WO2013142687A1 - Procédé d'extraction de lipides polaires et de lipides neutres avec deux solvants - Google Patents

Procédé d'extraction de lipides polaires et de lipides neutres avec deux solvants Download PDF

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Publication number
WO2013142687A1
WO2013142687A1 PCT/US2013/033301 US2013033301W WO2013142687A1 WO 2013142687 A1 WO2013142687 A1 WO 2013142687A1 US 2013033301 W US2013033301 W US 2013033301W WO 2013142687 A1 WO2013142687 A1 WO 2013142687A1
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Prior art keywords
extraction
phase
lipids
solvent set
solvent
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PCT/US2013/033301
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English (en)
Inventor
Aniket Kale
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Heliae Development, Llc
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Priority claimed from US13/540,182 external-priority patent/US8475660B2/en
Application filed by Heliae Development, Llc filed Critical Heliae Development, Llc
Publication of WO2013142687A1 publication Critical patent/WO2013142687A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0288—Applications, solvents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0215—Solid material in other stationary receptacles
    • B01D11/0223—Moving bed of solid material
    • B01D11/0234—Moving bed of solid material using other slow rotating arms or elements, whereby the general transport direction of the solids is not parallel to the rotation axis, e.g. perpendicular
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/04—Pretreatment of vegetable raw material
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B7/00—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
    • C11B7/0008—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents
    • C11B7/0041—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents in mixtures of individualized solvents (water is not taken into account)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B7/00—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
    • C11B7/0008—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents
    • C11B7/0058—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents in solvents or mixtures of solvents of different natures or compositions used in succession
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B7/00—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
    • C11B7/0008—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents
    • C11B7/0066—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents between two or more non-miscible solvent phases
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/003—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with alcohols

Definitions

  • the invention is concerned with extracting and fractionating algal products, including, but not limited to, oils and proteins.
  • the methods and systems disclosed herein relate to the separation of polar lipids and neutral lipids. More specifically, the systems and methods described herein utilize step extraction and fractionation with amphipath c and hydrophobic solvents to process wet algal biomass.
  • Petroleum is a natural resource composed primarily of hydrocarbons. Extracting petroleum oil from the earth is expensive, dangerous, and often at the expense of the environment. Furthermore, worldwide reservoirs of oil are dwindling rapidly. Costs also accumulate due to the transportation and processing required to convert petroleum oil into usable fuels such as gasoline and jet fuel. [0004] Algae have gained a significant importance in recent years given their ability to produce lipids, which can be used to produce sustainable biofuel. This ability can be exploited to produce renewable fuels, reduce global climate change, and treat wastewater.
  • Algae's superiority as a biofuel feedstock arises from a variety of factors, including high per-acre productivity compared to typical terrestrial oil crop plants, non-food based feedstock resources, use of otherwise non-productive, non-arable land, utilization of a wide variety of water sources (fresh, brackish, saline, and wastewater), production of both bio fuels and valuable co-products such as carotenoids and chlorophyll.
  • algal concentrations in cultures range from about 0.1- 1.0 % (w/v). This means that as much as 1000 times the amount of water per unit weight of algae must be removed before attempting oil extraction.
  • existing oil extraction methods for oleaginous materials strictly require almost completely dr feed to impro ve the yield and quality of the oil extracted. Due to the amount of energy required to heat the algal mass to dry it sufficiently, the algal feed to biofuel process is rendered uneconomical. Typically, the feed is extruded or flaked at high temperatures to enhance the extraction. These steps may not work with the existing equipment due to the single cell micrometnc nature of algae.
  • algal oil is very unstable due to the presence of double bonded long chain fatty acids. The high temperatures used in conventional extraction methods cause degradation of the oil, thereby increasing the costs of such methods.
  • Aigal oil extraction can be classified into two types: disruptive or non-disruptive methods.
  • Disruptive methods involve lysing cells by mechanical, thermal, enzymatic or chemical methods. Most disruptive methods result in emulsions, requiring an expensive cleanup process.
  • Algal oils contain a large percentage of polar lipids and proteins which enhance the emulsifi cation of the neutral lipids. The emulsification is further stabilized by the nutrient and salt components left in the solution.
  • the emulsion is a complex mixture, containing neutral lipids, polar lipids, proteins, and other algal products, which extensive refining processes to isolate the neutral lipids, which are the feed that is converted into biofuel.
  • Non-disruptive methods provide low yields. Milking is the use of solvents or chemicals to extract lipids from a growing algal culture. While sometimes used to extract algal products, milking may not work with some species of algae due to solvent toxicity and cell wall disruption. This complication makes the development of a generic process difficult. Furthermore, the volumes of solvents required would be astronomical due to the maximum attainable concentration of the solvent in the medium,
  • Embodiments described herein relate generally to systems and methods for extracting lipids of varying polarities from an oleaginous material, including for example, an algal biomass.
  • embodiments described herein concern extracting lipids of varying polarities from an algal biomass using solvents of varying polarity
  • a single solvent and water are used to extract and fractionate components present in an oleaginous material.
  • these components include, but are not limited to, proteins, polar lipids, and neutral lipids.
  • more than one solvent is used.
  • a mixture of solvents is used.
  • the disclosed methods allow for separation and fractionation of the components of the wet algal biomass.
  • Another aspect of the systems and methods described herein is the ability to accomplish preliminary refining, because the hydrophobic solvent in the system selectively separates the products further into lipids and non-lipid components.
  • the amount of amphipathic solvent used will depend on the amount of water in the system. The amount of hydrophobic solvent is minimized in order to reduce later processing to remove the solvent.
  • the methods and systems described herein are useful for extracting coproducts of lipids from oleaginous material.
  • coproducts include, without limitation, proteinaceous material, chlorophyll, and carotenoids.
  • Embodiments of the present invention allow for the simultaneous extraction and fractionation of algal products from algal biomass in a manner that allows for the production of both fuels and nutritional products.
  • a method of selectively removing products from an algal biomass comprising substantially intact algal cells includes combining an algal biomass and a first solvent set, to generate a first extraction mixture, the first extraction mixture including a first substantially solid phase and a first liquid phase; separating at least a portion of the first liquid phase of the first extraction mixture from the first substantially solid phase; combining the first extraction substantially solid phase and a second solvent set, to generate a second extraction mixture, the second extraction mixture including a second substantially solid phase and a second liquid phase, wherein the second extraction mixture is less polar than the first extraction mixture; separating at least a portion of the second liquid phase of the second extraction mixture from the second substantially solid phase: combining the second extraction substantially solid phase and a third solvent set, to generate a third extraction mixture, the third extraction mixture including a third substantially solid phase and a third liquid phase, wherein the third extraction mixture is less polar than the second extraction mixture; and separating at least a portion of the third liquid phase of the third extraction mixture from the third substantially solid phase
  • the method further comprises removing at least a portion of the first solvent set from the separated portion of the first liquid phase to obtain a first extraction product. In other aspects, the method further comprises removing at least a portion of the second solvent set from the separated portion of the second liquid phase to obtain a second extraction product. In still other aspects, the method further comprises removing at least a portion of the third solvent set from the separated portion of the third liquid phase to obtain a third extraction product.
  • the solvent set comprises a water miscible or water immiscible solvent.
  • the solvent set comprises two water miscible or two water immiscible solvents.
  • the solvent set comprises one or more water miscible solvents and one or more water immiscible solvents.
  • the first, second and'Or third extraction mixture is heated to a temperature below its boilmg point.
  • the extraction mixture is under a pressure greater than atmospheric pressure.
  • at least one of the first, second, and third solvent sets comprise one or more amphipathic solvents.
  • At least one of the one or more water miscible solvents is selected from the group consisting of methanol, ethanol, isopropanol, acetone, ethyl acetate, and acetonitrile.
  • at least one of the first, second, and third solvent sets comprises ethanol.
  • the solvent set is added to the biomass in a 1 : 1 weight,' ' weight ratio.
  • the cells comprising the algal biomass are not dried or disrupted.
  • the algal biomass is unfrozen.
  • the method further comprises adjusting the pH of at least one of the first, second, and third extraction mixtures to optimize protein extraction.
  • the algal biomass is simultaneously at least partially dewatered while products are selectively extracted from the algal biomass.
  • the first second, and third extraction substantially solid phases comprise substantially intact algal cells.
  • Embodiments of the instant invention include a method of separating polar lipids and neutral lipids from intact algal cells comprising: providing a wet algal biomass comprising intact algal cells which comprise proteins that are soluble in a mixture of an amphipathic solvent and water, polar lipids and neutral lipids; dewatering the wet algal biomass by removing extracellular water to increase the solid content of the wet algal biomass to between 5% and 50% to generate a partially dewatered wet algal biomass; mixing the partially dewatered wet algal biomass with a first amphipathic solvent set in a ratio of about one part first amphipathic solvent set to about one part wet algal biomass by weight, and a first hydrophobic solvent set to generate a first extraction mixture comprising a first heavier phase and a first lighter phase, wherein the first heavier phase comprises the first amphipathic solvent set, neutral lipids, proteins that are soluble in a mixture of an amphipathic solvent and water, and partially dewatere
  • the method of separating polar lipids and neutral lipids from intact algal cells does not include dewatering, the method comprises: providing a wet algal biomass comprising intact algal cells which comprise proteins that are soluble in a mixture of an amphipathic solvent and water, polar lipids and neutral lipids; mixing the wet algal biomass with a first amphipathic sol vent set in a ratio of about one part first amphipathic solvent set to about one part wet algal biomass by weight, and a first hydrophobic solvent set to generate a first extraction mixture comprising a first heavier phase and a first lighter phase, wherein the first heavier phase comprises the first amphipathic solvent set, neutral lipids, proteins that are soluble in a mixture of an amphipathic solvent and water, and wet algal biomass and the first lighter phase comprises the first hydrophobic solvent set and the polar lipids; separating the first lighter phase of the first extraction mixture from the first heavier phase of the first extraction mixture
  • Further embodiments of the instant invention include a method of separating polar lipids and neutral lipids from intact algal cells, comprising: providing a wet algal biomass comprising intact algal ceils which comprise proteins that are soluble in a mixture of an amphipathie solvent and water, polar lipids and neutral lipids; dewaiering the wet algal biomass by removing extracellular water to increase the solid content of the wet algal biomass to between 5% and 50% to generate a partially dewatered wet algal biomass; mixing the partially dewatered wet algal biomass with a first amphipathie solvent set in a ratio of about one part first amphipathie solvent set to about one part wet algal biomass by weight to generate a first extraction mixture; mixing a first hydrophobic solvent set with the first extraction mixture to generate a first heavier phase and a first lighter phase, wherein the first heavier phase comprises the first amphipathie solvent set, neutral lipids, proteins that are soluble in a mixture of an amphipathi
  • FIG. 1A is a flowchart of steps involved in a method according to an exemplary embodiment of the present disclosure.
  • FIG. I B is a schematic diagram of an exemplary embodiment of a dewatering process according to the present disclosure.
  • FIG. 2 is a schematic diagram of an exemplary embodiment of an extraction system according to the present disclosure.
  • FTG. 3 is a comparative graph showing Sohxlet extraction of freeze dried algae biomass using an array of solvents encompassing the complete polarity range showing maximum non -disrupti v e algae oil extraction efficiency and the effect of polarity on the polar and non-polar lipids extraction.
  • FIG. 4A and B are graphic representations showing neutral lipids (A) Purity and (B) Recovery in the two step solvent extraction process using methanol and petroleum ether at three different temperatures.
  • FTG. 5A and B are graphs showing neutral lipids (A) Purity and (B) Recovery in the two step solvent extraction process using aqueous methanol and petroleum ether at three different temperatures.
  • FIG. 6 is a graph showing lipid recovery in the two step solvent extraction process using aqueous methanol and petroleum ether at three different temperatures.
  • FIG. 7 is a graph showing the effect of solvents to solid biomass ratio on lipid recovery.
  • FTG. 8 is a graph showing the efficacy of different aqueous extraction solutions in a single step extraction recovery of aqueous methanol on dry biomass.
  • FIG. 9 is a graph showing the effect of multiple step methanol extractions on the cumulative total lipid yield and the neutral lipids purity.
  • FIG. 10 is a graph showing the cumulative recover ⁇ / of lipids using wet biomass and ethanol.
  • FIG. 1 1 is a graph showing a comparison of the extraction times of the micro wa ve assisted extraction and conventional extraction systems.
  • FIG. 12A is a flowchart of steps involved in a method according to an exemplary embodiment of the present disclosure which incorporates a step of protein extraction. Ail of the units in FIG. 12. A are in pounds.
  • FIG, 12B is a flowchart of steps involved in an exemplar extraction process according to the present disclosure.
  • FTG. 13 is a flowchart and mass balance diagram describing one of the
  • embodiments of the present invention wherein 1000 lbs. of algal biomass was processed through extraction and fractionation in order to separate neutral lipids, polar lipids, and protein from the algal biomass.
  • FIG. 14 is a flowchart describing one of the embodiments of the present invention wherein an algal mass can be processed to form various products.
  • FIG. 15 is a flowchart describing one of the embodiments of the present invention wherein algae neutral lipids are processed to form various products.
  • FIG. 16 is a flowchart describing one of the embodiments of the present invention wherein algae neutral lipids are processed to form fuel products.
  • FIG. 17 is a flowchart describing one of the embodiments of the present invention wherein algae proteins are selectively extracted from a freshwater algal biomass
  • FIG, 18 is a flowchart describing one of the embodiments of the present invention wherein algae proteins are selectively extracted from a saltwater algal biomass.
  • FTG. 19 is a flowchart describing one of the embodiments of the present invention wherein a selected algae protein is extracted from a saltwater or freshwater algal biomass.
  • FIG. 20 is a flowchart describing one of the embodiments of the present invention wherein a selected algae protein is extracted from a saltwater or freshwater algal biomass.
  • FIG. 21 is a photograph showing Scenedescemus sp. cells before and after extraction using the methods described herein. The cells are substantially intact both before and after extraction.
  • FTG. 22 is a schematic of an exemplary extraction scheme for the extraction of proteins by a single solvent (amphipathic) system.
  • FIG. 23 is a schematic of an exemplary extraction scheme for the extraction of polar lipids and proteins by a two-solvent (amphipathic/hydrophobic) system.
  • FIG. 24 is a schematic of an exemplary extraction scheme for the extraction of neutral lipids and proteins by a two-solvent (amphipathic/hydrophobic) system.
  • FIG. 25 is a schematic of an exemplary extraction scheme for the extraction of neutral lipids, polar lipids, and proteins by a two-solvent (amphipathic/hydrophobic) system.
  • FIG. 26 is a schematic demonstrating the eihanol extraction concept.
  • FIG, 27 is a schematic of an exemplary extraction scheme for the extraction of polar lipids and neutral lipids by a two-solvent (amphipathic/hydropbobic) system.
  • conduit or any variation thereof, as used herein, includes any stmcture through which a fluid may be conveyed.
  • Non-limiting examples of conduit include pipes, tubing, channels, or other enclosed structures.
  • reservoir or any variation thereof, as used herein, includes any body structure capable of retaining fluid.
  • Non- limiting examples of reservoirs include ponds, tanks, lakes, tubs, or other similar structures.
  • wet as used herein, is used to mean containing about 50% to about 99.9% water content. Water content may be located either intracellular ly or
  • solvent set is used to mean a composition comprising one or more solvents.
  • solvents can be amphipathic (also known as amphipathic or slightly nonpolar), hydrophilic, or hydrophobic (also known as lipophilic).
  • these solvents are water miscible and in others, they are immiscible in water.
  • Non-limiting example of sol vents that may be used to practice the methods of the instant invention include methanol, ethanol, isopropanol, acetone, ethyl acetate, and acetonitrile, alkanes (liexane, pentane, heptane, octane), esters (ethyl acetate, butyl acetate), ketones (methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK)), aromatics (toluene, benzene, cyclohexane, tetrahydrofuran), haloalkanes (chloroform, trichioroethyiene), ethers (diethyl ether), and mixtures (diesel, jet fuel, gasoline).
  • alkanes liexane, pentane, heptane, octane
  • esters ethyl acetate, buty
  • Non-limiting examples of amphipathic solvents include methanol, ethanol, isopropanol, acetone, ethyl acetate, acetonitrile, and combinations thereof. Food grade ethanols are also desirable for use in the disclosed methods.
  • Non-limiting examples of hydrophobic solvents include alkanes (hexane, pentane, heptane, octane), esters (ethyl acetate, butyl acetate), ketones (methyl ethyl ketone (MEK), methyl isobutyl keione (MIBK)), aromatics (toluene, benzene, cyclohexane, tetrahydrofuran), haloalkanes (chloroform, trichioroethyiene), ethers (diethyl ether), mixtures (diesel, jet fuel, gasoline), and combinations thereof.
  • alkanes hexane, pentane, heptane, octane
  • esters ethyl acetate, butyl acetate
  • ketones methyl ethyl ketone (MEK), methyl isobutyl keione (MIBK)
  • Non-limiting examples of sol vent reco very methods include evaporation, pervaporation, selective adsorption of the sol vent components, steam stripping, and combinations thereof.
  • Pervaporation is the process of selectively removing the vapors of a solvent component through a polymeric membrane by creating vacuum on the permeate side.
  • Selecti v e adsorption is used in the ethanol industry to remo v e small amounts of w ater from aqueous ethanol using zeolite.
  • Steam stripping can be used for non-temperature sensitive operations.
  • interfacial layer means the region comprising and adjoining the phase boundary within which the polarity index of a solvent set are significantly different from the polarity index of another, adjoining solvent set.
  • polarity index refers to the dimensionless Snyner Polarity Index as described by Snyder, L.R. "Classification of the Solvent Properties of Common Liquids” Journal of Chromatography. 92(2): 223-230 (May 1974).
  • oil as used herein includes compositions containing neutral lipids and polar lipids.
  • algae oil and “algal oil” as used herein are used interchangeably.
  • diffuse or “permeate” as used herein may refer to material that has passed through a separation device, including, but not limited to a filter or membrane.
  • retentate as used herein may refer to material that remains after the diffusate has passed through a separation device.
  • polar lipids or any variation thereof, as used herein, includes, but is not limited to, phospholipids and giycolipids.
  • the ierm "neutral lipids" or any variation thereof, as used herein, includes, bui is not limited to, triglycerides, diglycerides, monoglycerides, carotenoids, waxes, sterols.
  • solid phase refers to a collection of material that is generally more solid than not, and is not intended to mean that all of the material in the phase is solid. Thus, a phase having a substantial amount of solids, while retaining some liquids, is encompassed within the meaning of that term. Meanwhile, the term “liquid phase”, as used herein, refers to a collection of material that is generally more liquid than not, and such collection may include solid materials.
  • biodiesel refers to methyl or ethyl esters of fatty acids derived from algae
  • bitterai refers to a food product that provides health and/or medical benefits.
  • Non-limiting examples include carotenoids, carotenes, xanihophyils such as zeaxanthin, astaxanthin, and lutein.
  • biofuel refers to fuel derived from biological source.
  • Non-limiting examples include biodiesel, jet fuel, diesel, jet fuel blend stock and diesel blend stock.
  • impurities when used in connection with polar lipids, as used herein, refers to all components other than the products of interest that are coextracted or have the same properties as the product of interest.
  • lubricants when used in connection with polar lipids, as used herein refers to hydrotreated algal lipids such as C16-C20 alkanes.
  • detergents when used in connection with polar lipids, as used herein refers to giycolipids, phospholipids and derivatives thereof.
  • non-glycerin matter refers to any impurity that separates with the glycerin fraction. A further clean up step will remove most of what is present in order to produce pharmaceutical grade glycerin.
  • unsaturated fatty acids refers to fatty acids with at least one double carbon bond.
  • unsaturated fatty acids include palmitoleie acid, margaric acid, stearic acid, oleic acid, octadecenoic acid, linoleic acid, gamma-linoleic acid, alpha linoleic acid, arachidic acid, eicosenoic acid, homogamma linoleic acid, arachidonic acid, eicosapenenoie acid, behenic, docosadienoic acid, heneieosapeniaenoic, docosatetraenoic acid.
  • Fatty acids having 20 or more carbon atoms in the backbone are generally referred to as "long chain fatty acids”.
  • the fatty acids having 19 or fewer carbon atoms in the backbone are generally referred to as "short chain fatty acids”.
  • Unsaturated long chain fatty acids include, but are not limited to, omega-3 fatty acids, omega-6 fatty acids, and oniega-9 fatty acids.
  • omega-3 fatty acids refers to, but is not limited to the fatty acids listed in Table 1.
  • jet fuel blend stock refers to alkanes with the carbon chain lengths appropriate for use as jet fuels.
  • diesel blend stock refers to alkanes with the carbon chain lengths appropriate for use as diesel.
  • animal feed refers to algae-derived substances that can be consumed and used to provide nutritional support for an animal.
  • human food refers to algae-derived substances that can be consumed to provide nutritional support for people.
  • Algae-derived human food products can contain essential nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals.
  • bioremediation refers to use of algal growth to remove pollutants, such as, but not limited to, nitrates, phosphates, and heavy metals, from industrial wastewater or municipal wastewater.
  • the ierm "wastewater” as used herein refers to industrial wastewaier or municipal wastewater that contain a variety of contaminants or pollutants, including, but not limited to nitrates, phosphates, and heavy metals,
  • enriched shall mean about 50% or greater content.
  • globulin proteins refers to salt soluble proteins.
  • albumin proteins refers to water soluble proteins.
  • glycoprotein proteins refers to alkali soluble proteins.
  • prolamin proteins refers to alcohol soluble proteins.
  • Non-limiting examples of prolamin proteins are gliadin, zein, hordein, avenin.
  • algal culture refers to algal cells in culture medium.
  • algal biomass refers to an at least partially dewatered algal culture.
  • algal paste refers to a partially dewatered algal culture having fluid properties that allow it to flow. Generally an algal paste has a water content of about 90%.
  • algal cake refers to a partially dewatered algal culture that lacks the fluid properties of an algal paste and tends to clump. Generally an algal cake has a water content of about 60% or less,
  • Saltwater algal ceils include, but are not limited to, marine and brackish algal species. Saltwater algal ceils are found in nature in bodies of water such as, but not limited to, seas, oceans, and estuaries. Non-limiting examples of saltwater algal species include
  • Nannochloropsis sp. Dunaliell sp.
  • Freshwater algal cells are found in nature in bodies of w ater such as, b t not limi ted to, lakes and ponds.
  • Non-limiting examples of freshwater algal species include Scendescemus sp. , aemotococcus sp.
  • Non-limiting examples of m croalgae that can be used with the methods of the invention are members of one of the following divisions: Chlorophyta, Cyanophyta
  • the microalgae used with the methods of the invention are members of one of the following classes: Bacillariophyceae, Eusligmalophyceae, and Chrysophyceae.
  • the microalgae used with the methods of the invention are members of one of the following genera: Nannochloropsis, Chlorella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora, and Ochromonas.
  • Non-limiting examples of microalgae species that can be used with the methods of the present invention include: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora cqffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora cqffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicaiissima, Amphora delicaiissima var. capitaia. Amphora sp., Anabaena, Ankistrodesmus,
  • Chaetoceros sp. Chlamydoma perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorell aureoviridis, Chlorella Candida, Chlorella capsuiate, Chlorella desiccate, Chlorella eliipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella jusca var. vacuolata, ChloreUa glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var.
  • Chlorelia chosleri Chlorelia lobophora
  • Chlorelia luteoviridis Chlorelia luteoviridis var. aureoviridis
  • Chlorelia luteoviridis var. lutescens Chlorelia miniata, Chlorelia minutissima, Chlorelia mutabilis, Chlorelia nocturna, Chlorelia ovalis, Chlorelia parva, Chlorelia photophila, Chlorelia pringsheimii, Chlorelia proiothecoides, Chlorelia protothecoides var. acidicola, Chlorelia regularis, Chlorelia regularis var. minima, Chlorelia regularis var.
  • Stichococcus sp. Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula,
  • Tetraedron Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.
  • the biomass can be plant material, including but not limited to soy, corn, palm, camelina, jatropha, canofa, coconut, peanut, safflower, cottonseed, linseed, sunflower, rice bran, and olive.
  • amphipathic/hydrophobie solvent and water mixture that becomes progressively less polar ⁇ i.e., water in solvent/water ratio is progressively reduced as one proceeds from one extraction step to the next).
  • the interstitial water initially present in the algal in the algae (approximately 75% of its weight) is gradually replaced by the amphipamic/hydrophobic solvent to the azeotrope of the amphipathic/hydrophobie solvent. This results in the extraction of components soluble at the polarity developed at each step, thereby leading to simultaneous fractionation of the extracted components.
  • This process leads to a mixture containing two phases, one lighter and one heavier.
  • the lighter phases comprises the hydrophobic solvent and the polar lipids. Nonpolar lipids are also present in this phase.
  • the heavier phase comprises the amphipathic solvent, water, and the remaining algal biomass.
  • the presence of the hydrophobic solvent allows for selective extraction of the lipid components from the other algal components present in the total extract. This results in purer lipid product with almost no protein or water soluble algal product content.
  • a single solvent and water are used to extract and fractionate components present in an oleaginous material.
  • a solvent set and water are used to extract and fractionate components present in an oleaginous material.
  • the oleaginous material is wet.
  • the oleaginous material is algae.
  • Polar lipid recovery depends mainly on its ionic charge, water solubility, and location (intracellular, extracellular or membrane bound).
  • Examples of polar lipids include, but are not limited to, phospholipids and glycolipids.
  • Strategies that can be used to separate and purify polar lipids can roughly be divided into batch or continuous modes. Examples of batch modes include precipitation (pH, organic solvent), solvent extraction and crystallization. Examples of continuous modes include centrifuging, adsorption, foam separation and precipitation, and membrane technologies (tangential flow filtration, diafiftration and precipitation, ultra filtration).
  • the proposed non-disruptive extraction process results in over 90% recovery.
  • the small amount of polar lipids in the remaining bio mass enhances its value when the remaining biomass is used for feed. This is due, at least in part, to the high long chain unsaturated fatty acid content of the biomass.
  • ethanol extracts can further be directly transesterified.
  • the methods and systems described herein are generic for any algae, and enable recovery of a significant portion of the valuable components, including polar lipids, in the algae by the use of a water miscible organic solvent gradient.
  • the neutral lipid fraction obtained by the use of the present invention possesses a low metal content, thereby enhancing stability of the lipid fraction, and reducing subsequent processing steps, Metals tend to make neutral lipids unstable due to their ability to catalyze oxidation. Furthermore, metals inhibit hydrotreating catalysts, necessitating their removal before a neutral lipid mixture can be refined.
  • the systems and methods disclosed herein allow for the extraction of metals in the protein and/ or the polar lipid fractions. This is advantageous because proteins and polar lipids are not highly affected by metal exposure, and in some cases are actually stabilized by metals,
  • Degumming (physical and/or chemical) of vegetable oil is done in order to remove polar lipids (e.g., glycolipids and phospholipids). Vegetable oil that has been chemically degummed retains a significant quantity of neutral lipid. This neutral lipid fraction is further removed from the degummed material using solvent extraction or supercritical/subcritical fluid extraction or membrane technology, in contrast, separation of the neutral lipids from an oleaginous algal biomass is far more difficult than from a vegetable oil feedstock due to the presence of large quantities of polar lipids typically found in algal oil (see Table 2.). This is because the larger percentage of polar lipids present in algal oil enhances the emulsitication of the neutral lipids.
  • polar lipids e.g., glycolipids and phospholipids
  • the emulsitication is further stabilized by the nutrient and salt components left in the solution.
  • the presence of polar lipids, along with metals, results in processing difficulties for separation and utilization of neutral lipids.
  • polar lipids have an existing market, (heir reco very would add significant value to the use of algal oil to generate fuels.
  • Polar lipids are surfactants by nature due to their molecular structure and have a huge existing market. Many of the existing technologies for producing polar lipids are raw material or cost prohibitive.
  • Alternative feedstocks for glycoiipids and phospholipids are mainly algae oil, oat oil, wheat germ oil and vegetable oil.
  • Algae oil typically contains about 30-85 % (w/w) polar lipids depending on the species, physiological status of the ceil, culture conditions, time of harvest, and the solvent utilized for extraction. Further, the glycerol backbone of each polar lipid has two fatty acid groups attached instead of three in the neutral lipid triacylglycerol.
  • Transesterification of polar lipids may yield only two-thirds of the end product, i.e., esterified fatty acids, as compared to that of neutral lipids, on a per mass basis.
  • removal and recovery of the polar lipids would not only be highly beneficial in producing high quality biofuels or triglycerides from algae, but also generate value-added co- products glycoiipids and phospholipids, which in turn can offset the cost associated with algae- based biofuel production.
  • the ability to easily recover and fractionate the various oil and co- products produced by algae is advantageous to the economic success of the algae oil process.
  • a further aspect of the methods and systems described herein is the ability to extract proteins from an oleaginous material, such as algal biomass.
  • the methods disclosed herein of extraction of proteinaceous material from algal biomass comprise a flexible and highly customizable process of extraction and fractionation. For example, in some embodiments, extraction and fractionation occur in a single step, thereby providing a highly efficient process.
  • Proteins sourced from such biomass are useful for animal feeds, food ingredients and industrial products. For example, such proteins are useful in applications such as fibers, adhesives, coatings, ceramics, inks, cosmetics, textiles, chewing gum, and biodegradable plastics,
  • an algal biomass is mixed with an equal weight of solvent.
  • an algal biomass is mixed with a lesser weight of solvent.
  • an algal biomass is mixed with a greater weight of solvent.
  • the amount of solvent mixed with an algal biomass is calculated based on the solvent to be used and the desired polarity of the algal biomass/solvent mixture.
  • the algal mass is extracted in several steps. In an exemplary embodiment, an algal biomass is sequentially extracted, first with about 50-60% of its weight with a slightly nonpolar, water miscible solvent.
  • the remaining algal solids are extracted using about 70% of the solids' weight in solvent.
  • a third extraction is then performed using about 90% of the solid's weight in solvent.
  • the solvent used is ethanol.
  • Components may be selectively isolated by varying the ratio of solvent. Proteins can be extracted from an algal biomass with about 50% ethanol, polar lipids with about 80% ethanol, and neutral lipids with about 95% or greater ethanol. If methanol were to be used, the solvent concentration to extract proteins from an algal biomass would be about 70%. Polar lipids would require about 90% methanol, and neutral lipids would require about 100% methanol.
  • Embodiment s of the systems and methods described herein exhibit surprising and unexpected results.
  • the recovery/extraction process can be done on a wet biomass. This is a major economic advantage as exemplary embodiments avoid the use of large amounts of energy required to dry and disrupt the cells.
  • Extraction of neutral lipids from a dry algal biomass is far more effective using the systems and methods of the present invention.
  • the yields obtained from the disclosed processes are significantly higher and purer than those obtained by conventional extractions. This is because conventional extraction frequently results in emulsions, rendering component separations extremely difficult,
  • Exemplary embodiments may be applied to any algae or non-algae oleaginous material.
  • Exemplary embodiments may use any water-miscible slightly nonpolar solvent, including, but not limited to, methanol, ethanol, isopropanol, acetone, ethy l acetate, and acetonitrile.
  • Specific embodiments may use a green renewable solvent, such as ethanol.
  • the alcohol solvents tested resulted in higher yield and purity of isolated neutral lipids.
  • Ethanol is relatively economical to purchase as compared to other solvents disclosed herein.
  • extraction and fractionation can be performed in one step followed by membrane- based purification if needed.
  • the resulting biomass is almost devoid of water and can be completely dried with lesser energy than an aqueous algae slurry.
  • the solvent used to extract is ethanol.
  • Other embodiments include, but are not limited to, cyclohexane, petroleum ether, pentane, hexane, heptane, diethyl ether, toluene, ethyl acetate, chloroform, dicholoromethane, acetone, acetonitrile, isopropanol, and methanol.
  • the same solvent is used in sequential extraction steps.
  • different solvents are used in each extraction step.
  • two or more solvents are mixed and used in one or more extraction steps.
  • a mixture of two or more solvents used in any of the extraction steps includes at least one hydrophilic solvent and at least one hydrophobic solvent.
  • the hydrophilic solvent extracts the material from the biomass via diffusion.
  • a relatively small amount of hydrophobic solvent is used in combination and is involved in a liquid-liquid separation such that the material of interest is concentrated in the small amount of hydrophobic solvent.
  • the two different solvents then form a two-layer system, which can be separated using techniques known in the art.
  • the hydrophobic solvent can be any one or more of an alkane, an ester, a ketone, an aromatic, a haioaikane, an ether, or a commercial mixture (e.g., ciiesel, jet fuel, gasoline).
  • the extraction processes described herein incorporate pH excursion in one or more steps. Such pH excursion is useful for isolating proteinaceous material.
  • the pH of the extraction process is acid (e.g. , less than about 5).
  • the pH of the extraction process is alkaline (e.g., greater than about 10).
  • systems of the invention can be used to achieve methods of the invention.
  • a flowchart 100 provides an overvie of the steps involved in exemplary embodiments of methods used in the fractionation and purification of lipids from an algae-containing biomass.
  • a first step 1 algal cells are harvested.
  • water is removed from algal ceils to yield a 10-25% solid biomass.
  • a solvent-based extraction is performed on the biomass and the fractions are collected.
  • step 130 will also incorporate pH-based extraction and fraction collection.
  • a solid/liquid phase separation including, but not limited to techniques such as filtration, decanting, and centrifugation, may be performed in a step 140 to in order to separate out smaller lipid components.
  • the algae biomass when harvested in step 1 10 typically consists of about 1-5 g/L of total solids.
  • the biomass can be partially dewatered in step 120 using techniques including, but not limited to, dissolved air floatation, membrane filtration, fiocculation, sedimentation, filter pressing, decantatson or centrinigatiosi.
  • Dewatermg is the removal of some, most, or all of the water from a solid or semisolid substance.
  • Embodiments of the present invention utilize dewaiering techniques to remove water from a harvested algal biomass. Dewatermg can be carried out using any one of or a combination of any of the methods described herein, as well as by any other methods known to one of skill in the art.
  • the dewatered algae biomass resulting from step 120 typically consists of about 10- 30% solids.
  • This biomass can then be extracted with water miscible slightly nonpolar solvents (e.g., alcohols), in a multistage countercurrent solvent extraction process segregating the fractions at each stage. This type of process can reduce both capital and operating expenses.
  • the biomass also undergoes acid and/or alkaline extraction to fractionate protein material.
  • dewatermg of an algal biomass can be carried out by treating the harvested algal biomass with a solvent such as ethanol.
  • the algal biomass is then allowed to settle out of solution and the liquids may then be removed by methods such as, but not limited to, siphoning.
  • This novel method of dewatering has lower capital and operating costs than known methods, enables solvent recycling, reduces the cost of drying the biomass, and has the added benefit of decreasing the polarity of the algal biomass prior to beginning ex traction and/or separation of algal components.
  • the sol vent -based sedimentation processes described herein are effective, in part, due to the fact that organic solvents reduce or neutralize the negative charge on the algae surface.
  • dewatering methods are combined in order to remove even more water.
  • the addition of solvent during the dewatering process begins the process of extraction,
  • FIG. IB shows an illustrative implementation of a dewatering process 300.
  • An algal culture 310 having a final dry weight of about 1 g/L to about 10 g/L (i.e., 0.1 - 1% w/w) is subjected to a water separation process 320.
  • Process 320 can include centrifugation, decanting, settling, or filtration, in one embodiment, a sintered metal tube filter is used to separate the algal biomass from the water of the culture. When using such a filter, the recovered water 330 is recycled directed to other algae cultures.
  • the algal biomass recovered has been concentrated to an "algae paste" with a algae density as high as about 200 g L (i.e., 10-20% w/w).
  • This concentrated algae paste is then treated with a solvent 340 in a solvent-based sedimentation process 350.
  • Sedimentation process 350 involves adding solvent 340 to the algae paste to achieve a mixture having a weight/weight solvent to bio mass ratio of between about 1 : 1 to a bout 1 : 10.
  • the algae is allowed to settle in a settling vessel, and a solvent / ' water mixture 360 is removed by, for example, siphoning and/or decanting.
  • the solvent can be recovered and reused by well- known techniques, such as distillation and/or pervaporation.
  • the remaining wet biomass 370 is expected to have a solids content of about 30% to about 60% w/w in an alcohol and water solution.
  • Solvents ideal for dewatering are industrially common water-soluble solvents with densities over 1 .1 g/niL or below 0.9 g/niL. Examples include isopropanol, acetone, acetonitrile, t-butyl alcohol, ethanol, methanol, 1 -propanol, heavy water ⁇ D ), ethylene glycol, and/or glycerin. If the solvent density is o ver 1.1 g/mL then the algae biomass would float rather than create a sediment at the bottom of the settling vessel.
  • FTG. 2 is a schematic diagram of an exemplary embodiment of an extraction system 200.
  • the wet or dry algal biomass is transported using methods known in the art, including, but not limited to a moving belt, a screw conveyor, or through extraction chambers.
  • the solvent for extraction is recirculated from a storage tank assigned to each biomass slot position.
  • the extraction mixture is filtered, returning the biomass solids back into the slot and the extract into the storage tank.
  • the solids on the belt move periodically based on the residence time requirement for extraction.
  • the extracts in each storage tank may either be replenished at saturation or continuously replaced by fresh solvent. This would also reduce the downstream processing time and cost drastically.
  • This embodiment comprises a primary reservoir 210, a transport mechanism 220, a plurality of separation devices 241 -248 (e.g., membrane filtration devices), a plurality of extraction reservoirs 261-268, and a plurality of recycle pumps 281 -287.
  • primary reservoir 210 is divided up into a plurality of inlet reservoirs 21 1- 218.
  • algal biomass 201 is placed a first inlet reservoir 21 1 near a first end 221 of transport mechanism 220.
  • solvent 205 is placed into inlet reservoir 218 near a second end 222 of transport mechanism 220.
  • Transport mechanism 220 directs the algal biomass along transport mechanism 220 from first end 221 towards second end 222.
  • the algal biomass is iransported, it passes through the plurality of separation de vices 241 -248 and is separated into fractions of varying polarity.
  • the diffusate portions that pass through separation devices 241 -248 are directed to reservoirs 261-268.
  • the diffusate portion of the algal biomass that passes through the first separation device 241 (e.g., the portion containing liquid and particles small enough to pass through separation device 2 1) is directed to the first reservoir 261. From first reservoir 261 , the diffusate portion can be recycled back to first inlet reservoir 20.1. The retentate portion of the algal biomass that does not pass through first separation device 2.41 can then be directed by transport mechanism 220 to second inlet reservoir 212 and second separation device 242, which can comprise a finer separation or filtration media than the first separation device 241 .
  • the segment of the diffusate portion that passes through second separation device 242. can be directed to second reservoir 262, and then recycled back to second inlet reservoir 212 via recycle pump 282.
  • the retentate or extracted portion of the algal biomass that does not pass through second separation device 242 can be directed by transport mechanism 220 to third inlet reservoir 213. This process can be repeated for inlet reservoirs 213-218 and separation devices 243-248 such that the retentate portions at each stage are directed to the subsequent inlet reservoirs, while the diffusate portions are directed to the recycle reservoirs and recycled back to the current inlet reservoir.
  • the first fraction will be extracted with the highest water to slightly nonpolar solvent ratio, i.e., most polar mixture, while the last fraction will be extracted with the most pure slightly nonpolar solvent, i.e. the least polar mixture.
  • the process therefore extracts components in the order of decreasing polarity with the fraction.
  • the function of the first fraction is to remove the residual water and facilitate the sol vent extraction process.
  • the fractions that follow are rich in polar lipids, while the final fractions are rich in neutral lipids.
  • the oil fraction can be esterified to liberate the long chain unsaturated fatty acids.
  • the carotenoids and long chain unsaturated fatty acids can be separated from the oil using processes such as molecular distillation in conjunction with non-molecular distillation. All of the fatty acids can be separated from the carotenoids using the molecular distillation.
  • the distillates can be fractionated using a simple distillation column to separate the lower chain fatty acids for refining.
  • the long chain unsaturated fatty acids remain as high boiling residue in the column.
  • ihe extraction system and methods described herein incorporate one or more steps to isolate protein material from the oleaginous material (e.g., algal biomass).
  • Such protein extraction steps employ pH adjustments) to achieve isolation and extraction of protein.
  • the pH of the solvent in the first separation device is optimized for protein extraction, resulting in a first fraction that is rich in protein material.
  • the pH of the protein extraction step is adjusted depending on the pKa of the proteins of interest.
  • the pKa of a protein of interest may be ascertained using methods known to one of skill in the art, including, but not limited to using the Poisson-Boltzmann equation, empirical methods, molecular dynamics based methods, or the use of titration curves.
  • the solvent pH is alkaline.
  • the solvent pH is greater than about 10.
  • the solvent pH ranges from about 10 to about 12.
  • the solvent pH is about 10, about 1 1 , or about 12.
  • the solvent pH is acid.
  • the solvent pH is less than about 5.
  • the solvent pH ranges from about 2 to about 5.
  • the solvent pH is about 2, about 3, about 4, about 4.5, or about 5.
  • the extracted portion of the first separation device is then directed to subsequent inlet reservoirs to achieve extraction and fractionation based on polarity.
  • protein material is separated in the final separation device by similar means (i.e., solvent pH adjustment).
  • Adjustment of solvent pH is accomplished in accordance with methods known to those of skill in the art.
  • acid pH is achieved by mixture of an appropriate acid into the solvent stream.
  • acids useful for protein extraction include, without limitation, phosphoric acid, sulfuric acid, and hydrochloric acid.
  • alkaline pH is achieved by addition and mixture of an appropriate base into the solvent stream.
  • bases useful for protein extraction include, without limitation, potassium hydroxide, and sodium hydroxide.
  • protein extraction is performed in a system separate from the extraction and fractionation system described herein.
  • an algal biomass is soaked in a pH-adjusted solvent mixture, followed by isolation via an appropriate separation technique (e.g., centrifugation, or filtration).
  • the remaining solid is then introduced into an extraction and fractionation system based on polarity, as described herein.
  • the remaining extract from an extraction and fractionation process based on polarity is exposed to a pH-adjusted solvent mixture to isolate protein material at the end of the extraction process,
  • FIG. 3 presents the data gathered by extraction of a dry algal mass using various polar and nonpolar solvents combined with a Sohxlet extraction process.
  • 60-70% purity of neutral lipids and 15-45% of total lipid recovery can be achieved without disruption and solvent extraction.
  • the longest chain alkane solvent tested, heptane recovered 60% of the neutral lipids and 42% of the total lipid.
  • FIG. 3 shows, the results of extraction of dry algal mass using solvents and conventional extraction methods such as hexane are inefficient, expensive, and result in poor yields.
  • the methanol extractions were performed at different temperatures, 40 °C, 50 °C, and 65 °C, in order to determine which was optimal.
  • the petroleum ether extraction was performed at 35°C, close to the boilmg point of the solvent. Petroleum ether was chosen because of its high selectivity for neutral lipids, low boiling point, and the product quality observed after extraction.
  • FIG. 4 A shows that the neutral lipid purity in a petroleum ether extraction carried out after a methanol extraction step at 65' J C is over 80%, demonstrating that the combination of these two extraction steps enhanced the neutral lipid content of the final crude oil product
  • FIG. 4B shows that the total neutral lipid recovery was low and there was a significant amount of neutral lipid loss in the first step
  • FIG, 5A and 5B sho the results of extracting the aforementioned biomass with 70% v/ ' v aqueous methanol followed by extraction with petiOleum ether.
  • FTG. 5A shows that the neutral lipid purity was much higher in the petroleum ether extraction than was achieved by the use of pure methanol.
  • the loss of neutral lipids was greatly reduced by the use of aqueous methanol in the first extraction step.
  • methanol extraction at higher temperatures improved neutral lipid purity but slightly decreased the total lipid recovery in the subsequent step.
  • the temperature of the extraction process is controlled in order to ensure optimal stability of algal components present in the algal biomass.
  • Algal proteins, carotenoids, and chlorophyll are examples of algal components that exhibit temperature sensitivity.
  • the temperature is increased after the temperature sensitive algal components have been extracted from the algal biomass.
  • the temperature of the extraction process is adjusted in order to optimize the yield of the desired product. Extractions can be run from ambient temperature up to, but below, the boiling point of the extraction mixture. In still other embodiments, the temperature of the extraction process is changed depending on the solubility of the desired product. In still other embodiments, the extraction temperature is optimized depending on the algal strain of the biomass to be extracted. Elevated extraction temperatures increase the solubility of desired compounds and reduce the viscosity of the extraction mixture enhancing extraction recovery.
  • the extraction is run under pressure to elevate the boiling point of the extraction mixture.
  • the pressure is increased to the degree necessary to prevent boiling, while maintaining the temperature of the extraction mixture below a temperature at which any of the desired products would begin to degrade, denature, decompose, or be destroyed.
  • the extraction is performed near the boiling point of the solvent used, at the conditions under which the extraction is performed (e.g., atmospheric or elevated pressures). In other embodiments, the extraction is performed near the boiling point of the extraction mixture, again accounting for other extraction conditions. At such temperatures, vapor phase penetration of the solvent into the algal cells is faster due to lower mass transfer resistance. If the extraction temperature is allowed to significantly exceed the boiling point of the solvent, the solvent- water system can form an azeotrope. Thus, maintaining the sy stem at or near the boiling point of solvent would generate enough vapors to enhance the extraction, while reducing expense. In addition, the solubility of oil is increased at higher temperatures, which can further increase the effectiveness of extraction at temperatures close to the solvent boiling point. FIG.
  • the extraction is carried out under ambient lighting conditions.
  • the extraction is carried out in an opaque container such as, but not limited to, a steel tube or casing, in order to protect light sensitive algal components from degradation. Carotenoids are light sensitive algal components.
  • the extraction takes place under normal atmospheric conditions. In still other embodiments, the extraction takes place under a nitrogen atmosphere in order to protect algal components prone to oxidation. In still other
  • the extraction takes place under an atmosphere of inert gas in order to protect algal components prone to oxidation.
  • Algal components that might be prone to oxidation include carotenoids, chlorophyll, and lipids.
  • the solvent-to-solid ratio for the extraction is between 3-5 based on the dry weight of the solids in the biomass.
  • the residual algal biomass is rich in carbohydrates (e.g. , starch) and can be used as a feed stock to produce the solvent used for extraction.
  • FIG. 7 shows the effect of the sol v ent t o solid ratio on the total lipid recovery. As the solvent to solid ratio was increased, there was a corresponding and drastic increase in total lipid recoveiy. It is believed that this was because of the lower solubility of lipids in methanol as compared to other commonly used oil extraction solvents such as hexane.
  • the solubility of components is affected by the polarity of solvent used in an extraction process.
  • the solubility properties of a desired product can be used to determine the appropriate ratio of wet biomass to solvent in order to selectively extract the desired product.
  • the hydrophobic solvent amount is calculated based on the solubility of the amphipathic solvent and lipids in the hydrophobic solvent.
  • a polarity index of between about 6.5 to 6,7 extracts proteins and other alcohol soluble materials such sugars and salts. It has been found that after the removal of alcohol and proteins from such an exiraction mixtures, the remaining material is an excellent fermentation medium.
  • the polarity index of the mixture for extraction of polar lipids and neutral lipids is calculated to be between about 5.6 to 5,9 and 5.3 to 5.5 respectively.
  • the key to extracting the desired components is to manipulate the polarity of the extraction mixture by varying the amounts of hydrophobic and amphipathic solvents, always accounting for the water present in the wet algal biomass. By using the known polarity index of a solvent, the amount of the solvents can be adjusted to achieve the desired extraction mixture polarity.
  • a 40% w/w wet biomass has 40 g biomass and 60 g water for every 00 g of wet biomass.
  • f 100 g of ethanol is added to this mixture, the ratio of ethanol to wet biomass is 1 part wet biomass to 1 part ethanol and the concentration of ethanol in the mixture is 100/(100+60) equals about 62% w/w of ethanol in the liquid phase.
  • 62% w/w of eihanol in ethanol water mixture corresponds to a polarity index of 6.6, calculated by weight and averaging the polarities of the components, Ethanol, having a polarity index of 5.2, and water, having a polarity index of 9, in a mixture containing 62% ethanol and 38% water results in a polarity index of (0.62*5.2+38*9) about 6.6.
  • the polarity index of the mixture for extraction of polar lipids and neutral lipids is calculated to be about 5.8 and 5.4 respectively,
  • a 40% w/w wet biomass has 40 g biomass and 60 g water for every 100 g of wet biomass. If 100 g of eihanol is added to this mixture, the ratio of ethanol to wet biomass is 1 part wet biomass to 1 part ethanol and the concentration of ethanol in the mixture is 100/(100+60) equals about 62% w/w of ethanol in the liquid phase. 62% w/w of ethanol in ethanol water mixture corresponds to a polarity index of 6.6, calculated by weight and averaging the polarities of the components.
  • Ethanol having a polarity index of 5.2, and water, having a polarity index of 9, in a mixture containing 62% ethanol and 38% water results in a polarity index of (0.62*5.2+38*9) about 6.6.
  • the polarity index of the mixture for extraction of polar lipids and neutral lipids is calculated to be about 5.8 and 5.4 respectively. If acetonirrile is used as the hydrophobic solvent, the amount of acetonitrile would be between about 2% and 4% of the amount of ethanol, because there would be an interfacial layer volume almost twice the amount of water in the etihanol/acetonitrile/water azeotrope, which is about 2%.
  • a 40% w/w wet biomass has 40 g biomass and 60 g water for every 100 g of wet biomass. If 100 g of ethanol is added to this mixture, the ratio of ethanol to wet biomass is 1 part wet biomass to 1 part ethanol and the concentration of ethanol in the mixture is 100/(100+60) equals about 62% w/w of eihanol in the liquid phase, 62% w/ of ethanol in ethanol water mixture corresponds to a polarity index of 6.6, calculated by weight and averaging the polarities of the components.
  • Ethanol having a polarity index of 5,2, and water, having a polarity index of 9, in a mixture containing 62% eihanol and 38% water results in a polarity index of (0.62*5.2+38*9) about 6.6.
  • the polarity index of the mixture for extraction of polar lipids and neutral lipids is calculated to be about 5.8 and 5,4 respectively. If hexane is used as the hydrophobic solvent, the amount of hexane would be between about 6% and 12% of the amount of ethanol, because there would be an interfacial layer volume almost twice the amount of water in (he ethanol/hexane/water azeo trope, which is about 6%.
  • the size of the mterfacial layer is related to the miscibility of the two solvents used in the system, and is further affected by the detergent-like effects of the proteins and various lipids present in the extraction mixture.
  • the size of the mterfacial layer is also related to the temperature of the mixture. As the temperature is increased, the two solvents become more miscible, thereby increasing the size of the mterfacial layer.
  • the wet biomass to IPA ratio is (1 -0.55)/Q.6 ⁇ 0.75.
  • the extraction mixture described in all examples is made up of a substantially solid phase and a substantially liquid phase. These phases are then separated post extraction. This can then be followed by removal of the liquid solvent from the liquid phase, yielding an extraction product, in some embodiments, the solvent is evaporated. In such an
  • a liquid-liquid extraction technique can be used to reduce the amount of solvent that needs to be evaporated. Any solvents used can be recycled if conditions allow.
  • FIG, 9 is a chart showing the effect of an eight step methanol extraction on the cumulative total lipid yield and the purity of the extracted neutral lipid.
  • 1 12 grams of wet biomass 25.6% dry weight
  • FIG. 9 shows that it is possible to isolate high purity neutral lipid once the polar lipids are all extracted.
  • FIG. 10 shows that recovery of lipids can be made more efficient by the use of ethanol to extract lipids and protein from wet biomass.
  • 80% total lipid recovery can be achieved in about 4 steps rather than the 9 generally needed by using methanol.
  • This increase in recovery may be attributed to greater solubility of lipids in ethanol as compared to methanol.
  • the boiling point of aqueous ethanol is higher than aqueous methanol, facilitating further recovery of lipids. This is because the higher temperature renders the oil less viscous, thereby improving diffusability.
  • Another distinct advantage of this process is using the residual ethanol in the oil fraction for transesterification as well as lowering the heat load on the biomass drying operation.
  • FIG. 10 demonstrates that the initial fractions are non- lipid rich, containing proteins and other highly polar molecules, followed by the polar lipid rich fractions and finally the neutral lipid fractions.
  • Another embodiment of the current invention utilizes microwaves to assist extraction. Based on previously gathered data disclosed in this application, it is shown that methanol is the best single solvent for extraction of all lipids from algae. Hence, a single solvent multiple step extraction, as described in Example 1 of the instant application, was performed in order to gather data on the efficacy of a one solvent microwave extraction system.
  • FIG. 1 1 is a logarithmic plot comparing the extraction time and total lipid recovery of conventional extraction and microwave-assisted extraction. Based on the slope of the curve, it was calculated thai the microwave system reduces the extraction time by about five fold or more. While the conventional methods have a higher nei lipid recovery, this is due to higher recoveries of polar lipids. Based on these results, the conditions for extraction of dry algal biomass using solvents with and without microwave assistance have been optimized. Some embodiments of the invention use traditional microwave apparatus, which emit wavelengths that excite water molecules. Further embodiments of the invention utilize customized microwave apparatus capable of exciting different solvents. Still other embodiments of the invention utilize custom microwave apparatus capable of exciting the lipids present in the algal biomass. In some embodiments, the lipids present in the algal biomass are excited using microwaves, thereby enhancing the separation and extraction of the lipid components from the algal biomass.
  • Moisture content is another parameter of biomass that will influence the efficiency of oil extraction.
  • dry algal mass is extracted and fractionated.
  • the algal mass is wet. Biomass samples with algae mass contents of 10%, 25%, and 33% were used to investigate the influence of moisture on extraction performance.
  • FIG, 12 A shows an illustrative process 400 for a step-wise extraction of products from an algae biomass. All units in FIG. 12A are in pounds. FIG, 12 A shows a mass balance of the process 400, while the details of the equipment and/or systems for performing the process are described elsewhere herein.
  • a biomass containing 5 pounds of algae has about 0.63 pounds of polar lipids, 1.87 pounds neutral lipids, 1 pound protein, and 1.5 pounds carbohydrates.
  • the biomass and 1000 pounds of water is processed in a dewatering step 405, which separates 950 pounds of water from the mixture and passes 5 pounds of algae in 45 pounds of water to a fsrst extraction step 410.
  • any of the dewatering techniques discl osed herein can be used tin dewatering step 405.
  • the first extraction step 410 238 pounds of ethanol and 12 pounds of water are combined with the algae and water from the previous step.
  • the first extraction step 410 has a liquid phase of about 80.9% w/w ethanol.
  • a first liquid phase of 231 pounds of ethanol, 53 pounds of water, and 0.5 pounds of algal proteins are recovered, from which water and ethanol are removed by, e.g., evaporation, leaving a protein- rich product 415. Solvent recovered from the evaporation can be recycled to the first extraction step 410.
  • first solid phase from the first extraction step 410 is passed to a second extraction step 420; this first solid phase includes 4.5 pounds of algae, 2.6 pounds of water, and 10.9 pounds of ethanol. Eighty- six pounds of ethanol and 4 pounds of water are added to the first solid phase from the previous step.
  • the second extraction step 420 has a liquid phase of about 93.6% w/w ethanol.
  • a second liquid phase of 85.9 pounds ethanol, 5.9 pounds water, and 0.6 pounds polar lipids are recovered, from which water and ethanol are removed by, e.g., evaporation, leaving a polar lipid-rich product 425. Solvent recovered from the evaporation can be recycled to the second extraction step 420.
  • a second solid phase from the second extraction step 420 is passed to a third extraction step 430; this first solid phase includes 3.9 pounds of algae, 0.7 pounds of water, and 1 1 pounds of ethanol. Seventy-found and a half pounds of ethanol and 3.5 pounds of water are added to the second solid phase from the previous step.
  • the third extraction step 430 has a liquid phase of about 95.4% w/w ethanol, A third liquid phase of 78.9 pounds ethanol, 3.9 pounds water, and 1.6 pounds neutral lipids are recovered, from which water and ethanol are removed by, e.g., evaporation, leaving a neutral lipid-rich product 435. Solvent recovered from the evaporation can be recycled to the second extraction step 430 A solid phase of 2.3 pounds algae, 0.3 pounds water, and 6.6 pounds ethanol remain.
  • FIG. 12B shows an illustrative implementation 500 of one of the extraction steps of process 400.
  • An algae biomass and solvent mixture 505 is provided to an extraction vessel 510.
  • the mixture is provided to a coarse filtration system 515, such as a sintered metal tube filter, which separates the mixture into a liquid phase and a solid phase.
  • the solid phase is passed to a downstream extraction step.
  • the liquid phase is passed to a solvent removal system 520, e.g., an evaporator, to reduce the solvent (e.g., ethanol) content in the liquid phase.
  • the liquid phase remaining after solvent removal is, optionally, passed to a centrifuge 525. Any solids remaining in the solvent removal system are recycled or discarded. Centrifuge 525 assists in separating the desired algal product (e.g., proteins or lipids) from any remaining water and/or solids in the liquid phase.
  • FIG. 14 shows an example of a process 600 by which an algal mass can be processed to form or recover one or more algal products.
  • an algal biomass is extracted in a step-wise manner in a front-end process 605 using the methods disclosed herein. The extraction and separation steps are followed by an esterification process 610, a hydrolysis process 615, a hydrotreating process 620, and/or a distillation process 625 to further isolate components and products.
  • the components and products include algal lipids, algal proteins, glycerine, carotenoids, nutraceuticals (e.g., long chain unsaturated oils and/or esters), fuel esters (generally, the esters having chain lengths of C20 or shorter), fuels, fuel additives, naphtha, and/or liquid petroleum substitutes.
  • fuel esters generally, the esters having chain lengths of C20 or shorter
  • fuels fuel additives, naphtha, and/or liquid petroleum substitutes.
  • the fuel esters are C 16 chain lengths.
  • the fuel esters are CI 8 chain lengths.
  • fuel esters are a mixture of chain lengths, C20 or shorter.
  • the esterification process 610, hydrolysis process 615, hydrotreating process 620, and distillation process 625 are optional and can be used in various orders.
  • the dashed arrows and dotted arrows indicate some, but not all, of the options for when the hydrolysis, hydrotreating, and/or distillation processes may be performed in the processing of the lipid fractions.
  • the neutral lipids fraction can be directly hydrotreated in order to make fuel products and/or additives.
  • the neutral lipid fraction can be passed to esterification process 610.
  • Esterification process 610 can include techniques known in the art, such as acid / base catalysis, and can include transesterification. Although base catalysis is not excluded for producing some products, acid catalysis is preferred as those techniques avoid the soaps that are formed during base catalysis, which can complicated downstream processing. Enzymatic esterification tecimiques can also be used. Esterification can process subsianiially pure lipid material (over 75% lipid, as used herein). After esterification, glycerine byproduct can be removed. The esterified lipids can then undergo molecular and/or nonmolecular distillation (process 625) in order to separate esterified lipids of different chain lengths as well as carotenoids present in the lipid fraction.
  • process 625 molecular and/or nonmolecular distillation
  • the esterified lipids can then be passed to hydrotreating process 620 to generate jet fuel, biodiesel, and other fuel products.
  • Any hydrotreating process known in the art can he used; such a process adds hydrogen to the lipid molecules and removes oxygen molecules.
  • Exemplary conditions for hydrotreating comprise reacting the triglycerides, fatty acids, fatty acid esters with hydrogen under high pressure in the range of 600 psi and temperature in the range of 600T. Commonly used catalysts are NiMo or CoMo.
  • the esterification process 610 reduces the levels of certain phosphorus and metals compounds present in algal oils. These materials are poisons to catalysts typically used in hydrotreating processes. Thus, esterification prior to hydrotreating prolongs the life of the hydrotreating catalyst. Also, esterification reduces the molecular weight of the compounds being hydrotreated, thereby improving the performance of the hydrotreating process 620. Further still, it is advantageous to retain the fuel esters from the distillation process 625 to be hydrotreated in a vaporous form, as doing so reduces the energy needed for hydrotreating.
  • the neutral algal lipids are directly hydrotreated in order to convert the lipids into fuel products and additives. While in other implementations, the neutral lipids are esterified and separated into carotenoids, long chain unsaturated esters, eicosapentaenoie acid (EPA) esters, and/or fuel esters via distillation process 625. Distillation process 625 can include molecular distillation as well as any of the distillation techniques known in the art. For example, the distillates can be fractionated using a simple distillation column to separate the lower chain fatty acids for refining.
  • EPA eicosapentaenoie acid
  • the long chain unsaturated fatty acids remain as high boiling residue in the column, in some embodiments, the remaining vapor can then be sent to the hydrotreating process.
  • Two of the advantages of the present invention are that it yields pure feed as well as a vapor product, which favors the energy intensive hydrotreating reaction, as described above.
  • polar lipids (and, optionally, neutral lipids) are hydro lyzed in hydrolysis process 615 before being passed to the esterification process. Doing so unbinds the fatty acids of the algal lipids, and enables a greater amount of the algal lipids to be formed into useful products.
  • FIG, 15 is a flowchart showing a process 700 for producing nutraceutical products from neutral lipids.
  • neutral lipids are fed to an adsorption process 705 that separates carotenoids from EPA-rich oil.
  • the neutral lipids can be from an algae source generated by any of the selective extraction techniques disclosed herein. However, the neutral lipids can be from other sources, such as plant sources,
  • Adsorption process 705 includes contacting the neutral lipids with an adsorbent to adsorb the carotenoids, such as beta carotene and xanthophylls.
  • the adsorbent is Diaion HP20SS (commercially available from ITOCHU Chemicals America, Inc.).
  • the neutral lipids can contact the adsorbent in a batch-type process, in which the neutral lipid and adsorbent are held in a vessel for a selected amount of time. After the contact time, the absorbent and liquid are separated using techniques known in the art. In other
  • the adsorbent is held in an adsorbent bed, and the neutral lipids are passed through the adsorbent bed. Upon passing through the adsorbent bed, the carotenoids content of the neutral lipids is reduced, thereby producing an oil rich in EPA.
  • the carotenoids can be recovered from the adsorbent material by treating the adsorbent with an appropriate solvent, including, but not limited to, alcohols such as ethanoi, isopropyl alcohol , butanol, esters such as ethyl acetate or butyl acetate, alkanes such as hexane, and pentane.
  • an appropriate solvent including, but not limited to, alcohols such as ethanoi, isopropyl alcohol , butanol, esters such as ethyl acetate or butyl acetate, alkanes such as hexane, and pentane.
  • FIG, 16 is a flowchart showing a process 800 for producing fuel products 830 from neutral lipids 805.
  • the neutral lipids can be from an algae source generated by any of the selective extraction techniques disclosed herein. However, the neutral lipids can be from other sources, such as plant sources.
  • the neutral lipids are treated in a degumming process 810, in which the lipids are acid washed to reduce the levels of metals and phospholipids in the neutral lipids.
  • a relatively dilute solution of phosphoric acid is added to the neutral lipids, and the mixture is heated and agitated. The precipitated phospholipids and metals are then separated from the remaining oil, for example, by centrifuge.
  • bleaching process 815 to remove chlorophylls and other color compounds.
  • bleaching process 815 includes contacting the oil with clay and or other adsorbent material such as bleaching clay (i.e. benionite or fuller's earth), which reduce the levels of chlorophylls and other color compounds in the oil.
  • the treated oil then is passed to hydrotreating process 820, which hydrogenates and deoxygenates the components of the oil to form fuels products, for example, jet fuel mixtures, diesel fuel additive, and propane.
  • the hydrotreating process 820 also causes some cracking and the creation of smaller chain compounds, such as LPG and napfha.
  • hydrotreating process 820 Any of the hydrotreating processes described herein can be used for hydrotreating process 820.
  • the mixture of compounds created in (he hydrotreating process 820 are passed to a distillation process 82.5 to separate them into various fuel products 830, Distillation process 825 can include any of the molecular and non-molecular distillation techniques described herein or known in the art for separation of fuel compounds.
  • proteins may be selectively extracted from an algal biomass. Extraction of proteins using the disclosed methods offers many advantages. In particular, algal cells do not need to be iysed prior to extracting the desired proteins. This simplifies and reduces costs of extraction.
  • the methods of the instant invention exploit the solubility profiles of different classes of proteins in order to selectively extract and fractionate them from an algal culture, biomass, paste, or cake.
  • an algal biomass may be subjected to heating and mixing to extract water and salt soluble proteins called albumins and globulins. This mixture can then be subjected to a change in pH to recover the alkali soluble proteins called the glutelins. This step can then be followed by a solvent-based separation of the alcohol soluble proteins called prolamine. The remaining biomass would be rich in carbohydrates and lipids,
  • Proteins can be extracted from both saltwater and freshwater algal cells, as shown in FIGS. 17 and 18, The presence of salt in the saltwater algal culture or biomass affects the extraction of different classes of protein, but the methods disclosed herein enable one to selectively extract proteins from either fresh or saltwater algae.
  • extraction of proteins from freshwater algal cells is accomplished by the novel process shown in FIG. 17.
  • Freshwater algal cells or a freshwater algal biomass are heated and mixed.
  • Mixing can be accomplished by a variety of methods known in the art such as, but not limited to, stirring, agitation, and rocking.
  • This process generates a first heated extraction mixture or slurry, comprised of a first substantially liquid phase and a first substantially solid phase.
  • the solid and liquid phases are then separated. Separation can be accomplished by a variety of methods known in the art including, but not limited to, centrifugation, decantation, flotation, sedimentation, and filtration.
  • This first substantially liquid phase is enriched in albumin proteins.
  • the first substantially solid phase is then mixed with salt water and heated to generate a second heated extraction mixture or slurry, comprised of a second substantially liquid phase and a second substantially solid phase.
  • the salt water may be natural seawater or may be an aqueous salt solution. An example of such a solution would comprise about typically 35 g/L comprising mainly of NaCl.
  • the solid and liquid phases are then separated. This second substantially liquid phase is enriched in globulin proteins.
  • the second substantially solid phase is then mixed with water and heated to generate a third heated extraction mixture or slurry, comprised of a third substantially liquid phase and a third substantially sol id phase.
  • the pH of this third extraction mixture or slurry is then raised to about 9 or greater, enriching the third substantially liquid phase with glutelin proteins.
  • the solid and liquid phases are then separated, the third substantially liquid phase being enriched in glutelin proteins.
  • the third substantially solid phase is then mixed with a solvent set and heated to generate a fourth heated extraction mixture or slurry, comprised of a fourth substantially liquid phase and a fourth substantially solid phase.
  • the solvent set comprises ethanol.
  • the solvent set comprises one or more of the following solvents: methanol, isopropanol, acetone, ethyl acetate, and acetonitrile.
  • the solid and liquid phases are then separated. This fourth substantially liquid phase is enriched in prolamin proteins.
  • the remaining fourth substantially solid phase may be enriched in lipids, depending on the composition of the starting algal biomass.
  • extraction of proteins from saltwater algal cells is accomplished by the novel process shown in FIG. 18.
  • Saltwater algal cells or a saltwater algal biomass are heated and mixed.
  • Mixing can be accomplished by a variety of methods known in the art such as, but not limited to, stirring, agitation, and rocking.
  • This process generates a fsrst heated extraction mixture or slurry, comprised of a first substantially liquid phase and a first substantially solid phase.
  • the solid and liquid phases are then separated. Separation can be accompli shed by a vari ety of methods known in the art including, but not limited to, centrifugation, decantation, flotation, sedimentation, and filtration.
  • This first substantially liquid phase is enriched in globulin proteins.
  • the first substantially solid phase is then mixed with water and heated to generate a second heated extraction mixture or slurry, comprised of a second substantially liquid phase and a second substantially solid phase.
  • the solid and liquid phases are then separated.
  • This second substantially liquid phase is enriched in albumin proteins.
  • the second substantially solid phase is then mixed with water and heated to generate a third heated extraction mixture or slurry, comprised of a third substantiaiiy liquid phase and a third subs tan tially solid phase.
  • the pH of this third extraction mix ture or slurry is then raised to pH 9 or greater, enriching the third substantiaiiy liquid phase with glutelin proteins.
  • the solid and liquid phases are then separated, the third substantially liquid phase being enriched in glutelin proteins.
  • the third substantially solid phase is then mixed with a solvent set and heated to generate a fourth heated extraction mixture or slurry, comprised of a fourth substantially liquid phase and a fourth substantially solid phase.
  • the solvent set comprises ethanol.
  • the solvent set comprises one or more of the following solvents: methanol, isopropanol, acetone, ethyl acetate, and acetonitrile.
  • the solid and liquid phases are then separated.
  • This fourth substantially liquid phase is enriched in pro!;) mm proteins.
  • the remaining fourth substantially solid phase may be enriched in lipids, depending on the composition of the starting algal biomass.
  • the disclosed methods also provide for the selective extraction of different types of proteins, as shown in FIG. 17-20. Any of the steps of the a forementioned extraction process can be performed separately from the rest of the steps in order to selectively extract a single protein product. Two examples of this appear in FIG. 1 and 18, as the as demonstrated by the dashed box around extraction step 1 a.
  • globulin proteins can be selectively extracted from a freshwater algal biomass by mixing said biomass with salt water and heating to generate a heated extraciion mixture or slurry, comprised of a substantially liquid phase and a substantiaiiy solid phase. The solid and liquid phases can then be separated. The liquid phase is enriched in globulin proteins. See FIG. 17, extraction step la.
  • albumin proteins can be selectively extracted from a saltwater algal biomass by mixing said biomass with water and heating to generate a heated extraction mixture or sluny, comprised of a substantially liquid phase and a substantially solid phase. The solid and liquid phases can then be separated. The liquid phase is enriched in globulin proteins. See FIG. 18, extraction step la.
  • prolamin proteins can be selectively extracted from either a freshwater or saltwater algal biomass as shown in FIG. 19.
  • the selective extraction is accomplished by mixing the algal biomass with a solvent set and heating to generate a heated extraction mixture or slurry, comprised of a substantiaiiy liquid phase and a substantiaiiy solid phase.
  • the solid and liquid phases can then be separated.
  • the liquid phase is enriched in prolamin proteins.
  • a protein fraction can be selectively extracted from either a freshwater or saltwater algal biomass as shown in FIG. 20.
  • the selective extraction is accomplished by mixing the algal biomass with a solvent set to generate an extraction mixture or slurry and effecting a pH change in the mixture.
  • the mixture is comprised of a substantially liquid phase and a substantially solid phase.
  • the solid and liquid phases can then be separated.
  • the liquid phase is enriched in proteins.
  • FIG. 2.2 is a schematic of an exemplary extraction system for the extraction of proteins by a single solvent (amphipathic) system.
  • the exemplary system comprises an algae culture (2201 ), which may be pumped directly in or may be stored in a hold tank, a centrifuge (2202), an extractor (2203), a solids separation filter (2204), a heater (2205), a solvent recover system (2206), and a chiller (2207). Pumps (2213) assist in moving the product of each step to the next step.
  • FIG. 23 shows an exemplary system for extracting proteins and polar lipids from wet algal biomass, according to one exemplary embodiment.
  • the exemplary system comprises an algae culture, which may be pumped directly in or may be stored in a hold tank (2301), a centrifuge (2302), an extractor (2303), a decanter (2304), a heater (2305), a solvent recovery system (2306), a chilier (2307), a hold tank (2308), a solid separator (2309), a second heater (2310), a second solvent recovery system (231 1 ) and a second chiller (2312). Pumps (2313) assist in moving the product of each step to the next step.
  • the algae culture which may be pumped directly in or may be stored in a hold tank, (2301) is centrifuged (2302) to obtain a concentrated paste which is pumped to an extractor (2303).
  • the centrifuging unit (2302) may be replaced by a solid separator such as a membrane system or a dissolved air flotation unit or a flocculation sedimentation unit, or the like.
  • a mixture of ampliipaihic and hydrophobic solvent is added to the extractor.
  • the extractor mixes and heats the solution for a fixed tim e and at less than the boiling point of the sol vent mixture.
  • the heating can be achieved with various methods such as heating with microwaves, water, steam, hot oil, or electricity.
  • the extraction may be done at atmospheric pressure or under pressurized conditions to enhance the effectiveness of the extraction.
  • the extract is then pumped into a decanter to separate the lighter and heavier phases.
  • This step can also be performed using membrane filtration or by centrifuging.
  • the lighter phase comprises the hydrophobic solvent and the polar lipids.
  • the heavier phase consists of an ampliipaihic solvent, water and algae.
  • the hydrophobic layer is pumped through a heater (2305) and into a solvent recovery system (2306).
  • the vapors are passed through a chiller (2307) and recovered for recycle.
  • the residue is a concentrate comprising mostly polar lipids.
  • the heavier phase is collected in a hold tank (2308) and pumped through a solid liquid separator (2309).
  • the solids comprise the extracted algae biomass which can be further extracted or dried.
  • the liquid portion is passed through a second heater (2.310) and into a second solvent recovery system (2311 ).
  • the vapors from this system are passed through a second chiller (2312) to recover and recycle the amphipathic solvent.
  • the water and proteins form the residue in the solvent recovery unit.
  • FIG. 24 is a schematic of an exemplary extraction scheme for the extraction of neutral lipids and proteins by a two-solvent (amphipathic/hydrophobic) system.
  • the exemplary system comprises an algae culture (2401), which may be pumped directly in or may be stored in a hold tank, a centrifuge (2402), an extractor (2403), a decanter (2404), a heater (2405), a solvent recover system (2406), a chiller (2407), a hold tank (2408), a solid liquid separator (2409), a second heater (2410), a second solvent recover system (241 1 ) and a second chiller (2412).
  • Pumps (2413) assist in moving the product of each step to the next step.
  • FIG. 25 is a schematic of an exemplary system for extracting proteins, polar lipids and neutral lipids from wet algal biomass, according to one exemplary embodiment.
  • the exemplary system comprises an algae culture (2501), which may be pumped directly in or may be stored in a hold tank , a centrifuge (2502), an extractor (2503), a decanter (2504), a heater (2505), a solvent recover system (2506), a chiller (2507), a hold tank (2508), a solid liquid separator (2509), a second heater (2510), a second solvent recover system (251 1) and a second chiller (2512).
  • an algae culture (2501 which may be pumped directly in or may be stored in a hold tank , a centrifuge (2502), an extractor (2503), a decanter (2504), a heater (2505), a solvent recover system (2506), a chiller (2507), a hold tank (2508), a solid liquid separator (250
  • the algae culture (2501) is centrifuged (2502) to obtain a concentrated paste which is pumped to an extractor (2503).
  • the centrifuging unit (2502) may be replaced by a solid separator such as a membrane system or a dissolved air flotation unit or a iloeculation sedimentation unit, or the like.
  • a mixture of amphipathic and hydrophobic solvent is added to the extractor.
  • the extractor mixes and heats the solution for a fixed time and less than the boiling point of the solvent mixture.
  • the heating can be achieved with various methods such as heated with microwaves, water, steam, or hot oil or electricity.
  • the extxaction may be done at atmospheric pressure or under pressurized conditions to enhance the effectiveness of the extraction.
  • the extract is then pumped into a decanter to separate the lighter and heavier phases. This step can also be performed using membrane filtration or by centrifuging.
  • the lighter phase comprises of the hy drophobic solv ent and ihe polar lipids.
  • the heavier phase consists of a amphipathic solvent, water and algae.
  • the hydrophobic layer is pumped through a heater (2505) and into a solvent recover system (2506).
  • the vapors are passed through a chiller (2507) and recovered for recycle.
  • the residue is a concentrate comprising mostly of the polar lipids.
  • the heavier phase is collected in a hold tank (2508) and pumped through a solid liquid separator (2509).
  • the solids comprise of the extracted algae biomass which can be further extracted or dried.
  • the liquid portion is passed through a second heater (2510) and into a second solvent recover system (251 1).
  • the vapors from this unit are passed through a second chiller (2512) to recover and recycle the amphipathic solvent.
  • the water and proteins form the residue in the solvent recovery unit.
  • the extracted solids from the solid liquid separator (2509) are further extracted again with a ratio of the amphipathic solvent to the water in the system in the second extractor (2518).
  • a second mixture of amphipathic and hydrophobic solvent is added to the second extractor.
  • the second extractor mixes and heats the solution for a fixed time and less than the boiling point of the second solvent mixture.
  • the second extract is then pumped into a second decanter (2519) to separate the lighter and heavier pha ses.
  • the lighter phase comprises of the hydrophobic sol v ent and the neutral lipids.
  • the heavier phase consists of an amphipathic solvent, water and algae.
  • the hydrophobic layer is pumped through a heater (2520) and into a solvent recover system (2521).
  • the vapors are passed through a chiller (2522) and recovered for recycle.
  • the residue is a concentrate comprising mostly of the neutral lipids.
  • the heavier phase is recycled to the extractor (2518) or dried. Pumps (2513) assist in moving the product of each step to the next step.
  • FIG. 27 shows an exemplary system for extracting polar lipids and neutral lipids from wet algal biomass by a two-solvent (amphipathic/hydrophobic) system.
  • the exemplary system comprises an algae culture, which may be pumped directly in or may be stored in a hold tank (2701 ); a dewatering system (2702); a first extractor (2703); a first decanter (2704); a first heater (2705): a first solvent recovery sy stem (2706); a first chiller (2707); a second extractor (2708); a second decanter (2.709); a second heater (2710); a second solvent recovery system (2711 ); a second chiller (2712); a third heater (2714); a third solvent recovery system (2715); and a third chiller (2716).
  • Pumps (2713) assist in moving the product of each step to the next step.
  • the algae culture which may be pumped directly in or may be stored in a hold tank (2701), is dewatered to obtain a concentrated paste which is pumped to an extractor (2703).
  • the dewatering unit (2702) may comprise a centrifuge, a membrane system, a filter, dissolved air flotation unit, a flocculation and sedimentation unit, or the like.
  • a mixture of amphipathic and hydrophobic solvent is added to the first extractor (2703).
  • the first extractor mixes and heats the solution for a fixed time, and at less than the boiling point of the solvent mixture.
  • the heating can be achieved with various methods such as heating with microwaves, water, steam, hot oil, or electricity.
  • the extraction may be done at atmospheric pressure or under pressurized conditions to enhance the effectiveness of the extraction.
  • the extract is then pumped into a first decanter (2704) to separate the first lighter and first heavier phases.
  • This step can also be performed by centrifuging.
  • the first lighter phase comprises the hydrophobic solvent and the polar lipids.
  • the first heavier phase consists of an amphipathic solvent, water and algae.
  • the hydrophobic layer is pumped through a first heater (2705) and into a first solvent recovery system (2706).
  • the vapors are passed through a first chiller (2707) and recovered for recycle.
  • the residue is concentrate comprising mostly polar lipids.
  • the first heavier phase is pumped to a second extractor (2708) for further extraction with a ratio of the amphipathic solvent to the water in the system in a second extractor (2708).
  • a second mixture of amphipathic and hydrophobic solvent is added to the second extractor (2708).
  • the second extractor (2.708) mixes and heats the solution for a fixed time and at less than the boiling point of the second solvent mixture.
  • the heating can be achieved with various methods such as heating with microwaves, water, steam, hot oil, or electricity.
  • the extraction may be done at atmospheric pressure or under pressurized conditions to enhance the effectiveness of the extraction.
  • the second extract is then pumped into a second decanter (2709) to separate the second lighter and second heavier phases.
  • the second lighter phase comprises the hydrophobic solvent and the neutral lipids.
  • the second heavier phase consists of an amphipathic solvent, water, and algae.
  • the hydrophobic layer is pumped through a second heater (2710) and into a second solvent recovery system (271 1).
  • the vapors are passed through a second chiller (2712) and recovered for recycle.
  • the residue is concentrate comprising mostly neutral lipids.
  • the second heavier phase may be recycled to the second extractor (2708) for further extraction, or pumped to a third heater (2714) and into a third solvent recovery system (2715).
  • the vapors are passed through a third chiller (2716) and recovered for recycle.
  • the residue is concentrate comprising extracted biomass slurry.
  • the hydrophobic solvent instead of adding the hydrophobic solvent with the amphipathic solvent to the extractor, the hydrophobic solvent is added to the decanter after extraction has been performed with the amphipathic solvent.
  • the disclosed method of polar lipid extraction is performed using solvent to produce a residue concentrate comprising mostly polar lipids and a residue concentration of extracted biomass slurry without performing the method of neutral lipid extraction.
  • the disclosed method of neutral lipid extraction is performed using sol v ent to produce a residue concentrate comprising mostly neutral lipids and a residue concentration of extracted bioniass slurry without performing the method of polar lipid extraction, wherein the algae culture (2701 ) or algae exiting the dewatering system (2702) is pumped directly to the second extractor (2708) for the neutral lipid extraction method as described above.
  • the dewatering step is eliminated from the extraction method.
  • FIG. 26 is a schematic demonstrating the ethanol extraction concept. As the amount of ethanol is changed in proportion to the amount of water and hydrophobic solvent in the system, different components can be selectively extracted.
  • the extraction mixture/slurry may be maintained at a heated temperature for a period of time. In some embodiments, the extraction mixture is maintained at a heated temperature for between about 20 minutes to about 90 minutes, in some aspects, the extraction mixture is maintained at a heated temperature for between about 20 minutes and about 60 minutes. In other aspects, the extraction mixture is maintained at a heated temperature for between about 45 minutes to about 90 minutes.
  • the extraction mixture/slurry may be heated to temperatures less than about 50°C.
  • the albumin, globulin, and glutelin proteins are extracted at temperatures of less than about 50°C.
  • the extraction mixture/slurry is heated to a temperature close to the boiling point of extraction mixture/slurry.
  • the prolamin proteins are extracted at temperatures close to the boiling point of the extraction mixture/slurry .
  • the pressure is increased above atmospheric pressure, up to and including, 50psi, during the heating and mixing steps to enhance extraction.
  • the shelf life of an algal biomass can be increased by removal of interstitial and extracellular water. This results in less microbial growth and contamination of the biomass o v er a period of time. This can be achie v ed by mixing an amphipaihic solvent with an algal culture or biomass under unhealed conditions. No products are extracted by this process, but the water is removed from the biomass or culture and replaced with the solvent. In some embodiments, the solvent replaces 50-90% of the water in the algal culture or biomass. In some embodiments the amphipaihic solvent is ethanol, acetone, methanol, isopropanol, butanone, dimethyl ether, and propionafdehyde.
  • the amphipaihic solvent is ethanol denatured with isopropanol.
  • the solvent is isopropanol.
  • the solvent is aqueous.
  • the removal of mterstitial and extracellular water is performed under chilled conditions. The reduction in water content in the material leads to the enhanced shelf life.
  • Green microalgae Scendesmus dimorphus were cultured in outdoor panel phoiobioreaetors, SD samples of varying lipid contents were harvested. After removal of bulk water by centrifugation, the algal samples were stored as 3-5 cm algae cakes at -80°C until use. A pre-calculated amount of wet algal biomass (15 grams dry algae weight equivalent) and 90 mL of ethanol solvent was added into a three-neck flask equipped with condenser, mechanical stirring and a thermocouple. In one experiment, the mixture was refSuxed for 10 mm under microwave irradiance. In a second, the mixture was refiuxed for I h with electronic heating. Afterwards, the mixture was cooled to room temperature and separated into a diffusate and retaliate by filtration,
  • the total lipids of algal samples were analyzed using a chloroform-methanol-water system according to Bfigh and Dyer's lipid extraction method. This total lipid value was used as reference for the lipid recovery calculation.
  • Total lipids were further separated into neutral lipids and polar lipids by standard column chromatography method using 60-200 mesh silica gel (Merck Corp., Germany). Each lipid fraction was transferred into a pre-weighed vial, initially evaporated at 30 °C using a rotary evaporator (Buchi, Switzerland) and then dried under high vacuum. The dried retaliates were placed under nitrogen and then weighed.
  • Protein yield was calculated on a weight basis, comparing the weight of the freeze dried solids to the weight of the algal biomass prior to soaking in pH-adjusted water. Protein purity was determined by the Official Method of the American Oil Chemists' Society (Ba-2a- 38), measuring the amount of nitrogen in the freeze dried solids of each process. As proteins are an important product that adds to the value of algal product extraction, this information allows for the use of feedstocks with varying levels of protein in the systems and methods disclosed herein.
  • the saltwater algal culture initially made up of about 1-10% w/w solids in saltwater was heated to 50°C and maintained at this temperature for 1 hr.
  • the resulting slurry was centrifuged to separate the liquid phase from the solid phase.
  • the liquid extract was determined to be rich in globulin proteins (about 10% of the total proteins present in the original algal bioniass).
  • the solids were then suspended in fresh water and heated to about 50°C and maintained for about 1 hour.
  • the resulting slurry was centrifuged again to separate the liquid from the solid phase.
  • the liquid phase was determined to be rich in albumin proteins (about 10% of the total proteins present in the original algal biomass).
  • the solids were then suspended in ethanol to achieve a 70% w/w mixture. This mixture was heated to about 75°C and maintained at that temperature for about 1 hour. The resulting slurry was centrifuged to separate the liquid from the solid phase. The liquid phase was determined to be rich in albumin proteins (about 30% of the total proteins present in the original biomass).
  • the solids were then suspended in alkali solution (aqueous NaOH, pH 9) and heated to about 50°C and maintained at that temperature for about 1 hour. The resulting slurry was centrifuged to separate the liquid from the solid phase. The liquid phase was determined to be rich in glutelin proteins (about 50% of the total proteins present in the original biomass).
  • alkali solution aqueous NaOH, pH 9
  • Nannochloropsis biomass cultured from strain 202.0, obtained from Arizona State University, Laboratory for Algae Research and Biotechnology, ATCC Deposit Number PTA-1 1048, was harvested and dewatered until algae comprised about 35% w/w and then finally frozen.
  • the extraction steps were performed in a 400 gallon jacketed kettle with hinged lids.
  • the lids were tied down with straps and sealed with silicone.
  • the system also contained a mixer with a 2 horsepower explosion proof motor with a two blade shaft.
  • the frozen algae material was emptied into the tank and an equal weight of ethanol was pumped in using a pneumatic dram pump.
  • the material was stirred for 15 minutes and the jacket heated with steam to obtain the desired temperature at each extraction step.
  • the desired temperature is near, meaning within 3°C of the boiling point of the mixture, but not boiling. This desired temperature is different at each extraction step as the boiling point of the mixture changes as the proportion of ethanol is changed.
  • the system was stirred continuously held at the desired temperatui'e for 60 minutes to ensure that the contents of the kettle were uniformly heated.
  • the contents of the kettle were then pumped out of the extraction vessel and into a Sharpies decanter centrifuge, using a pneumatic Viking vane pump at about 1 gallon per minute.
  • the decanter centrifuge rotor speed was set to about 6000 rpm.
  • the solids were collected in an enclosed plastic drum and consisted of about 50% w/w solids to liquids. These solids were returned to the kettle, where the aforementioned extraction steps were repeated.
  • the liquid stream from the decanter was collected into a feed tank was and then fed to the membrane filtration system.
  • the membrane used was a 0.375 ft 2 SS membrane manufactured by Graver Technologies.
  • the operating conditions were 60°C ⁇ 5°C and with an average pressure gradient of 40 psi.
  • the membrane system was backwashed about every 15 minutes with compressed air to maintain the flux.
  • the permeate collected from the membrane system was free of any particulate matter.
  • the retentate was collected and recycled to the decanter.
  • Denatured ethanol containing 95% eihanol and 5% isopropyl alcohol was used in an extraction, but was found not to be as effective as 95% ethanol and 5% methanol.
  • Use of 100% ethanol is a preferred embodiment of the present invention, but is generally not available due to cost constraints.
  • Product 4 was the residual biomass, containing potential coproducts such as caret enoids.
  • algal biomass Upon harvesting, algal biomass typically contains between about 0.1 to 0.5 % (w/w) solids.
  • This can be dewatered using any of the methods known in the algae industry, including, but not limited to membrane filtration, centrifugation, heating, sedimentation or flotation. Flocculation can either assist in flotation or sedimentation.
  • the typical result of such methods is an algae slurry containing about 10% w/w solids.
  • another dewatering method may be used to remove some of the remaining free water to get the concentration of solids closer to 40% w/w.
  • the cost of dewatering increases exponentially after the first dewatering is carried out.
  • An advantage of the systems and methods disclosed herein is that the allow for the extrac tion and fractionation of an algal mass that has undergone only one round of dewatering,
  • An example of such a process might be that in the first extraction round, following the protocol described in Example 3, 1000 lbs. of wet biomass containing 90% pure water and is mixed with 1000 lbs. of denatured ethanol (95% EtOH and 5% MeOH), resulting in a solvent mixture of about 50% aqueous ethanol.
  • the resulting biomass 350 lbs. is 40% dry.
  • the solvent composition of these wet solids is 50% aqueous ethanol.
  • the composition of the mixture w ould be a bout 81% aqueo u s ethanol.
  • the resulting biomass (235 lbs.) is 40% dry.
  • the solvent composition of these wet solids is 81% aqueous ethanol.
  • the composition of the mixture would be about 95% aqueous ethanol.
  • the resulting solids would be 40% dry with about 95% ethanol.
  • This wet biomass requires 60% less energy to dry based on the latent heat of water and ethanol.
  • 100 lbs. of algae would have been extracted using 1820 lbs. ethanol.
  • 350 lbs. of the dry algae equivalent was extracted with 2085 lbs. ethanol.

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Abstract

L'invention concerne un procédé de séparation de lipides polaires et de lipides neutres à partir d'un matériau végétal, en particulier des cellules algales intactes, au moyen d'un jeu de solvants amphipathiques et un jeu de solvants hydrophobes. Certains modes de réalisation comprennent la déshydratation de cellules algales intactes, puis l'extraction des lipides polaires et des lipides neutres à partir des cellules algales. Les procédés constituent des processus d'extraction mono- et multi-étapes qui permettent une séparation efficace des lipides polaires et des lipides neutres algaux à partir d'une biomasse algale humide tout en évitant l'émulsification de mélanges d'extraction. Ces lipides polaires sont des produits de haute valeur qui peuvent être utilisés comme tensioactifs, détergents et additifs alimentaires. Ces lipides neutres sont également des produits de haute valeur qui peuvent être utilisés pour produire des combustibles renouvelables.
PCT/US2013/033301 2012-03-21 2013-03-21 Procédé d'extraction de lipides polaires et de lipides neutres avec deux solvants WO2013142687A1 (fr)

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US13/540,182 US8475660B2 (en) 2010-04-06 2012-07-02 Extraction of polar lipids by a two solvent method

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US8658772B2 (en) 2010-04-06 2014-02-25 Heliae Development, Llc Selective extraction of proteins from freshwater algae
US9120987B2 (en) 2010-04-06 2015-09-01 Heliae Development, Llc Extraction of neutral lipids by a two solvent method
US9200236B2 (en) 2011-11-17 2015-12-01 Heliae Development, Llc Omega 7 rich compositions and methods of isolating omega 7 fatty acids
CN105314739A (zh) * 2014-08-19 2016-02-10 湖北工业大学 一种微生物溶藻制剂同步脱氮、脱有机物的方法
CN105722573A (zh) * 2013-11-21 2016-06-29 株式会社神户制钢所 提取分离方法
CN107512810A (zh) * 2017-07-13 2017-12-26 安徽东至广信农化有限公司 一种硝基氯苯生产后废水处理方法

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WO2012138382A1 (fr) * 2011-04-06 2012-10-11 Heliae Development, Llc Extraction de lipides polaires à l'aide d'un procédé utilisant deux solvants

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US20110263886A1 (en) * 2010-04-06 2011-10-27 Heliae Development, Llc Methods of producing biofuels, chlorophylls and carotenoids
WO2012138382A1 (fr) * 2011-04-06 2012-10-11 Heliae Development, Llc Extraction de lipides polaires à l'aide d'un procédé utilisant deux solvants

Cited By (12)

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Publication number Priority date Publication date Assignee Title
US8658772B2 (en) 2010-04-06 2014-02-25 Heliae Development, Llc Selective extraction of proteins from freshwater algae
US8734649B2 (en) 2010-04-06 2014-05-27 Heliae Development, Llc Methods of and systems for dewatering algae and recycling water therefrom
US8741629B2 (en) 2010-04-06 2014-06-03 Heliae Development, Llc Selective heated extraction of globulin proteins from intact freshwater algal cells
US8741145B2 (en) 2010-04-06 2014-06-03 Heliae Development, Llc Methods of and systems for producing diesel blend stocks
US8748588B2 (en) 2010-04-06 2014-06-10 Heliae Development, Llc Methods of protein extraction from substantially intact algal cells
US8765923B2 (en) 2010-04-06 2014-07-01 Heliae Development, Llc Methods of obtaining freshwater or saltwater algae products enriched in glutelin proteins
US9120987B2 (en) 2010-04-06 2015-09-01 Heliae Development, Llc Extraction of neutral lipids by a two solvent method
US9200236B2 (en) 2011-11-17 2015-12-01 Heliae Development, Llc Omega 7 rich compositions and methods of isolating omega 7 fatty acids
CN105722573A (zh) * 2013-11-21 2016-06-29 株式会社神户制钢所 提取分离方法
CN105722573B (zh) * 2013-11-21 2017-11-24 株式会社神户制钢所 提取分离方法
CN105314739A (zh) * 2014-08-19 2016-02-10 湖北工业大学 一种微生物溶藻制剂同步脱氮、脱有机物的方法
CN107512810A (zh) * 2017-07-13 2017-12-26 安徽东至广信农化有限公司 一种硝基氯苯生产后废水处理方法

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