WO2016140374A1 - Technologie de production de biocarburant utilisant un liquide mixte, culture d'espèces mixtes - Google Patents

Technologie de production de biocarburant utilisant un liquide mixte, culture d'espèces mixtes Download PDF

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WO2016140374A1
WO2016140374A1 PCT/JP2016/057691 JP2016057691W WO2016140374A1 WO 2016140374 A1 WO2016140374 A1 WO 2016140374A1 JP 2016057691 W JP2016057691 W JP 2016057691W WO 2016140374 A1 WO2016140374 A1 WO 2016140374A1
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culture
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microorganism
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宗彦 朝山
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国立大学法人茨城大学
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    • 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
    • C12N1/20—Bacteria; 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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  • the present invention relates to a method for producing biofuel by, for example, co-culture of a photosynthetic microorganism and a non-photosynthetic microorganism.
  • Photosynthetic microorganisms including cyanobacteria belong to microalgae in a broad sense, but their photosynthetic ability is said to be several tens to one hundred times that of plants.
  • biofuels are roughly classified into neutral fats (triacylglycerol, TAG), fatty acids, hydrocarbons, and the like, which are raw materials for biodiesel.
  • TAG neutral fats
  • the former two have oxygen (O) molecules in their chemical structure, but hydrocarbons are composed only of carbon (C) and hydrogen (H), and are preferred in the petroleum industry as fuels that are friendly to engines, etc.
  • Hydrocarbon fuels are defined as fuel properties by the number of carbon atoms and the number and position of double bonds connecting them.
  • alkanes chain saturated hydrocarbons that do not contain double bonds
  • alkanes having 11 to 17 or 12 to 17 carbon atoms are liquid in a standard (normal temperature and normal pressure) state, and are positioned as fuel equivalent to jet fuel or light oil.
  • strains that produce alkanes having 15 carbon atoms (pentadecane, C 15 H 32 ) and 17 (heptadecane, C 17 H 36 ), which are some species of cyanobacterial natural algae among microalgae, are known. (Non-Patent Document 1).
  • Patent Document 1 The inventor group has so far succeeded in modifying cyanobacterial natural algae by genetic manipulation and obtaining mutant strains ranging from about 50 to 60% per dry cell weight.
  • Patent Document 1 biofuel production using natural algae of microalgae or genetically modified algae has been attempted.
  • biofuel has been produced in and out of algal cells by culturing isolated and purified algal cells in a sterile medium while being aseptically or close to aseptic conditions.
  • the target organic substance (biofuel, etc.) can be produced from glucose in one stage (while simultaneously culturing in one bioreactor).
  • examples of conventional methods for producing useful substances by co-cultivation include a system in which methyl halide is produced by co-culturing an actinotalea fermentus bacterium and yeast (Patent Document 2), and a green alga Botryococcus culture solution.
  • Patent Document 3 In a system that promotes the growth of green algae by adding and co-culturing a strain of Carius excentricus (Patent Document 3), a system that produces ethanol fuel from hydrolyzed biomass feedstock, or a clostridium phytofermentans cell or Examples thereof include a method by co-culture containing other fine substances (Patent Document 4).
  • an object of the present invention is to provide a novel technique for producing biofuel.
  • biofuels are produced by coexisting mixed species in a nitrogen source-deficient medium in the same space.
  • the present inventors have found that this can be done and have completed the present invention. That is, the present invention includes the following.
  • a method for producing a biofuel comprising co-culturing a mixed microorganism containing a photosynthetic microorganism and a non-photosynthetic microorganism in a nitrogen source-deficient medium.
  • ST1 strain in hydrocarbon production It is a figure which shows the GC-MS analysis result in the biofuel production of 5L scale by MCMS culture
  • the biofuel production method according to the present invention comprises co-culturing (culturing in the same space) a mixed microorganism containing a photosynthetic microorganism and a non-photosynthetic microorganism in a nitrogen source-deficient medium.
  • the photosynthetic microorganism produces sugar
  • the non-photosynthetic microorganism produces biofuel such as oil (hydrocarbon).
  • the biofuel to be produced include short-chain alkanes having 1 to 20 carbon atoms such as heptadecane.
  • FIG. 1 shows an outline of mixed-mixture mixed-species (MCMS).
  • a conventional cell culturing method is characterized by purifying a target cell and culturing one type of cell in a medium having a simple composition (UCUS: Uni-Culture Uni-Species). In this case, it may be difficult to purify the cell line, or the target outlet product and the culture conditions for producing it may be limited.
  • this method is characterized by mixed-mixture mixed-species (MCMS).
  • the mixed solution is a medium containing inorganic and organic substances as components, and the mixed species means that two or more types of cells including photosynthetic microorganisms coexist.
  • mixed seed culture is positioned as “co-culture”.
  • the photosynthetic microorganism is not particularly limited as long as it is a microorganism that fixes water and carbon dioxide by photosynthesis and produces organic substances such as glucose, and examples thereof include cyanobacteria (prokaryotic photosynthetic microorganisms).
  • cyanobacteria prokaryotic photosynthetic microorganisms.
  • Examples of cyanobacteria include microorganisms belonging to genera such as genus Halomicronema, genus Microkistis, genus Limnolix, genus Pseudoanabena and the like.
  • microorganisms belonging to the genus Halomicronema include, for example, Halomicronema sp. SZ2 strain (hereinafter sometimes referred to as “SZ2 strain” and the like) or mutants thereof having sugar-producing ability (for example, natural mutant strains, mutagenesis). Processing stocks).
  • SZ2 strain was founded in 2014 (November 2014) in the National Institute of Technology and Evaluation (NPMD) (Room 2-2-8-8 Kazusa Kamashi, Kisarazu, Chiba Prefecture, Japan 292-0818). Deposited on the 12th of the month, the deposit number is NITE P-01982.
  • the SZ2 strain was released in 2016 in the National Institute of Technology and Evaluation Patent Microorganisms Depository Center (NPMD) (Room 2-5-8 122, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818). ) As of February 12, it has been transferred to the International Deposit under the deposit number NITE BP-01982.
  • the SZ2 strain was identified as a new strain belonging to the genus Halomonema, a kind of filamentous cyanobacteria that produces sugar on the surface of cells by the bacteriological properties and 16S rRNA gene homology analysis shown in the Examples below.
  • the non-photosynthetic microorganism is not particularly limited as long as it is a microorganism that produces a biofuel using a carbon source such as sugar other than the photosynthetic microorganism.
  • microorganisms belonging to the genus Cinolizobium include, for example, Cinolizobium sp.
  • ST1 strain (hereinafter sometimes referred to as “ST1 strain” and the like) or mutants thereof having the ability to produce short-chain alkanes such as heptadecane (for example, natural mutants, Mutagenesis treated strain) and the like.
  • the ST1 strain was founded in 2014 (November 2014) in the National Institute of Technology and Evaluation Microbiology Depositary Center (NPMD) (Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818). Deposited on the 12th of the month, the deposit number is NITE P-01981.
  • NPMD National Institute of Technology and Evaluation Microbiology Depositary Center
  • ST1 strain was established in 2016 by the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818). ) As of February 12, it has been transferred to the International Deposit under the deposit number NITE BP-01981.
  • the ST1 strain was identified as a new strain belonging to the rhizobia Synorhizobium by the bacteriological properties shown in the examples below and the homology analysis of the 16S rRNA gene.
  • a mixed microorganism containing an SZ2 strain or a mutant strain thereof capable of producing a sugar and an ST1 strain or a mutant strain thereof capable of producing a short-chain alkane is preferably used.
  • a photosynthetic microorganism and a non-photosynthetic microorganism are prepared.
  • the photosynthetic microorganism can be prepared by culturing (pre-culture) in an inorganic medium.
  • the inorganic medium means a medium that does not contain saccharide as a nutrient source, such as BG11 medium.
  • a BG11 liquid medium [medium composition: 0.003 mM Na 2 -Mg EDTA, 0.029 mM citric acid, 0.18 mM K 2 HPO 4 , 0.30 mM MgSO 4 .7H 2 O as an inorganic medium] 0.25 mM CaCl 2 ⁇ 2H 2 O, 0.19 mM Na 2 CO 3 (anhydrous), 0.03 mM ammonium iron citrate, 1 ml / L micronutrients (micronutrient composition: 2.86 g / L boric acid, 1 .81 g / L MnCl 2 .4H 2 O, 0.22 g / L ZnSO 4 .7H 2 O, 0.39 g / L Na 2 MoO 4 .2H 2 O, 0.08 g / L CuSO 4 .5H 2 O, 0 049 g / L Co (NO 3 ) 2 ⁇ 6H 2 O), 1.5
  • the SZ2 strain culture can be prepared by shaking (1 to 200 rpm, preferably 40 to 110 rpm) or stationary culture for 7 to 90 days (preferably 10 to 60 days) under low temperature.
  • non-photosynthetic microorganisms can be prepared by culturing (preculture) in an organic medium.
  • the organic medium means a medium containing a saccharide such as glucose (or a compound containing carbon and oxygen among compounds containing carbon excluding carbon monoxide and carbon dioxide) as a nutrient source.
  • an ST1 strain for example, an LB liquid (agar) medium (medium composition: NaCl 10 g / L, Bacto Tryptone Peptone 10 g / L, powdered yeast extract 5 g / L, or Bacto Agar as described above is used as an organic medium. Is added at a rate of 15 g / L, and this is poured into a petri dish after autoclaving) and shaken at a temperature of 28 to 30 ° C. for 1 to several days (a few days to 60 days in the case of an agar medium) By culturing (0 to 200 rpm, preferably 90 to 120 rpm), an ST1 strain culture can be prepared.
  • agar medium medium composition: NaCl 10 g / L, Bacto Tryptone Peptone 10 g / L, powdered yeast extract 5 g / L, or Bacto Agar as described above is used as an organic medium. Is added at a rate of 15 g / L, and this is poured into
  • a photosynthetic microorganism and a non-photosynthetic microorganism are combined to form a mixed microorganism, and the mixed microorganism is co-cultured, for example, in a nitrogen source-deficient medium.
  • the SZ2 strain coexists with the ST1 strain as a coexisting bacterium in nature, and a mixed microorganism under the coexistence may be used.
  • the photosynthetic microorganism and the non-photosynthetic microorganism may be isolated from each other, or the above-mentioned preculture itself may be used as each microorganism.
  • the nitrogen source deficient medium in the co-culture contains the inorganic medium and / or the organic medium used for the preculture.
  • the ratio of the inorganic medium and / or the organic medium used for the preculture relative to the nitrogen source-deficient medium is, for example, 0 to 100% by volume (v / v), preferably 0.4 to 20% by volume (v / v). It is done.
  • the ST1 strain collected from 5 mL is 1: 1
  • SZ2 strain: ST1 strain 1: 0.04 to 1: 2, preferably about 1: 1.
  • the nitrogen source-deficient medium used for the co-culture does not contain a nitrogen source or is poor in nitrogen source relative to the nitrogen source in a normal medium (for example, BG11 liquid medium) (for example, 0 to 50%, Preferably, the medium contains a nitrogen source of 0 to 10%.
  • a normal medium for example, BG11 liquid medium
  • the medium contains a nitrogen source of 0 to 10%.
  • the nitrogen source include ammonia, ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium iron citrate, sodium nitrate, potassium nitrate, and calcium nitrate.
  • BG11 0 liquid medium as a nitrogen source deficient medium (medium composition: that remove all NaNO 3 from BG11 medium composition of the) or partially NaNO 3 ( The BG11 medium extracted in about 10% to 90%)
  • the nitrogen source-deficient medium may be prepared using non-sterile distilled water or the like. By using non-sterile distilled water, the culture cost can be reduced.
  • a carbon source and / or a sugar source to the nitrogen source-deficient medium.
  • the carbon source can be further reinforced by adding the carbon source. Examples of the carbon source to be added include sodium acetate, potassium acetate, sodium carbonate and the like.
  • the amount of the carbon source added to the nitrogen source-deficient medium is, for example, an amount that gives a final concentration of 1 to several hundred mM, preferably about 10 mM, in the nitrogen source-deficient medium.
  • the added sugar source can be used to allow the non-photosynthetic microorganism to produce biofuel and improve biofuel production. it can.
  • sugar source examples include monosaccharides such as glucose, xylose, arabinose, xylose, ribose, deoxyribose, fructose, galactose, mannose, etc., disaccharides such as lactose, maltose, trehalose, etc., trisaccharides such as maltotrise, raffinose, etc.
  • Oligosaccharides such as fructo-oligo (FOS) sugar, galactooligosaccharide (GOS), mannan oligo (MOS) sugar, etc., polysaccharides such as glycogen, starch, cellulose, dextrin, glucan, fructan, chitin, and other industrial waste ( Waste molasses, fermentation liquid, manure liquid, sewage, etc.).
  • the amount of the sugar source added to the nitrogen source-deficient medium is, for example, an amount that provides a final concentration of 0.01 to several hundred mM, preferably 0.1 to 20 mM (about 0.5 mM in this method) in the nitrogen source-deficient medium.
  • Co-culture for example, temperature of 20 ⁇ 30 ° C.
  • the culture cost can be reduced by providing a predetermined dark period.
  • co-cultures can be performed under illuminated 12 hour intervals (ie, light / dark (12 hours / 12 hours) cycle) conditions. Further, after the co-culture, the co-culture is further cultured under drought stress (for example, natural drying of the medium liquid), so that the biosynthetic microorganism itself can produce biofuel.
  • the culture is performed by static culture for several days to several tens of days (preferably about 2 weeks to 2 months) under the conditions according to the above-mentioned co-culture conditions.
  • biofuels such as hydrocarbons (alkanes) can be produced in high yield.
  • biofuel can be recovered by subjecting the co-culture to solvent extraction such as ethyl acetate.
  • the SZ2 cell mass grown on the agar medium was scraped off, transferred to a new BG11 liquid medium, and subcultured.
  • the isolate obtained as described above (including ST1 cells) was named SZ2. 1-2.
  • Physiological properties of SZ2 strain When halomicronema sp. SZ2 strain is cultivated in a BG11 liquid medium at 30 ° C (under white fluorescent light) (stirring once a day), the long side per cell is about 3 Shows filamentous cell morphology with ⁇ 5 ⁇ m cells connected.
  • PAS Periodic acid-Schiff stain
  • SZ2 strain is a new species of algae that is positioned as a kind of filamentous cyanobacteria Halomicrone genus cluster. It became clear that there was.
  • the long side per cell is unicellular with about 0.7 to 2 ⁇ m. 2-3.
  • Classification and identification of ST1 strain by 16S rRNA gene sequence Total cell DNA was extracted from ST1 isolated (purified) strain, and 16S rDNA base sequence (SEQ ID NO: 2, 475 base pairs) was decoded.
  • a brute force search with the gene DNA database using this sequence as a query revealed that it had 99% or more homology with 16S rDNA possessed by several strains of the genus Sinorhizobium as of March 2015.
  • FIG. 2 is a micrograph of the SZ2 strain (x 1,000: (A) observation under an optical microscope, (B) observation under a fluorescence microscope). In FIG. 2, the bar is 10 ⁇ m.
  • SZ2 strain was cultured with shaking (100 rpm) in a 100 mL Erlenmeyer flask containing 50 mL of BG11 liquid medium for 3 weeks. Under the above conditions, the preferential species was SZ2 algal cells, and the coexisting bacteria such as ST1 cells (arrows) were only slightly observed.
  • FIG. 3 is a photograph showing the sugar production of SZ2 strain.
  • PAS staining is a method for detecting neutral polysaccharides (glycogen, chitin, heparin, mucus protein, glycoprotein, glycolipid, etc.). Periodic acid selectively oxidizes glucose residues to produce aldehydes, which are reddish purple by the Schiff reagent.
  • FIG. 3 (A) shows a state where the cells shown in FIG. 3 (A) were further cultured for 2 months, and then the cells producing the polysaccharide were transferred together with the lump to a petri dish. It is a polysaccharide in which the white film-like part around the SZ2 cell mass is accumulated. (In FIG. 3B, the bar is 1 cm).
  • FIG. 4 is a photograph showing hydrocarbon production by the ST1 strain in the presence of SZ2 strain and ST1 strain.
  • the SZ2 algae cells ST1 strain coexist in BG11 medium ( Figure 2), culture 50mL harvested by centrifugation, new nitrogen-deficient cell mass BG11 0 liquid medium 50mL (100 mL Erlenmeyer flask ).
  • BG11 0 to liquid medium reinforced carbon source had been added sodium acetate (pH 7.0) so as to advance final concentration 10 mM.
  • the above medium was allowed to stand for 10 days in a normal atmosphere under irradiation of a white fluorescent lamp (30 ⁇ mol photons / m 2 / s 1 ), and then 1 mL of a liquid medium was recovered.
  • the need for a carbon source for the production of hydrocarbons in ST1 cells means that when only isolated and purified ST1 cells are cultured, no sodium acetate as a carbon source is added to the medium regardless of whether or not nitrogen deficiency is present. Is consistent with the fact that is not recognized (lower left and right in Fig. 6). In FIG. 6, the bar is 10 ⁇ m. Moreover, in FIG. 6, the presence or absence of hydrocarbon (oil) production by ST1 stock: Yes, +; No,-. 3-6.
  • MCMS culture 1 L of SZ2 algae strain culture medium cultured in BG11 liquid medium (inorganic medium) for one month was inoculated into LB liquid medium (organic medium) with ST1 strain isolated and purified overnight at 30 ° C. culture (110 rpm) was ST1 cells (OD 660 ⁇ 2) 0.2 L were mixed, (prepared distilled water that is not in sterilized base) the 1.2L mixture species was new BG11 0 liquid medium 3.8L (Sodium acetate was added to a concentration of 10 mM with respect to the MCMS medium having a total medium volume of 5 L).
  • the culture apparatus was a box-shaped plastic container (length 20 cm ⁇ width 12 cm ⁇ height 16 cm) having a thickness of 2 mm.
  • the white circle graph shows the amount of heptadecane accumulated in the collected cells, and the black diamond graph shows the amount of heptadecane contained in the cell supernatant (culture solution) at that time. As a result, the accumulation rate was 37% on the 12th day, 133% on the 17th day, and 79% on the 22nd day.
  • the cells were collected by centrifugation, samples were prepared by the method described in Sections 3-6 and 3-7, and the amount of heptadecane produced was measured by GC-MS analysis. When the isolated and purified ST1 strain was not added to the medium, the amount of heptadecane produced was set to 100.
  • FIG. 9 (A) the vertical axis represents the relative value of heptadecane produced relative to eicosane (20 ppm) added as an internal standard substance, and the horizontal axis represents the SZ2 strain (50 mL).
  • the amount of ST1 strain added to the corresponding amount of cells was collected by centrifugation and added to the SZ2 culture solution.
  • the relative amount of heptadecane synthesized with respect to the internal standard substance added at a constant concentration (20 ppm) was examined.
  • the ratio of SZ2 amount: ST1 amount 1: 1 (50 mL SZ2 When mixed with 5 mL of ST1 culture solution), the maximum production amount (harvest rate) was shown.
  • biofuel was produced in the MCMS culture system, it was revealed that a certain ratio in the amount of ST1 cells added to the SZ2 algal cell culture solution is effective. 3-9.
  • the dried cells containing only a small amount of water were similarly observed with a microscope (lower part of FIG. 10).
  • a fluorescent dye Nile Red to a final concentration of 10 ⁇ M to the medium (or after mixing by adding a small amount of BG11 0 media naturally dried cells).
  • the cells were collected by centrifugation and observed with a fluorescence microscope (OLYMPUS BX53 / DP72) (in FIG. 10, the bar is 10 ⁇ m).
  • the left column is the result of observation with an optical filter (BF)
  • the right column is the result of observation with a blue fluorescent filter (BW).
  • FIG. 10 indicate the cells that mainly accumulate oil under the conditions.
  • ST1 was seen in the upper part of FIG. 10, and SZ2 cells were preferentially shining yellow in the lower part, indicating accumulation of oil.
  • ST1 strains preferentially produce hydrocarbons under normal MCMS culture conditions, but it became clear that drought stress enables oil production by SZ2 algae itself. Therefore, oil components accumulated in SZ2 algae due to drought stress were analyzed by FID (Frame Ionization Detector, hydrogen flame ionization detector). The result is shown in FIG. FIG. 11 (A) shows the result of FID analysis of the SZ2 algal intracellular oil composition shown in the lower part of FIG.
  • FIG. 11B shows the accumulated amount of fatty acid and heptadecane (biofuel) detected from the results of panel A as relative values (%).
  • biofuels such as C16 and C18 fatty acid methyl ester compounds and heptadecane (C 17 H 36 ) were detected in SZ2 algae under drought stress.
  • the present application enables efficient fuel production by ST1 in the MCMS culture system. Furthermore, after MCMS culture, biofuel can be produced by SZ2 algae itself by drought stress.
  • biofuels such as hydrocarbons (alkanes) can be produced in high yield.

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Abstract

Le but de la présente invention est de fournir une nouvelle technologie de production de biocarburant, et, spécifiquement, la présente invention concerne un procédé de fabrication de biocarburant qui comprend la coculture de micro-organismes mixtes comprenant des micro-organismes photosynthétiques et des micro-organismes non photosynthétiques en utilisant un milieu sans source d'azote.
PCT/JP2016/057691 2015-03-05 2016-03-04 Technologie de production de biocarburant utilisant un liquide mixte, culture d'espèces mixtes WO2016140374A1 (fr)

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