DECONSTRUCTING ALGAE USING IONIC LIQUIDS

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] The invention relates generally to industrial biotechnology and more specifically to the processing of algae.

BACKGROUND INFORMATION

[0002] On a global scale, an alternative to fossil fuels is widely accepted as being of critical importance. Fossil fuels are nonrenewable resources that are in increasingly short supply with ever increasing demand. On a national scale, the reduction of energy dependency from foreign oil is now viewed as essential to ensure the long-term security and economic stability of the United States or any industry based country. To achieve economic sustainability as well as environmental security, fuel production processes are required that are not only renewable, but also capable of sequestering the atmospheric greenhouse gas carbon dioxide. Further, nearly all of the current renewable energy sources, such as hydroelectric, solar, wind, tidal, and geothermal, target the electricity market. However, fuels make up a much larger share of the global energy demand. Hence, development of renewable biofuels is a strategic imperative.

[0003] Algae are used to produce certain nutrients and cosmetic components, and are now being developed to produce renewable energy, fuels and chemicals. However, processing algae remains a substantial challenge limiting the utilization of algae as fuel. There exists in the art a need to, in an energy efficient manner, release algae constituents.

SUMMARY OF THE INVENTION

[0004] The present invention is based on the discovery that ionic liquids can lyse algae cells and extract lipids, optionally from algae that have not been dried. In one aspect, the invention provides a method for processing algae. The method includes mixing algae cells with an ionic liquid solution, wherein the algae cells are processed into at least two constituents.

[0005] In one embodiment, the ionic liquid solution comprises an ionic liquid and water. In another embodiment, at least one constituent is a lipid. In some embodiments, the at least one constituent is about immiscible in the ionic liquid solution. In some embodiments, a carbohydrate is released from the algae cells.

[0006] In another aspect, the invention provides a method for deconstructing algae cells. The method includes contacting algae cells with a solution comprising an ionic liquid and water, wherein the algae cell wall is lysed, and wherein algae lipid is about immiscible in the solution. In some embodiments the ionic liquid may be l-butyl-3-methylimidazolium chloride, l-propyl-3-methylimidazolium chloride, l-allyl-3-methylimidazolium chloride, 1- ethyl-3-methylimidazolium chloride, l-(2-hydroxylethyl)-3-methylimidazolium chloride, 1- butyl-l-methylpyrrolidinium decanoate and any combination thereof.

[0007] In some embodiments, the algae cells are microalgae, diatoms, cyanobacteria, macroalgae, or any combination thereof. In some embodiments, the algae cells are

Chlamydomonas moewusii, Chlamydomonas reinhardii, Neochloris pseudostigmata, Scenedesmus quadricauda, Chlorella vulgaris, Chlorococcum hypnosporum, Dunaliella salina and Chlorella pyrenoidosa.

[0010] In some embodiments, the ionic liquid solution is a pure ionic liquid with at least 99.9% of an ionic liquid. In other embodiments, the ionic liquid solution is an active ionic liquid with at least 50% of an ionic liquid. In yet other embodiments, the ionic liquid solution is an inactive ionic liquid, wherein the method further includes activating the ionic liquid solution by removing a sufficient quantity of water to process the algae cells. In some embodiments, the ionic liquid solution has a melting temperature below about 150 degrees C

[0011] In another aspect, the invention provides a method for lysing algae cells including inputting less than about 5 megajoules of energy per kilogram of algae cells.

[0012] In another aspect, the invention includes the products resulting from the methods of the invention. In some embodiments, the products include oil, triglyceride, free fatty acids, water soluble sugars, water insoluble carbohydrates, protein, hydrogen, methane, ethanol, biodiesel, green diesel, biosynthetic liquid fuel, gasoline, kerosene, or any combination thereof. [0013] In some embodiments the algae cells include water, whereby mixing algae cells with the ionic solution results in the ionic solution including an ionic liquid and water. In other embodiments, the algae cells are substantially dry. In some embodiments the water is fresh water, salt water, brackish water or waste water.

[0014] In some embodiments the constituent can be DNA, RNA, protein, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows a pure ionic liquid element of an active ionic liquid.

[0016] Figure 2 provides a chemical structure of a particular pure ionic liquid element.

[0017] Figure 3 illustrates cell disruption.

[0018] Figure 4 illustrates cell lysis.

[0019] Figure 5 provides a process of obtaining products via lysis of an algae using an ionic liquid.

[0020] Figure 6 provides a process of heating and lysing algae using a recoverable ionic liquid.

[0021] Figure 7 provides a process for dewatering and/or drying algae.

[0022] Figure 8 provides a process of separating algae constituents.

[0023] Figure 9 illustrates separation of phases in deriving product and/or an ionic solution from lysed algae.

[0024] Figure 10 illustrates separation of lysed algae constituents.

[0025] Figure 11 illustrates a reactor system for converting algae to an algae component and/or product.

[0026] Figure 12 illustrates the structure of alginic acid. [0027] Figure 13 illustrates the structure of agarose. [0028] Figure 14 illustrates the structure of cellulose.

[0029] Figure 15A is a picture of N. pseudostigmata directly sampled from harvest and before reaction with ionic liquid.

[0030] Figure 15B is a picture of N. pseudostigmata after reaction with ionic liquid.

[0031] Figure 16 is a mass spectra of lipids extracted from N. pseudostigmata using [BMIM]C1.

[0032] Figure 17A is a picture of C. reinhardii after harvesting.

[0033] Figure 17B is a picture of C. reinhardii in [BMIMJCl at 110° C and 1 : 10 mixing ratio after 3 minutes.

[0034] Figure 18A is a picture of washed and harvested Chlorella pyrenoidosa and Chlorella vulgaris.

[0035] Figure 18B is a picture of Chlorella pyrenoidosa and Chlorella vulgaris in [PMIMJC1 at 120° C and 1 : 10 mixing ratio after 15 minutes.

[0036] Figure 19A is a picture of washed and harvested Chlorella pyrenoidosa and Chlorella vulgaris.

[0037] Figure 19B is a picture of Chlorella pyrenoidosa and Chlorella vulgaris in [AMIMJCl at 140° C and 1 : 10 mixing ratio after 25 minutes.

[0038] Figure 20A is a picture of washed and harvested Chlorella pyrenoidosa.

[0039] Figure 20B is a picture of Chlorella pyrenoidosa in [BMIMJCl at 110° C and 1 : 10 mixing ratio after 15 minutes.

[0040] Figure 21 A is a picture of C. moewusii after harvesting.

[0041] Figure 21B is a picture of C. moewusii in [BMIMJCl at 100-130° C and 1 : 10 mixing ratio after 2 minutes.

[0042] Figure 22A is a picture of C. hypnosporum after harvesting. [0043] Figure 22B is a picture of C. hypnosporum in [BMIM]C1 at 120° C and 1 :20 mixing ratio after 30 minutes.

[0044] Figure 23 A is a picture of S. quadricauda after harvesting.

[0045] Figure 23B is a picture of 5". quadricauda in [BMIM]C1 at 130° C and 1 : 10 mixing ratio after 30 minutes.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention is based on the discovery that ionic liquids can lyse algae cells and extract lipids, optionally from algae that have not been dried.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, a plurality of terms are defined below.

[0048] The term "invention" or "present invention" as used herein is not meant to be limiting to any one specific embodiment of the invention but applies generally to any and all embodiments of the invention as described in the claims and specification.

[0049] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0050] Ionic Liquids

[0051] An "ionic liquid" refers to liquids composed primarily of ions that are fluid at temperatures below about 150° C. For example, molten sodium chloride, which is commonly referred to as table salt, is not an ionic liquid in is molten form as the melting point of sodium chloride is 801° C, which is above the herein defined melting point of an ionic liquid of less than about 150° C. However, l-propyl-3-methylimidazolium chloride, which has an anion, a cation, and a melting point of 74° C is an ionic liquid when above 74° C.

[0052] Herein, for clarity and without limitation, l-propyl-3-methylimidazolium chloride is used as an example of an ionic liquid 100. Many salts exist that are ionic liquids, which are usable in the methods, apparatus, and processes herein. Some further examples include l-allyl-3-methylimidazolium chloride, l-butyl-3-methylimidazolium chloride, l-ethyl-3- methylimidazolium chloride, l-(2-hydroxylethyl)-3-methylimidazolium chloride, 1 -butyl- 1- methylpyrrolidinium decanoate. For clarity, examples of additional ionic liquids may be found in (U.S. Pub. No. 2009/0234146, U.S. Pub. No. 2003/0157351, and U.S. Pub. No. 2009/00201 12, each of which is incorporated herein by reference in their entirety).

[0053] Herein, an ionic liquid 100 may refer to the pure ionic liquid component of an ionic liquid solution. For the purposes of the present invention, definitions are provided for compositions containing various amounts of further components such as water along with the ionic liquid. For clarity, "ionic liquid solution" shall mean any solution that contains any amount of one or more ionic liquids. Within ionic liquid solutions, definitions are provided for "pure ionic liquid", "active ionic liquid", "inactive ionic liquid" and "ionic solution".

[0054] Ionic Liquid Solutions

[0055] Some ionic liquids are hygroscopic, which results in the hygroscopic liquids absorbing, attracting, or scavenging moisture from the air. Hence, an ionic liquid may have a small percentage of water in the liquid solution. Herein, a "pure ionic liquid" (PIL) contains less than about one-tenth of one percent water by mass. Therefore, a pure ionic liquid contains at least about 99.9% ionic liquid. In a pure ionic liquid, the solvent is the salt or ions.

[0056] The invention also distinguishes between "active ionic liquids" and "inactive ionic liquids" based on their ability to lyse an algae cell. The primary test to distinguish one from another is to contact the ionic liquid solution with algae cells using the methods of the invention. If the cells are lysed, the solution is an "active ionic liquid". If the cells are not lysed it is an "inactive ionic liquid". It is further understood that "pure ionic liquids" may also be able to lyse algae cells. The concentration of water in the ionic solution further provides some guidance as to whether the ionic liquid solution is likely to be an active or inactive ionic liquid.

[0057] An "active ionic liquid" (AIL) is composed primarily of an ionic liquid with water comprising a smaller fraction of the liquid. Particularly, an active ionic liquid comprises about 50% to 99.9% ion constituent and 0.1% to about 50% water, where the ion constituent when isolated comprises a melting point of less than about 150° C. Additional soluble components are optionally present in the active ionic liquid, but the salt

concentration is at least about 50% of the soluble active ionic liquid components and water is less than about 50% of the soluble active ionic liquid components. If an insoluble component is present, the percentage of active ionic liquid refers to the percentage of the soluble components only. For example, if insoluble, or not yet dissolved, cell walls are present, then the cell walls are not a portion of the active ionic liquid. A pure ionic liquid in a solution of 0.1% to about 50% water is an active ionic liquid.

[0058] An "inactive ionic liquid" is composed primarily of water with an ionic liquid comprising some of the liquid. Particularly, an inactive ionic liquid comprises at least about 50% water and less than about 50% ionic liquid, where the ion constituent when isolated comprises a melting point of less than about 150° C. Additional soluble components are optionally present in the inactive ionic liquid, but the ionic liquid concentration is at most about 50% of the soluble inactive ionic liquid components and water is at least about 50% of the soluble inactive ionic liquid components. If an insoluble component is present, the percentage of inactive ionic liquid refers to the percentage of the soluble components only. For example, if insoluble, or not yet dissolved, cell walls are present, then the cell walls are not a portion of the inactive ionic liquid. A pure ionic liquid in a solution of 50% to about 100% water is an inactive ionic liquid.

[0059] An inactive ionic liquid is distinguishable from an ionic solution in that ionic solutions do not necessarily contain any ionic liquid, while inactive ionic liquids must contain some ionic liquid. In that regard, a pure ionic liquid is changeable to an active ionic liquid and/or inactive ionic solution and back, such as by the additional or removal of water.

[0060] An "ionic solution" is also a solution where the solvent is water. However, an ionic solution does not necessarily contain any ionic liquid. For example, a solution of sodium chloride in water is an ionic solution. Ionic solutions cannot be converted to ionic liquid solutions by the addition or removal of water.

[0061] In contrast, it is recognized that the various ionic liquid solutions may be converted between one another by the addition or removal of water. Adding water to a pure ionic liquid will convert it to an active ionic liquid, with further addition of water to an inactive ionic liquid. Removal of water from an inactive ionic liquid will convert it to an active ionic liquid, with further removal of water converting it to a pure ionic liquid. The present invention allows for, and provides methods for such conversions.

[0062] In one embodiment, a pure ionic liquid and/or an active ionic liquid is used to, in an energy efficient manner, break, dissolve, disrupt, solubilize, and/or lyse an algae cell wall, which releases algae constituents used in the creation of energy, fuel, nutrients, drugs, and/or cosmetic components.

[0063] In another embodiment, a reactor is used to extract energy or cosmetic components from harvested algae using a pure ionic liquid and/or an active ionic liquid.

[0064] A l-propyl-3-methylimidazolium chloride and water example is used to clarify the differences between a pure ionic liquid, an active ionic liquid, and an inactive ionic liquid. Referring now to Table 1, the percentages of l-propyl-3-methylimidazolium chloride and water in each of a pure ionic liquid, an active ionic liquid, and an inactive ionic liquid is provided. If in the presence of un-dissolved components, then the percentages refer to only the liquid components of the solution. In the provided example, the solvent is l-propyl-3- methylimidazolium chloride in the pure ionic liquid. Conversely, the solvent is water in an inactive ionic liquid. For the intermediate case of the active ionic liquid, the solvent is 1- propyl-3-methylimidazolium chloride, but the percentage of the l-propyl-3- methylimidazolium chloride ranges from about 50% to 99.9% of the liquid elements of the solution. Table 1: Active Ionic Liquid vs. Pure Ionic Liquids and Ionic Solutions

[0065] An additional example is used to clarify an active ionic liquid. Referring now to Table 2, the percentages of ionic liquid components are provided in solutions (a) in the presence of water and (b) in the presence of both water and additional liquid components. In either case, the ionic liquid components comprise at least 50% of the solution.

Table 2: Active Ionic Liquid

[0066] Mixtures of Ionic Liquids

[0067] Herein, an active ionic liquid refers to a single ionic liquid or a combination of 2, 3, 4, 5, or more separate ionic liquids. The total active ionic liquid percentage is a sum of the individual separate ionic liquid percentages in the active ionic liquid.

[0068] Ionic Liquid Structure

[0069] Referring now to FIG. 1, an ionic liquid element of an active ionic

liquid 100 optionally includes three structural regions: a charge-rich region 110, Ri, a symmetry-breaking region 120, R2, that decreases the melting point; and a hydrophobic region 130, R3, that increases the melting point. Referring now to FIG. 2, each of the charge rich region 110, the symmetry-breaking region 120, and the hydrophobic region 130 of 1- propyl-3-methylimidazolium chloride, [PMIM] CI, are illustrated. The charge-rich region 110 is optionally integrated or adjacent to one or both of the symmetry breaking

region 120 or the hydrophobic region 130. Optionally, the ionic liquid 100 contains two or more charge rich regions. Optionally, one or more of the charge rich regions contains three or more charge centers, where a charge center is a negatively charged region and/or a positively charged region. A salt of l -propyl-3-methylimidazolium is an example of a specific ionic liquid element of an active ionic liquid 100.

[0070] The salt, l -propyl-3-methylimidazolium chloride, is an example of an ionic liquid 100. However, many ionic liquids exist and ionic liquids 100 are further described, infra.

[0071] The charge rich region 110 of the active ionic liquid 100 contains an anion and a cation. Optionally, one or more of the charge region regions 110 are present in the ionic liquid 100 and each charge rich region 110 optionally contains multiple anions and/or cations. Examples of anions include: a chloride, bromide, iodide, perchlorate, a thiocyanate, cyanate, carboxylate, or any negatively charged element or group. Examples of cations include any positively charged atom or group. Optionally, the cation is part of a ring structure, such as in the symmetry-breaking region 120. An example of a cation, which is also a symmetry breaking element is a ring structure containing nitrogen, such as any molecule having a base imidazolium ring. The symmetry breaking region 120 is optionally any structure that hinders a first ionic liquid element from laying in flat contact with a second ionic liquid element, which reduces the melting point of the ionic liquid element of an active ionic liquid 100. The hydrophobic region 130 is a Ci-C ₆ alkyl group, but is optionally a carbon based chain of any length.

[0072] Ionic Liquid Melting Point

[0073] Ionic liquids are known with high melting points, such as above 150° C. Herein, the ionic liquid element of an active ionic liquid 100, hereinafter an ionic liquid 100, is preferably used in a low temperature reaction, such as below 150° C. Hence a low melting point ionic liquid is preferred, such as an ionic liquid having a melting point of less than about 150, less than about 140, less than about 130, less than about 120, less than about 110, less than about 100, or less than about 90 degrees centigrade. The ionic liquid 100 is also referred to herein as a molten liquid when at or above its melting point. Optionally, the ionic liquid is used herein at temperatures below the ionic liquid's melting point, such as at a glass transition temperature, where the ionic liquid contains properties that are a blend of its solid salt form and molten salt form.

[0074] Ionic liquids typically have negligible vapor pressures at operating temperatures under 150° C, are not flammable, and are thermally stable, which makes the ionic liquids suitable for low temperature extraction and/or separation techniques.

[0075] Algae

[0076] It should be understood that "algae" refers to a family of aquatic, eukaryotic or prokaryotic single cell or multicellular organisms, that are typically autotrophic, photosynthetic, contain chlorophyll, and grow in bodies of water, including fresh water, sea water, and brackish water. Algae do not generally have stems, roots or leaves. For at least those reasons, algae are distinguishable from plants. Algae may also be distinguishable from the generic term "biomass", with biomass generally meaning plant matter.

[0077] For the purposes of the present invention, "algae" includes both "microalgae" and "macroalgae". Included in microalgae are both eukaryotic microalgae, and prokaryotic species (cyanobacteria). Any algae species, mixture of algae species, or mixture of algae with other biomass are suitable for practicing the methods of the present invention.

Therefore, the invention includes eukaryotic microalgae, cyanobacteria, and macroalgae.

[0078] The term "microalgae" refers to photosynthetic organisms that include a variety of unicellular, coenocytic, colonial, and multicellular organisms, such as the protozoans, slime molds, brown and red algae, algal strains, diatoms, dinoflagellates, cyanobacteria and such.

[0079] Exemplary algae include, but are not limited to, organisms of the division of Chlorophya (green algae), Rhodophyta (red algae), Phaeophyceae (brown algae),

Bacillariophyceae (diatoms), or Dinoflagellata (dinoflagellates). In one embodiment, the algae is a species of Chlamydomonas, Neochloris, Dunallela, Botrycoccus, Chlorella, Gracilaria, Sargassum, Pleurochrysis, Porphyridium, Phaeodactylum, Haematococcus, Isochrysis, Scenedesmus, Monodus, Chlorococcum, Cyclotella, Nitzschia, or Parietochloris. In another embodiment, the algae is Chlamydomonas reinhardtii. In yet other embodiments, the algae is Chlamydomonas moewusii, Neochloris pseudostigmata, Scenedesmus quadricauda, Chlorella vulgaris, Chlorococcum hypnosporum, Dunaliella salina or Chlorella pyrenoidosa. In some embodiments, the algae is a cyanobacterium. Exemplary cyanobacteria include, but are not limited to, organisms from the genus Spirulina,

Synechococcus or Synechocystis. In one embodiment the cyanobacterium is Spirulina platensis. Also included in algae are the macroalgae or "seaweeds". Also included in algae are the green non-sulfur bacteria, the green sulfur bacteria, the purple sulfur bacteria and the purple non-sulfur bacteria.

[0080] Further examples of algae species may be found in (U.S. Pub. No. 2009/0203070, which is incorporated herein by reference in its entirety). The methods of the present invention can be practiced with either wild-type or genetically modified algae.

[0081] In yet another embodiment of the invention, the algae is a synthetic organism. As used herein, the term "synthetic organism" refers to an organism that is created by man and not found in nature. While it is well known that all transgenic organisms are man-made and not found in nature, synthetic organisms differ in the methods of modification and extensive degree to which they are modified or holistically designed. Typically, synthetic organisms, rather than a simple transgenic organism, are created to provide an organism of minimal genome size and complexity, which exhibits predictable properties. Use products can then be made in a precise manner. In one example of a synthetic organism, an entire genome is synthesized from chemical building blocks and transplanted into a naturally occurring organism, replacing the natural genome and therefore changing the species identity of the resulting cell. As an example of this method, researchers created Mycoplasma laboratorium from Mycoplasma genitalium through genome transplantation (U.S. Pub. No.

2007/0122826, incorporated herein by reference. Other types of synthetic organisms might include organisms with a large numbers of accumulated changes to a genome such that the genome is of a new species, or an organism with a large numbers of genes deleted to create a minimal genome size. Synthetic organisms also include cell-like systems as described in (US Pub. No. 2007/0269862).

[0082] Cell Walls of Algae

[0083] Algae have a simpler organization than plants. Many algae are single-celled and some have no cell wall. Other algae have cell walls, though the wall's composition and structure differ strongly from that of higher plants. Furthermore, algal cell walls do not fulfill the same requirements as those of higher plants.

[0084] Plant cell walls develop in the course of tissue formation in contact with neighboring cells. Strength of the plant cell wall is a decisive and limiting criterion. In contrast with plants, a main function of the algae cell wall is to mediate between the cell and its surrounding.

[0085] The algae cell wall not only protects the cell, but serves to communicate with cells of the same or other types. In contrast, communication via the whole cell surface is largely restricted in plants.

[0086] The algae cell wall has to be permeable for the transport of metabolites and regulators. In plants, the exchange of compounds between cells occurs via specific openings in the wall. It must also carry receptor molecules for contacting other cells. Plant cell walls do not generally perform these functions.

[0087] The diversity of these functions (and their specificity) caused the evolution of a variety of differently structured algae cell wall than that of a plant. [0088] All archaebacteria, eubacteria and blue-green algae (cyanobacteria or blue-green algae) have complex walls with an energetically rather costly biosynthesis. Neither in composition nor in biosynthesis do they have any common ground with the cell walls of plants.

[0089] Although the evolution of plants from early eukaryotic cells is not known in detail, is it commonly agreed that primitive algae are flagellates closely related to the non- green flagellates. Many, though not all species of this stage of evolution, among which the euglenophyta are typical green representatives, have no cell wall. These species have not only a simple membrane, but a pellicle of quite complex organization, that separates them from their surroundings. They comprise mainly glycoproteins organized in regular patterns such as helical ribs wind round the cell's surface.

[0090] Most single-celled algae like the Volvocales possess real cell walls. The most- studied species is Chlamydomonas reinhardii. Quite distinct from a plant cell wall, its wall lacks long, fibrillary carbohydrates. Most of it is made up by glycoproteins, such as an extensin-like protein rich in hydroxyproline. Among the identified sugar residues are arabinosyl-, galactosyl- and mannosyl residues. This is distinct from plant walls, which comprise cellulose, which comprises glucose residues. The algae cell wall may comprise many layers, and in one example seven layers. The middle layer may contain an extensive grid-shaped framework of polygonal plates comprising the mentioned glycoproteins, while the layers above and below display fiber-like structures. The thickness of the outer layer varies since it includes components that the cell takes up from its surrounding.

[0091] Algae is distinct from lignocellulose. Plants contain lignin, which is a structural support in plant cell walls along with cellulose. Algae does not contain lignin so is not lignocellulose.

[0092] Algae is also distinct from cellulose. Cellulose is an organic compound with the formula (CeHioOs , a polysaccharide consisting of a linear chain of several hundred to over ten thousand β(1→4) linked D-glucose units (Figure 14). Cellulose is the structural component of the primary cell wall of green plants, such as in wood or cotton. [0093] Some classes of algae contain cellulose, however there are remarkable differences between the cellulose fiber structure between plants and algae. In algae, cellulose may aggregate in many more or less uniform crystalline structures. The periodic distances at the molecular level may differ considerably from species to species and are especially large in Rhodophyta. In other classes of algae, cellulose may exist only in disperse textures. Many Chlorophyta species have a higher degree of organization (layers of parallel microfibrils). Such layers do usually alternate with layers of an amorphous material. In contrast with plants, no clear difference between primary and secondary cell wall exists in most algae. Where such a distinction is possible, the details differ from that in higher plants. Algae also lack the various structures that characterize cellulose based land plants, such as the lignin, and phyllids, leaves, rhizoids in nonvascular plants, leaves, roots, and other organs that are found in tracheophytes, vascular plants.

[0094] In contrast with cellulose, algae cell walls contain structural materials such as mannanes, xylanes, alginic acid, agarose, sulfonated polysaccharides and further cell wall components such as silicon or calcium. These materials all have a structure distinct from that of cellulose (Figure 14). For example, Figure 12 shows the structure of alginic acid, which is different than that of cellulose. For a second example, Figure 13 shows the structure of agarose, which is different than cellulose.

[0095] To further point out distinctions:

[0096] Mannanes constitute the main structural elements in a number of marine green algae (Codium, Dasycladus, Acetabularia, etc.) as well as in the walls of some red algae (Porphyra, Bangia). They are linear and the mannosyl residues are 1 > 4 glycosidically linked. Hydrogen bonds are the cause of the partially crystalline organization of microfibrils. In Codium the carbohydrates are tightly associated with protein.

[0097] Xylanes are polymers where the teto-D-xylosyl residues are linked via 1 > 3 and 1 > 4 glycosidic bonds. In contrast to the polymers discussed until now, xylans are partially ramified. In species with xylan-containing walls, there exist a layered structure.

[0098] Alginic Acid and its salts, the alginates are important components of the walls of phaeophyta (brown algae). They contain uronic acids: mannuronic acid and beta-L- glucuronic acid in changing ratios, along with small amounts of teto-D-glucuronic acid. The alginates of brown algae exist both within the cell wall and in the intercellular substance. Their part in the cell wall may be as high as 40 per cent of the dry matter.

[0099] Sulfonated Polysaccharides are polysaccharides whose monomers are esterized to sulfuric acid residues and are moreover partially methylated. They have been detected in nearly all marine algae. They occur partially in the cell wall itself and partially in the intercellular substance. Sulfonated galactanes are typical for many red algae, depending on their origin are they called agarose, carrageenan, porphyran, furcelleran and funoran. The extraordinary binding types of agarose and carrageenan lead to specific tertiary structures.

[0100] Further Cell Wall Compounds. A number of algae contain mineral cell wall components. Silicon, for example, is the main component of the diatom shell, though it occurs also in the cell walls of other groups of algae. Silicon-containing scales enclose the chrysophyt Synura. Silicon is a cell wall component in some brown algae and in the green algae Hydrodictyon. Diatoms take silicon up as silicate. The process is dependent on oxygen and temperature, it consumes energy and it is dependent on the presence of divalent sulphur.

[0101] Sporopollenin is an isoprene derivative. It is a component of pollen cell walls, but was also detected in the walls of some green algae (Chlorella, Scenedesmus, etc.).

[0102] Calcium encrustations of cell walls have on several occasions been described. They seem to be especially common in species of tropical, marine waters. Some species participate in reef formation. Calcium is generally deposited as calcium carbonate. Calcium carbonate occurs in two different crystalline states: calcite and argonite. Calcite is produced in the walls of some groups of red algae and in charophycea, while argonite is produced by some green (Acetabularia, etc.), brown and red algae.

[0103] In addition to the homopolymers described above, heteropolymers also exist in many algal groups.

[0104] Biofuel Production Using Algae [0105] Herein, solvating, dissolving, or lysing algae is described. For clarity, lysing algae is herein described for fuel component isolation or the breaking apart of algae. Lysing refers to breaking of the cell wall. However, the techniques described herein apply to release of any algae constituent contained within an algae cell wall for use in any application, such as in energy production, to acquire starting reagents, or in the cosmetic or pharmaceutical industries.

[0106] Algae Lysing

[0107] Referring now to FIG. 3 and FIG. 4, use of an ionic liquid 100 in the breakdown of an algae cell 310 is contrasted with use of elevated temperature, elevated pressure, sonication, and/or use of radiation in the disruption of an algae cell.

[0108] Referring now to FIG. 3, it is observed that pressure, temperature, and radiation inputs are dissipated by water about the algae cell before the algae cell is disrupted. Using pressure, temperature, and/or radiation, considerable energy is required to disrupt the cell 310. The dissipation of energy means that more energy must be put in to disrupt the cell or the matrix holding the cell needs to be treated to remove the dissipating medium. In yet another embodiment, use of an ionic liquid 100 is combined with any of pressure, temperature, and radiation inputs as the ionic liquid 100 lowers the required energy levels and the combined techniques optionally enhance processing of the algae 510.

[0109] In stark contrast, referring now to FIG. 4, in a process 400 the ionic

liquid 100 contacts the cell and lyses, dissolves, degrades, or solvates the cell, which releases the cell constituents to the surrounding liquid. In the lysing process, the ionic liquid interacts with the cell walls and pulls the cell wall components into solution forming a lysed cell 315. The process of lyses or dissolution using the ionic liquid 100 has a small energy barrier compared to traditional cell disruption processes. Traditional cell disruption processes require about a mega Joule per kilogram of algae (MJ kg). In stark contrast, the inventor has discovered processing techniques that require about a kilo Joule per kilogram of algae to lyse the algae cell wall. Generally, the traditional MJ/kg techniques are replaceable with the techniques taught herein, which require less than about 1000 kJ/kg to as low as less than about 0.1 kJ/kg. , less than about 500, less than about 400, less than about 300, less than about 200, less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, less than about 50, less than about 40, less than about 30, less than about 20, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, less than about 1, less than about 0.5 or less than about 0.1 kJ/kg. Accordingly, the dissolution process using the ionic liquid 100 is operable at mild temperatures, such as at or below about 150° C to below about 80° C. An ionic liquid 100 with a melting point of less than 110° C is referred to herein as a low melting point ionic liquid. An ionic

liquid 100 with a melting point of less than 90° C is referred to herein as a very low melting point ionic liquid. An ionic liquid 100 with a melting point of less than 75° C is referred to herein as an ultra low melting point ionic liquid. Use of an ionic liquid 100 with a lower melting point requires less energy to maintain the ionic liquid 100 at its melting point, which can aid in energy efficiency of the process 500 of converting algae 510 to product 530. Further, the dissolution process using the ionic liquid is operable at low pressures, such as at about one atmosphere, though running the process under elevated pressures is an alternative embodiment. An ionic liquid with a melting point of greater than 150° C is referred to herein as a high melting point ionic liquid. It should be understood that the melting point of an ionic liquid 100 decreases sharply in the presence of water. For clarity, herein the melting point of the ionic liquid 100 refers to the melting point of the pure ionic liquid. For example, a pure ionic liquid with a melting point of 150° C may be fully in liquid form at a lower temperature, such as at 125° C, at 100° C, at 75° C or at 50° C.

However, for clarity and definiteness of description, the ionic liquid melting point of the active ionic liquid refers to the pure ionic liquid melting point.

[0110] Algae Products

[0111] Referring now to FIG. 5, a process of product creation 500 from algae 510 by the dissolution of algae 510 to release cell components 520, which are converted directly and/or directly into products 530 is described. In a process 515, the algae 510 is dissolved by the ionic liquid 100, as described supra, resulting in cell components, such as hydrogen, lipids, hydrocarbons, carbohydrates, and biomass, to be released into the solution. Subsequently, in a process 525, the cell components are separated and/or transformed into products 530, such as hydrogen, methane, ethanol, biodiesel, green diesel, and/or biosynthetic liquid fuel, oil, triglyceride, free fatty acids, water soluble sugars, water insoluble carbohydrates, protein, hydrogen, methane, ethanol, biodiesel, green diesel, biosynthetic liquid fuel, gasoline, kerosene, or any combination thereof.

[0112] Processes described herein operate on single cell algae species, multi-cellular species, wild type algae, engineered algae, and combination thereof.

[0113] In another embodiment, the use of the ionic liquid 100 to release cell components of an algae having a silica layer in the cell wall is described. In one example, Scenedesmus, an algae, was analyzed and treated with an ionic liquid 100. The cell wall of Scenedesmus has an elaborate chemical and three-dimensional structure that is uncharacteristic compared to single-cell microalgae such as Chlorella or Chlamydomonas. The cell wall of

Scenedesmus includes three layers. An inner layer is a thick layer of cellulose microfibrils bounding the coenobium. The middle layer is very thin and is made of silica. The outer layer is a very elaborate pectic layer having a system of hexagonal nets on the surface and is supported by a system of cylindrical props. At various places, the pectic layer is interrupted by circular openings. Tests reported here show that the ionic liquid 100 dissolved both the exopolysaccharide pectic layer and the inner cellulosic layer, but left the middle layer, which is a silica layer, intact. However, the silica layer is discontinuous and all or most of the algae organelles were washed out of the algae cell in the presence of the ionic liquid 100 in a period of less than about twenty minutes, which demonstrates effective lysing of the algae cell for extraction of algae constituents for fuel production. Generally, the use of an ionic liquid 100 to lyse or dissolve algae cell walls and internal components is found effective for diatoms, which are a major group of eukaryotic photosynthetic algae having large potential for lipid production. The lysing leaves the silica layer behind, but substantially lyses or dissolves the rest of the cell wall, having carbohydrates and proteins, and also lyses or dissolves elements inside the cell wall.

[0114] Reactor

[0115] Referring now to FIG. 6, an overview of a reactor system 600 is provided. Any of the reactor elements or subsystems described, infra, are optionally used independently. However, the reactor elements and subsystems are preferably used together to process algae 510 to form products 530. [0116] Generally, the reactor system 600 processes algae 510 to products 530.

Algae 510 is heated in a heat exchanger 610 driving off some water 615. The heated and moist algae is subsequently lysed in a lysing reactor 620 using an active ionic liquid 100. Optionally, components of the lysing reactor 620 are heated and/or agitated 625. The ionic liquid 100 is optionally and preferably recovered 630, such as by separation, as are components dissolved in the ionic liquid. Energy 635 drives the lysing reactor 620 and/or the recovery 630. Products, 530, such as biofuels 532, biodiesel and/or bioalcohols are separated from the lysing reactor and/or ionic liquid recovery chamber. For clarity, subsystems of the reactor system 600 are described, FIGS. 7, 8,9, and 10.

[0117] Algae Harvesting, Dewatering, and Drying

[0118] Referring now to FIG. 7, in a process 700, algae 500 is dewatered and/or heated. The initial processing of algae 700 prior to placement in the reactor system 600 and/or after placing the algae within the reactor system 600 is further described. In a first process 710, the algae is harvested. In a second process 720, the harvested algae is preferably partially dewatered. Partial dewatering refers to increasing the solid concentration in the water from a concentration of about 0.1% to about 0.15%, typical of a photoreactor or pond

suspensions, up to a solid concentration of between about 5% to about 15%. Dewatering includes a low energy input step, such as settling or filtration. In a third process 730, the dewatered algae is dried, such as in the heat exchanger 610. Herein, increasing the solid concentration of algae above about 10% is referred to as drying. Optionally, the harvested algae 510 is placed into the heat exchanger 610, either before or after dewatering. In the drying process 730, energy is used to partially evaporate water about the incoming algae 510. In one partial drying example, in a process 735, steam at about 100° C and at about one atmosphere pressure is driven into the heat exchanger, which results in partial evaporation of water in the heat exchanger 610. The evaporated water is

extracted 615 yielding water at about 100° C. Since the extracted water 615 is about 100° C, the heated water is optionally and preferably used as input water in a fourth process 735 of generating steam, which is input into the heat exchanger 610 as one energy supply of the aforementioned energy input into the heat exchanger. Optionally and typically in combination, an outside water source 738 is also converted to steam in the fourth process 735. Optionally, contents inside the heat exchanger 610 are continuously, periodically, and/or intermittently stirred or agitated, which facilitates the drying step 730. While, for clarity of presentation, steam is described as the energy source to the heat exchanger 610 in the drying process 730, any form of energy is optionally used to dry the algae, such as solar, photonic, radiant, convective, and/or electrical. In a fifth process, 740, the resulting heated and concentrated algae is moved to a lysing reactor 620, described infra. Although not preferred, the lysing step described, infra, optionally occurs in the heat exchanger 610.

[0119] Lysing

[0120] Lysing of algae 800 in the reactor system 600 is further described. The now heated and concentrated algae is moved to the lysing reactor 620, which contains pure ionic liquid or active ionic liquid 100. Once algae is introduced into the lysing reactor, any pure ionic liquid is converted, as described supra, into active ionic liquid. The active ionic liquid 100 lyses the algae, as describes supra.

[0121] Lysing of algae 510 with an active ionic solution 100 typically occurs very rapidly, such as within a second. However, in a lysing reactor 620, lysing time may be extended to enhance ionic liquid 100 / algae 510 contact to ensure lysing, to time periods of between about 1 minute to about 200 minutes..

[0122] The contents of the lysing reactor 620 are optionally continuously, periodically, and/or intermittently stirred and/or agitated to facilitate the lysing and/or dissolution.

[0123] Heat is optionally input into the lysing reactor 620. Preferably, the lysing reactor 620 is maintained via heat input at between about 80° C to about 120° C. Optionally, the lysing reactor 620 is maintained at a higher temperature using any heat source, such as steam or microwave radiation. The heat of the lysing reactor 620 is optionally maintained and/or initially set at a temperature within about 10° C to about 100° C of the melting point of the ionic liquid 100.

[0124] The pressure placed on the contents of the lysing reactor 620 is preferably about one atmosphere. However, the contents of the lysing reactor 620 are optionally maintained at higher or lower pressures, between about 0.5 to about 3, or more atmospheres of pressure. [0125] In various embodiments, the algae 510 is lysed using the ionic solution 100 with any combination of lysing time, agitation, heat, and/or pressure.

[0126] Product Separation

[0127] Referring now to FIG. 8, processing elements 800 of the lysing reactor 620 are described. After lying the algae 510, the lysing reactor 620 contains a plurality of lysing reactor constituents 910, such as one or more of: triacylglycerides, free fatty acids, carbohydrates, proteins, cellulose, water, the ionic liquid 100, or any other pond water or algae constituents. In a task 820, the lysing reactor constituents 910 are separated or partially separated, such as into a first phase 830 or a first state and into a second phase 840 or a second state.

[0128] In a first example, the separation 820 uses chemical forces to separate the lysing reactor constituents 910, where the first phase 830 are polar compounds and the second phase 840 are non-polar compounds. Referring now to FIG. 9, the first example is illustrated figuratively and the lysing reactor constituents 910 is an emulsion of fatty elements in the ionic liquid, which spontaneously separates into an upper non-polar solution, such as fats, and a lower polar solution, such as ionic liquid 100 and water. Still referring to FIG. 9, the separation is optionally performed in the lysing reactor 620 and/or in a separate separation container or system 920.

[0129] In a second example, the separation 820 uses magnetic forces to separate the lysing reactor constituents 910, where the first phase 830 contains magnetically susceptible constituents and the second phase 840 are non-magnetic constituents.

[0130] In a third example, the separation step 820 uses density differences to separate the lysing reactor constituents 910, where the first phase 830 are lower density constituents and the second phase 840 are higher density constituents. In practice, the separation

step 820 separates the constituents into any number of phases or states, not just the illustrated two phases 830,840. Additional separation methods 820 are described, infra.

[0131] Generally, solutions, solutions with suspended particles, biphasic solutions, multiphasic solutions, and solutions with settled solids exist in one or more of the heat exchanger 610, lysing reactor 620, collection container or stream 1030, and/or an ionic liquid separator 1040, which are collectively referred to herein as ionic liquid containing solutions. Optionally, one or more of a number of separation techniques 820 are used to process any of these ionic liquid containing solutions to at least partially separate out or extract a contained constituent, such as triacylglycerides, free fatty acids, ionic liquid, carbohydrates, proteins, water, cellulose, or other pond water constituent. Separation techniques include: settling and decantation; precipitation with an organic solvent, described supra; formation of an aqueous biphasic solution via addition of a kosmotropic salt, described supra; a liquid-liquid extraction, described supra; foam fractionation or air flotation rising select groups to an upper surface with subsequent removal by skimming; electrochemical separation; dialysis; electrophoresis; electrofiltration; crystallization;

distillation; thermal conversion and separation; enzymatic conversion and separation;

adsorption; chromatography; moving a mixture dissolved in a mobile phase through a stationary phase; centrifugation; ultrafiltration; flocculation; stripping; a separation using a magnetic field to extract magnetically susceptible solute molecules or structures; and a low Energy process requiring less than about IMJ/kg and as low as below about 1 kJ/kg of total solute, and could be less than about 500 kJ/kg, less than about 100 kJ/kg, or less than about lO kJ/kg.

[0132] To further clarify, additional separation techniques 820 are described, infra. [0133] Specific gravity and/or solubility separation

[0134] Lipids have about 0.2 to about 0.4 lower specific gravities than l-propyl-3- methylimidazolium chloride. In addition, l-propyl-3-methylimidazolium chloride is about lipophilic. Hence lipids spontaneously phase-separate from the ionic liquid 100. It is clear that as the cell wall is removed, lipid vesicles present inside the cell are freed, forming emulsion droplets in the ionic liquid 100, which can spontaneously coalesce to form one layer or phase 830 at the top of the reaction mixture and the ionic liquid will form a second layer or phase 840 below the spontaneously formed lipophilic layer. Other cell constituents are either dissolved in the ionic liquid 100 or precipitate out of solution.

[0135] Liquid/Liquid Extraction Based Separation [0136] In some cases, the emulsion 910 is stable, for instance due to interaction with surfactants also present in the cell. If lipid emulsions are sufficiently stable, a liquid-liquid (L-L) extraction step for the lipid is optionally used. In this example, the ionic liquid lysate solution from the dissolution and/or lysis is treated with a liquid-liquid extraction step. In the liquid-liquid extraction step, the liquid extractant is brought into contact with the emulsified lipid to extract the lipid components from the ionic liquid based solution. In one case, the extractant is the same lipid produced by algae due to its poor miscibility in ionic liquid, which is much less than 1%, and is an exact polarity match resulting in maximizing extraction efficiency. Optionally, any lipophilic solution is used as the extractant, such as an organic solvent. Similarly, the extractant is optionally hydrophobic and is used to extract the ionic liquid 100. For example, the extractant is a second distinct ionic liquid.

[0137] A specific example of lysing of algae 510 and product separation is further described. In practice, the lysing and separation steps are optionally done on an industrial scale in a continuous flow system, an intermittent flow system, and/or in a batch system. Referring now to FIG. 10, for clarity, an example of a separation processes using laboratory type equipment is illustrated figuratively. As described, supra, the lysing

reactor 620 contains the heated and partially dried algae 510 and the ionic liquid 100. Upon lysing and/or dissolution of the algae, the lysing reactor contains triacylglycerides, free fatty acids, carbohydrates, proteins, cellulose, water, and/or the ionic liquid 100. Additional components of lesser concentration are optionally present in the lysed solution 315. Herein, a triacylglyceride (TAG) or triacylglycerol is an ester composed of a glycerol bound to three fatty acids, which is a constituent of algae, vegetable oil, and/or animal fats. In the lysing reactor, or any container to which the lysed components are transferred, the lysed solution spontaneously separates based on density, such as into two or more layers or phases.

Generally, the ionic liquid 100 and/or certain constituents settle toward the bottom to form a lower layer 1010, which in an example of a first phase 830, and hydrophobic and/or ionic liquid phobic constituents rise toward the top of the reaction mixture to form an upper layer 1020, which is an example of a second phase 840. A phase or density separation or partial separation layer 1015 forms a partial boundary between the lower layer 1010 and upper layer 1020. In practice, multiple density layers are optionally formed. Examples of constituents in the upper layer include the triacylglycerides and/or the fatty acids. Examples of constituents in the lower layer include the ionic liquid(s) 100, the carbohydrates, the proteins, the cellulose, and the water. It is observed that the lower density constituents in the upper layer 1020 are readily removed, such as via an overflowl022, via a mechanical pump pulling from the upper layer 1020, via decanting, or by any automated or manual removal of at least a portion of the upper layer 1020 to a collection container or stream 1030, which contains lower density constituents 1025, such as the triacylglycerides and/or free fatty acids. Similarly, the higher density constituents in the lower layer 1010 are readily removed, such as via a mechanical pump 1030 pulling from the lower layer 1010, via decanting and keeping the lower layer 1010, or by and automated or manual removal of at least a portion of the lower layer 1010 to a second container, stream, or ionic liquid separator system 1040, which contains higher density constituents 1045, such as the ionic liquid 100,

carbohydrates, proteins, cellulose, and/or water. The ionic liquid concentration and product recovery system 630 is optionally continuously run by providing a semi-dry algae stream 1050 to the lysing reactor 620. The solution in the lysing reactor is optionally continuously, periodically, or discontinuously stirred and/or agitated with a stirrer 1012. Components in the upper layer collection container or stream are optionally further processed, such as by transesterification to form biodiesel. Notably, the lower density constituents 1025 are optionally available in the collection container or stream 1030 at about 100° C, which is an adequate starting temperature for the transesterification process.

[0138] Ionic Liquid Recovery

[0139] The ionic liquid separator 1040 of the ionic liquid separation and/or recovery process 630 is further described. Generally, any of the separation techniques, described supra, used in separation of the lysing reactor 620 constituents 910 are used in the ionic liquid separation and/or recovery process 630, in which generally: ionic liquid is recovered; and/or carbohydrates, proteins, cellulose, and/or water are separated.

[0140] Optionally, the separation step 820 occurs prior to or at the same time as the ionic liquid separation and/or recovery process 630. Examples are provided of processes used in the ionic liquid separator step 740 as part of the ionic liquid separation and/or recovery process 630.

[0141] In a first example of ionic liquid recovery, the ionic liquid 100 is recovered through the addition of salt. In one case, a kosmotropic salt is added to the collected ionic liquid containing solution. Particularly, separation of the ionic liquid 100 is achieved through the formation of an aqueous biphasic system (ABS). Certain ionic liquids 100, such as l-propyl-3-methylimidazolium chloride, form an aqueous biphasic system upon addition of a salt, such as any of K ₃ PO4, KOH, K2CO ₃ , Na ₂ HP0 ₄ , and a ₂ S ₂ 03. The process is referred to herein as salting out. In salting out, addition of a suitable concentrated salt introduces electrostatic and/or hydrophobic forces that spontaneously separate the solution into an ionic liquid-rich and a salt-rich phase. These two phases, in turn, separate-out species present in the original solution before salting out. For instance, substances more soluble in water tend to stay in the water phase and substances more soluble in the ionic liquid tend to stay in the ionic liquid phase. Optionally, the exploitation of aqueous biphasic system formation on the ionic liquid/water solutions takes place in liquid-liquid extraction steps, as described supra. Optionally, the extraction of the ionic liquid 100 is achieved using ion exchange extraction and/or ion pair extraction.

[0142] In a second example, an organic solvent is used to precipitate a constituent of the ionic liquid containing solution, such as precipitation of carbohydrates, celluloses and/or proteins. The removal of the precipitate results in a renewed or concentrated ionic liquid 100. Examples of organic solvents used to precipitate a liquid constituent include: methanol, methanol/water mixtures, chloroform, dichloromethane, or ethyl acetate.

Optionally, this, or any of the extraction techniques described herein, are performed in a continuous solid-liquid or liquid-liquid extraction step, such as with the volatile organic or volatile organic chemical input.

[0143] In a third example, a physical property of the particular ionic liquid 100 to be separated is used in the separation process.

[0144] Harvesting Algae Biofuel

[0145] In yet another embodiment, use of an ionic liquid to aid in recovery of an algae component is combined with any other algae constituent extraction technique, such as use of solar, ultrasound, elevated temperature, elevated pressure, sonication, microwave heating, and/or radiation. [0146] Referring now to FIG. 11, a reactor system 1100 for converting algae 510 to an algae component 1130 and/or product 532 is illustrated. One or more of the ionic liquid 100, and extractant 1110, water 615, energy 635, and heat and agitation 625 are added to the reaction chamber 1120 and at least one algae component is extracted.

[0147] Although the invention has been described herein with reference to certain preferred embodiments, one skilled in the art will readily appreciate that other applications may be substituted for those set forth herein without departing from the spirit and scope of the present invention. The various embodiments described above can also be combined to provide further embodiments.

[0148] All publications including all of the U.S. patents, all of the U.S. patent applications, all of the foreign patents, all of the foreign patent applications and all of the non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. No admission is made that any reference constitutes prior art.

[0149] The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1

GENERAL PROCEDURES

[0150] This example illustrates general procedures used to carry out the Examples 2 through 9.

[0151] Algae Preparation:

[0152] Microscopic algae were grown in laboratory conditions to maintain a controlled source for cells used in all subsequent experiments. Culture batches consisted of 2-Liter Erlenmeyer flasks with 1 L of standard culture media containing nitrates, phosphates and other minerals necessary or conducive for growth and diluted in ultra-filtered and deionized water. Broad-spectrum fluorescent light bulbs placed above and sideways relative to the culture flasks provided continuous illumination for photosynthesis. A two-hole rubber stopper capped each culture flask with glass tubes through each hole. A small air pump connected by flexible Tygon tubing to one glass tube provided bubbling of ambient air through the culture to deliver sufficient C0 ₂ and maintain cells suspended. The other glass tube had a cotton insert to allow air out while preventing contamination. Cultures were maintained between 25 and 30 C and greening peaked 1 to 2 weeks after inoculation. Before harvesting, cultures were inspected under a brightfield microscope for signs of contamination (bacterioplankton) and discarded if contaminated (~ 1 out of 10 cultures). Cultured algae were allowed to settle in a beaker for 12 hours, and supernatant media was removed by vacuuming. Recovered cells were washed by three rounds of suspension in 10- fold ultra-filtered and deionized water by volume, followed by centrifugation and removal of supernatant. Typical yields were ~ 1 g/L and final harvested cells had ~ 90% water.

[0153] Lysis Experiments:

[0154] In preparation for each experiment, 50 μΐ., of harvested cells were sampled, diluted 10-fold in water, and inspected under a microscope to: i) certify the absence of

fragmentation, and ii) set baselines of cell number density and appearance for a given culture batch. Flat-bottom, 4-mL glass reaction vials were loaded with 0.50 or 1.0 mL of ionic liquid heated to a set temperature on a custom-made heating block designed to minimize temperature gradients and provide accurate temperature monitoring. Reaction temperatures from 70 to 140 °C were investigated in 10-degree increments. Harvested cells were added to reaction vials at 1 :20 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Reaction vials were left partially capped at the set temperature for up to 2 hours. Reaction aliquots were sampled at varying intervals for microscopic visualization. At the end of the reaction, vials were allowed to equilibrate with room temperature. The ionic liquid lysate mixture was extracted by adding an equal volume of chloroform and vortexing for 2 min. The result was centrifuged at ~ 2000 x g for a few minutes to accelerate phase separation. The chloroform extract was kept refrigerated for chemical analysis.

[0155] Microscopic Visualization:

[0156] Reaction aliquots of 50 μΐ., were quickly diluted 5-fold in water to quench the reaction and precipitate any dissolved cells, cell walls, or other remains out of solution. Dilution also lowered the refractive index of the surrounding medium to enhance contrast. All visualizations were done in a brightfield microscope (Fisher Scientific). After inspection and annotation, cell number densities were estimated from recorded images sampled at random positions throughout the microscope slide.

[0157] Mass Spectra Analysis:

[0158] ESI-TOF. Mass spectra were acquired by an electrospray ionization quadrupole time-of-flight (ESI-TOF)unit (Q-TOF Premier, Waters Corporation). Samples either in chloroform or methanol were injected at a flow rate of 50 μΙ7ηιίη with a mobile phase of acetonitrile, or 1 : 1 acetonitrile in water. The ion source voltage was set to 3 KV for positive and negative ion acquisitions modes. Also, the sampling cone was at 37 V and the extraction cone was at 3 V. For both modes, the source and desolvation temperatures were maintained at 120 °C and 225 °C, respectively. The desolvation gas flowrate was 200 L/h. TOF scanning was set from mlz = 100 to 1400 at 1 s with 0.1 s inter-scan delay using extended dynamic range acquisition with centroid data format. Putative identification of unknowns relied on primary ion peaks compared to the LipidMaps library

( ^' http://www.lipidmaps.org/).

[0159] MALDI-TOF. Mass spectra were also acquired by a matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) unit (Microflex LT, Bruker Daltonics). For sample preparation, one microliter of chloroform extract was pipetted onto the target plate in triplicate for drying at room temperature. The samples and calibration standard were overlaid with 1 \L of saturated a-cyano-4-hydroxycinnamic acid (HCCA) solution in 50% acetonitrile and 2.5% trifluoroacetic acid (v/v) prepared daily. Ablation and soft ionization was carried out by Nitrogen laser (337.1 nm) pulsations at 250 μΐ. Detection occurred in linear mode, summing over 500 laser pulsations in both positive and negative ion reflector modes. The acceleration voltage was set to 25 kV, giving good resolution over a m/z range between 50 and 1400. Bruker Daltonics proprietary noise reduction software was applied. Putative identification of unknowns relied on primary ion peaks and comparison to the LipidMaps library (http://www.lipidmaps.org/). EXAMPLE 2

REACTION BETWEEN WET NEOCHLORIS PSEUDOSTIGMATA AND 1-BUTYL- 3-METHYLIMIDAZOLIUM CHLORIDE

[0160] This example illustrates washed harvested N. pseudostigmata cells (Figure 15A) with ~ 90% water content were mixed with l-butyl-3-methylimidazolium chloride, or

[BMIMJC1. Reaction temperatures from 70 to 140 °C were investigated in 10-degree increments. Harvested cells were added to reaction vials at 1 :20 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Below 80 °C, cells appeared intact regardless of mixing ratio and residence time. Above 100 °C over ~ 95% of cells vanished within 15 minutes. To certify whether cells walls were lysed and not simply dissolved, reaction aliquots were diluted 5-fold in water to precipitate celluloses. The absence of cells confirmed lysing (Figure 15B). Freed lipid droplets could be distinguished resting against the top-oriented glass coverslip. Cooled lysate was extracted with neat chloroform and its mass spectra confirmed the presence of lipids. Substituting ionic liquid for water in otherwise identical experiments did not result in extraction, confirming the role of the ionic liquid (Figure 16). Spectra in the 50 to 1400 m/z range and peak identification revealed the whole range of lipids expected from algae, starting with low MW (C3-C4) acids and fatty acids (CI 2) to high molecular weight triacylglycerides and glycero lipids (Figure 16).

EXAMPLE 3

REACTION BETWEEN WET CHLAMYDOMONAS REINHARDII AND 1-BUTYL- 3-METHYLIMIDAZOLIUM CHLORIDE

[0161] This example illustrates washed harvested C. reinhardii cells (Figure 17A) with ~ 90% water content were mixed with l-butyl-3-methylimidazolium chloride, or [BMIMJC1. Reaction temperatures from 70 to 140 °C were investigated in 10-degree increments.

Harvested cells were added to reaction vials at 1 :20 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Below 80 °C , cells appeared intact regardless of mixing ratio and residence time. Approaching 100 °C, reactions longer than 15 minutes resulted in the appearance of round dark spots inside cells, possibly dispersed nucleic material or another altered organelle. Above 100 °C over ~ 95% of cells vanished within 15 minutes. To certify whether cells walls were lysed and not simply dissolved, reaction aliquots were diluted 5-fold in water to precipitate celluloses. The absence of cells confirmed lysing (Figure 17B). Freed lipid droplets could be distinguished resting against the top-oriented glass coverslip.

EXAMPLE 4

REACTION BETWEEN WET CHLORELLA PYRENOIDOSA AND CHLORELLA VULGARIS AND l-PROPYL-3-METHYLIMIDAZOLIUM CHLORIDE

[0162] This example illustrates washed harvested C. pyrenoidosa and C. vulgaris cells (Figure 18A) with > 80% water content were mixed with l-propyl-3-methylimidazolium chloride, or [PMIMJC1. Reaction temperatures from 70 to 140 °C were investigated in 10- degree increments. Harvested cells were mixed and added to reaction vials at 1 :20 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Below 80 °C, cells appeared intact regardless of mixing ratio and residence time. Approaching 100 °C, reactions longer than 15 minutes resulted in the appearance of round dark spots inside cells, possibly dispersed nucleic material or another altered organelle. Above 100 °C over ~ 95% of cells vanished within 45 minutes. To certify whether cells walls were lysed and not simply dissolved, reaction aliquots were diluted 5- fold in water to precipitate celluloses. The absence of cells confirmed lysing (Figure 18B). Freed lipid droplets could be distinguished resting against the top-oriented glass coverslip. Cooled lysate was extracted with neat chloroform and its mass spectra confirmed the presence of lipids.

EXAMPLE 5

REACTION BETWEEN WET CHLORELLA PYRENOIDOSA AND CHLORELLA VULGARIS AND l-ALLYL-3-METHYLIMIDAZOLIUM CHLORIDE

[0163] This example illustrates washed harvested C. pyrenoidosa and C. vulgaris cells (Figure 19A) with > 80% water content were mixed with l-allyl-3-methylimidazolium chloride, or [AMIM]C1. Reaction temperatures from 70 to 140 °C were investigated in 10- degree increments. Harvested cells were mixed and added to reaction vials at 1 :20 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Below 80 °C, cells appeared intact regardless of mixing ratio and residence time. Approaching 100 °C, reactions longer than 15 minutes resulted in the appearance of round dark spots inside cells, possibly dispersed nucleic material or another altered organelle. Above 100 °C over ~ 95% of cells vanished within 45 minutes. To certify whether cells walls were lysed and not simply dissolved, reaction aliquots were diluted 5- fold in water to precipitate celluloses. The absence of cells confirmed lysing (Figure 19B). Freed lipid droplets could be distinguished resting against the top-oriented glass coverslip. Cooled lysate was extracted with neat chloroform and its mass spectra confirmed the presence of lipids.

EXAMPLE 6

REACTION BETWEEN WET CHLORELLA PYRENOIDOSA AND l-BUTYL-3- ME THYLIMID AZOLIUM CHLORIDE

[0164] This example illustrates washed harvested C. pyrenoidosa cells (Figure 20A) with ~ 90% water content were mixed with l-butyl-3-methylimidazolium chloride, or

[BMIMJC1. Reaction temperatures from 70 to 140 °C were investigated in 10-degree increments. Harvested cells were added to reaction vials at 1 :20 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Below 80 °C, cells appeared intact regardless of mixing ratio and residence time. Approaching 100 °C, reactions longer than 15 minutes resulted in the appearance of round dark spots inside cells, possibly dispersed nucleic material or another altered organelle. Above 100 °C over ~ 95% of cells vanished within 15 minutes. To certify whether cells walls were lysed and not simply dissolved, reaction aliquots were diluted 5-fold in water to precipitate celluloses. The absence of cells confirmed lysing (Figure 20B). Freed lipid droplets could be distinguished resting against the top-oriented glass coverslip. Cooled lysate was extracted with neat chloroform and its mass spectra confirmed the presence of lipids (Figure 21). Analysis showed no appreciable degradation or derivatization had occurred compared to a Bligh & Dyer extract of the same batch, and somewhat higher yields (Figure 21). EXAMPLE 7

REACTION BETWEEN WET CHLAMYDOMONAS MOEWUSII AND l-BUTYL-3- ME THYLIMID AZOLIUM CHLORIDE

[0165] This example illustrates washed harvested C. moewusii cells (Figure 22A) with ~ 95% water content were mixed with l-butyl-3-methylimidazolium chloride, or [BMIMJC1. Reaction temperatures from 70 to 140 °C were investigated in 10-degree increments. Harvested cells were added to reaction vials at 1 :50 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Below 80 °C, cells appeared intact regardless of mixing ratio and residence time. Approaching 100 °C, reactions longer than 15 minutes resulted in the appearance of round dark spots inside cells, possibly dispersed nucleic material or another altered organelle. Above 100 °C over ~ 95% of cells vanished within 5 minutes. To certify whether cells walls were lysed and not simply dissolved, reaction aliquots were diluted 5-fold in water to precipitate celluloses. The absence of cells confirmed lysing (Figure 22B). Freed lipid droplets could be distinguished resting against the top-oriented glass coverslip.

EXAMPLE 8

REACTION BETWEEN WET CHLOROCOCCUM HYPNOSPORUM AND 1- BUTYL-3-METHYLIMIDAZOLIUM CHLORIDE

[0166] This example illustrates washed harvested C. hypnosporum cells (Figure 23A) with ~ 90% water content were mixed with l-butyl-3-methylimidazolium chloride, or [BMIMJC1. Reaction temperatures from 70 to 140 °C were investigated in 10-degree increments. Harvested cells were added to reaction vials at 1 :20 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Below 80 °C, cells appeared intact regardless of mixing ratio and residence time. Approaching 100 °C, reactions longer than 15 minutes resulted in the appearance of round dark spots inside cells, possibly dispersed nucleic material or another altered organelle. Above 100 °C over ~ 95% of cells vanished within 5 - 15 minutes. To certify whether cells walls were lysed and not simply dissolved, reaction aliquots were diluted 5-fold in water to precipitate celluloses. The absence of cells confirmed lysing (Figure 23B). Freed lipid droplets could be distinguished resting against the top-oriented glass coverslip. EXAMPLE 9

REACTION BETWEEN WET SCENEDESMUS QUADRICA UDA AND l-BUTYL-3- ME THYLIMID AZOLIUM CHLORIDE

[0167] This example illustrates washed harvested S. quadricauda cells (Figure 24A) with ~ 95% water content were mixed with l-butyl-3-methylimidazolium chloride, or

[BMIMJC1. Reaction temperatures from 70 to 140 °C were investigated in 10-degree increments. Harvested cells were added to reaction vials at 1 :20 to 1 : 1 volume ratios and hand-tumbled gently, causing water and algae to disperse quickly throughout the ionic liquid. Below 80 °C, cells appeared intact regardless of mixing ratio and residence time. Approaching 100 °C, reactions did not affect cells appreciably. Above 100 °C and after 30 minutes, cell outlines remained intact but cell contents vanished. S. quadricauda is a silicaceous species, that is, its cell wall contains a layer of mineral silica, similar to diatoms and other families of algae. In conclusion, the ionic liquid was able to dissolve all cell wall components except for the silica layer. However, because the silica layer is porous, cell contents were similarly extracted. This was confirmed, as before, by diluting reaction aliquots 5-fold in water to precipitate celluloses. The absence of other fragments confirmed the cell contents were extracted even though the silicaceous part of the cell wall survived (Figure 24B).

[0168] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.