WO2019078398A2 - Chlorella genus abc-001 strain having excellent lipid productivity and cell growth rate under high carbon dioxide and salt concentration conditions, and use thereof - Google Patents

Chlorella genus abc-001 strain having excellent lipid productivity and cell growth rate under high carbon dioxide and salt concentration conditions, and use thereof Download PDF

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WO2019078398A2
WO2019078398A2 PCT/KR2017/012373 KR2017012373W WO2019078398A2 WO 2019078398 A2 WO2019078398 A2 WO 2019078398A2 KR 2017012373 W KR2017012373 W KR 2017012373W WO 2019078398 A2 WO2019078398 A2 WO 2019078398A2
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strain
culture
carbon dioxide
lipid
abc
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Korean (ko)
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WO2019078398A3 (en
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장용근
조준묵
이봉수
김은경
문명훈
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재단법인 탄소순환형 차세대 바이오매스 생산전환 기술연구단
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9706—Algae
    • A61K8/9722—Chlorophycota or Chlorophyta [green algae], e.g. Chlorella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10G—CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION; RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
    • C10G3/00—Production of liquid hydrocarbon mixtures from oxygen-containing organic materials, e.g. fatty oils, fatty acids
    • 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
    • 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
    • C12N1/125—Unicellular algae isolates
    • 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
    • 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
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • 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
    • 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
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/202—Algae extracts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P30/00—Technologies relating to oil refining and petrochemical industry
    • Y02P30/20—Technologies relating to oil refining and petrochemical industry using bio-feedstock
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02T—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO TRANSPORTATION
    • Y02T50/00—Aeronautics or air transport
    • Y02T50/60—Efficient propulsion technologies, e.g. for aircraft
    • Y02T50/678—Aviation using fuels of non-fossil origin

Definitions

  • the present invention relates to chlorella ABC-001 strain having excellent lipid productivity and cell growth rate under a high concentration of carbon dioxide and high salt and its use.
  • microalgae produce useful materials such as lipids, carbohydrates and proteins by using carbon dioxide directly or using reducing sugar obtained through photosynthesis using carbon dioxide. These materials can be used as raw materials for biofuels, health functional foods, cosmetics and feeds, and are also highly applicable to microalgae biomass itself.
  • production of biofuels using microalgae lipids can reduce the use of fossil fuels, which can contribute to reducing atmospheric CO2.
  • the lipids of microalgae also have nutritional value because they contain ⁇ 3 and ⁇ 6 fatty acids, which are known to be good for health. Because of these advantages, much research has been done on the lipid accumulation of microalgae.
  • microalgae There are various ways to promote lipid accumulation in microalgae, but one of the well known methods is salt stress. When cultured under high salt conditions, microalgae are stressed and microalgae accumulate more lipid in this process. However, this kind of stress increases the lipid accumulation in the microalgae, but the growth of the cells decreases and eventually the productivity is deteriorated. Therefore, it is important to find microalgae strains that have good lipid accumulation rate and maintain growth even at high salt stress.
  • Such a strain that maintains its growth ability even at high salinity has an advantage that it can be cultured in sea water or sea water. Compared to freshwater, seawater is absolutely large in quantity and does not overlap with drinking water or living water, so there is no great burden to supply in large quantities. In addition, most coal-fired power plants are located close to the coast, so it is easy to supply sea water when cultivating salt-resistant micro-tanks to remove carbon dioxide from the thermal power plant exhaust gas. Therefore, a strain having a high salt content and a high lipid productivity under a high concentration of carbon dioxide can effectively remove carbon dioxide from the exhaust gas of the power plant, and can contribute greatly to the production of biomass and the production of useful materials by using it as a raw material.
  • Korean Patent No. 1743232 discloses 'microalgae with increased lipid content using salt stress and method for producing the microalgae'
  • Korean Patent Publication No. 2016-0143327 discloses a method for culturing microalgae
  • the chlorella ABC-001 strain having excellent lipid productivity and cell growth rate under high concentration of carbon dioxide and high salt conditions and its use There is nothing to be found.
  • the present invention has been made in view of the above-described needs.
  • a novel chlorella ABC-001 strain having high salt tolerance and high growth and lipid productivity under a high concentration of carbon dioxide was isolated.
  • the chlorella ABC-001 strain of the present invention exhibited a high growth rate when 10% or 15% of carbon dioxide was mixed with air, and the growth rate of the cell even at the same salinity (35 g / L) (0 g / L) of fresh water culture medium.
  • the chlorella ABC-001 strain having high salt tolerance and high lipid productivity under the high-concentration carbon dioxide condition of the present invention can effectively remove carbon dioxide from the exhaust gas of the power plant adjacent to the coast, and can produce biomass and use it as a raw material
  • the present invention has been completed.
  • the present invention provides a Chlorella sp. ABC-001 strain having excellent lipid productivity and cell growth rate under a high concentration of carbon dioxide and high salt.
  • the present invention also provides a microorganism preparation for lipid production comprising the strain or a culture thereof as an active ingredient.
  • the present invention also provides a method for producing a lipid characterized in that the strain is cultured and lipid is separated from the culture.
  • the present invention also relates to a method for producing a microorganism, which comprises culturing the strain;
  • the present invention also provides a food, a feed composition or a cosmetic composition containing the strain or a culture thereof, a concentrate of the culture solution or a dried product of the culture as an active ingredient.
  • the present invention also relates to a method for producing a microorganism, which comprises culturing the strain;
  • the present invention is to provide a novel chlorella ABC-001 strain which is resistant to salt, grows well under a high concentration of carbon dioxide, and accumulates a large amount of lipid.
  • the novel chlorella ABC-001 strain of the present invention exhibits almost the same cell growth and lipid productivity in fresh water and salty water and sea water, and is easily harvested by natural precipitation when cultured in seawater.
  • the chlorella ABC-001 strain having high salt tolerance and high lipid productivity under the high carbon dioxide condition of the present invention can effectively remove carbon dioxide from the exhaust gas of the power plant adjacent to the coast, and produces biomass and a useful substance by using it as a raw material It is very useful for the related industries.
  • SEQ ID NO: 1 shows the phylogenetic tree prepared based on the 18s rRNA gene sequence (SEQ ID NO: 1) information obtained from the microalgae selected in the present invention.
  • FIG. 2 shows that the microalgae selected in the present invention were cultured in an incubator in which air or air was mixed with carbon dioxide at a concentration of 10% or 15% at a rate of 300 mL / min, and then the cell growth rate was calculated as dry cell weight, DCW).
  • FIG. 3 is a graph showing the results of salt resistance test of microalgae selected in the present invention by making a seawater culture supplemented with 10% carbon dioxide under the condition of the highest growth rate in Example 3 of the present invention. Under the same culture conditions, the culture was supplemented with 0, 7, 21, and 35 g / L of mannitol in the culture medium, followed by independent culture twice.
  • FIG. 4 is a result of confirming the cell growth rate and lipid productivity after culturing the microalgae selected in the present invention in an incubator in which air or air was mixed with carbon dioxide at a concentration of 10% or 15% at a rate of 300 mL / min.
  • FIG. 5 shows the results of confirming the cell growth rate and lipid productivity after the salt-resistance test with the salt water added with 10% carbon dioxide under the condition of the highest growth rate in Example 3 of the present invention. Under the same culture conditions, the culture was supplemented with 0, 7, 21, and 35 g / L of mannitol in the culture medium, followed by independent culture twice.
  • FIG. 6 shows the fatty acid composition after culturing the microalgae selected in the present invention at various salt concentrations under 10% carbon dioxide.
  • FIG. 7 shows the result of observing the natural sedimentation of cells after 24 hours of incubation, after transferring about 12 mL of the culture solution to a 15 mL falcon tube after fixing the falcon tube.
  • the present invention provides a Chlorella sp. ABC-001 strain excellent in lipid productivity and cell growth rate under high concentration of carbon dioxide and high salt.
  • Chlorella genus (Chlorella sp.) ABC-001 strain showed higher growth rate when the mixture of carbon dioxide than if supplied only was identified, removed from the stream of Youngwol area, air with 10% or 15%, and salts, such as water ( 35 g / L), the growth rate of the cells was maintained, and no significant difference was observed in the dry weight of the cells compared to fresh water medium (0 g / L).
  • the Chlorella sp. ABC- (KCTC) on October 30, 2017 (Accession No .: KCTC13386BP).
  • the high concentrations of carbon dioxide and high salt may be 1 to 18% carbon dioxide and 33 to 40 g / l of salt, respectively, and preferably 1 to 15% carbon dioxide and 33 to 37 g / And most preferably 10% carbon dioxide and 35 g / l salt, but is not limited thereto.
  • the present invention also provides a microorganism preparation for lipid production comprising the strain or a culture thereof as an active ingredient.
  • the microorganism preparation may contain Chlorella sp. ABC-001 as an active ingredient and may be effectively used for mass production of bio-oil.
  • the mass produced bio-oil can be used to produce biodiesel through a trans-esterification process.
  • the present invention also provides a method for producing a lipid characterized in that the strain is cultured and lipid is separated from the culture.
  • the strain can produce lipids at a high level of 1 to 18% carbon dioxide and 33 to 40 g / l of salt, preferably 1 to 15% carbon dioxide and 33 to 37 g / l of salt, most preferably 10% And 35 g / l salt, but is not limited thereto. Any method known in the art can be used as a method for separating lipids from the cell culture medium. Since the cell line of the present invention can harvest the strain through natural precipitation after the culture of sea water, there is an advantage that the process after lipid extraction and extraction after cell harvesting can be minimized.
  • the culture of the strain may be culturing in fresh water, seawater or nodules, and preferably, culturing in seawater, but is not limited thereto.
  • the method according to an embodiment of the present invention may include, but is not limited to, harvesting the strain through natural precipitation after the seawater culture.
  • the strain is cultured under the conditions of 1 to 18% carbon dioxide and 33 to 40 g / l of the culture.
  • the culture medium for the strain may be a medium commonly used in the art.
  • the extraction solvent may be selected from the group consisting of pentane, hexane, cyclohexane, toluene, methylene chloride, ethyl acetate, acetone, chloroform, methanol, ethanol and mixed solvents thereof.
  • the present invention can transesterify the triglyceride produced from the Chlorella sp. ABC-001 strain to separate the fatty acid ester and glycerol to produce biodiesel.
  • the fatty acid ester may preferably be a fatty acid methyl ester or a fatty acid ethyl ester, but is not limited thereto.
  • the biodiesel can be produced in the form of a methyl ester or an ethyl ester by transesterifying the fatty acid contained in the biomass.
  • the transesterification process refers to a method of transforming a large branched molecular structure of fat contained in biomass into a small, linear chain molecule required by a regular diesel engine.
  • Triton X-100 or Tween 60 may be added, but the present invention is not limited thereto.
  • the emulsifier can be used to stabilize the interface of the bio-oil and mix well, thereby increasing the reaction yield and thus saving the biodiesel recovery cost.
  • sodium hydroxide or potassium hydroxide may be used as a reaction catalyst in the transesterification process, but the present invention is not limited thereto.
  • the present invention provides a food containing the strain, a culture solution thereof, a concentrate of the culture solution, or a dried product of the culture solution as an active ingredient.
  • the Chlorella sp. ABC-001 strain has a high lipid content including an unsaturated fatty acid
  • the food containing the strain or the culture medium thereof as an active ingredient is highly nutritious.
  • the food can be used in the form of a processed food or can be used with other food or food ingredients as it is, or can be suitably used according to conventional methods. There is no particular limitation on the kind of the processed food. Examples of foods to which the Chlorella sp.
  • ABC-001 strain powder can be added include dairy products including meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, Products, various soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes, and includes processed foods in a conventional sense.
  • the present invention also provides a feed composition comprising the strain, a culture thereof, a concentrate of the culture solution, or a dried product of the culture solution as an active ingredient.
  • the content of the Chlorella sp. ABC-001 strain, the culture medium thereof, the concentrate of the culture medium or the dried material of the culture medium in the feed composition can be appropriately selected according to the kind, age, body weight, .
  • the feed composition of the present invention can be produced according to a feed production method known in the art, and can be produced, for example, by using various feed ingredients or compound feeds and the strain of the present invention, a culture thereof, a concentrate of the culture solution, And further performing additional processing steps such as forming into pellets or cutting in the form of granules and the like. It is apparent to those skilled in the art that the composition, composition, manufacturing method, feeding method and the like of the feed composition of the present invention can be appropriately selected from conventional techniques known in the art.
  • the present invention also provides a cosmetic composition
  • a cosmetic composition comprising the strain, the culture liquid thereof, the concentrated liquid of the culture liquid, or the dried material of the culture liquid as an active ingredient.
  • the components contained in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to the above-mentioned Chlorella sp. ABC-001 strain as an active ingredient, a culture thereof, a concentrate of the culture solution or a dried product of the culture solution , Customary adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers.
  • the cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a flexible lotion (skin), a nutritional lotion (milk lotion), a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder .
  • the present invention also relates to a method for producing a microorganism, which comprises culturing the strain;
  • the culture medium of the present invention may be a culture medium commonly used in the art, and any method known in the art may be used as a method for separating lipids from the culture medium of the present invention.
  • the method for producing bio-aerosol oil according to the present invention is characterized in that deoxygenation, isomerization and deoxygenation, isomerization and cracking reactions are carried out from lipids in the presence of a catalyst, To form paraffinic hydrocarbons, and distilling the reaction product to extract aerial oil fractions.
  • the present invention is not limited thereto.
  • NiMo, CoMo, Pt, and Pd may be used, but the present invention is not limited thereto.
  • composition of the N8 culture used in the experiment is as follows; 5 mM KNO 3 , 5.44 mM KH 2 PO 4 , 1.83 mM Na 2 HPO 4 , 0.2 mM MgSO 4 .7H 2 O, 0.12 mM CaCl 2 .2H 2 O, FeNaEDTA 3H 2 O and 1 ml micronutrient 4 ⁇ 7H 2 O, 0.07 mM MnCl 2 ⁇ 4H 2 O, 0.07 mM CuSO 4 ⁇ 5H 2 O, 0.01 mM Al 2 (SO 4) 3 ⁇ 18H 2 O)
  • the 18s rRNA gene sequence was analyzed to identify the novelty of the selected microalgae and to identify the species to which the microalgae belong.
  • the DNA of the strain was extracted using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany).
  • primer 18SF (5'-CCT GGT TGA TCC TGC CAG-3 ' : SEQ ID NO: 2)
  • 18SR 5'-TTG ATC CTT CTG CAG GTT CA-3 ': SEQ ID NO: 3
  • the obtained nucleotide sequence (SEQ ID NO: 1) was blasted on the basis of the NCBI database.
  • the weight of the dried cells was calculated by measuring the weight of the dried cells by washing the cells with GF / C filter paper, washing with distilled water more than 2 times, drying at 70 ° C for 24 hours, and passing the 3-5 mL of the culture solution through the GF / C filter paper.
  • the carbon dioxide was mixed at 10% and 15%, it showed a high growth rate and reached the highest at 2.9 g / L at 10% (FIG. 2).
  • the results showed that the strain showed high growth even at high concentration of carbon dioxide.
  • Example 3 a salt water culture medium containing 10% carbon dioxide at which the growth rate was highest was added to the sea salt medium, and the salt resistance test was conducted. Under the same culture conditions, the culture was supplemented with 0, 7, 21, and 35 g / L of mannitol in the culture medium, followed by independent culture twice. 7 days. As a result, it was confirmed that this strain maintained cell growthability even at the same salinity (35 g / L) as seawater and showed no significant difference in dry cell weight as compared to fresh water medium (0 g / L) ). In addition, salt tolerance of this strain was verified by using NaCl - added saline and actual seawater as well as sun - salt.
  • the culture was centrifuged at 4000 rpm for 1 minute to separate the cells.
  • the separated cells were washed twice with distilled water, and the final biomass was lyophilized for 3 days to facilitate extraction.
  • the dried cells were weighed 10 mg into a glass tube and used in the analysis of fatty acid (Fatty Acid Methyl Ester, FAME), and 50 mg was used to measure total lipid content.
  • the lipid content was higher in the high concentration carbon dioxide condition (FIG. 4). Also, the biomass concentration was higher than that in the high concentration CO2 condition, so the total fat productivity (g / L / d) was 4.5 times higher at 10% CO2 and 8% and 15% than the air condition.
  • Example 4 After the culture was completed in Example 4, about 12 mL of each culture was transferred to a 15 mL Falcon tube, and the cells were settled for 24 hours after fixing the Falcon tube (FIG. 7). After about 2 hours, it was observed that the cells incubated at 35 g / L of sun saliva were noticeably submerged. After 24 hours, most of the cells were observed to stick to the bottom of the tube. In other conditions, we could confirm the difference in the rate at which the cells settled after about 6 hours. After 24 hours, it was verified that the cells cultured at high concentration of sodium salt rapidly precipitated rapidly. It was shown that when the strain was cultured in seawater, it could be harvested while retaining similar growth and lipid contents compared to culturing in freshwater.

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Abstract

The present invention provides a novel Chlorella genus ABC-001 strain that has salt resistance, can grow well under high carbon dioxide concentration conditions, and accumulates a large amount of lipids. A novel Chlorella genus ABC-001 strain according to the present invention exhibits almost identical cell growth and lipid productivity in brackish water and saltwater, which are salty, as in freshwater, and is particularly easy to harvest through natural sedimentation when cultured in saltwater. The Chlorella genus ABC-001 strain having salt resistance and having high lipid productivity under high carbon dioxide concentration conditions of the present invention can effectively remove carbon dioxide in exhaust gas from power plants near the shoreline, and greatly contribute to biomass production and processes for producing useful materials using biomass as a raw material, thus being extremely useful in industries related thereto.

Description

고농도의 이산화탄소 및 고염 조건에서 지질 생산성 및 세포 생장률이 우수한 클로렐라 속 ABC-001 균주 및 이의 용도A chlorella ABC-001 strain excellent in lipid productivity and cell growth rate under a high concentration of carbon dioxide and high salt and its use
본 발명은 고농도의 이산화탄소 및 고염 조건에서 지질 생산성 및 세포 생장률이 우수한 클로렐라 속 ABC-001 균주 및 이의 용도에 관한 것이다.The present invention relates to chlorella ABC-001 strain having excellent lipid productivity and cell growth rate under a high concentration of carbon dioxide and high salt and its use.
범지구적인 환경문제인 지구 온난화는 대기 중의 이산화탄소가 주원인으로 알려져 있다. 대기 중 이산화탄소 농도를 증가시키는데 가장 큰 기여를 한 것은 화력발전소이다. 화력발전소에서 배출되는 배기가스에는 5-15%의 고농도 이산화탄소가 포함되어 있는데 아직까지 이를 제거할 수 있는 상용화 기술이 없다. 전 세계적으로 대기 중의 이산화탄소를 제거하기 위해 많은 투자와 개발이 이뤄지고 있고 그러한 방법들 중 하나로 미세조류가 각광을 받고 있다. 이산화탄소와 빛을 이용하는 광합성을 통해 성장하는 미세조류는 육상식물에 비해 생장율이 월등히 높고 이산화탄소 이용률 또한 높다. 그러나 미세조류가 잘 자랄 수 있는 일반적인 이산화탄소의 농도는 2~5%이며 고농도의 이산화탄소 조건에서는 생장에 저해를 받는다. 하지만 효율적으로 화력발전소에서 배출되는 배기가스 내 이산화탄소를 제거하기 위해서는 고농도 이산화탄소 조건에서 높은 생장성을 보이는 미세조류 균주가 필요하다.Global warming, a global environmental problem, is known to be mainly caused by atmospheric carbon dioxide. The biggest contributor to increasing the atmospheric carbon dioxide concentration is the thermal power plant. Exhaust gases from thermal power plants contain 5-15% of high-concentration carbon dioxide, but there is no commercially available technology to remove them. There are many investments and developments in the world to remove atmospheric carbon dioxide, and microalgae is attracting attention as one such method. The microalgae that grow through photosynthesis using carbon dioxide and light have higher growth rate and higher carbon dioxide utilization than land plants. However, the concentration of common carbon dioxide that microalgae can grow well is 2 ~ 5%, and it is inhibited by growth under the high concentration of carbon dioxide. However, in order to efficiently remove carbon dioxide from the exhaust gas discharged from a thermal power plant, a microalgae strain showing a high growth rate under a high concentration of carbon dioxide is required.
미세조류는 이산화탄소를 직접적으로 이용하거나 이산화탄소를 이용하는 광합성을 통해 얻어진 환원당을 사용하여 지질, 탄수화물, 단백질과 같은 유용한 물질을 만들어낸다. 이러한 물질들은 바이오연료, 건강기능식품, 화장품, 사료 등의 원료로 활용 가능하며, 미세조류 바이오매스 자체로도 활용도가 매우 높다. 이 중에서도 미세조류의 지질을 이용하여 바이오연료를 생산하면 화석연료의 사용을 줄일 수 있기 때문에 대기 중 이산화탄소 증가를 줄이는데 기여할 수 있다. 또한 미세조류의 지질에는 건강에 좋다고 알려진 ω3와 ω6 지방산들이 포함되어 있어 영양학적으로도 가치가 있다. 이러한 장점들 때문에 미세조류의 지질 축적에 대한 많은 연구가 이뤄져왔다. 미세조류의 지질 축적을 촉진시키는데 다양한 방법이 있지만 그 중 잘 알려진 방법 중 하나가 염 스트레스를 주는 것이다. 높은 염 조건에서 배양을 하게 되면 미세조류가 스트레스를 받게 되고 이 과정에서 미세조류는 지질을 더 많이 축적한다. 하지만 이런 종류의 스트레스를 받게 되면 미세조류 내 지질축적은 높아지나 세포의 성장은 떨어지게 되고 결국에는 생산성에 악영향을 끼치게 된다. 이 때문에 높은 염 스트레스에서도 지질 축적률이 좋으며 생장을 유지하는 미세조류 균주를 찾는 것이 중요하다.The microalgae produce useful materials such as lipids, carbohydrates and proteins by using carbon dioxide directly or using reducing sugar obtained through photosynthesis using carbon dioxide. These materials can be used as raw materials for biofuels, health functional foods, cosmetics and feeds, and are also highly applicable to microalgae biomass itself. Among these, production of biofuels using microalgae lipids can reduce the use of fossil fuels, which can contribute to reducing atmospheric CO2. The lipids of microalgae also have nutritional value because they contain ω3 and ω6 fatty acids, which are known to be good for health. Because of these advantages, much research has been done on the lipid accumulation of microalgae. There are various ways to promote lipid accumulation in microalgae, but one of the well known methods is salt stress. When cultured under high salt conditions, microalgae are stressed and microalgae accumulate more lipid in this process. However, this kind of stress increases the lipid accumulation in the microalgae, but the growth of the cells decreases and eventually the productivity is deteriorated. Therefore, it is important to find microalgae strains that have good lipid accumulation rate and maintain growth even at high salt stress.
이렇게 높은 염도에서도 생장성을 유지하는 균주는 해수나 기수에서도 배양이 가능하다는 장점이 있다. 담수에 비해 해수는 절대적으로 양이 많고 식수나 생활수와 겹치지 않기 때문에 대량으로 공급되는데 큰 부담이 없다. 또한 대부분 화력발전소는 해안과 인접한 곳에 위치해 있어서, 화력발전소 배기가스 내 이산화탄소를 제거하기 위해 내염성을 가진 미세조를 배양할 때 해수의 공급이 수월하다. 그러므로 내염성을 가지며 고농도 이산화탄소 조건에서 높은 지질 생산성을 가진 균주는 발전소 배기가스 내 이산화탄소를 효과적으로 제거하며 바이오매스 생산 및 이를 원료로 사용하여 유용한 물질을 생산하는 공정에 큰 기여를 할 수 있다.Such a strain that maintains its growth ability even at high salinity has an advantage that it can be cultured in sea water or sea water. Compared to freshwater, seawater is absolutely large in quantity and does not overlap with drinking water or living water, so there is no great burden to supply in large quantities. In addition, most coal-fired power plants are located close to the coast, so it is easy to supply sea water when cultivating salt-resistant micro-tanks to remove carbon dioxide from the thermal power plant exhaust gas. Therefore, a strain having a high salt content and a high lipid productivity under a high concentration of carbon dioxide can effectively remove carbon dioxide from the exhaust gas of the power plant, and can contribute greatly to the production of biomass and the production of useful materials by using it as a raw material.
한편, 한국등록특허 제1743232호에서는 '염류 스트레스를 이용하여 지질함량을 증가시킨 미세조류 및 그의 제조방법'이 개시되어 있고, 한국공개특허 제2016-0143327호에서는 '미세조류의 배양방법 및 이를 이용한 미세조류의 세포 내에 축적되는 영양 성분의 함량을 조절하는 방법'이 개시되어 있으나, 본 발명에서와 같이, 고농도의 이산화탄소 및 고염 조건에서 지질 생산성 및 세포 생장률이 우수한 클로렐라 속 ABC-001 균주 및 이의 용도에 대해서는 밝혀진 바가 전혀 없다.Korean Patent No. 1743232 discloses 'microalgae with increased lipid content using salt stress and method for producing the microalgae', Korean Patent Publication No. 2016-0143327 discloses a method for culturing microalgae, Discloses a method for controlling the content of nutrients accumulated in cells of microalgae. However, as in the present invention, the chlorella ABC-001 strain having excellent lipid productivity and cell growth rate under high concentration of carbon dioxide and high salt conditions and its use There is nothing to be found.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 내염성을 가지면서도 고농도 이산화탄소 조건에서 높은 생장성과 지질 생산성을 보이는 신규한 클로렐라 속 ABC-001 균주를 분리하였다. 구체적으로, 본 발명의 클로렐라 속 ABC-001 균주는 공기만 공급했을 때보다 이산화탄소를 10% 또는 15%로 혼합하였을 때 높은 성장률을 보였고, 해수와 같은 염도(35 g/L)에서도 세포의 생장성을 유지하며 담수배지(0 g/L) 대비 건조균체중량에서 큰 차이를 보이지 않은 것을 확인하였다. The present invention has been made in view of the above-described needs. In the present invention, a novel chlorella ABC-001 strain having high salt tolerance and high growth and lipid productivity under a high concentration of carbon dioxide was isolated. Specifically, the chlorella ABC-001 strain of the present invention exhibited a high growth rate when 10% or 15% of carbon dioxide was mixed with air, and the growth rate of the cell even at the same salinity (35 g / L) (0 g / L) of fresh water culture medium.
따라서, 본 발명의 내염성을 가지며 고농도 이산화탄소 조건에서 높은 지질 생산성을 가진 클로렐라 속 ABC-001 균주는 해안에 인접한 발전소 배기가스 내 이산화탄소를 효과적으로 제거할 수 있으며, 바이오매스 생산 및 이를 원료로 사용하여 유용한 물질을 생산하는 공정에 큰 기여를 할 수 있음을 확인함으로써, 본 발명을 완성하였다.Therefore, the chlorella ABC-001 strain having high salt tolerance and high lipid productivity under the high-concentration carbon dioxide condition of the present invention can effectively remove carbon dioxide from the exhaust gas of the power plant adjacent to the coast, and can produce biomass and use it as a raw material The present invention has been completed.
상기 목적을 달성하기 위하여, 본 발명은 고농도의 이산화탄소 및 고염 조건에서 지질 생산성 및 세포 생장률이 우수한 클로렐라 속(Chlorella sp.) ABC-001 균주를 제공한다.In order to achieve the above object, the present invention provides a Chlorella sp. ABC-001 strain having excellent lipid productivity and cell growth rate under a high concentration of carbon dioxide and high salt.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 지질 생산용 미생물제제를 제공한다.The present invention also provides a microorganism preparation for lipid production comprising the strain or a culture thereof as an active ingredient.
또한, 본 발명은 상기 균주를 배양하고, 그 배양액으로부터 지질을 분리하는 것을 특징으로 하는 지질의 제조 방법을 제공한다.The present invention also provides a method for producing a lipid characterized in that the strain is cultured and lipid is separated from the culture.
또한, 본 발명은 상기 균주를 배양하는 단계;The present invention also relates to a method for producing a microorganism, which comprises culturing the strain;
상기 배양액으로부터 지질을 분리하는 단계;Separating the lipid from the culture liquid;
상기 지질을 트랜스에스테르화시켜 지방산 에스테르를 생산하는 단계; 및Transesterifying the lipid to produce a fatty acid ester; And
상기 생산된 지방산 에스테르를 바이오디젤로 전환하는 단계를 포함하는 바이오디젤의 생산 방법을 제공한다.And converting the produced fatty acid ester into biodiesel.
또한, 본 발명은 상기 균주 또는 이의 배양액, 상기 배양액의 농축물 또는 상기 배양액의 건조물을 유효성분으로 함유하는 식품, 사료 조성물 또는 화장료 조성물을 제공한다.The present invention also provides a food, a feed composition or a cosmetic composition containing the strain or a culture thereof, a concentrate of the culture solution or a dried product of the culture as an active ingredient.
또한, 본 발명은 상기 균주를 배양하는 단계;The present invention also relates to a method for producing a microorganism, which comprises culturing the strain;
상기 배양액으로부터 지질을 분리하는 단계; 및Separating the lipid from the culture liquid; And
상기 분리한 지질을 촉매전환시켜 항공유로 전환하는 단계를 포함하는 바이오항공유의 생산 방법을 제공한다.And converting the separated lipid to catalytic conversion into aviation oil.
본 발명은 내염성을 가지며 고농도 이산화탄소 조건에서 잘 자라고, 지질을 많이 축적하는 신규한 클로렐라 속 ABC-001 균주를 제공하는 것이다. 본 발명의 신규한 클로렐라 속 ABC-001 균주는 담수와 염도가 있는 기수 및 해수에서 거의 동일한 세포 생장과 지질 생산성을 보이며 특히, 해수에서 배양했을 시 자연침전으로 수확이 용이하다. 본 발명의 내염성을 가지며 고농도 이산화탄소 조건에서 높은 지질 생산성을 가진 클로렐라 속 ABC-001 균주는 해안에 인접한 발전소 배기가스 내 이산화탄소를 효과적으로 제거할 수 있으며, 바이오매스 생산 및 이를 원료로 사용하여 유용한 물질을 생산하는 공정에 큰 기여를 할 수 있어 관련 산업에 매우 유용하다.The present invention is to provide a novel chlorella ABC-001 strain which is resistant to salt, grows well under a high concentration of carbon dioxide, and accumulates a large amount of lipid. The novel chlorella ABC-001 strain of the present invention exhibits almost the same cell growth and lipid productivity in fresh water and salty water and sea water, and is easily harvested by natural precipitation when cultured in seawater. The chlorella ABC-001 strain having high salt tolerance and high lipid productivity under the high carbon dioxide condition of the present invention can effectively remove carbon dioxide from the exhaust gas of the power plant adjacent to the coast, and produces biomass and a useful substance by using it as a raw material It is very useful for the related industries.
도 1은 본 발명에서 선별한 미세조류로부터 얻은 18s rRNA 유전자 서열(서열번호 1) 정보를 토대로 작성된 계통수를 나타낸다.1 shows the phylogenetic tree prepared based on the 18s rRNA gene sequence (SEQ ID NO: 1) information obtained from the microalgae selected in the present invention.
도 2는 본 발명에서 선별한 미세조류를 공기 또는 공기와 10% 또는 15%의 농도로 이산화탄소를 혼합한 배양기에 300 mL/min를 넣어주면서 배양한 후 세포 성장률을 건조균체중량(Dry cell weight, DCW)으로 나타낸 결과이다.FIG. 2 shows that the microalgae selected in the present invention were cultured in an incubator in which air or air was mixed with carbon dioxide at a concentration of 10% or 15% at a rate of 300 mL / min, and then the cell growth rate was calculated as dry cell weight, DCW).
도 3은 본 발명의 실시예 3에서 성장률이 가장 우수하였던 10% 이산화탄소 조건에서 천일염이 첨가된 해수 배지를 만들어 본 발명에서 선별한 미세조류의 내염성 테스트를 수행한 결과이다. 동일한 배양조건 아래 천일염을 0, 7, 21, 35 g/L 농도로 맞추어 배지에 첨가한 후 독립적인 배양을 2회씩 실시하였다.FIG. 3 is a graph showing the results of salt resistance test of microalgae selected in the present invention by making a seawater culture supplemented with 10% carbon dioxide under the condition of the highest growth rate in Example 3 of the present invention. Under the same culture conditions, the culture was supplemented with 0, 7, 21, and 35 g / L of mannitol in the culture medium, followed by independent culture twice.
도 4는 본 발명에서 선별한 미세조류를 공기 또는 공기와 10% 또는 15%의 농도로 이산화탄소를 혼합한 배양기에 300 mL/min를 넣어주면서 배양한 후 세포 성장률 및 지질생산성을 확인한 결과이다.FIG. 4 is a result of confirming the cell growth rate and lipid productivity after culturing the microalgae selected in the present invention in an incubator in which air or air was mixed with carbon dioxide at a concentration of 10% or 15% at a rate of 300 mL / min.
도 5는 본 발명의 실시예 3에서 성장률이 가장 우수하였던 10% 이산화탄소 조건에서 천일염이 첨가된 해수 배지를 만들어 내염성 테스트를 수행한 후 세포 성장률 및 지질생산성을 확인한 결과이다. 동일한 배양조건 아래 천일염을 0, 7, 21, 35 g/L 농도로 맞추어 배지에 첨가한 후 독립적인 배양을 2회씩 실시하였다.FIG. 5 shows the results of confirming the cell growth rate and lipid productivity after the salt-resistance test with the salt water added with 10% carbon dioxide under the condition of the highest growth rate in Example 3 of the present invention. Under the same culture conditions, the culture was supplemented with 0, 7, 21, and 35 g / L of mannitol in the culture medium, followed by independent culture twice.
도 6은 본 발명에서 선별한 미세조류를 10% 이산화탄소 조건에서 다양한 염 농도에서 배양 후 지방산 조성을 나타낸다.FIG. 6 shows the fatty acid composition after culturing the microalgae selected in the present invention at various salt concentrations under 10% carbon dioxide.
도 7은 실시예 4에서 배양이 끝난 후 배양액 각각 약 12 mL를 15 mL 팔콘 튜브에 옮겨 담고 팔콘 튜브를 고정시킨 후 24시간 동안 세포가 가라앉는 자연침전을 관찰한 결과이다.FIG. 7 shows the result of observing the natural sedimentation of cells after 24 hours of incubation, after transferring about 12 mL of the culture solution to a 15 mL falcon tube after fixing the falcon tube.
본 발명의 목적을 달성하기 위하여, 본 발명은 고농도의 이산화탄소 및 고염 조건에서 지질 생산성 및 세포 생장률이 우수한 클로렐라 속(Chlorella sp.) ABC-001 균주를 제공한다.In order to achieve the object of the present invention, the present invention provides a Chlorella sp. ABC-001 strain excellent in lipid productivity and cell growth rate under high concentration of carbon dioxide and high salt.
상기 클로렐라 속(Chlorella sp.) ABC-001 균주는 영월지역의 하천에서 분리하여 동정하였으며, 공기만 공급했을 때보다 이산화탄소를 10% 또는 15%로 혼합하였을 때 높은 성장률을 보였고, 해수와 같은 염도(35 g/L)에서도 세포의 생장성을 유지하며 담수배지(0 g/L) 대비 건조균체중량에서 큰 차이를 보이지 않은 것을 확인하였으며, 상기 클로렐라 속(Chlorella sp.) ABC-001 균주는 한국생명공학연구원(KCTC)에 2017년 10월 30일자로 기탁하였다(기탁번호: KCTC13386BP).The Chlorella genus (Chlorella sp.) ABC-001 strain showed higher growth rate when the mixture of carbon dioxide than if supplied only was identified, removed from the stream of Youngwol area, air with 10% or 15%, and salts, such as water ( 35 g / L), the growth rate of the cells was maintained, and no significant difference was observed in the dry weight of the cells compared to fresh water medium (0 g / L). The Chlorella sp. ABC- (KCTC) on October 30, 2017 (Accession No .: KCTC13386BP).
본 발명의 일 구현 예에 따른 균주에서, 상기 고농도의 이산화탄소 및 고염은 각각 1~18% 이산화탄소 및 33~40g/l 염일 수 있고, 바람직하게는, 1~15% 이산화탄소 및 33~37g/l 염일 수 있고, 가장 바람직하게는 10% 이산화탄소 및 35g/l 염일 수 있으나, 이에 제한되지 않는다.In a strain according to an embodiment of the present invention, the high concentrations of carbon dioxide and high salt may be 1 to 18% carbon dioxide and 33 to 40 g / l of salt, respectively, and preferably 1 to 15% carbon dioxide and 33 to 37 g / And most preferably 10% carbon dioxide and 35 g / l salt, but is not limited thereto.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 지질 생산용 미생물제제를 제공한다.The present invention also provides a microorganism preparation for lipid production comprising the strain or a culture thereof as an active ingredient.
상기 미생물 제제는 클로렐라 속(Chlorella sp.) ABC-001를 유효성분으로 포함할 수 있으며, 바이오오일의 대량 생산에 효과적으로 이용될 수 있다. 상기 대량 생산된 바이오오일은 트랜스에스테르화 과정을 통해 바이오디젤을 생산하는데 이용될 수 있다.The microorganism preparation may contain Chlorella sp. ABC-001 as an active ingredient and may be effectively used for mass production of bio-oil. The mass produced bio-oil can be used to produce biodiesel through a trans-esterification process.
또한, 본 발명은 상기 균주를 배양하고, 그 배양액으로부터 지질을 분리하는 것을 특징으로 하는 지질의 제조 방법을 제공한다. 상기 균주는 1~18% 이산화탄소 및 33~40g/l 염인 조건에서 지질을 고생산할 수 있으며, 바람직하게는, 1~15% 이산화탄소 및 33~37g/l 염일 수 있고, 가장 바람직하게는 10% 이산화탄소 및 35g/l 염일 수 있으나, 이에 제한되지 않는다. 세포주 배양액으로부터 지질을 분리하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다. 본 발명의 세포주는 해수 배양 후 자연침전을 통해 균주를 수확할 수 있으므로, 세포 수확 후 지질 추출 및 추출 후 공정을 최소화할 수 있는 장점이 있다.The present invention also provides a method for producing a lipid characterized in that the strain is cultured and lipid is separated from the culture. The strain can produce lipids at a high level of 1 to 18% carbon dioxide and 33 to 40 g / l of salt, preferably 1 to 15% carbon dioxide and 33 to 37 g / l of salt, most preferably 10% And 35 g / l salt, but is not limited thereto. Any method known in the art can be used as a method for separating lipids from the cell culture medium. Since the cell line of the present invention can harvest the strain through natural precipitation after the culture of sea water, there is an advantage that the process after lipid extraction and extraction after cell harvesting can be minimized.
본 발명의 일 구현 예에 따른 방법에서, 상기 균주의 배양은 담수, 해수 또는 기수에서 배양하는 것일 수 있고, 바람직하게는 해수에서 배양하는 것일 수 있으나, 이에 제한되지 않는다.In the method according to an embodiment of the present invention, the culture of the strain may be culturing in fresh water, seawater or nodules, and preferably, culturing in seawater, but is not limited thereto.
본 발명의 일 구현 예에 따른 방법은 상기 해수 배양 후 자연침전을 통해 균주를 수확하는 것일 수 있으나, 이에 제한되지 않는다.The method according to an embodiment of the present invention may include, but is not limited to, harvesting the strain through natural precipitation after the seawater culture.
또한, 본 발명은 In addition,
상기 균주를 배양하는 단계;Culturing the strain;
상기 배양액으로부터 지질을 분리하는 단계;Separating the lipid from the culture liquid;
상기 지질을 트랜스에스테르화시켜 지방산 에스테르를 생산하는 단계; 및Transesterifying the lipid to produce a fatty acid ester; And
상기 생산된 지방산 에스테르를 바이오디젤로 전환하는 단계를 포함하는 바이오디젤의 생산 방법을 제공한다.And converting the produced fatty acid ester into biodiesel.
상기 균주의 배양 조건으로, 1~18% 이산화탄소 및 33~40g/l 염인 조건에서 배양된다. 균주 배양 배지는 당업계에서 일반적으로 통용되는 배지를 이용할 수 있다.The strain is cultured under the conditions of 1 to 18% carbon dioxide and 33 to 40 g / l of the culture. The culture medium for the strain may be a medium commonly used in the art.
본 발명의 균주 배양액으로부터 지질을 추출하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다. 상기 추출용매는 펜탄, 헥산, 사이클로헥산, 톨루엔, 메틸렌클로라이드, 에틸아세테이트, 아세톤, 클로로포름, 메탄올, 에탄올 및 이들의 혼합용매로 이루어진 군으로부터 선택될 수 있다. Any method known in the art can be used as a method for extracting lipids from the culture broth of the present invention. The extraction solvent may be selected from the group consisting of pentane, hexane, cyclohexane, toluene, methylene chloride, ethyl acetate, acetone, chloroform, methanol, ethanol and mixed solvents thereof.
또한, 본 발명은 클로렐라 속(Chlorella sp.) ABC-001 균주로부터 생산된 트라이글리세리드를 트랜스에스테르화시켜 지방산 에스테르와 글리세롤을 분리하여 바이오디젤을 제조할 수 있다. 상기 지방산 에스테르는 바람직하게는, 지방산 메틸 에스테르 또는 지방산 에틸 에스테르일 수 있으나, 이에 제한되지는 않는다. 상기 바이오디젤은 바이오매스에 포함된 지방산의 트랜스에스테르화 과정에 의해 메틸 에스테르 또는 에틸 에스테르 형태로 생산될 수 있다. 상기 트랜스에스테르화 과정은 바이오매스에 포함된 지방의 크고 가지를 낸 분자 구조를 정규 디젤 엔진이 요구하는 작고 직선 사슬의 분자로 변형시키는 방법을 말한다. 상기 트랜스에스테르화 과정의 유화제로서 Triton X-100 또는 Tween 60 등을 첨가할 수 있으나, 이에 제한되지는 않는다. 상기 유화제는 바이오오일의 계면 안정을 도모하고 잘 혼합되도록 하여 반응 수율을 높여서 바이오디젤의 회수 비용을 절약할 수 있게 한다. 또한, 상기 트랜스에스테르화 과정의 반응 촉매제로서 수산화나트륨 또는 수산화칼륨을 사용할 수 있으나, 이에 제한되지는 않는다.In addition, the present invention can transesterify the triglyceride produced from the Chlorella sp. ABC-001 strain to separate the fatty acid ester and glycerol to produce biodiesel. The fatty acid ester may preferably be a fatty acid methyl ester or a fatty acid ethyl ester, but is not limited thereto. The biodiesel can be produced in the form of a methyl ester or an ethyl ester by transesterifying the fatty acid contained in the biomass. The transesterification process refers to a method of transforming a large branched molecular structure of fat contained in biomass into a small, linear chain molecule required by a regular diesel engine. As the emulsifier in the transesterification process, Triton X-100 or Tween 60 may be added, but the present invention is not limited thereto. The emulsifier can be used to stabilize the interface of the bio-oil and mix well, thereby increasing the reaction yield and thus saving the biodiesel recovery cost. In addition, sodium hydroxide or potassium hydroxide may be used as a reaction catalyst in the transesterification process, but the present invention is not limited thereto.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 식품을 제공한다. 상기 클로렐라 속(Chlorella sp.) ABC-001 균주는 불포화 지방산을 포함한 지질함량이 높기 때문에, 상기 균주 또는 이의 배양액을 유효성분으로 함유하는 식품은 영양학적으로 매우 가치가 높다. 상기 식품은 상기 균주를 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있으며 가공식품 형태로 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 상기 가공식품의 종류에는 특별한 제한은 없다. 상기 클로렐라 속(Chlorella sp.) ABC-001 균주 분말을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 가공식품을 모두 포함한다.Further, the present invention provides a food containing the strain, a culture solution thereof, a concentrate of the culture solution, or a dried product of the culture solution as an active ingredient. Since the Chlorella sp. ABC-001 strain has a high lipid content including an unsaturated fatty acid, the food containing the strain or the culture medium thereof as an active ingredient is highly nutritious. The food can be used in the form of a processed food or can be used with other food or food ingredients as it is, or can be suitably used according to conventional methods. There is no particular limitation on the kind of the processed food. Examples of foods to which the Chlorella sp. ABC-001 strain powder can be added include dairy products including meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, Products, various soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes, and includes processed foods in a conventional sense.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 사료 조성물을 제공한다. 사료 조성물 중의 상기 클로렐라 속(Chlorella sp.) ABC-001 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물의 함량은 급여 가축의 종, 주령, 체중, 및 사육 조건 등에 따라 적절히 선택될 수 있다. 본 발명의 사료 조성물은 기술분야에 공지된 사료 제조방법에 따라 제조될 수 있으며, 예를 들어, 각종 사료 원료 또는 배합사료와 본 발명의 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 혼합한 후, 추가적인 가공 공정, 예를 들어 펠렛 형태로의 성형 또는 과립 등의 형태로의 절단 단계 등을 더 수행함으로써 제조될 수 있다. 본 발명의 사료 조성물의 구성성분, 조성, 제조방법, 급여방법 등은 기술분야에 공지된 통상의 기술로부터 적절히 선택될 수 있음이 통상의 기술자에게 명백하다.The present invention also provides a feed composition comprising the strain, a culture thereof, a concentrate of the culture solution, or a dried product of the culture solution as an active ingredient. The content of the Chlorella sp. ABC-001 strain, the culture medium thereof, the concentrate of the culture medium or the dried material of the culture medium in the feed composition can be appropriately selected according to the kind, age, body weight, . The feed composition of the present invention can be produced according to a feed production method known in the art, and can be produced, for example, by using various feed ingredients or compound feeds and the strain of the present invention, a culture thereof, a concentrate of the culture solution, And further performing additional processing steps such as forming into pellets or cutting in the form of granules and the like. It is apparent to those skilled in the art that the composition, composition, manufacturing method, feeding method and the like of the feed composition of the present invention can be appropriately selected from conventional techniques known in the art.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 화장료 조성물을 제공한다. 본 발명의 화장료 조성물에 포함되는 성분은 유효 성분으로서의 상기 클로렐라 속(Chlorella sp.) ABC-001 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물 이외에 화장품 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함한다. 본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연 화장수(스킨), 영양 화장수(밀크로션), 영양 크림, 마사지 크림, 에센스, 아이크림, 클렌징 크림, 클렌징 폼, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The present invention also provides a cosmetic composition comprising the strain, the culture liquid thereof, the concentrated liquid of the culture liquid, or the dried material of the culture liquid as an active ingredient. The components contained in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to the above-mentioned Chlorella sp. ABC-001 strain as an active ingredient, a culture thereof, a concentrate of the culture solution or a dried product of the culture solution , Customary adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers. The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a flexible lotion (skin), a nutritional lotion (milk lotion), a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder .
또한, 본 발명은 상기 균주를 배양하는 단계;The present invention also relates to a method for producing a microorganism, which comprises culturing the strain;
상기 배양액으로부터 지질을 분리하는 단계; 및Separating the lipid from the culture liquid; And
상기 분리한 지질을 촉매전환시켜 항공유로 전환하는 단계를 포함하는 바이오항공유의 생산 방법을 제공한다.And converting the separated lipid to catalytic conversion into aviation oil.
본 발명의 상기 균주 배양 배지는 당업계에서 일반적으로 통용되는 배지를 이용할 수 있으며, 본 발명의 균주 배양액으로부터 지질을 분리하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다. The culture medium of the present invention may be a culture medium commonly used in the art, and any method known in the art may be used as a method for separating lipids from the culture medium of the present invention.
본 발명에 따른 바이오항공유 제조방법은 통상적으로 수소를 첨가하지 않고 촉매의 존재 하에서 지질로부터 탈산소(deoxygenation), 이성화(isomerization) 및 탈산소(deoxygenation), 이성화(isomerization) 및 크래킹(cracking) 반응을 통해 파라핀계 탄화수소를 형성시키고, 상기 반응생성물을 증류시켜 항공유분을 추출하는 단계를 포함하여 이루어질 수 있으나, 이에 제한되지 않는다.The method for producing bio-aerosol oil according to the present invention is characterized in that deoxygenation, isomerization and deoxygenation, isomerization and cracking reactions are carried out from lipids in the presence of a catalyst, To form paraffinic hydrocarbons, and distilling the reaction product to extract aerial oil fractions. However, the present invention is not limited thereto.
본 발명의 상기 촉매반응을 위해 사용되는 촉매로는 NiMo, CoMo, Pt, Pd가 사용될 수 있으나, 이에 제한되지 않는다.As the catalyst used for the catalytic reaction of the present invention, NiMo, CoMo, Pt, and Pd may be used, but the present invention is not limited thereto.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
실시예Example 1. 미세조류 채취 및 선별 1. Collection and selection of microalgae
영월지역의 하천에서 시료를 채취한 뒤, 이산화탄소 혼합가스(CO2 10%, 공기 90%)를 공급하며 N8 액체배지에서 형광등을 광원으로 3주간 정치 배양한 후 100 ㎍/mL의 앰피실린을 포함한 동일 고체배지 위에 100 ㎕ 만큼 떨어뜨려 넓게 퍼뜨려 주었다. 그 후, 형성된 콜로니들 중 단일 콜로니들을 선별하여 계대하였다. 실험에서 사용된 N8 배양액의 조성은 다음과 같다; 5 mM KNO3, 5.44 mM KH2PO4, 1.83 mM Na2HPO4, 0.2 mM MgSO4·7H2O, 0.12 mM CaCl2·2H2O, FeNaEDTA·3H2O 및 1 ml micronutrient (0.01 mM ZnSO4·7H2O, 0.07 mM MnCl2·4H2O, 0.07 mM CuSO4·5H2O, 0.01 mM Al2(SO4)3·18H2O)After collecting samples from rivers in Yeongwol area, a CO2 gas mixture (10% CO 2 , 90% air) was supplied and the fluorescent lamps were incubated in a N8 liquid medium for 3 weeks with a light source. Then, 100 μg / ml ampicillin 100 ㎕ on the same solid medium was spread widely. Thereafter, single colonies among the formed colonies were sorted and transferred. The composition of the N8 culture used in the experiment is as follows; 5 mM KNO 3 , 5.44 mM KH 2 PO 4 , 1.83 mM Na 2 HPO 4 , 0.2 mM MgSO 4 .7H 2 O, 0.12 mM CaCl 2 .2H 2 O, FeNaEDTA 3H 2 O and 1 ml micronutrient 4 · 7H 2 O, 0.07 mM MnCl 2 · 4H 2 O, 0.07 mM CuSO 4 · 5H 2 O, 0.01 mM Al 2 (SO 4) 3 · 18H 2 O)
실시예Example 2. 분류학적 분석 2. Taxonomic analysis
선별한 미세조류의 신규성을 확인하고, 미세조류가 속한 종을 확인하기 위해 18s rRNA 유전자 서열을 분석하였다. 상기 균주의 DNA는 DNeasy Plant Mini kit (Qiagen, Hilden, Germany)를 사용하여 추출하였으며, 서열 분석을 위해 클로렐라 18s rRNA 유전자 분석용으로 사용되는 프라이머 18SF(5'-CCT GGT TGA TCC TGC CAG-3': 서열번호 2)와 18SR(5'-TTG ATC CTT CTG CAG GTT CA-3': 서열번호 3)를 사용하여 염기서열 정보를 읽었다. 얻은 염기서열(서열번호 1)에 대하여 NCBI 데이터베이스를 기반으로 Blast한 결과, 현재까지 보고되지 않은 신규 종임을 확인할 수 있었고, 클로렐라 종과 유연관계가 깊은 것으로 판단되었다. 따라서 관련된 종들의 18s rRNA 유전자 정보와 함께 계통수를 그려 조사하였으며, 이를 근거로 신규 미세조류 종이 클로렐라(Chlorella)에 속하는 균주임을 밝혔다(도 1). 따라서 상기 균주를 KCTC 생물자원센터에 기탁하였다.(기탁번호 KCTC13386BP)The 18s rRNA gene sequence was analyzed to identify the novelty of the selected microalgae and to identify the species to which the microalgae belong. The DNA of the strain was extracted using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany). For the sequencing, primer 18SF (5'-CCT GGT TGA TCC TGC CAG-3 ' : SEQ ID NO: 2) and 18SR (5'-TTG ATC CTT CTG CAG GTT CA-3 ': SEQ ID NO: 3). The obtained nucleotide sequence (SEQ ID NO: 1) was blasted on the basis of the NCBI database. As a result, it was confirmed that the nucleotide sequence was a new species not yet reported, and it was determined that the nucleotide sequence was closely related to the chlorella species. Therefore, we examined the 18S rRNA gene information and the phylogenetic tree of the related species. Based on this, we found that the new microalgae belong to the genus Chlorella (Fig. 1). Thus, the strain was deposited at the KCTC BRC (Deposit No. KCTC13386BP)
실시예Example 3. 이산화탄소 농도에 따른 생장량 측정 3. Measurement of growth by carbon dioxide concentration
신규 클로렐라 속 ABC-001의 이산화탄소 농도별 생장속도를 보기 위하여, 다양한 이산화탄소 농도에서 배양을 실시하였다. 배양액 부피를 500 mL로 하는 Pyrex 유리재질의 기포탑 광생물반응기에 초기 세포농도를 660 nm 파장대의 흡광도를 기준으로 0.2에 맞추었다. 이산화탄소 공급은 공기 또는 공기와 이산화탄소를 혼합한 10% 또는 15%의 농도로 배양기에 300 mL/min를 넣어주었다. 배양액으로는 N8 배지를 사용했으며, 170 μmol photons/m2/s의 빛 세기에서 30℃의 조건을 각각 유지하며 독립적인 배양을 2회씩 실시하였다. 7일간 건조균체중량(Dry cell weight, DCW)을 매일 측정하여 미세조류의 생장성을 확인하였다. 건조균체중량은 3-5 mL의 배양액을 GF/C 여과지를 통과시켜 세포만 분리한 후 증류수로 2번 이상 세척하고 70℃에서 24시간 건조 후 무게를 측정하여 계산하였다. 그 결과 공기만 공급했을 때보다 이산화탄소를 10%, 15%로 혼합하였을 때 높은 성장률을 보였고 10%일 때 가장 높은 2.9 g/L까지 도달하였다(도 2). 이를 통해 본 균주가 이산화탄소 고농도에서도 높은 생장성을 보인다는 것을 확인할 수 있었다.To observe the growth rate of the new chlorella ABC-001 by carbon dioxide concentration, cultivation was carried out at various carbon dioxide concentrations. The initial cell concentration was adjusted to 0.2 based on the absorbance of the 660 nm wavelength band in a Pyrex glass bubble column photobioreactor having a volume of 500 mL. Carbon dioxide was fed to the incubator at a concentration of 10% or 15% in air or air and carbon dioxide at 300 mL / min. N8 medium was used as the culture medium, and independent culturing was carried out twice at a light intensity of 170 μmol photons / m 2 / s and 30 ° C., respectively. Dry cell weight (DCW) was measured daily for 7 days to confirm the growth of microalgae. The weight of the dried cells was calculated by measuring the weight of the dried cells by washing the cells with GF / C filter paper, washing with distilled water more than 2 times, drying at 70 ° C for 24 hours, and passing the 3-5 mL of the culture solution through the GF / C filter paper. As a result, when the carbon dioxide was mixed at 10% and 15%, it showed a high growth rate and reached the highest at 2.9 g / L at 10% (FIG. 2). The results showed that the strain showed high growth even at high concentration of carbon dioxide.
실시예Example 4. 천일염 농도에 따른 생장량 측정 4. Measurement of growth rate by salinity
실시예 3에서 성장률이 가장 우수하였던 10% 이산화탄소 조건에서 천일염이 첨가된 해수 배지를 만들어 내염성 테스트를 수행하였다. 동일한 배양조건 아래 천일염을 0, 7, 21, 35 g/L 농도로 맞추어 배지에 첨가한 후 독립적인 배양을 2회씩 실시하였다. 7일간 배양한 결과 본 균주가 해수와 같은 염도(35 g/L)에서도 세포의 생장성을 유지하며 담수배지(0 g/L) 대비 건조균체중량에서 큰 차이를 보이지 않은 것을 확인하였다(도 3). 또한 천일염뿐만 아니라 NaCl을 첨가한 염수 그리고 실제 해수를 사용하여 동일한 염도에서 배양했을 시에도 높은 생장성을 보이며 이를 통해 본 균주의 내염성을 검증하였다.In the case of Example 3, a salt water culture medium containing 10% carbon dioxide at which the growth rate was highest was added to the sea salt medium, and the salt resistance test was conducted. Under the same culture conditions, the culture was supplemented with 0, 7, 21, and 35 g / L of mannitol in the culture medium, followed by independent culture twice. 7 days. As a result, it was confirmed that this strain maintained cell growthability even at the same salinity (35 g / L) as seawater and showed no significant difference in dry cell weight as compared to fresh water medium (0 g / L) ). In addition, salt tolerance of this strain was verified by using NaCl - added saline and actual seawater as well as sun - salt.
실시예Example 5. 지질 추출 및 성분 분석 5. Lipid extraction and component analysis
배양이 끝난 후 배양액을 4000 RPM에서 1분간 원심분리하여 세포들을 분리하였다. 분리한 세포들은 증류수로 2번 씻어준 후 최종적으로 얻어진 바이오매스는 3일간 동결건조를 진행하여 추출이 용이하게 하였다. 건조된 세포를 유리 튜브에 10 mg씩 무게를 재어 지방산 (Fatty Acid Methyl Ester, FAME) 분석에 사용하였고 50 mg씩은 총지방량 (Total lipid content)을 측정하는데 사용하였다.After the incubation, the culture was centrifuged at 4000 rpm for 1 minute to separate the cells. The separated cells were washed twice with distilled water, and the final biomass was lyophilized for 3 days to facilitate extraction. The dried cells were weighed 10 mg into a glass tube and used in the analysis of fatty acid (Fatty Acid Methyl Ester, FAME), and 50 mg was used to measure total lipid content.
지방산 분석을 위에 건조된 세포에 클로로포름-메탄올 (2:1 v/v) 혼합액 2 mL을 주입 후 섞어준 후에 내부표준물질 (100 mg heptadecanoic acid/200 mL chloroform) 1 mL, 메탄올 1 mL와 황산 0.3 mL를 추가하고 섞어주었다. 그리고 100℃에서 10분간 반응시키고 상온까지 식힌 후 증류수 1 mL를 첨가한 후 섞어주었다. 층 분리를 위하여 4000 RPM에서 10분간 원심분리를 한 후 주사기를 이용하여 하층액의 유기상 약 1 mL를 뽑아 여과 후 GC (Gas Chromatograph) 분석용 바이알에 담았다. 추출한 지방산 분석은 Flame ionized detector (FID)가 장착된 GC (HP 6890 Agilent, USA)을 이용하여 분석하였다.After the fatty acid analysis, add 2 mL of chloroform-methanol (2: 1 v / v) mixture to the dried cells, mix 1 mL of internal standard (100 mg heptadecanoic acid / 200 mL chloroform), 1 mL of methanol and 0.3 mL of sulfuric acid mL was added and mixed. The reaction was carried out at 100 ° C for 10 minutes. After cooling to room temperature, 1 ml of distilled water was added and mixed. For layer separation, centrifugation was carried out at 4,000 rpm for 10 minutes. Then, about 1 mL of the organic phase of the lower layer was extracted by using a syringe, and the resultant was filtered and packed in a GC (Gas Chromatograph) analysis vial. The extracted fatty acids were analyzed by GC (HP 6890 Agilent, USA) equipped with a Flame ionized detector (FID).
총지방량 분석을 위해 건조된 세포에 클로로포름-메탄올 (2:1 v/v) 혼합액 10 mL을 주입 후 4시간 동안 Bath sonicator를 사용하여 음파처리(sonication)를 하였다. 그리고 증류수 2.5 mL를 넣고 섞어준 후 층 분리를 위하여 4000 RPM에서 10분간 원심분리를 하였다. 그 후 주사기를 이용하여 하층액의 유기상 5 mL를 뽑아 미리 무게를 잰 유리튜브에 옮겨 담았다. 튜브에 질소를 불어주며 용매를 모두 증발시킨 후 지질만 남아있는 유리튜브의 무게를 재어 총지질량을 계산하였다.For total fat analysis, 10 mL of chloroform-methanol (2: 1 v / v) mixture was added to the dried cells and sonication was performed using a bath sonicator for 4 hours. Then, 2.5 mL of distilled water was added, and the mixture was centrifuged at 4,000 rpm for 10 minutes for layer separation. Then, 5 mL of the organic phase of the lower layer was drawn using a syringe and transferred to a pre-weighed glass tube. Nitrogen was injected into the tube and all of the solvent was evaporated. The total mass of the glass tube was calculated by weighing the glass tube with only the lipid.
고농도 이산화탄소 조건에서 배양한 세포들과 공기만 불어 넣어 배양한 세포들의 총 지질함량을 비교해보았을 때 고농도 이산화탄소 조건에서 지질 함유량이 더 높은 것을 확인할 수 있다 (도 4). 또한 바이오매스 농도 또한 고농도 이산화탄소 조건에서 더 높기 때문에 공기 조건에 비해 총 지방 생산성 (g/L/d)이 10% 이산화탄소 조건에서 약 8배, 15% 조건에서 4.5배 높았다.Comparing the total lipid content of the cells cultured in the high-concentration carbon dioxide atmosphere and the culture of the air-insoluble culture, the lipid content was higher in the high concentration carbon dioxide condition (FIG. 4). Also, the biomass concentration was higher than that in the high concentration CO2 condition, so the total fat productivity (g / L / d) was 4.5 times higher at 10% CO2 and 8% and 15% than the air condition.
또한 해수에서 배양한 세포들로 총 지방량 및 지방산 조성 분석을 하였다. 그 결과 총 지질함량은 천일염 농도 변화에 영향을 받지 않는 것을 볼 수 있었다 (도 5). 이 결과를 통해 본 균주가 담수와 해수 그리고 담수와 해수의 중간인 기수에서도 지질 생산성을 거의 동일하게 유지시킬 수 있는 것을 확인하였다. 지방산 조성도 크게 달라지지 않았으며 팔미트산 (C16:0), 스테아르산 (C18:0), 올레산 (C18:1n9c), 리놀레산 (C18:2n6c), 리놀렌산 (C18:3n3c) 이 공통적으로 지질의 주 성분을 이루었다 (도 6). 이들은 모두 바이오연로로 전환이 가능하기 때문에 본 균주의 배양을 통해서 얻어진 지질의 가치를 검증할 수 있다.The total fat content and fatty acid composition of the cells were also analyzed. As a result, it was found that the total lipid content was not affected by the change of the salinity (FIG. 5). These results confirm that the strain can maintain the lipid productivity almost equally in the fresh water, the seawater, and the paddy between the fresh water and the seawater. (C18: 0), oleic acid (C18: 1n9c), linoleic acid (C18: 2n6c) and linolenic acid (C18: 3n3c) were common lipid (Fig. 6). Since all of these can be converted into biofuels, the value of the lipid obtained through cultivation of this strain can be verified.
실시예Example 6. 천일염 농도에 따른 침전율 6. Settling rate according to the concentration of salt
실시예 4에서 배양이 끝난 후 배양액 각각 약 12 mL를 15 mL 팔콘 튜브에 옮겨 담고 팔콘 튜브를 고정시킨 후 24시간 동안 세포가 가라앉는 것을 관찰하였다 (도 7). 약 2시간이 지난 후 천일염 35 g/L 조건에서 배양한 세포들이 눈에 띄게 가라앉는 것을 확인할 수 있었고 24시간 후에는 대부분의 세포들이 튜브의 바닥에 뭉쳐있는 것을 관찰하였다. 그 외의 조건들에서는 약 6시간이 지난 후 세포들이 침전하는 속도에서 차이를 확인할 수 있었다. 24시간이 지난 후 천일염의 농도가 높은 농도에서 배양한 세포들이 빨리 자연침전되는 것을 검증할 수 있었다. 이를 통해 본 균주를 해수에서 배양했을 시 담수에서 배양했을 때에 비해 비슷한 생장과 지질 함량을 유지하면서도 수확이 용이하다는 것을 보였다.After the culture was completed in Example 4, about 12 mL of each culture was transferred to a 15 mL Falcon tube, and the cells were settled for 24 hours after fixing the Falcon tube (FIG. 7). After about 2 hours, it was observed that the cells incubated at 35 g / L of sun saliva were noticeably submerged. After 24 hours, most of the cells were observed to stick to the bottom of the tube. In other conditions, we could confirm the difference in the rate at which the cells settled after about 6 hours. After 24 hours, it was verified that the cells cultured at high concentration of sodium salt rapidly precipitated rapidly. It was shown that when the strain was cultured in seawater, it could be harvested while retaining similar growth and lipid contents compared to culturing in freshwater.
[수탁번호][Access number]
기탁기관명 : 한국생명공학연구원Institution name: Korea Biotechnology Research Institute
수탁번호 : KCTC13386BPAccession number: KCTC13386BP
수탁일자 : 20171030Checked on: 20171030

Claims (12)

  1. 고농도의 이산화탄소 및 고염 조건에서 지질 생산성 및 세포 생장률이 우수한 클로렐라 속(Chlorella sp.) ABC-001 균주. Chlorella sp. ABC-001 strain with excellent lipid productivity and cell growth rate under high concentration of carbon dioxide and high salt condition.
  2. 제1항에 있어서, 상기 균주는 기탁번호가 KCTC13386BP인 것을 특징으로 하는 균주.The strain according to claim 1, wherein the strain has the deposit number KCTC13386BP.
  3. 제1항에 있어서, 상기 고농도의 이산화탄소 및 고염은 각각 1~18% 이산화탄소 및 33~40g/l 염인 것을 특징으로 하는 균주.The strain according to claim 1, wherein the high concentration of carbon dioxide and high salt are respectively 1 to 18% carbon dioxide and 33 to 40 g / l salt.
  4. 제1항 내지 제3항 중 어느 한 항에 따른 균주 또는 이의 배양액을 유효성분으로 포함하는 지질 생산용 미생물제제.A microorganism preparation for lipid production comprising the strain according to any one of claims 1 to 3 or a culture thereof as an active ingredient.
  5. 제1항 내지 제3항 중 어느 한 항에 따른 균주를 배양하고, 그 배양액으로부터 지질을 분리하는 것을 특징으로 하는 지질의 제조 방법.A method for producing a lipid comprising culturing a strain according to any one of claims 1 to 3 and separating lipid from the culture.
  6. 제5항에 있어서, 상기 균주의 배양은 담수, 해수 또는 기수에서 배양하는 특징으로 하는 방법.The method according to claim 5, wherein the culture of the strain is cultured in fresh water, seawater, or nursery.
  7. 제6항에 있어서, 상기 해수 배양 후 자연침전을 통해 균주를 수확하는 것을 특징으로 하는 방법.7. The method according to claim 6, wherein the strain is harvested through natural precipitation after the seawater culture.
  8. 제1항 내지 제3항 중 어느 한 항에 따른 균주를 배양하는 단계;Culturing the strain according to any one of claims 1 to 3;
    상기 배양액으로부터 지질을 분리하는 단계;Separating the lipid from the culture liquid;
    상기 지질을 트랜스에스테르화시켜 지방산 에스테르를 생산하는 단계; 및Transesterifying the lipid to produce a fatty acid ester; And
    상기 생산된 지방산 에스테르를 바이오디젤로 전환하는 단계를 포함하는 바이오디젤의 생산 방법.And converting the produced fatty acid ester to biodiesel.
  9. 제1항 내지 제3항 중 어느 한 항에 따른 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 식품.A food comprising the strain according to any one of claims 1 to 3, a culture thereof, a concentrate of the culture solution or a dried product of the culture solution as an active ingredient.
  10. 제1항 내지 제3항 중 어느 한 항에 따른 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 사료 조성물.A feed composition comprising a strain according to any one of claims 1 to 3, a culture thereof, a concentrate of the culture solution or a dried product of the culture solution as an active ingredient.
  11. 제1항 내지 제3항 중 어느 한 항에 따른 균주, 이의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 화장료 조성물.A cosmetic composition comprising the strain according to any one of claims 1 to 3, a culture thereof, a concentrate of the culture solution or a dried product of the culture solution as an active ingredient.
  12. 제1항 내지 제3항 중 어느 한 항에 따른 균주를 배양하는 단계;Culturing the strain according to any one of claims 1 to 3;
    상기 배양액으로부터 지질을 분리하는 단계; 및Separating the lipid from the culture liquid; And
    상기 분리한 지질을 촉매전환시켜 항공유로 전환하는 단계를 포함하는 바이오항공유의 생산 방법.And converting the separated lipid to catalytic conversion to aviation oil.
PCT/KR2017/012373 2017-10-17 2017-11-03 Chlorella genus abc-001 strain having excellent lipid productivity and cell growth rate under high carbon dioxide and salt concentration conditions, and use thereof WO2019078398A2 (en)

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KR100265034B1 (en) * 1998-03-27 2000-09-01 김충섭 A highly co2 tolerant chlorella sp. kr-1 and co2 fixation technology using chlorella sp. kr-1
EP2555633B1 (en) * 2010-04-06 2014-06-11 Heliae Development LLC Selective extraction of proteins from freshwater or saltwater algae
KR101424852B1 (en) * 2010-11-26 2014-08-01 한국생명공학연구원 Chlorella vulgaris CV-16 producing biodiesel, and method for producing biodiesel using the strain
KR101273234B1 (en) * 2011-08-29 2013-06-11 한국에너지기술연구원 Chlorella sp. M082 having excellent carbon dioxide fixation ability at high temperature and uses thereof
KR101575208B1 (en) * 2013-01-09 2015-12-07 한국생명공학연구원 Microalgae Chlorella strain high-producing starch and lipid isolated from arctic ocean and uses thereof

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CN113980815A (en) * 2021-12-15 2022-01-28 海南大学 Chlorella vulgaris with broad salinity and application thereof
CN113980815B (en) * 2021-12-15 2024-01-26 海南大学 Chlorella with characteristic of guangsalt and application thereof

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