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WO2016163382A1 - Procédé de production d'acide organique - Google Patents

Procédé de production d'acide organique Download PDF

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WO2016163382A1
WO2016163382A1 PCT/JP2016/061216 JP2016061216W WO2016163382A1 WO 2016163382 A1 WO2016163382 A1 WO 2016163382A1 JP 2016061216 W JP2016061216 W JP 2016061216W WO 2016163382 A1 WO2016163382 A1 WO 2016163382A1
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organic acid
microalga
microalgae
aqueous medium
acid
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誠久 蓮沼
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Kobe University NUC
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Kobe University NUC
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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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for producing an organic acid using microalgae.
  • Succinic acid belongs to organic acids and is used as a raw material for polymers such as polyester and polyamide.
  • Organic acids such as lactic acid and succinic acid are widely used as synthetic raw materials for foods, pharmaceuticals, and other chemicals. These organic acids are currently produced industrially from raw materials derived from fossil fuel resources, but there are concerns about the recent depletion of fossil fuel resources and an increase in atmospheric carbon dioxide (CO 2 ) on a global scale. From the background of the problem, production from carbohydrate biomass such as starch and cellulose is expected as one of renewable energy.
  • Microalgae are aquatic organisms that can produce carbohydrate energy from CO 2 using light. Because it is aquatic, it can avoid competition with food and land use, and is attracting attention as a promising biological system for bioenergy production. However, a common challenge in the production of bio-based chemicals is to enable mass production and lower prices. For this reason, although productivity efficiency improvement is calculated
  • a microalgae photosynthesis evaluation system has been developed by the present inventors (Non-patent Documents 1 and 2), and a method for increasing the cell density by identifying and enhancing the factors that determine the microalgae's growth potential has been sought. Various studies are underway.
  • Patent Document 1 There is a disclosure of a method for producing succinic acid including a step of converting an organic raw material into succinic acid in the presence of a microorganism or a processed product thereof in an aqueous medium.
  • Patent Document 1 it is disclosed that the production rate of succinic acid is increased by setting the concentration of the alkali metal succinate in the aqueous medium to a specific range and then adding ammonia and / or ammonium salt.
  • methods using microalgae have not been studied.
  • An object of the present invention is to provide an organic acid production method that is environmentally friendly and effective without using fossil fuel resources.
  • the present inventors have effectively converted CO 2 and light energy directly from microalgae to effectively recover organic acids that are intracellular metabolites. It has been found that an organic acid can be produced, and as a result, a method for producing an organic acid that is environmentally friendly and effective can be provided, and the present invention has been completed. Specifically, the microalgae is cultured in an aqueous medium containing carbonate ions and / or bicarbonate ions, and an organic acid that is an intracellular metabolite is recovered.
  • microalgae with enhanced NADPH-O 2 oxidoreductase function and / or rate-limiting enzyme function in glycolysis from glycogen to citrate cycle are cultured in aqueous medium, and are organic metabolites that are intracellular metabolites. By recovering the acid.
  • this invention consists of the following.
  • the manufacturing method of the organic acid from a micro algae including the process of the following (A) and (B): (A) a step of culturing microalgae in an aqueous medium having a carbonate ion and / or bicarbonate ion content of 20 to 2000 mM; (B) The process of collect
  • An aqueous medium having a carbonate ion and / or bicarbonate ion content of 20 to 2000 mM is (1) filled with carbon dioxide and / or (2) sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, 2.
  • the production method according to item 1 which is an aqueous medium obtained by adding any one or two or more carbonates selected from magnesium carbonate. 3.
  • the production method according to item 2 or 3 wherein the carbon dioxide is filled until saturated in an aqueous medium. 5.
  • the manufacturing method of the organic acid from a micro algae including the process of the following (a) and (b): (A) culturing a microalga with enhanced function of PEP carboxylase in an aqueous medium; (B) A step of recovering the organic acid produced from the microalga of (a). 11. 9. The method for producing an organic acid according to any one of 1 to 8 above, wherein the microalga is a microalga in which the function of pyruvate ferredoxin oxidoreductase is enhanced.
  • the manufacturing method of the organic acid from a micro algae including the process of the following (c) and (d): (C) culturing a microalga with enhanced function of pyruvate ferredoxin oxidoreductase in an aqueous medium; (D) A step of recovering the organic acid produced from the microalga of (c). 13 9. The method for producing an organic acid according to any one of 1 to 8 above, wherein the microalga is a microalga in which the function of phosphoglucomutase is enhanced.
  • the manufacturing method of the organic acid from a micro algae including the process of the following (e) and (f): (E) culturing a microalga with enhanced function of phosphoglucomutase in an aqueous medium; (F) The process of collect
  • a method for activating a citric acid cycle in a microalgae comprising culturing the microalgae in an aqueous medium having a carbonate ion and / or bicarbonate ion content of 20 to 2000 mM. 16. 9.
  • the manufacturing method of the organic acid from a micro algae including the process of the following (C) and (D): (C) culturing a microalga with enhanced function of NADPH-O 2 oxidoreductase in an aqueous medium; (D) The process of collect
  • a method for producing an organic acid from microalgae comprising the following steps (g) to (i): (G) transforming microalgae so that NADPH-O 2 oxidoreductase is expressed or enhanced; (H) culturing a microalga in which the NADPH-O 2 oxidoreductase of (g) is expressed or enhanced in an aqueous medium; (I) The process of collect
  • the citrate cycle in the microalgae can be activated by culturing the microalgae in an aqueous medium containing carbonate ions and / or bicarbonate ions.
  • an organic acid that is an intracellular metabolite is effectively produced by effectively utilizing glycogen synthesized by photosynthesis of microalgae and a carbon source taken up from an aqueous medium.
  • culturing microalgae with enhanced NADPH-O 2 oxidoreductase function and / or rate-limiting enzyme function in the glycolysis system from glycogen to citrate cycle in an aqueous medium more effective organic An acid can be produced.
  • organic acids can be produced effectively and environmentally friendly without using fossil fuel resources.
  • Example 1 It is a figure which shows the production result of the organic acid by microalgae (cinekocystis).
  • Example 1 It is a figure which shows the production result of the organic acid by microalgae (cinekocystis).
  • Example 1 It is a figure which shows the production result of the organic acid by a micro algae (Euglena).
  • Example 2 It is a figure which shows the production result of the organic acid by a micro algae (Chlamydomonas).
  • Example 3 By CO 2 aeration in the microalgae (Synechocystis) is a diagram showing the production results of the organic acid.
  • Example 4 It is a figure which shows the production pathway of succinic acid by a micro algae.
  • Example 5 It is a figure which shows the production result of the organic acid by the micro algae (Synecocystis) which overexpressed PEP carboxylase. (Example 5) It is a figure which shows the preparation methods of PCC6803 (PGMox) which overexpressed PGM. (Example 6) It is a figure which shows the production result of the organic acid by PCC6803 (PGMox). (Example 6) It is a figure which shows the preparation methods of PCC6803 (PFORox) which overexpressed PFO. (Example 7) It is a figure which shows the production result of the organic acid by PCC6803 (PFORox).
  • Example 7 It is a figure which confirmed the production of lactic acid and succinic acid by culturing Flv3 overexpressing microalgae.
  • Example 8) It is a figure which confirmed the intracellular accumulation amount of glycogen and ATP by culture
  • Example 9 It is a figure which shows a time-dependent change of the 13 C labeling rate of a micro algae (cinekocystis) intracellular metabolite.
  • Reference Example 1 It is a result figure which measured with time the cell density and the amount of intracellular accumulation of glycogen by culture of micro algae.
  • (Reference Example 2) It is a result figure which measured intracellular accumulation amount of acetic acid, lactic acid, and succinic acid by cultivation of micro algae with time.
  • (Reference Example 3) It is a figure which shows the result of having investigated the intracellular metabolic profile by culture
  • the present invention relates to a method for producing an organic acid, and particularly relates to a method for producing an organic acid from microalgae including the following steps.
  • A a step of culturing microalgae in an aqueous medium containing 20 to 2000 mM carbonate ions and / or bicarbonate ions;
  • B The process of collect
  • microalgae refers to a microorganism having chlorophyll (chlorophyll) and performing photosynthesis. Microalgae can synthesize saccharides (eg, glycogen) by immobilizing CO 2 in the atmosphere by photosynthesis, while generating oxygen (O 2 ) from water (H 2 O) (“oxygen-generating photosynthesis”) Also called).
  • the microalgae may have a single cell morphology or may have a colony morphology (eg, filaments, sheets or balls). The microalgae may be propagated in either the ocean or fresh water.
  • the microalgae of the present invention may be any of prokaryotic cyanobacteria (Cyanobacteria) and eukaryotes (for example, green algae, diatoms, dinoflagellates, red algae, prasino algae, Euglena algae, true ocular algae, etc.) There may be.
  • prokaryotic cyanobacteria Cyanobacteria
  • eukaryotes for example, green algae, diatoms, dinoflagellates, red algae, prasino algae, Euglena algae, true ocular algae, etc.
  • Cyanobacteria examples include, for example, Synechocystis, Arthrospira, Spirulina, Anabaena, Synechococcus, Thermosynechococcus, Thermosynechococcus, Stock genus (Nostoc), Prochlorococcus (Prochlorococcu), Microcystis (Microcystis), Gloeobacter (Gloeobacter) etc. are mentioned.
  • Examples of eukaryotes include green algae such as Chlamydomonas, Chlorella, Dunaliella, Hematococcus, Volvox, and Botryococcus; Rhizosolenia Rhizosolenia, Chaetoceros, Cyclotella, Cylindrotheca, Navicula, Phaeodactylum, Thalassiosira, Fitzlifera Genus; Amphidinium, Symbiodinium and other dinoflagellates; Cyanidioschyzon, Phorphyridium and other red algae; Ostreococcus, etc.
  • Plasinophytic algae such as Euglena -Grenade algae
  • true eye spot algae such as Nannochloropsis.
  • microbial species of microalgae are Synechocystis PCC6803 (Synechocystis sp. PCC6803), Synecococcus PCsp. Spirulina maxima, Spirulina subsalsa, Anabaena PCC7120 (Anabaena sp.
  • the microalgae that can be used in the method of the present invention may be a wild type or a microalga modified so as to effectively produce an organic acid.
  • a modification method a method known per se or any method developed in the future can be applied.
  • the modification can be performed by a technique such as gene recombination.
  • examples of such microalgae include genetically modified microalgae that can overexpress an enzyme capable of enhancing the production of organic acids.
  • NADPH-O 2 oxidoreductase refers to converting reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP + ), and from O 2 to H
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • NADP + oxidized nicotinamide adenine dinucleotide phosphate
  • O 2 O An enzyme that catalyzes the production of 2 O.
  • the enzyme can convert NADPH generated by conversion from NADP + by electron transfer from reduced ferredoxin to NADP + again, and generate water from oxygen.
  • Examples of the NADPH-O 2 oxidoreductase of the present invention include flavodi iron protein (hereinafter also referred to as “Flv”).
  • Examples of the flavodi iron protein include Flv3 and Flv1, and Flv3 is particularly preferable.
  • Examples of the flavodi iron protein include those derived from the above-mentioned microalgae.
  • Flv3 and Flv1 are involved in the photoreduction of O 2 in the photosynthetic photosystem I.
  • examples of Flv3 include those derived from the above-mentioned microalgae.
  • PCC6803 species (sll0550: the base sequence and amino acid sequence of the coding region are shown in SEQ ID NOs: 1 and 2) can be used.
  • Flv3 or Flv1 is specified by the amino acid sequence disclosed in the database for Flv3 or Flv1, and one or several amino acids in the disclosed amino acid sequence are deleted, substituted or added It is also possible to specify an amino acid sequence having an enzyme activity necessary for the present invention. Alternatively, for example, an amino acid sequence having 70% or more sequence identity with the amino acid sequence disclosed in the database for Flv3 or Flv1 and having the enzyme activity required in the present invention can be specified.
  • the gene related to NADPH-O 2 oxidoreductase used in the present invention refers to a gene capable of expressing the above flavodi iron protein, specifically refers to a gene capable of expressing Flv3 or Flv1, particularly preferably Flv3.
  • a gene that can be expressed may be a DNA having a base sequence disclosed in a database, or a gene that hybridizes with a DNA having a base sequence complementary to the DNA under stringent conditions.
  • Stringent conditions refer to the conditions disclosed on pages 1.101 to 1.104 of Molecular Cloning, 2nd. Ed., Cold Spring Harbor Laboratory 1989, New York, for example.
  • sequence-specific binding is brought about, so such a functional oligonucleotide is also included in the gene related to NADPH-O 2 oxidoreductase of the present invention.
  • Such a gene for example, using primers designed based on the disclosed or known base sequences, DNA extracted from various organisms, various cDNA libraries or genomic DNA libraries etc. as a template, For example, it can be obtained as a nucleic acid fragment by PCR amplification.
  • a nucleic acid fragment can be obtained by performing hybridization using a nucleic acid derived from the above library as a template and a DNA fragment that is a part of a gene encoding an enzyme to be expressed or expressed in the present invention as a probe. Can do.
  • the gene may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.
  • the gene may be codon optimized to optimize expression in the host microorganism. Codon optimization can be performed using any means and apparatus commonly used by those skilled in the art.
  • fine algae function of NADPH-O 2 oxidoreductase is enhanced refers to a "transformed microalgae to express or enhanced expression of NADPH-O 2 oxidoreductase".
  • expression or enhancement of expression of NADPH-O 2 oxidoreductase means that expression of a gene related to NADPH-O 2 oxidoreductase is enhanced.
  • the form in which the expression of the gene of the NADPH-O 2 oxidoreductase is enhanced, as compared with the prior modified to enhance expression of these genes in microalgae of the invention is carried out, the NADPH-O 2 oxide
  • the modification that enhances gene expression may be a method known per se, or any method developed in the future.
  • any endogenous gene is linked under the control of a stronger promoter (which can be either a constitutive promoter or an inducible promoter).
  • a stronger promoter which can be either a constitutive promoter or an inducible promoter.
  • an embodiment in which either an endogenous gene and / or an exogenous gene is additionally introduced can be mentioned.
  • Any additionally introduced gene is preferably operably retained by a strong promoter, such as a constitutive promoter.
  • the enhancement of expression is also referred to as “overexpression” in the present specification.
  • any promoter that functions in microalgae can be used.
  • the microalgae are cyanobacteria (Cyanobacteria)
  • promoters such as sbDII, psbA3, psbA2, nirA, petE, nrsRS, nrsABCD, ndhF3, rbcL, rbcX, glnA, atp1, atp2, petF1, etc. Is mentioned.
  • a plasmid vector for introducing the above gene into microalgae is the pTCP2031V vector.
  • the pTCP2031V vector include a psbA2 (slr1311) promoter, a part of the coding region of slr2030 and slr2031 (as a platform for homologous recombination), and a recombinant plasmid containing a chloramphenicol resistance cassette (Satoh S et al., 2001, J. Biol. Chem. 276, 4293-4297; Horiuchi M et al., 2010, Biochem. J. 431, 135-140).
  • a recombination construct for example, an expression vector or a chromosome-integrated vector prepared as described above can be introduced into a host microalgae to produce a transformed microalgae.
  • gene homologous recombination methods can be used for transformation of transformed microalgae (particularly cyanobacteria).
  • the pTCP2031V vector can be preferably used.
  • a method known per se or any method developed in the future can be applied. Examples thereof include electroporation method, protoplast-PEG method, microinjection method, particle gun method, calcium phosphate method, lipofection method, calcium ion method and the like.
  • the transformed strain is selected by using a selection marker or the like possessed by the expression vector used for gene transfer or the chromosome integration type vector.
  • Antibiotics or drugs corresponding to the selection marker can be added to a medium suitable for each host microorganism.
  • a selective medium any medium suitable for the growth of microalgae can be used.
  • BG-11 agar eg described in Rippka R et al., 1979, J Gen Microbiol 111: 1-61: can be used for cyanobacteria
  • HSM agar and TAP agar (these are for example Fukuzawa et al., 2009, Cryogenic Science, 67: 17-21: Can be used for eukaryotes such as green algae).
  • transformants are selected based on this selection marker, and then transformants are selected by analyzing the expression of the target gene (ie, NADPH-O 2 oxidoreductase gene) or its product. Can do.
  • the NADPH-O 2 oxidoreductase expression product can be confirmed, for example, by Western blotting.
  • rate-limiting enzymes in the glycolysis system in this specification include phosphoenolpyruvate carboxylase (PEP carboxylase: PEPC), phosphoglucomutase (PGM), and pyruvate ferredoxin oxidoreductase (PFO). (See FIG. 6).
  • PEP carboxylase refers to an enzyme that synthesizes oxaloacetate from phosphoenolpyruvate and CO 2 through the C 4 pathway of the carbonic acid fixation pathway.
  • PGM phosphoglucomutase
  • G1P glucose-1-phosphate
  • G6P glucose-6-phosphate
  • PFO pyruvate ferredoxin oxidoreductase
  • aqueous medium refers to an aqueous solution used for seed culture and / or main culture.
  • an aqueous solution containing a nitrogen source, an inorganic salt and the like is preferable.
  • artificial or natural seawater or fresh water for example, distilled water
  • BG-11 medium J Gen Microbiol 111: 1-61 (1979)
  • HSM medium and TAP medium Cellular Science, 67: 17-21 (2009)
  • Cramer-Myers medium CM medium
  • a medium having the composition shown in Table 1 below may be used.
  • an organic raw material may be added to the medium as a carbon source in the aqueous medium.
  • the organic raw material used in the main culture is not particularly limited as long as the microalgae can be assimilated and proliferated.
  • carbohydrates such as galactose, lactose, glucose, fructose, sucrose, saccharose, starch, and cellulose; glycerol, Fermentable carbohydrates such as polyalcohols such as mannitol, xylitol, and ribitol are used, can be selected according to the target organic acid, and can be selected from general organic raw materials.
  • glucose, sucrose, or fructose is preferable, and glucose or sucrose is particularly preferable.
  • sugar are also used,
  • organic materials can be used alone or in combination.
  • the aqueous medium can contain carbonate ions, bicarbonate ions or CO 2 .
  • the culture temperature of the microalgae is usually 15 to 40 ° C, preferably 20 to 37 ° C, more preferably 25 to 35 ° C.
  • the pH of the aqueous medium can be adjusted to any pH suitable for microalgae growth, such as pH 5-10, preferably pH 6-9, more preferably pH 6-8.
  • the pH can be appropriately adjusted by adding sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide or the like.
  • microalgae can be pre-cultured in advance. For example, it can be cultured through steps such as (1) pre-culture, (2) pre-culture, (3) main culture (photoautotrophic), (4) main culture (anaerobic, dark).
  • aeration culture can be performed, and the culture period is preferably 3 to 14 days, more preferably 3 to 5 days.
  • air or air mixed with CO 2 can be aerated through an aqueous medium.
  • photoautotrophic In this specification (3) Main culture (photoautotrophic), “photoautotrophic” is used in a general sense, and microalgae make sugar from CO 2 and water by photosynthesis, and use this as an energy source. A mechanism for growth.
  • the light irradiation condition at the time of photoautotrophic may be either natural light or artificial light, and the intensity thereof can be appropriately adjusted depending on the algal body density in the aqueous medium, the depth of the culture tank, and the like.
  • natural or artificial light of 30 to 2000 ⁇ mol photons m ⁇ 2 s ⁇ 1 , preferably 30 to 1000 ⁇ mol photons m ⁇ 2 s ⁇ 1 , more preferably 50 to 600 ⁇ mol photons m ⁇ 2 s ⁇ 1 may be used.
  • microalgae can perform photosynthesis and grow smoothly.
  • the light irradiation may be continuous or periodic.
  • a light / dark cycle may be provided to minimize costs and avoid the additional cost of artificial lighting.
  • anaerobic culture refers to culturing while keeping the dissolved oxygen concentration in the solution low.
  • the container is sealed and reacted without aeration, supplied with an inert gas such as nitrogen gas (N 2 ), or reacted with a CO 2 -containing inert gas. This method can be used.
  • an inert gas such as nitrogen gas (N 2 )
  • CO 2 -containing inert gas This method can be used.
  • the organic acid is discharged into the aqueous medium.
  • carbonate ions, bicarbonate ions and / or CO 2 are contained. It is preferable that the carbonate ions and bicarbonate ions contain 20 to 2000 mM, preferably 20 to 500 mM, more preferably 20 to 150 mM.
  • the introduction of carbonate ions and / or bicarbonate ions into the aqueous medium is selected from (1) filling of CO 2 and / or (2) sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate. Any one or two or more carbonates can be added. In the case of filling with CO 2 , the filling can be performed until saturation occurs. When CO 2 is saturated, the carbonate ion concentration is 20 to 2000 mM.
  • Organic acid produced by the above method is separated and purified from an aqueous medium as necessary by a method known per se or any separation and purification method developed in the future. can do. Specifically, after separation from microalgae and their products by ultrafiltration membrane separation, centrifugation, concentration, etc., purification by known methods such as column method, crystallization method, etc., and drying as crystals The method of collecting is mentioned.
  • the organic acid to be produced is not particularly limited, but is an intracellular metabolic organic acid produced in a citric acid cycle, specifically, an organic carboxylic acid.
  • organic carboxylic acid examples include succinic acid, lactic acid, acetic acid, fumaric acid, 2-ketoglutaric acid, malic acid, citric acid and the like.
  • organic acids aliphatic dicarboxylic acids such as succinic acid, fumaric acid, 2-ketoglutaric acid and malic acid are preferred, and succinic acid is particularly preferred.
  • organic acid By collecting the organic acid produced from the microalgae by the above method, the organic acid can be produced effectively and environmentally friendly without using fossil fuel resources. That is, organic acid can be produced from biomass by photosynthesis of microalgae and a carbon source taken into the microalgae, and the organic acid can be produced effectively and environmentally.
  • the supply of carbonate ions and / or bicarbonate ions to the aqueous medium can be effectively utilized using, for example, CO 2 in the atmosphere discharged industrially in the manufacturing process of electricity, steel, and the like. It has an excellent effect on the natural environment in that it can effectively utilize CO 2 in the atmosphere.
  • the aqueous medium used as biomass can utilize not only fresh water but also seawater, and can be stably and effectively utilized regardless of the depletion of water resources or the limits of cultivated land. be able to.
  • the present invention relates to a method for activating a citric acid circuit in a microalgae, which comprises culturing microalgae in an aqueous medium having a carbonate ion and / or bicarbonate ion content of 20 to 2000 mM. It also extends. According to the method of the present invention, the citric acid cycle is activated, and as a result, the production of organic acids that are intracellular metabolites is enhanced.
  • Example 1 Production of organic acid by microalgae (Sinechocystis)
  • Synechocystis sp. PCC6803 glucose tolerance (GT) (Williams JGK, 1988, Methods Enzymol 167: 766-778) (hereinafter referred to as “PCC6803 (GT)” in this example and each example).
  • GT glucose tolerance
  • Pre-culture PCC6803 (GT) colonies grown on BG-11 agar medium (BG-11 containing 1.5% Agar) were picked with a platinum loop and added to an aqueous medium (70 mL). Then, the cells were cultured at 30 ° C. for 4-5 days under 50 ⁇ mol photons m ⁇ 2 s- 1 under aeration conditions.
  • a culture solution containing 17.6 mM NaNO 3 and 20 mM Hepes-KOH in a BG-11 liquid medium (Rippka R et al., 1J Gen Microbiol 111: 1-61 (1979)) was used as an aqueous medium.
  • the algal density was measured by OD 750 using a Shimadzu UV mini spectrophotometer (ultraviolet visible spectrophotometer: manufactured by Shimadzu Corporation).
  • the OD 750 after culture was 1 to 1.5.
  • the ventilation means air ventilation unless otherwise specified. The same applies to the following embodiments.
  • Pre-culture PCC6803 (GT) pre-cultured in (1) above is added to an aqueous medium (150 mL) so that OD 750 is 0.1, and 50 ⁇ mol photons m ⁇ at pH 7.8 under aeration conditions. They were cultured for 4 to 5 days at 30 °C in 2 s- 1.
  • a culture solution containing 17.6 mM NaNO 3 and 20 mM Hepes-KOH in a BG-11 liquid medium was used as an aqueous medium.
  • the OD 750 after culture was 1 to 1.5.
  • PCC6803 (GT) pre-cultured in (2) above is added to an aqueous medium (70 mL) so that the OD 750 is 0.4, and 120 ⁇ mol photons m -2 s- 1 under CO 2 aeration conditions at pH 7.8. And then cultured at 30 ° C. for 3 days.
  • a culture solution containing 5 mM NH 4 Cl and 50 mM Hepes-KOH in a BG-11 liquid medium was used as an aqueous medium.
  • the OD 750 after culture was 6-7.
  • PCC6803 (GT) was collected by filtration with a filter (polytetrafluoroethylene membrane, pore size 1 ⁇ m).
  • the aqueous medium recovered in (4) above is centrifuged at 14000 g and 4 ° C. for 5 minutes, respectively, and the supernatant is recovered and filtered using 0.45 ⁇ m pore size Mini-UniPrep (manufactured by GE Healthcare Japan). did.
  • the production amount of succinic acid, lactic acid, acetic acid and glycogen was measured using a high performance liquid chromatography (HPLC) column (Aminex HPX-87H; manufactured by Bio-Rad) and a refractive index detector (RID-10A; Shimadzu Corporation). Measured by HPLC equipped with Seisakusho.
  • CE capillary electrophoresis / mass spectrometry
  • LC Liquid chromatography-triple quadrupole mass spectrometry (LC / QqQ-MS) controlled by Agilent G7100; MS, Agilent G6224AA LC / MSD TOF; Agilent Technologies) and MassHunter Workstation Data Acquisition software (Agilent Technologies) Analysis and measurement were performed using a system (LC: Agilent 1200 series; MS, Agilent 6460 with Jet Stream Technology; manufactured by Agilent Technologies).
  • succinic acid, lactic acid, acetic acid, fumaric acid, 2-ketoglutaric acid, malic acid, citric acid tended to increase as the NaHCO 3 concentration increased (FIG. 1, 2). It was confirmed that succinic acid, lactic acid and acetic acid can be effectively produced by PCC6803 (GT) by adjusting the NaHCO 3 concentration.
  • Example 2 Production of organic acid by microalgae (Euglena)
  • Euglena gracilis (NIES-48) strain was used as a microbial species of microalgae.
  • NIES-48 Hereinafter referred to as “NIES-48”.
  • CM medium containing 1.5% Agar aqueous medium (70 mL), and aerated at pH 7.8. Under the conditions, the cells were cultured for 4 to 5 days at 30 ° C. in 50 ⁇ mol photons m ⁇ 2 s- 1 .
  • an aqueous medium a culture solution containing 7.5 mM (NH 4 ) 2 HPO 4 in a CM medium having the composition shown in Table 2 was used. The OD 750 after culture was 1 to 1.5.
  • Pre-culture NIES-48 previously cultured in (1) above is added to an aqueous medium (150 mL) so that the OD 750 is 0.1, and 50 ⁇ mol photons m -2 at pH 3.5 under aeration conditions.
  • the cells were cultured for 4 to 5 days at 30 ° C. with s- 1 .
  • a culture medium containing 7.5 mM (NH 4 ) 2 HPO 4 in a CM medium was used as an aqueous medium.
  • the OD 750 after culture was 1 to 1.5.
  • NIES-48 pre-cultured in (2) above is added to an aqueous medium (70 mL) so that OD 750 is 0.4, and 120 ⁇ mol photons m -2 s- 1 at pH 3.5 under CO 2 aeration conditions.
  • the cells were cultured at 30 ° C for 5 days.
  • a culture medium containing 3 mM (NH 4 ) 2 HPO 4 in CM medium was used as an aqueous medium.
  • the OD 750 after culture was 6-7.
  • NIES-48 was recovered by centrifugation (3000 g, 5 minutes, 4 ° C.).
  • the aqueous medium containing NIES-48 cultured in (4) above was centrifuged at 14000 g and 4 ° C. for 5 minutes, respectively, and the supernatant was collected and filtered by the same method as in Example 1. Further, production amounts of succinic acid, lactic acid, acetic acid and citric acid were measured by the same method as in Example 1. As a result, when the NaHCO 3 concentration was 100 mM, the production amounts of succinic acid, lactic acid, and acetic acid increased, and when the concentration was 200 mM, the production amounts of lactic acid and acetic acid tended to decrease (FIG. 3). On the other hand, the production amount of citric acid tended to increase as the concentration of NaHCO 3 increased (FIG. 3). It was confirmed that organic acids (succinic acid, lactic acid, acetic acid, citric acid) can be effectively produced by Euglena by adjusting the NaHCO 3 concentration.
  • Example 3 Production of organic acid by microalgae (Chlamydomonas)
  • a Chlamydomonas reinhardtii strain was used as a microbial species of microalgae.
  • C. reinhardtii a Chlamydomonas reinhardtii strain
  • TAP agar medium TAP medium containing 1.5% Agar
  • aqueous medium 70 mL
  • CO 2 CO 2 at pH 7.8.
  • the cells were cultured at 30 ° C. for 4 to 5 days at 100 ⁇ mol photons m ⁇ 2 s- 1 .
  • MB6N medium was used as an aqueous medium.
  • the OD 750 after culture was 2.5-3.5.
  • the composition of the TAP medium and MB6N medium is shown in Table 3.
  • Pre-culture C. reinhardtii previously cultured in (1) above is added to an aqueous medium (150 mL) so that the OD 750 is 0.1, and 120 ⁇ mol photons m at pH 7.8 under CO 2 aeration conditions.
  • the cells were cultured at ⁇ 2 s- 1 at 30 ° C. for 3 days.
  • MB6N medium was used as an aqueous medium.
  • the OD 750 after culture was 2.5-3.5.
  • C. reinhardtii pre-cultured in (2) above is added to an aqueous medium (70 mL) so that the OD 750 is 0.4, and 120 ⁇ mol photons m ⁇ 2 s- 1 at pH 7.8 under CO 2 aeration.
  • the cells were cultured at 30 ° C for 5 days.
  • MB6N medium was used as an aqueous medium.
  • the OD 750 after culture was 3.5 to 4.5.
  • C. reinhardtii was recovered by centrifugation (14000 g, 5 minutes, 4 ° C.).
  • the aqueous medium recovered in (4) above was centrifuged at 14000 g and 4 ° C. for 5 minutes, respectively, and the supernatant was recovered by the same method as in Example 1. 0.45 ⁇ m pore size Mini-UniPrep (GE Healthcare Japan Co., Ltd.) (Manufactured by company). With respect to the filtrate, the production amounts of succinic acid, lactic acid and acetic acid were further measured by the same method as in Example 1. As a result, when the NaHCO 3 concentration was 100 mM, the production amounts of succinic acid, lactic acid, and acetic acid increased (FIG. 4). It was confirmed that organic acids (succinic acid, lactic acid, acetic acid) can be effectively produced by Chlamydomonas by adjusting the NaHCO 3 concentration.
  • Example 4 Production of organic acid by microalgae (cinekocystis)
  • production of organic acid was confirmed when microalgae were cultured under saturated CO 2 conditions.
  • PCC6803 (GT) was used as in Example 1.
  • Pre-culture PCC6803 (GT) colonies grown on BG-11 agar medium (BG-11 containing 1.5% Agar) were picked with a platinum loop and added to an aqueous medium (70 mL). Then, the cells were cultured at 30 ° C. for 4-5 days under 50 ⁇ mol photons m ⁇ 2 s- 1 under aeration conditions.
  • a culture solution containing 17.6 mM NaNO 3 and 20 mM Hepes-KOH in a BG-11 liquid medium (Rippka R et al., 1J Gen Microbiol 111: 1-61 (1979)) was used as an aqueous medium.
  • the algal density was measured by OD 750 using a Shimadzu UV mini spectrophotometer (ultraviolet visible spectrophotometer: manufactured by Shimadzu Corporation).
  • the OD 750 after culture was 1 to 1.5.
  • the ventilation means air ventilation unless otherwise specified. The same applies to the following embodiments.
  • Pre-culture PCC6803 (GT) pre-cultured in (1) above is added to an aqueous medium (150 mL) so that OD 750 is 0.1, and 50 ⁇ mol photons m ⁇ at pH 7.8 under aeration conditions. They were cultured for 4 to 5 days at 30 °C in 2 s- 1.
  • a culture solution containing 17.6 mM NaNO 3 and 20 mM Hepes-KOH in a BG-11 liquid medium was used as an aqueous medium.
  • the OD 750 after culture was 1 to 1.5.
  • PCC6803 (GT) pre-cultured in (2) above is added to an aqueous medium (70 mL) so that the OD 750 is 0.4, and 120 ⁇ mol photons m -2 s- 1 under CO 2 aeration conditions at pH 7.8. And then cultured at 30 ° C. for 3 days.
  • a culture solution containing 5 mM NH 4 Cl and 50 mM Hepes-KOH in a BG-11 liquid medium was used as an aqueous medium.
  • the OD 750 after culture was 6-7.
  • PCC6803 (GT) was collected by filtration with a filter (polytetrafluoroethylene membrane, pore size 1 ⁇ m).
  • the aqueous medium recovered in (4) above is centrifuged at 14000 g and 4 ° C. for 5 minutes, respectively, and the supernatant is recovered and filtered using 0.45 ⁇ m pore size Mini-UniPrep (manufactured by GE Healthcare Japan). did.
  • the production amount of succinic acid, lactic acid, acetic acid and glycogen was measured using a high performance liquid chromatography (HPLC) column (Aminex HPX-87H; manufactured by Bio-Rad) and a refractive index detector (RID-10A; Shimadzu Corporation). Measured by HPLC equipped with Seisakusho.
  • the production amounts of fumaric acid, 2-ketoglutaric acid, malic acid, and citric acid were extracted from intracellular metabolites as described in Non-Patent Document 2, and a capillary electrophoresis / mass spectrometry (CE / MS) system (CE: Liquid chromatography-triple quadrupole mass spectrometry (LC / QqQ-MS) controlled by Agilent G7100; MS, Agilent G6224AA LC / MSD TOF; Agilent Technologies) and MassHunter Workstation Data Acquisition software (Agilent Technologies) Analysis and measurement were performed using a system (LC: Agilent 1200 series; MS, Agilent 6460 with Jet Stream Technology; manufactured by Agilent Technologies). As a result, the production amount of succinic acid and acetic acid tended to increase under the culture conditions with CO 2 (FIG. 5).
  • Example 5 Production of organic acid by microalgae overexpressing PEP carboxylase
  • production of organic acid by microalgae overexpressing PEP carboxylase was confirmed.
  • a PEP carboxylase overexpression strain PCC6803 (PEPox) was prepared by gene recombination using PCC6803 (GT) shown in Example 1.
  • the production method of PCC6803 (PEPox) is as follows. The production route of succinic acid by microalgae is shown in FIG. 6, and the PEP carboxylase action point is shown.
  • the rbcL terminator and a part of the coding region downstream of the slr0168 region use the oligonucleotides shown in SEQ ID NOs: 1 and 2 and the oligonucleotides shown in SEQ ID NOs: 3 and 4 as a primer set from genomic DNA extracted from PCC6803 (GT) And amplified by PCR.
  • the obtained amplified fragment was inserted into PstI and HindIII digested pBluescriptBlueII SK (+) (AgilentgilTechnologies, Palo Alto, CA) using In-Fusion HD Cloning Kit (manufactured by Clonetech, Takara Bio Inc.).
  • PBluescript-TrbcL-slr0168 was obtained.
  • Sequence number 3 5'-CCTCTAGAGTCGACCTGCAGGTTACAGTTTTGGCAATTAC-3 ' Sequence number 4: 5'-GCCAGCCCCAACACCTGACGCGTTTCCCCACTTAGATAAAAAATCC-3 ' Sequence number 5: 5'-TCTAAGTGGGGAAACGCGTCAGGTGTTGGGGCTGGC-3 ' Sequence number 6: 5'-TGATTACGCCAAGCTTCTAAGTCAGCGTAAATCTGACAATG-3 '
  • the oligonucleotides shown in SEQ ID NOs: 5 and 6 and the oligonucleotides shown in SEQ ID NOs: 7 and 8 were used as primer sets from the genomic DNA of pCRII-TOPO (Invitrogen, Carlsbad, CA) and PCC6803 (GT). And amplified by PCR.
  • Sequence number 7 5'-CGGGCCCCCCCTCGAGCCGGAATTGCCAGCTGGGGC-3 ' Sequence number 8: 5'-TGGACTTTCTAATTAGAGCGGCCGCTCAGAAGAACTCGTCAAGA-3 ' Sequence number 9: 5'-TCTTGACGAGTTCTTCTGAGCGGCCGCTCTAATTAGAAAGTCCA-3 ' SEQ ID NO: 10: 5'-CCGGGGATCCTCTAGACATATGGGTCAGTCCTCCAT-3 '
  • a part of the coding region upstream of the slr0168 region was amplified by PCR from the genomic DNA extracted from PCC6803 (GT) using the oligonucleotides shown in SEQ ID NOs: 9 and 10 as primer sets.
  • the obtained amplified fragment was inserted into In-Fusion HD Cloning Kit KpnI and XhoI digested pBluescript-Km r with (Clonetech Inc., Takara available from Bio Inc.) -PrbcL-TrbcL-slr0168, pBluescript -slr0168- Km r -PrbcL-TrbcL-slr0168 was obtained.
  • Sequence number 11 5'-TATAGGGCGAATTGGGTACCATGACTATTCAATACACCCCCCTAG-3 ' Sequence number 12: 5'-TACCGTCGACCTCGAGCACCAGACCAAAGCCGGGAATTTC-3 '
  • CACATG digested with AatII and EcoRI was substituted for the NdeI site (CATATG) of pUC19 (Takara Bio), and the synthetic DNA was inserted.
  • the fragment containing slr0168 was inserted into the KpnI / HindIII site of the pUC19 vector prepared above to prepare pSKrbcL-slr0168.
  • Ppc (sll0920) encoding PEP carboxylase was amplified by PCR from genomic DNA extracted from PCC6803 (GT) using the oligonucleotides shown in SEQ ID NO: 11 and SEQ ID NO: 12 as primer sets. The obtained amplified fragment was inserted into NdeI / SalI-digested pSKtrc-slr0168 using In-Fusion®HD®Cloning®Kit (manufactured by Takalon Bio Inc., Clontech) to obtain pSKtrc-slr0168 / sll0920.
  • SEQ ID NO: 13 5'-AGGAAACAGACCCATATGAACTTGGCAGTTCCTGC-3 '
  • SEQ ID NO: 14 5'-AACCTGCAGGTCGACTCAACCAGTATTACGCA-3 '
  • PCC6803 (GT) was transformed with the obtained plasmid pSKtrc-slr0168 / sll0920 vector (including the sll0920 coding region). As a control, transformation with an empty vector (plasmid pSKtrc-slr0168 vector not containing the sll0920 coding region) was performed.
  • PCC6803 (GT) transformed so as to overexpress sll0920 is referred to as PCC6803 (PEPox).
  • PCC6803 (VC) wild-type PCC6803
  • Example 6 Production of organic acid by PGM overexpressing microalgae
  • production of organic acid by PGM overexpressing microalgae was confirmed.
  • PCC6803 (GT) shown in Example 1 a PGM overexpression strain PCC6803 (PGMox) was produced by gene recombination.
  • a method for producing PCC6803 (PGMox) is shown in FIG.
  • the production route of succinic acid by microalgae is shown in FIG.
  • Example 5-1 ⁇ 5-3.
  • PCC6803 (GT) prepared by transforming PCC6803 (GT) with pSKtrc-slr0168 / sll0726 and overexpressing sll0726 is referred to as PCC6803 (PGMox) in this example.
  • PCC6803 (PGMox) untransformed wild-type PCC6803 (GT) was prepared in Example 5-6.
  • PCC6803 (VC) Like PCC6803 (VC).
  • PCC6803 (PGMox) and PCC6803 (VC) were cultured based on the same culture conditions as in (1) to (3) of Example 1.
  • an aqueous medium containing PCC6803 (PGMox) or PCC6803 (VC) is added. It was collected.
  • recovered aqueous medium the production amount of succinic acid, lactic acid, and acetic acid was measured by the same method as Example 1.
  • FIG. 9 the PGM overexpressing strain PCC6803 (PGMox) showed higher productivity in all cases (FIG. 9).
  • Example 7 Production of organic acid by PFOR overexpressing microalgae
  • production of organic acid by PFOR overexpressing microalgae was confirmed.
  • PCC6803 (GT) shown in Example 1 a strain PCC6803 (PFORox) overexpressing the PFOR gene (A1443) derived from Synechococcus sp. PCC7002 was prepared by gene recombination.
  • the method for producing PCC6803 (PFORox) is shown in FIG.
  • the production route of succinic acid by microalgae is shown in FIG. 6, and the action point of PFO is shown.
  • Example 5-1 ⁇ 5-3.
  • PCC6803 (GT) was transformed with pSKtrc-PFOR (A1443).
  • PCC6803 (GT) prepared so that PFOR (A1443) is overexpressed is referred to as PCC6803 (PFORox) in this example.
  • untransformed wild-type PCC6803 (GT) was used in Example 5-6.
  • PCC6803 (VC) Like PCC6803 (VC).
  • PCC6803 PFORox
  • PCC6803 (VC) PCC6803
  • 0 or 100 mM NaHCO 3 was added to the aqueous medium, and after 10 minutes of N 2 aeration, the culture was started under anaerobic conditions, and after 72 hours.
  • An aqueous medium containing PCC6803 (PFORox) or PCC6803 (VC) was recovered. About the collect
  • the obtained 1.7 kb amplified fragment was inserted into the NdeI digested pTCP2031V vector using In-Fusion Cloning Kit (available from Clonetech, Takara Bio Inc.).
  • the pTCP2031V vector is a recombinant plasmid containing the psbA2 (slr1311) promoter and part of the coding region of slr2030 and slr2031 (as a platform for homologous recombination) and a chloramphenicol resistance cassette (Satoh S et al., 2001, J. Biol. Chem. 276, 4293-4297; Horiuchi M et al., 2010, Biochem. J. 431, 135-140).
  • PCC6803 (GT) was transformed with the obtained plasmid pTCP2031V vector (including flv3 coding region) to prepare PCC6803 (Flv3ox).
  • PCC6803 (VC) wild-type PCC6803 (GT) that is not transformed.
  • Example 9 Intracellular glycogen and ATP by culture of PCC6803 (Flv3ox)
  • PCC6803 (Flv3ox) cultured under anaerobic / dark conditions in Example 8 (8-2) and PCC6803 (VC) as a comparative example the amounts of intracellular glycogen and ATP accumulated were measured.
  • Ammonium chloride (NH 4 Cl) was used as a nitrogen source in the aqueous medium.
  • PCC6803 (Flv3ox) overexpressed in Flv3 showed a high accumulation amount for both glycogen and ATP (FIG. 13).
  • ATP quantification As described in Non-Patent Document 2, the amount of ATP is obtained by extracting intracellular metabolites and capillary electrophoresis / mass spectrometry (CE / MS) system (CE: Agilent G7100; MS, Agilent G6224AA LC / MSD TOF; Agilent Technologies) and liquid chromatography-triple quadrupole mass spectrometry (LC / QqQ-MS) system (LC: Agilent 1200 series) controlled by MassHunter Workstation Data Acquisition software (Agilent Technologies) MS, Agilent 6460 with Jet Stream Technology (manufactured by Agilent Technologies) and analyzed.
  • CE / MS capillary electrophoresis / mass spectrometry
  • LC / QqQ-MS liquid chromatography-triple quadrupole mass spectrometry
  • PCC6803 (GT) shown in Example 1 was used as a microalgae, and 13 C labeling rate of intracellular metabolites was changed over time. It was confirmed.
  • PCC6803 (GT) was suspended in 100 mM Hepes-KOH aqueous medium (pH 7.8) containing 100 mM NaH 13 CO 3 and cultured under anaerobic conditions after aeration with N 2 .
  • PCC6803 5 mg dry weight PCC6803 (GT) was collected using a 1 ⁇ m pore size polytetrafluoroethylene (PTEE) membrane filter (Omnipore; manufactured by Millipore). Immediately after filtration, the cells were washed with 20 mM pre-chilled ammonium carbonate. Immediately, PCC6803 (GT) on the membrane filter was frozen in liquid nitrogen and lyophilized in a lyophilizer (Labconco).
  • PTEE polytetrafluoroethylene
  • biomass can be effectively utilized by culturing microalgae by the method of the present invention.
  • Organic acids can be produced from biomass by photosynthesis of microalgae and carbon sources incorporated into microalgae.
  • organic metabolites that are intracellular metabolites are more effective.
  • the acid can be recovered.
  • the supply of carbonate ions and / or bicarbonate ions to the aqueous medium can be effectively utilized using, for example, CO 2 in the atmosphere discharged industrially in the manufacturing process of electricity, steel, and the like.
  • succinic acid among organic acids has been produced using petroleum or the like as a raw material, but CO 2 was emitted at that time, whereas according to the method of the present invention, the production process of organic acid in not only does not emit CO 2, in that it can effectively utilize the CO 2 in the atmosphere, it has an excellent effect against the natural environment. Furthermore, according to the microalgae of the present invention, the aqueous medium used as biomass can utilize not only fresh water but also seawater, and can be stably and effectively utilized regardless of the depletion of water resources or the limits of cultivated land. be able to.
  • the organic acid produced is effectively used in food, pharmaceuticals and other various fields.
  • succinic acid is used for pharmaceutical excipients, pH adjusters, seasonings as foods, other food additives, and industrially for plating. It is also used as a component such as a bathing agent that foams carbon dioxide gas.

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Abstract

La présente invention concerne un procédé de production d'un acide organique, moyennant quoi il devient possible de produire efficacement l'acide organique à partir d'une microalgue. Un acide organique peut être produit de manière efficace en permettant du dioxyde de carbone et une énergie optique d'être utilisés directement par une microalgue et ensuite en recueillant de l'acide organique, qui est un métabolite intercellulaire de la microalgue, à partir de la microalgue. Plus particulièrement, un acide organique est produit par culture d'une microalgue dans un milieu aqueux contenant des ions carbonate et/ou des ions bicarbonate et ensuite par recueil de l'acide organique qui est un métabolite intercellulaire de la microalgue. En variante, un acide organique est produit par culture d'une microalgue, dans laquelle la fonction d'une NADPH-O2 oxydoréductase et/ou la fonction d'une enzyme à régulation de vitesse dans le système glycolytique à partir de glycogène vis-à-vis du cycle acide citrique est améliorée, dans un milieu aqueux et ensuite par recueil de l'acide organique qui est un métabolite intercellulaire de la microalgue. Par exemple, l'acide succinique, parmi les acides organiques, a été produit en utilisant du pétrole ou similaire comme matière première. Selon le procédé de la présente invention, un acide organique peut être produit en utilisant efficacement une biomasse. L'acide organique produit par le procédé peut être utilisé efficacement dans les aliments, les médicaments et divers autres domaines.
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