WO2019131502A1 - Méthode de production de lipides - Google Patents

Méthode de production de lipides Download PDF

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WO2019131502A1
WO2019131502A1 PCT/JP2018/047230 JP2018047230W WO2019131502A1 WO 2019131502 A1 WO2019131502 A1 WO 2019131502A1 JP 2018047230 W JP2018047230 W JP 2018047230W WO 2019131502 A1 WO2019131502 A1 WO 2019131502A1
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acetic acid
microorganism
producing
culture
lipid
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PCT/JP2018/047230
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English (en)
Japanese (ja)
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秋　庸裕
研志 渡邉
豊 中島田
幸彦 松村
好子 岡村
誉久 田島
蘭 廣谷
元務 石垣
新造 黛
吉田　和広
健 沢田
祐介 角田
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国立大学法人広島大学
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Priority to PL434591A priority Critical patent/PL434591A1/pl
Priority to JP2019561642A priority patent/JP7048056B2/ja
Publication of WO2019131502A1 publication Critical patent/WO2019131502A1/fr

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    • 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
    • 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
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • 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
    • 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

Definitions

  • the present invention relates to a method for producing a lipid using a Labyrinthula aurantiochytrium microorganism.
  • Labyrinthula aurantiochytrium microbes are expected as industrial microbes applicable to various industries because they have properties of producing remarkable amounts of various hydrocarbons in addition to polyunsaturated fatty acids and carotenoids.
  • the heterotrophic microorganisms such as Aurantiochytrium microorganisms are efficiently cultured on the heterotrophic microorganisms by stepwise culturing them using two types of media having different contents of nitrogen and carbon. It is described that it can produce fats and oils well and can produce fats and oils in a large amount in a short time.
  • heterotrophic microorganisms such as Auranthiochytrium microorganisms are cultured in a stepwise manner using two types of media having different contents of saccharides and glycerol, thereby achieving a large amount of in a short time. It is described that oil and fat can be produced.
  • Japanese Patent Publication Japanese Unexamined Patent Publication No. 2013-126404 (published on June 27, 2013)
  • Japanese patent publication Japanese Patent Application Laid-Open No. 2013-183687 (Sep. 19, 2013)
  • carbohydrates are mainly used as a carbon source in lipid production such as fats and oils by aurantiochytrium microbes, and the raw material cost due to the supply of the carbohydrates is a problem.
  • green algae capable of producing similar lipids such as fat and oil autonomously produce carbohydrates by photosynthesis but their growth rate is generally low due to conditions of light irradiation or equipment restrictions, and as a result, production of lipids Low efficiency.
  • one aspect of the present invention aims to achieve both reduction in raw material cost and high-efficient production of lipids in lipid production such as fats and oils by Aurantiochytrium microbes.
  • Another object of the present invention is to provide a novel lipid production technology that can contribute to environmental conservation and a low carbon society by using unused biomass, exhaust gas, and the like as raw materials.
  • the method for producing a lipid of the present invention is characterized in that it comprises a culture step of culturing the Aurantiochytrium microorganism in a medium containing acetic acid.
  • the acetic acid concentration in the above-mentioned medium is preferably 10 g / L or more.
  • the acetic acid may be acetic acid produced by an acetic acid-producing bacterium.
  • an acetic acid producing bacterium culture step of culturing an acetic acid producing bacterium in a culture medium may be further included prior to the culture step.
  • the above-mentioned acetic acid-producing bacteria are preferably anaerobic microorganisms.
  • an alunch is produced in an acetic acid-producing bacterial culture step of culturing an anaerobic acetic acid-producing bacterium in a culture medium under anaerobic conditions, and in a medium containing acetic acid after the above acetic acid-producing bacterial culture step. It is preferable to include a culture step of culturing an Ochrium microorganism.
  • the above-mentioned acetic acid-producing bacteria is a microorganism of the genus Morella (Moorella), a microorganism of the genus Clostridium (Clostridium), a microorganism of the genus Acetobacterium (Acetobacterium), a microorganism of the genus Blautia, Eubacteria It is preferable that it is any one or more selected from the group consisting of a microorganism belonging to the genus Eubacterium and a microorganism belonging to the genus Sporomusa.
  • the culture of the acetic acid-producing bacteria in the above-mentioned acetic acid-producing bacteria culture step is preferably performed in an anaerobic atmosphere containing carbon dioxide.
  • lipids such as fats and oils by Aurangthiochytrium microorganisms
  • highly efficient production of lipids can be performed by using acetic acid as a raw material.
  • acetic acid produced from raw materials such as unused biomass and exhaust gas for the lipid production method of the present invention, it is possible to reduce the cost of lipid production while contributing to environmental conservation and a low carbon society can do.
  • (A) is a graph which shows the dry-cell body quantity of the Oranthiochytrium microorganism strain
  • (b) is a graph which shows the composition and the production amount of the fatty acid which the Oranthiochytrium microorganism produced. It is a graph which shows the dry-cell body amount and fatty-acid production amount of aurantiochytrium microbe by two-step fermentation of an acetobacterium genus microbe and auranthiochytrium microbe. It is a graph which shows the fatty acid content rate (total fatty acid / dry cell amount) by two-step fermentation of an acetobacterium genus microorganism and an aurantiochytrium microorganism.
  • Embodiment 1 A method for producing a lipid according to an aspect of the present invention (hereinafter referred to as "the production method of the present invention") is characterized by comprising a culture step of culturing alanthiochytrium microorganism in a medium containing acetic acid. .
  • lipid as used herein means a lipid that can be produced by Aurantiochytrium microorganisms.
  • lipids that can be produced by Aurantiochytrium microorganisms include saturated fatty acids such as myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid; docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, oleic acid, Unsaturated fatty acids such as linoleic acid, ⁇ -linolenic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid; Astaxanthin, phenicoxanthin, canthaxanthin, equinenone, ⁇ -carotene, carotenoids such as lycopene; squalene, sterol, etc.
  • the microorganism of the genus Orangiochytrium is cultured in a medium containing acetic acid. It will not be limited especially if it is a microorganism classified into Labyrinthula aurantithiochytrium (Aurantiochytrium) genus as the above-mentioned "Aurantiochytrium microorganism".
  • Aurantiochytrium microorganism For example, Aurantiochytrium sp. SR21, Aurantiochytrium sp. KH105 (reception number: FERM AP-22267) etc. can be mentioned.
  • said "Orranthiochytrium microbe” is also called "Schizochytrium microbe".
  • the culture medium used in the above-mentioned culture step is a culture medium in which the Aurantiochytrium microorganism can grow, and contains acetic acid.
  • the above-mentioned culture medium is not particularly limited, but is preferably a liquid culture medium because it is suitable for mass culture and that culture conditions can be easily controlled.
  • the concentration of acetic acid contained in the culture medium is preferably 1 g / L or more, more preferably 5 g / L or more, and still more preferably 10 g / L or more.
  • the Aurantiochytrium microorganism can efficiently produce a lipid.
  • the upper limit of the acetic acid concentration is not particularly limited as long as it is a concentration at which the Aurantiochytrium microorganism can grow and can produce a lipid, but preferably 40 g / L or less, for example, 35 g / L. It is preferably L or less, more preferably 30 g / L or less.
  • acetic acid commercially available ones may be used, or acetic acid produced using an acetic acid-producing bacterium may be used.
  • an acetic acid-producing bacterium means a microorganism capable of fermentatively producing acetic acid.
  • the above-mentioned acetic acid-producing bacteria may be either an anaerobic microorganism or an aerobic microorganism, and it is not particularly limited.
  • a microorganism of the genus Morella a microorganism of the genus Clostridium
  • a microorganism of the genus Acetobacterium a microorganism of the genus Blautia
  • a eubacteria Anaerobic microbes such as Ummus microbes and Sporomyces microbes are preferred.
  • Acetobacterium microbes which are aerobic microorganisms and Gluconacetobacter microbes etc. exist as acetic acid-producing bacteria, but aerobic acetic acid-producing bacteria can not produce acetic acid from hydrogen and carbon dioxide.
  • aerobic acetic acid-producing bacteria can not produce acetic acid from hydrogen and carbon dioxide.
  • it is an anaerobic acetic acid producing bacterium, it is possible to produce acetic acid from hydrogen and carbon dioxide, and it is possible to reduce the raw material cost associated with the supply of a carbon source.
  • Morella Morella
  • Morella microorganism examples include Moorella thermoacetica, Moorella thermoautotrophica and the like.
  • Clostridium microorganism is not particularly limited as long as it is an acetic acid-producing bacterium belonging to the genus Clostridium thermoaceticum.
  • Examples of the Clostridium microorganism include Clostridium thermoaceticum, Clostridium aceticum, Clostridium cellulolyticum, Clostridium ljungdahlii and the like.
  • Acetobacterium an acetic acid producing microbe which belongs to Acetobacterium (Acetobacterium) as a microorganism which belongs to said Acetobacter microorganism.
  • Acetobacter microorganism examples include Acetobacterium woodii, Acetobacterium dehalogenans and the like.
  • the above-mentioned Blautia microorganism is not particularly limited as long as it is an acetic acid-producing bacterium belonging to Blautia.
  • Examples of the above-mentioned Blautia microbes include Blautia hydrogenotrophica, Blautia producta, and the like.
  • the above-mentioned Eubacteria is not particularly limited as long as it is an acetic acid-producing bacterium belonging to the genus Eubacterium.
  • Examples of the above-mentioned Eubacterium microorganism include Eubacterium aggregans, Eubacterium limosum and the like.
  • Sporommus Sporomusa
  • Sporomomas genus microorganisms examples include Sporomusa acidovorans, Sporomusa malonica and the like.
  • the microorganism belonging to the Acetobacter microorganism is not particularly limited as long as it is an acetic acid-producing microorganism belonging to Acetobacter.
  • Examples of the Acetobacter microorganism include Acetobacter aceti, Acetobacter pasteurianus, Acetobacter xylinus and the like.
  • the microorganism belonging to the above Gluconacetobacter microorganism is not particularly limited as long as it is an acetic acid producing bacterium belonging to Gluconacetobacter.
  • Examples of the above-mentioned Gluconacetobacter microorganism include Gluconacetobacter xylinus, Gluconacetobacter liquefaciens and the like.
  • any one of the above-mentioned acetic acid-producing bacteria may be used, or a plurality of them may be used in combination.
  • the culture solution after cultivation of the above-mentioned acetic acid-producing bacteria is used as it is.
  • the culture solution may be used as a medium of (1), or acetic acid purified from a culture solution after culture of an acetic acid-producing bacterium may be used.
  • the components contained in the above medium may include a carbon source, a nitrogen source and sodium chloride necessary for the growth of Aurantiochytrium microorganisms.
  • acetic acid is contained in the medium as a carbon source, but in the production method of the present invention, other carbon sources other than acetic acid may be contained.
  • the above-mentioned "other carbon source” is not particularly limited as long as it is capable of assimilating the microorganism of the genus Oranthiochytrium.
  • carbohydrates monosaccharides, oligosaccharides, polysaccharides, sugar alcohols, etc.
  • organic acids alcohols And lipids, etc.
  • As another carbon source only one type may be used, or a plurality of types may be used in combination.
  • nitrogen source is not particularly limited as long as it can be utilized by aurantiochytrium microorganism, for example, nitrogen-containing compounds such as polypeptone, yeast extract, peptone, soybean flour, corn steep liquor and the like; ammonium sulfate And ammonium salts of inorganic acids such as ammonium nitrate and ammonium phosphate or organic acids, and the like.
  • nitrogen source only one type may be used, or a plurality of types may be used in combination.
  • the concentration of the nitrogen source added to the culture medium is preferably 1 to 50 g / L, more preferably 5 to 20 g / L. If the nitrogen source concentration in the culture medium is in the above range, the Aurantiochytrium microorganism can be grown well. When a plurality of types of nitrogen sources are used in combination, the nitrogen source may be added to the culture medium so that the total concentration of the plurality of types of nitrogen sources is in the above range.
  • Sodium chloride is contained in the above-mentioned culture medium in order for the above-mentioned Aurantiochytrium microorganism which is a marine eukaryotic microorganism to stably grow in the above-mentioned culture medium.
  • the concentration of sodium chloride that can be contained in the above medium is preferably 0.5 to 4% (w / v), and more preferably 1.5 to 3% (w / v).
  • the Auranthiochytrium microorganism can be suitably grown.
  • seawater other than sodium is also included in the seawater in which the Auranthiochytrium microorganism naturally grows
  • seawater including artificial seawater
  • seawater may be used as a source of sodium chloride.
  • seawater may be used in such a manner that the concentration of sodium chloride falls within the above-mentioned preferred range.
  • the medium may contain other components other than the carbon source and the nitrogen source.
  • the above-mentioned “other components” are not particularly limited as long as they are generally used for culturing a microorganism, and examples thereof include inorganic salts, coenzymes such as vitamins, dyes and the like.
  • concentration of the above-mentioned “other components” in the culture medium can be set appropriately.
  • the medium becomes acidic if it is not adjusted.
  • the pH of the culture medium is preferably adjusted with sodium hydroxide or the like so as to fall within the above range.
  • the temperature during culture is preferably 20 ° C. to 40 ° C., More preferably, the temperature is 25 to 35 ° C.
  • the culture step in the production method of the present invention is carried out under aerobic conditions.
  • a known culture apparatus such as a jar fermenter can be suitably used for the culture in the culture step.
  • the operating conditions (temperature, aeration amount, pH, stirring speed, etc.) of the culture apparatus such as the jar fermenter may be appropriately set according to the culture scale and the growth state of the Auranthochytrium microorganism.
  • an acetic acid-producing bacterium is cultured in a culture medium prior to the above-mentioned culturing step, and an acetic acid-producing bacterium culturing step, and a lipid recovery for recovering the lipid accumulated in Auranthiochytrium microorganisms after the culturing step Process, lipid purification process to purify lipid recovered by lipid recovery process, substance conversion process to convert obtained lipid to other substance, pre-culture process to pre-culture various microorganisms before main culture, etc. Any one or more steps may be included as appropriate.
  • the lipid recovery step, lipid purification step, and substance conversion step are described below.
  • the produced lipid may be taken out of the cell of Oranthiochytrium microorganism, and (b) the produced lipid may be Orangthiochytrium microorganism. It may be recovered in the state of being accumulated in the cells of
  • the method of recovering the lipid is not particularly limited as long as the lipid can be removed from the extracellular part of the Aurantiochytrium microorganism.
  • chloroform / methanol mixture (2: 1, v / v)
  • t-butyl methyl ether / methanol / water 2: 1: 0.5, v / v
  • carotenoids can be extracted from Aurantiochytrium microorganisms.
  • squalene can be extracted from Alanthiochytrium microorganisms by using hexane or a mixture of chloroform / methanol (2: 1, v / v).
  • the recovery method is not particularly limited as long as high value added lipid can be recovered in a state where it is accumulated in the cells of Alanthiochytrium microorganism.
  • high-added-value lipid-containing bacterial cells can be recovered by discarding the culture supernatant by centrifuging, filtering, etc., the culture solution in which the Aurantiochytrium microorganism is cultured.
  • the method for purifying the lipid in the lipid purification step is not particularly limited.
  • the lipid can be purified by methods such as solvent fractionation, supercritical fluid extraction, adsorption chromatography and the like.
  • the conversion step is a step of converting the obtained lipid into another substance, but for example, the obtained triacylglycerol or isoprenoid hydrocarbon is converted into jet fuel using a known method May be Alternatively, the obtained triacylglycerol may be converted into diesel fuel using a known method. In addition, the obtained isoprenoid hydrocarbon may be converted to a chemical material using a known method.
  • the method for producing a lipid according to an aspect of the present invention may further include an acetic acid producing bacterium culture step of culturing an acetic acid producing bacterium in a culture medium before the culture step. That is, the production method of the present invention may be a two-step fermentation including an acetic acid-producing bacterial culture step and a culture step of culturing the above Aurantiochytrium microorganism.
  • the method of producing a lipid which contributes to the reduction of the cost involved in supplying the carbon source used for the production of lipid and the realization of environmental preservation by the two-step fermentation of the above-mentioned culture step and the above-mentioned acetic acid producing bacteria culture step. realizable.
  • the description of Embodiment 1 is incorporated into the description common to the present embodiment and [Embodiment 1].
  • the culture medium for acetic acid-producing bacteria culture used in the above-mentioned acetic acid-producing bacteria culture step is not particularly limited as long as it is a culture medium in which the acetic acid-producing bacteria can grow, and carbon sources, nitrogen sources and other components may be included.
  • the description of [Embodiment 1] can be incorporated for the carbon source, the nitrogen source, and the other components.
  • acetic acid-producing bacteria belonging to anaerobic microorganisms are used in the acetic acid-producing bacteria culture step, since acetic acid can be produced using carbon dioxide as a carbon source, carbon sources such as carbohydrates other than carbon dioxide are contained in the medium. It does not have to be included.
  • a liquid culture medium is preferable because it is suitable for a large-scale culture medium and that culture conditions can be easily controlled.
  • an anaerobic microorganism When using an anaerobic microorganism as an acetic acid-producing bacterium, culture (anaerobic culture) must be performed under anaerobic conditions.
  • an anaerobic gas such as nitrogen or carbon dioxide other than oxygen is generally supplied under a culture atmosphere to achieve an anaerobic atmosphere.
  • it is preferable to achieve an anaerobic atmosphere in particular, by aerating an anaerobic gas containing hydrogen and carbon dioxide and containing no oxygen under a culture atmosphere.
  • Acetic acid can be produced from the hydrogen and carbon dioxide of the anaerobic gas used to achieve the anaerobic atmosphere, as long as it is an acetic acid-producing bacterium belonging to the anaerobic microorganism.
  • the anaerobic atmosphere means that the oxygen concentration in the atmosphere is as low as 0.1% (v / v) or less as close to 0 as possible.
  • the partial pressure of hydrogen and carbon dioxide in the anaerobic gas supplied to the culture atmosphere is preferably 1: 1 to 8: 1, more preferably 2: 1 to 8: 1, and still more preferably 4: 1 to 8: 1.
  • Anaerobic gases supplied to the culture atmosphere may contain, in addition to hydrogen and carbon dioxide, gases such as carbon monoxide, methane and water vapor.
  • the above-mentioned anaerobic gas may be prepared by appropriately mixing hydrogen and carbon dioxide etc.
  • it is prepared by gasifying various biomass such as rice straw, wheat straw, bagasse etc.
  • an anaerobic gas is used.
  • anaerobic gas prepared from biomass effective utilization of biomass can be performed.
  • by-product gases generated when purifying hydrogen for fuel cells from naphtha and methane derived from fossil fuel mainly hydrogen and carbon dioxide are included, and other components include methane, carbon monoxide and water vapor
  • this by-product gas as an anaerobic gas in the present invention, it is possible to reduce emissions of greenhouse gases such as carbon dioxide.
  • the culture conditions in the above-mentioned acetic acid producing bacteria culture step are appropriately determined according to the genus of the acetic acid producing bacteria to be cultured.
  • acetic acid-producing bacteria that are anaerobic microorganisms such as the above-mentioned Morella microorganism, Clostridium microorganism, and Acetobacter microorganism can be cultured according to a known anaerobic culture method.
  • acetic acid-producing bacteria that are aerobic microorganisms such as the above-mentioned Acetobacter microorganism or Gluconacetobacter microorganism can be cultured according to a known aerobic culture method.
  • FIG. 1 shows a flow diagram of a method for producing lipid according to an embodiment of the present invention.
  • the production method of the present invention is not limited to the invention shown in FIG.
  • Anaerobic gases obtained by gasifying various biomasses such as rice straw, straw and bagasse, and by-product gases generated when hydrogen is produced using naphtha or methane as a raw material can be a Morella microorganism, Clostridium microorganism, aceto It is supplied to a culture tank of an acetic acid-producing bacterium that is an anaerobic microorganism such as a bacterium belonging to the genus Bacteria to perform anaerobic culture (anaerobic fermentation).
  • various types of biomass may be subjected to pressurized hot water treatment and the like, and various degradation products obtained may be supplied to a culture tank for acetic acid-producing bacteria to perform anaerobic fermentation.
  • Acetic acid-producing bacteria produce acetic acid by this anaerobic fermentation, and acetic acid is secreted into the medium.
  • Sodium chloride etc. which are essential for the growth of Aurantiochytrium microbes are added to the culture medium containing this acetic acid (if necessary, a nitrogen source, a carbon source, and other components may be further added. Moreover, it is necessary if necessary.
  • the culture medium may be diluted with water etc.), pH is adjusted to a suitable range, and then the Alanthiochytrium microorganism is inoculated and aerobic culture is performed.
  • aerobic culture acetic acid-producing bacteria that are anaerobic microorganisms are killed and lysed.
  • the bacterial cell component of the lysed acetic acid-producing bacteria can be used as the nitrogen source of the Aurantiochytrium microorganism, and the like.
  • lipids such as polyunsaturated fatty acids (PUFAs), long-chain fatty acids, hydrocarbons, and carotenoids are accumulated in the cells of Aurantiothiotrium microorganisms.
  • the cells of the Auranthiochytrium microorganism having accumulated lipids may be collected by centrifugation or the like, and the produced lipid may be recovered from the obtained cells.
  • any one or more of the lipid recovery step, lipid purification step, substance conversion step, pre-culture step, etc. described in [Embodiment 1] is appropriately included. It may be
  • Example 1 Production of fatty acid by Aurantiochytrium microorganism
  • Aurantiochytrium microorganism Aurantiochytrium sp. SR21 strain ATCC HYA-1381
  • 0-30 g / L acetic acid Nacalai Tesque
  • 6 g / L high polypeptone Nippon Pharmaceutical Co., Ltd.
  • 2 g / L yeast extract Kyuto Pharmaceutical Co., Ltd.
  • the cells contained in the culture solution after culture were collected by centrifugation and lyophilized to determine the amount of dry cells.
  • FIG. (A) of FIG. 2 is a graph showing the dry cell mass (g / L) of Aurantiochytrium SR21 strain recovered from various culture solutions
  • (b) of FIG. 2 is a genus of Auranthiochytrium. It is a graph which shows the composition and production amount of fatty acid in SR21 strain.
  • Auranthiochytrium is The SR21 strain was found to be able to grow vigorously in a medium containing acetic acid.
  • the total fatty acid production amount increases as the concentration of acetic acid contained in the culture medium increases, and the total fatty acid production amount is 4.8 g / l at an acetic acid concentration of 30 g / L. It became L.
  • the dry cell weight was 0.59 g / g-cell, that is, the lipid content accumulated in the cells of the Aurantiochytrium SR21 strain was 59%.
  • the Aurantiochytrium sp. SR21 is capable of using acetic acid as a main carbon source and capable of accumulating a large amount of lipid in the cell (that is, capable of producing lipid with high efficiency).
  • acetic acid is produced in the culture medium by the anaerobic acetic acid-producing bacteria in the culture medium containing hydrogen and carbon dioxide.
  • H 2 -CO 2 medium NH 4 Cl 1.0 g / L, KH 2 PO 4 0.33 g, K 2 HPO 4 0.45 g, MgSO 4 ⁇ 7 H 2 O 0.16 g, NaHCO 3 10.0 g, yeast extract 2.0 g, Na 2 S ⁇ 9 H 2 O 0.5 g, cysteine-HCl ⁇ H 2 O 0.5 g, various It is described that 50 g / L of acetic acid was produced by cultivating Acetobacterium woodii in metal salts ⁇ 10 mg, vitamin B group ⁇ 0.1 mg).
  • Example 2 Fats and oils production from CO 2 by two-step fermentation of Acetobacter microorganism and Aurantiochytrium microorganism [Method] ⁇ Acetate formation by Acetobacter bacteria>
  • a pre-culture medium for Acetobacter sp. was prepared anaerobically according to the method of Miller et al. (Appl. Microbiol. 27: 985, 1974). Briefly, the culture medium of Table 1 is prepared, and 50 ml of the culture medium is placed in a vial and filled so that the H 2 -CO 2 gas (80:20 (v / v)) is 0.20 MPa. did.
  • the above-mentioned vial was inoculated with the acetic acid-producing bacterium Acetobacterium woodii, and cultured at 30 ° C., 180 rpm, until the OD 600 reached 0.15 to 0.25.
  • 40 ml of this pre-culture solution was inoculated into 500 L of the main culture medium for acetobacterium (Table 1) and anaerobically treated in a 1 L reactor treated with H 2 -CO 2 gas (60:40 (v / v)) While culturing at 30 ° C. and 700 rpm while aeration at 1.5 L / h.
  • Aurantiochytrium microbe Aurantiochytrium limacinum SR21 strain is reciprocally shaken at 250C and 250 rpm using GPY medium (3% glucose, 0.2% yeast extract, 0.6% high polypeptone, 2% artificial seawater salt) While culturing for 48 hours. This was used as a pre-culture solution of the Aurantiochytrium microorganism Aurantiochytrium limacinum SR21 strain.
  • acetic acid is added to the main culture medium for Acetobacterium (0% acetic acid) prepared based on Table 1 and the main culture medium for Acetobacterium prepared based on Table 1 to a final concentration of 30 g / L.
  • Three test tubes were prepared, each containing 5 mL of culture medium (3% acetic acid) and culture solution 1 (2.9% acetic acid) prepared above.
  • the preculture liquid of the Aurantiochytrium microorganism Aurantiochytrium limacinum SR21 strain was inoculated in each of the test tubes so that the OD600 was 0.1, and cultured for 48 hours while reciprocating at 28 ° C. and 250 rpm. .
  • the amount of acetic acid remaining in the culture solution was measured, the cells were collected, and the amount of dried cells and the fatty acid composition of the Orangiochytrium microorganism Aurantiochytrium limacinum SR21 strain were measured.
  • lipid production can be performed from CO 2 with high efficiency by two-step fermentation of an acetobacter microorganism (Acetobacterium woodii) and an Aurantithiochytrium microorganism (Aurantiochytrium limacinum).
  • the production method of the present invention can be used in a wide range of industrial fields, such as food, cosmetics, pharmaceuticals, feeds, chemicals, fuels, etc. using lipids.

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Abstract

Le but de la présente invention est d'obtenir à la fois une réduction du coût de matière première et une production hautement efficace de lipides dans la production de lipides tels que des graisses et des huiles par des micro-organismes du genre Aurantiochytrium. La méthode de production de lipides dans un mode de réalisation de la présente invention est une méthode de fermentation en deux étapes qui comprend : une étape de production par fermentation, par des bactéries produisant de l'acide acétique, d'acide acétique à partir d'un sous-produit gazeux comprenant du dioxyde de carbone ; et une étape de production par fermentation, par des micro-organismes du genre Aurantiochytrium, de lipides à partir de l'acide acétique produit par fermentation dans l'étape précédente.
PCT/JP2018/047230 2017-12-26 2018-12-21 Méthode de production de lipides WO2019131502A1 (fr)

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WO2020071444A1 (fr) * 2018-10-02 2020-04-09 国立研究開発法人科学技術振興機構 Procédé de culture de microalgue d'eau douce

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