WO2016140374A1 - Biofuel production technology using mixed-liquid, mixed-species culturing - Google Patents

Biofuel production technology using mixed-liquid, mixed-species culturing 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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strain
medium
culture
cells
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

Definitions

  • 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.

Abstract

The purpose of the present invention is to provide a novel technology for producing biofuel, and, specifically, the present invention relates to a biofuel manufacturing method that includes coculturing of mixed microorganisms including photosynthetic microorganisms and non-photosynthetic microorganisms by using a nitrogen-source deprived medium.

Description

混合液混合種培養によるバイオ燃料生産技術Biofuel production technology by mixed liquid mixed seed culture
　本発明は、例えば光合成微生物と非光合成微生物との共培養により、バイオ燃料を生産する方法に関する。 The present invention relates to a method for producing biofuel by, for example, co-culture of a photosynthetic microorganism and a non-photosynthetic microorganism.
　エネルギーを化石燃料（天然ガス・石油・石炭等）に依存する日本の現状を鑑みると、将来、自国の技術により燃料を自力で生産できることが重要である。そこで、水と二酸化炭素を光合成により固定し、ブドウ糖等の有機物を生産することのできる光合成生物を利用してバイオ燃料を生産する試みが日本内外で大変注目されている。
　シアノバクテリア（原核光合成微生物）を含む光合成微生物は、広義の意味で微細藻類に属するが、光合成能は植物のそれと比較して数十倍から百倍とも言われている。加えて、バイオ燃料生産に微細藻類を使用する場合、植物と比べて土や根茎等といった栽培後に不要となる廃棄物も出ない事から、次世代バイオ燃料生産に使用される生物として有望視されている。
　ところで、バイオ燃料は、バイオディーゼルの原料となる中性脂肪（トリアシルグリセロール，ＴＡＧ）や脂肪酸、炭化水素等に大別される。前者２つは化学構造に酸素（Ｏ）分子を有しているが、炭化水素は炭素（Ｃ）と水素（Ｈ）のみで構成されており、エンジン等に優しい燃料として石油産業界で好まれている。炭化水素燃料は炭素数とそれらを繋ぐ二重結合の数と位置により、燃料としての性質が規定される。このうち二重結合を含まない鎖式飽和炭化水素は、アルカン（ＣｎＨ２ｎ＋２）と呼ばれ、炭素数が１~２０までの短鎖アルカンは、私達の身の回りで様々な燃料として役立っている。とりわけ炭素数が１１~１７又は１２~１７のアルカンは、標準（常温常圧）状態で液体であり、ジェット燃料や軽油相当の燃料として位置づけられる。
　これまで微細藻類のうち、シアノバクテリア天然藻の一部の種で、炭素数１５（ペンタデカン，Ｃ１５Ｈ３２）や１７（ヘプタデカン，Ｃ１７Ｈ３６）のアルカンを生産する株が知られている（非特許文献１）。本発明者グループは、これまでシアノバクテリア天然藻を遺伝子操作により改変し、細胞乾燥重量当たり約５０~６０％に及ぶ変異株を取得することに成功している（特許文献１）。これに限らず微細藻類の天然藻や遺伝子組換藻を用いたバイオ燃料生産が試みられている。
　それら従来の技術では、単離純化された藻細胞を滅菌した培地中で無菌的あるいは無菌状態に近づけながら培養することによって、藻細胞内外へバイオ燃料を生産してきた。また、バイオエタノール生産等に見られるように、植物から生産されるブドウ糖を回収した後、酵母の発酵により目的物を得る２段階生産技術と比較して、光合成微生物を利用することのメリットは、前述したようにブドウ糖から目的有機物質（バイオ燃料等）を１段階（一つのバイオリアクター内で同時に培養しながら）生産できる点にある。
　一方、共培養による有用物質生産の従来法の例としては、アクチノタレア・ファーメンタス細菌と酵母を共培養することによりハロゲン化メチルを生産する系（特許文献２）、緑藻ボトリオコッカス培養液にアスティカカリウス・エキセントリカス菌株を添加し共培養することにより緑藻の増殖を促進させる系（特許文献３）、加水分解されたバイオマス原料からエタノール燃料を生産する系においてクロストリジウム・フィトフェルメンタンス細胞あるいは更に他の微細物を含む共培養による方法（特許文献４）等が挙げられる。 In view of the current situation in Japan that relies on fossil fuels (natural gas, oil, coal, etc.) for energy, it is important that in the future it will be possible to produce fuel on its own with its own technology. Attempts to produce biofuels using photosynthetic organisms that can fix water and carbon dioxide by photosynthesis and produce organic substances such as glucose have attracted a great deal of attention both inside and outside Japan.
Photosynthetic microorganisms including cyanobacteria (prokaryotic photosynthetic microorganisms) belong to microalgae in a broad sense, but their photosynthetic ability is said to be several tens to one hundred times that of plants. In addition, when microalgae are used for biofuel production, waste that is unnecessary after cultivation, such as soil and rhizomes, does not come out compared to plants. ing.
By the way, biofuels are roughly classified into neutral fats (triacylglycerol, TAG), fatty acids, hydrocarbons, and the like, which are raw materials for biodiesel. 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. ing. Hydrocarbon fuels are defined as fuel properties by the number of carbon atoms and the number and position of double bonds connecting them. Of these, chain saturated hydrocarbons that do not contain double bonds are called alkanes (C n H 2n + 2 ), and short-chain alkanes with 1 to 20 carbon atoms are useful as various fuels around us. Yes. In particular, 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.
So far, 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). 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). In addition to this, biofuel production using natural algae of microalgae or genetically modified algae has been attempted.
In these conventional techniques, 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. In addition, as seen in bioethanol production, etc., the merit of using photosynthetic microorganisms compared to the two-stage production technology in which the target product is obtained by fermentation of yeast after recovering glucose produced from plants, As described above, the target organic substance (biofuel, etc.) can be produced from glucose in one stage (while simultaneously culturing in one bioreactor).
On the other hand, 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. 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).
特開２０１３−１９８４７３号公報JP 2013-198473 A 特表２０１１−５０５１４８号公報Special table 2011-505148 gazette 特開２０１０−２５２７００号公報JP 2010-252700 A 特表２００９−５２４４３２号公報Special table 2009-524432
　上述の実情に鑑み、本発明は、バイオ燃料を生産する新規の技術を提供することを目的とする。
　上記課題を解決するため鋭意研究を行った結果、光合成微生物と非光合成微生物とを同時に存在させ、混合種同士を同一空間で窒素源欠乏培地において培養（共培養）することにより、バイオ燃料を生産できることを見出し、本発明を完成するに至った。
　すなわち、本発明は、以下を包含する。
（１）光合成微生物と非光合成微生物とを含む混合微生物を、窒素源欠乏培地において共培養することを含む、バイオ燃料の製造方法。
（２）光合成微生物がハロミクロネマ（Ｈａｌｏｍｉｃｒｏｎｅｍａ）属に属する微生物である、（１）記載の方法。
（３）ハロミクロネマ属に属する微生物が、受託番号ＮＩＴＥ　ＢＰ−０１９８２で特定されるハロミクロネマ・エスピー（Ｈａｌｏｍｉｃｒｏｎｅｍａ　ｓｐ．）ＳＺ２菌株又は糖生産能を有するその変異株である、（２）記載の方法。
（４）非光合成微生物がシノリゾビウム（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ）属に属する微生物である、（１）~（３）のいずれか１記載の方法。
（５）シノリゾビウム属に属する微生物が、受託番号ＮＩＴＥ　ＢＰ−０１９８１で特定されるシノリゾビウム・エスピー（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ　ｓｐ．）ＳＴ１菌株又は短鎖アルカン生産能を有するその変異株である、（４）記載の方法。
（６）培地が無機物培地と有機物培地とをさらに含有する、（１）~（５）のいずれか１記載の方法。
（７）培地が炭素源をさらに含有する、（１）~（６）のいずれか１記載の方法。
（８）炭素源が酢酸ナトリウムである、（７）記載の方法。
（９）培地が糖源をさらに含有する、（１）~（８）のいずれか１記載の方法。
（１０）糖源がグルコースである、（９）記載の方法。
（１１）共培養において所定の暗黒期間を設ける、（１）~（１０）のいずれか１記載の方法。
（１２）共培養後、共培養物を乾燥ストレス下で更に培養することを含む、（１）~（１１）のいずれか１記載の方法。
（１３）バイオ燃料が短鎖アルカンである、（１）~（１２）のいずれか１記載の方法。
（１４）短鎖アルカンがヘプタデカンである、（１３）記載の方法。
（１５）受託番号ＮＩＴＥ　ＢＰ−０１９８２で特定されるハロミクロネマ・エスピーＳＺ２菌株又は糖生産能を有するその変異株。
（１６）受託番号ＮＩＴＥ　ＢＰ−０１９８１で特定されるシノリゾビウム・エスピーＳＴ１菌株又は短鎖アルカン生産能を有するその変異株。
（１７）（１５）記載のハロミクロネマ・エスピーＳＺ２菌株又は糖生産能を有するその変異株と（１６）記載のシノリゾビウム・エスピーＳＴ１菌株又は短鎖アルカン生産能を有するその変異株とを含む混合微生物。
　本明細書は本願の優先権の基礎となる日本国特許出願番号２０１５−０４３８１７号の開示内容を包含する。 In view of the above circumstances, an object of the present invention is to provide a novel technique for producing biofuel.
As a result of diligent research to solve the above problems, 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.
(1) 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.
(2) The method according to (1), wherein the photosynthetic microorganism is a microorganism belonging to the genus Halomicronema.
(3) The method according to (2), wherein the microorganism belonging to the genus Halomicronema is a Halomicronema sp. SZ2 strain specified by the accession number NITE BP-01982, or a mutant strain thereof having sugar-producing ability.
(4) The method according to any one of (1) to (3), wherein the non-photosynthetic microorganism is a microorganism belonging to the genus Sinorhizobium.
(5) The method according to (4), wherein the microorganism belonging to the genus Sinorizobium is a Sinorhizobium sp. ST1 strain specified by the accession number NITE BP-01981 or a mutant strain thereof capable of producing a short-chain alkane. .
(6) The method according to any one of (1) to (5), wherein the medium further contains an inorganic medium and an organic medium.
(7) The method according to any one of (1) to (6), wherein the medium further contains a carbon source.
(8) The method according to (7), wherein the carbon source is sodium acetate.
(9) The method according to any one of (1) to (8), wherein the medium further contains a sugar source.
(10) The method according to (9), wherein the sugar source is glucose.
(11) The method according to any one of (1) to (10), wherein a predetermined dark period is provided in co-culture.
(12) The method according to any one of (1) to (11), further comprising culturing the coculture under a drought stress after the coculture.
(13) The method according to any one of (1) to (12), wherein the biofuel is a short-chain alkane.
(14) The method according to (13), wherein the short-chain alkane is heptadecane.
(15) A halomicronema sp. SZ2 strain specified by the accession number NITE BP-01982, or a mutant strain thereof having sugar-producing ability.
(16) A Synorhizobium sp. ST1 strain identified by the accession number NITE BP-01981 or a mutant strain thereof capable of producing a short-chain alkane.
(17) A mixed microorganism comprising the halomicronema sp. SZ2 strain described in (15) or a mutant strain thereof having sugar-producing ability, and the Sinolizobium sp. ST1 strain or the mutant strain capable of producing short-chain alkanes described in (16).
This specification includes the disclosure of Japanese Patent Application No. 2015-043817 which is the basis of the priority of the present application.
ＭＣＭＳ培養の概要を示す模式図である。It is a schematic diagram which shows the outline | summary of MCMS culture | cultivation. ハロミクロネマ・エスピーＳＺ２菌株の顕微鏡写真である。It is a microscope picture of Halomonema sp. SZ2 strain. ハロミクロネマ・エスピーＳＺ２菌株の糖生産を示す写真である。It is a photograph showing the sugar production of Halomicronema sp. SZ2 strain. ハロミクロネマ・エスピーＳＺ２菌株とシノリゾビウム・エスピーＳＴ１菌株共存下でのシノリゾビウム・エスピーＳＴ１菌株による炭化水素生産を示す写真である。It is a photograph which shows the hydrocarbon production by Sinorizobium sp. ST1 strain in the presence of Halomicronema sp. SZ2 strain and Sinorizobium sp. ST1 strain. 炭化水素生産におけるハロミクロネマ・エスピーＳＺ２菌株とシノリゾビウム・エスピーＳＴ１菌株の相性を示す図である。It is a figure which shows the compatibility of the Halomonema sp. SZ2 strain and the Sinorizobium sp. ST1 strain in hydrocarbon production. 炭化水素生産におけるシノリゾビウム・エスピーＳＴ１菌株の培養のための培地組成条件の検討結果を示す図である。It is a figure which shows the examination result of the culture medium composition conditions for culture | cultivation of Sinorizobium sp. ST1 strain in hydrocarbon production. ＭＣＭＳ培養による５Ｌ規模のバイオ燃料生産におけるＧＣ−ＭＳ分析結果を示す図である。It is a figure which shows the GC-MS analysis result in the biofuel production of 5L scale by MCMS culture | cultivation. ＭＣＭＳ培養による燃料蓄積の経時変化を示すグラフである。It is a graph which shows a time-dependent change of the fuel accumulation | storage by MCMS culture | cultivation. ＭＣＭＳ培養による油（ヘプタデカン）生産量を示すグラフである。It is a graph which shows the oil (heptadecane) production amount by MCMS culture | cultivation. 乾燥ストレスによるＳＺ２藻の油生産を示す図である。It is a figure which shows the oil production of SZ2 algae by drought stress. 乾燥ストレス藻ＳＺ２細胞内の脂肪酸と炭化水素Ｃ１７Ｈ３６の蓄積を示す図である。Drought algae SZ2 fatty acids within cells and is a diagram showing the accumulation of hydrocarbon C 17 H 36.
　本発明に係るバイオ燃料の製造方法（以下、「本方法」と称する）は、光合成微生物と非光合成微生物とを含む混合微生物を、窒素源欠乏培地において共培養する（同一空間で培養する）ことで、光合成微生物が糖を生産する一方、当該生産された糖を炭素源として利用し、非光合成微生物が油（炭化水素）等のバイオ燃料を生産する方法である。生産されるバイオ燃料としては、例えばヘプタデカン等の炭素数１~２０個の短鎖アルカンが挙げられる。
　図１に、混合液混合種培養（ＭＣＭＳ：Ｍｉｘｅｄ−Ｃｕｌｔｕｒｅ　Ｍｉｘｅｄ−Ｓｐｅｃｉｅｓ）の概要を示す。従来の細胞の培養方法は、目的とする細胞を純化し一種類の細胞を単純組成の培地で培養する（ＵＣＵＳ：Ｕｎｉ−Ｃｕｌｔｕｒｅ　Ｕｎｉ−Ｓｐｅｃｉｅｓ）ことを特徴としている。この場合、細胞株の純化が困難であったり、目的とする出口産物やそれを生産する培養条件が限定される場合がある。一方、本方法は、混合液混合種培養（ＭＣＭＳ：Ｍｉｘｅｄ−Ｃｕｌｔｕｒｅ　Ｍｉｘｅｄ−Ｓｐｅｃｉｅｓ）を特徴とする。混合液とは、無機物と有機物を成分とする培地であり、混合種とは、光合成微生物を含む２種類以上の細胞が共存することを意味する。特に、混合種培養は「共培養」と位置づけられる。共培養では、単一種純粋培養よりも培養条件や目的生産物の種類に多様性が与えられ、さらには昼夜を問わず油等のバイオ燃料の生産が可能であるので、バイオ燃料の生産量を向上させ、また経費を削減することができる。
　本方法において、光合成微生物としては、水と二酸化炭素とを光合成により固定し、ブドウ糖等の有機物を生産する微生物であれば特に限定されないが、例えばシアノバクテリア（原核光合成微生物）が挙げられる。シアノバクテリアとしては、例えばハロミクロネマ属、マイクロキスティス属、リムノスリックス属、シュードアナベナ属等の属に属する微生物が挙げられる。また、ハロミクロネマ属に属する微生物としては、例えば、ハロミクロネマ・エスピーＳＺ２菌株（以下、「ＳＺ２菌株」等と称する場合がある）又は糖生産能を有するその変異株（例えば自然突然変異株、突然変異誘発処理株）等が挙げられる。
　ＳＺ２菌株は、独立行政法人製品評価技術基盤機構特許微生物寄託センター（ＮＰＭＤ）（〒２９２−０８１８　日本国千葉県木更津市かずさ鎌足２−５−８　１２２号室）に平成２６年（２０１４年）１２月１２日付で寄託されており、その受託番号は、ＮＩＴＥ　Ｐ−０１９８２である。さらに、ＳＺ２菌株は、独立行政法人製品評価技術基盤機構　特許微生物寄託センター（ＮＰＭＤ）（〒２９２−０８１８　日本国千葉県木更津市かずさ鎌足２−５−８　１２２号室）に平成２８年（２０１６年）２月１２日付けで受託番号ＮＩＴＥ　ＢＰ−０１９８２として国際寄託へ移管されている。ＳＺ２菌株は、下記の実施例に示す菌学的性質及び１６ＳｒＲＮＡ遺伝子の相同性分析等により、菌体表面に糖を生産する糸状性シアノバクテリアの一種ハロミクロネマ属に属する新菌株であると同定した。
　一方、非光合成微生物としては、光合成微生物以外の糖等の炭素源を利用してバイオ燃料を生産する微生物であれば特に限定されないが、例えばシノリゾビウム属、シノリゾビウム属と近属のリゾビウム属、アゾリゾビウム属、メソリゾビウム属、アグロバクテリム属等に属する微生物が挙げられる。シノリゾビウム属に属する微生物としては、例えば、シノリゾビウム・エスピーＳＴ１菌株（以下、「ＳＴ１菌株」等と称する場合がある）又はヘプタデカン等の短鎖アルカン生産能を有するその変異株（例えば自然突然変異株、突然変異誘発処理株）等が挙げられる。
　ＳＴ１菌株は、独立行政法人製品評価技術基盤機構　特許微生物寄託センター（ＮＰＭＤ）（〒２９２−０８１８　日本国千葉県木更津市かずさ鎌足２−５−８　１２２号室）に平成２６年（２０１４年）１２月１２日付で寄託されており、その受託番号は、ＮＩＴＥ　Ｐ−０１９８１である。さらに、ＳＴ１菌株は、独立行政法人製品評価技術基盤機構　特許微生物寄託センター（ＮＰＭＤ）（〒２９２−０８１８　日本国千葉県木更津市かずさ鎌足２−５−８　１２２号室）に平成２８年（２０１６年）２月１２日付けで受託番号ＮＩＴＥ　ＢＰ−０１９８１として国際寄託へ移管されている。ＳＴ１菌株は、下記の実施例に示す菌学的性質及び１６ＳｒＲＮＡ遺伝子の相同性分析等により、根粒菌シノリゾビウムに属する新菌株であると同定した。
　本方法においては、好ましくはＳＺ２菌株又は糖生産能を有するその変異株とＳＴ１菌株又は短鎖アルカン生産能を有するその変異株とを含む混合微生物を使用する。
　先ず、本方法においては、光合成微生物と非光合成微生物とを準備する。
　光合成微生物は、無機物培地で培養（前培養）し、調製することができる。ここで、無機物培地とは、例えばＢＧ１１培地のように栄養源として糖類を含まない培地を意味する。ＳＺ２菌株の場合には、例えば無機物培地としてＢＧ１１液体培地［培地組成：０．００３ｍＭ　Ｎａ２−Ｍｇ　ＥＤＴＡ、０．０２９ｍＭ　クエン酸、０．１８ｍＭ　Ｋ２ＨＰＯ４、０．３０ｍＭ　ＭｇＳＯ４・７Ｈ２Ｏ、０．２５ｍＭ　ＣａＣｌ２・２Ｈ２Ｏ、０．１９ｍＭ　Ｎａ２ＣＯ３（無水）、０．０３ｍＭ　クエン酸鉄アンモニウム、１ｍｌ／Ｌ　微量栄養素（微量栄養素の組成：２．８６ｇ／Ｌ　ホウ酸、１．８１ｇ／Ｌ　ＭｎＣｌ２・４Ｈ２Ｏ、０．２２ｇ／Ｌ　ＺｎＳＯ４・７Ｈ２Ｏ、０．３９ｇ／Ｌ　Ｎａ２ＭｏＯ４・２Ｈ２Ｏ、０．０８ｇ／Ｌ　ＣｕＳＯ４・５Ｈ２Ｏ、０．０４９ｇ／Ｌ　Ｃｏ（ＮＯ３）２・６Ｈ２Ｏ）、１．５ｇ／Ｌ　ＮａＮＯ３］を使用し、例えば２０~４０℃（好ましくは２７~３３℃）の温度下で７~９０日間（好ましくは１０~６０日間）振盪（１~２００ｒｐｍ、好ましくは４０~１１０ｒｐｍ）又は静置培養することで、ＳＺ２菌株培養物を準備することができる。
　一方、非光合成微生物は、有機物培地で培養（前培養）し、調製することができる。ここで、有機物培地とは、栄養源としてグルコースなどの糖類（あるいは一酸化炭素や二酸化炭素などを除いた炭素を含む化合物の中で炭素と酸素から成るもの）を含有する培地を意味する。ＳＴ１菌株の場合には、例えば有機物培地としてＬＢ液体（寒天）培地（培地組成：ＮａＣｌ　１０ｇ／Ｌ、Ｂａｃｔｏ　Ｔｒｙｐｔｏｎｅ　Ｐｅｐｔｏｎｅ　１０ｇ／Ｌ、粉末酵母エキス５ｇ／Ｌ、寒天培地にする場合は以上にＢａｃｔｏ　Ａｇａｒを１５ｇ／Ｌの割合で添加し、これをオートクレーブ後、シャーレに注ぎ固化させる）を使用し、２８~３０℃の温度下で１~数日間（寒天培地の場合は数日~６０日間）振盪（０~２００ｒｐｍ、好ましくは９０~１２０ｒｐｍ）培養することで、ＳＴ１菌株培養物を準備することができる。
　次いで、本方法では、光合成微生物と非光合成微生物とを組み合わせて混合微生物とし、当該混合微生物を例えば窒素源欠乏培地において共培養する。なお、ＳＺ２菌株には、自然界において共存菌としてＳＴ１菌株が共存しており、当該共存下の混合微生物を使用してもよい。
　また、光合成微生物と非光合成微生物は、それぞれ単離したものであってよく、あるいは上述の前培養物自体を各微生物として使用することができる。前培養物を使用する場合には、共培養における窒素源欠乏培地に前培養に使用した無機物培地及び／又は有機物培地とが含有されることとなる。窒素源欠乏培地に対する前培養に使用した無機物培地及び／又は有機物培地の割合としては、例えば０~１００容量％（ｖ／ｖ）、好ましくは０．４~２０容量％（ｖ／ｖ）が挙げられる。
　混合微生物における光合成微生物と非光合成微生物との混合比としては、例えばＳＺ２菌株とＳＴ１菌株との場合において、２ヶ月間前培養した培養液５０ｍＬから集菌したＳＺ２菌株と前培養した培養液（ＯＤ６６０＝２）５ｍＬから集菌したＳＴ１菌株とを１：１とすると、ＳＺ２菌株：ＳＴ１菌株＝１：０．０４~１：２、好ましくは１：１程度が挙げられる。
　共培養に使用される窒素源欠乏培地としては、窒素源を含まないか、又は通常の培地（例えば、ＢＧ１１液体培地）中の窒素源に対して窒素源が乏しい（例えば、０~５０％、好ましくは０~１０％の窒素源を含む）培地である。窒素源としては、例えばアンモニア、硫酸アンモニウム、炭酸アンモニウム、塩化アンモニウム、クエン酸鉄アンモニウム、硝酸ナトリウム、硝酸カリウム、硝酸カルシウム等が挙げられる。例えばＳＺ２菌株とＳＴ１菌株との混合微生物の共培養においては、窒素源欠乏培地としてＢＧ１１０液体培地（培地組成：前記のＢＧ１１培地組成よりＮａＮＯ３を全部抜いたもの）或いはＮａＮＯ３を部分的（１割~９割程度）に抜いたＢＧ１１培地を使用することができる。なお、窒素源欠乏培地は、非滅菌の蒸留水等を使用して調製したものであってもよい。非滅菌の蒸留水を使用することで、培養コストを削減することができる。
　さらに、窒素源欠乏培地には、炭素源及び／又は糖源を添加することが好ましい。炭素源を添加することで、更に炭素源を補強することができる。添加する炭素源としては、例えば酢酸ナトリウム、酢酸カリウム、炭酸ナトリウム等が挙げられる。窒素源欠乏培地への炭素源の添加量は、例えば窒素源欠乏培地における最終濃度１~数１００ｍＭ、好ましくは１０ｍＭ程度となる量である。一方、糖源を添加することで、光合成微生物が生産する糖に加えて、添加した糖源を利用し、非光合成微生物がバイオ燃料を生産することができ、バイオ燃料生産量を向上させることができる。糖源としては、例えば単糖類であるグルコース、キシロース、アラビノース、キシロース、リボース、デオキシリボース、フルクトース、ガラクトース、マンノース等、二糖類であるラクトース、マルトース、トレハロース等、三糖類であるマルトトリース、ラフィノース等、オリゴ糖である、フラクトオリゴ（ＦＯＳ）糖、ガラクトオリゴ糖（ＧＯＳ）、マンナンオリゴ（ＭＯＳ）糖等、多糖類であるグリコーゲン、デンプン、セルロース、デキストリン、グルカン、フルクタン、キチン質等、その他産業廃棄物（廃糖蜜、発酵液、糞尿液、下水等）に含まれる廃棄糖等が挙げられる。窒素源欠乏培地への糖源の添加量は、例えば窒素源欠乏培地における最終濃度０．０１~数百ｍＭ、好ましくは０．１~２０ｍＭ（本方法では０．５ｍＭ程度）となる量である。
　共培養は、例えば２０~３０℃（好ましくは２５~３０℃）の温度条件、０~５００μｍｏｌｐｈｏｔｏｎｓ／ｍ２／ｓ１（好ましくは１０~１００μｍｏｌ　ｐｈｏｔｏｎｓ／ｍ２／ｓ１）の光強度条件（例えば、白色光照射下）、０．０４~１２％（好ましくは０．５~３％）の二酸化炭素ガス供給（０．０１~５０Ｌ／ｍｉｎ、好ましくは０．１~２０Ｌ／ｍｉｎ）下等の条件下で数日~３０日間（好ましくは５日間程度~２０日間程度）静置又は振盪（１０~２００ｒｐｍ、好ましくは３０~５０ｒｐｍ）培養により行われる。
　また、共培養において、所定の暗黒期間を設けることで、培養コストを削減することができる。例えば、照明１２時間間隔（すなわち、明／暗（１２時間／１２時間）サイクル）条件下で共培養を行うことができる。
　さらに、共培養後、共培養物を乾燥ストレス（例えば、培地液体の自然乾燥）下で更に培養することで、光合成微生物自身でもバイオ燃料の生産が可能となる。当該培養は、上述の共培養条件に準じた条件下で数日~数十日間（好ましくは２週間程度~２ヶ月間程度）静置培養により行われる。
　以上に説明した本方法によれば、炭化水素（アルカン）等のバイオ燃料を高収量で生産することができる。例えば、共培養物を酢酸エチル等の溶媒抽出に供することで、バイオ燃料を回収することができる。また、ＭＣＭＳ系で連続的にヘプタデカン等を液体燃料として回収し、その後、培養の最後は共培養物を乾燥させると、乾燥菌体を固形燃料として使用でき、経済的にコスト安な燃料製造法となる。
　以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。 The biofuel production method according to the present invention (hereinafter referred to as “the present method”) 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. In this method, the photosynthetic microorganism produces sugar, while the produced sugar is used as a carbon source, and the non-photosynthetic microorganism produces biofuel such as oil (hydrocarbon). Examples of 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. On the other hand, 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. In particular, mixed seed culture is positioned as “co-culture”. In co-culture, the variety of culture conditions and target product types are more diverse than single-type pure culture, and furthermore, biofuels such as oil can be produced day and night. Can improve and reduce costs.
In the present method, 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). Examples of cyanobacteria include microorganisms belonging to genera such as genus Halomicronema, genus Microkistis, genus Limnolix, genus Pseudoanabena and the like. In addition, examples of 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).
The 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. In addition, 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.
On the other hand, 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. For example, the genus Synorizobium, the Synozobium genus, and the related genus Rhizobium genus, Azorizobium , Microorganisms belonging to the genus Mesozobium, Agrobacterium, and the like. Examples of 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. Furthermore, 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.
In the present method, 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.
First, in this method, a photosynthetic microorganism and a non-photosynthetic microorganism are prepared.
The photosynthetic microorganism can be prepared by culturing (pre-culture) in an inorganic medium. Here, the inorganic medium means a medium that does not contain saccharide as a nutrient source, such as BG11 medium. In the case of the SZ2 strain, for example, 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 g / L NaNO 3 ], for example, a temperature of 20 to 40 ° C. (preferably 27 to 33 ° C.) 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.
On the other hand, non-photosynthetic microorganisms can be prepared by culturing (preculture) in an organic medium. Here, 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. In the case of the 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.
Next, in this method, 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.
In addition, 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. When the preculture is used, 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.
As a mixing ratio of the photosynthetic microorganism and the non-photosynthetic microorganism in the mixed microorganism, for example, in the case of the SZ2 strain and the ST1 strain, the culture solution pre-cultured with the SZ2 strain collected from 50 mL of the culture solution precultured for 2 months (OD 660 = 2) When 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%. Examples of the nitrogen source include ammonia, ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium iron citrate, sodium nitrate, potassium nitrate, and calcium nitrate. For example, in the co-culture of a mixed microorganism and SZ2 strains and ST1 strain, 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%) can be used. 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.
Furthermore, it is preferable to add 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. On the other hand, by adding a sugar source, in addition to the sugar produced by the photosynthetic microorganism, the added sugar source can be used to allow the non-photosynthetic microorganism to produce biofuel and improve biofuel production. it can. Examples of the sugar source 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. (preferably 25 ~ 30 ℃), 0 ~ 500μmolphotons / m 2 / s 1 ( preferably 10 ~ 100μmol photons / m 2 / s 1) of the light intensity conditions (e.g. White light irradiation), 0.04 to 12% (preferably 0.5 to 3%) carbon dioxide gas supply (0.01 to 50 L / min, preferably 0.1 to 20 L / min), etc. Under conditions, it is carried out by standing or shaking (10 to 200 rpm, preferably 30 to 50 rpm) for several days to 30 days (preferably about 5 days to 20 days).
Further, in the co-culture, the culture cost can be reduced by providing a predetermined dark period. For example, 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.
According to the method described above, biofuels such as hydrocarbons (alkanes) can be produced in high yield. For example, biofuel can be recovered by subjecting the co-culture to solvent extraction such as ethyl acetate. In addition, when heptadecane and the like are continuously recovered as a liquid fuel in the MCMS system, and then the co-culture is dried at the end of the culture, the dried cells can be used as a solid fuel, and an economically inexpensive fuel production method It becomes.
EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
ハロミクロネマ・エスピーＳＺ２菌株の分離・単離及び当該菌株の分類学的性質
１−１．ＳＺ２菌株の分離
　日本国静岡県修善寺市の道端石垣より数グラム（一塊）の試料を採取し、これをＢＧ１１液体培地と混ぜて１ヶ月程度静置培養して藻細胞の生育を促した。この藻細胞液からＳＺ２株細胞を混釈重層法（Ｎｉｓｈｉｚａｗａら２０１０年，Ｂｉｏｓｃｉｅｎｃｅ，Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ａｎｄ　Ｂｉｏｃｈｅｍｉｓｔｒｙ　２７巻１８２７−１８３５頁）により分離し、ＢＧ１１寒天培地へ塗抹し、３０℃（白色蛍光灯下）で培養した。寒天培地上に生育したＳＺ２株細胞塊をかき取り、新しいＢＧ１１液体培地に移して継代培養した。以上により得られた分離株（ＳＴ１細胞含む）をＳＺ２と命名した。
１−２．ＳＺ２菌株の生理学的性質
　ハロミクロネマ・エスピーＳＺ２菌株をＢＧ１１液体培地で３０℃（白色蛍光灯下）で静置培養（一日一回程度の撹拌を行う）すると、１細胞当たりの長辺が約３~５μｍの細胞が連なった糸状性細胞形態を示す。ＳＺ２株は、さらに上記培養条件下で約２週間以上培養すると、ＰＡＳ（Ｐｅｒｉｏｄｉｃ　ａｃｉｄ−Ｓｃｈｉｆｆ　ｓｔａｉｎ）染色で観察される中性多糖を菌体外に生産する。この多糖の生産が数ヶ月続くとＳＺ２株細胞塊の回りは、バイオフィルムで覆われ肉眼でも顕著な特徴として観察される。
１−３．１６Ｓ　ｒＲＮＡ遺伝子（１６Ｓ　ｒＤＮＡ）配列によるＳＺ２菌株の分類特定
　ＳＺ２株を優先種とする培養液より菌体を回収し、細胞全ＤＮＡを抽出して１６Ｓ　ｒＤＮＡ塩基配列（配列番号１：８６２塩基対）を解読した。この配列をＱｕｅｒｙとして遺伝子ＤＮＡデータベースと総当たり検索をしたところ、２０１５年３月時点でシアノバクテリアＨａｌｏｍｉｃｒｏｎｅｍａ属株数種の有する１６Ｓ　ｒＤＮＡと９２％以上の相同性を有していた。またコンピューター解析ソフトＭＥＧＡ４（Ｔａｍｕｒａら２００７年，Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ　ａｎｄ　Ｅｖｏｌｕｔｉｏｎ，２４巻１５９６−１５９９頁）により分子系統樹を作成すると、ＳＺ２株が糸状性シアノバクテリアの一種Ｈａｌｏｍｉｃｒｏｎｅｍａ属クラスターに位置付けされる新種藻であることが明らかとなった。 1. Isolation and isolation of Halomicronema sp. SZ2 strain and taxonomic properties of the strain 1-1. Isolation of SZ2 strain A few grams (one block) of a sample was collected from a roadside stone wall in Shuzenji, Shizuoka, Japan, and this was mixed with a BG11 liquid medium and allowed to stand for about 1 month to promote the growth of algal cells. The SZ2 strain cells were separated from this algal cell solution by the piling layer method (Nishizawa et al. 2010, Bioscience, Biotechnology, and Biochemistry 27: 1827-1835), and smeared on BG11 agar medium at 30 ° C. (under white fluorescent light) ). 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 μm cells connected. When the SZ2 strain is further cultured for about 2 weeks or more under the above-described culture conditions, it produces neutral polysaccharides observed by PAS (Periodic acid-Schiff stain) staining outside the cells. When this polysaccharide production continues for several months, the SZ2 cell mass is covered with a biofilm and is observed as a remarkable feature with the naked eye.
1-3. Classification and identification of SZ2 strain by 16S rRNA gene (16S rDNA) sequence Bacterial cells are collected from a culture solution with SZ2 strain as a preferred species, and total cell DNA is extracted to obtain a 16S rDNA base sequence (SEQ ID NO: 1). 862 base pairs). When this sequence was used as a query and a brute force search was performed with the gene DNA database, as of March 2015, it had 92% or more homology with 16S rDNA possessed by several strains of the genus Cyanobacteria Halomicronema. In addition, when a molecular phylogenetic tree is created by computer analysis software MEGA4 (Tamura et al. 2007, Molecular Biology and Evolution, Vol. 24, pages 1596-1599), 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.
シノリゾビウム・エスピーＳＴ１菌株の単離及び当該菌株の分類学的性質
２−１．ＳＴ１菌株の単離
　実施例１において前述した混釈重層法により得られたＳＺ２分離株（ＳＴ１株細胞を含む）のＢＧ１１培養液から約１０マイクロリットルを採取し、これをＬＢ寒天培地にクロスストリークしながら塗り広げた。この寒天培地を３０℃で数日間培養し、生育したＳＴ１のコロニーを単離した。以上により得られた単離（純化）株をＳＴ１と命名した。
２−２．ＳＴ１菌株の生理学的性質
　ＬＢ培地あるいはＬＢ寒天培地で生育させる温度は、通常３０℃前後である（３７℃培養で生育は認められない）。１細胞当たりの長辺は、約０．７~２μｍの単細胞性である。
２−３．１６Ｓ　ｒＲＮＡ遺伝子配列によるＳＴ１菌株の分類特定
　ＳＴ１単離（純化）株より細胞全ＤＮＡを抽出し、１６Ｓ　ｒＤＮＡ塩基配列（配列番号２：１，４７５塩基対）を解読した。この配列をＱｕｅｒｙとして遺伝子ＤＮＡデータベースと総当たり検索をしたところ、２０１５年３月時点でＳｉｎｏｒｈｉｚｏｂｉｕｍ属株数種の有する１６Ｓ　ｒＤＮＡと９９％以上の相同性を有していた。またコンピューター解析ソフトＭＥＧＡ４（前述）により分子系統樹を作成すると、ＳＴ１株がシノリゾビウムやリゾビウム属クラスターに位置付けされる新種株であることが明らかとなった。 1. Isolation of Synorizobium sp. ST1 strain and taxonomic properties of the strain 2-1. Isolation of ST1 strain About 10 microliters was collected from the BG11 culture solution of the SZ2 isolate (including ST1 strain cells) obtained by the pour-layer method described above in Example 1, and this was cross-streaked on an LB agar medium. While spreading. This agar medium was cultured at 30 ° C. for several days, and the grown ST1 colonies were isolated. The isolated (purified) strain obtained as described above was named ST1.
2-2. Physiological properties of ST1 strain The temperature for growth on LB medium or LB agar medium is usually around 30 ° C. (no growth is observed at 37 ° C. culture). The long side per cell is unicellular with about 0.7 to 2 μm.
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. Moreover, when a molecular phylogenetic tree was created with the computer analysis software MEGA4 (described above), it became clear that the ST1 strain is a new strain that is positioned in the Synorhizobium or Rhizobium genus cluster.
ハロミクロネマ・エスピーＳＺ２菌株とシノリゾビウム・エスピーＳＴ１菌株との共培養（ＭＣＭＳ培養）によるバイオ燃料生産
３−１．ＳＺ２菌株
　図２は、ＳＺ２菌株の顕微鏡写真（ｘ　１，０００：（Ａ）光学顕微鏡観察、（Ｂ）蛍光顕微鏡観察）である。図２において、棒は１０μｍである。ＳＺ２菌株を、ＢＧ１１液体培地５０ｍＬを含有する１００ｍＬ容三角フラスコで３週間振盪（１００ｒｐｍ）培養した。以上の条件下では、優先種はＳＺ２藻細胞であり、ＳＴ１細胞（矢印）等の共存菌は僅かに観察される程度であった。１細胞当たりの長辺は、ＳＺ２菌株では約３~５μｍであり、ＳＴ１菌株では約０．７~２μｍであった。蛍光顕微鏡（励起４６０−４９５ｎｍ／放射５１０ｎｍ，オリンパスＢＸ５３／ＤＰ７２）観察では、光合成微生物特有の自家蛍光が認められた。ＳＺ２細胞の凝集体では、糸状性細胞の周りに多糖と思われる物質の蓄積が認められた。
３−２．ＳＺ２菌株の糖生産
　図３は、ＳＺ２菌株の糖生産を示す写真である。
　ＰＡＳ染色（Ｐｅｒｉｏｄｉｃ　ａｃｉｄ−Ｓｃｈｉｆｆ　ｓｔａｉｎ）は、中性多糖類（グリコーゲン、キチン、ヘパリン、粘液タンパク質、糖タンパク質、糖脂質等）の検出法である。過ヨウ素酸はグルコース残基を選択的に酸化してアルデヒドを生成し、シッフ試薬によって赤紫色を呈す。
　図３（Ａ）：ＳＺ２菌株をＢＧ１１液体培地で４週間静置培養した後、培養液１ｍＬを回収して遠心し、藻菌体を集菌した。蒸留水で藻を洗浄後、再び遠心して蒸留水を捨てた。集菌細胞に０．５％（ｗ／ｖ）過ヨウ素酸（ｐｅｒｉｏｄｉｃ　ａｃｉｄ，和光純薬製）水溶液を３００μＬ加え懸濁後、３分放置した。その後、過ヨウ素酸水溶液を遠心して捨てた。これに蒸留水を加え洗浄し、遠心して蒸留水を捨てた。シッフ試薬（Ｓｃｈｉｆｆ’ｓ　ｒｅａｇｅｎｔ，和光純薬製）を加え、藻を懸濁した。１３分放置後、シッフ試薬を遠心して捨てた。これを３回繰り返した後、蒸留水を加え洗浄し遠心して、顕微鏡観察の試料とした。これを光学顕微鏡（オリンパスＢＸ５３／ＤＰ７２）観察した。細胞内、細胞外表面に多糖の蓄積が観察された（図３（Ａ）において、棒は１０μｍである）。
　図３（Ｂ）：図３（Ａ）に示す試料を更に２ヶ月間静置培養した後、多糖を生産した細胞を塊ごとシャーレに移した時の様子を示す。ＳＺ２細胞塊の周りの白いフィルム様部分が蓄積した多糖である。（図３（Ｂ）において、棒は１ｃｍである）。
３−３．ＳＺ２菌株とＳＴ１菌株共存下でのＳＴ１菌株による炭化水素生産
　図４は、ＳＺ２菌株とＳＴ１菌株共存下でのＳＴ１菌株による炭化水素生産を示す写真である。
　ＳＴ１菌株が共存しているＳＺ２藻細胞をＢＧ１１培地で３週間培養後（図２）、培養液５０ｍＬを回収し遠心して、細胞塊を新たな窒素欠乏ＢＧ１１０液体培地５０ｍＬ（１００ｍＬ用三角フラスコ内）へ移植した。ＢＧ１１０液体培地には炭素源の補強として、予め最終濃度１０ｍＭになるように酢酸ナトリウム（ｐＨ７．０）を添加しておいた。以上の培地を通常大気中で白色蛍光灯照射下（３０μｍｏｌ　ｐｈｏｔｏｎｓ／ｍ２／ｓ１）で１０日間静置培養した後、液体培地１ｍＬを回収した。炭化水素の蓄積を確認するため、Ｎｉｌｅ　Ｒｅｄ（和光純薬）を最終濃度１０μＭになるよう培地に添加し細胞を染色し、これを光学顕微鏡（図４（Ａ））並びに蛍光顕微鏡（励起４６０−４９５ｎｍ／放射５１０ｎｍ，図４（Ｂ））で観察した。
　光学顕微鏡観察の結果、窒素欠乏条件下で培養すると糸状性ＳＺ２藻細胞を取り囲むようにしてＳＴ１細胞の増殖が認められた。これによりＳＺ２細胞とＳＴ１細胞の比率は、ＢＧ１１培地での培養では圧倒的にＳＺ２細胞が優先種で占められているのに対し、ＢＧ１１０培地では相対的にＳＴ１細胞が増加していることが明らかとなった。一方、蛍光顕微鏡観察の結果、ＳＴ１細胞内に顕著な炭化水素の蓄積が認められた（図４（Ａ）及び（Ｂ）において、棒は１０μｍである）。
３−４．炭化水素生産におけるＳＺ２菌株とＳＴ１菌株の相性
　ＳＺ２株培養液より単離純化したＳＴ１株細胞の培養液を３種類のシアノバクテリア藻培養液に添加混合し、ＳＴ１株の相手となる藻細胞と「共培養」することにより、ＳＴ１株による炭化水素生産における相手藻細胞の相性を検証した。
　結果を図５に示す。使用したシアノバクテリア３種のうち、ＳＺ２株以外の２種はそれぞれ純化株を用いた。３週間ＢＧ１１培地で前培養した各種藻細胞液５０ｍＬに相当する菌体を遠心回収し、第３−３節で説明したＢＧ１１０液体培地（酢酸ナトリウム＝酢酸Ｎａ添加）へ懸濁した。これに予め一晩ＬＢ液体培地で培養（３０℃、１１０ｒｐｍ振盪培養）したＳＴ１細胞培養液１ｍＬ（ＯＤ６６０＝２）を遠心分離して集菌した量に相当する湿潤菌体を直接とって、上記５０ｍＬ　ＢＧ１１０液体培地へ移植した。これを第３−３節で記述したように大気中（０．０４％ＣＯ２ガス含む）で５日間静置培養した。その後、細胞をＮｉｌｅ　Ｒｅｄ染色して蛍光顕微鏡観察を行った。
　その結果、ＢＧ１１液体培地を使用してＳＴ１株と各種藻を共培養した場合は、いずれの共培養下でも炭化水素生産は殆ど認められなかった。一方、ＢＧ１１０（窒素源欠乏）培地で共培養した場合、ＳＴ１株による炭化水素生産量は、ＳＺ２株＞ＰＣＣ６８０３株＞ＡＢＲＧ５−３株との共培養の順に高かった。この結果から、ＳＴ１株による炭化水素生産時に共存させる相手藻に関しては特異性が認められ、元々自然界から分離された時のＳＴ１株と共存するＳＺ２藻との相性が一番良い事が明らかとなった。
　図５において略語は以下を示す：ＳＺ２，Ｈａｌｏｍｉｃｒｏｎｅｍａ　ｓｐ．ＳＺ２；ＰＣＣ６８０３，Ｓｙｎｅｃｈｏｃｙｓｔｉｓ　ｓｐ．ＰＣＣ６８０３（パスツール研究所（仏国，パリ）保存株；かずさＤＮＡ研究所（千葉県木更津市かずさ鎌足２−６−７）より分譲）；ＡＢＲＧ５−３，Ｌｉｍｎｏｔｈｒｉｘ／Ｐｓｅｕｄａｎａｂａｅｎａ　ｓｐ．ＡＢＲＧ５−３（ＦＥＲＭ　Ｐ−２２１７２；特開２０１３−１９８４７３号公報）。また、図５において、棒は１０μｍである。さらに、図５において、ＳＴ１株による炭化水素（油）生産の有無：　有，＋；無，−。
３−５．炭化水素生産における培地組成条件
　ＳＺ２株とＳＴ１株の菌体組み合わせ、培地中の窒素源の有無並びに酢酸ナトリウム（酢酸Ｎａ，最終濃度１０ｍＭ）添加の有無について、第３−４節に示す培養条件により飼育した菌体をＮｉｌｅ　Ｒｅｄにより染色し、ＳＴ１株の炭化水素生産における培地組成条件について検証した。
　結果を図６に示す。先ず、ＳＴ１細胞が炭化水素を一番効率良く生産する条件としては、ＳＺ２藻細胞との共培養（ＭＳ：Ｍｉｘｅｄ−Ｓｐｅｃｉｅｓ）で窒素欠乏（ＢＧ１１０培地使用）条件下且つ培地に炭素源の補強剤として酢酸ナトリウムを添加した場合であった（図６上段左）。この組み合わせより培地を窒素欠乏にしなければ（ＢＧ１１培地使用）、ＳＴ１細胞の炭化水素の顕著な蓄積は認められなかった（図６上段右）。このことは、ＳＴ１細胞の効率的な炭化水素生産にはＳＺ２藻細胞との共培養が有効であり、且つ窒素源欠乏培地で培養することが必須ということが明らかとなった。
　次に、単離純化されたＳＴ１株のみを用いて同様の実験を行ったところ、窒素源欠乏培地で酢酸ナトリウムを添加した場合は、ＳＴ１株単独培養（ＵＣＵＳ：Ｕｎｉ−Ｃｕｌｔｕｒｅ　Ｕｎｉ−Ｓｐｅｃｉｅｓ）でも炭化水素を生産していた（図６中段左）。しかしながら、その生産能は、ＳＺ２株と共培養した場合に比べて低かった。また酢酸ナトリウムを添加していても、培地が窒素欠乏になっていなければＳＴ１株の炭化水素生産は認められなかった（図６中段右）。
　以上により、ＳＴ１株の炭化水素生産は、炭素源を供給し、且つ窒素源を欠乏した場合に限り可能なことが示唆された。従って、ＳＺ２藻細胞との共培養では、ＳＺ２藻細胞表面に生産された多糖をＳＴ１株が炭素源として利用し、炭化水素生産の効率を高めている（図６上段左）のではないかと推察された。ＳＴ１細胞の炭化水素生産に炭素源が必要な事は、単離純化したＳＴ１細胞のみを培養した場合、窒素欠乏の有無にかかわらず、培地へ炭素源となる酢酸ナトリウムを添加しなければ、生産が認められない事と矛盾しない（図６下段左右）。
　なお、図６において、棒は１０μｍである。また、図６において、ＳＴ１株による炭化水素（油）生産の有無：　有，＋；無，−。
３−６．ＭＣＭＳ培養による５Ｌ規模のバイオ燃料生産
　ＳＺ２株とＳＴ１株との共培養によるバイオ燃料生産の実用化に向けた基盤技術を確立するため更に、（１）５リットル（５Ｌ）規模でのＭＣＭＳ培養技術、（２）ＭＣＭＳ培養により生産される油の成分について検証した。（１）に関しては、特に経費軽減のため、ＢＧ１１０培地作製には滅菌水の代わりに蒸留水を使用し、照明も１２時間（１２ｈ）間隔の暗黒を設けることとした。以下に、培養と燃料成分分析について記述する。
　藻株には本来ＳＴ１株が共存しているが、予め培養したＳＺ２株培養液に単離したＳＴ１株培養液を所定の濃度で添加することにより、炭化水素を蓄積生産することが可能であることは図５で示されている。ＭＣＭＳ培養に関しては、一ヶ月間ＢＧ１１液体培地（無機物培地）で培養したＳＺ２藻株培養液１Ｌに対し、単離純化したＳＴ１株をＬＢ液体培地（有機物培地）に接種し一晩３０℃で振盪培養（１１０ｒｐｍ）したＳＴ１細胞（ＯＤ６６０≒２）０．２Ｌを混合し、この１．２Ｌ混合種液を新しいＢＧ１１０液体培地３．８Ｌ（滅菌処理はしていない蒸留水をベースに作製）へ移した（培地総量５ＬのＭＣＭＳ培地に対し、１０ｍＭとなるよう酢酸ナトリウムを添加）。培養装置は、厚さ２ｍｍの箱型プラスチック容器（縦２０ｃｍｘ横１２ｃｍｘ高さ１６ｃｍ）であった。これに０．５％二酸化炭素ガス（１５Ｌ／ｍｉｎ）をシリコンチューブで供給した。この培地を、白色蛍光灯（３０μｍｏｌ　ｐｈｏｔｏｎｓ／秒／ｍ２）下、明暗（１２ｈ／１２ｈ）サイクル条件で１９日間培養した。
　培養後、培養液５０ｍＬを回収し、遠心により菌体を集めた。以後、酢酸エチルを用いて回収菌体から炭化水素を回収し（特開２０１３−１９８４７３号公報参照）、そのうち一部の試料をとってＧＣ−ＭＳ分析に供した（特開２０１３−１９８４７３号公報参照）。
　結果を図７に示す。上記試料液中に内標準物質（対照区）として最終濃度２０ｐｐｍになるよう調製して添加されたエイコサン（Ｃ２０Ｈ４２）のピーク（ｒｅｔｅｎｔｉｏｎ　ｔｉｍｅ＝１８．１８ｍｉｎ）に対し、アルカン（ヘプタデカン、Ｃ１７Ｈ３６）を示すｒｅｔｅｎｔｉｏｎ　ｔｉｍｅ（１４．６０ｍｉｎ）にメジャーなピークが観察された。このピーク面積と内標準物質のピーク面積との比［ｒｅｌａｔｉｖｅ　ｖａｌｕｅ（％）＝（ｖａｌｕｅ　ｏｆ　Ｃ１７Ｈ３６／ｖａｌｕｅ　ｏｆ　Ｃ２０Ｈ４２）ｘ１００％］は１２２％であり、これより計算されたＳＺ２＋ＳＴ１細胞乾燥重量当たりに占めるヘプタデカンの生産量は約５％であった。これに別の燃料物質と思われるマイナーなピークが幾つか観察され、これらを足し合わせると細胞乾燥重量当たり~約１０％のバイオ燃料の生産量が確認された。乾燥重量当たりのヘプタデカンを含む燃料の生産量を１０％とし、１トン規模のＭＣＭＳ液体培地からの生産量を試算すると０．１ｋｇのバイオ燃料生産量に相当する。ヘプタデカンは軽油相当であるので、１％燃料に混ぜる場合には約５~１０Ｌの燃料としてトラック等の走行に使用可能と考えられる。
　以上により、５Ｌ規模ＭＣＭＳ培養によりバイオ燃料アルカンを生産するに至った。培地の大部分を占めるＢＧ１１０は未滅菌の蒸留水を用いたことからその分、経費軽減が達成された。また、その培養条件下では、ＳＺ２株とＳＴ１株の両者が常に培養液中で優先種であった。更に、培地には０．５％（大気中の１２．５倍）二酸化炭素ガスを培地に供給することにより、温室効果ガスの有効利用も期待された。一方、ＢＧ１１０（無機物培地）へのＬＢ培地（有機物培地）の混合（＝ＭＣ、Ｍｉｘｅｄ　Ｃｕｌｔｕｒｅ）では、１２時間間隔の暗黒を入れながらでもＳＺ２＋ＳＴ１混合種（＝ＭＳ、Ｍｉｘｅｄ　Ｓｐｅｃｉｅｓ）培養でも燃料生産が成立する事から、これも培養経費軽減に繋がるものとして多いに期待された。
３−７．燃料蓄積の経時変化
　第３−６節に示したＭＣＭＳ培養条件下でのバイオ燃料の経時的な蓄積に関して検証し、その結果を図８に示す。
　図８において、グラフの横軸は培養日数を、縦軸は内標準物質であるエイコサン（最終濃度２０ｐｐｍ）に対するヘプタデカンの生産量の相対値［ｒｅｌａｔｉｖｅ　ｖａｌｕｅ（％）＝（ｖａｌｕｅ　ｏｆ　Ｃ１７Ｈ３６／ｖａｌｕｅ　ｏｆ　Ｃ２０Ｈ４２）ｘ１００％］を示す。また、白抜き丸のグラフは集菌細胞内のヘプタデカン蓄積量を示し、黒塗りの菱形のグラフはその時の細胞上澄液（培養液）に含まれるヘプタデカン量である。これにより１２日目では３７％、１７日目では１３３％、２２日目では７９％の蓄積率を示した。以上により、５Ｌ規模でのＭＣＭＳ培養では、第３−６節に示した培養装置と培養条件下では、１７日前後でバイオ燃料の蓄積が良く、その辺りの時期が回収に適していることが明らかとなった。
３−８．ＳＺ２菌株とＳＴ１菌株の混合比と燃料生産量
　第３−４節に示す培地と培養条件を以下のように改変して試験した。２ヶ月間ＢＧ１１培地で前培養したＳＺ２藻細胞液５０ｍＬに相当する菌体を遠心回収し、ＢＧ１１０液体培地（酢酸ナトリウム添加）へ懸濁した。これに予め一晩ＬＢ液体培地で培養（３０℃、１１０ｒｐｍ振盪培養）したＳＴ１細胞培養液５ｍＬ（ＯＤ６６０＝２）を遠心分離して集菌した量に相当する湿潤菌体を直接とって上記５０ｍＬ　ＢＧ１１０液体培地へ移植した。これをＳＺ２量：ＳＴ１量＝１：１とした。例えば、ＳＺ２量：ＳＴ１量＝１：２の場合は、上記ＳＴ１細胞培養液１０ｍＬ（ＯＤ６６０＝２）を遠心分離し集菌した。以上を２％ＣＯ２ガス濃度に制御された培養インキュベーター内（１２ｈ−明／１２ｈ−暗）でレシプロ式振盪（４０ｒｐｍ）しながら５日間培養した。培養後は、菌体を遠心して回収し、第３−６節と第３−７節で記述した方法により試料を調製し、ＧＣ−ＭＳ分析によってヘプタデカンの生産量を測定した。なお、単離純化したＳＴ１株を培地に添加しない場合、生産されるヘプタデカンの量を値１００とした。
　結果を図９（Ａ）に示す。なお、図９（Ａ）において、縦軸は、内標準物質として添加したエイコサン（２０ｐｐｍ）に対して生産されたヘプタデカンの生産量の相対値を示し、また、横軸は、ＳＺ２株（５０ｍＬに相当する菌体量）に添加したＳＴ１株量（培養液ＸｍＬを遠心して集菌し、これをＳＺ２培養液に添加）を示す。
　図９（Ａ）に示すように、濃度一定（２０ｐｐｍ）で添加している内標準物質に対する相対的なヘプタデカンの合成量を調べたところ、ＳＺ２量：ＳＴ１量＝１：１の比率（５０ｍＬ　ＳＺ２培養液に対し５ｍＬのＳＴ１培養液に相当）で混合した場合が、最大の生産量（収穫率）を示した。これによりバイオ燃料をＭＣＭＳ培養系で生産させる場合、ＳＺ２藻細胞培養液に添加するＳＴ１細胞量にある特定の比率が有効であることが明らかとなった。
３−９．糖添加培地／明暗条件での油生産
　ＭＣＭＳ培養において培地に外部から糖（有機物）を添加し、暗黒１２時間を含む（培養時の電力の節約に繋がる）１２ｈ−明／１２ｈ−暗（１２時間ずつ明暗）培養条件下での油生産量を検証した。
　図９（Ａ）に示す結果から、ＳＺ２量（５０ｍＬからの菌体量に相当）：ＳＴ１量（５ｍＬからの菌体量に相当）＝１：１、あるいはＳＴ１株のみ（５ｍＬからの菌体量に相当）に設定し、第３−８節に示す培地と条件で培養する際、グルコース（Ｇｌｕｃｏｓｅ＝Ｇｌｃ）を所定の濃度で添加した。培養後は、菌体を遠心して回収し、第３−６~３−８節で記述した方法により試料を調製し、ＧＣ−ＭＳ分析によってヘプタデカンの生産量を測定した。なお糖無添加の場合、生産されるヘプタデカンの量を値１００とした。
　結果を図９（Ｂ）及び（Ｃ）に示す。なお、図９（Ｂ）及び（Ｃ）において、縦軸は、内標準物質として添加したエイコサン（２０ｐｐｍ）に対して生産されたヘプタデカンの生産量の相対値を示し、また、横軸は、培地へのグルコース（Ｇｌｃ）添加量（最終濃度）を示す。
　図９（Ｂ）に示すように、濃度一定（２０ｐｐｍ）で添加している内標準物質に対する相対的なヘプタデカンの合成量を調べたところ、ＳＺ２＋ＳＴ１混合培養系においては、糖無添加の場合と比較して、ブドウ糖を最終濃度で０．５ｍＭ添加した明暗培養条件で約１．５倍増加を示した。以上によりＭＣＭＳ培養では、ＳＺ２藻が生産する多糖に加え、培地の外から添加する糖も燃料生産量を向上させる一因となり、且つ暗黒（照明電力の軽減に繋がる）を取り入れながら従属栄養条件下でも燃料生産が可能であることが実証された。
　さらに、図９（Ｃ）に示すように、上記の培養条件下で、純化したＳＴ１株のみの培地（ＳＴ１単一培養系）にグルコースを添加した場合、同様に最終濃度０．５ｍＭの時にヘプタデカン生産量が無添加の約１．６倍となった。また糖添加量と生産されるヘプタデカン生産量との関係は、ＳＺ２＋ＳＴ１混合培養系とＳＴ１単一培養系で似ていた。この結果は、ＳＺ２＋ＳＴ１混合培養系で主にヘプタデカンを生産しているのは、ＳＴ１細胞の方であるとする図４~７の結果を強く支持するものであった。またＳＴ１株単独でも窒素源欠乏培地に炭素源（酢酸Ｎａ）が添加されれば、炭化水素（ヘプタデカン）の蓄積量が増加するという図６の結果と矛盾しない。
　以上により、ＭＣＭＳ培養系においては、ＢＧ１１培地で生育した５０ｍＬ相当に対するＳＺ２株の液体培地（ＳＴ１株が潜在的に共存している）にＬＢ培地で予め培養したＳＴ１株を５ｍＬ（ＳＺ２培養液の１０分の１に相当する菌体量）を添加し、これに有機物栄養源として酢酸Ｎａを１０ｍＭ、糖源としてグルコースを０．５ｍＭ程度で同時に混ぜ、さらに窒素欠乏培地で２％炭酸ガスを供給すると、共存菌であるＳＴ１株から昼夜（明暗）を問わず、ヘプタデカン油を効率良く生産する新技術が確立された。
３−１０．乾燥ストレスによるＳＺ２藻の油生産
　図９（Ｂ）の試験後、綿栓付フラスコ内のＳＺ２＋ＳＴ１菌体培養液から１週間ほど培養した菌体を回収し、顕微鏡観察を行い、炭化水素生産を確認した（図１０の上段）。一方、ＳＺ２（＋ＳＴ１）藻を含む培養液を２ヶ月程度静置し、綿栓付フラスコ内の培地液体の自然乾燥を促した。その後、ほんの少しだけ水分を含んだ乾燥菌体を同様に顕微鏡観察した（図１０の下段）。顕微鏡観察では、培養液（あるいは自然乾燥した菌体に少量のＢＧ１１０培地を添加して混ぜた後）に蛍光染色剤Ｎｉｌｅ　Ｒｅｄを最終濃度１０μＭになるように添加した。数分間放置後、菌体を遠心により集め、これを蛍光顕微鏡（ＯＬＹＭＰＵＳ　ＢＸ５３／ＤＰ７２）で観察した（図１０において、棒は１０μｍである）。図１０において、左列は光学フィルター（ＢＦ）で観察した結果であり、右列は青色蛍光フィルター（ＢＷ）で観察した結果である。図１０中株名の太字は、その条件下で油を主に蓄積している菌体を示す。
　Ｎｉｌｅ　Ｒｅｄ染色による蛍光顕微鏡観察では、図１０の上段ではＳＴ１が、下段でＳＺ２細胞が優先して黄色に光って見え、油の蓄積が伺えた。
　以上の結果、通常のＭＣＭＳ培養条件ではＳＴ１菌株が炭化水素生産を優先して行うが、乾燥ストレスはＳＺ２藻自身による油の生産を可能にすることが明らかとなった。
　それ故、乾燥ストレスによりＳＺ２藻内に蓄積された油成分をＦＩＤ（Ｆｌａｍｅ　Ｉｏｎｉｚａｔｉｏｎ　Ｄｅｔｅｃｔｏｒ，水素炎イオン化型検出器）により分析した。その結果を図１１に示す。
　図１１（Ａ）は、図１０の下段に示したＳＺ２藻細胞内油組成をＦＩＤにより分析した結果を示す。上３段は脂肪酸並びにヘプタデカンの内標準を供した。４段目は、ＳＺ２株が優先種の試料をＦＩＤ分析した結果である。検出されたバイオ燃料を「＊」で示した。
　図１１（Ｂ）は、パネルＡの結果より検出された脂肪酸並びにヘプタデカン（バイオ燃料）の蓄積量を相対値（％）で示した。
　以上の結果、乾燥ストレス下のＳＺ２藻では、Ｃ１６やＣ１８の脂肪酸メチルエステル化合物及びヘプタデカン（Ｃ１７Ｈ３６）等バイオ燃料が検出された。
　以上、本願によりＭＣＭＳ培養系でＳＴ１による効率の良い燃料の生産が可能である。更にＭＣＭＳ培養後、乾燥ストレスによりＳＺ２藻自身でもバイオ燃料の生産が可能である。実用化を考えると、ＭＣＭＳ系で連続的にヘプタデカン等を液体燃料として回収し、その後、培養の最後はＳＺ２（＋ＳＴ１）を乾燥させると、固形燃料として使用できる可能性があり、経済的にコスト安な燃料製造法となりえる。 3. Biofuel production by co-culture (MCMS culture) of halomicronema sp. SZ2 and sinorizobium sp. ST1. 3-1. SZ2 strain 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 μm. 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. The long side per cell was about 3-5 μm for the SZ2 strain and about 0.7-2 μm for the ST1 strain. In fluorescence microscope (excitation 460-495 nm / radiation 510 nm, Olympus BX53 / DP72) observation, autofluorescence unique to photosynthetic microorganisms was observed. In the aggregate of SZ2 cells, accumulation of a substance considered to be a polysaccharide was observed around the filamentous cells.
3-2. Sugar Production of SZ2 Strain FIG. 3 is a photograph showing the sugar production of SZ2 strain.
PAS staining (Periodic acid-Schiff stain) 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): The SZ2 strain was statically cultured in a BG11 liquid medium for 4 weeks, and then 1 mL of the culture solution was collected and centrifuged to collect algal cells. The algae were washed with distilled water, and then centrifuged again to discard the distilled water. 300 μL of 0.5% (w / v) periodic acid (periodic acid, manufactured by Wako Pure Chemical Industries) aqueous solution was added to the collected cells and suspended, and the cells were left for 3 minutes. Thereafter, the periodic acid aqueous solution was centrifuged and discarded. Distilled water was added to this for washing, centrifuged, and the distilled water was discarded. A Schiff's reagent (manufactured by Wako Pure Chemical Industries) was added to suspend the algae. After leaving for 13 minutes, the Schiff reagent was centrifuged and discarded. After repeating this three times, distilled water was added, washed and centrifuged to obtain a sample for microscopic observation. This was observed with an optical microscope (Olympus BX53 / DP72). Accumulation of polysaccharide was observed on the intracellular and extracellular surfaces (in FIG. 3 (A), the bar is 10 μm).
FIG. 3 (B): 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).
3-3. Hydrocarbon production by ST1 strain in the presence of SZ2 strain and ST1 strain FIG. 4 is a photograph showing hydrocarbon production by the ST1 strain in the presence of SZ2 strain and ST1 strain.
After 3 weeks of culture 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 μmol photons / m 2 / s 1 ), and then 1 mL of a liquid medium was recovered. In order to confirm the accumulation of hydrocarbons, Nile Red (Wako Pure Chemical Industries) was added to the medium to a final concentration of 10 μM to stain the cells, and this was stained with an optical microscope (FIG. 4A) and a fluorescence microscope (excitation 460−). Observation was performed at 495 nm / radiation 510 nm, FIG.
As a result of observation under an optical microscope, when cultured under a nitrogen-deficient condition, proliferation of ST1 cells was recognized so as to surround the filamentous SZ2 algal cells. Thus SZ2 cells and ST1 proportion of cells, whereas the culture in BG11 medium are overwhelmingly SZ2 cells occupied by dominant species, be relatively ST1 cells are increased in BG11 0 Medium It became clear. On the other hand, as a result of observation with a fluorescence microscope, significant hydrocarbon accumulation was observed in ST1 cells (in FIGS. 4A and 4B, the bar is 10 μm).
3-4. Compatibility of SZ2 and ST1 strains in hydrocarbon production The culture solution of ST1 strain cells isolated and purified from the SZ2 strain culture solution is added to and mixed with three types of cyanobacterial algae culture solutions. By co-culturing, the compatibility of the partner algal cells in the hydrocarbon production by the ST1 strain was verified.
The results are shown in FIG. Of the three types of cyanobacteria used, two types other than the SZ2 strain used purified strains. Cells corresponding to various algae cell solution 50mL were precultured in 3 weeks BG11 medium was collected by centrifugation and suspended to BG11 0 liquid medium described (sodium acetate = Na acetate added) at 3-3 Section. To this, the wet cells corresponding to the amount collected by centrifuging 1 mL (OD 660 = 2) of ST1 cell culture previously cultured overnight in LB liquid medium (30 ° C., 110 rpm shaking culture) were directly collected, It was transplanted into the 50mL BG11 0 liquid medium. This was statically cultured for 5 days in air (containing 0.04% CO 2 gas) as described in Section 3-3. Thereafter, the cells were stained with Nile Red and observed with a fluorescence microscope.
As a result, when the ST1 strain and various algae were co-cultured using the BG11 liquid medium, almost no hydrocarbon production was observed under any co-culture. On the other hand, when co-cultured in BG11 0 (nitrogen source deficient) medium, the amount of hydrocarbon produced by the ST1 strain was higher in the order of co-culture with SZ2 strain> PCC6803 strain> ABRG5-3 strain. From this result, specificity is recognized for the partner algae that coexist during the hydrocarbon production by the ST1 strain, and it becomes clear that the compatibility with the SZ2 algae coexisting with the ST1 strain when originally isolated from the natural world is the best. It was.
In FIG. 5, abbreviations indicate the following: SZ2, Halomicronema sp. SZ2; PCC6803, Synechocystis sp. PCC6803 (stored in Pasteur Research Institute (Paris, France); Kazusa DNA Research Institute (distributed from 2-6-7, Kazusa Kamashizu, Chiba Prefecture)); ABRG5-3; Limnothrix / Pseudanabaena sp. ABRG5-3 (FERM P-22172; JP 2013-198473 A). In FIG. 5, the bar is 10 μm. Furthermore, in FIG. 5, the presence or absence of hydrocarbon (oil) production by ST1 strain: Yes, +; No,-.
3-5. Medium composition conditions for hydrocarbon production Regarding the combination of cells of strains SZ2 and ST1, the presence or absence of a nitrogen source in the medium, and the presence or absence of sodium acetate (Na acetate, final concentration 10 mM), the culture conditions shown in Section 3-4 The cultivated cells were stained with Nile Red, and the medium composition conditions in the hydrocarbon production of the ST1 strain were verified.
The results are shown in FIG. First, as a condition for ST1 cells most efficiently produce hydrocarbons, co-culture with SZ2 algae cells (MS: Mixed-Species) Nitrogen deficiency in (BG11 0 medium used) conditions and reinforcing carbon source to the culture medium This was the case when sodium acetate was added as an agent (upper left in FIG. 6). If the medium was not deficient in nitrogen from this combination (BG11 medium was used), no significant accumulation of hydrocarbons in ST1 cells was observed (upper right in FIG. 6). This indicates that co-culture with SZ2 algal cells is effective for efficient hydrocarbon production of ST1 cells, and it is essential to culture in a nitrogen source-deficient medium.
Next, the same experiment was performed using only the isolated and purified ST1 strain. When sodium acetate was added in a nitrogen source-deficient medium, ST1 strain single culture (UCUS: Uni-Culture Uni-Species) was also used. Hydrocarbons were produced (the middle left of FIG. 6). However, its productivity was lower than that when co-cultured with the SZ2 strain. In addition, even when sodium acetate was added, hydrocarbon production of ST1 strain was not observed unless the medium was deficient in nitrogen (FIG. 6, middle right).
From the above, it was suggested that hydrocarbon production of ST1 strain is possible only when a carbon source is supplied and a nitrogen source is deficient. Therefore, in the co-culture with SZ2 algal cells, it is inferred that the ST1 strain uses the polysaccharide produced on the surface of SZ2 algal cells as a carbon source to increase the efficiency of hydrocarbon production (upper left in FIG. 6). It was done. 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 μm. Moreover, in FIG. 6, the presence or absence of hydrocarbon (oil) production by ST1 stock: Yes, +; No,-.
3-6. 5L scale biofuel production by MCMS culture To establish a basic technology for practical use of biofuel production by co-culture of SZ2 and ST1 strains, (1) MCMS culture technology at 5 liter (5L) scale (2) The components of the oil produced by MCMS culture were verified. Regarding (1), especially for cost mitigation, in BG11 0 medium prepared using distilled water instead of sterile water, lighting also was the provision of darkness for 12 hours (12h) intervals. The following describes the culture and fuel component analysis.
Although the ST1 strain originally coexists in the algae strain, it is possible to accumulate and produce hydrocarbons by adding the ST1 strain culture solution isolated to the SZ2 strain culture solution previously cultured at a predetermined concentration. This is illustrated in FIG. For 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. 0.5% carbon dioxide gas (15 L / min) was supplied to this with a silicon tube. This medium was cultured for 19 days under light / dark (12 h / 12 h) cycle conditions under a white fluorescent lamp (30 μmol photons / sec / m 2 ).
After the culture, 50 mL of the culture solution was collected, and the cells were collected by centrifugation. Thereafter, hydrocarbons were recovered from the recovered cells using ethyl acetate (see JP 2013-198473), and a part of the sample was taken for GC-MS analysis (JP 2013-198473 A). reference).
The results are shown in FIG. With respect to the peak (retention time = 18.18 min) of eicosane (C 20 H 42 ) prepared and added to the final concentration of 20 ppm as an internal standard substance (control group) in the sample solution, alkane (heptadecane, C 17 H 36 ), a major peak was observed at retention time (14.60 min). The ratio [relative value (%) = (value of C 17 H 36 / value of C 20 H 42 ) × 100%] of this peak area to the peak area of the internal standard substance is 122%, and SZ2 + ST1 calculated from this ratio The amount of heptadecane production per cell dry weight was about 5%. Several minor peaks that seemed to be another fuel substance were observed, and when these were added together, a production amount of about 10% biofuel per cell dry weight was confirmed. When the production amount of fuel containing heptadecane per dry weight is 10% and the production amount from a 1-ton scale MCMS liquid medium is estimated, this corresponds to a biofuel production amount of 0.1 kg. Since heptadecane is equivalent to light oil, when mixed with 1% fuel, it is considered that about 5 to 10 L of fuel can be used for running a truck or the like.
As described above, biofuel alkanes were produced by 5 L scale MCMS culture. BG11 0 occupying most of the medium is that amount because using distilled water unsterilized, expense relief was achieved. Also, under the culture conditions, both SZ2 and ST1 strains were always preferred species in the culture solution. Furthermore, it was expected that greenhouse gases would be effectively used by supplying 0.5% (12.5 times the air) carbon dioxide gas to the medium. On the other hand, the mixing of BG11 0 LB medium to the (inorganic medium) (organics medium) (= MC, Mixed Culture) in, even while taking the darkness of the 12-hour intervals SZ2 + ST1 mixed species (= MS, Mixed Species) fuel production in culture Therefore, this was also expected to be a great way to reduce culture costs.
3-7. Changes in fuel accumulation over time The biofuel accumulation over time under the MCMS culture conditions shown in Section 3-6 was verified, and the results are shown in FIG.
In FIG. 8, the horizontal axis of the graph represents the number of days of culture, and the vertical axis represents the relative value of heptadecane production relative to eicosan (final concentration 20 ppm) as an internal standard substance [relative value (%) = (value of C 17 H 36 / value of C 20 H 42 ) × 100%]. Moreover, 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. As described above, in MCMS culture on a 5 L scale, biofuel accumulation is good around 17 days under the culture apparatus and culture conditions described in Section 3-6, and the time around that is suitable for recovery. It became clear.
3-8. Mixing ratio of SZ2 strain and ST1 strain and fuel production amount The medium and culture conditions shown in Section 3-4 were modified as follows and tested. Cells corresponding to SZ2 algal cell solution 50mL preincubated for 2 months BG11 medium was collected by centrifugation and suspended to BG11 0 liquid medium (sodium acetate added). To this, 5 mL (OD 660 = 2) of ST1 cell culture previously cultured overnight in an LB liquid medium (30 ° C., 110 rpm shaking culture) was centrifuged and the wet cells corresponding to the amount collected were collected directly. It was transplanted to 50mL BG11 0 liquid medium. This was SZ2 amount: ST1 amount = 1: 1. For example, when SZ2 amount: ST1 amount = 1: 2, 10 mL of the ST1 cell culture solution (OD 660 = 2) was centrifuged and collected. The above was cultured in a culture incubator (12h-light / 12h-dark) controlled to 2% CO 2 gas concentration for 5 days with reciprocal shaking (40 rpm). After culturing, 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.
The results are shown in FIG. In 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 (the culture solution XmL was collected by centrifugation and added to the SZ2 culture solution).
As shown in FIG. 9A, 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. As a result, when 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. Sugar-added medium / oil production under light / dark conditions In MCMS culture, sugar (organic matter) is added to the medium from the outside and includes 12 hours of darkness (leading to power saving during cultivation) 12 h-light / 12 h-dark (12 hours The amount of oil produced under culture conditions was verified.
From the results shown in FIG. 9 (A), SZ2 amount (corresponding to the amount of cells from 50 mL): ST1 amount (corresponding to the amount of cells from 5 mL) = 1: 1, or ST1 strain alone (cells from 5 mL) When culturing under the medium and conditions shown in Section 3-8, glucose (Glucose = Glc) was added at a predetermined concentration. After the culture, the cells were collected by centrifugation, a sample was prepared by the method described in Sections 3-6 to 3-8, and the production amount of heptadecane was measured by GC-MS analysis. When no sugar was added, the amount of heptadecane produced was set to 100.
The results are shown in FIGS. 9 (B) and (C). 9B and 9C, the vertical axis represents the relative value of heptadecane produced with respect to eicosane (20 ppm) added as an internal standard substance, and the horizontal axis represents the culture medium. The glucose (Glc) addition amount (final concentration) is shown.
As shown in FIG. 9 (B), the relative amount of heptadecane synthesized relative to the internal standard substance added at a constant concentration (20 ppm) was examined. In the SZ2 + ST1 mixed culture system, it was compared with the case where no sugar was added. Thus, an increase of about 1.5 times was observed under the light and dark culture conditions in which glucose was added at a final concentration of 0.5 mM. As described above, in MCMS culture, in addition to the polysaccharides produced by SZ2 algae, sugars added from outside the medium also contribute to improving fuel production, and in the heterotrophic conditions while incorporating darkness (leading to reduction of lighting power). But it was proven that fuel production was possible.
Furthermore, as shown in FIG. 9 (C), when glucose was added to the purified ST1 strain-only medium (ST1 single culture system) under the above-described culture conditions, heptadecane was similarly obtained at a final concentration of 0.5 mM. The production amount was about 1.6 times that of no additive. The relationship between the amount of added sugar and the amount of heptadecane produced was similar between the SZ2 + ST1 mixed culture system and the ST1 single culture system. This result strongly supported the results of FIGS. 4 to 7 in which it was ST1 cells that mainly produced heptadecane in the SZ2 + ST1 mixed culture system. Further, even with the ST1 strain alone, if a carbon source (Na acetate) is added to the nitrogen source-deficient medium, this is consistent with the result of FIG. 6 that the amount of accumulated hydrocarbon (heptadecane) increases.
As described above, in the MCMS culture system, 5 mL of the S1 strain previously cultured in the LB medium in the liquid medium of the SZ2 strain corresponding to 50 mL grown in the BG11 medium (ST1 strain potentially coexists) The amount of bacterial cells corresponding to 1/10) is added, and 10 mg of sodium acetate as an organic nutrient source and glucose of 0.5 mM as a sugar source are mixed simultaneously, and 2% carbon dioxide gas is supplied in a nitrogen-deficient medium. Then, a new technology for efficiently producing heptadecane oil was established regardless of day or night (light and dark) from the ST1 strain, which is a coexisting bacterium.
3-10. Oil production of SZ2 algae by drought stress After the test of Fig. 9 (B), the cells cultured for about a week from the SZ2 + ST1 cell culture solution in the flask with cotton plugs are collected and observed under a microscope to confirm hydrocarbon production (The upper part of FIG. 10). On the other hand, the culture solution containing SZ2 (+ ST1) algae was allowed to stand for about 2 months to promote natural drying of the medium liquid in the flask with cotton plug. Thereafter, the dried cells containing only a small amount of water were similarly observed with a microscope (lower part of FIG. 10). In microscopic observation, it was added a fluorescent dye, Nile Red to a final concentration of 10μM to the medium (or after mixing by adding a small amount of BG11 0 media naturally dried cells). After standing for several minutes, the cells were collected by centrifugation and observed with a fluorescence microscope (OLYMPUS BX53 / DP72) (in FIG. 10, the bar is 10 μm). In FIG. 10, the left column is the result of observation with an optical filter (BF), and the right column is the result of observation with a blue fluorescent filter (BW). The bold letters in the strain names in FIG. 10 indicate the cells that mainly accumulate oil under the conditions.
In the fluorescence microscope observation by Nile Red staining, 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.
As a result, 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. The upper three stages provided internal standards for fatty acids and heptadecane. The fourth row shows the result of FID analysis of the sample of the priority species of the SZ2 strain. The detected biofuel is indicated by “*”.
FIG. 11B shows the accumulated amount of fatty acid and heptadecane (biofuel) detected from the results of panel A as relative values (%).
As a result, 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.
As described above, 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. Considering practical application, if heptadecane, etc. is continuously recovered as a liquid fuel in the MCMS system, and then SZ2 (+ ST1) is dried at the end of the culture, it may be used as a solid fuel, which is economically costly. It can be a cheap fuel production method.
　本発明によれば、炭化水素（アルカン）等のバイオ燃料を高収量で生産することができる。 According to the present invention, biofuels such as hydrocarbons (alkanes) can be produced in high yield.
　ＮＩＴＥ　ＢＰ−０１９８２
　ＮＩＴＥ　ＢＰ−０１９８１
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。
NITE BP-01982
NITE BP-01981
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (17)

  1. 　光合成微生物と非光合成微生物とを含む混合微生物を、窒素源欠乏培地において共培養することを含む、バイオ燃料の製造方法。 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.
  2. 　光合成微生物がハロミクロネマ（Ｈａｌｏｍｉｃｒｏｎｅｍａ）属に属する微生物である、請求項１記載の方法。 The method according to claim 1, wherein the photosynthetic microorganism is a microorganism belonging to the genus Halomicronema.
  3. 　ハロミクロネマ属に属する微生物が、受託番号ＮＩＴＥ　ＢＰ−０１９８２で特定されるハロミクロネマ・エスピー（Ｈａｌｏｍｉｃｒｏｎｅｍａ　ｓｐ．）ＳＺ２菌株又は糖生産能を有するその変異株である、請求項２記載の方法。 The method according to claim 2, wherein the microorganism belonging to the genus Halomicronema is a Halomicronema sp. SZ2 strain specified by the accession number NITE BP-01982, or a mutant strain thereof having sugar-producing ability.
  4. 　非光合成微生物がシノリゾビウム（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ）属に属する微生物である、請求項１~３のいずれか１項記載の方法。 The method according to any one of claims 1 to 3, wherein the non-photosynthetic microorganism is a microorganism belonging to the genus Sinorhizobium.
  5. 　シノリゾビウム属に属する微生物が、受託番号ＮＩＴＥ　ＢＰ−０１９８１で特定されるシノリゾビウム・エスピー（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ　ｓｐ．）ＳＴ１菌株又は短鎖アルカン生産能を有するその変異株である、請求項４記載の方法。 The method according to claim 4, wherein the microorganism belonging to the genus Synorizobium is a Sinorhizobium sp. ST1 strain identified by the accession number NITE BP-01981 or a mutant strain thereof capable of producing a short-chain alkane.
  6. 　培地が無機物培地と有機物培地とをさらに含有する、請求項１~５のいずれか１項記載の方法。 6. The method according to any one of claims 1 to 5, wherein the medium further contains an inorganic medium and an organic medium.
  7. 　培地が炭素源をさらに含有する、請求項１~６のいずれか１項記載の方法。 The method according to any one of claims 1 to 6, wherein the medium further contains a carbon source.
  8. 　炭素源が酢酸ナトリウムである、請求項７記載の方法。 The method according to claim 7, wherein the carbon source is sodium acetate.
  9. 　培地が糖源をさらに含有する、請求項１~８のいずれか１項記載の方法。 The method according to any one of claims 1 to 8, wherein the medium further contains a sugar source.
  10. 　糖源がグルコースである、請求項９記載の方法。 The method according to claim 9, wherein the sugar source is glucose.
  11. 　共培養において所定の暗黒期間を設ける、請求項１~１０のいずれか１項記載の方法。 The method according to any one of claims 1 to 10, wherein a predetermined dark period is provided in the co-culture.
  12. 　共培養後、共培養物を乾燥ストレス下で更に培養することを含む、請求項１~１１のいずれか１項記載の方法。 The method according to any one of claims 1 to 11, further comprising further culturing the co-culture under drought stress after the co-culture.
  13. 　バイオ燃料が短鎖アルカンである、請求項１~１２のいずれか１項記載の方法。 The method according to any one of claims 1 to 12, wherein the biofuel is a short-chain alkane.
  14. 　短鎖アルカンがヘプタデカンである、請求項１３記載の方法。 The method according to claim 13, wherein the short-chain alkane is heptadecane.
  15. 　受託番号ＮＩＴＥ　ＢＰ−０１９８２で特定されるハロミクロネマ・エスピーＳＺ２菌株又は糖生産能を有するその変異株。 Halomicronema sp. SZ2 strain specified by the accession number NITE BP-01982, or a mutant strain thereof having a sugar-producing ability.
  16. 　受託番号ＮＩＴＥ　ＢＰ−０１９８１で特定されるシノリゾビウム・エスピーＳＴ１菌株又は短鎖アルカン生産能を有するその変異株。 Sinorizobium sp. ST1 strain identified by the accession number NITE BP-01981, or a mutant strain having the ability to produce short-chain alkanes.
  17. 　請求項１５記載のハロミクロネマ・エスピーＳＺ２菌株又は糖生産能を有するその変異株と請求項１６記載のシノリゾビウム・エスピーＳＴ１菌株又は短鎖アルカン生産能を有するその変異株とを含む混合微生物。 A mixed microorganism comprising the halomicronema sp. SZ2 strain according to claim 15 or a mutant strain thereof having sugar-producing ability, and the Sinolizobium sp. ST1 strain according to claim 16 or a variant strain having short-chain alkane producing ability.
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