Methods of recovering oil from microorganisms

Abstract

To provide methods of recovering oil from microorganisms.SOLUTION: The methods of recovering oil described herein include contacting a population of microorganisms with one or more enzymes under conditions that cause disruption of the microorganisms, concentrating the disrupted microorganisms, and extracting lipids from the disrupted microorganisms at high temperature in the presence of a salt and in the absence of solvent. The methods are useful, for example, in obtaining nutritional oils and / or lipid biofuels.SELECTED DRAWING: Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 918,886, filed December 20, 2013, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Oil can be recovered from microorganisms (such as microalgae) using wet or dry extraction methods. In dry extraction, the microorganisms are typically harvested and dried before the oil is extracted. However, drying is an expensive and energy-intensive process. Also, if the oil is rich in polyunsaturated fatty acids (PUFAs) (e.g., for food and nutritional supplement applications), the process can cause significant oxidation of the PUFAs due to the high temperatures involved in drying.

[0003] Furthermore, dry extraction methods for recovering oil from microorganisms are typically performed with organic solvents (e.g., hexane) and require coupling with mechanical cell disruption methods to achieve adequate oil yields. However, mechanical disruption methods are expensive and energy-intensive, while organic solvents are flammable and toxic and must be removed from the final oil product.

[0004] [Summary of the Invention] Provided herein are wet, solvent-free methods for recovering oil (i.e., lipids) from microorganisms. The methods are useful, for example, for obtaining nutrient oils and / or lipid biofuels. The oil recovery methods described herein can optionally be carried out as integrated bioprocesses (i.e., "one-pot" methods).

[0005] The methods for recovering lipids from a population of microorganisms described herein include contacting a population of microorganisms with one or more enzymes under conditions that cause disruption of the microorganisms, concentrating the disrupted microorganisms, and extracting lipids from the disrupted microorganisms at elevated temperatures in the presence of salt and in the absence of an organic solvent. Optionally, the contacting step occurs in a fermentation medium. Optionally, the contacting step can be performed at a pH within 5 to 8.5 (e.g., about 8). Optionally, the contacting step can be performed at a temperature within about 50°C to about 70°C. The contacting step can be performed for 1 to 20 hours (e.g., 1 to 8 hours or 4 hours), inclusive.

[0006] Optionally, the enzyme used in the contacting step is a protease. Optionally, the enzyme is Alcalase 2.4L. Optionally, the enzyme is at a concentration of about 0.001% to about 0.4% volume / volume (inclusive) (e.g., between 0.05 and 0.2%, or about 0.05, 0.1, or 0.2%). Optionally, the contacting step can be carried out at 55°C for 1 to 8 hours in the presence of 0.05 to 0.2% enzyme (inclusive). For example, the contacting step can be carried out at 55°C for 4 hours in the presence of 0.1% enzyme. Optionally, the contacting step is carried out at 55°C for 6 hours in the presence of 0.05% enzyme. The pH can be titrated to a pH of about 8.0 during the contacting step.

[0007] Optionally, the contacting step can include mixing that occurs by aeration or recirculation. Optionally, the mixing does not occur by agitation.

[0008] The contacting step can be performed in the absence of a surfactant. Optionally, the population of microorganisms is not concentrated prior to the contacting step. The method can further include a pretreatment step, which includes disrupting the cells prior to the contacting step. The pretreatment step can be performed using chemical, mechanical, or enzymatic cell disruption methods.

[0009] The concentration step optionally includes centrifugation. Optionally, the concentration step includes 25% to 95% aqueous removal, inclusive (e.g., 50% to 95% or 85% aqueous removal).

[0010] The elevated temperature during the extraction process can be 55°C to 95°C (e.g., 75°C to 95°C, or 85°C or 90°C). Optionally, the concentration of salt added during the extraction process is 1% to 5% or 3% to 5% (e.g., 3% or 5%). The salt in the extraction process can be sodium sulfate. At least 80% of the lipids can be extracted from the disrupted microorganisms. The extraction process can be performed in the presence of oil (e.g., coconut oil) or biofuel, or in the absence of oil or biofuel.

[0011] Optionally, the methods of extracting lipids from a population of microorganisms described herein lack a pasteurization step. Optionally, the methods of extracting lipids from a population of microorganisms described herein lack a drying step.

[0012] The population of microorganisms can be selected from the group consisting of algae, fungi, bacteria, and protists. Optionally, the population of microorganisms is selected from the genus Thraustochytrium, Schizochytrium, or a mixture thereof. Optionally, the population of microorganisms is a species of the genus Thraustochytrium, for example, as deposited under ATCC Accession No. PTA-6245.

[0013] The details of one or more embodiments are set forth in the drawings and description which follow. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0014] [Figure 1]1 is a graph showing the percentage of oil recovered from cells hydrolyzed with 40 mM (left bar), 100 mM (middle bar), and 160 mM (right bar) hydrochloric acid, phosphoric acid, sulfuric acid, sodium hydroxide, and potassium hydroxide. [Figure 2] 1 is a graph showing the percentage of oil recovered from cells enzymatically hydrolyzed with 0.2% (left bar) and 0.4% (right bar) Viscozyme, Alcalase, Flavorzyme and Mannaway enzymes. [Figure 3] Graph showing the percentage of oil recovered from unwashed cells (left bar) and washed cells (right bar) after hydrolysis with 40 mM H2SO4, 160 mM H2SO4 and 0.2% (v / v) Alcalase. [Figure 4] 1 is a graph showing the percentage of oil recovered from cells enzymatically hydrolyzed at 55° C. for 18 hours and at 70° C. for 4 hours. [Figure 5] 1 is a graph showing the lipid class profiles of TG algal oil, biofuel, and EE algal oil. [Figure 6] Depiction (from left to right) of biomass feed hydrolyzed with 0.4% (v / v) Alcalase at 55°C for 18 hours, concentrated hydrolyzed biomass after centrifugation at 55°C, and medium after centrifugation at 55°C. [Figure 7] Figure 7A is a depiction of concentrated hydrolyzed biomass feed treated with NaSO and heating at 90° C. Figure 7B is a photograph showing the waste biomass after centrifugation at 90° C (left) and the recovered oil after centrifugation at 90° C (right). [Figure 8] 1 is a graph showing the percentage of oil recovered from cells and the amino acid concentration in the medium after enzymatic hydrolysis with 0.1% (v / v) Alcalase at 55° C. and pH 8.0 as a function of hydrolysis time. [Figure 9]1 is a graph showing the percentage of oil recovered from cells and the amino acid concentration in the medium after enzymatic hydrolysis with 0.05% (v / v) Alcalase at 55° C. and pH 8.0 as a function of hydrolysis time. [Figure 10] 1 is a graph showing the percentage of oil recovered from cells enzymatically hydrolyzed at pH 3.5, 4.5, 5.5, 6.5, 7.5 and 8.0. DETAILED DESCRIPTION OF THE INVENTION

[0015] Described herein is a wet, solvent-free method for recovering lipids from a population of microorganisms. The lipid recovery method includes contacting a population of microorganisms with one or more enzymes under conditions that cause microorganism disruption, concentrating the disrupted microorganisms, and extracting lipids from the disrupted microorganisms. Cell disruption is wet. Extraction is carried out at high temperature in the presence of salt and in the absence of solvent. Because microbial oil production and cell disruption can be carried out in the same vessel, and oil can be released at will, the method described herein can be referred to as a "one-pot" or "integrated" process. Thus, downstream processing steps (e.g., oil extraction and recovery) can be integrated at the end of upstream processing steps (e.g., fermentation).

[0016] I. Microorganisms The methods described herein include recovering lipids from a population of microorganisms. The population of microorganisms described herein can be algae (e.g., microalgae), fungi (including yeast), bacteria, or protists. Optionally, the microorganisms include the Thraustochytrids of the order Thraustochytriales, more specifically the genera Thraustochytrium and Schizochytrium of the order Thraustochytriales. Optionally, the population of microorganisms includes the order Thraustochytriales as described in U.S. Patent Nos. 5,340,594 and 5,340,742 (incorporated herein by reference in their entireties). The microorganism can be a species of the genus Thraustochytrium (such as the species of the genus Thraustochytrium deposited under ATCC Accession No. PTA-6245 (i.e., ONC-T18)).

[0017] Microorganisms for use in the methods described herein can produce a variety of lipid compounds. As used herein, the term lipid includes phospholipids, free fatty acids, esters of fatty acids, triacylglycerols, sterols and sterol esters, carotenoids, xanthophylls (e.g., oxycarotenoids), hydrocarbons, and other lipids known to those of skill in the art.

[0018] Optionally, the lipid compounds include saturated fatty acids, monounsaturated fatty acids and / or polyunsaturated fatty acids.

[0019] Optionally, lipid compounds include unsaturated lipids.Unsaturated lipids can include polyunsaturated lipids (i.e., lipids that contain at least two unsaturated carbon-carbon bonds (e.g., double bonds)) or highly unsaturated lipids (i.e., lipids that contain four or more unsaturated carbon-carbon bonds).Examples of unsaturated lipids include omega-3 polyunsaturated fatty acids and / or omega-6 polyunsaturated fatty acids (docosahexaenoic acid (i.e., DHA), eicosapentaenoic acid (i.e., EPA) and other naturally occurring unsaturated compounds and polyunsaturated compounds, etc.).

[0020] II. Process fermentation The microorganisms described herein can be cultured according to methods known in the art. For example, Thraustochytrids (e.g., species of the genus Thraustochytrium) can be cultured according to the methods described in U.S. Patents US2009 / 0117194 or US2012 / 0244584 (the entire contents of which are incorporated herein by reference). The microorganisms are grown in a growth medium (also known as a "culture medium"). Any of a variety of media may be suitable for use in culturing the microorganisms described herein. Optionally, the medium provides various nutritional components for the microorganisms, including a carbon source and a nitrogen source.

[0021] Optionally, the microorganisms provided herein are cultured under conditions that increase biomass and / or production of a compound of interest (e.g., oil or total fatty acid (TFA) content). Thraustochytrids are typically cultured, for example, in saline medium. Optionally, Thraustochytrids can be cultured in a medium having a salt concentration of about 2.0 g / L to about 50.0 g / L. Optionally, Thraustochytrids are cultured in a medium having a salt concentration of about 2 g / L to about 35 g / L (e.g., about 18 g / L to about 35 g / L). Optionally, Thraustochytrids described herein can be grown in low salt conditions. For example, Thraustochytrids can be cultured in a medium having a salt concentration of about 5 g / L to about 20 g / L (e.g., about 5 g / L to about 15 g / L). The culture medium optionally contains NaCl. Optionally, the medium includes natural or artificial sea salt and / or artificial seawater.

[0022] Compared to conventional methods, the chloride concentration in the culture medium can be reduced (i.e., lower). The culture medium can include a chloride-free sodium salt (e.g., sodium sulfate) as a sodium source. For example, the majority of the total sodium can be provided by a non-chloride salt, such that less than about 100%, 75%, 50%, or 25% of the total sodium in the culture medium is provided by sodium chloride.

[0023] Optionally, the culture medium has a chloride concentration of less than about 3 g / L, 500 mg / L, 250 mg / L, or 120 mg / L. For example, the culture medium has a chloride concentration of between about 60 mg / L and 120 mg / L. Examples of suitable non-chloride sodium salts for use in accordance with the present methods include, but are not limited to, soda ash (a mixture of sodium carbonate and sodium oxide), sodium carbonate, sodium bicarbonate, sodium sulfate, and mixtures thereof. See, e.g., U.S. Patent Nos. 5,340,742 and 6,607,900, the entire contents of each of which are incorporated herein by reference.

[0024] Media for the cultivation of Thraustochytrids can include any of a variety of carbon sources. Exemplary carbon sources include fatty acids; lipids; glycerol; triglycerol; carbohydrates (glucose, starch, cellulose, hemicellulose, fructose, dextrose, xylose, lactulose, galactose, maltotriose, maltose, lactose, glycogen, gelatin, starch (corn or wheat), acetate, m-inositol (from corn steep liquor), galacturonic acid (from pectin), L-fucose (from galactose), gentiobiose, glutathione, glutathione, glutathione, glutathione-1, glutathione-2, glutathione-3, glutathione-4, glutathione-5, glutathione-6, glutathione-7, glutathione-8, glutathione-9, glutathione-10, glutathione-11, glutathione-12, glutathione-13, glutathione-14, glutathione-15, glutathione-16, glutathione-17, glutathione-18, glutathione-19, glutathione-20, glutathione-21, glutathione-22, glutathione-23, glutathione-24, glutathione-25, glutathione-26, glutathione-27, glutathione-28, glutathione-29, glutathione-30, glutathione-31, glutathione-32, glutathione-33, glutathione-34, glutathione-35, glutathione-36, glutathione-37, glutathione-38, glutathione-39, glutathione-40, glutathione-41, glutathione-42, glutathione-43, glut sugars (from molasses); polyols (such as maltitol, erythritol, adonitol, and oleic acid (such as glycerol and Tween 80)); amino sugars (such as N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and N-acetyl-β-D-mannosamine); and any type of biomass or waste stream.

[0025] Optionally, the medium includes a carbon source at a concentration of about 5 g / L to about 200 g / L. The medium can have a C:N (carbon to nitrogen) ratio of between about 1:1 and about 40:1. When using a two-phase culture, the medium can have a C:N ratio of about 1:1 to about 5:1 for the first phase, and then within about 1:1 to about 1:0 (i.e., no or minimal nitrogen) in the second phase. As used herein, the term minimal refers to less than about 10% (e.g., 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 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, or less than about 0.1%). For example, minimal nitrogen in the medium can refer to less than about 10% (e.g., 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 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, or less than about 0.1%) of nitrogen in the medium.

[0026] Media for culturing Thraustochytrids can include any of a variety of nitrogen sources. Exemplary nitrogen sources include ammonium solutions (e.g., NH in HO), ammonium or amine salts (e.g., (NH)SO, (NH)PO, NHNO, NHOOCHCH(NHAc)), peptone, tryptone, yeast extract, malt extract, fish meal, monosodium glutamate, soybean extract, casamino acids, and grain meal. The concentration of the nitrogen source in a suitable medium typically ranges from about 1 g / L to about 25 g / L.

[0027] The medium optionally contains a phosphate salt (such as potassium phosphate or sodium phosphate). Inorganic salts and micronutrients in the medium may include ammonium sulfate, sodium bicarbonate, sodium orthovanadate, potassium chromate, sodium molybdate, selenite, nickel sulfate, copper sulfate, zinc sulfate, cobalt chloride, iron chloride, manganese chloride, calcium chloride, and EDTA. Vitamins (such as pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, para-aminobenzoic acid, riboflavin, nicotinic acid, biotin, folic acid, and vitamin B12) may be included.

[0028] The pH of the medium can be adjusted to within 3.0 to 10.0 using an acid or base and / or a nitrogen source as needed. Optionally, the medium is adjusted to a pH of 4.0 to 6.5, inclusive. The medium can be sterilized.

[0029] Generally, the medium used for culturing microorganisms is a liquid medium. However, the medium used for culturing microorganisms can be a solid medium. In addition to the carbon source and nitrogen source discussed herein, the solid medium can contain one or more components (e.g., agar or agarose) that provide structural support and / or allow the medium to be in solid form.

[0030] Cells can be cultured for anywhere from 1 day to 60 days. Optionally, culturing is carried out for 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less. Culturing is optionally carried out at a temperature of about 4°C to about 30°C (e.g., about 18°C ​​to about 28°C). Culturing can include aeration-shaking culture, shaking culture, static culture, batch culture, semi-continuous culture, continuous culture, rolling batch culture, wave culture, or the like. Culturing can be carried out using conventional stirred tank fermentors, bubble column fermentors (batch or continuous culture), wave fermentors, etc.

[0031] The culture can be aerated by one or more of a variety of methods, including shaking. Optionally, the shaking is in the range of about 100 rpm to about 1000 rpm (e.g., about 350 rpm to about 600 rpm or about 100 to about 450 rpm). Optionally, the culture is aerated using different shaking speeds during the biomass production phase and during the lipid production phase. Alternatively, or additionally, the shaking speed can vary depending on the type of culture vessel (e.g., the shape or size of the flask).

[0032] Optionally, the dissolved oxygen (DO) level is higher during the biomass production phase than during the lipid production phase. Thus, the DO level is reduced during the lipid production phase (i.e., the DO level is less than the amount of dissolved oxygen during the biomass production phase). Optionally, the dissolved oxygen level is reduced below saturation. For example, the dissolved oxygen level can be reduced to a very low or even undetectable level.

[0033] To obtain higher amounts of desired compounds, the production of desired lipids can be promoted by culturing cells according to a method that includes shifting one or more culture conditions. Optionally, cells are first cultured under conditions that maximize biomass, and then one or more culture conditions are shifted to conditions that support lipid productivity. The shifted conditions can include oxygen concentration, C:N ratio, temperature, and combinations thereof. Optionally, a two-stage culture is performed, where the first stage supports biomass production (e.g., using conditions of high oxygen (e.g., generally or compared to the second stage), low C:N ratio, and ambient temperature), and then the second stage supports lipid production (e.g., oxygen is reduced, C:N ratio is increased, and temperature is reduced).

[0034] pasteurization Optionally, the resulting biomass is pasteurized to kill cells and inactivate undesirable substances present in the biomass. For example, the biomass can be pasteurized to inactivate compounds that degrade the substances. The biomass can be present in the fermentation medium or can be isolated from the fermentation medium for the pasteurization step. The pasteurization step can be accomplished by heating the biomass and / or fermentation medium to an elevated temperature. For example, the biomass and / or fermentation medium can be heated to a temperature of about 55°C to about 121°C (e.g., about 55°C to about 90°C, or about 65°C to about 80°C). Optionally, the biomass and / or fermentation medium can be heated for about 4 minutes to about 120 minutes (e.g., about 30 minutes to about 120 minutes, about 45 minutes to about 90 minutes, or about 55 minutes to about 75 minutes). Pasteurization can be accomplished using any suitable heating means known to those skilled in the art (e.g., by direct steam injection).

[0035] Optionally, no pasteurization step is performed (ie, the method lacks a pasteurization step).

[0036] Harvesting and cleaning Optionally, the biomass can be harvested according to methods known to those skilled in the art. For example, the biomass can optionally be collected from the fermentation medium using a variety of conventional methods, such as centrifugation (e.g., solids-discharge centrifuge) or filtration (e.g., cross-flow filtration), which can also include the use of precipitating agents (e.g., sodium phosphate or calcium chloride) to accelerate the collection of the cellular biomass.

[0037] Optionally, the biomass is washed with water. Optionally, the biomass can be concentrated to about 30% solids. For example, the biomass can be concentrated to about 5% to about 30% solids, about 7.5% to about 15% solids, or about 15% to about 20% solids, or any percentage within the recited range. Optionally, the biomass can be concentrated to about 30% solids or less, about 29% solids or less, about 28% solids or less, about 27% solids or less, about 26% solids or less, about 25% solids or less, about 24% solids or less, about 23% solids or less, about 22% solids or less, about 21% solids or less, about 20% solids or less, about 19% solids or less, about 18% solids or less, about 17% solids or less, about 16% solids or less, about 15% solids or less, about 14% solids or less, about 13% solids or less, about 12% solids or less, about 11% solids or less, about 10% solids or less, about 9% solids or less, about 8% solids or less, about 7% solids or less, about 6% solids or less, about 5% solids or less, about 4% solids or less, about 3% solids or less, about 2% solids or less, or about 1% solids or less.

[0038] Hydrolysis Cell hydrolysis (i.e., cell disruption) can be accomplished using chemical, enzymatic, and / or mechanical methods. Optionally, the methods described herein lack a drying step. For example, the biomass is optionally not dried prior to cell hydrolysis. Optionally, the biomass is not concentrated after fermentation is completed and prior to the contacting step.

[0039] Chemical methods for cell hydrolysis can include adding acid to the cells and are referred to herein as acid hydrolysis. In acid hydrolysis, the biomass can be washed with water, for example, using centrifugation, and concentrated as described above before cell hydrolysis. Optionally, the biomass is concentrated to about 15% solids with water.

[0040] An acid is then added to the washed wet biomass. Optionally, the biomass is not dried before the addition of the acid. Suitable acids for use in the acid hydrolysis step include sulfuric acid, hydrochloric acid, phosphoric acid, hydrobromic acid, nitric acid, perchloric acid, and other strong acids as known to those skilled in the art. An appropriate amount of acid can be added to the washed wet biomass to achieve a final concentration of about 100 mM to about 200 mM (e.g., about 120 mM to about 180 mM, or about 140 mM to about 160 mM). Sulfuric acid can be added to the washed wet biomass to a final concentration of 160 mM.

[0041] The resulting mixture containing water, biomass, and acid can then be incubated for a period of time to hydrolyze the cells. Optionally, the mixture can be incubated at a temperature of about 30°C to about 200°C. For example, the mixture can be incubated at a temperature of about 45°C to about 180°C, about 60°C to about 150°C, or about 80°C to about 130°C. Optionally, the mixture is incubated in an autoclave at a temperature of 121°C. The mixture can be incubated for a period of time appropriate to hydrolyze at least 50% of the cells (e.g., at least 60% of the cells, at least 70% of the cells, at least 80% of the cells, at least 90% of the cells, at least 95% of the cells, or 100% of the cells). The period for cell incubation depends on the culture temperature. Incubating the mixture at a higher temperature can result in hydrolysis that proceeds at a faster rate (i.e., a shorter period of time is required for hydrolysis). In some examples, the cells can be incubated at 60°C for 1 hour.

[0042] As described above, cell hydrolysis (i.e., cell disruption) can be accomplished using enzymatic methods. Specifically, a population of microorganisms can be contacted with one or more enzymes under conditions that cause disruption of the microorganisms. Optionally, the enzyme is a protease. An example of a suitable protease is Alcalase 2.4L FG (Novozymes; Franklinton, North Carolina). Optionally, the cells are not washed with water before enzymatic hydrolysis. Optionally, the population of microorganisms is not concentrated before enzymatic hydrolysis.

[0043] Prior to contacting the microorganism with one or more enzymes, the pH of the fermentation medium can optionally be adjusted to about 5 to 8.5, e.g., about 5.5 to 8.0 or about 6.5 to 7.5, or any value within the recited range. For example, the pH of the fermentation medium can optionally be adjusted to 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. The pH can be adjusted using, for example, a base, such as sodium hydroxide (e.g., 1N NaOH), ammonium hydroxide, calcium hydroxide, magnesium hydroxide, or potassium hydroxide. The pH of the fermentation medium can also be adjusted during the contacting step. Optionally, the pH is adjusted to a pH of about 8.0 before or during the contacting step.

[0044] While the population of microorganisms is in a fermentation medium, the microorganisms can be contacted with one or more enzymes (i.e., the contacting step occurs in the fermentation medium). Optionally, the fermentation medium is concentrated after fermentation and before the contacting step. Optionally, the fermentation medium is diluted after fermentation and before the contacting step. Optionally, the enzymes added to the fermentation medium are at a concentration of about 0.001% to about 0.4% volume / volume (v / v). For example, the enzymes added to the fermentation medium can be at a concentration of 0.05% (v / v) to 0.4% (v / v), 0.05% to 0.2% (v / v), or 0.1% to 0.2% (v / v). In some embodiments, the enzyme is added to the fermentation medium at a concentration of 0.05% (v / v), 0.1% (v / v), 0.15% (v / v), 0.2% (v / v), 0.25% (v / v), 0.30% (v / v), 0.35% (v / v), or 0.4% (v / v).

[0045] The contacting step can be carried out at a temperature of at least about 45°C. Optionally, the contacting step is carried out at a temperature of about 45°C to about 70°C, about 50°C to about 70°C, about 55°C to about 70°C, or 55°C. The contacting step is carried out for a suitable period of time to result in disruption of the microorganisms. For example, the contacting step can be carried out for about 1 hour to about 20 hours, e.g., 1 hour to 18 hours, 1 hour to 6 hours, 4 hours to 6 hours, or any time frame within the recited range. Optionally, the contacting step can be carried out for about 4 hours.

[0046] The optimum temperature, time, pH and enzyme concentration will depend on the particular enzyme, and one skilled in the art will be able to modify the temperature, time, pH and enzyme concentration as appropriate for a given enzyme.

[0047] Optionally, the contacting step is carried out in the presence of either about 0.05% (v / v) or about 0.2% (v / v) enzyme at about 55° C. for about 1-6 hours. For example, the contacting step can be carried out in the presence of 0.1% (v / v) enzyme at 55° C. for 4 hours. Optionally, the contacting step is carried out in the absence of a detergent (i.e., no detergent is present).

[0048] Optionally, cell disruption can be accomplished using other chemical and mechanical methods known to those skilled in the art, for example, alkaline hydrolysis, bead milling, sonication, detergent hydrolysis, solvent extraction, rapid decompression (i.e., cell bombing), or high shear mechanical methods, contact with chemicals, homogenization, ultrasound, grinding, shear force, French press, cold press, heating, drying, osmotic shock, pressure vibration, expression of autolytic genes, or combinations thereof.

[0049] The method can further include a pretreatment step, which includes disrupting cells before the contacting step. The pretreatment step can be performed using a chemical, mechanical, or enzymatic cell disruption method. In other words, cell disruption can be performed using a combination of two or more of the chemical, enzymatic, and / or mechanical methods described herein (e.g., enzymatic hydrolysis combined with bead milling). Cell disruption methods can be performed sequentially (e.g., bead milling followed by enzymatic hydrolysis). Optionally, a chemical or mechanical method can be performed as the first hydrolysis step, followed by enzymatic cell disruption as the second hydrolysis step. In these examples, a lower amount of enzyme can be used in the enzymatic cell disruption step compared to the amount of enzyme used when only one cell disruption method is performed.

[0050] concentrated The disrupted microorganisms resulting from enzymatic hydrolysis can be concentrated by separation and removal of the fermentation medium to provide a desired concentration of disrupted microorganisms for subsequent processing. Optionally, the disrupted microorganisms are concentrated by centrifugation and removal of one or more substances to provide a desired concentration. If centrifugation is used, optionally, the centrifugation can provide two or more layers, including a heavy layer and a light layer. The heavy layer contains soluble fermentation medium components and water, while the light layer contains the disrupted microorganisms in the form of lipids and waste biomass. The light layer may further contain some water. The light layer may contain at least a portion of the lipids and biomass, as well as water, in the form of an emulsion.

[0051] The concentrating step includes removing one or more substances present in the contacting step. For example, the concentrating step can include aqueous removal. Optionally, the concentrating step includes removing about 25% to about 95% of the water by volume (e.g., about 85% of the water). Optionally, the concentrating step includes removing at least a portion of the heavy layer, for example, by draining the heavy layer through a valve or by aspirating or decanting the heavy layer.

[0052] Optionally, the concentrated disrupted microorganisms can be allowed to rest for a period of time before recovering oil from the disrupted microorganisms according to the extraction process described below. The concentrated disrupted microorganisms can be allowed to rest for up to 24 hours (e.g., 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, or 24 hours).

[0053] extraction As described above, lipids are extracted from disrupted microorganisms at high temperatures in the presence of salt and in the absence of organic solvents.

[0054] The extraction process can be carried out at elevated temperatures. As used herein, elevated temperatures refer to temperatures of at least about 55°C (e.g., temperatures of about 55°C to about 95°C). For example, the lipid and biomass mixture can be contacted with salt, oil, or biofuel at temperatures of about 65°C or higher, about 70°C or higher, about 75°C or higher, about 80°C or higher, about 85°C or higher, about 90°C or higher, or about 95°C or higher.

[0055] The extraction step is carried out in the presence of a salt. The salt can be, for example, sodium sulfate. The concentration of the salt added during the extraction step can be about 1% to about 5% (v / v) (e.g., about 3% to about 5% (v / v)) based on the volume of the extraction mixture. For example, the concentration of the salt added during the extraction step can be about 1%, about 2%, about 3%, about 4%, or about 5%.

[0056] Lipids are dissolved in organic solvents (e.g., C5-C 12The microorganism is extracted from the disrupted microorganism in the absence of an organic solvent (e.g., an alkane, a chlorinated C1-C6 alkane, a C1-C6 alcohol, or supercritical carbon dioxide). As used herein, in the absence of an organic solvent means less than about 0.5% (e.g., less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.05%, less than about 0.01%, less than about 0.005%, or 0%) of the solvent based on the weight of the disrupted microorganism.

[0057] Optionally, lipids can be extracted from the disrupted microorganisms by the addition of oil (e.g., coconut oil) or biofuel.

[0058] Optionally, the oil added during the extraction process can be a nutritional oil (for example, oil derived from or obtained from a nutritional source).Examples of nutritional oils suitable for use in the methods described herein include coconut oil, palm oil, canola oil, sunflower oil, soybean oil, corn oil, olive oil, safflower oil, palm kernel oil, cottonseed oil, and combinations thereof.Any derivative of these oils can also be used, such as alkylated derivatives (for example, methylated oil or ethylated oil).

[0059] As used herein, biofuel refers to any fuel, fuel additive, aromatic and / or aliphatic compound derived from a biomass starting material. For example, biofuels suitable for use in the methods described herein can be derived from plant or algal sources. Examples of suitable sources for biofuels include algae, corn, switchgrass, sugarcane, sugar beet, rapeseed, soybeans, and the like.

[0060] Optionally, biofuel can be obtained by harvesting oil from biological sources and converting the oil into biofuel. Methods for converting oil obtained from biological sources (e.g., oil obtained from plant and / or algal sources) are known to those skilled in the art. Optionally, a method for obtaining biofuel can include culturing oil-producing biomass (e.g., algae), extracting oil (e.g., algal oil), and converting the oil (e.g., algal oil) to form biofuel. Optionally, oil can be converted into biofuel using transesterification. As used herein, transesterification refers to a process of exchanging an alkoxy group of an ester with another alcohol. For example, a transesterification process for use in the methods described herein can include converting algal oil (e.g., triglycerides) into biodiesel (e.g., fatty acid alkyl esters and glycerol). Transesterification can be accomplished using conventional chemical processes (such as acid- or base-catalyzed reactions) or by using enzyme-catalyzed reactions.

[0061] As used herein, the term organic solvent is defined herein to exclude vegetative oils (such as coconut oil, palm oil, canola oil, sunflower oil, soybean oil, corn oil, olive oil, safflower oil, palm kernel oil, cottonseed oil, or alkylated (e.g., methylated or ethylated) derivatives thereof), and therefore does not include biofuels.

[0062] Optionally, the oil or biofuel used to extract lipids from the disrupted microorganisms is not subsequently removed from the extracted lipids. Subsequent fractionation of the extracted oil (the added oil or biofuel remains with only one of the oil fractions) is not considered to remove the oil or biofuel from the extracted lipids. For example, after recovery, the oils described herein can be combined with other oils for use as one or more of the products described herein, or can be incorporated into one or more of the products described herein. Any one of these other oils or products (such as biofuels) can be added to the mixture of lipids and biomass during the extraction process, as an alternative to or in addition to combining with the recovered oil after the end of the recovery process. Adding other oils during the extraction process can aid in the demulsification and separation of lipids from used biomass.

[0063] In the conventional method that relies on organic solvent extraction to separate lipid from biomass, organic solvent must be removed from lipid after recovery, but typically at least a trace amount of solvent remains behind.However, in the method described herein, optionally, when the oil or biofuel that is added during extraction process is used as or incorporated into final product, more than about 80% of it remains in the recovered oil.That is, optionally, less than about 20% of the oil or biofuel that is added during extraction process is removed from the recovered oil before being used as or incorporated into final product.For example, optionally, less than about 15%, less than about 10%, less than about 5%, less than about 2% or 0% of the oil or biofuel that is added during extraction process is removed from the recovered oil before being used as or incorporated into final product.

[0064] The disrupted microorganisms or biomass can be mixed with salt, oil, and / or biofuel for a period of time suitable for extracting lipids from the disrupted microorganisms or biomass. For example, the salt, oil, and / or disrupted microorganisms or biomass can be mixed for about 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 1 hour or more, or 2 hours or more. The lipids can then be separated from the remaining components of the mixture by centrifuging the solution.

[0065] Optionally, at least 65% of the lipids theoretically produced by the microorganisms are extracted from the disrupted microorganisms using this method (i.e., the method provides a yield of at least about 65%). For example, the yield of lipids extracted from the disrupted microorganisms can be at least about 70%, at least 75%, at least 80%, or at least 85%.

[0066] Alternatively, the extraction process can be carried out in the absence of oil or biofuel. For example, lipids can be extracted using mechanical methods. The hydrolyzed biomass and microorganisms can be centrifuged, and the lipids can be separated from the rest of the components. Separating the oil by centrifugation can optionally include adding sodium sulfate to the biomass emulsion. Optionally, the centrifugation can be carried out at an elevated temperature (e.g., about 55°C or higher, about 80°C, etc.) as described herein. Optionally, the lipids are contained in the upper layer of the centrifuged material and can be removed from the other materials, for example, by suction or decantation.

[0067] Optionally, at least about 65% of the lipids produced by the microorganisms are extracted from the disrupted microorganisms using this method (i.e., the method provides a yield of at least 65%). For example, the yield of lipids extracted from the disrupted microorganisms is at least 60%, at least 70%, at least 80%, or at least 90% of the total amount produced.

[0068] III.Product Polyunsaturated fatty acids (PUFAs) (e.g., DHA, EPA) and other lipids produced according to the methods described herein can be used in any of a variety of applications, for example, by utilizing their biological or nutritional properties. Optionally, the compounds can be used in pharmaceuticals, nutritional oils (e.g., nutritional oil supplements), food supplements, animal feed additives, cosmetics, biofuels, and the like. Lipids produced according to the methods described herein can also be used as intermediates in the production of other compounds. Optionally, lipids produced according to the methods described herein can be incorporated into final products (e.g., food or feed supplements, infant formula, pharmaceuticals, fuels (e.g., biofuels), etc.).

[0069] Suitable food or feed supplements into which the lipids described herein can be incorporated include beverages (such as milk, water, sports drinks, energy drinks, tea, and juice); confectioneries (such as jellies and biscuits); fat-containing foods and beverages (such as dairy products); processed food products (such as soft rice (or porridge)); infant formula; breakfast cereals; or the like. Optionally, one or more produced lipids can be incorporated into a food supplement (such as a multivitamin). Optionally, the lipids produced according to the methods described herein can be included in a food supplement, or can be directly incorporated into a food or feed component (e.g., a food supplement).

[0070] Examples of feed ingredients into which the lipids produced by the methods described herein can be incorporated include pet food (cat food; dog food and the like; feed for ornamental fish, farmed fish or crustaceans, etc.); feed for farmed animals (including livestock and fish or crustaceans raised in aquaculture). The food or feed material into which the lipids produced according to the methods described herein can be incorporated is preferably palatable to the intended recipient organism. The food or feed material can have any physical properties currently known for food materials (e.g., solid, liquid, soft).

[0071] Optionally, one or more of the produced compounds (e.g., PUFAs) can be incorporated into a pharmaceutical product. Examples of such pharmaceutical products include various types of tablets, capsules, ingestible medicines, etc. Optionally, the pharmaceutical product is suitable for topical application. Dosage forms can include, for example, capsules, oils, particles, granules, powders, tablets, pills, lozenges, or the like.

[0072] Optionally, one or more of the compounds produced can be used as biofuel or incorporated into biofuel.For example, biofuel can be produced by transesterifying one or more of the compounds produced.Biofuel can be produced by base-catalyzed transesterification of the produced oil, acid-catalyzed transesterification of the oil, or by converting the oil into its fatty acid and then biofuel.

[0073] The lipids produced according to the methods described herein can be incorporated into products such as those described herein in combination with any of a variety of agents. For example, such compounds can be combined with one or more binders or fillers. In some embodiments, the product can include one or more chelating agents, pigments, salts, surfactants, moisturizers, viscosity adjusters, thickeners, emollients, fragrances, preservatives, etc., and combinations thereof.

[0074] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims. [Example]

[0075] Example 1. Pasteurization, Harvesting and Cleaning, and Chemical Hydrolysis pasteurization The T18 biomass was heated at 60°C for 1 hour with stirring to pasteurize the cells.

[0076] Harvesting and cleaning The pasteurized T18 biomass was centrifuged at 4150 rpm for 20 minutes at ambient temperature to separate the final medium from the cell paste. The medium was removed and an equal mass of water was added to the cell paste to wash the cells. The cell paste-water mixture was shaken for 1 minute, recentrifuged, and the aqueous phase was removed.

[0077] chemical hydrolysis Water-washed T18 cell paste was adjusted to 150 g / L with water. Subsamples (10 mL) were removed and added to 50 mL centrifuge tubes. Each subsample was treated with acid or base to the final concentration according to Table 1. The mixture was autoclaved at 121 °C for 15 minutes to hydrolyze the cells. After hydrolysis, the samples were extracted with hexane, and the percentage of oil recovered was determined by mass balance (Figure 1). Hydrolysis with 160 mM HCl and H2SO4 resulted in greater than 85% oil recovery. [Table 1]

[0078] Example 2. Enzymatic hydrolysis Water-washed T18 cell paste was adjusted to 220 g / L with water. The pH was adjusted to 7.5 with 1 N NaOH. Subsamples (10 mL) were removed and added to 50 mL centrifuge tubes. Each subsample was treated with enzymes according to Table 2. The mixture was incubated with shaking at 50°C for 22 hours to hydrolyze the cells. After hydrolysis, the samples were extracted with hexane, and the percentage of oil recovered was determined by mass balance (Figure 2). Hydrolysis with Alcalase alone or in combination with another enzyme resulted in greater than 85% oil recovery. [Table 2]

[0079] Example 3. Acid hydrolysis and enzymatic hydrolysis, effect of washing Subsamples of pasteurized, unwashed T18 biomass (10 mL) were added to 50 mL centrifuge tubes. The control was water-washed, adjusted to 170 g / L with water, and subsampled into 50 mL centrifuge tubes. Each subsample was treated with acid or enzyme according to Table 3. Acid-hydrolyzed samples were autoclaved at 121°C for 15 minutes to hydrolyze the cells. Enzymatically hydrolyzed samples were adjusted to pH 7.5 with 1 N NaOH and incubated at 50°C for 26 hours with shaking to hydrolyze the cells. After acid or enzymatic hydrolysis, samples were extracted with hexane, and the percentage of recovered oil was determined by mass balance (Figure 3). Recovery of unwashed oil equivalent to that of washed oil was achieved with 0.2% Alcalase hydrolysis. [Table 3]

[0080] Example 4. Enzymatic Hydrolysis, Temperature / Time Effect Water-washed T18 cell paste was adjusted to 210 g / L with water. The pH was adjusted to 7.5 with 1 N NaOH. Subsamples (10 mL) were added to 50 mL centrifuge tubes. Each subsample was treated with 0.2% v / v Alcalase. The mixture was incubated at 70°C for 4 hours with shaking to hydrolyze the cells. A control was incubated at 55°C for 18 hours with shaking. After hydrolysis, samples were extracted with hexane, and the percentage of oil recovered was determined by mass balance (Figure 4). By increasing the temperature to 70°C, oil recovery equivalent to 18 hours of hydrolysis at 55°C was achieved in 4 hours.

[0081] Example 5. Enzymatic hydrolysis, extraction with biofuels The water-washed T18 cell paste was adjusted to 200 g / L with water, and the pH was adjusted to 7.5 with 1 N NaOH. Subsamples (10 mL) were removed and added to 50 mL centrifuge tubes. Each subsample was treated with 0.2% v / v Alcalase. The mixture was incubated with shaking at 55°C for 18 hours to hydrolyze the cells. After hydrolysis, each subsample was extracted with biofuel according to Table 5, and the oil percentage was determined by mass balance and based on the lipid class profile of the pure oil (Figure 5 and Table 5). Extraction with a 1:0.4 (wet biomass:biofuel) ratio resulted in greater than 85% oil recovery based on the lipid class profile. Triglyceride (TG) algal oil was ethylated (EE) and used in addition to the parent TG oil for oil extraction (Table 6). All ratios of wet biomass:EE algal oil resulted in greater than 85% oil recovery based on the lipid class profile. [Table 4] [Table 5]

[0082] Example 6. Enzymatic Hydrolysis, Solvent-Free Oil Extraction 200 mL of unwashed T18 biomass with a solids concentration of 224 g / L (biomass concentration of 159 g / L) was heated to 55°C and adjusted to pH 8 with 1 N NaOH. The sample was treated with 0.4% (v / v) Alcalase and incubated at 55°C for 18 hours with shaking to hydrolyze the cells. After hydrolysis, the sample was centrifuged at 4600 rpm for 20 minutes at 40°C to separate the medium from the concentrated hydrolyzed biomass. 85% of the medium was removed. After water removal, the remaining sample was centrifuged at 4600 rpm for 20 minutes at 40°C to separate the oil from the waste biomass. The oil was recovered, and the percentage of oil recovered was determined by mass balance. By reducing the water concentration after hydrolysis, greater than 90% oil recovery was achieved by solvent-free oil extraction.

[0083] Example 7. Solvent-free oil extraction, large scale 151,400 kg of unwashed T18 biomass with a biomass concentration of 111 g / L was heated to 55°C and adjusted to pH 8 with 50 N NaOH. 606 L of Alcalase was added, the pH was adjusted back to 8, and the mixture was recirculated between the two vessels and mixed at 55°C for 18 hours for hydrolysis. After hydrolysis, the broth weighed 160,400 kg. The hydrolyzed biomass was held at 55°C for centrifugation to separate the medium from the concentrated hydrolyzed biomass (Figure 6). 134,400 kg of medium was removed, and 26,000 kg of concentrated hydrolyzed biomass was recovered. The concentrated hydrolyzed biomass was treated with 1300 kg of Na2SO4, heated with mixing at 90°C, and then centrifuged to separate the oil from the waste biomass (Figure 7). 7606 kg of oil was recovered for a total oil recovery of 82%. The peroxide value (PV) and acid value (AV) of the recovered oil were 0.4 meq / kg and 0.38 mg KOH / g, respectively.

[0084] Example 8. Optimized enzyme concentration / time (0.1% enzyme) Unwashed T18 biomass at a biomass concentration of 143 g / L was heated to 55°C and adjusted to pH 8.0 with 1 N NaOH. 25 mL subsamples were removed into 50 mL centrifuge tubes. Each subsample was treated according to Table 7. The mixture was incubated with shaking at 55°C to hydrolyze the cells. After hydrolysis, the sample was placed in a 100°C bath for 20 minutes to inactivate the enzyme. After enzyme inactivation, the sample was centrifuged at 4600 rpm for 20 minutes at 40°C to separate the medium from the concentrated hydrolyzed biomass. Approximately 80% of the medium was removed. The medium was passed through a 0.25 μm filter and the degree of hydrolysis was determined by the o-phthaldialdehyde (OPA) method (Spellman, D., McEvoy, E., O'Cuinn, G., and Fitz, GRJ 2003. Proteinase and exopeptidase hydrolysis of whey protein: Comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis. International Dairy Journal, 13:447-453). See Table 7.

[0085] The concentrated hydrolyzed biomass was treated with 5% (w / v) NaSO and heated at 70°C for 60 minutes with shaking. After treatment, the sample was centrifuged at 4600 rpm for 20 minutes at 40°C to separate the oil from the waste biomass. The oil was recovered and the % of oil recovered was determined by mass balance (Figure 8). Hydrolysis with 0.1% Alcalase was complete after 4 hours. [Table 6]

[0086] Example 9: Optimized enzyme concentration / time (0.05% enzyme) Unwashed T18 biomass at a biomass concentration of 143 g / L was heated to 55°C and adjusted to pH 8.0 with 1 N NaOH. 25 mL subsamples were removed into 50 mL centrifuge tubes. Each subsample was treated according to Table 8. The mixture was incubated with shaking at 55°C to hydrolyze the cells. After hydrolysis, the sample was placed in a 100°C bath for 20 minutes to inactivate the enzyme. After enzyme inactivation, the sample was centrifuged at 4600 rpm for 20 minutes at 40°C to separate the medium from the concentrated hydrolyzed biomass. Approximately 80% of the medium was removed. The medium was passed through a 0.25 μm filter, and the degree of hydrolysis was determined by the OPA method (Table B).

[0087] The concentrated hydrolyzed biomass was treated with 5% (w / v) NaSO and heated with shaking at 70°C for 60 minutes. After treatment, the sample was centrifuged at 4600 rpm for 20 minutes at 40°C to separate the oil from the waste biomass. The oil was recovered and the % of oil recovered was determined by mass balance (Figure 9). Hydrolysis with 0.05% Alcalase was complete after 6 hours. [Table 7]

[0088] Example 10. Enzymatic hydrolysis, low pH A 150 mL subsample of unwashed T18 was removed into a 250 mL beaker. Each subsample was treated with 1 M HCl or NaOH to adjust to the desired pH according to Table 9. A 30 mL subsample of each pH condition was removed into a 50 mL centrifuge tube. Each subsample was treated with 0.5% v / v Alcalase. The mixture was incubated at 55°C with shaking for 16 hours to hydrolyze the cells. After hydrolysis, the sample was centrifuged at 4600 rpm for 20 minutes at 40°C to separate the medium from the concentrated hydrolyzed biomass. Approximately 60% of the medium was removed. The concentrated hydrolyzed biomass was treated with 5% (w / v) Na2SO4 and heated at 70°C with shaking for 60 minutes to separate the oil from the waste biomass. The oil was recovered, and the percentage of oil recovered without solvent was determined by mass balance. Figure 10. Equivalent oil recovery was achieved at pH 5.5 and 8. [Table 8]

[0089] Example 11. Enzymatic hydrolysis, alternative enzymes Subsamples of 30 mL of unwashed T18 biomass were removed into 50 mL centrifuge tubes. Each subsample was treated with 0.5% (v / v) enzyme and according to Table 9. The mixture was incubated at 55°C with shaking to hydrolyze the cells. After hydrolysis, the samples were centrifuged at 4600 rpm for 20 minutes at 40°C to separate the medium from the concentrated hydrolyzed biomass. Approximately 50% of the medium was removed. The concentrated hydrolyzed biomass was treated with 5% (w / v) NaSO and heated to 70°C for 60 minutes with shaking. After treatment, the samples were extracted with hexane, and the percentage of oil recovered was determined by mass balance. Although alternative enzymes were not identified as being as efficient as Alcalase for hydrolysis, the enzyme of choice (Protease 'M', Savinase Ultra, Blaze Evity, or Polarzyme) could be used in place of Alcalase if necessary. [Table 9]

[0090] Example 12. Mechanical Hydrolysis An unwashed T18 biomass sample with a biomass concentration of 180 g / L was fed into a 300 mL Dyno-Mill Multi Lab stainless steel chamber loaded with 0.6-0.8 mm zirconium oxide grinding media (80% v / v bead volume). The bead mill was operated in continuous mode with an impeller tip speed of 10 m / s and a feed flow rate of 80 mL / min. A subsample of the partially crushed biomass slurry was passed back through the crushing chamber for a second pass. The biomass slurry was centrifuged at 4600 rpm for 20 minutes at 40 °C to separate the oil from the waste biomass. The oil was recovered, and the percentage of oil recovered was determined by mass balance. One pass and two passes resulted in 70% oil recovery.

[0091] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims; any functionally equivalent compositions and methods are within the scope of this disclosure. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Furthermore, while only certain representative compositions, methods, and aspects of these compositions and methods have been specifically described, other compositions and methods, and combinations of various features of the compositions and methods, even if not specifically recited, are intended to fall within the scope of the appended claims. Thus, although combinations of steps, elements, components, or ingredients may be explicitly recited herein, all other combinations of steps, elements, components, and ingredients are included, even if not expressly stated.

Claims

1. 1. A method for recovering lipids from a population of microorganisms, comprising: (a) contacting a population of microorganisms with one or more enzymes under conditions that cause disruption of the microorganisms; (b) concentrating the disrupted microorganisms; (c) extracting lipids from the disrupted microorganisms at elevated temperatures in the presence of salt and in the absence of organic solvents; A method comprising:

2. 10. The method of claim 1, wherein the contacting step is carried out at a pH of from 5 to 8.

5.

3. 3. The method of claim 1 or 2, wherein the contacting step is carried out at a temperature of from about 50°C to about 70°C.

4. The method of any one of claims 1 to 3, wherein the contacting step is carried out for 1 to 20 hours.

5. 5. The method of claim 4, wherein the contacting step is carried out for 1 to 8 hours.

6. The method according to any one of claims 1 to 5, wherein the enzyme is a protease.

7. 7. The method of claim 6, wherein the enzyme is Alcalase 2.4L.

8. 8. The method of any one of claims 1 to 7, wherein the enzyme is at a concentration of between 0.001 and 0.4% volume / volume.

9. 9. The method of claim 8, wherein the enzyme is at a concentration of between 0.05 and 0.2% volume / volume.

10. 10. The method of any one of claims 1 to 9, wherein the contacting step is carried out in the presence of 0.05% to 0.2% enzyme at about 55°C for 1 to 8 hours.

11. The method of any one of claims 1 to 10, wherein the contacting step is carried out in the absence of a surfactant.

12. The method according to any one of claims 1 to 11, further comprising a pretreatment step, wherein the pretreatment step comprises disrupting the cells before the contacting step.

13. The method of any one of claims 1 to 12, wherein the concentration step comprises centrifugation.

14. 14. The method of any one of claims 1 to 13, wherein the concentration step comprises removing 25% to 90% of the water.

15. 15. The method of claim 14, wherein the concentration step comprises removing 85% of the water.

16. 16. The method of any one of claims 1 to 15, wherein the elevated temperature during the extraction step is from 75°C to 95°C.

17. 17. The method of claim 16, wherein the elevated temperature during the extraction step is 85°C.

18. 18. The method according to any one of claims 1 to 17, wherein the concentration of salt added during the extraction step is between 1% and 5%.

19. 19. The method of claim 18, wherein the concentration of salt added during the extraction step is between 3% and 5%.

20. 20. The method according to any one of claims 1 to 19, wherein the salt added during the extraction step is sodium sulfate.

21. 21. The method according to any one of claims 1 to 20, wherein the extraction step is carried out in the presence of oil.

22. 22. The method of claim 21, wherein the oil is coconut oil.

23. 21. The method of any one of claims 1 to 20, wherein the extraction step is carried out in the presence of biofuel.

24. 21. The method of any one of claims 1 to 20, wherein the extraction step is carried out in the absence of oil or biofuel.

25. 25. The method of any one of claims 1 to 24, wherein the population of microorganisms is selected from the group consisting of algae, fungi, bacteria and protists.

26. 26. The method of claim 25, wherein the population of microorganisms is selected from the genera Thraustochytrium, Schizochytrium, and mixtures thereof.

27. 27. The method of claim 26, wherein the population of microorganisms is a species of the genus Thraustochytrium deposited under ATCC Accession No. PTA-6245.

28. 28. The method of any one of claims 1 to 27, wherein the lipid comprises docosahexaenoic acid.

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