlet 150 for storage, additional purification or use.

[0043] As illustrated in FIG. 4, embodiments of the present disclosure can include a system 400 for extracting one or more products from biomass materials or removing undesirable agents from an extract using organic solvents and ion-exchange resins. System 400 is substantially similar to part of system 200 where an ion exchange resin and a filter aid are housed in separate extraction vessels 415A, 415B connected by an optional enclosed channel 490. The enclosed channels 490 facilitate transfer or flow of a solution including an organic solvent/extract from one vessel to the next extraction vessel as applicable. Enclosed channels 190, 290, 490 can be constructed of various high strength corrosion resistant materials (e.g., metals or resistant composite plastics or the like) to resist adverse effects of agents such as organic solvents disclosed herein, and can contain valves or other mechanisms to ensure proper flow, as would be readily recognized by one of skill in the art and based on the present disclosure.

[0044] System 400 represented in Fig. 4 can include two or more extraction vessels 415 A, 415B. In a first extraction vessel 415A, organic solvent/extract of the first extraction vessel 4 5A can be introduced by fluid connection, manually or by enclosed channel to the first extraction vessel 41 A having a compartment 125 for receiving the organic solvent/extract and a compartment 185 having an ion exchange resin for purifying out certain undesirable agents from an extract. The at least first extraction vessel 415 A can include one or more filter/separators before and/or after a compartment or region containing the ion- exchange resin 185. Each extraction vessel 415 A, 415B of system 400 performs a predetermined functional feature of further purification and/or separation of a biomass, in parallel vessels or in series, In the first extraction vessel 415 A of system 400, an extract solute/organic solvent composition can be produced and held or transition through organic solvent/extract solute tailspace 245. An extract solute/organic solvent composition can then be introduced either manually or by fluid connection or enclosed channel to at least a second extraction vessel 41 5B through an inlet 255 and optionally held or transitioned through headspace 225. Then extract solute/organic solvent can then transition through filter aid 1 10 which can include one or more of a separator /filter 240, 235 in fluid communication with filter aid 110. Once an extract solute/organic solvent is transitioned through at least a filter aid of system 400, a purified extract can be held or transitioned through a purified extract tailspace 145 to a purified extract outlet 50 for further purification and/or use.

[0045] Extraction vessels disclosed herein may be arranged to improve further operation queuing and/or to reduce the time required to remove and/or supplement or replace biomass, organic solvents, and/or ion exchange resins. Systems of the present disclosure may also include at least one biomass material entry and exit port, at least one essentially insoluble purification or ion exchange matrix entry and exit port, and at least one organic solvent entry and exit port, in order to facilitate the recharging of the system for further rounds of extraction and purification.

[0048] Embodiments disclosed herein can also include methods for extracting a product or removing at least one undesirable agent from a biomass material. In accordance with these embodiments, method disclosed herein can include combining biomass material with one or more organic solvents, (e.g. acetone and/or ethanol), in one or more biomass material solubilization compartments to form a solution. In accordance with these embodiments, methods can also include causing extracts to flow from the one or more extract vessel compartments to one or more biomass material purification compartments that can contain an essentially insoluble purification matrix, for example, an ion-exchange resin, and in certain embodiments, at least one filter. In other embodiments, essentially insoluble purification matrix can be used to isolate and/or separate any undesirable and/or harmful agents (e.g., pheophorbide, arsenic, and the like) from a solubilized biomass solution, so that the desirable products can then be collected in a substantially purified form and/or undesirable agents removed.

EXAMPLES [0047] 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.

[0048] As illustrated in 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% in astaxanthin, while the allowable threshold for astaxanthin for arsenic contamination is 2.0 ppm (0.0002%).

[0049] Unprocessed biomass from H. pluvialis ("Solix-11") was used as the starting biomass material, containing approximately 0.41% pheophorbide and 1.7 ppm arsenic. Using extraction methods disclosed herein with acetone as an exemplary organic solvent, biomass from If, pluvialis was processed in three 16 g loads, for a total of 48 g of oleoresin processed per 8.7 g of resin. As illustrated in Table 1 , pheophorbide contamination was significantly reduced, while arsenic contamination was not undetectable.

Table 1: Extraction of pheophorbide and arsenic from microorganism biomass

[0051] 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.

[0062] 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.

[0053] Moreover, though the description of the disclosure has included descripti on 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.