NZ615225A - Methods of producing biofuels, chlorophylls and carotenoids - Google Patents

Abstract

Disclose a method of isolating chlorophylls and omega-3 rich oil from algae, comprising: a. dewatering substantially intact algal cells to make an algal biomass; b. adding a first ethanol fraction to the algal biomass in a ratio of about 1 part ethanol to about 1 part algal biomass by weight; c. separating a first substantially solid biomass fraction from a first substantially liquid fraction comprising proteins; d. combining the first substantially solid biomass fraction with a second ethanol fraction in a ratio of about 1 part ethanol to about 1 part solids by weight; e. separating a second substantially solid biomass fraction from a second substantially liquid fraction comprising polar lipids; f. combining the second substantially solid biomass fraction with a third ethanol solvent fraction in a ratio of about 1 part ethanol to about 1 part substantially solid biomass by weight; g. separating a third substantially solid biomass fraction from a third substantially liquid fraction comprising neutral lipids, including omega-3 fatty acids, carotenoids, and chlorophyll, wherein the third substantially solid biomass fraction comprises carbohydrates; and h. isolating at least one of carotenoids, chlorophyll, and omega-3 fatty acids from the third substantially liquid fraction.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §120 of US. Patent Application No. 13/149,595, filed May 31, 2011, entitled Methods of ing Biofuels, Chlorophyiis and Carotenoids, which is a continuation~in—part of and claims the benefit of US. Application No 13/081,221, filed April 6, 2011, entitled Methods of and Systems for Isolating Nutraceutical Products from Algae, which claims priority to US. Provisional Application No. 61/321,290, filed April 6, 2010, entitled tion with Fractionation of Oil and Proteinaceous Material from Oleaginous Material, and US. Provisional Application No. 61/321,286, filed April 6, 2010, entitled Extraction With Fractionation of Oil and Co-Products from Oleaginous Material, the entire contents of which are hereby incorporated by reference herein.

FIELD OF THE INVENTION The invention is ned with extracting and fractionating algal products, including, but not limited to, oils and proteins. More specifically, the systems and methods described herein utilize step extraction and fractionation with a slightly nonpolar solvent process wet algal biomass.

BACKGROUND OF THE INVENTION eum 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, world wide 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.

Algae have gained a significant importance in recent years given their ability to e lipids, which can be used to produce nable biofuel. This ability can be exploited to produce renewable fuels, reduce global e 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 , od based ock resources, use of otherwise non-productive, non-arable land, utilization of a wide variety ofwater sources W0 2012/138438 PCT/U52012/027537 (fresh, brackish, saline, and wastewater), production of both biofuels and valuable co—products such as carotenoids and chlorophyll.

Several thousand species of algae have been screened and studied for lipid production ide over the past several decades. Of these, about 300 species rich in lipid production have been identified. The lipid composition and content vary at ent stages of the life cycle and are ed by environmental and culture conditions. The strategies and approaches for extraction are rather different depending on individual algal species/strains ed because of the considerable variability in biochemical composition and the physical properties of the algae cell wall. Conventional al extraction processes, such as extrusion, do not work well with algae given the thickness of the cell wall and the small size (about 2 to about 20nm) of algal cells. Furthermore, the large amounts of polar lipids in algal oil, as compared to the typical oil red from seeds, lead to refining issues.

Upon harvesting, typical algal concentrations in cultures range from about 01-10 ”/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. Currently, existing oil extraction methods for oleaginous materials ly require almost completely dry feed to improve the yield quality of the oil ted. Due to the amount ofenergy required to heat the algal mass to dry it sufficiently, the algal feed to biofuel process is rendered uneconomical. Typically, the feed is ed 0r flaked at high temperatures to enhance the extraction. These steps may not work with the existing equipment due to the single cell micrometric nature of algae.

Furthermore, algal oil is very unstable due to the presence of double bonded long chain fatty acids. The high temperatures used in tional extraction methods cause degradation ofthe oil, thereby increasing the costs of such methods.

It is known in the art to extract oil from dried algal mass by using hexane as a solvent. This process is energy intensive. The use of heat to dry and hexane to t produces product of lower quality as this type of processing causes lipid and protein ation.

Algal oil extraction can be classified into two types: disruptive or non-disruptive methods.

Disruptive methods involve cell lies 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 W0 2012/138438 PCT/U52012/027537 emulsification of the neutral lipids. The fication is further stabilized by the nutrient and salt components left in the solution. The emulsion is a complex mixture, containing neutral lipids, polar , proteins, and other algal products, which ive refining processes to isolate the l lipids, which are the feed that is converted into biofucl.

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 lt.

Furthermore, the volumes of solvents ed would be astronomical due to the maximum attainable concentration of the solvent in the medium.

Multiphase extractions would require extensive distillations, using complex solvent es, and necessitating mechanisms for solvent recovery and recycle. This makes such extractions impractical and uneconomical for use in algal oil technologies.

Accordingly, to overcome these deficiencies, there is a need in the art for improved methods and systems for extraction and nating algal products, in particular algal oil, algal proteins, and algal noids.

BRIEF SUMMARY OF THE INVENTION Embodiments bed herein relate generally to systems and methods for extracting lipids ofvarying polarities from an oleaginous material, including for example, an algal biomass. In particular, embodiments described herein concern extracting lipids of varying polarities from an algal biomass using solvents of varying polarity and/or a series of membrane filters. In some ments, the filter is a mierofilter.

In some ments of the invention, a single solvent and water are used to t and fractionate components present in an oleaginous material. In other embodiments, these components include, but are not limited to, proteins, polar , and neutral lipids. In still other embodiments, more than one solvent is used. In still other embodiments, a mixture of solvents is used.

In some embodiments, the methods and s described herein are useful for extracting coproducts of lipids from oleaginous material. Examples of such coproducts include, without limitation, proteinaceous material, chlorophyll, and carotenoids.

W0 2012/138438 PCT/U52012/027537 Embodiments of the present invention allow for the aneous extraction and onation of algal products from algal biomass in a manner that allows for the production of both fiiels and nutritional ts.

In another embodiment of the invention, a method of isolating nutraeeuticals products from algae is provided.

In a further embodiment of the invention, a method of isolating carotenoids and omega-3 rich oil from algae includes dewatering substantially intact algal cells to make an algal biomass and adding a first ethanol fraction to the algal biomass in a ratio of about 1 part ethanol to about 1 part algal biomass by weight. The method also includes separating a first ntially solid biomass fraction from a first substantially liquid fraction comprising proteins and combining the first ntially solid biomass fraction with a second ethanol fraction in a ratio of about 1 part ethanol to about 1 part solids by . The method further includes separating a second substantially solid biomass fraction from a second substantially liquid fraction comprising polar lipids and combining the second substantially solid biomass fraction with a third ethanol solvent fraction in a ratio of about 1 part ethanol to about 1 part substantially solid biomass by . The method also includes separating a third substantially solid biomass fraction from a third substantially liquid fraction comprising neutral lipids, wherein the third substantially solid biomass fraction comprises carbohydrates and separating the neutral lipids into carotenoids and omega-3 rich oil.

In yet r embodiment of the invention, the method also includes isolating carotenoids, omega—3 rich oil, carbohydrates and polar lipids; isolating the polar lipids from components that are not polar lipid ents; and/or isolating carotenoids from non— carotenoid components.

In still a further embodiment of the invention, the method also includes processing the polar lipids into at least one of lubricants, detergents, and food additives. [0020} In another ment of the ion, at least one of the first, second, and third solvent sets comprises an alcohol. ally, the alcohol is ethanol.

Embodiments ofthe present in invention include a method of isolating chlorophylls and omega-3 rich oil from algae, comprising dewatering substantially intact algal cells ded in a fluid medium to generate an algal biomass; extracting a substantially liquid fraction comprising neutral lipids, carotenoids, and chlorophylls from the algal biomass, the W0 2012/138438 PCT/U82012/027537 l lipids including 3 fatty acids; and separating the carotenoids and chlorophylls from the neutral lipids. 1n some embodiments, the separating is carried out by at least one of adsorption and membrane diafiltration.

In other embodiments, the methods further comprise esterifying the neutral lipids with a catalyst in the presence of an alcohol, and separating a water soluble on comprising glycerin from a water insoluble fraction comprising fuel esters. In still other embodiments, the separating is carried out by adsorption with an adsorbent material. In still others, the separating is carried out by adsorption with a clay. In some embodiments, the clay is selected from the following group consisting of bleaching clay, bentonite, and fuller’s earth.

Further embodiments of the invention include a method of isolating chlorophylls and omega-3 rich oil from algae, comprising dewatering substantially intact algal cells to make an algal biomass; adding a first ethanol fraction to the algal s in a ratio of about 1 part ethanol to about 1 part algal biomass by weight; separating a first ntially solid s fraction from a first substantially liquid fraction comprising proteins; combining the first substantially solid s fraction with a second ethanol fraction in a ratio of about 1 part ethanol to about 1 part solids by weight; separating a second substantially solid biomass fraction from a second ntially liquid fraction comprising polar lipids; combining the second substantially solid biomass fraction with a third ethanol solvent fraction in a ratio of about 1 part ethanol to about 1 part substantially solid biomass by ; separating a third substantially solid biomass fraction from a third substantially liquid fraction sing neutral lipids, including omega-3 fatty acids, carotenoids, and chlorophyll, wherein the third ntially solid biomass fraction comprises carbohydrates; and isolating at least one of carotenoids, chlorophyll, and 3 fatty acids from the third substantially liquid fraction.

In some embodiments of the , at least one of the first, second, and third solvent sets comprises an ethanol. In others, at least one of the first, second, and third solvent sets comprises an alcohol.

In yet other ments, the method r comprises esterifying the neutral lipids with a catalyst in the presence of an alcohol, and separating a water soluble fraction comprising glycerin from a water insoluble fraction comprising fuel esters. In still other embodiments, the method further comprises distilling the fuel esters under vacuum to obtain a C16 or shorter fuel ester fraction, a C16 or longer fuel ester fraction, and a e comprising omega—3 fatty acids. Some embodiments flirther comprise deoxygenating the C16 or shorter W0 2012/138438 2012/027537 fuel ester fraction to obtain a jet fuel blend stock and/or the C16 or longer fuel fraction obtain a diesel blend stock. In still other embodiments, the isolating is carried out by adsorption with an ent material. In still other embodiments, the isolating is carried out by adsorption with a clay. In some embodiments, the clay is selected from the following group consisting of bleaching clay, bentonite, and fuller’s earth.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. IA is a rt of steps ed in a method according to an exemplary embodiment of the present disclosure. is a schematic diagram of an exemplary embodiment of a dewatering process according to the present disclosure. is a schematic diagram of an exemplary embodiment of an extraction system according to the present disclosure. 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-disruptive algae oil extraction efficiency and the effect of polarity on the polar and non—polar lipids extraction. &B are graphic representations showing neutral lipids (A) Purity and (B) Recovery in the two step solvent tion process using methanol and eum ether at three different temperatures. &B are graphs showing neutral lipids (A) Purity and (B) Recovery in two step solvent extraction process using aqueous methanol and petroleum ether at three different temperatures. is a graph g lipid recovery in the two step solvent extraction s using aqueous ol and petroleum other at three different temperatures. is a graph showing the effect of solvents to solid biomass ratio on lipid recovery. is a graph showing the efficacy of different aqueous extraction solutions in a single step extraction ry of aqueous methanol on dry biomass.

PCT/U82012/027537 is a graph g the effect of multiple step methanol extractions on the cumulative total lipid yield and the neutral lipids purity. is a graph g the cumulative recovery of lipids using wet biomass and ethanol. is a graph showing a comparison of the extraction times of the ave assisted extraction and conventional extraction systems.

A is a flowchart of steps involved in a method according to an exemplary embodiment ofthe t disclosure which incorporates a step of n extraction. All of the units in A are in pounds.

B is a flowchart of steps involved in an exemplary extraction process according to the present disclosure. is a flowchart and mass balance diagram describing one ofthe embodiments ofthe present invention wherein 1000 lbs. of algal s was processed through extraction and onation in order to separate neutral lipids, polar lipids, and protein from the algal biomass. is a flowchart describing one of the embodiments of the present ion wherein an algal mass can be processed to form various products. is a flowchart bing one of the embodiments of the present invention wherein algae neutral lipids are processed to form various products. is a flowchart describing one of the embodiments of the present invention wherein algae neutral lipids are processed to form fuel products. is a flowchart describing one of the embodiments of the present invention n algae proteins are selectively extracted from a freshwater algal biomass. is a flowchart describing one of the embodiments of the present invention wherein algae proteins are ively extracted from a ter algal biomass. is a flowchart describing one of the embodiments of the present invention wherein a selected algae n is extracted from a saltwater or freshwater algal biomass. 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.

W0 2012/138438 PCT/U52012/027537 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.

A is a flowchart of steps involved in an method of chlorophyll extraction.

B is a schematic diagram of an exemplary embodiment of chlorophyll extraction of the present disclosure.

DETAILED DESCRIPTION Definitions The term “conduit” or any variation thereof, as used herein, includes any structure h which a fluid may be conveyed. Non—limiting examples of conduit include pipes, , channels, or other enclosed structures.

The term “reservoir” or any variation thereof, as used , includes any body structure capable of retaining fluid. Non-limiting examples of reservoirs include ponds, tanks, lakes, tubs, or other similar structures.

The term “about” or ximately,” as used herein, are defined as being close to as tood by one of ry skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

The terms “inhibiting” or “reducing” or any variation of these terms, as used herein includes any measurable decrease or complete inhibition to achieve a desired result.

The term “effective,” as used herein, means adequate to lish a desired, expected, or intended .

The use of the word “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of “one “ more, at least one,” and “one or more than one.” The term “or” as used herein, means “and/or” unless itly indicated to refer to alternatives only or the atives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” W0 2012/138438 PCT/U82012/027537 The use of the term “wet” as used herein, is used to mean containing about 50% to about 99.9% water t. Water content may be located either intracellularly or extracelluarly.

The use of the term “solvent set” as used herein, is used to mean composition comprising one or more solvents. These solvents can be amphipathic (also known as amphiphilic or slightly nonpolar), hilic, or hydrophobic. In some ment, thcsc solvents are water miscible and in others, they are ible in water. Non—limiting example of solvents that may be used to practice the methods of the instant invention include methanol, ethanol, isopropanol, acetone, ethyl acetate, and acetonitrile, alkanes (hexane, pentane, heptane, octane), esters (ethyl acetate, butyl acetate), ketones (methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK)), ics (toluene, e, cyclohexane, tetrahydrofuran), haloalkanes (chloroform, trichloroethylene), ethers (diethyl ether), and mixtures (diesel, jet fuel, gasoline).

The term “oil” as used herein es compositions containing neutral lipids and polar lipids. The terms “algae oil” and “algal oil” as used herein are used interchangeably.

The term “diffusate” or “permeate" as used herein may refer to al that has passed through a separation device, including, but not limited to a filter or membrane.

The term “retentate” as used herein may refer to material that remains after the diffusate has passed through a separation device.

As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of ning, such as “contains” and in”) are inclusive or nded and do not exclude additional, unrecited elements or method steps.

The term “polar ” or any variation thereof, as used herein, includes, but is not limited to, phospholipids and glycolipids.

The term “neutral lipids” or any variation thereof, as used , includes, but not limited to, triglycerides, diglycerides, monoglycerides, carotenoids, waxes, sterols.

The term “solid phase” as used herein 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 tion may include solid materials.

The term “biodiesel” as used herein refers to methyl or ethyl esters of fatty acids derived from algae.

The term eeutical” as used herein refers to a food product that provides health and/or medical benefits. Non-limiting es include carotenoids, carotenes, xanthophylls such as zeaxanthin, astaxanthin, and lutein.

The term “biofuel” as used herein refers to fuel derived from biological source.

Non-limiting examples include biodiesel, jet fuel, diesel, jet fuel blend stock and diesel blend stock.

The term “impurities”, when used in connection with polar lipids, as used herein, refers to all components other than the products of interest that are eoextracted or have the same properties as the product of interest.

The term “lubricants”, when used in connection with polar , as used herein refers to hydrotreated algal lipids such as C16-C20 alkanes.

The term “detergents”, when used in tion with polar lipids, as used herein refers to glycolipids, phospholipids and derivatives thereof.

The term “food additives”, when used in connection with polar lipids, as used herein refers to soy lecithin substitutes or phospholipids derived from algae.

The term lycerin ” as used herein 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.

The term “unsaturated fatty acids” as used herein refers to fatty acids with at least one double carbon bond. Non-limiting examples ofunsaturated fatty acids include palmitoleie acid, margaric acid, stearic acid, oleie acid, octadeeenoic acid, ic acid, gamma-linoleic acid, alpha linoleic acid, arachidic acid, eicosenoic acid, homogamma linoleic acid, arachidonie acid, penenoic acid, behenic, doeosadienoic acid, heneieosapentaenoic, tetraenoie acid. Fatty acids having 20 or more carbon atoms in the backbone are generally referred to as PCT/U52012/027537 “long chain fatty acids”. The fatty acids having 19 or fewer carbon atoms in the backbone are generally ed to as “short chain fatty acids”. rated long chain fatty acids include, but are not limited to, omega-3 fatty acids, omega-6 fatty acids, and omega-9 fatty acids. The term —3 fatty acids” as used herein refers to, but is not d to the fatty acids listed in Table 1. "{£135"Z'iiléiél'i‘i"iii’i7""éi'é'c'iééifiéfiéié'éaéi"""""" """""3: The term “jet fuel blend stock” as used herein refers to alkanes with the carbon chain lengths appropriate for use as jet fuels.

The term “diesel blend stock” as used herein refers to alkanes with the carbon chain lengths appropriate for use as diesel.

The term “animal feed” as used herein refers to algae-derived substances that can be consumed and used to provide nutritional support for an animal.

The term “human food” as used herein refers to algae—derived substances that 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.

The term “bioremediation” as used herein refers to use of algal growth to remove pollutants, such as, but not limited to, nitrates, ates, and heavy metals, from industrial wastewater or municipal wastewater.

The term “wastewater” as used herein refers to industrial wastewater or municipal wastewater that contain a variety of contaminants or pollutants, including, but not limited to nitrates, phosphates, and heavy .

The term “enriched”, as used herein, shall mean about 50% or greater t.

The term “substantially”, as used herein, shall mean mostly.

The term lin proteins” as used herein refers to salt soluble proteins.

The term “albumin proteins” as used herein refers to water soluble proteins.

The term “glutelin proteins” as used herein refers to alkali soluble proteins.

The term “prolamin proteins” as used herein refers to alcohol soluble proteins.

Non-limiting examples ofprolamin ns are gliadin, zein, hordein, avenin.

The term “algal culture” as used herein refers to algal cells in culture medium.

The term “algal biomass” as used herein refers to an at least lly dewatered algal culture.

The term “dewatered” as used herein refers to the l of at least some water.

The term “algal paste” as used herein 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%.

The term “algal cake” as used herein 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 cells include, but are not limited to, marine and brackish algal species. Saltwater algal cells are found in nature in bodies ofwater such as, but not limited to, seas, oceans, and estuaries. Non-limiting examples of ter algal s include Nannochloropsz’s sp., Dumzlz’ella Sp.

PCT/U82012/027537 Freshwater algal cells are found in nature in bodies of water such as, but not limited to, lakes and ponds. Non-limiting examples of freshwater algal species include Scendescemus 5p. , Haemotococcus Sp.

Non—limiting examples ofmicroalgae that can be used with the s of the invention are members of one of the following divisions: Chlorophyta, Cyanophyta (Cyanobaeteria), and Heterokontophyta. In certain embodiments, the microalgae used with the methods of the ion are members of one of the following classes: Bacillarz’ophyceae, Eustz‘gmatophyceae, and Chrysophyceae. In certain embodiments, the microalgae used with the methods ofthe invention are members of one of the following : Nannochloropsz’s, Chlorella, Dunalz‘ella, Scenedesmus, Selenastrum, Oscillatoria, Phormz'a’z‘um, Spirulz'na, Amphora, and Ochromonas. miting examples of microalgae species that can be used with the s of the t invention include: Achnanrhes orientalz's, llum $1717., mra hya/z'ne, Amp/mm j‘brmz‘s, Amp/mm coffézj‘brmis var. Zinea, Amphora cofl‘eiformis var. punctata, Amphora cofi’eiformis var. taylori, m cofi’ez'formis var. , Amphora delicatl'ssz'ma, Amp/10m delicarz‘ssz’ma var. . capitata, Amphora S11, Anabaena, Ankz'stmdesmus, Ankz‘strodesmusfalcatus, Boekelovz’a hooglandz'i, Borodinella Sp., Botryococcus braum'z', Bonyococcus sudetz'cus, Bracteococcus minor, Bracteococcus medionucleatus, Carterz'a, Chaetoceros gracilis, Chaetoceros muellerz‘, Chaetoceros muellerz' var. subsalsum, Chaetoceros sp., Chlamydomas perigranulata, Chlorella anitrata, Chlorella tica, Chlorella aureovz'ridz's, Chlorella Candida, Chlorella capsulate, Chlorella ate, Chlorella ellipsoidea, Chlorella emersanii, Chlorellafusca, Chlorellafusca var. vacuolata, Chlorella glucotropha, Chlorella infitsz‘onum, lla infiisz‘onum var. actophz'la, Chlorella infirsz'onum var. auxenophila, Chlorella kessleri, Chlorella lobaphora, Chlorella luteoviridis, Chlorella luteovz‘rz'dis var. aureovz‘rz’dz‘s, Chlore/la luteovz'ridz‘s var. lutescens, Chlorella miniata, Chlorella minutissz'ma, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella paiva, Chlorella plwtophz'la, Chlorella pringsheimii, Chlarella protothecoz‘des, Chlorella protothecoz‘a’es var. ola, Chlorella regularz's, Chlorella regularis var. minima, lla regularis var. umbrz’cata, Chlorella reisz'gliz‘, Chlorella saccharophz‘la, lla rophz'la var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella 'm‘ana, Chlorella Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannz‘e/lz‘z', Chlorella vulgarz's, Chlorella vulgarisfo. terria, Chlorella vulgaris var. autotrophz‘ca, Chlorella vulgaris var.

W0 2012/138438 2012/027537 vz'rz‘dz'S, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgarisfo. tertia, Chlorella iS var. vulgarisfo. viridz‘s, Chlorella xanthella, Chlorella zofingierzSiS, Chlorella xz'oz'des, Chlorella vulgariS, Chlorococcum infusionum, Chlorococcum Sp, Chlorogorzz'um, Chroomonas Sp, CthSOSphaera Sp, Cricosphaera Sp, Crwthecodz’m’um colmz'z', Cryptomonas Sp, Cyclotella cryptica, Cyclolella menegkim'ana, Cyclotella Sp, Dzmalz‘ella Sp, Dzmalz'ella bardawil, Dunaliella bz'oculata, Dunaliella ate, Dunaliella maritime, Dunalz’ella mimlta, Dunalz'elia parva, Dzmalz’ella pez‘rcez‘, Dunalz’ella primolecta, ella salina, Dzmaliella terricola, Dzmalz'ella terliolecta, Dunalz'ella viridis, Dunalz’ella terliolecta, Eremosphaera viridz's, Eremasphaera Sp, Ellipsoidon Sp, Euglena Spp, z'a Sp, Fragilarz'a crotonensz’s, Fragilarz’a Sp, Gleocapsa Sp, Gloeothamniorz Sp, Haematococcus pluvz'alz'S, Hymenomonas Sp, [sochrysz's ajj‘. galbana, [sochrysz's galbana, Lepocinclis, filicractinium, Alicractiniwn, Alonoraphz’dium minutum, phidium Sp, Nannochloris Sp, Nannochloropsis salina, hlampsz‘s Sp, Navicula accepzata, Navicula biskamerae, Navicula pseudotenelloz‘des, Navz'cula culosa, Navicula saprophila, Navicula chloris Sp, Nephroselmis Sp, Nz'tschia communis, Nitzschz'a alexandrz‘na, Nitzschia closterz'um, Nitzschia communis, Nitzschz‘a dissipata, Nitzschz‘afiustulum, Nitzschia chz’ana, Nitzschz’a inconspicua, Nitzschz'a intermedia, Nitzschz‘a microcephala, Nitzschz'a pusilla, Nitzschia a elliptica, Nitzschia pusz‘lla monoenSZS, Nitzschia quadrangular, Nitzschz‘a Sp, Ochromonas Sp, OocyStz'S parva, Oocystis pusz'lla, Oocystis Sp, Oscillatorz'a limaetz’ca, Oscz'llatorz'a Sp, Oscillatoria subbrevis, Parachlorella kesslerz', Pascheria hila, a Sp, Phaeodacgzlum tricomutum, Phagus, Phormz’dium, Platymonas Sp, chrysis ae, Pleurochrysis dentate, Pleurochljzsz‘s Sp, Protoz‘heca wickel‘hamii, Prototheca Stagnora, Prototheca portorz'censz's, Protorheca morzformis, Prototheca zap/ii, Pseudochlorella aquatica, Pyramimonas Sp, erobotrys, Rhodococcus opacus, Sarcz'noz’a’ phyte, Scenedesmus armatuS, Schizochytrium, yra, Spirulina platenSis, Stz'chococcus Sp, Synechococcus Sp, Synechocystz’sf, TageteS erecta, T ageteS patula, Tetraedron, Tetraselmz‘s Sp, Tetraselmis suecz’ca, Thalassz'osz'ra weissflogz‘z’, and Viridz'ella fridericz'ana.

In other embodiments, the s can be plant material, including but not limited to soy, corn, palm, camelina, jatropha, canola, coconut, peanut, safflower, seed, linseed, sunflower, rice bran, and olive.

Systems and methods for extracting lipids and coproducts (e.g., proteins) of varying polarity from a wet oleaginous material, including for e, an algal biomass, are disclosed.

In ular, the methods and systems described herein concern the ability to both extract and fractionate the algae components by doing sequential extractions with a hydrophilic solvent/water mixture that becomes progressively less polar , water in t/water ratio is progressively reduced as one proceed from one extraction step to the next). In other words, the interstitial solvent in the algae (75% of its weight) is initially water and is replaced by the slightly nonpolar solvent gradually to the azeotrope of the organic solvent. This s in the extraction of components soluble at the polarity developed at each step, thereby leading to simultaneous fractionation of the extracted components. Extraction ofproteinaceous byproducts by acid ng and/or alkaline extraction is also disclosed.

In some embodiments of the invention, a single solvent and water are used to t and fractionate components present in an oleaginous material. In other embodiments, a solvent set and water are used to extract and fractionate components present in an oleaginous material. In some embodiments the oleaginous material is wet. In other embodiments, the oleaginous material is algae.

Polar lipid recovery depends mainly on its ionic , water solubility, and location (intracellular, extracellular or membrane bound). es ofpolar lipids include, but are not d to, phospholipids and ipids. Strategies that can be used to te and purify polar lipids can roughly be divided into batch or continuous modes. Examples ofbatch modes include precipitation (pH, organic solvent), t extraction and crystallization.

Examples of continuous modes include centrifuging, adsorption, foam separation and precipitation, and membrane technologies (tangential flow filtration, diafiltration and precipitation, ultra filtration).

Other objects, features and advantages of the present invention will become apparent from the ing detailed description. It should be understood, however, that the detailed description and the examples, while indicating specific embodiments of the invention, are given by way of ration only. onally, it is plated that changes and modifications within the range and scope of the invention will become apparent to those skilled in the art from this detailed description.

Surprisingly, the proposed non-disruptive extraction process results in over 90% recovery. The small amount of polar lipids in the remaining biomass enhances its value when PCT/USZOIZ/027537 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. In on, ethanol extracts can further be directly transesterified. Furthermore, unlike the existing tional methods, the s and systems described herein are generic for any algae, and enable recovery of a icant portion of the le components, ing 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 t, thereby enhancing stability of the lipid fraction, and ng subsequent processing steps. Metals tend to make neutral lipids unstable due to their ability to catalyze oxidation. Furthermore, metals inhibit hydrotreating sts, 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 .

The systems and methods disclosed herein can start with wet biomass, reducing drying and dewaterin g costs. Compared to conventional extraction processes, the disclosed extraction and fractionation processes should have relatively low operating costs due to the moderate temperature and pressure conditions, along with the solvent recycle.

Furthermore, conventional tion processes are cost prohibitive and cannot meet the demand ofthe market.

Another aspect of the systems and methods bed herein is the ability accomplish preliminary refining, which is the separation of polar lipids from neutral lipids during the extraction process. The differences between algal oil used in exemplary embodiments and vegetable oils used in previous embodiments e the percentage of individual classes of lipids. An exemplary algal crude oil composition is compared with vegetable oil shown in Table 2 below: Table 2 Algal Crude Oil (w/W) Vegetable Oil (W/w) Neutral lipids 30-90% 90-98% PCT/U52012/027537 Phospholipids 10-40% Glycolipids 10-40% Free fatty acids l-10% Pigments 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/subcritieal fluid extraction or membrane technology. In st, separation of the neutral lipids from an oleaginous algal s is far more lt 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 fication of the neutral lipids. The emulsification 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. However, because polar lipids have an existing market, their recovery would add significant value to the use of algal oil to generate fuels.

Polar lipids are tants by nature due to their lar structure and have huge existing market. Many of the existing technologies for ing polar lipids are raw material or cost prohibitive. Alternative feedstocks for glycolipids and phospholipids are mainly algae oil, oat oil, wheat germ oil and vegetable oil. Algae oil typically contains about —85 % (w/w) polar lipids depending on the species, physiological status of the cell, culture conditions, time of t, 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 ofthe end product, z’.e., esterified fatty acids, as compared to that of neutral lipids, on a per mass basis.

Hence, removal and recovery of the polar lipids would not only be highly beneficial in W0 2012/138438 PCT/U52012/027537 producing high quality biofuels or triglycerides from algae, but also te value-added co- products glycolipids and olipids, which in turn can offset the cost associated with algae- based biofuel production. The ability to easily recover and fractionate the various oil and products produced by algae is advantageous to the economic success of the algae oil process.

A r aspect of the s and systems described herein is the ability to extract proteins from an oleaginous material, such as algal biomass. The s disclosed herein of extraction of proteinaceous material from algal biomass comprise a flexible and highly customizable s of extraction and fractionation. For example, in some embodiments, extraction and fractionation occur in a single step, thereby ing a highly efficient process.

Proteins sourced from such biomass are useful for animal feeds, food ingredients and rial products. For example, such proteins are useful in applications such as fibers, adhesives, coatings, ceramics, inks, cosmetics, textiles, chewing gum, and biodegradable plastics.

Another aspect of the methods and systems described herein involves varying the ratio of algal biomass to t based on the components to be extracted. In one embodiment, an algal biomass is mixed with an equal weight of t. In another embodiment, an algal biomass is mixed with a lesser weight of solvent. In yet r embodiment, an algal biomass is mixed with a greater weight of solvent. In some embodiments, the amount of solvent mixed with an algal biomass is calculated based on the solvent to be used and the desired polarity the algal biomass/solvent mixture. In still other embodiments, 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. Second, the remaining algal solids are extracted using about 70% of the ’ weight in solvent. A third extraction is then performed using about 90% ofthe solid’s weight in solvent. Having been informed of these aspects of the invention, one of skill in the art would be able to use ent solvents of different polarities by adjusting the ratios of algal biomass and/or solid residuals the d polarity in order to selectively extract algal products.

For example, in preferred embodiment, 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. anol 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 e about 100% methanol.

W0 2012/138438 PCT/U82012/027537 Embodiments ofthe systems and methods described herein exhibit surprising and unexpected s. First of all, the ry/extraction s can be done on a wet biomass.

This is a major economic advantage as exemplary embodiments avoid the use of large amounts of energy ed to dry and t 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. ary 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, panol, acetone, ethyl 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 l lipids.

Ethanol is relatively economical to purchase as compared to other solvents disclosed herein. In some ary embodiments, extraction and fractionation can be performed in one step followed by membrane-based purification ifneeded. The resulting biomass is almost devoid of water and can be tely dried with lesser energy than an aqueous algae slurry.

In some exemplary embodiments, the solvent used to extract is ethanol. Other embodiments include, but are not limited to, cyclohexane, eum ether, pentane, hexane, e, diethyl ether, toluene, ethyl acetate, chloroform, dicholoromethane, acetone, acetonitrile, isopropanol, and methanol. In some embodiments, the same solvent is used in tial extraction steps. In other embodiments, different solvents are used in each extraction step. In still other embodiments, two or more solvents are mixed and used in one or more extraction steps.

In some embodiments of the methods described , a mixture oftwo or more ts used in any of the extraction steps includes at least one hydrophilic solvent and at least one hydrophobic solvent. When using such a mixture, the hydrophilic solvent ts the material from the biomass via diffusion. Meanwhile, a relatively small amount of hydrophobic solvent is used in combination and is involved in a liquid—liquid separation such that material of interest is concentrated in the small amount of hydrophobic solvent. The different solvents then form a two-layer system, which can be separated using techniques known in the art. In such an implementation, the hydrophobic solvent can be any one or more W0 2012/138438 PCT/U$2012/027537 of an alkane, an ester, a ketone, an aromatic, a haloalkane, an ether, or a commercial mixture (e.g., diesel, jet fuel, gasoline).

In some embodiments, the extraction processes described herein incorporate pH ion in one or more steps. Such pH excursion is useful for isolating proteinaceous material. In some embodiments, the pH of the extraction process is acid (e. g., less than about ). In some embodiments, the pH of the extraction process is alkaline (e.g., greater than about ).

The use of hexane in conventional extraction procedures contaminates algal s such that coproducts may not be used in food products. Embodiments ofthe present invention are or to those known in the art as they require the use of far less energy and render products suitable for use as fuels as well as foodstuffs and nutrient supplements.

It is contemplated that any ment discussed in this specification can be implemented with respect to any method or system of the invention, and vice versa. rmore, systems of the invention can be used to achieve s of the invention.

DESCRIPTION OF ARY EMBODIMENTS For solvent extraction of oil from algae the best case scenario is a solvent which selectively extracts triacylglycerols (TAG) and leaving all polar lipids and non—TAG neutral lipids such as waxes, sterols in the algal cell with high recoveries. The second option would be selectively extract polar lipids and then extract purer neutral lipids devoid of polar lipids, resulting in high recovery. The last option would be to extract all the lipids and e very high ry in one or two steps.

Referring now to FIG. IA, a flowchart 100 provides an overview of the steps involved in exemplary ments ofmethods used in the fractionation and purification of lipids from an algae-containing s. In a first step 110, algal cells are harvested. In a subsequent step 120, water is removed from algal cells to yield a 10-25% solid s. In step 130, a solvent-based extraction is performed on the biomass and the fractions are collected. In some embodiments, step 130 will also incorporate pH-based extraction and fraction collection. Finally, 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.

W0 2012/138438 PCT/U82012/027537 The algae biomass when harvested in step 110 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, flocculation, sedimentation, filter pressing, decantation or centrifiigation. Dewatering is the removal of some, most, or all of the water from a solid or semisolid substance. Embodiments of the present ion utilize dewatering techniques to remove water from a harvested algal biomass. Dewatering can be carried out using any one of or a combination of any of the methods described herein, as well as by any other s known to one of skill in the art.

The dewatered algae biomass resulting from step 120 typically consists of about 10— % solids. This s can then be extracted with water miscible slightly nonpolar solvents (e.g. , alcohols), in a multistage countercurrent solvent extraction s ating the fractions at each stage. This type of process can reduce both capital and operating In some embodiments, the biomass also undergoes acid and/or alkaline extraction to fractionate n material. [0122} In some embodiments, dewatering 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 on and the liquids may then be removed by methods such as, but not d to, siphoning. This novel method of dewatering has lower capital and opcrating costs than known methods, enables solvent recycling, reduces the cost of drying the biomass, and has the added benefit of sing the polarity of the algal biomass prior to beginning tion and/or separation of algal components. In fact, it is theorized that the solvent—based sedimentation processes described herein are effective, in part, due to the fact that organic ts reduce or neutralize the negative charge on the algae surface. In some embodiments ofthe invention, ring methods are combined in order to remove even more water. In some embodiments, the addition of solvent during the dewatering process begins the process of tion. 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 (226., 0.1-l% 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 ed directed to other algae cultures. Meanwhile, the algal biomass recovered has been W0 2012/138438 2012/027537 concentrated to an “algae paste” with a algae density as high as about 200 g/L (226., 10-20% w/w). This trated 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 biomass ratio of between about 1:1 to about 1:10.

The algae is allowed to settle in a ng 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 ing 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 ts with densities over 1.1 g/mL or below 0.9 g/mL. Examples include isopropanol, acetone, acetonitrile, t-butyl alcohol, ethanol, methanol, 1-propanol, heavy water (D20), ne , and/or glycerin. If the solvent density is over 1.1 g/mL then the algae biomass would float rather than create a sediment at the bottom of the settling vessel. is a schematic diagram of an ary 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 e 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 ement for extraction. The extracts in each e tank may either be replenished at saturation or uously replaced by fresh solvent. This would also reduce the downstream processing time and cost drastically. This embodiment ses a primary reservoir 210, a transport mechanism 220, a plurality of separation devices 241-248 (2.g. , membrane filtration devices), a ity of extraction reservoirs 261—268, and a plurality of recycle pumps 281—287.

In this embodiment, y reservoir 210 is divided up into a plurality of inlet reservoirs 211- 218.

During operation, algal biomass 201 is placed a first inlet reservoir 211 near a first end 221 of transport mechanism 220. In addition, 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. As the PCT/USZOIZ/027537 algal biomass is transported, it passes through the plurality of tion devices 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.

For example, the diffusate portion of the algal biomass that passes h the first separation device 241 (e. g., the portion ning liquid and particles small enough to pass through tion device 241) is directed to the first reservoir 261. From first reservoir 261, the diffusate portion can be recycled back to first inlet reservoir 201. The retentate portion of the algal biomass that does not pass through first separation device 241 can then be directed by transport mechanism 220 to second inlet reservoir 212 and second separation device 242, which can comprise a finer tion 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 pass through second separation device 242 can be directed by transport mechanism 220 to third inlet reservoir 213. This process can be ed for inlet oirs 213-218 and separation devices 243-248 such that the retentate portions at each stage are directed to the uent inlet reservoirs, while the diffusate portions are directed to the recycle oirs and ed back to the current inlet reservoir.

In exemplary embodiments, the first fraction will be extracted with the highest water to slightly nonpolar solvent ratio, i.e., most polar mixture, while the last on 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 ofthe first fraction is to remove the residual water and facilitate the solvent 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 noids and long chain rated fatty acids can be separated from the oil using processes such as molecular lation 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 rated fatty acids remain as high boiling residue in the column.

W0 2012/138438 In some non-limiting embodiments, the tion 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 ment(s) to e ion and extraction of protein. For example, in one non-limiting ment, the pH of the solvent in the first separation device is zed 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, the use oftitration curves.

In some embodiments, the solvent pH is alkaline. For example, in some embodiments, the t pH is greater than about 10. In other embodiments, the solvent pH ranges from about 10 to about 12. In further embodiments, the solvent pH is about l0, about 11, or about 12. In other embodiments, the t pH is acid. For example, in some embodiments, the solvent pH is less than about 5. In other embodiments, the solvent pH ranges from about 2 to about 5. In further ments, 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 subsequent inlet reservoirs to achieve extraction and fractionation based on polarity. In another non—limiting embodiment, protein material is separated in the final separation device by similar means (i.e., solvent pH adjustment).

Adjustment of solvent pH is accomplished in ance with methods known to those of skill in the art. For example, acid pH is achieved by mixture of an appropriate acid into the solvent stream. Exemplary acids useful for protein extraction include, t limitation, phosphoric acid, sulfuric acid, and hydrochloric acid. Similarly, alkaline pH is achieved by addition and mixture of an appropriate base into the solvent stream. Exemplary bases useful for protein extraction include, without limitation, potassium hydroxide, sodium hydroxide.

In some embodiments, protein tion is performed in a system separate from the extraction and fractionation system described . For example, in some embodiments, an algal biomass is soaked in a pH-adj usted solvent mixture, followed by isolation via an riate separation technique (e.g, centrifugation, or filtration). The remaining solid is then introduced into an extraction and fractionation system based on polarity, as bed herein.

W0 38438 PCT/U82012/027537 Similarly, in some embodiments, 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.

As shown in the solvent selection and the theory of fractionation based polarity were developed by extensive analysis of ts and the effect on extraction using the Sohxlet extraction s, which allows the separation of lipids from a solid material. The t tion system was utilized for rapid screening solvents for lipid class selectivity and recovery. Solvents from various chemical classes encompassing a wide range of polarities such as s, cycloalkane, alkyl halides, esters, ketones, were tested. Prior to the extraction, the lipid content and composition of the biomass to be extracted was tested in triplicate using the standard methods for algae oil estimation such as the Bligh-Dyer lipid extraction method.

The biomass contained 22.16% total lipid, ofwhich 49.52% was neutral lipid. ts the data gathered by extraction of a dry algal mass using s polar and nonpolar solvents combined with a Sohxlet extraction process. ing on the chain length of the alkane solvent, 60-70% purity ofneutral lipids and 15-45% of total lipid recovery can be achieved without disruption and solvent extraction. The longest chain alkane solvent tested, e, red 60% of the neutral lipids and 42% of the total lipid. As 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 . The systems and methods discloses herein address these inefficiencies by controlling the tion of slightly nonpolar solvent to water in order to separate out components of differing polarities with minimal loss of components. [0138} The lower carbon alcohol solvents were more selective for polar lipids. The neutral lipid purity was 22% for methanol and 45% for ethanol. pyl alcohol did not show any selectivity between polar and nonpolar lipids, resulting in a 52% pure neutral lipid product.

Methanol recovered 67% of the total lipids and more than 90% of the polar lipids. Therefore, methanol is an excellent candidate for an ment of the present invention wherein methanol can be used to selectively extract polar lipids from an oleaginous material prior extracting the l lipids using heptane or hexane. The other solvent classes tested did not show any selectivity towards lipid class since the neutral lipid purity was close to 49%, similar to the lipid composition present in the original biomass. Furthermore, the total lipid recovery W0 2012/138438 PCT/U82012/027537 achieved with these solvents ranged from about 15—35%, rendering these solvents unsuitable for the ive extraction of particular lipid classes or total lipid extraction.

The results from the Sohxlct is were confirmed using the standard bench scale batch solvent extraction apparatus described below in Example 1. The solvents selected were methanol for the first step to recover polar , and eum ether in the second step for recovery of neutral lipids. All of the extractions were performed with a 1:10 solidzsolvent ratio. Each extraction step in this experiment was 1 hour long. Other ments done (data not shown) indicate that about 45 minutes or longer is long enough for the extraction to be successful. This retention time is dependent on the heat and mass transfer ofthe system.

The methanol extractions were performed at different temperatures, 40 0C, 50 OC, and 65 °C, in order to determine which was l. The petroleum ether extraction performed at 35°C, close to the boiling point of the t. Petroleum ether was chosen e of its high selectivity for neutral lipids, low boiling point, and the product quality observed after extraction. shows that the neutral lipid purity in a petroleum ether extraction carried out after a ol extraction step at 65°C is over 80%, demonstrating that the combination of these two extraction steps enhanced the neutral lipid content of the final crude oil product. shows that the total neutral lipid recovery was low and there was a significant amount ofneutral lipid loss in the first step.

To minimize the loss of neutral lipids in the methanol extraction step, the polarity of the solvent can be increased by adding water to the solvent. and 5B show the results of extracting the entioned biomass with 70% v/v aqueous methanol followed by extraction with petroleum ether. shows that the neutral lipid purity was much higher in the petroleum ether extraction than was achieved by the use ofpure methanol. Moreover, the loss of neutral lipids was greatly reduced by the use of aqueous methanol in the first extraction step. As seen in , methanol extraction at higher temperatures improved neutral lipid purity but slightly decreased the total lipid recovery in the subsequent step.

In some exemplary embodiments the temperature of the extraction process is controlled in order to ensure optimal stability of algal components present in the algal s.

Algal proteins, carotenoids, and chlorophyll are examples of algal components that exhibit ature sensitivity. In other embodiments, the temperature is increased after the temperature sensitive algal components have been extracted from the algal biomass.

PCT/U82012/027537 In still other exemplary embodiments, 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 ofthe extraction process is changed depending on the solubility ofthe desired product. In still other embodiments, the extraction temperature is optimized depending on the algal strain of the biomass to be extracted. ed extraction temperatures increase the solubility of d nds and reduce the viscosity of the tion mixture enhancing extraction ry.

In some embodiments, the extraction is run under re to e the boiling point of the extraction mixture. In these implementations, the pressure is increased to the degree ary 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.

In some exemplary embodiments, the extraction is performed near the boiling point ofthe 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 g 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 cantly exceed the boiling point of the solvent, the solvent—water system can form an azeotrope. Thus, maintaining the system at or near the boiling point of solvent would generate enough vapors to e the extraction, while ng expense. In addition, the solubility of oil is increased at higher temperatures, which can further increase the effectiveness of extraction at atures close to the solvent boiling point. shows the total lipid recovery in the aqueous ol-petroleum ether extraction scheme. Although performing the methanol extraction near its boiling temperature slightly decreases the neutral lipid recovery as observed in , it enhances the total lipid recovery.

In other embodiments, the extraction is carried out under ambient lighting conditions. In other ments, the extraction is carried out in an opaque container such as, but not d to, a steel tube or casing, in order to protect light sensitive algal components from degradation. Carotenoids are light sensitive algal components.

W0 2012/138438 PCT/U52012/027537 In other exemplary ments, 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 embodiments, 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.

In exemplary embodiments, the t-to-solid ratio for the tion is between 3—5 based on the dry weight of the solids in the biomass. The residual algal biomass is rich carbohydrates (e.g., starch) and can be used as a feed stock to produce the solvent used for extraction. shows the effect of the solvent to 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 recovery. It is believed that this was because of the lower solubility of lipids in methanol as compared to other commonly used oil tion solvents such as hexane.

The solubility of components is affected by the polarity of solvent used in extraction process. The solubility properties can be used to determine the ratio of wet biomass to solvent. For example, a 40% w/w wet biomass has 40 g biomass and 60 g water for every 100 g ofwet 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 l 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 ty index of 6.6, calculated by weight averaging the polarities of the ents. Ethanol, having a polarity index of 5.2, and water, having a ty 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 e for extraction ofpolar lipids and neutral lipids is calculated to be about 5.8 and 5.4 respectively.

In light of the instant disclosure, one of skill in the art would be able to formulate a solvent set that can ively extract these components.

In r e, if the extraction t is a 1:1 mixture of isopropyl alcohol and ethanol, the polarity of this solvent is ((3.9+5.4)/2) which is about 4.65. The ratio of solvent to wet biomass would be calculated to match the polarities. To get a 6.6 polarity index, we would need to make a 55% w/w of IPA-water mixture calculated by solving the following algebraic equation: W0 2012/138438 PCT/U$2012/027537 X*4.65+(1—x)*9=6.6 x = (9-6.6)/(9-4.65) = 0.55 55% w/w*ofsolvent mix in t ter For a 40% w/w wet biomass, the wet biomass to IPA ratio is (l-0.55)/0.6 ~ 0.75.

With a 40% w/w wet biomass this would correspond to a ratio of 100 parts wet biomass to 75 parts solvent mixture. A 40% W/w wet biomass has 40 g biomass and 60 g water for every 100 g of wet biomass. If 75 g of solvent mixture is added to this e, the concentration of solvent in the mixture is (75/(75+60)) is about 55% w/w of solvent mixture in the solvent mixture—water solution. This calculation can be used to obtain the solvent s ratio at each extraction stage and for each product. A few nonlimiting examples of solvent sets appear in Table 3.

Table 3 ......................Extraction"? ; g s 3 5 §Solvent- solvent :parts wet2parts EBiomass Parts Dry EParts iwater :polarity §95% ethanol 5% methanol mixture 395% ethanol 5% IPA mixture One step Polar lipids tion EEthanol gPropanol 21:1 lPA EtOH mixture 95% ethanol water mixture i 95% ethanol 5% methanol mixture ' 5% ethanol 5%lPA mixture 95% l 5% met §95% ethanol 5%IPA mixture PCT/U52012/027537 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 tion. This can then be followed by removal of the liquid solvent from the liquid phase, yielding extraction product. In some embodiments, the solvent is evaporated. In such an implementation, 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.

It was theorized that treatment of the algal biomass prior to extraction would enhance the productivity and efficiency of lipid extraction. In this direction an experiment was done comparing the effect of adding a base or another organic solvent to an algal biomass to change the surface properties and enhance extraction. A variety of treatments ing aqueous ol, aqueous sodium hydroxide, and aqueous DMSO were attempted. As demonstrates, the addition of 5% DMSO increases the lipid ry 3-fold. These tion steps may be exploited to dramatically reduce the methanol extraction steps. However, the solutions used in the above experiments may not be ideal for use on larger scales due to the high cost, viscosity, and ability to r and recycle DMSO. is a chart showing the effect of an eight step methanol extraction on the cumulative total lipid yield and the purity of the ted neutral lipid. In this embodiment, 112 grams of wet biomass (25.6% dry weight), was extracted with 350 mL pure methanol and heating for 10 minutes at 160 W irradiance power in each step. This ed in an extraction ature of about 75°C, which was near the boiling point of the extraction mixture. Using this process, it was determined that it is possible to obtain highly pure neutral lipids from algal oil once the majority of the polar lipids have been ted. shows that it is possible to isolate high purity neutral lipid once the polar lipids are all extracted. In this case a 5% yield of total biomass was achieved with over 90% neutral lipids purity in methanol extraction steps 5 through 8. Furthermore, due to the boiling point of the extraction mixture, most of the water in the s is completely extracted in the first extraction step, along with carbohydrates, ns and metals. shows that ry of lipids can be made more efficient by the use of ethanol to extract lipids and protein from wet biomass. By using ethanol, 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 ed to methanol. Furthermore, the boiling point of aqueous ethanol is higher than W0 2012/138438 PCT/U82012/027537 aqueous ol, facilitating fiirther ry of lipids. This is e the higher temperature renders the oil less viscous, thereby improving ability. 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.

Further, FIG. lO demonstrates that the initial ons are pid rich, containing proteins and other highly polar molecules, followed by the polar lipid rich ons and finally the neutral lipid fractions. Hence with a proper design of the extraction apparatus, one can recover all the three products in a single extraction and fractionation process. r 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 le step tion, as described in Example 1 of the instant application, was med in order to gather data on the efficacy of a one solvent microwave extraction system. 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 that the microwave system reduces the extraction time by about five fold more. While the conventional methods have a higher net 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 ofthe invention use traditional microwave apparatus, which emit wavelengths that excite water molecules. r 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 s.

Moisture content is another parameter of biomass that will influence the efficiency of oil extraction. In some embodiments of the present invention, dry algal mass is extracted and onated. In other embodiments, the algal mass is wet. Biomass samples with algae mass contents of 10%, 25%, and 33% were used to investigate the influence of moisture extraction performance.

PCT/U52012/027537 A shows an illustrative s 400 for a step—wise extraction of products from an algae biomass. All units in FlG. 12A are in pounds. A shows a mass e ofthe 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 ofpolar lipids, 1.87 pounds l 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 first extraction step 410. Any of the dewatering techniques disclosed herein can be used tin dewatering step 405. In the first extraction step 410, 238 pounds of ethanol and 12 pounds ofwater 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 of231 pounds of ethanol, 53 pounds of water, and 0.5 pounds of algal proteins are red, from which water and ethanol are removed by, e.g. , evaporation, g a protein- rich product 415. Solvent red from the evaporation can be recycled to the first extraction step 410.

A 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 ation 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 11 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, g 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 algac, 0.3 pounds water, and 6.6 pounds l rcmain.

W0 2012/138438 PCT/U52012/027537 As demonstrated in A, the resulting lipid profile with each sequential ethanol extraction step was largely ced by the moisture content in the starting algae.

Models of process 400 were run on three different biomass collections, each having a different initial water content. As the initial water content decreased, the m lipid recovery step changed from the third extraction step to a fourth (not shown). However, the overall lipid ry from these three biomass samples were quite similar, all above 95% of the total lipid content of the algal biomass.

When algal mass with higher moisture content was used, the ethanol concentration in the s ethanol mixture was much lower, and consequently the neutral lipid percentage in the crude extract was also lower. It has been reported that dewatering an algae paste with 90% water is a very energy intensive process. The methods described herein unexpectedly can be used to successfully extract and onate an algal mass containing mostly water. As overall lipid recovery was not significantly influenced by ng from an algae paste containing 90% water (10% algal solids), unlike conventional extraction methods, the methods disclosed herein do not require the use of an energy intensive drying step.

B shows an illustrative implementation 500 of one of the extraction steps of process 400. An algae biomass and t mixture 505 is provided to an extraction vessel 510. After the algae is extracted (as described elsewhere herein), the mixture is provided to a coarse filtration system 515, such as a ed 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 l 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 t removal system are recycled or discarded. Centrifuge 525 assists in separating the desired algal product (e.g., ns or lipids) from any remaining water and/or solids in the liquid phase. shows an example of a process 600 by which an algal mass can be processed to form or recover one or more algal products. In this example, an algal biomass is extracted in a step-wise manner in a front-end process 605 using the methods disclosed .

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 e components and products. The components and products e 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. In preferred embodiments the fuel esters are C16 chain lengths. In others, the fuel esters are C18 chain lengths. In still other embodiments, fuel esters are a mixture of chain lengths, C20 or shorter.

The esterification process 610, ysis process 615, hydrotreating s 620, and distillation process 625 are optional and can be used in various orders. The dashed arrows and dotted arrows te 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. For example, in some embodiments of the ion, after extraction and/or separation are carried out, the neutral lipids fraction can be directly hydrotreated in order to make fuel products and/or additives. Alternatively, in other embodiments, the neutral lipid fraction can be passed to esterification process 610.

Esterification s 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 ream processing. Enzymatic esterification ques can also be used. fication can process substantially 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 on. The esterified lipids can then be passed to reating process 620 to generate jet fuel, biodiesel, and other fuel products. Any hydrotreating process known in the art can be 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 600°F. Commonly used sts are NiMo or CoMo.

Hydrotreating the fiiel esters rather than the raw lipids has several advantages.

First, the fication s 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 W0 38438 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.

In some embodiments ofthe invention, 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 cstcrificd and separated into carotenoids, long chain rated esters, eicosapentaenoic acid (EPA) esters, and/or fuel esters via distillation process 625. Distillation process 625 can include molecular lation 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. 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 ive hydrotreating reaction, as described above.

In some embodiments ofthe invention, polar lipids (and, optionally, l lipids) are yzed in hydrolysis process 615 before being passed to the esterification process.

Doing so unbinds the fatty acids of the algal lipids, and s a greater amount of the algal lipids to be formed into useful products. is a flowchart showing a process 700 for producing nutraceutical products from neutral lipids. In one implementation ofprocess 700, 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 s, such as plant sources.

Adsorption process 705 es contacting the l lipids with an adsorbent to adsorb the carotenoids, such as beta carotene and phylls. In one implementation, the adsorbent is Diaion HP2OSS (commercially available from ITOCHU als 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 implementations, 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.

W0 2012/138438 PCT/U52012/027537 The carotenoids can be recovered from the adsorbent material by ng the adsorbent with an appropriate t, ing, but not limited to, alcohols such as ethanol, isopropyl alcohol , butanol, esters such as ethyl acetate or butyl acetate, alkanes such as hexane, and e.

In yet another embodiment of the invention, chlorophylls and carotenoids isolated from the neutral lipids. The adsorption process can be performed using a hydrophobic adsorbent such as polystyrene-divinylbenzene resin (PS-DVB) or with higher carbon loading resin. Some examples of the resins are XAD 16, XAD 4 from Rohm and Hass (Philadelphia, PA) or HP20, SP—850 from Mitsubishi Chemical ries Limited (Tokyo, Japan) distributed by ltochu Chemicals America, Inc, White Plains, NY). The extraction on dissolved in aqueous ethanol would be passed over a packed column to selectively retain the chlorophylls and noids. Upon reaching the adsorption loading capacity, the column would be washed with a hydrophobic solvent such as hexane. The pure noids and chlorophyll on dissolved in hexane is evaporated under mild conditions to obtain a concentrate. The pure carotenoids and chlorophyll could also be crystallized and precipitated in the solution.

In yet another embodiment of the ion, extracting neutral lipids from the algal biomass, followed by a membrane diafiltration step for the separation of chlorophylls and carotenoids from the neutral lipids. Membrane diafiltration works based on the principle of selectively g the product through a membrane and retaining the remaining components of the feed mixture. In this case, we would use a solvent stable nanofiltration membrane such SelRO ts from Koch membrane systems ngton, MA). The neutral lipids extract suspended in aqueous ethanol would be filtered through the membrane to selectively separate the unbound carotenoids and chlorophylls. The feed is continuously diluted with ethanol until all the carotenoids and chlorophylls are washed off and collected in In yet another embodiment of the invention, chlorophylls and carotenoids isolated from the polar lipids. The adsorption process can be performed using a hydrophobic adsorbent such as polystyrene-divinylbenzene resin (PS—DVB) or with higher carbon loading resin. Some examples of the resins are XAD 16, XAD 4 from Rohm and Hass (Philadelphia, PA) or HP20, SP—850 from Mitsubishi Chemical Industries Limited (Tokyo, Japan) distributed by ltochu Chemicals a, Inc, White Plains, NY). The polar lipids extract is dissolved in aqueous ethanol would be passed over a packed column to selectively retain the chlorophylls and carotenoids. Upon reaching the adsorption loading capacity, the column would be washed W0 2012/138438 with a hydrophobic solvent such as . The pure carotenoids and phyll fraction dissolved in hexane is evaporated under mild conditions to obtain a concentrate. The pure carotenoids and chlorophyll could also be llized and precipitated in the solution.

In yet another embodiment of the invention, extracting polar lipids from the algal biomass, followed by a membrane diafiltration step for the separation of phylls and carotenoids from the polar lipids. Membrane diafiltration works based on the principle of selectively eluting the product through a membrane and retaining the remaining components of the feed mixture. In this case, we would use a solvent stable nanofiltration membrane such SelRO from Koch membrane systems (Wilmington, MA). The polar lipids extract suspended in aqueous ethanol would be filtered through the membrane to selectively separate the unbound carotenoids and chlorophylls. The feed is continuously diluted with ethanol until all the carotenoids and chlorophylls are washed off and collected in permeate.

In yet another embodiment of the invention, a method of making biofiiels includes dewatering substantially intact algal cells to make an algal biomass, extracting neutral lipids from the algal biomass, followed by an adsorption step for the separation of chlorophylls and carotenoids from the neutral lipids. The method also includes esterifying the neutral lipids with a catalyst in the presence of an alcohol, and separating a water soluble fraction comprising glycerin from a water insoluble fraction comprising fuel esters. The method still further es ling the fuel esters under vacuum to obtain a C16 or shorter fuel esters on, a C16 or longer fuel ester fraction, and a residue comprising omega—3 fatty acids and enating and deoxygenating at least one of (i) the C16 or shorter fuel esters to obtain a jet fuel blend stock and (ii) the C16 or longer fuel esters to obtain a diesel blend stock.

In yet another embodiment of the invention, a method ofmaking biofuels includes dewatering substantially intact algal cells to make an algal s, extracting neutral lipids from the algal biomass, followed by a membrane diafiltration step for the separation of chlorophylls and carotenoids from the l . The method also includes esterifying the neutral lipids with a catalyst in the presence of an alcohol, and separating a water soluble fraction comprising glycerin from a water insoluble on sing fuel esters. The method still further includes distilling the fuel esters under vacuum to obtain a C16 or shorter fuel esters fraction, a C16 or longer fuel ester fraction, and a residue comprising 3 fatty acids and hydrogenating and deoxygenating at least one of (i) the Cl 6 or shorter fuel esters to obtain a jet fuel blend stock and (ii) the C16 or longer fuel esters to obtain a diesel blend stock.

W0 2012/138438 PCT/U52012/027537 In yet another ment of the invention, a method of making nutraceuticals and health foods es dewatering substantially intact algal cells to make an algal biomass, ting neutral lipids from the algal biomass, followed by an adsorption step for the tion of chlorophylls and carotenoids from the l lipids. The neutral lipids resulting from this step would be rich in omega-3 fatty acids with a balanced ratio of monounsaturated, polyunsaturated and saturated fatty acids. This can be sold as high quality oil for better health.

In yet another embodiment of the invention, a method of making nutraceuticals and health foods includes dewatering substantially intact algal cells to make an algal biomass, extracting l lipids from the algal biomass, followed by a ne diafiltration step for the separation of chlorophylls and carotenoids from the neutral lipids. The neutral lipids resulting from this step would be rich in omega-3 fatty acids with a balanced ratio of monounsaturated, saturated and saturated fatty acids. This can be sold as high quality oil for better health. is a flowchart showing a process 800 for producing fuel products 830 from neutral lipids 805. The l lipids can be from an algae source generated by any of the selective extraction techniques disclosed herein. However, the l 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 l lipids. In some implementations, a relatively dilute solution of phosphoric acid is added to the neutral lipids, and the mixture is heated and agitated. The precipitated olipids and metals are then separated from the remaining oil, for example, by centrifuge.

The treated oil is then passed to bleaching process 815 to remove chlorophylls and other color compounds. In some implementations, bleaching process 815 includes contacting the oil with clay and or other adsorbent material such as ing clay (Le. bentonite 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 es, diesel fuel additive, and propane. In addition, the hydrotreating process 820 also causes some cracking and the creation of smaller chain compounds, such as LPG and naptha. Any of the hydrotreating processes described herein can be used for hydrotreating process 820.

The mixture of compounds created in the hydrotreating process 820 are passed to a distillation process 825 to separate them into various fuel products 830. Distillation process W0 2012/138438 PCT/U82012/027537 825 can include any of the molecular and non-molecular distillation ques described herein or known in the art for separation of fuel compounds.

In some embodiments of the instant ion, proteins may be selectively ted from an algal biomass. Extraction of proteins using the disclosed methods offers many advantages. In particular, algal cells do not need to be lysed prior to extracting the desired proteins. This simplifies and reduces costs of extraction. The methods of the instant invention exploit the solubility s of different classes of proteins in order to selectively extract and fractionate them from an algal culture, biomass, paste, or cake.

For example, an algal biomass may be subjected to heating and mixing to extract water and salt soluble proteins called albumins and globulins. This e can then be subjected to a change in pH to recover the alkali soluble ns called the glutelins. This step can then be followed by a solvent-based separation of the alcohol soluble proteins called prolamins. The remaining s 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 s 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.

In some embodiments, extraction of proteins from freshwater algal cells is accomplished by the novel process shown in . 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. tion can be accomplished by a variety of methods known in the art including, but not limited to, eentrifugation, decantation, flotation, sedimentation, and filtration. This first substantially liquid phase is enriched in n ns.

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 se about typically 35 g/L comprising mainly . The solid and liquid phases are then ted.

This second ntially liquid phase is enriched in globulin proteins.

PCT/U52012/027537 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 solid 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 generate a fourth heated tion mixture or , sed of a fourth substantially liquid phase and a fourth substantially solid phase. In one red embodiment, the solvent set comprises ethanol. In other non-limiting embodiments, the solvent set ses one or more ofthe ing ts: 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 ng algal biomass.

In some embodiments, tion of proteins from saltwater algal cells is accomplished by the novel process shown in . Saltwater algal cells or a saltwater algal biomass are heated and mixed. Mixing can be accomplished by a variety of s known in the art such as, but not limited to, stirring, agitation, and g. 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 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 generate a third heated extraction mixture or slurry, comprised of a third substantially liquid phase and a third substantially solid phase. The pH of this third extraction mixture or slurry is then raised to pH 9 or r, enriching the third substantially liquid phase with glutelin W0 2012/138438 PCT/U52012/027537 proteins. The solid and liquid phases are then separated, the third ntially liquid phase being enriched in glutelin proteins.

The third substantially solid phase is then mixed with a t 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. In one preferred embodiment, the solvent set comprises ethanol. In other non-limiting embodiments, the solvent set comprises one or more ofthe following solvents: methanol, isopropanol, acetone, ethyl acetate, and itrile. 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.

The disclosed methods also provide for the selective extraction of different types of proteins, as shown in -20. Any of the steps of the entioned 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 and 18, as the as demonstrated by the dashed box around extraction step la.

In a non-limiting e, globulin proteins can be selectively extracted from freshwater algal biomass by mixing said biomass with salt water and heating to generate a heated extraction mixture or slurry, comprised of a ntially liquid phase and substantially solid phase. The solid and liquid phases can then be separated. The liquid phase is enriched in globulin proteins. See , extraction step la.

In another non—limiting example, 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 slurry, 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 ns. See , extraction step la.

In a further non~limiting example, prolamin proteins can be ively extracted from either a freshwater or saltwater algal biomass as shown in . The ive 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 substantially liquid phase and a substantially solid phase. The solid and liquid phases can then be ted. The liquid phase is ed in prolamin proteins.

W0 2012/138438 PCT/U52012/027537 In yet another non-limiting e, a protein fraction can be selectively extracted from either a freshwater or ter algal biomass as shown in . The selective extraction is lished 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.

Having been informed of these aspects of the invention, one of skill in the art would be able to selectively extract a d protein from either a freshwater or saltwater algal s by either a single step extraction process, or a multi—step extraction process. In light of the instant disclosure, one of skill in the art would be able to hange the order of the above disclosed multi—step extraction schemes, provided that the protein content of the algal mass and the solubility properties of the proteins of st are taken into account. Other embodiments ofthe disclosed methods may incorporate a wash step between each extraction step.

For any of the disclosed protein extraction methods, 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 tion mixture is maintained at a heated temperature for between about 20 minutes and about 60 minutes. In other aspects, the tion mixture is maintained at a heated temperature for between about 45 minutes to about 90 minutes.

In some embodiments, the extraction mixture/slurry may be heated to atures less than about 50°C. In some aspects, the albumin, globulin, and glutelin proteins extracted at temperatures of less than about 50°C. In other embodiments the tion mixture/slurry is heated to a temperature close to the boiling point of extraction mixture/slurry.

In some aspects, the prolamin proteins are extracted at atures close to the boiling point ofthe extraction mixture/slurry. In other embodiments, the pressure is increased above heric pressure, up to and including, 5Opsi, during the heating and mixing steps to enhance extraction.

Example 1 Green microalgae Scena’esmus dz'morphus (SD) were cultured in outdoor panel ioreactors. 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.

W0 2012/138438 2012/027537 A pie-calculated amount ofwet 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 refluxed for 10 min under microwave irradiance. In a second, the e was refluxed for 1h with electronic heating.

Afterwards, the e was cooled to room temperature and separated into a diffusate and retentate by filtration.

The total lipids of algal samples were ed using a chloroforrn—methanol—water system according to Bligh 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 (Biichi, Switzerland) and then dried under high vacuum. The dried retentates were placed under nitrogen and then weighed. The fatty acid profile of each sample was quantified by GC-MS after derivatization into fatty acid methyl esters using ecanoic acid (C17:0) as the internal standard.

The results (data not shown) ted that microwave assisted extraction was best for removal of the polar lipids in the first extraction step, and somewhat less effective for separation of l lipids. Electronic heating is more consistent in extraction effectiveness.

The final yield is comparable n microwave assisted extraction and electronic heating assisted extraction, but, microwave assisted extraction is significantly faster.

Example 2 Protein extraction from algal biomass (1) Acid Leaching: Algal biomass was soaked in water at pH 4.5 for 1 hour. The s were then centrifuged at 3000 rpm for three minutes, and the supernatant d.

The remaining solids were washed 3 times with dilute acid (pH 4.5) and freeze dried. (2) Alkaline extraction: Algal biomass was soaked in water at pH 11 for 1 hour. following the addition of pH-adjusted water. The s were then centrifuged at 3000 for three minutes, and the supernatant removed. The supernatant was neutralized with acid (pH 4.5) following the centrifugation. The remaining solids were washed 3 times with dilute acid (pH 4.5) and freeze dried.

The results of acid leaching and alkaline extraction are shown below in Table 4.

W0 2012/138438 LIME! Process Protein Yield Protein Purity (% weight) (% weight of protein yield) Alkaline tion 16 45 Acid Leaching 70 32.5 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), ing 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.

Example 3 Extraction of ns from Saltwater Algal Biomass The saltwater algal culture lly made up of about 1—10% w/w solids in saltwater was heated to 50°C and ined at this temperature for 1 hr. The resulting slurry was fuged 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 biomass).

The solids were then suspended in fiesh 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 % of the total proteins present in the original algal biomass).

The solids were then suspended in ethanol to achieve a 70% w/w e. This e 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 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).

Example 4 Step Fractionation and Extraction of Algal Biomass by Ethanol One thousand pounds ofNaimochloropsz's biomass (cultured from strain 202.0, ed from Arizona State University, Laboratory for Algae Research and Biotechnology, ATCC Deposit Number PTA-l 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 proofmotor with a two blade shaft. The frozen algae al was emptied into the tank and an equal weight of l was pumped in using a pneumatic drum pump. The material was stirred for 15 minutes and the jacket heated with steam to obtain the desired ature 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 ofthe e changes as the tion of ethanol is changed. Upon reaching the desired temperature, the system was d continuously held at the desired temperature for 60 minutes to ensure that the contents ofthe kettle were uniformly heated. {0220] The contents of the kettle were then pumped out of the extraction vessel and into Sharples decanter centrifuge, using a tic 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 ed plastic drum and consisted of about 50% w/w solids to s. 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 ft2 SS ne manufactured by Graver Technologies. The operating ions were 60°C i 5°C and with an average W0 2012/138438 PCT/U52012/027537 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 ne system was free of any particulate matter. The retentate was collected and recycled to the decanter.

This extraction and fractionation is due to the change in polarity of the solvent through the process in each extraction. in the extraction shown in F1G. 13, the process began with about 1000 lbs. ofwet algal biomass containing about 65% pure water (35% w/w algal solids). This was mixed with 860 lbs. of denatured ethanol (95% ethanol and 5% methanol), resulting in a mixture containing about 55% aqueous l. The solids and liquids were separated using a decanter as described above. The wet solid portion weighed 525 lbs. and was 40% dry mass. A total of 525 lbs. of 95% the denatured ethanol was added to the solids, ing in a mixture made up of about 85% aqueous ethanol. The solids and liquids were separated using a decanter as described above. The solid portion weighed 354.5 lbs. and was 40% dry mass. To this mass, r 700 lbs. of denatured ethanol was added, resulting in mixture of about 95% aqueous ethanol. The solids and liquids were separated using a decanter as described above. The resulting solids were about 40% dry mass. This biomass requires 60% less energy to dry, ated based on the latent heat ofwater and ethanol.

In some experiments (data not shown) other types of denatured ethanol were tried.

Denatured l containing 95% ethanol 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 ment of the present ion, but is generally not available due to cost constraints.

The permeate stream from the membrane system was evaporated using an in-house fabricated batch still. The operating conditions were about 80°C during the vacuum distillation.

All of the ethanol in the permeate was evaporated. These extraction steps were repeated three times, resulting in four product pools, as shown in . This is because with each extraction step, the polarity d with the addition of water to the mixture, allowing for the extraction of ent ents with each step. t 1 contained the algal proteins, and as a result, retained excess water in the system that could not be vaporized under the operating conditions. Product 2 contained the polar lipids. Product 3 ned the neutral lipids.

Finally, Product 4 was the residual biomass, containing potential coproducts such as carotenoids.

W0 2012/138438 Example 5 Dewatering and tion of Algal Biomass by Ethanol Upon harvesting, algal biomass typically contains n 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. To dewater further, 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. r, the cost of ring increases exponentially after the first dewatering is carried out. An advantage of the s and s disclosed herein is that the allow for the extraction 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), ing 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. With another 350 lbs. of denatured l, the composition of the mixture would be about 81% aqueous ethanol. The resulting biomass (235 lbs.) is 40% dry. The solvent composition of these wet solids is 81% aqueous ethanol. With another 470 lbs. of denatured 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. In this case, 100 lbs. of algae would have been extracted using 1820 lbs. ethanol.

When compared with e 3, wherein the starting material was 40% algal solids, 350 lbs. ofthe dry algae equivalent was extracted with 2085 lbs. ethanol.

PCT/U52012/027537 nces The following references are herein incorporated by reference in their entirety: US. Patent 7,148,366 Rhodes, Science Progress, 92(1):39—90, 2009. Generic review on using algae to produce biodieselChisti, Y. . Biodiesel from lgae. Biotechnol Adv 25, 294-306. - Generic review on using algae to produce biodiesel Amin, Energy Convers. Manage, 4—1840, 2009. Generic review on using algae to produce biofuel and gas Catchpole et al., J. of Supercritical Fluids, 47:591—597, 2009. SCF C02 based extraction of specialty lipids Bligh E G & Dyer W J. A rapid method of total lipid extraction and purification. Can. J.

Biochem. Physiol. 37: 911-917, 1959.

Christie, W. W., Lipid Analysis, 3rd ed., Oily Press, Bridgewarer, UK, 2003, 416.

Approved Methods of the AACC, 9th ed, American Association of Cereal Chemists. St. Paul, MN, 1995 AACC Method 58-19.

Claims (3)

1. A method of isolating chlorophylls and omega-3 rich oil from algae, comprising: a. dewatering substantially intact algal cells to make an algal s; b. adding a first ethanol fraction to the algal biomass in a ratio of about 1 part ethanol to about 1 part algal biomass by weight; c. separating a first substantially solid biomass fraction from a first substantially liquid fraction comprising proteins; d. combining the first substantially solid biomass fraction with a second ethanol fraction in a ratio of about 1 part ethanol to about 1 part solids by weight; e. separating a second substantially solid biomass on from a second substantially liquid fraction comprising polar lipids; f. combining the second substantially solid biomass fraction with a third ethanol solvent fraction in a ratio of about 1 part l to about 1 part substantially solid biomass by weight; g. ting a third substantially solid biomass on from a third substantially liquid fraction comprising neutral , including omega-3 fatty acids, carotenoids, and chlorophyll, wherein the third substantially solid biomass fraction comprises carbohydrates; and h. isolating at least one of noids, chlorophyll, and omega-3 fatty acids from the third substantially liquid fraction.

2. The method of claim 1, further comprising esterifying the neutral lipids with a catalyst in the presence of an l, and separating a water soluble fraction comprising glycerin from a water insoluble on comprising fuel esters.

3. The method of claim 2, further comprising distilling the fuel esters under vacuum to obtain a C16 or shorter fuel ester fraction, a C16 or longer fuel ester fraction, and a residue comprising omega-3 fatty acids. . The method of claim 3, further comprising deoxygenating the C16 or shorter filel ester fraction to obtain a jet fuel blend stock and/or the C16 or longer fuel on to obtain a diesel blend stock. . The method of any one of the preceding claims, wherein the isolating is carried out by adsorption with an adsorbent material. . The method of claim 5, wherein the isolating is carried out by tion with a clay. . The method of claim 6, wherein the clay is selected from the following group consisting ofbleaching clay, bentonite, and fuller’s earth. . The method of claim 1, substantially as herein described with reference to any one of the es and/or