US20170049132A1 - Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors - Google Patents

Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors Download PDF

Info

Publication number
US20170049132A1
US20170049132A1 US15/344,960 US201615344960A US2017049132A1 US 20170049132 A1 US20170049132 A1 US 20170049132A1 US 201615344960 A US201615344960 A US 201615344960A US 2017049132 A1 US2017049132 A1 US 2017049132A1
Authority
US
United States
Prior art keywords
biomass
microorganisms
lipids
fermentation medium
fatty acids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US15/344,960
Inventor
Richard B. Bailey
Don DiMasi
Jon Milton Hansen
Peter J. MIRRASOUL, III
Craig M. Ruecker
George T. Veeder
Tatsuo Kaneko
William R. Barclay
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=22653135&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20170049132(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by DSM IP Assets BV filed Critical DSM IP Assets BV
Priority to US15/344,960 priority Critical patent/US20170049132A1/en
Publication of US20170049132A1 publication Critical patent/US20170049132A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi

Definitions

  • U.S. application Ser. No. 11/352,421, filed 2006 Apr. 10 is a Continuation of U.S. application Ser. No. 10/371,394, filed 2003 Feb. 21, now Abandoned.
  • U.S. application Ser. No. 10/371,394, filed 2003 Feb. 21 is a Continuation of U.S. application Ser. No. 09/771,352, filed 2001 Jan. 26, now U.S. Pat. No. 6,607,900, issued 2011 Dec. 28.
  • U.S. application Ser. No. 09/771,352, filed 2001 Jan. 26 is a Non-Provisional of U.S. application No. 60/178,588, filed 2000 Jan. 28, now Expired.
  • the present invention is directed to a novel process for growing microorganisms and recovering microbial lipids.
  • the present invention is directed to producing microbial polyunsaturated lipids.
  • polyenoic fatty acids fatty acids containing 2 or more unsaturated carbon-carbon bonds
  • eukaryotic microorganisms Production of polyenoic fatty acids (fatty acids containing 2 or more unsaturated carbon-carbon bonds) in eukaryotic microorganisms is generally known to require the presence of molecular oxygen (i.e., aerobic conditions). This is because it is believed that the cis double bond formed in the fatty acids of all non-parasitic eukaryotic microorganisms involves a direct oxygen-dependent desaturation reaction (oxidative microbial desaturase systems).
  • Eukaryotic microbes such as algae; fungi, including yeast; and protists
  • Eukaryotic microbes have been demonstrated to be good producers of polyenoic fatty acids in fermentors.
  • very high density cultivation greater than about 100 g/L microbial biomass, especially at commercial scale
  • polyenoic fatty acid contents can lead to decreased polyenoic fatty acid contents and hence decreased polyenoic fatty acid productivity. This may be due in part to several factors including the difficulty of maintaining high dissolved oxygen levels due to the high oxygen demand developed by the high concentration of microbes in the fermentation broth.
  • Methods to maintain higher dissolved oxygen level include increasing the aeration rate and/or using pure oxygen instead of air for aeration and/or increasing the agitation rate in the fermentor.
  • Microbial cell breakage is an increased problem in cells that have undergone nitrogen limitation or depletion to induce lipid formation, resulting in weaker cell walls.
  • lipid producing eukaryotic microbes when lipid producing eukaryotic microbes are grown at very high cell concentrations, their lipids generally contain only very small amounts of polyenoic fatty acids.
  • the yeast Lipomyces starkeyi has been grown to a density of 153 g/L with resulting lipid concentration of 83 g/L in 140 hours using alcohol as a carbon source.
  • the polyenoic fatty acid content of the yeast at concentration greater than 100 g/L averaged only 4.2% of total fatty acids (dropping from a high of 11.5% of total fatty acid at a cell density of 20-30 g/L). Yamauchi et al., J. Ferment. Technol., 1983, 61, 275-280.
  • Rhodotorula glutinus has been demonstrated to have a lipid productivity of about 0.49 g/L/hr, but also a low overall polyenoic fatty acid content in its lipids (15.8% of total fatty acids, 14.7% C18:2 and 1.2% C18:3) resulting in a polyenoic fatty acid productivity in fed-batch culture of only about 0.047 g/L/hr and 0.077 g/L/hr in continuous culture.
  • Thraustochytriales can be excellent producers of polyenoic fatty acids in fermentors, especially when grown at low salinity levels and especially at very low chloride levels.
  • Others have described Thraustochyrids which exhibit a polyenoic fatty acid (DHA, C22 6n-3, and DPA, C22 5n-6) productivity of about 0.158 g/L/hr, when grown to cell density of 59 g/L/hr in 120 hours.
  • DHA polyenoic fatty acid
  • C22 6n-3 DPA, C22 5n-6
  • productivity productivity was only achieved at a salinity of about 50% seawater, a concentration that would cause serious corrosion in conventional stainless steel fermentors.
  • the present invention provides a process for growing eukaryotic microorganisms which are capable of producing at least about 20% of their biomass as lipids and a method for producing the lipids.
  • the lipids contain one or more polyenoic fatty acids.
  • the process comprises adding to a fermentation medium comprising eukaryotic microorganisms a carbon source, preferably a non-alcoholic carbon source, and a nitrogen source.
  • the carbon source and the nitrogen source are added at a rate sufficient to increase the biomass density of the fermentation medium to at least about 100 g/L.
  • the fermentation condition comprises a biomass density increasing stage and a lipid production stage, wherein the biomass density increasing stage comprises adding the carbon source and the nitrogen source, and the lipid production stage comprises adding the carbon source without adding the nitrogen source to induce nitrogen limiting conditions which induces lipid production.
  • the amount of dissolved oxygen present in the fermentation medium during the lipid production stage is lower than the amount of dissolved oxygen present in the fermentation medium during the biomass density increasing stage.
  • microorganisms are selected from the group consisting of algae, fungi, protists, and mixtures thereof, wherein the microorganisms are capable of producing polyenoic fatty acids or other lipids which requires molecular oxygen for their synthesis.
  • a particularly useful microorganisms of the present invention are eukaryotic microorganisms which are capable of producing lipids at a fermentation medium oxygen level of about less than 3% of saturation.
  • microorganisms are grown in a fed-batch process. Moreover,
  • Yet still another aspect of the present invention provides maintaining an oxygen level of less than about 3% of saturation in the fermentation medium during second half of the fermentation process.
  • lipids wherein greater than about 15% of said lipids are polyunsaturated lipids.
  • Another aspect of the present invention provides a lipid recovery step which comprises:
  • the water removal step comprises contacting the fermentation medium directly on a drum-dryer without prior centrifugation.
  • FIG. 1 is a table and a plot of various lipid production parameters of a microorganism versus the amount of dissolved oxygen level in a fermentation medium.
  • microorganisms such as, for example, fungi (including yeast), algae, and protists.
  • microorganisms are selected from the group consisting of algae, protists and mixtures thereof More preferably, microorganisms are algae.
  • the process of the present invention can be used to produce a variety of lipid compounds, in particular unsaturated lipids, preferably polyunsaturated lipids (i.e., lipids containing at least 2 unsaturated carbon-carbon bonds, e.g., double bonds), and more preferably highly unsaturated lipids (i.e., lipids containing 4 or more unsaturated carbon-carbon bonds) such as omega-3 and/or omega-6 polyunsaturated fatty acids, including docosahexaenoic acid (i.e., DHA); and other naturally occurring unsaturated, polyunsaturated and highly unsaturated compounds.
  • unsaturated lipids preferably polyunsaturated lipids (i.e., lipids containing at least 2 unsaturated carbon-carbon bonds, e.g., double bonds), and more preferably highly unsaturated lipids (i.e., lipids containing 4 or more unsaturated carbon-carbon bonds)
  • lipid includes phospholipids; free fatty acids; esters of fatty acids; triacylglycerols; sterols and sterol esters; carotenoids; xanthophyls (e.g, oxycarotenoids); hydrocarbons; and other lipids known to one of ordinary skill in the art.
  • processes of the present invention are useful in producing eukaryotic microbial polyenoic fatty acids, carotenoids, fungal sterols, phytosterols, xanthophyls, ubiquinones, other compounds which require oxygen for producing unsaturated carbon-carbon bonds (i.e., aerobic conditions), and secondary metabolites thereof
  • processes of the present invention are useful in growing microorganisms which produce polyenoic fatty acid(s) and for producing microbial polyenoic fatty acid(s).
  • processes of the present invention can be used to grow a wide variety of microorganisms and to obtain polyunsaturated lipid containing compounds produced by the same, for the sake of brevity, convenience and illustration, this detailed description of the invention will discuss processes for growing microorganisms which are capable of producing lipids comprising omega-3 and/or omega-6 polyunsaturated fatty acids, in particular microorganisms which are capable of producing DHA.
  • Thraustochytrids of the order Thraustochytriales more specifically Thraustochytriales of the genus Thraustochytrium and Schizochytrium, including Thraustochytriales which are disclosed in commonly assigned U.S. Pat. Nos. 5,340,594 and 5,340,742, both issued to Barclay, all of which are incorporated herein by reference in their entirety.
  • processes of the present invention produces biomass density of at least about 100 g/L, more preferably at least about 130 g/L, still more preferably at least about 150 g/L, yet still more preferably at least about 170 g/L, and most preferably greater than 200 g/L.
  • biomass density at least about 100 g/L, more preferably at least about 130 g/L, still more preferably at least about 150 g/L, yet still more preferably at least about 170 g/L, and most preferably greater than 200 g/L.
  • Processes of the present invention for growing microorganisms of the order Thraustochytriales include adding a source of carbon and a source of nitrogen to a fermentation medium comprising the microorganisms at a rate sufficient to increase the biomass density of the fermentation medium to those described above.
  • This fermentation process where a substrate (e.g., a carbon source and a nitrogen source) is added in increments, is generally referred to as a fed-batch fermentation process. It has been found that when the substrate is added to a batch fermentation process the large amount of carbon source present (e.g., about 200 g/L or more per 60 g/L of biomass density) had a detrimental effect on the microorganisms.
  • Processes of the present invention for growing microorganisms can include a biomass density increasing stage.
  • the primary objective of the fermentation process is to increase the biomass density in the fermentation medium to obtain the biomass density described above.
  • the rate of carbon source addition is typically maintained at a particular level or range which does not cause a significant detrimental effect on productivity of microorganisms.
  • An appropriate range of the amount of carbon source needed for a particular microorganism during a fermentation process is well known to one of ordinary skill in the art.
  • a carbon source of the present invention is a non-alcoholic carbon source, i.e., carbon source that does not contain alcohol.
  • an “alcohol” refers to a compound having 4 or less carbon atoms with one hydroxy group, e.g., methanol, ethanol and isopropanol. More preferably, a carbon source of the present invention is a carbohydrate, including, but not limited to, fructose, glucose, sucrose, molasses, and starch. Other suitable simple and complex carbon sources and nitrogen sources are disclosed in the above-referenced patents. Typically, however, a carbohydrate, preferably corn syrup, is used as the primary carbon source.
  • a particularly preferred nitrogen source is inorganic ammonium salt, more preferably ammonium salts of sulfate, hydroxide, and most preferably ammonium hydroxide.
  • ammonium When ammonium is used as a nitrogen source the fermentation medium becomes acidic if it is not controlled by base addition or buffers. When ammonium hydroxide is used as the primary nitrogen source, it can also be used to provide a pH control.
  • the microorganisms of the order Thraustochytriales, in particular Thraustochytriales of the genus Thraustochytrium and Schizochytrium will grow over a wide pH range, eg., from about pH 5 to about pH 11. A proper pH range for fermentation of a particular microorganism is within the knowledge of one skilled in the art.
  • Processes of the present invention for growing microorganisms can also include a production stage.
  • the primary use of the substrate by the microorganisms is not increasing the biomass density but rather using the substrate to produce lipids.
  • lipids are also produced by the microorganisms during the biomass density increasing stage; however, as stated above, the primary goal in the biomass density increasing stage is to increase the biomass density.
  • the addition of the nitrogen substrate is reduced or preferably stopped.
  • the dissolved oxygen level in the fermentation medium during the biomass density increasing stage is at least about 8% of saturation, and preferably at least about 4% of saturation
  • the dissolved oxygen level in the fermentation medium is reduced to about 3% of saturation or less, preferably about 1% of saturation or less, and more preferably about 0% of saturation.
  • the amount of dissolved oxygen level in the fermentation medium is varied during the fermentation process. For example, for a fermentation process with total fermentation time of from about 90 hours to about 100 hours, the dissolved oxygen level in the fermentation medium is maintained at about 8% during the first 24 hours, about 4% from about 24 th hour to about 40 th hour, and about 0.5% or less from about 40 th hour to the end of the fermentation process.
  • the amount of dissolved oxygen present in the fermentation medium can be controlled by controlling the amount of oxygen in the head-space of the fermentor, or preferably by controlling the speed at which the fermentation medium is agitated (or stirred). For example, a high agitation (or stirring) rate results in a relatively higher amount of dissolved oxygen in the fermentation medium than a low agitation rate.
  • the agitation rate is set at from about 50 rpm to about 70 rpm during the first 12 hours, from about 55 rpm to about 80 rpm during about 12 th hour to about 18 th hour and from about 70 rpm to about 90 rpm from about 18 th hour to the end of the fermentation process to achieve the dissolved oxygen level discussed above for a total fermentation process time of from about 90 hours to about 100 hours.
  • a particular range of agitation speeds needed to achieve a particular amount of dissolved oxygen in the fermentation medium can be readily determined by one of ordinary skill in the art.
  • a preferred temperature for processes of the present invention is at least about 20° C., more preferably at least about 25° C., and most preferably at least about 30° C. It should be appreciated that cold water can retain a higher amount of dissolved oxygen than warm water. Thus, a higher fermentation medium temperature has additional benefit of reducing the amount of dissolved oxygen, which is particularly desired as described above.
  • Certain microorganisms may require a certain amount of saline minerals in the fermentation medium. These saline minerals, especially chloride ions, can cause corrosion of the fermentor and other downstream processing equipment. To prevent or reduce these undesired effects due to a relatively large amount of chloride ions present in the fermentation medium, processes of the present invention can also include using non-chloride containing sodium salts, preferably sodium sulfate, in the fermentation medium as a source of saline (i.e., sodium). More particularly, a significant portion of the sodium requirements of the fermentation are supplied as non-chloride containing sodium salts.
  • non-chloride containing sodium salts preferably sodium sulfate
  • less than about 75% of the sodium in the fermentation medium is supplied as sodium chloride, more preferably less than about 50% and more preferably less than about 25%.
  • the microorganisms of the present invention can be grown at chloride concentrations of less than about 3 g/L, more preferably less than about 500 mg/L, more preferably less than about 250 mg/L and more preferably between about 60 mg/L and about 120 mg/L.
  • Non-chloride containing sodium salts can include soda ash (a mixture of sodium carbonate and sodium oxide), sodium carbonate, sodium bicarbonate, sodium sulfate and mixtures thereof, and preferably include sodium sulfate. Soda ash, sodium carbonate and sodium bicarbonate tend to increase the pH of the fermentation medium, thus requiring control steps To maintain the proper pH of the medium.
  • the concentration of sodium sulfate is effective to meet the salinity requirements of the microorganisms, preferably the sodium concentration is (expressed as g/L of Na) at least about 1 g/L, more preferably in the range of from about 1 g/L to about 50 g/L and more preferably in the range of from about 2.0 g/L to about 25 g/L.
  • the microorganisms are recovered in a dry form from the fermentation medium by evaporating water from the fermentation medium, for example, by contacting the fermentation medium directly (i.e, without pre-concentration, for example, by centrifugation) with a dryer such as a drum-dryer apparatus, i.e., a direct dram-dryer recovery process.
  • a dryer such as a drum-dryer apparatus, i.e., a direct dram-dryer recovery process.
  • a steam heated drum-dryer is employed.
  • the biomass density of the fermentation medium is preferably at least about 130 g/L, more preferably at least about 150 g/L, and most preferably at least about 180 g/L.
  • This high biomass density is generally desired for the direct drum-dryer recovery process because at a lower biomass density, the fermentation medium comprises a sufficient amount of water to cool the drum significantly, thus resulting in incomplete drying of microorganisms.
  • Other methods of drying cells, including spray-drying, are well known to one of ordinary skill in the art.
  • Processes of the present invention provide a lipid production rate of at least about 0.5 g/L/hr, preferably at least about 0.7 g/L/hr, more preferably at least about 0.9 g/L/hr, and most preferably at least about 1.0 g/L/hr.
  • lipids produced by processes of the present invention contain polyunsaturated lipids in the amount greater than about 15%, preferably greater than about 20%, more preferably greater than about 25%, still more preferably greater than about 30%, and most preferably greater than about 35%.
  • Lipids can be recovered from either dried microorganisms or from the microorganisms in the fermentation medium.
  • At least about 20% of the lipids produced by the microorganisms in the processes of the present invention are omega-3 and/or omega-6 polyunsaturated fatty acids, preferably at least about 30% of the lipids are omega-3 and/or omega-6 polyunsaturated fatty acids, more preferably at least about 40% of the lipids are omega-3 and/or omega-6 polyunsaturated fatty acids, and most preferably at least about 50% of the lipids are omega-3 and/or omega-6 polyunsaturated fatty acids.
  • processes of the present invention provides a DHA production rate of at least about 0.2 g of DHA/L/hr, preferably at least about 0.3 g of DHA/L/hr, more preferably at least about 0.4 g of DHA/L/hr, and most preferably at least about 0.5 g of DHA/L/hr.
  • at least about 25% of the lipid is DHA (based on total fatty acid methyl ester), preferably at least about 30%, more preferably at least about 35%, and most preferably at least about 40%.
  • Microorganisms, lipids extracted therefrom, the biomass remaining after lipid extraction or combinations thereof can be used directly as a food ingredient, such as an ingredient in beverages, sauces, dairy based foods (such as milk, yogurt, cheese and ice-cream) and baked goods; nutritional supplement (in capsule or tablet forms); feed or feed supplement for any animal whose meat or products are consumed by humans; food supplement, including baby food and infant formula; and pharmaceuticals (in direct or adjunct therapy application).
  • the term “animal” means any organism belonging to the kingdom Animalia and includes, without limitation, any animal from which poultry meat, seafood, beef, pork or lamb is derived. Seafood is derived from, without limitation, fish, shrimp and shellfish.
  • product includes any product other than meat derived from such animals, including, without limitation, eggs, milk or other products.
  • polyunsaturated lipids can be incorporated into the flesh, milk, eggs or other products of such animals to increase their content of these lipids.
  • the strain of Schizochytrium used in these examples produces two primary polyenoic acids, DHAn-3 and DPAn-6 in the ratio of generally about 3:1, and small amounts of other polyenoic acids, such as EPA and C20:3, under a wide variety of fermentation conditions.
  • DHAn-3 and DPAn-6 in the ratio of generally about 3:1
  • other polyenoic acids such as EPA and C20:3
  • This example illustrates the affect of oxygen content in a fermentation medium on lipid productivity.
  • Microorganisms were produced using fermentors with a nominal working volume of 1,200 gallons. The resulting fermentation broth was concentrated and microorganisms were dried using a drum-dryer. Lipids from aliquots of the resulting microorganisms were extracted and purified to produce a refined, bleached, and deodorized oil. Approximately 3,000 ppm of d-l- ⁇ -tocopheryl acetate was added for nutritional supplementation purposes prior to analysis of the lipid.
  • Corn syrup was fed until the volume in the fermentor reached about 1,200 gallons, at which time the corn syrup addition was stopped. The fermentation process was stopped once the residual sugar concentration fell below 5 g/L. The typical age, from inoculation to final, was about 100 hours.
  • the fermentation broth i.e., fermentation medium
  • the concentrated cell paste was heated to 160° F. (about 71° C.) and dried on a Blaw Knox double-drum dryer (42′′ ⁇ 36′′).
  • microorganisms are dried directly on a drum-dryer without prior centrifugation.
  • the fermentation medium used throughout the Examples section includes the following ingredients, where the first number indicates nominal target concentration and the number in parenthesis indicates acceptable range: sodium sulfate 12 g/L (11-13); KCl 0.5 g/L (0.45-0.55); MgSO 4 .7H 2 O 2 g/L (1.8-2.2); Hodag K-60 antifoam 0.35 g/L (0.3-0.4); K 2 SO 4 0.65 g/L (0.60-0.70); KH 2 PO 4 1 g/L (0.9-1.1); (NH 4 ) 2 SO 4 1 g/L (0.951.1); CaCl 2 .2H 2 O 0.17 g/L (0.15-0.19); 95 DE corn syrup (solids basis) 4.5 g/L (2-10); MnCl 2 .4H 2 O 3 mg/L (2.7-3.3); ZnSO 4 .7H 2 O 3 mg/L (2.7-3.3); CoCl 2 .6H 2 O 0.04 mg/L (0.035-
  • the ash content of the dried microorganisms is about 6% by weight.
  • This example illustrates the effect of reduced dissolved oxygen level in the fermentation medium on the productivity of microorganisms using G-tank scale.
  • Example 2 Using the procedure described in Example 2, a 14,000 gallon nominal volume fermentation was conducted using a wild-type strain Schizochytrium, which can be obtained using isolation processes disclosed in the above mentioned U.S. Pat. Nos. 5,340,594 and 5,340,742.
  • the dissolved oxygen level in the fermentation medium was about 8% during the first 24 hours, about 4% from the 24 th hour to the 40 th hour and about 0.5% from the 40 th hour to the end of fermentation process. Results of this lower dissolved oxygen level in fermentation medium processes are shown in Table 3.
  • This example illustrates the effect of reduced dissolved oxygen level in the fermentation medium on the productivity of microorganisms on a 41,000 gallon scale.
  • This example illustrates the affect of extra nitrogen on the fermentation process of the present invention.
  • This example shows a kinetic profile of a fermentation process of the present invention.
  • a 1000 gallon scale fed-batch experiment was conducted using a procedure similar to Example 3.
  • Kinetic profile of the fermentation process is shown in Table 6.
  • This example illustrates effect of the amount of carbon source on productivity.
  • the present invention in various embodiments, includes components, methods, processes, systems and/or apparatus substantially as depicted and described herein, including various embodiments, subcombinations, and subsets thereof. Those of skill in the art will understand how to make and use the present invention after understanding the present disclosure.
  • the present invention in various embodiments, includes providing devices and processes in the absence of items not depicted and/or described herein or in various embodiments hereof, including in the absence of such items as may have been used in previous devices or processes, e.g., for improving performance, achieving ease and ⁇ or reducing cost of implementation.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Polymers & Plastics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Nutrition Science (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Birds (AREA)
  • Insects & Arthropods (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Obesity (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention provides a process for growing eukaryotic microorganisms which are capable of producing lipids, in particular lipids containing polyenoic fatty acids. The present invention also provides a process for producing eukaryotic microbial lipids.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • The present application is a Continuation of U.S. application Ser. No. 11/745,490, filed 2007 May 8, Ser. No. 11/745,498, filed 2007 May 8, Ser. No. 11/745,500, filed 2007 May 8, Ser. No. 11/745,502, filed 2007 May 8, Ser. No. 11/745,506, filed 2007 May 8, 11/745,511, filed 2007 May 8, Ser. No. 11/745,513, filed 2007 May 8, Ser. No. 11/745,526, filed 2007 May 8, Ser. No. 11/745,531, filed 2007 May 8, and Ser. No. 11/745,533, filed 2007 May 8, currently pending. U.S. application Ser. No. 11/745,490, filed 2007 May 8, Ser. No. 11/745,498, filed 2007 May 8, Ser. No. 11/745,500, filed 2007 May 8, Ser. No. 11/745,502, filed 2007 May 8, and Ser. No. 11/745,506, filed 2007 May 8 are Continuations of U.S. application Ser. No. 11/352,421, filed 2006 Apr. 10, now U.S. Pat. No. 7,579,174, issued 2011 Dec. 28. U.S. application Ser. No. 11/745,511, filed 2007 May 8, Ser. No. 11/745,513, filed 2007 May 8, Ser. No. 11/745,526, filed 2007 May 8, Ser. No. 11/745,531, filed 2007 May 8, and Ser. No. 11/745,533, filed 2007 May 8 are Divisionals of U.S. application Ser. No. 11/352,421, filed 2006 Apr. 10, now U.S. Pat. No. 7,579,174, issued 2011 Dec. 28. U.S. application Ser. No. 11/352,421, filed 2006 Apr. 10 is a Continuation of U.S. application Ser. No. 10/371,394, filed 2003 Feb. 21, now Abandoned. U.S. application Ser. No. 10/371,394, filed 2003 Feb. 21 is a Continuation of U.S. application Ser. No. 09/771,352, filed 2001 Jan. 26, now U.S. Pat. No. 6,607,900, issued 2011 Dec. 28. U.S. application Ser. No. 09/771,352, filed 2001 Jan. 26 is a Non-Provisional of U.S. application No. 60/178,588, filed 2000 Jan. 28, now Expired.
    • FIELD OF THE INVENTION
  • The present invention is directed to a novel process for growing microorganisms and recovering microbial lipids. In particular, the present invention is directed to producing microbial polyunsaturated lipids.
    • BACKGROUND OF THE INVENTION
  • Production of polyenoic fatty acids (fatty acids containing 2 or more unsaturated carbon-carbon bonds) in eukaryotic microorganisms is generally known to require the presence of molecular oxygen (i.e., aerobic conditions). This is because it is believed that the cis double bond formed in the fatty acids of all non-parasitic eukaryotic microorganisms involves a direct oxygen-dependent desaturation reaction (oxidative microbial desaturase systems). Other eukaryotic microbial lipids that are known to require molecular oxygen include fungal and plant sterols, oxycarotenoids (i.e., xanthophyls), ubiquinones, and compounds made from any of these lipids (i.e., secondary metabolites).
  • Eukaryotic microbes (such as algae; fungi, including yeast; and protists) have been demonstrated to be good producers of polyenoic fatty acids in fermentors. However, very high density cultivation (greater than about 100 g/L microbial biomass, especially at commercial scale) can lead to decreased polyenoic fatty acid contents and hence decreased polyenoic fatty acid productivity. This may be due in part to several factors including the difficulty of maintaining high dissolved oxygen levels due to the high oxygen demand developed by the high concentration of microbes in the fermentation broth. Methods to maintain higher dissolved oxygen level include increasing the aeration rate and/or using pure oxygen instead of air for aeration and/or increasing the agitation rate in the fermentor. These solutions generally increase the cost of lipid production and can cause additional problems. For example, increased aeration can easily lead to severe foaming problems in the fermentor at high cell densities and increased mixing can lead to microbial cell breakage due to increased shear forces in the fermentation broth (this causes the lipids to be released in the fermentation broth where they can become oxidized and/or degraded by enzymes). Microbial cell breakage is an increased problem in cells that have undergone nitrogen limitation or depletion to induce lipid formation, resulting in weaker cell walls.
  • As a result, when lipid producing eukaryotic microbes are grown at very high cell concentrations, their lipids generally contain only very small amounts of polyenoic fatty acids. For example, the yeast Lipomyces starkeyi has been grown to a density of 153 g/L with resulting lipid concentration of 83 g/L in 140 hours using alcohol as a carbon source. Yet the polyenoic fatty acid content of the yeast at concentration greater than 100 g/L averaged only 4.2% of total fatty acids (dropping from a high of 11.5% of total fatty acid at a cell density of 20-30 g/L). Yamauchi et al., J. Ferment. Technol., 1983, 61, 275-280. This results in a polyenoic fatty acid concentration of only about 3.5 g/L and a polyenoic fatty acid productivity of only about 0.025 g/L/hr. Additionally, the only polyenoic fatty acid reported in the yeast lipids was C18:2.
  • Another yeast, Rhodotorula glutinus, has been demonstrated to have a lipid productivity of about 0.49 g/L/hr, but also a low overall polyenoic fatty acid content in its lipids (15.8% of total fatty acids, 14.7% C18:2 and 1.2% C18:3) resulting in a polyenoic fatty acid productivity in fed-batch culture of only about 0.047 g/L/hr and 0.077 g/L/hr in continuous culture.
  • Present inventors have previously demonstrated that certain marine microalgae in the order Thraustochytriales can be excellent producers of polyenoic fatty acids in fermentors, especially when grown at low salinity levels and especially at very low chloride levels. Others have described Thraustochyrids which exhibit a polyenoic fatty acid (DHA, C22 6n-3, and DPA, C22 5n-6) productivity of about 0.158 g/L/hr, when grown to cell density of 59 g/L/hr in 120 hours. However, this productivity was only achieved at a salinity of about 50% seawater, a concentration that would cause serious corrosion in conventional stainless steel fermentors.
  • Costs of producing microbial lipids containing polyenoic fatty acids, and especially the highly unsaturated fatty acids, such as C18 4n-3, C20 4n-6, C20 5n3, C22:5n-3, C22:5n-6 and C22:6n-3, have remained high in part due to the limited densities to which the high polyenoic fatty acid containing eukaryotic microbes have been grown and the limited oxygen availability both at these high cell concentrations and the higher temperatures needed to achieve high productivity.
  • Therefore, there is a need for a process for growing microorganisms at high concentration which still facilitates increased production of lipids containing polyenoic fatty acids.
    • SUMMARY OF THE INVENTION
  • The present invention provides a process for growing eukaryotic microorganisms which are capable of producing at least about 20% of their biomass as lipids and a method for producing the lipids. Preferably the lipids contain one or more polyenoic fatty acids. The process comprises adding to a fermentation medium comprising eukaryotic microorganisms a carbon source, preferably a non-alcoholic carbon source, and a nitrogen source. Preferably, the carbon source and the nitrogen source are added at a rate sufficient to increase the biomass density of the fermentation medium to at least about 100 g/L.
  • In one aspect of the present invention, the fermentation condition comprises a biomass density increasing stage and a lipid production stage, wherein the biomass density increasing stage comprises adding the carbon source and the nitrogen source, and the lipid production stage comprises adding the carbon source without adding the nitrogen source to induce nitrogen limiting conditions which induces lipid production.
  • In another aspect of the present invention, the amount of dissolved oxygen present in the fermentation medium during the lipid production stage is lower than the amount of dissolved oxygen present in the fermentation medium during the biomass density increasing stage.
  • In yet another aspect of the present invention, microorganisms are selected from the group consisting of algae, fungi, protists, and mixtures thereof, wherein the microorganisms are capable of producing polyenoic fatty acids or other lipids which requires molecular oxygen for their synthesis. A particularly useful microorganisms of the present invention are eukaryotic microorganisms which are capable of producing lipids at a fermentation medium oxygen level of about less than 3% of saturation.
  • In still another aspect of the present invention, microorganisms are grown in a fed-batch process. Moreover,
  • Yet still another aspect of the present invention provides maintaining an oxygen level of less than about 3% of saturation in the fermentation medium during second half of the fermentation process.
  • Another embodiment of the present invention provides a process for producing eukaryotic microbial lipids comprising:
      • (a) growing eukaryotic microorganisms in a fermentation medium to increase the biomass density of said fermentation medium to at least about 100 g/L;
      • (b) providing a fermentation condition sufficient to allow said microorganisms to produce said lipids; and
      • (c) recovering said lipids,
  • wherein greater than about 15% of said lipids are polyunsaturated lipids.
  • Another aspect of the present invention provides a lipid recovery step which comprises:
      • (a) removing water from said fermentation medium to provide dry microorganisms; and
      • (b) isolating said lipids from said dry microorganisms.
  • Preferably, the water removal step comprises contacting the fermentation medium directly on a drum-dryer without prior centrifugation.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a table and a plot of various lipid production parameters of a microorganism versus the amount of dissolved oxygen level in a fermentation medium.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides a process for growing microorganisms, such as, for example, fungi (including yeast), algae, and protists. Preferably, microorganisms are selected from the group consisting of algae, protists and mixtures thereof More preferably, microorganisms are algae. Moreover, the process of the present invention can be used to produce a variety of lipid compounds, in particular unsaturated lipids, preferably polyunsaturated lipids (i.e., lipids containing at least 2 unsaturated carbon-carbon bonds, e.g., double bonds), and more preferably highly unsaturated lipids (i.e., lipids containing 4 or more unsaturated carbon-carbon bonds) such as omega-3 and/or omega-6 polyunsaturated fatty acids, including docosahexaenoic acid (i.e., DHA); and other naturally occurring unsaturated, polyunsaturated and highly unsaturated compounds. As used herein, the term “lipid” includes phospholipids; free fatty acids; esters of fatty acids; triacylglycerols; sterols and sterol esters; carotenoids; xanthophyls (e.g, oxycarotenoids); hydrocarbons; and other lipids known to one of ordinary skill in the art.
  • More particularly, processes of the present invention are useful in producing eukaryotic microbial polyenoic fatty acids, carotenoids, fungal sterols, phytosterols, xanthophyls, ubiquinones, other compounds which require oxygen for producing unsaturated carbon-carbon bonds (i.e., aerobic conditions), and secondary metabolites thereof Specifically, processes of the present invention are useful in growing microorganisms which produce polyenoic fatty acid(s) and for producing microbial polyenoic fatty acid(s).
  • While processes of the present invention can be used to grow a wide variety of microorganisms and to obtain polyunsaturated lipid containing compounds produced by the same, for the sake of brevity, convenience and illustration, this detailed description of the invention will discuss processes for growing microorganisms which are capable of producing lipids comprising omega-3 and/or omega-6 polyunsaturated fatty acids, in particular microorganisms which are capable of producing DHA. More particularly, preferred embodiments of the present invention will be discussed with reference to a process for growing marine microorganisms, in particular algae, such as Thraustochytrids of the order Thraustochytriales, more specifically Thraustochytriales of the genus Thraustochytrium and Schizochytrium, including Thraustochytriales which are disclosed in commonly assigned U.S. Pat. Nos. 5,340,594 and 5,340,742, both issued to Barclay, all of which are incorporated herein by reference in their entirety. It is to be understood, however, that the invention as a whole is not intended to be so limited, and that one skilled in the art will recognize that the concept of the present invention will be applicable to other microorganisms producing a variety of other compounds, including other lipid compositions, in accordance with the techniques discussed herein.
  • Assuming a relatively constant production rate of lipids by an algae, it is readily apparent that the higher biomass density will lead to a higher total amount of lipids being produced per volume. Current conventional fermentation processes for growing algae yield a biomass density of from about 50 to about 80 g/L or less. The present inventors have found that by using processes of the present invention, a significantly higher biomass density than currently known biomass density can be achieved. Preferably, processes of the present invention produces biomass density of at least about 100 g/L, more preferably at least about 130 g/L, still more preferably at least about 150 g/L, yet still more preferably at least about 170 g/L, and most preferably greater than 200 g/L. Thus, with such a high biomass density, even if the lipids production rate of algae is decreased slightly, the overall lipids production rate per volume is significantly higher than currently known processes.
  • Processes of the present invention for growing microorganisms of the order Thraustochytriales include adding a source of carbon and a source of nitrogen to a fermentation medium comprising the microorganisms at a rate sufficient to increase the biomass density of the fermentation medium to those described above. This fermentation process, where a substrate (e.g., a carbon source and a nitrogen source) is added in increments, is generally referred to as a fed-batch fermentation process. It has been found that when the substrate is added to a batch fermentation process the large amount of carbon source present (e.g., about 200 g/L or more per 60 g/L of biomass density) had a detrimental effect on the microorganisms. Without being bound by any theory, it is believed that such a high amount of carbon source causes detrimental effects, including osmotic stress, for microorganisms and inhibits initial productivity of microorganisms. Processes of the present invention avoid this undesired detrimental effect while providing a sufficient amount of the substrate to achieve the above described biomass density of the microorganisms.
  • Processes of the present invention for growing microorganisms can include a biomass density increasing stage. In the biomass density increasing stage, the primary objective of the fermentation process is to increase the biomass density in the fermentation medium to obtain the biomass density described above. The rate of carbon source addition is typically maintained at a particular level or range which does not cause a significant detrimental effect on productivity of microorganisms. An appropriate range of the amount of carbon source needed for a particular microorganism during a fermentation process is well known to one of ordinary skill in the art. Preferably, a carbon source of the present invention is a non-alcoholic carbon source, i.e., carbon source that does not contain alcohol. As used herein, an “alcohol” refers to a compound having 4 or less carbon atoms with one hydroxy group, e.g., methanol, ethanol and isopropanol. More preferably, a carbon source of the present invention is a carbohydrate, including, but not limited to, fructose, glucose, sucrose, molasses, and starch. Other suitable simple and complex carbon sources and nitrogen sources are disclosed in the above-referenced patents. Typically, however, a carbohydrate, preferably corn syrup, is used as the primary carbon source.
  • A particularly preferred nitrogen source is inorganic ammonium salt, more preferably ammonium salts of sulfate, hydroxide, and most preferably ammonium hydroxide.
  • When ammonium is used as a nitrogen source the fermentation medium becomes acidic if it is not controlled by base addition or buffers. When ammonium hydroxide is used as the primary nitrogen source, it can also be used to provide a pH control. The microorganisms of the order Thraustochytriales, in particular Thraustochytriales of the genus Thraustochytrium and Schizochytrium will grow over a wide pH range, eg., from about pH 5 to about pH 11. A proper pH range for fermentation of a particular microorganism is within the knowledge of one skilled in the art.
  • Processes of the present invention for growing microorganisms can also include a production stage. In this stage, the primary use of the substrate by the microorganisms is not increasing the biomass density but rather using the substrate to produce lipids. It should be appreciated that lipids are also produced by the microorganisms during the biomass density increasing stage; however, as stated above, the primary goal in the biomass density increasing stage is to increase the biomass density. Typically, during the production stage the addition of the nitrogen substrate is reduced or preferably stopped.
  • It was previously generally believed that the presence of dissolved oxygen in the fermentation medium is crucial in the production of polyunsaturated compounds by eukaryotic microorganisms including omega-3 and/or omega-6 polyunsaturated fatty acids. Thus, a relatively large amount of dissolved oxygen in the fermentation medium was generally believed to be preferred. Surprisingly and unexpectedly, however, the present inventors have found that the production rate of lipids is increased dramatically when the dissolved oxygen level during the production stage is reduced. Thus, while the dissolved oxygen level in the fermentation medium during the biomass density increasing stage is at least about 8% of saturation, and preferably at least about 4% of saturation, during the production stage the dissolved oxygen level in the fermentation medium is reduced to about 3% of saturation or less, preferably about 1% of saturation or less, and more preferably about 0% of saturation. In one particular embodiment of the present invention, the amount of dissolved oxygen level in the fermentation medium is varied during the fermentation process. For example, for a fermentation process with total fermentation time of from about 90 hours to about 100 hours, the dissolved oxygen level in the fermentation medium is maintained at about 8% during the first 24 hours, about 4% from about 24th hour to about 40th hour, and about 0.5% or less from about 40th hour to the end of the fermentation process.
  • The amount of dissolved oxygen present in the fermentation medium can be controlled by controlling the amount of oxygen in the head-space of the fermentor, or preferably by controlling the speed at which the fermentation medium is agitated (or stirred). For example, a high agitation (or stirring) rate results in a relatively higher amount of dissolved oxygen in the fermentation medium than a low agitation rate. For example, in a fermentor of about 14,000 gallon capacity the agitation rate is set at from about 50 rpm to about 70 rpm during the first 12 hours, from about 55 rpm to about 80 rpm during about 12th hour to about 18th hour and from about 70 rpm to about 90 rpm from about 18th hour to the end of the fermentation process to achieve the dissolved oxygen level discussed above for a total fermentation process time of from about 90 hours to about 100 hours. A particular range of agitation speeds needed to achieve a particular amount of dissolved oxygen in the fermentation medium can be readily determined by one of ordinary skill in the art.
  • A preferred temperature for processes of the present invention is at least about 20° C., more preferably at least about 25° C., and most preferably at least about 30° C. It should be appreciated that cold water can retain a higher amount of dissolved oxygen than warm water. Thus, a higher fermentation medium temperature has additional benefit of reducing the amount of dissolved oxygen, which is particularly desired as described above.
  • Certain microorganisms may require a certain amount of saline minerals in the fermentation medium. These saline minerals, especially chloride ions, can cause corrosion of the fermentor and other downstream processing equipment. To prevent or reduce these undesired effects due to a relatively large amount of chloride ions present in the fermentation medium, processes of the present invention can also include using non-chloride containing sodium salts, preferably sodium sulfate, in the fermentation medium as a source of saline (i.e., sodium). More particularly, a significant portion of the sodium requirements of the fermentation are supplied as non-chloride containing sodium salts. For example, less than about 75% of the sodium in the fermentation medium is supplied as sodium chloride, more preferably less than about 50% and more preferably less than about 25%. The microorganisms of the present invention can be grown at chloride concentrations of less than about 3 g/L, more preferably less than about 500 mg/L, more preferably less than about 250 mg/L and more preferably between about 60 mg/L and about 120 mg/L.
  • Non-chloride containing sodium salts can include soda ash (a mixture of sodium carbonate and sodium oxide), sodium carbonate, sodium bicarbonate, sodium sulfate and mixtures thereof, and preferably include sodium sulfate. Soda ash, sodium carbonate and sodium bicarbonate tend to increase the pH of the fermentation medium, thus requiring control steps To maintain the proper pH of the medium. The concentration of sodium sulfate is effective to meet the salinity requirements of the microorganisms, preferably the sodium concentration is (expressed as g/L of Na) at least about 1 g/L, more preferably in the range of from about 1 g/L to about 50 g/L and more preferably in the range of from about 2.0 g/L to about 25 g/L.
  • Various fermentation parameters for inoculating, growing and recovering microorganisms are discussed in detail in U.S. Pat. No. 5,130,242, which is incorporated herein by reference in its entirety. Any currently known isolation methods can be used to isolate microorganisms from the fermentation medium, including centrifugation, filtration, decantation, and solvent evaporation. It has been found by the present inventors that because of such a high biomass density resulting from processes of the present invention, when a centrifuge is used to recover the microorganisms it is preferred to dilute the fermentation medium by adding water, which reduces the biomass density, thereby allowing more effective separation of microorganisms from the fermentation medium.
  • Preferably, the microorganisms are recovered in a dry form from the fermentation medium by evaporating water from the fermentation medium, for example, by contacting the fermentation medium directly (i.e, without pre-concentration, for example, by centrifugation) with a dryer such as a drum-dryer apparatus, i.e., a direct dram-dryer recovery process. When using the direct drum-dryer recovery process to isolate microorganisms, typically a steam heated drum-dryer is employed. In addition when using the direct drum-dryer recovery process, the biomass density of the fermentation medium is preferably at least about 130 g/L, more preferably at least about 150 g/L, and most preferably at least about 180 g/L. This high biomass density is generally desired for the direct drum-dryer recovery process because at a lower biomass density, the fermentation medium comprises a sufficient amount of water to cool the drum significantly, thus resulting in incomplete drying of microorganisms. Other methods of drying cells, including spray-drying, are well known to one of ordinary skill in the art.
  • Processes of the present invention provide a lipid production rate of at least about 0.5 g/L/hr, preferably at least about 0.7 g/L/hr, more preferably at least about 0.9 g/L/hr, and most preferably at least about 1.0 g/L/hr. Moreover, lipids produced by processes of the present invention contain polyunsaturated lipids in the amount greater than about 15%, preferably greater than about 20%, more preferably greater than about 25%, still more preferably greater than about 30%, and most preferably greater than about 35%. Lipids can be recovered from either dried microorganisms or from the microorganisms in the fermentation medium. Generally, at least about 20% of the lipids produced by the microorganisms in the processes of the present invention are omega-3 and/or omega-6 polyunsaturated fatty acids, preferably at least about 30% of the lipids are omega-3 and/or omega-6 polyunsaturated fatty acids, more preferably at least about 40% of the lipids are omega-3 and/or omega-6 polyunsaturated fatty acids, and most preferably at least about 50% of the lipids are omega-3 and/or omega-6 polyunsaturated fatty acids. Alternatively, processes of the present invention provides a DHA production rate of at least about 0.2 g of DHA/L/hr, preferably at least about 0.3 g of DHA/L/hr, more preferably at least about 0.4 g of DHA/L/hr, and most preferably at least about 0.5 g of DHA/L/hr. Still alternatively, at least about 25% of the lipid is DHA (based on total fatty acid methyl ester), preferably at least about 30%, more preferably at least about 35%, and most preferably at least about 40%.
  • Microorganisms, lipids extracted therefrom, the biomass remaining after lipid extraction or combinations thereof can be used directly as a food ingredient, such as an ingredient in beverages, sauces, dairy based foods (such as milk, yogurt, cheese and ice-cream) and baked goods; nutritional supplement (in capsule or tablet forms); feed or feed supplement for any animal whose meat or products are consumed by humans; food supplement, including baby food and infant formula; and pharmaceuticals (in direct or adjunct therapy application). The term “animal” means any organism belonging to the kingdom Animalia and includes, without limitation, any animal from which poultry meat, seafood, beef, pork or lamb is derived. Seafood is derived from, without limitation, fish, shrimp and shellfish. The term “products” includes any product other than meat derived from such animals, including, without limitation, eggs, milk or other products. When fed to such animals, polyunsaturated lipids can be incorporated into the flesh, milk, eggs or other products of such animals to increase their content of these lipids.
  • Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting.
    • EXAMPLES
  • The strain of Schizochytrium used in these examples produces two primary polyenoic acids, DHAn-3 and DPAn-6 in the ratio of generally about 3:1, and small amounts of other polyenoic acids, such as EPA and C20:3, under a wide variety of fermentation conditions. Thus, while following examples only list the amount of DHA, one can readily calculate the amount of DPA produced by using the above disclosed ratio.
    • Example 1
  • This example illustrates the affect of oxygen content in a fermentation medium on lipid productivity.
  • Fermentation results of Schizochytrium ATCC No. 20888 at various levels of dissolved oxygen content were measured. The results are shown in FIG. 1, where RCS is residual concentration of sugar, and DCW is dry-cell weight.
    • Example 2
  • This example illustrates the reproducibility of processes of the present invention.
  • Microorganisms were produced using fermentors with a nominal working volume of 1,200 gallons. The resulting fermentation broth was concentrated and microorganisms were dried using a drum-dryer. Lipids from aliquots of the resulting microorganisms were extracted and purified to produce a refined, bleached, and deodorized oil. Approximately 3,000 ppm of d-l-α-tocopheryl acetate was added for nutritional supplementation purposes prior to analysis of the lipid.
  • Nine fermentations of Schizochytrium ATCC No. 20888 were run and the results are shown in Table 1. The dissolved oxygen level was about 8% during the first 24 hours and about 4% thereafter.
  • TABLE 1
    Fed-batch fermentation results for the production of DHA.
    Age Yield1 DHA FAME2
    Entry (Hrs) (g/L) (%) (%) Productivity3
    1 100.3 160.7 17.8 49.5 0.285
    2 99.8 172.4 19.4 51.3 0.335
    3 84.7 148.7 14.4 41.4 0.253
    4 90.2 169.5 19.7 53.9 0.370
    5 99.0 164.1 12.5 38.9 0.207
    6 113.0 187.1 19.7 47.2 0.326
    7 97.0 153.5 13.7 41.0 0.217
    8 92.8 174.8 16.4 48.6 0.309
    Avg4 97.1 166.4 16.7 46.5 0.288
    Std5 8.4 12.3 2.9 5.4 0.058
    CV6(%) 8.7 7.4 17.3 11.7 20.2
    1actual yield of biomass density.
    2total fatty acid methyl esters.
    3(grams of DHA)/L/Hr.
    4average.
    5standard deviation.
    6coefficients of variability. Coefficients of variability values below 5% indicates a process which has excellent reproducibility, values between 5% and 10% indicates a process which has good reproducibility and values between 10% and 20% indicates a process which has reasonable reproducibility.
  • Corn syrup was fed until the volume in the fermentor reached about 1,200 gallons, at which time the corn syrup addition was stopped. The fermentation process was stopped once the residual sugar concentration fell below 5 g/L. The typical age, from inoculation to final, was about 100 hours.
  • The fermentation broth, i.e., fermentation medium, was diluted with water using approximately a 2:1 ratio to reduce the ash content of the final product and help improve phase separation during the centrifugation step. The concentrated cell paste was heated to 160° F. (about 71° C.) and dried on a Blaw Knox double-drum dryer (42″×36″). Preferably, however, microorganisms are dried directly on a drum-dryer without prior centrifugation.
  • The analysis result of lipids extracted from aliquots of each entries in Table 1 is summarized in Table 2.
  • TABLE 2
    Analysis of lipids from microorganisms of Table 1.
    % DHA relative Total Lipid %
    Entry to FAME1 by wt.
    1 36.0 72.3
    2 37.8 70.3
    3 34.8 61.5
    4 36.5 74.8
    5 32.1 52.8
    6 41.7 67.7
    7 33.4 49.9
    8 33.7 61.4
    Avg 35.8 63.8
    Std. 3 3.0 9.1
    CV4(%) 8.5 14.2
    1see Table 1
    2see discussion above
    3standard deviation
    4coefficients of variability Coefficients of variability values below 5% indicates a process which has excellent reproducibility, values between 5% and 10% indicates a process which has good reproducibility and values between 10% and 20% indicates a process which has reasonable reproducibility.
  • Unless otherwise stated, the fermentation medium used throughout the Examples section includes the following ingredients, where the first number indicates nominal target concentration and the number in parenthesis indicates acceptable range: sodium sulfate 12 g/L (11-13); KCl 0.5 g/L (0.45-0.55); MgSO4.7H2O 2 g/L (1.8-2.2); Hodag K-60 antifoam 0.35 g/L (0.3-0.4); K2SO4 0.65 g/L (0.60-0.70); KH2PO4 1 g/L (0.9-1.1); (NH4)2SO4 1 g/L (0.951.1); CaCl2.2H2O 0.17 g/L (0.15-0.19); 95 DE corn syrup (solids basis) 4.5 g/L (2-10); MnCl2.4H2O 3 mg/L (2.7-3.3); ZnSO4.7H2O 3 mg/L (2.7-3.3); CoCl2.6H2O 0.04 mg/L (0.035-0.045); Na2 MoO4.2H2O 0.04 mg/L (0-0.045); CuSO4.5H2O 2 mg/L (1.8-2.2); NiSO4.6H2O 2 mg/L (1.8-2.2); FeSO4.7H2O 10 mg/L (9-11); thiamine 9.5 mg/L (4-15); vitamin B120.15 mg/L (0.05-0.25) and Ca1/2Pantothenate 3.2 mg/L (1.3-5.1). In addition, 28% NH4OH solution is used as the nitrogen source.
  • The ash content of the dried microorganisms is about 6% by weight.
    • Example 3
  • This example illustrates the effect of reduced dissolved oxygen level in the fermentation medium on the productivity of microorganisms using G-tank scale.
  • Using the procedure described in Example 2, a 14,000 gallon nominal volume fermentation was conducted using a wild-type strain Schizochytrium, which can be obtained using isolation processes disclosed in the above mentioned U.S. Pat. Nos. 5,340,594 and 5,340,742. The dissolved oxygen level in the fermentation medium was about 8% during the first 24 hours, about 4% from the 24th hour to the 40th hour and about 0.5% from the 40th hour to the end of fermentation process. Results of this lower dissolved oxygen level in fermentation medium processes are shown in Table 3.
  • TABLE 3
    14,000 gallon scale fermentation of Schizochytrium.
    DHA
    Produc-
    % DHA tivity
    Age Yield % % rel. to (g of
    Entry (Hrs) (g/L) DHA FAME FAME DHA/L/hr)
    1 82.0 179.3 21.7 52.4 41.4 0.474
    2 99.0 183.1 22.3 55.0 40.5 0.412
    3 72.0 159.3 — — 40.9 —
    4 77.0 161.3 — — 43.2 —
    5 100.0 173.0 23.9 53.3 44.9 0.413
    6 102.0 183.3 21.6 50.8 42.6 0.388
    7 104.0 185.1 23.7 55.0 43.1 0.422
    8 88.0 179.3 22.3 52.6 42.4 0.454
    9 100.0 166.4 22.5 53.5 42.1 0.374
    10 97.0 182.6 22.8 51.6 44.1 0.429
    11 87.5 176.5 19.8 45.6 43.5 0.399
    12 67.0 170.8 18.8 48.1 39.1 0.479
    13 97.0 184.9 23.2 52.7 44.0 0.442
    14 102.0 181.9 23.6 52.9 44.6 0.421
    15 102.0 186.9 19.9 47.8 41.8 0.365
    16 97.0 184.4 19.6 45.5 43.0 0.373
    17 98.0 174.7 19.7 45.1 43.7 0.351
    18 103.5 178.8 18.3 44.5 41.2 0.316
    19 102.0 173.7 15.8 43.1 36.7 0.269
    20 94.0 190.4 19.3 46.9 41.1 0.391
    21 72.0 172.5 22.8 52.8 43.2 0.546
    22 75.0 173.1 21.0 51.7 40.8 0.485
    23 75.0 152.7 20.3 50.3 40.4 0.413
    24 75.5 172.5 21.9 51.7 42.3 0.500
    25 61.0 156.4 17.3 45.7 37.8 0.444
    26 74.5 150.6 20.2 50.1 40.2 0.408
    27 70.5 134.3 14.8 40.6 36.6 0.282
    28 75.5 146.1 21.3 49.7 42.8 0.412
    29 82.0 174.3 21.4 50.4 42.5 0.455
    30 105.0 182.3 21.7 50.7 42.8 0.377
    31 66.0 146.2 16.4 44.6 36.7 0.363
    Avg 87.2 171.5 20.6 49.5 41.6 0.409
    Std 13.9 14.1 2.4 3.8 2.3 0.061
    CV 16.0% 8.2% 11.6% 7.7% 5.5% 15.0%
    • Example 4
  • This example illustrates the effect of reduced dissolved oxygen level in the fermentation medium on the productivity of microorganisms on a 41,000 gallon scale.
  • Same procedure as Example 3 in a 41,000 gallon fermentor was performed. Results are shown in Table 4.
  • TABLE 4
    41,000 gallon scale fermentation of Schizochytrium
    DHA
    Produc-
    % DHA tivity
    Age Yield % % rel. to (g of
    Entry (Hrs) (g/L) DHA FAME FAME DHA/L/hr)
    1 75.0 116.1 17.3 46.1 37.4 0.268
    2 99.0 159.3 17.4 47.0 37.1 0.280
    3 103.0 152.6 16.0 47.2 33.8 0.237
    4 68.0 136.8 17.9 45.9 39.1 0.360
    5 84.0 142.0 17.5 47.0 37.2 0.296
    Avg 85.8 141.4 17.2 46.6 36.9 0.288
    Std 15.1 16.6 0.7 0.6 1.9 0.046
    CV 17.5% 11.8 4.2% 1.3% 5.2% 15.8%
    • Example 5
  • This example illustrates the affect of extra nitrogen on the fermentation process of the present invention.
  • Four sets of 250-L scale fed-batch experiments were conducted using a procedure similar to Example 3. Two control experiments and two experiments containing extra ammonia (1.15× and 1.25× the normal amount) were conducted. Results are shown in Table 5.
  • TABLE 5
    Affects of extra ammonia on fermentation of Schizochytrium.
    Age Yield Biomass Conversion DHA FAME DHA
    (hrs) (g/L) Productivity Efficiency Content Content Productivity
    Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.0X NH3
    48 178 3.71 g/L/hr 51.5% 10.7% 37.8% 0.40 g/L/hr
    60 185 3.08 g/L/hr 46.9% 16.3% 47.2% 0.50 g/L/hr
    72 205 2.85 g/L/hr 45.2% 17.4% 47.4% 0.50 g/L/hr
    84 219 2.61 g/L/hr 43.8% 17.1% 45.5% 0.45 g/L/hr
    90 221 2.46 g/L/hr 44.1% 18.4% 48.9% 0.45 g/L/hr
    Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.15X NH3
    48 171 3.56 g/L/hr 55.6% 12.0% 36.3% 0.43 g/L/hr
    60 197 3.28 g/L/hr 54.6% 9.4% 38.4% 0.31 g/L/hr
    72 191 2.65 g/L/hr 52.8% 9.4% 40.0% 0.25 g/L/hr
    84 190 2.26 g/L/hr 52.5% 10.0% 42.5% 0.23 g/L/hr
    90 189 2.10 g/L/hr 52.2% 9.2% 43.3% 0.19 g/L/hr
    Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.25X NH3
    48 178 3.71 g/L/hr 56.4% 11.5% 33.7% 0.43 g/L/hr
    60 179 2.98 g/L/hr 48.6% 10.3% 36.0% 0.31 g/L/hr
    72 180 2.50 g/L/hr 48.8% 12.0% 37.6% 0.30 g/L/hr
    84 181 2.15 g/L/hr 46.1% 13.6% 40.1% 0.29 g/L/hr
    90 185 2.06 g/L/hr 45.7% 12.6% 40.7% 0.26 g/L/hr
    Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.0X NH3
    48 158 3.29 g/L/hr 55.7% 13.1% 36.5% 0.43 g/L/hr
    60 174 2.90 g/L/hr 48.9% 17.9% 39.2% 0.52 g/L/hr
    72 189 2.63 g/L/hr 45.7% 21.0% 39.4% 0.55 g/L/hr
    84 196 2.33 g/L/hr 44.1% 22.4 40.1% 0.52 g/L/hr
    90 206 2.29 g/L/hr 44.8% 22.1% 40.3% 0.51 g/L/hr

    In general, extra nitrogen has a negative effect on fermentation performance, as significant reductions were observed in the DHA productivity for the two batches where extra ammonia were added. As shown on Table 5, the control batches resulted in final DHA levels of 18.4% and 22.1% versus the 9.2% (1.15× ammonia) and 12.6% (1.25× ammonia) for extra nitrogen supplemented batches.
    • Example 6
  • This example shows a kinetic profile of a fermentation process of the present invention. A 1000 gallon scale fed-batch experiment was conducted using a procedure similar to Example 3. Kinetic profile of the fermentation process is shown in Table 6.
  • TABLE 6
    Kinetic Profile for a 1,000 gallon scale Fed-Batch fermentation of Schizochytrium.
    Age Yield Biomass Conversion % DHA % FAME DHA
    (hrs) (g/L) Productivity Efficiency Content Content Productivity
    24 118 4.92 g/L/hr 78.2% 7.4 18.8 0.36 g/L/hr
    30 138 4.60 g/L/hr 60.3% 10.6 30.9 0.49 g/L/hr
    36 138 3.83 g/L/hr 46.6% 11.6 36.5 0.44 g/L/hr
    42 175 4.17 g/L/hr 49.8% 13.4 41.7 0.56 g/L/hr
    48 178 3.71 g/L/hr 45.1% 18.7 52.8 0.69 g/L/hr
     48* 164 3.42 g/L/hr 41.5% 15.3 33.1 0.52 g/L/hr
    54 196 3.63 g/L/hr 45.7% 16.6 51.2 0.60 g/L/hr
    60 190 3.17 g/L/hr 41.7% 16.9 33.9 0.54 g/L/hr
    72 189 2.62 g/L/hr 39.1% 15.6 31.8 0.41 g/L/hr
    84 195 2.32 g/L/hr 38.5% 16.4 32.7 0.38 g/L/hr
    90 200 2.22 g/L/hr 39.0% 18.8 33.3 0.42 g/L/hr
    90 171 1.90 g/L/hr 33.3% 22.2 61.6  0.42 g/L/hr**
    *Two separate samples were analyzed at 48 hrs.
    **This is for a washed dry-cell weights (DCW) sample. Other reported values are for unwashed samples.
    • Example 7
  • This example illustrates effect of the amount of carbon source on productivity.
  • Three different fermentation processed using the process of Example 3 were conducted using various amounts of carbon source. Results are shown on Table 7.
  • TABLE 7
    Fermentation results for various amounts of carbon
    source on fermentation of Schizochytrium.
    Age Yield Carbon Conversion % DHA % FAME Productivity
    (hrs) (q/L) Charge Efficiency Content Content (g/L/hr)
    90 171 51.3% 33.3% 22.2 61.6 0.42
    94 122 40.5% 30.1% 19.1 57.3 0.25
    59 73 20.0% 36.5% 11.9 40.8 0.15
  • The present invention, in various embodiments, includes components, methods, processes, systems and/or apparatus substantially as depicted and described herein, including various embodiments, subcombinations, and subsets thereof. Those of skill in the art will understand how to make and use the present invention after understanding the present disclosure. The present invention, in various embodiments, includes providing devices and processes in the absence of items not depicted and/or described herein or in various embodiments hereof, including in the absence of such items as may have been used in previous devices or processes, e.g., for improving performance, achieving ease and\or reducing cost of implementation.
  • The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

Claims (20)

1.-22. (canceled)
23. A biomass of microorganisms having highly unsaturated fatty acids, wherein said biomass is produced by a process of conducting a fermentation of the microorganisms in a fermentation medium comprising a carbon source and a limiting nutrient source, the process comprising:
a) providing the fermentation medium comprising the carbon source and the limiting nutrient source;
b) adding the microorganisms to the fermentation medium;
c) adding an additional amount of the carbon source and limiting nutrient source to the fermentation medium to increase the biomass density of the fermentation medium to at least about 100 g/L on a dry cell weight basis;
d) adding an additional amount of the carbon source while limiting the amount of limiting nutrient source added to the formulation medium to induce lipid production by the microorganisms; and
wherein the microorganisms are of the order Thraustochytriales, and produce at least 15% by weight of the dry cell weight of the biomass as lipids.
24. The biomass of claim 23, wherein the process further comprises e) recovering at least some of the lipids from the microorganisms.
25. The biomass of claim 23, wherein the microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof
26. The biomass of claim 23, wherein the highly unsaturated fatty acids are selected from the group consisting of docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, arachidonic acid and mixtures thereof.
27. The biomass of claim 26, wherein the highly unsaturated fatty acids are eicosapentaenoic acid.
28. The biomass of claim 23, wherein at least 25% of the lipids is docosahexaenoic acid.
29. The biomass of claim 1, wherein the process produces lipids at an average rate of at least 0.5 g/L/hr and wherein at least 25% of the lipids is docosahexaenoic acid.
30. A biomass of microorganisms having highly unsaturated fatty acids, wherein said biomass has a density of 100 g/L on a dry cell weight basis of the fermentation medium that it is isolated from, and wherein at least 15% by weight of the dry cell weight of the biomass is lipids.
31. The biomass of claim 30, wherein said biomass has a density of 130 g/L on a dry cell weight basis of the fermentation medium that it is isolated from.
32. The biomass of claim 30, wherein said biomass has a density of 150 g/L on a dry cell weight basis of the fermentation medium that it is isolated from.
33. The biomass of claim 30, wherein the microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof
34. The biomass of claim 30, wherein the highly unsaturated fatty acids are selected from the group consisting of docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, arachidonic acid and mixtures thereof.
35. The biomass of claim 30, wherein at least 30% by weight of the dry cell weight of the biomass is lipids.
36. An oil extracted from a biomass of claim 23 or claim 30.
37. The oil as recited in claim 36, wherein said oil is a crude oil extracted from the biomass without further processing.
38. A feed product that contains a biomass of any one of claims 23-35 or an oil of any one of claims 36-37.
39. The feed product of claim 38, wherein said feed product is a feed or feed supplement.
40. The feed of claim 38, wherein said feed is an animal feed.
41. The feed of claim 38, wherein said feed is an aquaculture feed.
US15/344,960 2000-01-28 2016-11-07 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors Abandoned US20170049132A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US15/344,960 US20170049132A1 (en) 2000-01-28 2016-11-07 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors

Applications Claiming Priority (17)

Application Number Priority Date Filing Date Title
US17858800P 2000-01-28 2000-01-28
US09/771,352 US6607900B2 (en) 2000-01-28 2001-01-26 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US10/371,394 US20030180898A1 (en) 2000-01-28 2003-02-21 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/352,421 US7579174B2 (en) 2000-01-28 2006-02-10 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,498 US8288133B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,526 US8133706B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,513 US8206956B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,490 US8216812B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,506 US8124384B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,502 US20080032381A1 (en) 2000-01-28 2007-05-08 Enhanced Production of Lipids Containing Polyenoic Fatty Acid by Very High Density Cultures of Eukaryotic Microbes in Fermentors
US11/745,511 US8124385B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,500 US8288134B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,533 US8187846B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,531 US8187845B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US13/342,623 US9848623B2 (en) 2000-01-28 2012-01-03 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors
US14/711,979 US20150320084A1 (en) 2000-01-28 2015-05-14 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors
US15/344,960 US20170049132A1 (en) 2000-01-28 2016-11-07 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US14/711,979 Division US20150320084A1 (en) 2000-01-28 2015-05-14 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors

Publications (1)

Publication Number Publication Date
US20170049132A1 true US20170049132A1 (en) 2017-02-23

Family

ID=22653135

Family Applications (18)

Application Number Title Priority Date Filing Date
US09/771,352 Expired - Lifetime US6607900B2 (en) 2000-01-28 2001-01-26 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US10/371,394 Abandoned US20030180898A1 (en) 2000-01-28 2003-02-21 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/352,421 Expired - Fee Related US7579174B2 (en) 2000-01-28 2006-02-10 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/399,588 Expired - Fee Related US7732170B2 (en) 2000-01-28 2006-04-05 Enhanced production of lipids containing polyenoic fatty acid by very hugh density cultures of eukaryotic microbes in fermentors
US11/745,533 Expired - Fee Related US8187846B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,513 Expired - Fee Related US8206956B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,500 Expired - Lifetime US8288134B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,498 Expired - Lifetime US8288133B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,502 Abandoned US20080032381A1 (en) 2000-01-28 2007-05-08 Enhanced Production of Lipids Containing Polyenoic Fatty Acid by Very High Density Cultures of Eukaryotic Microbes in Fermentors
US11/745,526 Expired - Fee Related US8133706B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,506 Expired - Fee Related US8124384B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,490 Expired - Fee Related US8216812B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,511 Expired - Fee Related US8124385B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,531 Expired - Fee Related US8187845B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US13/342,623 Expired - Fee Related US9848623B2 (en) 2000-01-28 2012-01-03 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors
US14/711,979 Abandoned US20150320084A1 (en) 2000-01-28 2015-05-14 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors
US15/344,948 Abandoned US20170049131A1 (en) 2000-01-28 2016-11-07 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors
US15/344,960 Abandoned US20170049132A1 (en) 2000-01-28 2016-11-07 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors

Family Applications Before (17)

Application Number Title Priority Date Filing Date
US09/771,352 Expired - Lifetime US6607900B2 (en) 2000-01-28 2001-01-26 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US10/371,394 Abandoned US20030180898A1 (en) 2000-01-28 2003-02-21 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/352,421 Expired - Fee Related US7579174B2 (en) 2000-01-28 2006-02-10 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/399,588 Expired - Fee Related US7732170B2 (en) 2000-01-28 2006-04-05 Enhanced production of lipids containing polyenoic fatty acid by very hugh density cultures of eukaryotic microbes in fermentors
US11/745,533 Expired - Fee Related US8187846B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,513 Expired - Fee Related US8206956B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,500 Expired - Lifetime US8288134B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,498 Expired - Lifetime US8288133B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,502 Abandoned US20080032381A1 (en) 2000-01-28 2007-05-08 Enhanced Production of Lipids Containing Polyenoic Fatty Acid by Very High Density Cultures of Eukaryotic Microbes in Fermentors
US11/745,526 Expired - Fee Related US8133706B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,506 Expired - Fee Related US8124384B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,490 Expired - Fee Related US8216812B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,511 Expired - Fee Related US8124385B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US11/745,531 Expired - Fee Related US8187845B2 (en) 2000-01-28 2007-05-08 Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
US13/342,623 Expired - Fee Related US9848623B2 (en) 2000-01-28 2012-01-03 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors
US14/711,979 Abandoned US20150320084A1 (en) 2000-01-28 2015-05-14 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors
US15/344,948 Abandoned US20170049131A1 (en) 2000-01-28 2016-11-07 Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors

Country Status (27)

Country Link
US (18) US6607900B2 (en)
EP (7) EP1251744B2 (en)
JP (9) JP2004501603A (en)
KR (6) KR20100116233A (en)
CN (8) CN101519678B (en)
AT (1) ATE374531T1 (en)
AU (4) AU785124B2 (en)
BR (1) BRPI0107832B1 (en)
CA (3) CA2760879C (en)
CY (1) CY1107114T1 (en)
CZ (1) CZ301130B6 (en)
DE (2) DE60130737T3 (en)
DK (1) DK1251744T4 (en)
ES (4) ES2545492T3 (en)
HK (1) HK1050611A1 (en)
HU (1) HUP0301794A3 (en)
IL (3) IL150770A0 (en)
MX (2) MX345559B (en)
NO (1) NO20023588L (en)
NZ (1) NZ520420A (en)
PL (1) PL362620A1 (en)
PT (1) PT1251744E (en)
RU (1) RU2326171C2 (en)
TR (1) TR200302163T3 (en)
TW (3) TWI310788B (en)
WO (1) WO2001054510A1 (en)
ZA (1) ZA200205957B (en)

Families Citing this family (182)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5340742A (en) * 1988-09-07 1994-08-23 Omegatech Inc. Process for growing thraustochytrium and schizochytrium using non-chloride salts to produce a microfloral biomass having omega-3-highly unsaturated fatty acids
US6451567B1 (en) * 1988-09-07 2002-09-17 Omegatech, Inc. Fermentation process for producing long chain omega-3 fatty acids with euryhaline microorganisms
US20060094089A1 (en) * 1988-09-07 2006-05-04 Martek Biosciences Corporation Process for the heterotrophic production of microbial products with high concentrations of omega-3 highly unsaturated fatty acids
EP0823475B1 (en) 1995-04-17 2009-06-17 National Institute of Advanced Industrial Science and Technology Novel microorganisms capable of producing highly unsaturated fatty acids and process for producing highly unsaturated fatty acids by using the microorganisms
US20080175953A1 (en) * 1995-06-07 2008-07-24 Martek Biosciences Corporation Process for the Heterotrophic Production of Microbial Products with High Concentrations of Omega-3 Highly Unsaturated Fatty Acids
HUP0301794A3 (en) 2000-01-28 2011-04-28 Martek Biosciences Corp Enhanced production of lipids containing polyenoic fatty acids by high density cultures of eukaryotic microbes in fermentors
DK2330407T3 (en) * 2001-09-14 2013-06-24 Arkray Inc Method, tool and device for measuring a concentration
US7560123B2 (en) * 2004-08-12 2009-07-14 Everett Laboratories, Inc. Compositions and methods for nutrition supplementation
CA2484334C (en) * 2002-05-03 2013-01-22 Martek Biosciences Corporation High-quality lipids and methods for producing by enzymatic liberation from biomass
US6814983B2 (en) * 2002-12-10 2004-11-09 Everett Laboratories, Inc. Compositions and methods for nutrition supplementation
US8617617B2 (en) * 2002-12-10 2013-12-31 Everett Laboratories, Inc. Methods and kits for co-administration of nutritional supplements
US20050019880A1 (en) * 2003-03-31 2005-01-27 Council Of Scientific And Industrial Research Method of enhancing levels of polyunsaturated fatty acids in thraustochytrid protists
BRPI0410491A8 (en) 2003-04-24 2017-10-24 Univ Vanderbilt COMPOSITIONS AND METHODS FOR INSECT CONTROL
US7622269B2 (en) * 2004-03-19 2009-11-24 Tyratech, Inc. Methods of screening tyramine- and octopamine-expressing cells for compounds and compositions having potential insect control activity
US20060263403A1 (en) * 2003-04-24 2006-11-23 Essam Enan Compositions and methods for controlling insects involving the tyramine receptor
AU2003902794A0 (en) * 2003-06-03 2003-06-19 Agresearch Limited Improvements in grass endophytes
US7267976B2 (en) * 2003-07-02 2007-09-11 E.I. Du Pont De Nemours And Company Acyltransferases for alteration of polyunsaturated fatty acids and oil content in oleaginous yeasts
WO2005021735A1 (en) * 2003-09-01 2005-03-10 Novozymes A/S Method for increasing yield of biomass of and/or components of biomass from marine microorganisms
JP4733043B2 (en) 2003-10-02 2011-07-27 マーテック バイオサイエンシーズ コーポレーション Method for producing high levels of DHA in microalgae using modified amounts of chloride and potassium
WO2005047480A2 (en) * 2003-11-12 2005-05-26 E.I. Dupont De Nemours And Company Delta-15 desaturases suitable for altering levels of polyunsaturated fatty acids in oleaginous plants and yeast
CN103357199B (en) * 2003-12-30 2019-02-01 帝斯曼知识产权资产管理有限公司 Degassing method
US8101587B2 (en) 2004-08-12 2012-01-24 Everett Laboratories, Inc. Kits for nutrition supplementation
US8241868B2 (en) 2005-02-08 2012-08-14 Nippon Suisan Kaisha, Ltd. Production of polyunsaturated fatty acids using cell treatment method
JP4849806B2 (en) * 2005-02-08 2012-01-11 日本水産株式会社 Method for producing polyunsaturated fatty acids using novel cell treatment method
EP1899453B1 (en) 2005-06-07 2013-12-18 Ocean Nutrition Canada Limited Eukaryotic microorganisms for producing lipids and antioxidants
WO2007005725A2 (en) 2005-07-01 2007-01-11 Martek Biosciences Corporation Polyunsaturated fatty acid-containing oil product and uses and production thereof
US8177262B2 (en) * 2005-07-28 2012-05-15 Hydril Company Lp Mid-seal for expandable connections
WO2007074479A1 (en) * 2005-12-29 2007-07-05 Abl Biotechnologies Ltd Novel strain of schizochytrium limacinum useful in the production of lipids and extracellular polysaccharides and process thereof
MY160041A (en) * 2006-06-27 2017-02-15 Tyratech Inc Compositions and methods for treating parasitic infections
MX2009000548A (en) * 2006-07-17 2009-05-28 Tyratech Inc Compositions and methods for controlling insects.
US9023616B2 (en) 2006-08-01 2015-05-05 Dsm Nutritional Products Ag Oil producing microbes and method of modification thereof
US9637714B2 (en) * 2006-12-28 2017-05-02 Colorado State University Research Foundation Diffuse light extended surface area water-supported photobioreactor
AU2008205516A1 (en) * 2007-01-16 2008-07-24 Tyratech, Inc. Pest control compositions and methods
KR20100039846A (en) * 2007-06-14 2010-04-16 닉콜라오스 미트로포울로스 Algae growth for biofuels
WO2008155410A1 (en) * 2007-06-21 2008-12-24 Novozymes A/S Production of lipids containing poly-unsaturated fatty acids
CA2692355C (en) * 2007-06-29 2018-09-11 Martek Biosciences Corporation Production and purification of esters of polyunsaturated fatty acids
GB0713121D0 (en) * 2007-07-06 2007-08-15 Univ Keele Refrigerated gas equilibration device
AU2008300002B2 (en) * 2007-09-12 2014-04-10 Dsm Ip Assets B.V. Biological oils and production and uses thereof
ITMI20072343A1 (en) 2007-12-14 2009-06-15 Eni Spa PROCESS FOR THE PRODUCTION OF ALGAL BIOMASS WITH HIGH LIPID CONTENT
US7989195B2 (en) * 2008-02-20 2011-08-02 Washington State University Research Foundation Heterotrophic algal high cell density production method and system
ES2326022B1 (en) 2008-03-25 2010-06-07 Neuron Biopharma, S.A. IMPROVED PROCEDURE FOR BIODIESEL PRODUCTION.
EP2265724A4 (en) * 2008-04-09 2013-01-23 Solazyme Inc Direct chemical modification of microbial biomass and microbial oils
JP5171422B2 (en) * 2008-06-19 2013-03-27 ルネサスエレクトロニクス株式会社 Photosensitive composition, pattern forming method using the same, and method for producing semiconductor element
US20100050502A1 (en) * 2008-08-21 2010-03-04 LiveFuels, Inc. Systems and methods for hydrothermal conversion of algae into biofuel
US20100236137A1 (en) * 2008-09-23 2010-09-23 LiveFuels, Inc. Systems and methods for producing eicosapentaenoic acid and docosahexaenoic acid from algae
US20100081835A1 (en) * 2008-09-23 2010-04-01 LiveFuels, Inc. Systems and methods for producing biofuels from algae
US20100077654A1 (en) * 2008-09-23 2010-04-01 LiveFuels, Inc. Systems and methods for producing biofuels from algae
CN102271525B (en) 2008-10-14 2015-01-07 索拉兹米公司 Food compositions of microalgal biomass
US8809037B2 (en) 2008-10-24 2014-08-19 Bioprocessh20 Llc Systems, apparatuses and methods for treating wastewater
WO2010059598A1 (en) * 2008-11-18 2010-05-27 LiveFuels, Inc. Methods for producing fish with high lipid content
WO2010080377A1 (en) * 2008-12-19 2010-07-15 Alpha-J Research Limited Partnership Optimization of algal product production through uncoupling cell proliferation and algal product production
EP2401386A4 (en) 2009-02-25 2013-03-13 Vb Medicare Pvt Ltd Improved methods for fermentative production of docosahexaenoic acid
US8207363B2 (en) * 2009-03-19 2012-06-26 Martek Biosciences Corporation Thraustochytrids, fatty acid compositions, and methods of making and uses thereof
JP5636039B2 (en) 2009-04-14 2014-12-03 ソラザイム、インクＳｏｌａｚｙｍｅ Ｉｎｃ Novel microalgal food composition
WO2010121094A1 (en) 2009-04-17 2010-10-21 Livefuels. Inc. Systems and methods for culturing algae with bivalves
EP2427420A1 (en) 2009-05-04 2012-03-14 Primafuel, Inc. Improved recovery of desired co-products from fermentation stillage streams
CN101899481A (en) * 2009-05-25 2010-12-01 华盛顿州立大学 Method and system for producing heterotrophic alga in high density
JP2013505024A (en) 2009-09-18 2013-02-14 フィコイル バイオテクノロジー インターナショナル，インコーポレイテッド Fermentation of microalgae using controlled lighting
JP5920890B2 (en) 2010-01-19 2016-05-18 ディーエスエム アイピー アセッツ ビー．ブイ． Eicosapentaenoic acid producing microorganisms, fatty acid compositions, and methods for making them and their use
AU2011224449A1 (en) 2010-03-11 2012-09-27 BP Biofuels UK Limited Methods, biological oils, biofuels, units, and organisms related to use in compression engines
US9145539B2 (en) 2010-03-12 2015-09-29 Solix Algredients, Inc. Systems and methods for positioning flexible floating photobioreactors
CN103025862B (en) * 2010-05-04 2015-02-11 韩国生命工学研究院 Novel thraustochytrid-based microalgae, and method for preparing bio-oil by using same
KR101147450B1 (en) 2010-05-04 2012-05-21 한국생명공학연구원 Novel Fatty Oilic Microalgae KRS101 and Preparing Method for Biooil Using Thereof
AU2011261455B2 (en) 2010-06-01 2016-03-24 Dsm Ip Assets B.V. Extraction of lipid from cells and products therefrom
MY163938A (en) 2010-06-14 2017-11-15 Io-Mega Holding Corp Method for the production of algae derived oils
US10479969B2 (en) 2010-10-11 2019-11-19 Phycoil Biotechnology International. Inc. Utilization of wastewater for microalgal cultivation
CN102485898A (en) * 2010-12-02 2012-06-06 丰益(上海)生物技术研发中心有限公司 Method for producing lipids through microbial fermentation
WO2012109375A2 (en) * 2011-02-08 2012-08-16 Phycal Inc. Methods for improved mixed trophic algal culture
CN103649313B (en) 2011-03-07 2017-10-24 Dsm营养产品股份公司 It is engineered thraustochytriale microorganism belonging to genus
US9487716B2 (en) 2011-05-06 2016-11-08 LiveFuels, Inc. Sourcing phosphorus and other nutrients from the ocean via ocean thermal energy conversion systems
BR112013033021A2 (en) 2011-06-23 2017-01-31 Rho Renewables Inc "host cell and methods for producing phenolic compound using aromatic polyketide synthases"
US8183227B1 (en) 2011-07-07 2012-05-22 Chemo S. A. France Compositions, kits and methods for nutrition supplementation
EP3978597A1 (en) * 2011-07-13 2022-04-06 Alltech, Inc. Algal lipid compositions and methods of preparing and utilizing the same
AU2012285803B2 (en) 2011-07-21 2016-08-25 Dsm Ip Assets B.V. Eicosapentaenoic acid-producing microorganisms, fatty acid compositions, and methods of making and uses thereof
US9222112B2 (en) 2011-07-21 2015-12-29 Dsm Ip Assets B.V. Eicosapentaenoic acid-producing microorganisms, fatty acid compositions, and methods of making and uses thereof
WO2013032333A1 (en) * 2011-09-01 2013-03-07 Algae Biotech S.L. Oral dosage units containing astaxanthin, phospholipids and omega-3 fatty acids
US8168611B1 (en) 2011-09-29 2012-05-01 Chemo S.A. France Compositions, kits and methods for nutrition supplementation
CA2869020A1 (en) 2012-04-09 2013-10-17 BP Biofuels UK Limited Low polysaccharide microorganisms for production of biofuels and other renewable materials
US10100338B2 (en) * 2012-05-22 2018-10-16 Ineos Bio S.A. Method of operation of a syngas fermentation process
RU2539766C2 (en) * 2012-08-03 2015-01-27 Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Arthrospira platensis (Nordst) Geitl rsemsu T/05-117 STRAIN - LIPID-CONTAINING BIOMASS PRODUCER
CN102864111B (en) * 2012-10-10 2013-07-17 江南大学 Schizochytrium limacinum strain for producing docosahexaenoic acid
BR112015008569B1 (en) 2012-10-17 2020-12-29 Corbion Biotech., Inc microalgae flour granule, process for preparing granules, method for controlling one or more particle size, flowability and wettability of a microalgae flour, and microalgae flour
EP2724625A1 (en) 2012-10-26 2014-04-30 Roquette Freres Microalgal flour granules and process for preparation thereof
US9394505B2 (en) 2012-12-04 2016-07-19 Flint Hills Resources, Lp Recovery of co-products from fermentation stillage streams
EP2947141B1 (en) * 2013-01-18 2019-11-06 Kyowa Hakko Bio Co., Ltd. Microorganisms producing docosahexaenoic acid and utilization thereof
DE102013201978A1 (en) 2013-02-07 2014-08-07 Evonik Industries Ag Disruption of cells which contains recyclable material, comprises allowing cells to swell before cell disruption
EP2762008A1 (en) 2013-02-05 2014-08-06 Evonik Industries AG Improving bioavailability of valuable materials from microorganisms by use of a rotor-stator system for cell disruption
EP2953480B1 (en) 2013-02-05 2020-06-03 Evonik Operations GmbH Improving bioavailability of valuable materials from microorganisms
EP2762009A1 (en) 2013-02-05 2014-08-06 Evonik Industries AG Improving bioavailability of valuable materials from microorganisms
US20160024125A1 (en) * 2013-03-11 2016-01-28 Life Science Nutrition As Natural lipids containing non-oxidizable fatty acids
PT2970926T (en) 2013-03-13 2018-03-22 Dsm Nutritional Products Ag Engineering microorganisms
EP2777400A1 (en) 2013-03-15 2014-09-17 Roquette Freres Microalgal flour granules and process for preparation thereof
EP3067414B1 (en) 2013-03-29 2018-10-31 Corbion Biotech, Inc. Microalgal biomass protein enrichment method
EP2826384A1 (en) 2013-07-16 2015-01-21 Evonik Industries AG Method for drying biomass
FR3008581B1 (en) 2013-07-19 2016-11-04 Roquette Freres LIPID RICH MICROALGUE FLOUR AND PROCESS FOR PREPARING THE SAME
CN105636456A (en) 2013-10-18 2016-06-01 罗盖特公司 Process for texturing a microalgal biomass
MY177808A (en) 2013-11-12 2020-09-23 Roshan Viswanath Makam A process of production and extra-cellular secretion of lipids
KR101541521B1 (en) 2013-11-28 2015-08-03 롯데케미칼 주식회사 Method of producing microalgal dha using microalgae
ES2793935T3 (en) 2013-11-29 2020-11-17 Corbion Biotech Inc Procedure for the enrichment of microalgae biomass in carotenoids and proteins
WO2015079169A1 (en) 2013-11-29 2015-06-04 Roquette Freres Granules of protein-rich microalgal biomass flour and method for preparing same
FR3015516B1 (en) 2013-12-19 2016-01-22 Roquette Freres PROCESS FOR ENHANCING DHA BIOMASS OF MICROALGUES OF THE GENUS THRAUSTOCHYTRIUM
CA2934491C (en) 2013-12-20 2023-09-26 Dsm Ip Assets B.V. Processes for obtaining microbial oil from microbial cells
MX2016008228A (en) 2013-12-20 2016-11-28 Dsm Ip Assets Bv Processes for obtaining microbial oil from microbial cells.
EP3083545B1 (en) 2013-12-20 2023-08-02 DSM IP Assets B.V. Processes for obtaining microbial oil from microbial cells
PT3626806T (en) 2013-12-20 2024-07-31 Mara Renewables Corp Methods of recovering oil from microorganisms
NZ721417A (en) 2013-12-20 2022-07-01 Dsm Ip Assets Bv Processes for obtaining microbial oil from microbial cells
BR112016014208B1 (en) 2013-12-20 2022-09-20 Dsm Nutricional Products Ag METHOD OF EXTRACTION OF LIPIDS FROM A POPULATION OF MICRO-ORGANISMS
TWI646189B (en) 2013-12-20 2019-01-01 荷蘭商Ｄｓｍ智慧財產有限公司 Method for obtaining microbial oil from microbial cells (5)
CN107075547A (en) 2014-01-20 2017-08-18 罗盖特兄弟公司 Method for the protein-enriched of microalgae biomass
CA2941102C (en) * 2014-02-28 2022-01-04 Technische Universiteit Delft Process for the recovery of lipids or hydrocarbons
BR112017002791B1 (en) 2014-05-22 2024-03-05 MARA Renewables Corporation METHOD FOR OIL PRODUCTION IN MICRO-ORGANISMS
US20150352034A1 (en) 2014-06-08 2015-12-10 Solazyme, Inc. Personal Care Products Containing Microalgae or Extracts Thereof
CN110835606B (en) * 2014-07-03 2020-12-22 可持续生物制品公司 Fusarium oxysporum strain and methods of making and using same
CA2958457C (en) 2014-10-02 2022-10-25 Evonik Industries Ag Process for producing a pufa-containing biomass which has high cell stability
EP3200604B1 (en) 2014-10-02 2021-11-03 Evonik Operations GmbH Method for preparing an animal feed
US11464244B2 (en) 2014-10-02 2022-10-11 Evonik Operations Gmbh Feedstuff of high abrasion resistance and good stability in water, containing PUFAs
CN106793803B (en) * 2014-10-02 2021-03-09 赢创运营有限公司 Method for producing PUFA-containing feed by extruding PUFA-containing biomass
MX2017002150A (en) * 2014-10-16 2017-08-21 Mara Renewables Corp Repeated fed-batch culture methods.
CA2959860C (en) 2014-10-16 2023-04-18 MARA Renewables Corporation Semi-continuous culture methods
US10570427B2 (en) * 2014-10-31 2020-02-25 Lanzatech New Zealand Limited Fermentation process for the production of lipids
JP6711278B2 (en) * 2014-11-28 2020-06-17 味の素株式会社 Method for producing isoprenoid compound
FR3030191B1 (en) 2014-12-18 2018-03-23 Corbion Biotech, Inc. COMPOSITION FOR FRESH FAT OIL PRODUCT AND METHOD OF MANUFACTURE
EP3098318A1 (en) 2015-01-24 2016-11-30 Indian Oil Corporation Limited Thraustochytrid based process for treating waste effluents
FR3031984B1 (en) * 2015-01-27 2019-05-24 Roquette Freres PROCESS FOR ENRICHING THE BIOMASS OF MICROALGUES OF THE GENUS TRAUSTOCHYTRIUM IN DHA AND IN AMINO ACIDS ARG AND GLU
DK3274430T3 (en) 2015-03-24 2022-10-03 Corbion Biotech Inc Microalgal compositions and their uses
AR104042A1 (en) * 2015-03-26 2017-06-21 Mara Renewables Corp HIGH-DENSITY PRODUCTION OF BIOMASS AND OIL USING GLUCEROL IN GROSS
CN107849514A (en) 2015-07-13 2018-03-27 玛拉可再生能源公司 Strengthen the microalgae metabolism of xylose
CN104974944B (en) * 2015-07-15 2018-05-25 南京工业大学 Schizochytrium limacinum genetic engineering strain for producing DHA (docosahexaenoic acid), and construction method and application thereof
ITUB20152958A1 (en) 2015-08-06 2017-02-06 Eni Spa Method for concentrating a cell suspension comprising a mucilaginous biomass of oleaginous yeasts.
CN105018539B (en) * 2015-08-24 2019-02-12 青岛旭能生物工程有限责任公司 A method of culture schizochytrium limacinum high yield DHA
CN105002227B (en) * 2015-08-24 2018-09-07 青岛旭能生物工程有限责任公司 A method of shake flask fermentation schizochytrium limacinum is improved by feed-batch process and produces docosahexaenoic acid
CN105420122A (en) * 2015-12-23 2016-03-23 通威股份有限公司 Schizochytrium limacinum suitable for high-density culture and method for producing grease rich in DHA
WO2017131188A1 (en) * 2016-01-28 2017-08-03 日本水産株式会社 Production method for fats and oils containing highly unsaturated fatty acid
KR20180117131A (en) 2016-03-01 2018-10-26 서스테이너블 바이오프로덕츠, 인크. Biomat of filamentous fungi, its production method and its use
US10851395B2 (en) * 2016-06-10 2020-12-01 MARA Renewables Corporation Method of making lipids with improved cold flow properties
AU2017297760B2 (en) 2016-07-13 2021-09-23 Dsm Ip Assets B.V. Method of separating lipids from a lysed lipids containing biomass
US11946017B2 (en) 2016-07-13 2024-04-02 Evonik Operations Gmbh Method of separating lipids from a lysed lipids containing biomass
WO2018011275A1 (en) 2016-07-13 2018-01-18 Evonik Degussa Gmbh Method for isolating lipids from lipid-containing cells
US10676709B2 (en) 2016-07-20 2020-06-09 MARA Renewables Corporation Flowable microbial oil
CN109415654A (en) 2016-07-20 2019-03-01 玛拉可再生能源公司 For carrying out two step fractional methods of winterization to oil
CN106359930A (en) * 2016-08-29 2017-02-01 界首市任寨乡天佑家庭农场 Feed for producing high EPA and DPA content functional beef
US11352651B2 (en) 2016-12-27 2022-06-07 Evonik Operations Gmbh Method of isolating lipids from a lipids containing biomass
WO2018122057A1 (en) 2016-12-27 2018-07-05 Evonik Degussa Gmbh Method of isolating lipids from a lipids containing biomass
CN106636235B (en) * 2016-12-30 2020-08-04 内蒙古金达威药业有限公司 Method for producing DHA (docosahexaenoic acid) by microbial fermentation
TWI661780B (en) * 2017-02-10 2019-06-11 台原藥股份有限公司 Vegetarian composition containing unsaturated fatty acids
BR112020000421A2 (en) 2017-07-12 2020-09-01 Bunge Global Innovation Llc process of oil extraction from algae biomass
CN111356767A (en) * 2017-08-17 2020-06-30 赢创运营有限公司 Enhancing lipid production by limiting at least two limiting nutrient sources
KR20230127261A (en) 2017-08-30 2023-08-31 더 파인더 그룹, 인크. Edible composition with filamentous fungi and bioreactor system for the cultivation thereof
EP3470502A1 (en) 2017-10-13 2019-04-17 Evonik Degussa GmbH Method of separating lipids from a lysed lipids containing biomass
WO2019048327A1 (en) 2017-09-05 2019-03-14 Evonik Degussa Gmbh Method of separating lipids from a lysed lipids containing biomass
EA202090440A1 (en) 2017-09-21 2020-07-17 Эвоник Оперейшенс ГмбХ IMPROVED LIPID PRODUCTION BY LIMITING TWO LIMITING NUTRIENT SOURCES
EP3527664A1 (en) 2018-02-15 2019-08-21 Evonik Degussa GmbH Method of isolating lipids from a lipids containing biomass
WO2019121752A1 (en) 2017-12-20 2019-06-27 Evonik Degussa Gmbh Method of isolating lipids from a lipids containing biomass
WO2019122030A1 (en) 2017-12-22 2019-06-27 Dsm Ip Assets B.V. Method of separating lipids from a lysed lipids containing biomass
BR112020019783A2 (en) 2018-03-29 2021-01-05 Dsm Ip Assets B.V. INNOVATIVE USE OF 2H-REPLACED CHROMENES AND THEIR DERIVATIVES
EP3774757A1 (en) 2018-03-29 2021-02-17 DSM IP Assets B.V. Chroman-6-ols with an extended lipophilic side chain in position 2, their manufacture and use
WO2019185889A1 (en) 2018-03-29 2019-10-03 Dsm Ip Assets B.V. Novel use of carnosic acid
WO2019185941A1 (en) 2018-03-29 2019-10-03 Dsm Ip Assets B.V. Novel use of substituted chroman-6-ols with extended lipophilic side chains
WO2019185891A1 (en) 2018-03-29 2019-10-03 Dsm Ip Assets B.V. Novel use of substituted chroman-6-ols
US20210037855A1 (en) 2018-03-29 2021-02-11 Dsm Ip Assets B.V. Use of twin-chromanols as antioxidants
WO2019185939A1 (en) 2018-03-29 2019-10-03 Dsm Ip Assets B.V. Use of twin-chromanols as antioxidants in oil
WO2019185942A1 (en) 2018-03-29 2019-10-03 Dsm Ip Assets B.V. Use of tocotrienols as antioxidants
WO2019185894A1 (en) 2018-03-29 2019-10-03 Dsm Ip Assets B.V. Novel use of substituted chroman-6-ols
WO2019185910A2 (en) 2018-03-29 2019-10-03 Dsm Ip Assets B.V. Novel use of substituted 2h-chromens and their derivatives
WO2019185888A1 (en) 2018-03-29 2019-10-03 Dsm Ip Assets B.V. Novel use of tocopherols
US11447459B2 (en) 2018-03-29 2022-09-20 Dsm Ip Assets B.V. Use of substituted chroman-6-ols with extended lipophilic side chains
RU2760575C1 (en) 2018-05-15 2021-11-29 Эвоник Оперейшнс Гмбх Method for isolating lipids from lipid-containing biomass using hydrophobic silicon dioxide
US11976253B2 (en) 2018-05-15 2024-05-07 Evonik Operations Gmbh Method of isolating lipids from a lysed lipids containing biomass by emulsion inversion
CN109022284B (en) * 2018-09-03 2021-05-21 杭州园泰生物科技有限公司 Method for improving isochrysis galbana biomass and DHA yield
FR3085825B1 (en) 2018-09-14 2021-07-16 Fermentalg MICROORGANISM OIL RICH IN DOCOSAHEXAENOIC ACID
FR3085962B1 (en) 2018-09-14 2021-06-18 Fermentalg PROCESS FOR EXTRACTING AN OIL RICH IN PUFA
EP3877535A1 (en) * 2018-11-09 2021-09-15 Evonik Operations GmbH Method for producing a biomass which can be easily broken down and which has an increased content of polyunsaturated fatty acids
CN112969796A (en) * 2018-11-09 2021-06-15 赢创运营有限公司 Method for producing biomass with increased polyunsaturated fatty acid content
KR102286636B1 (en) * 2019-01-31 2021-08-05 한국생명공학연구원 Novel microalgae having high productivity for loliolide
TW202103564A (en) 2019-02-27 2021-02-01 美商可持續生物股份有限公司 Food materials comprising filamentous fungal particles and membrane bioreactor design
US11649586B2 (en) 2019-06-18 2023-05-16 The Fynder Group, Inc. Fungal textile materials and leather analogs
EP3933016A1 (en) 2020-06-30 2022-01-05 Evonik Operations GmbH Method of isolating lipids from a lipids containing biomass
WO2022228687A1 (en) 2021-04-29 2022-11-03 S2B Gmbh & Co. Kg Biotechnological production of terpenes
TWI766669B (en) * 2021-04-29 2022-06-01 大自然環保科技有限公司 Organic liquid biological fermentation tank device
EP4180513A1 (en) 2021-11-15 2023-05-17 Indian Oil Corporation Limited An improved process for production of enriched algal biomass
EP4198136A3 (en) 2021-12-16 2023-08-30 Indian Oil Corporation Limited Methods and formulations for enhancing high value lipids
WO2023144707A1 (en) 2022-01-25 2023-08-03 Dsm Ip Assets B.V. Media refinement and nutrient feeding approaches to increase polyunsaturated fatty acid production

Family Cites Families (140)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2879162A (en) * 1956-10-04 1959-03-24 Du Pont Augmenting poultry feed
US2890989A (en) 1957-07-01 1959-06-16 Ralph F Anderson Method for the production of carotenes
GB857161A (en) 1958-01-07 1960-12-29 Boots Pure Drug Co Ltd Improvements in the propagation of fungi
US3108402A (en) * 1961-03-16 1963-10-29 Grain Processing Corp Production of carotenoid pigments
US3444647A (en) * 1961-08-08 1969-05-20 Masahito Takahashi Process of cultivating algae
US3142135A (en) * 1962-02-13 1964-07-28 Grain Processing Corp Production of carotenoids by the cultivation of algae
US3282794A (en) * 1963-09-19 1966-11-01 Ajinomoto Kk Method of producing citrulline by bacterial fermentation
US3296079A (en) * 1963-12-09 1967-01-03 Pfizer & Co C Products sweetened without sugar and characterized by freedom from aftertaste
US3316674A (en) 1964-09-11 1967-05-02 Yakult Honsha Kk Method of new industrial cultivation of unicellular green algae such as chlorella
GB1143405A (en) 1965-02-25 1969-02-19 Unilever Ltd Processing of foodstuffs and the like
GB1123884A (en) 1966-05-23 1968-08-14 Pfizer & Co C Animal feed compositions
FR1557635A (en) 1967-04-20 1969-02-21
US3661663A (en) 1968-08-21 1972-05-09 Owens Corning Fiberglass Corp Method of producing siliceous fiber corrosion inhibiting composites
DE1901980A1 (en) * 1969-01-16 1970-09-03 Wunder Kg Heinrich Safety ski binding with swiveling pressure jaws
IL33732A (en) 1969-02-06 1973-05-31 Meiji Seika Kaisha The new antibiotics sf-837,sf-837-a2,sf-837-a3 and sf-837-a4 and their production
US3617299A (en) 1969-09-15 1971-11-02 Abbott Lab Animal feed premix resistant to static charge and method of making same
US3647482A (en) * 1970-03-04 1972-03-07 Gen Mills Inc Reduction and modification of the unpleasant aftertaste of saccharin
GB1401956A (en) 1971-09-11 1975-08-06 Marmite Ltd Growing of yeast and other microorganisms
US3924017A (en) 1972-07-28 1975-12-02 Gen Foods Corp Sweetness inducer
JPS564233B2 (en) 1973-03-30 1981-01-29
US3908026A (en) * 1973-04-19 1975-09-23 Procter & Gamble Culinary composition containing paramethoxycinnamaldehyde as a flavoring agent and sweetener
US3908028A (en) 1973-04-19 1975-09-23 Procter & Gamble Soft drink composition containing paramethoxycinnamaldehyde as a flavoring agent and sweetner
GB1466853A (en) 1973-05-22 1977-03-09 Simon Rosedowns Ltd Extraction
US3879890A (en) 1974-02-06 1975-04-29 Canadian Patents Dev Algal polysaccharide production
US4162324A (en) * 1975-11-21 1979-07-24 Merck & Co., Inc. Antibiotics 890A1 and 890A3
IT1109471B (en) * 1976-08-17 1985-12-16 Deral Sa PROCEDURE AND PRODUCT FOR THE PRESERVATION AND ENHANCEMENT OF GREEN VEGETABLES AND OF THE WET PRODUCTS UNDER AGRO-FOOD INDUSTRIES
JPS5944020B2 (en) 1978-01-27 1984-10-26 協和醗酵工業株式会社 How to raise marine fish using oleaginous yeast
US4229544A (en) * 1978-08-08 1980-10-21 Payfer Laboratories Inc. Living organism packaging
US4304794A (en) 1978-12-14 1981-12-08 Chimicasa Gmbh Artificial-sweetener composition and process of preparing and using same
US4232122A (en) 1979-01-17 1980-11-04 Z-L Limited Partnership Antioxidants, antioxidant compositions and methods of preparing and using same
JPS55111794A (en) 1979-02-19 1980-08-28 Agency Of Ind Science & Technol Production of lipid having high linolic acid content
US4405649A (en) * 1979-05-07 1983-09-20 Marvin Dudley Process for producing premium quality fish meal from whole fish
IL57712A (en) * 1979-07-03 1984-02-29 Yissum Res Dev Co Cultivation of halophilic algae of the dunaliella species for the production of fuel-like product
DE3011185A1 (en) * 1980-03-22 1981-10-01 Kali-Chemie Pharma Gmbh, 3000 Hannover METHOD FOR OBTAINING DIRECTLY APPLICABLE RIN (PARAGRAPH) LECITHIN FOR PHYSIOLOGICAL PURPOSES
GB2098065A (en) 1981-04-14 1982-11-17 Nippon Suisan Kaisha Ltd Antithrombotic compositions containing docosahexaenoic acid
US4426396A (en) * 1981-06-11 1984-01-17 Union Oil Company Of California Preservation of harvested crops and animal feedstuffs
IL63734A (en) 1981-09-04 1985-07-31 Yeda Res & Dev Lipid fraction,its preparation and pharmaceutical compositions containing same
EP0089983B1 (en) * 1981-10-07 1986-01-29 Commonwealth Scientific And Industrial Research Organisation Method for harvesting algae
US4383038A (en) 1981-12-10 1983-05-10 Ethyl Corporation Process for the preparation of L-proline by cultivating algae
DE3368377D1 (en) 1982-04-16 1987-01-29 Nestle Sa Lipid composition for oral, enteral or parenteral feeding
JPS58196068A (en) 1982-05-12 1983-11-15 Nec Corp Electrostrictive effect element
JPS58213613A (en) 1982-06-03 1983-12-12 Sumitomo Bakelite Co Ltd Preparation of spherical active carbon
US4670285A (en) * 1982-08-06 1987-06-02 The University Of Toronto Innovations Foundation Infant formula
US4425396A (en) 1982-09-28 1984-01-10 The B. F. Goodrich Company Insulative panel
US4588600A (en) 1983-04-18 1986-05-13 Scm Corporation Dry premix composition for imparting a fried appearance to baked foods
JPS6087798A (en) 1983-10-21 1985-05-17 Meiji Milk Prod Co Ltd Production of eicosapentaenoic acid by algae
JPS60105471A (en) 1983-11-14 1985-06-10 Nisshin Flour Milling Co Ltd Production of health food egg
JPS60133094A (en) * 1983-12-21 1985-07-16 日清製油株式会社 Manufacture of high purity eicosapentaenoic acid
EP0155420B1 (en) * 1984-02-09 1988-03-23 The Agency of Industrial Science and Technology A method for the preparation of a fungal body and a lipid rich in gamma-linolenic acid therefrom
JPS61170366A (en) 1985-01-21 1986-08-01 Nisshin Kararingu Kk Production of food comprising fish containing polyfunctional unsaturated fatty acid as main component
IL74497A (en) 1985-03-05 1990-02-09 Proterra Ag Pharmaceutical compositions containing phenyl carbamate derivatives and certain phenyl carbamate derivatives
US4634533A (en) * 1985-04-26 1987-01-06 Somerville Robert L Method of converting brines to useful products
US4792418A (en) 1985-08-14 1988-12-20 Century Laboratories, Inc. Method of extraction and purification of polyunsaturated fatty acids from natural sources
US4749522A (en) * 1985-10-31 1988-06-07 Angio-Medical Corporation Supercritical fluid extraction of animal derived materials
DE3603000A1 (en) 1986-01-31 1987-08-06 Milupa Ag NEW FATTY MIXTURE OF POLYENIC ACID AND THEIR USE IN THE PRODUCTION OF INFANT FOODS
JPS6340711A (en) 1986-08-06 1988-02-22 Ngk Insulators Ltd Production of beta-type silicon nitride
US4758438A (en) 1986-10-14 1988-07-19 Stroz John J Sweetener composition
US4804555A (en) 1986-10-21 1989-02-14 General Mills, Inc. Physical process for simultaneous deodorization and cholesterol reduction of fats and oils
US5064665A (en) 1987-03-23 1991-11-12 The Board Of Regents Of The University Of Nebraska Method of making and using a ruminant feed
US4957748A (en) 1987-03-23 1990-09-18 The Board Of Regents Of The University Of Nebraska Ruminant feed, method of making and method of using
US5023091A (en) 1987-03-23 1991-06-11 The Board Of Regents Of The University Of Nebraska Ruminant feed method of making and method of using
JPS63237745A (en) 1987-03-27 1988-10-04 Harumi Okuyama Production of animal food with enriched content of alpha-linolenic acid based fatty acid
US4764392A (en) 1987-04-01 1988-08-16 Q.P. Corporation Margarine containing fish oil
US4918104A (en) 1987-06-16 1990-04-17 Weiss Howard S Method and composition for increasing the concentration of omega-3 polyunsaturated fatty acids in poultry and poultry eggs and poultry and eggs resulting therefrom
AU2122688A (en) * 1987-07-20 1989-02-13 Maricultura, Incorporated Microorganism production of omega-3 (n-3) lipids
CA1263270A (en) 1987-08-19 1989-11-28 Bruce J. Holub Animal feed supplement
US4871551A (en) * 1988-02-08 1989-10-03 Microbio Resources, Inc. Pigmentation supplements for animal feed compositions
JP2723243B2 (en) 1988-02-25 1998-03-09 サントリー株式会社 Animal feed with polyunsaturated fatty acids
JPH01218573A (en) * 1988-02-25 1989-08-31 Yoshio Tanaka Dunaliella algae-containing solid food and production thereof
US4822500A (en) * 1988-02-29 1989-04-18 Texas United Chemical Corporation Saturated brine well treating fluids and additives therefore
US4874629A (en) 1988-05-02 1989-10-17 Chang Stephen S Purification of fish oil
US6977167B2 (en) 1988-09-07 2005-12-20 Martek Biosciences Corporation Mixtures of omega-3 and omega-6 highly unsaturated fatty acids from euryhaline microorganisms
US5130242A (en) 1988-09-07 1992-07-14 Phycotech, Inc. Process for the heterotrophic production of microbial products with high concentrations of omega-3 highly unsaturated fatty acids
US5340742A (en) 1988-09-07 1994-08-23 Omegatech Inc. Process for growing thraustochytrium and schizochytrium using non-chloride salts to produce a microfloral biomass having omega-3-highly unsaturated fatty acids
US5340594A (en) * 1988-09-07 1994-08-23 Omegatech Inc. Food product having high concentrations of omega-3 highly unsaturated fatty acids
US20060094089A1 (en) 1988-09-07 2006-05-04 Martek Biosciences Corporation Process for the heterotrophic production of microbial products with high concentrations of omega-3 highly unsaturated fatty acids
US7033584B2 (en) * 1988-09-07 2006-04-25 Omegatech, Inc. Feeding Thraustochytriales to poultry for increasing omega-3 highly unsaturated fatty acids in eggs
US6451567B1 (en) * 1988-09-07 2002-09-17 Omegatech, Inc. Fermentation process for producing long chain omega-3 fatty acids with euryhaline microorganisms
US5985348A (en) 1995-06-07 1999-11-16 Omegatech, Inc. Milk products having high concentrations of omega-3 highly unsaturated fatty acids
US5698244A (en) 1988-09-07 1997-12-16 Omegatech Inc. Method for raising animals having high concentrations of omega-3 highly unsaturated fatty acids
US5012761A (en) 1988-11-17 1991-05-07 Oh Suk Y Chicken egg having relatively high percentage of long chain fatty acids and method of reducing heart related disease in humans using such eggs
JP2664452B2 (en) 1988-12-23 1997-10-15 サントリー株式会社 Fish and shellfish feed
DE3920679A1 (en) 1989-06-23 1991-01-10 Milupa Ag FAT MIXTURE FOR THE MANUFACTURE OF FOOD, ESPECIALLY SUGAR FOODS
US5272085A (en) 1989-10-31 1993-12-21 Queen's University Sodium tolerance genes derived from schizosaccharomyces pombe
US5407957A (en) * 1990-02-13 1995-04-18 Martek Corporation Production of docosahexaenoic acid by dinoflagellates
US5244921A (en) * 1990-03-21 1993-09-14 Martek Corporation Eicosapentaenoic acids and methods for their production
JPH0458847A (en) 1990-06-23 1992-02-25 Nippon Oil & Fats Co Ltd Obesity-resistant fat and oil and obesity-resistant food
US5297625A (en) 1990-08-24 1994-03-29 Associated Universities, Inc. Biochemically enhanced oil recovery and oil treatment
JP3102645B2 (en) 1990-10-15 2000-10-23 雪印乳業株式会社 Nutritional composition for nutritional support
US5133963A (en) 1990-12-21 1992-07-28 Shuntaro Ise Method of producing commercially useful poultry products with increased concentrations of Omega-3 polyunsaturated fatty acids
US5658767A (en) * 1991-01-24 1997-08-19 Martek Corporation Arachidonic acid and methods for the production and use thereof
DE4101976C2 (en) 1991-01-24 1995-09-21 Adatomed Pharma Chiron Treatment system for retinal development
EP1787532A3 (en) 1991-01-24 2010-03-31 Martek Biosciences Corporation Microbial oil mixtures and uses thereof
PH11992043811B1 (en) * 1991-01-24 2002-08-22 Martek Corp Arachidonic acid and methods for the production and use thereof
JPH0728677B2 (en) 1991-01-25 1995-04-05 理研ビタミン株式会社 Oil and fat composition for fish feed and fish feed using the same
JPH04271754A (en) 1991-02-26 1992-09-28 Nippon Nousan Kogyo Kk Preparation of cattle and poultry feed and cattle meat
KR940005180B1 (en) 1991-08-20 1994-06-13 주식회사 우방랜드 Feed composition for breeding pigs with n-3 fatty acid-accumulated pork
FR2686619B1 (en) 1992-01-28 1995-07-13 Commissariat Energie Atomique PROCESS FOR THE SELECTIVE PRODUCTION OF POLYUNSATURATED LIPIDS FROM A CULTURE OF PORPHYRIDIUM MICROALGAE AND TANK USED IN THIS PROCESS.
GB2266652B (en) 1992-05-06 1995-11-22 Woobang Land Company Ltd Feed composition for a broiler
US6410281B1 (en) 1992-07-10 2002-06-25 Omegatech, Inc. Reducing corrosion in a fermentor by providing sodium with a non-chloride sodium salt
KR940007396A (en) 1992-09-30 1994-04-27 스마 요시츠기 Constant Groove Joint of Cross Groove Type
JP2558050B2 (en) 1993-02-18 1996-11-27 イセ食品株式会社 Chicken feed
JPH078215A (en) 1993-04-30 1995-01-13 Kawasaki Steel Corp Marine microalgal food raw material containing docosahexaenoic acid and its production
JPH07255387A (en) 1994-03-24 1995-10-09 Morinaga Milk Ind Co Ltd Feed for edible poultry and breeding of edible poultry by the same feed
DE59506314D1 (en) 1994-08-16 1999-08-05 Frische Gmbh METHOD FOR OBTAINING NON-WATER-SOLUBLE, NATURAL PRODUCTS FROM NATURAL MIXTURES WITH THE AID OF THE CENTRIFUGAL FORCE
JP2764572B2 (en) * 1995-04-17 1998-06-11 工業技術院長 Novel microorganism having docosahexaenoic acid-producing ability and method for producing docosahexaenoic acid using the same
EP0823475B1 (en) * 1995-04-17 2009-06-17 National Institute of Advanced Industrial Science and Technology Novel microorganisms capable of producing highly unsaturated fatty acids and process for producing highly unsaturated fatty acids by using the microorganisms
JPH08322475A (en) 1995-05-29 1996-12-10 Nisshin Flour Milling Co Ltd Feed for fowls
MY121221A (en) 1995-05-30 2006-01-28 Suntory Holdings Ltd Domestic fowl eggs having a high content of highly unsaturated fatty acid, their production process and their use
US20080175953A1 (en) 1995-06-07 2008-07-24 Martek Biosciences Corporation Process for the Heterotrophic Production of Microbial Products with High Concentrations of Omega-3 Highly Unsaturated Fatty Acids
JPH0965971A (en) * 1995-09-04 1997-03-11 Mitsuo Fukushige Thermal cooking vessel
JPH0965871A (en) * 1995-09-04 1997-03-11 Kawasaki Steel Corp Culture of maritime fine algae
JPH0984590A (en) 1995-09-21 1997-03-31 Itouen:Kk Production of alpha-linolenic acid
JPH09110888A (en) 1995-10-17 1997-04-28 Nippon Oil & Fats Co Ltd Phospholipid composition
US6255505B1 (en) * 1996-03-28 2001-07-03 Gist-Brocades, B.V. Microbial polyunsaturated fatty acid containing oil from pasteurised biomass
EP0821068A3 (en) * 1996-03-29 1999-06-02 Rohm And Haas Company Novel sphingolipids and a process thereto
CA2261231C (en) * 1996-07-23 2012-11-27 Nagase Biochemicals, Ltd. Process using ulkenia sp. to prepare docosahexaenoic acid and docosapentaenoic acid
FI964692A0 (en) * 1996-11-25 1996-11-25 Primalco Ltd Cellulose-based cellulose processing
PL335227A1 (en) * 1997-02-20 2000-04-10 Dsm Nv Industrial-scale production of valuable compounds by fermentation in a chemically defined medium
US6566583B1 (en) 1997-06-04 2003-05-20 Daniel Facciotti Schizochytrium PKS genes
US6140486A (en) 1997-06-04 2000-10-31 Calgene Llc Production of polyunsaturated fatty acids by expression of polyketide-like synthesis genes in plants
HUP0003079A3 (en) * 1997-08-14 2001-02-28 Martek Biosciences Corp Columb A method for increasing incorporation efficiency of omega-3 highly unsaturated fatty acid in poultry meat
DE19749413A1 (en) 1997-11-07 1999-05-12 Hoechst Ag Novel sophorose lipids, process for their preparation and use
DE19838011C2 (en) * 1998-08-21 2000-01-13 Christoph Syldatk Process for the biotechnical production of fatty acid methyl esters ("biodiesel") based on whey
US7271315B2 (en) 1999-01-14 2007-09-18 Martek Biosciences Corporation PUFA polyketide synthase systems and uses thereof
US7247461B2 (en) 1999-01-14 2007-07-24 Martek Biosciences Corporation Nucleic acid molecule encoding ORFA of a PUFA polyketide synthase system and uses thereof
DE19903095C2 (en) * 1999-01-27 2003-05-22 Nutrinova Gmbh Obtaining gamma-linolenic acid from protozoa of the genus Colpidium
WO2000051444A1 (en) 1999-03-04 2000-09-08 Suntory Limited Utilization of material containing docosapentaenoic acid
HUP0301794A3 (en) 2000-01-28 2011-04-28 Martek Biosciences Corp Enhanced production of lipids containing polyenoic fatty acids by high density cultures of eukaryotic microbes in fermentors
US6338866B1 (en) 2000-02-15 2002-01-15 Applied Food Biotechnology, Inc. Pet foods using algal or fungal waste containing fatty acids
TWI337619B (en) 2001-04-16 2011-02-21 Martek Biosciences Corp Pufa polyketide synthase systems and uses thereof
KR101234200B1 (en) 2003-03-26 2013-02-19 마텍 바이오싸이언스스 코포레이션 Pufa polyketide synthase systems and uses thereof
US7208160B2 (en) 2003-08-26 2007-04-24 Sol Katzen Process of treating sea algae and halophytic plants
DE102004017370A1 (en) 2004-04-08 2005-10-27 Nutrinova Nutrition Specialties & Food Ingredients Gmbh PUFA-PKS Genes from Ulkenia
JP4821409B2 (en) * 2006-03-31 2011-11-24 アイシン・エィ・ダブリュ株式会社 Hydraulic control device for automatic transmission
US8585552B2 (en) * 2006-08-01 2013-11-19 GM Global Technology Operations LLC Torque converter clutch lock on method and low slip regulation
US7485073B2 (en) * 2006-08-02 2009-02-03 Horng-Jiun Chang Muscle exerciser
EP2197962B1 (en) 2007-09-25 2017-08-30 Monsanto Technology, LLC Use of oils with high concentrations of polyunsaturated fatty acids in plastics and surface coatings
CA3012998C (en) 2009-03-19 2021-09-07 Dsm Ip Assets B.V. Polyunsaturated fatty acid synthase nucleic acid molecules and polypeptides, compositions, and methods of making and uses thereof
FR3015516B1 (en) 2013-12-19 2016-01-22 Roquette Freres PROCESS FOR ENHANCING DHA BIOMASS OF MICROALGUES OF THE GENUS THRAUSTOCHYTRIUM

Also Published As

Publication number Publication date
EP2341127B1 (en) 2015-05-27
EP2960325B1 (en) 2017-09-27
US8216812B2 (en) 2012-07-10
TWI354702B (en) 2011-12-21
ES2653545T3 (en) 2018-02-07
JP2005270115A (en) 2005-10-06
HUP0301794A2 (en) 2003-09-29
JP2019088319A (en) 2019-06-13
CN101003821A (en) 2007-07-25
IL196776A (en) 2011-09-27
US7732170B2 (en) 2010-06-08
EP2338974A2 (en) 2011-06-29
US20010046691A1 (en) 2001-11-29
US8187845B2 (en) 2012-05-29
ES2208141T1 (en) 2004-06-16
TR200302163T3 (en) 2004-02-23
US20080032387A1 (en) 2008-02-07
PL362620A1 (en) 2004-11-02
CA2396691A1 (en) 2001-08-02
US20080057551A1 (en) 2008-03-06
ES2545492T3 (en) 2015-09-11
DE01903376T1 (en) 2004-07-08
ZA200205957B (en) 2003-11-19
US6607900B2 (en) 2003-08-19
BRPI0107832B1 (en) 2015-07-07
CN105567752A (en) 2016-05-11
EP1251744A4 (en) 2004-02-25
KR20100116233A (en) 2010-10-29
DK1251744T3 (en) 2007-10-29
MXPA02007321A (en) 2003-01-28
JP6534156B2 (en) 2019-06-26
CZ20022508A3 (en) 2003-02-12
PT1251744E (en) 2007-11-08
CA2396691C (en) 2012-09-18
US8124385B2 (en) 2012-02-28
JP6134136B2 (en) 2017-05-24
US20060286648A1 (en) 2006-12-21
EP1251744A1 (en) 2002-10-30
MX345559B (en) 2017-02-03
EP1251744B2 (en) 2015-08-26
EP2341127A2 (en) 2011-07-06
KR20090064603A (en) 2009-06-19
EP2338974A3 (en) 2011-10-05
US8124384B2 (en) 2012-02-28
US20080032360A1 (en) 2008-02-07
US20060286649A1 (en) 2006-12-21
CN101519679B (en) 2013-11-13
CN101519678A (en) 2009-09-02
HK1050611A1 (en) 2003-07-04
CN105112463A (en) 2015-12-02
US20150320084A1 (en) 2015-11-12
US8288133B2 (en) 2012-10-16
EP2341125A3 (en) 2011-10-05
RU2326171C2 (en) 2008-06-10
AU3120101A (en) 2001-08-07
US20080032381A1 (en) 2008-02-07
DK1251744T4 (en) 2015-11-16
EP2960325A1 (en) 2015-12-30
CN101519680A (en) 2009-09-02
IL171408A (en) 2009-09-01
AU2013205894A1 (en) 2013-06-06
TWI310788B (en) 2009-06-11
US8206956B2 (en) 2012-06-26
EP2341127A3 (en) 2011-10-05
CN101519680B (en) 2013-11-13
NZ520420A (en) 2005-03-24
US20030180898A1 (en) 2003-09-25
CA2760879A1 (en) 2001-08-02
AU2013205894B2 (en) 2016-06-02
RU2002120481A (en) 2004-02-20
EP2341126A2 (en) 2011-07-06
ES2208141T3 (en) 2008-04-01
EP1707055A2 (en) 2006-10-04
US20080032361A1 (en) 2008-02-07
US20080032362A1 (en) 2008-02-07
ATE374531T1 (en) 2007-10-15
KR20110139767A (en) 2011-12-29
KR101293135B1 (en) 2013-09-12
WO2001054510A1 (en) 2001-08-02
KR20030013368A (en) 2003-02-14
WO2001054510A9 (en) 2002-10-17
HUP0301794A3 (en) 2011-04-28
CN101519677A (en) 2009-09-02
US8288134B2 (en) 2012-10-16
JP2014087375A (en) 2014-05-15
JP2010057508A (en) 2010-03-18
CN1416320A (en) 2003-05-07
JP2016202189A (en) 2016-12-08
JP2004501603A (en) 2004-01-22
EP1707055A3 (en) 2009-01-14
DE60130737T3 (en) 2016-01-14
DE60130737T8 (en) 2008-10-30
EP2341125B1 (en) 2017-09-27
CA2786722A1 (en) 2001-08-02
AU2009213103A1 (en) 2009-10-08
AU2006207882A1 (en) 2006-10-05
US20170049131A1 (en) 2017-02-23
US20120178135A1 (en) 2012-07-12
US7579174B2 (en) 2009-08-25
US8187846B2 (en) 2012-05-29
TWI301509B (en) 2008-10-01
TW200513535A (en) 2005-04-16
IL150770A0 (en) 2003-02-12
EP2341126A3 (en) 2011-10-05
ES2208141T5 (en) 2015-11-26
US8133706B2 (en) 2012-03-13
KR20070073986A (en) 2007-07-10
DE60130737T2 (en) 2008-07-17
CN100491519C (en) 2009-05-27
CA2760879C (en) 2018-02-27
US20080032364A1 (en) 2008-02-07
ES2654384T3 (en) 2018-02-13
US20080032363A1 (en) 2008-02-07
CN101519678B (en) 2013-11-13
KR20080086938A (en) 2008-09-26
US9848623B2 (en) 2017-12-26
CN101519679A (en) 2009-09-02
KR100938945B1 (en) 2010-01-26
BR0107832A (en) 2004-04-27
EP2341125A2 (en) 2011-07-06
EP2338974B1 (en) 2017-09-27
CZ301130B6 (en) 2009-11-11
CY1107114T1 (en) 2012-10-24
JP2013051973A (en) 2013-03-21
AU785124B2 (en) 2006-09-28
EP1251744B1 (en) 2007-10-03
AU2006207882B2 (en) 2009-06-11
US20080032366A1 (en) 2008-02-07
NO20023588D0 (en) 2002-07-26
JP2013099366A (en) 2013-05-23
TW200911995A (en) 2009-03-16
AU2009213103B2 (en) 2013-06-06
DE60130737D1 (en) 2007-11-15
JP2012019802A (en) 2012-02-02
US20080032365A1 (en) 2008-02-07
NO20023588L (en) 2002-09-26
KR100925290B1 (en) 2009-11-04

Similar Documents

Publication Publication Date Title
US20170049132A1 (en) Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION