WO2023074226A1 - Method for culturing chlamydomonas reinhardtii - Google Patents
Method for culturing chlamydomonas reinhardtii Download PDFInfo
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- green alga
- alga chlamydomonas
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- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000012258 culturing Methods 0.000 title claims abstract description 19
- 241000195597 Chlamydomonas reinhardtii Species 0.000 title abstract description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 78
- 239000007788 liquid Substances 0.000 claims abstract description 51
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 39
- 229920002472 Starch Polymers 0.000 claims abstract description 35
- 239000008107 starch Substances 0.000 claims abstract description 35
- 235000019698 starch Nutrition 0.000 claims abstract description 35
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 16
- 239000011574 phosphorus Substances 0.000 claims abstract description 16
- 241000195585 Chlamydomonas Species 0.000 claims description 66
- 239000004202 carbamide Substances 0.000 claims description 26
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea group Chemical group NC(=O)N XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 26
- 229910019142 PO4 Inorganic materials 0.000 claims description 11
- 235000021317 phosphate Nutrition 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 9
- 239000010452 phosphate Substances 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000009630 liquid culture Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 239000007789 gas Substances 0.000 description 8
- 239000001569 carbon dioxide Substances 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 206010021033 Hypomenorrhoea Diseases 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000195493 Cryptophyta Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229910000365 copper sulfate Inorganic materials 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 229910000358 iron sulfate Inorganic materials 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- 229960001763 zinc sulfate Drugs 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004931 aggregating effect Effects 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
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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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to a method for culturing the green alga Chlamydomonas.
- Algae biofuels are being developed as alternative fuels to fossil fuels. Specifically, a method in which the green alga Chlamydomonas reinhardtii immobilizes carbon dioxide (CO 2 ) to accumulate starch, and then the green alga Chlamydomonas reinhardtii self-ferments under dark anaerobic conditions to produce ethanol. is known (see, for example, Patent Document 1).
- CO 2 carbon dioxide
- An object of the present invention is to provide a method for culturing the green alga Chlamydomonas that can increase the amount of starch produced without exchanging the medium.
- One aspect of the present invention is a method for cultivating the green alga Chlamydomonas, comprising a step of culturing the green alga Chlamydomonas in a liquid medium to accumulate starch in the green alga Chlamydomonas, wherein the liquid medium contains nitrogen element contained in a nitrogen source.
- the content is 35 ppm or more and 70 ppm or less, and the content of elemental phosphorus is 420 ppm or more.
- the nitrogen source may be urea.
- the phosphorus element may be contained in the phosphate.
- the phosphate may be dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
- the liquid medium may have a content of dipotassium hydrogen phosphate of 1440 ppm or more and a content of potassium dihydrogen phosphate of 720 ppm or more.
- the green alga Chlamydomonas may be Hyundai DREAMO strain (accession number FERM BP-22306).
- 1 is a graph showing the relationship of starch production to urea content in a liquid medium.
- 1 is a graph showing the relationship between the production amount of Chlamydomonas green algae and the amount of accumulated starch in Chlamydomonas green algae with respect to the content of urea in a liquid medium.
- 1 is a graph showing the relationship between the amount of phosphate in a liquid medium and the amount of production of the green alga Chlamydomonas.
- 1 is a graph showing starch production amounts of Examples 1 and 2 and Comparative Examples 1 and 2.
- the method for culturing the green alga Chlamydomonas of the present embodiment includes a step of culturing the green alga Chlamydomonas in a liquid medium to accumulate starch in the green alga Chlamydomonas. At this time, in order to increase the amount of starch produced, it is necessary to accumulate starch in the green alga Chlamydomonas and grow the green alga Chlamydomonas in a liquid medium.
- the content of nitrogen element contained in the nitrogen source in the liquid medium is 35 ppm or more and 70 ppm or less. If the nitrogen element content in the nitrogen source in the liquid medium is less than 35 ppm, the growth of the green alga Chlamydomonas becomes difficult. On the other hand, when the nitrogen element content in the nitrogen source in the liquid medium exceeds 70 ppm, the amount of starch accumulated in the green alga Chlamydomonas decreases. That is, by setting the content of nitrogen element contained in the nitrogen source in the liquid medium to 70 ppm or less, more ethanol can be produced from the green alga Chlamydomonas, and bioethanol can be efficiently produced.
- the nitrogen source is not particularly limited, but examples include inorganic nitrogen sources such as ammonium salts and nitrates, and organic nitrogen sources such as peptone and urea.
- the content of urea in the liquid medium is preferably 150 ppm or less, more preferably 80 ppm or more and 150 ppm or less.
- the content of elemental phosphorus in the liquid medium is 420 ppm or more, preferably 500 ppm or more. If the content of elemental phosphorus in the liquid medium is less than 420 ppm, the growth of the green alga Chlamydomonas becomes difficult.
- the phosphorus element is preferably contained in the phosphate. This facilitates the growth of the green alga Chlamydomonas.
- the phosphate is not particularly limited, but examples thereof include dipotassium hydrogen phosphate (K 2 HPO 4 ), potassium dihydrogen phosphate (KH 2 PO 4 ), calcium phosphate (Ca 3 (PO 4 ) 2 ), and the like. be done.
- the content of dipotassium hydrogen phosphate in the liquid medium is preferably 1440 ppm or more, preferably 1730 ppm or more. More preferred. Also, the content of potassium dihydrogen phosphate in the liquid medium is preferably 720 ppm or more, more preferably 860 ppm or more.
- the content of the sulfur source in the liquid medium is not particularly limited, but is, for example, 4.5 ppm or more.
- the sulfur source is not particularly limited, but examples include magnesium sulfate, zinc sulfate, iron sulfate, copper sulfate, calcium sulfate, and the like.
- the liquid medium may further contain minerals such as calcium chloride, boric acid, manganese chloride, hexaammonium heptamolybdate, zinc sulfate, iron sulfate, and copper sulfate.
- minerals such as calcium chloride, boric acid, manganese chloride, hexaammonium heptamolybdate, zinc sulfate, iron sulfate, and copper sulfate.
- the method for culturing the green alga Chlamydomonas in a liquid medium is not particularly limited, and examples include stationary culture, shaking culture, submerged culture, and aeration culture.
- an artificial climate vessel can be used when culturing the green alga Chlamydomonas in a liquid medium.
- the temperature for culturing the green alga Chlamydomonas in the liquid medium is not particularly limited, but is, for example, 2°C or higher and 38°C or lower.
- the light-dark cycle when culturing the green algae Chlamydomonas in a liquid medium is not particularly limited, but is, for example, a 24-hour cycle with a light period of 6 hours or more and 24 hours or less.
- the photosynthetically effective photon flux density in the light period is not particularly limited, but is, for example, 50 ⁇ mol/m 2 /s or more and 2000 ⁇ mol/m 2 /s or less.
- the method of supplying the gas composition containing carbon dioxide is not particularly limited, and known methods can be used.
- the content of carbon dioxide gas in the gas composition is not particularly limited, but is, for example, 0.5% by volume or more and 5% by volume or less.
- the amount of permeation of the gas composition is not particularly limited, but is, for example, 0.05 vvm or more and 0.5 vvm or less.
- the green alga Chlamydomonas is not particularly limited, but includes, for example, the Hyundai DREAMO strain (accession number FERM BP-22306).
- the Honda DREAMO strain is a mutant strain that has the ability to produce ethanol under dark anaerobic conditions and has acquired the ability to grow while aggregating (see Patent Document 1).
- the method for cultivating the green alga Chlamydomonas may further include a step of culturing the Hyundai DREAMO strain under dark anaerobic conditions to cause the Hyundai DREAMO strain to produce ethanol. good.
- the method for collecting the cultured green alga Chlamydomonas is not particularly limited, but examples include centrifugation and filtration.
- Starch, ethanol, etc. can be extracted from the collected Chlamydomonas algae.
- the method of producing ethanol from the green alga Chlamydomonas is not particularly limited, but includes, for example, a method of self-fermentation using the inherent ethanol-producing ability of the green alga Chlamydomonas, and a method of saccharifying and fermenting starch extracted from the green alga Chlamydomonas.
- the collected green alga Chlamydomonas may be dried if necessary.
- the method for drying the green alga Chlamydomonas is not particularly limited, but includes, for example, a freeze-drying method.
- Example 1 The content of nitrogen element contained in the nitrogen source in the liquid medium is 1/3 (43.6 ppm; urea 93.5 ppm), and the content of phosphorus element is 1.2 times (504 ppm; dipotassium hydrogen phosphate 1728 ppm, phosphorus).
- the green alga Chlamydomonas was cultured in the same manner as in Comparative Example 1, except that the amount of potassium dihydrogen acid was 864 ppm).
- Example 2 The green alga Chlamydomonas was cultured in the same manner as in Comparative Example 1, except that the nitrogen element content in the nitrogen source in the liquid medium was halved (65.5 ppm; urea: 140.3 ppm).
- FIG. 1 shows the relationship between the amount of starch produced and the content of urea in a liquid medium in which the elemental phosphorus content was set to 420 ppm.
- the contents of nitrogen elements contained in urea which is the nitrogen source in the liquid medium, are 32.7 ppm (70.1 ppm of urea), 43.6 ppm (93.5 ppm of urea), 65.5 ppm (140 ppm of urea). 3 ppm) and 130.9 ppm (urea 280.5 ppm), it can be seen that when the nitrogen element content is 43.6 ppm, the amount of starch production is maximized. In addition, when the nitrogen element content is 65.5 ppm, the starch production amount is the second largest, and when the nitrogen element content is 32.7 ppm and 130.9 ppm, the starch production amount decreases. It can be seen that
- FIG. 2 shows the relationship between the production amount of green alga Chlamydomonas and the amount of starch accumulated in Chlamydomonas green algae with respect to the content of urea in a liquid medium in which the elemental phosphorus content was set to 420 ppm.
- the contents of nitrogen elements contained in urea which is the nitrogen source in the liquid medium, are 32.7 ppm (urea 70.1 ppm), 43.6 ppm (urea 93.5 ppm), 65.5 ppm (urea 140.3 ppm). ), 130.9 ppm (urea 280.5 ppm), the production of the green alga Chlamydomonas decreased as the nitrogen element content decreased.
- the accumulated amount of starch increases as the nitrogen element content decreases.
- Fig. 3 shows the relationship between the production amount of the green alga Chlamydomonas and the phosphate content in the liquid medium.
- FIG. 4 shows the production amounts of starch in Examples 1 and 2 and Comparative Examples 1 and 2.
- Example 2 produced a large amount of starch compared to Comparative Examples 1 and 2. Moreover, it can be seen that in Example 1 in which the content of elemental phosphorus contained in urea in the liquid medium was adjusted to 1.2 times, the amount of starch produced was the highest.
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Abstract
Provided is a method for culturing Chlamydomonas reinhardtii, the method comprising a step for culturing Chlamydomonas reinhardtii in a liquid medium and causing Chlamydomonas reinhardtii to accumulate starch. This liquid culture medium has a nitrogen source having an elemental nitrogen content of 35 ppm to 70 ppm and has an elemental phosphorus content of 420 ppm or greater.
Description
本発明は、緑藻クラミドモナスの培養方法に関する。
The present invention relates to a method for culturing the green alga Chlamydomonas.
化石燃料の代替燃料として、藻類バイオ燃料の開発が進められている。具体的には、緑藻クラミドモナス（Ｃｈｌａｍｙｄｏｍｏｎａｓ ｒｅｉｎｈａｒｄｔｉｉ）が炭酸ガス（ＣＯ２）を固定化することにより、デンプンを蓄積させた後、暗嫌気下で緑藻クラミドモナスに自己発酵させることにより、エタノールを産生する方法が知られている（例えば、特許文献１参照）。
Algae biofuels are being developed as alternative fuels to fossil fuels. Specifically, a method in which the green alga Chlamydomonas reinhardtii immobilizes carbon dioxide (CO 2 ) to accumulate starch, and then the green alga Chlamydomonas reinhardtii self-ferments under dark anaerobic conditions to produce ethanol. is known (see, for example, Patent Document 1).
緑藻クラミドモナスにデンプンを蓄積させる方法としては、最小培地中で緑藻クラミドモナスを培養し、増殖させた後、窒素源が欠乏している欠乏培地中で緑藻クラミドモナスを培養し、緑藻クラミドモナスにデンプンを蓄積させる方法が知られている（例えば、特許文献２参照）。
As a method for accumulating starch in the green alga Chlamydomonas, after culturing the green alga Chlamydomonas in a minimal medium and growing it, the green alga Chlamydomonas is cultured in a nitrogen-deficient medium, and starch is accumulated in the green alga Chlamydomonas. A method is known (see, for example, Patent Document 2).
しかしながら、培地を交換する際に、回収、遠心分離、洗浄等の工程が必要となるため、培地を交換せずに、デンプンの生産量を増大させることが望まれている。
However, since steps such as recovery, centrifugation, and washing are required when changing the medium, it is desired to increase the amount of starch produced without changing the medium.
本発明は、培地を交換せずに、デンプンの生産量を増大させることが可能な緑藻クラミドモナスの培養方法を提供することを目的とする。
An object of the present invention is to provide a method for culturing the green alga Chlamydomonas that can increase the amount of starch produced without exchanging the medium.
本発明の一態様は、緑藻クラミドモナスの培養方法において、液体培地中で緑藻クラミドモナスを培養して、前記緑藻クラミドモナスにデンプンを蓄積させる工程を含み、前記液体培地は、窒素源に含まれる窒素元素の含有量が３５ｐｐｍ以上７０ｐｐｍ以下であり、リン元素の含有量が４２０ｐｐｍ以上である。
One aspect of the present invention is a method for cultivating the green alga Chlamydomonas, comprising a step of culturing the green alga Chlamydomonas in a liquid medium to accumulate starch in the green alga Chlamydomonas, wherein the liquid medium contains nitrogen element contained in a nitrogen source. The content is 35 ppm or more and 70 ppm or less, and the content of elemental phosphorus is 420 ppm or more.
前記窒素源は、尿素であってもよい。
The nitrogen source may be urea.
前記リン元素は、リン酸塩に含まれてもよい。
The phosphorus element may be contained in the phosphate.
前記リン酸塩は、リン酸水素二カリウムおよびリン酸二水素カリウムであってもよい。
The phosphate may be dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
前記液体培地は、リン酸水素二カリウムの含有量が１４４０ｐｐｍ以上であり、リン酸二水素カリウムの含有量が７２０ｐｐｍ以上であってもよい。
The liquid medium may have a content of dipotassium hydrogen phosphate of 1440 ppm or more and a content of potassium dihydrogen phosphate of 720 ppm or more.
前記緑藻クラミドモナスは、Ｈｏｎｄａ ＤＲＥＡＭＯ株（受託番号ＦＥＲＭ ＢＰ－２２３０６）であってもよい。
The green alga Chlamydomonas may be Honda DREAMO strain (accession number FERM BP-22306).
本発明によれば、培地を交換せずに、デンプンの生産量を増大させることが可能な緑藻クラミドモナスの培養方法を提供することができる。
According to the present invention, it is possible to provide a method for culturing the green alga Chlamydomonas that can increase the amount of starch produced without exchanging the medium.
以下、本発明の実施形態について説明する。
Embodiments of the present invention will be described below.
本実施形態の緑藻クラミドモナスの培養方法は、液体培地中で緑藻クラミドモナスを培養して、緑藻クラミドモナスにデンプンを蓄積させる工程を含む。このとき、デンプンの生産量を増大させるためには、液体培地中で、緑藻クラミドモナスにデンプンを蓄積させるとともに、緑藻クラミドモナスを増殖させる必要がある。
The method for culturing the green alga Chlamydomonas of the present embodiment includes a step of culturing the green alga Chlamydomonas in a liquid medium to accumulate starch in the green alga Chlamydomonas. At this time, in order to increase the amount of starch produced, it is necessary to accumulate starch in the green alga Chlamydomonas and grow the green alga Chlamydomonas in a liquid medium.
液体培地中の窒素源に含まれる窒素元素の含有量は、３５ｐｐｍ以上７０ｐｐｍ以下である。液体培地中の窒素源に含まれる窒素元素の含有量が３５ｐｐｍ未満であると、緑藻クラミドモナスが増殖しにくくなる。一方、液体培地中の窒素源に含まれる窒素元素の含有量が７０ｐｐｍを超えると、緑藻クラミドモナス中のデンプンの蓄積量が低下する。すなわち、液体培地中の窒素源に含まれる窒素元素の含有量を７０ｐｐｍ以下とすることで、緑藻クラミドモナスからより多くのエタノールを生成できるため、効率的にバイオエタノールを生産することができる。
The content of nitrogen element contained in the nitrogen source in the liquid medium is 35 ppm or more and 70 ppm or less. If the nitrogen element content in the nitrogen source in the liquid medium is less than 35 ppm, the growth of the green alga Chlamydomonas becomes difficult. On the other hand, when the nitrogen element content in the nitrogen source in the liquid medium exceeds 70 ppm, the amount of starch accumulated in the green alga Chlamydomonas decreases. That is, by setting the content of nitrogen element contained in the nitrogen source in the liquid medium to 70 ppm or less, more ethanol can be produced from the green alga Chlamydomonas, and bioethanol can be efficiently produced.
窒素源としては、特に限定されないが、例えば、アンモニウム塩、硝酸塩等の無機窒素源、ペプトン、尿素等の有機窒素源が挙げられる。
The nitrogen source is not particularly limited, but examples include inorganic nitrogen sources such as ammonium salts and nitrates, and organic nitrogen sources such as peptone and urea.
例えば、窒素源として、尿素を用いる場合、液体培地中の尿素の含有量は、１５０ｐｐｍ以下であることが好ましく、８０ｐｐｍ以上１５０ｐｐｍ以下であることがさらに好ましい。
For example, when urea is used as the nitrogen source, the content of urea in the liquid medium is preferably 150 ppm or less, more preferably 80 ppm or more and 150 ppm or less.
液体培地中のリン元素の含有量は、４２０ｐｐｍ以上であり、５００ｐｐｍ以上であることが好ましい。液体培地中のリン元素の含有量が４２０ｐｐｍ未満であると、緑藻クラミドモナスが増殖しにくくなる。
The content of elemental phosphorus in the liquid medium is 420 ppm or more, preferably 500 ppm or more. If the content of elemental phosphorus in the liquid medium is less than 420 ppm, the growth of the green alga Chlamydomonas becomes difficult.
リン元素は、リン酸塩に含まれることが好ましい。これにより、緑藻クラミドモナスが増殖しやすくなる。
The phosphorus element is preferably contained in the phosphate. This facilitates the growth of the green alga Chlamydomonas.
リン酸塩としては、特に限定されないが、例えば、リン酸水素二カリウム（Ｋ２ＨＰＯ４）、リン酸二水素カリウム（ＫＨ２ＰＯ４）、リン酸カルシウム（Ｃａ３（ＰＯ４）２）等が挙げられる。
The phosphate is not particularly limited, but examples thereof include dipotassium hydrogen phosphate (K 2 HPO 4 ), potassium dihydrogen phosphate (KH 2 PO 4 ), calcium phosphate (Ca 3 (PO 4 ) 2 ), and the like. be done.
例えば、リン酸塩として、リン酸水素二カリウムおよびリン酸二水素カリウムを用いる場合、液体培地中のリン酸水素二カリウムの含有量は、１４４０ｐｐｍ以上であることが好ましく、１７３０ｐｐｍ以上であることがさらに好ましい。また、液体培地中のリン酸二水素カリウムの含有量は、７２０ｐｐｍ以上であることが好ましく、８６０ｐｐｍ以上であることがさらに好ましい。
For example, when dipotassium hydrogen phosphate and potassium dihydrogen phosphate are used as phosphates, the content of dipotassium hydrogen phosphate in the liquid medium is preferably 1440 ppm or more, preferably 1730 ppm or more. More preferred. Also, the content of potassium dihydrogen phosphate in the liquid medium is preferably 720 ppm or more, more preferably 860 ppm or more.
液体培地中の硫黄源の含有量は、特に限定されないが、例えば、４.５ｐｐｍ以上である。
The content of the sulfur source in the liquid medium is not particularly limited, but is, for example, 4.5 ppm or more.
硫黄源としては、特に限定されないが、例えば、硫酸マグネシウム、硫酸亜鉛、硫酸鉄、硫酸銅、硫酸カルシウム等が挙げられる。
The sulfur source is not particularly limited, but examples include magnesium sulfate, zinc sulfate, iron sulfate, copper sulfate, calcium sulfate, and the like.
液体培地は、塩化カルシウム、ホウ酸、塩化マンガン、七モリブデン酸六アンモニウム、硫酸亜鉛、硫酸鉄、硫酸銅等のミネラルをさらに含んでいてもよい。
The liquid medium may further contain minerals such as calcium chloride, boric acid, manganese chloride, hexaammonium heptamolybdate, zinc sulfate, iron sulfate, and copper sulfate.
液体培地中で緑藻クラミドモナスを培養する方法としては、特に限定されず、例えば、静置培養法、振盪培養法、深部培養法、通気培養法等が挙げられる。
The method for culturing the green alga Chlamydomonas in a liquid medium is not particularly limited, and examples include stationary culture, shaking culture, submerged culture, and aeration culture.
液体培地中で緑藻クラミドモナスを培養する際に、例えば、人工気象器を用いることができる。
For example, an artificial climate vessel can be used when culturing the green alga Chlamydomonas in a liquid medium.
液体培地中で緑藻クラミドモナスを培養する際の温度は、特に限定されないが、例えば、２℃以上３８℃以下である。
The temperature for culturing the green alga Chlamydomonas in the liquid medium is not particularly limited, but is, for example, 2°C or higher and 38°C or lower.
液体培地中で緑藻クラミドモナスを培養する際の明暗周期は、特に限定されないが、例えば、明期が６時間以上２４時間以下の２４時間周期である。
The light-dark cycle when culturing the green algae Chlamydomonas in a liquid medium is not particularly limited, but is, for example, a 24-hour cycle with a light period of 6 hours or more and 24 hours or less.
明期の光合成有効光量子束密度は、特に限定されないが、例えば、５０μｍｏｌ／ｍ２／ｓ以上２０００μｍｏｌ／ｍ２／ｓ以下である。
The photosynthetically effective photon flux density in the light period is not particularly limited, but is, for example, 50 μmol/m 2 /s or more and 2000 μmol/m 2 /s or less.
液体培地中で緑藻クラミドモナスを通気培養する際に、炭酸ガスを含むガス組成物を通気することが好ましい。
When aerating the green alga Chlamydomonas in a liquid medium, it is preferable to aerate a gas composition containing carbon dioxide gas.
炭酸ガスを含むガス組成物の供給方法としては、特に限定されず、公知の方法を用いることができる。
The method of supplying the gas composition containing carbon dioxide is not particularly limited, and known methods can be used.
ガス組成物中の炭酸ガスの含有量は、特に限定されないが、例えば、０.５体積％以上５体積％以下である。
The content of carbon dioxide gas in the gas composition is not particularly limited, but is, for example, 0.5% by volume or more and 5% by volume or less.
ガス組成物の通気量は、特に限定されないが、例えば、０.０５ｖｖｍ以上０.５ｖｖｍ以下である。
The amount of permeation of the gas composition is not particularly limited, but is, for example, 0.05 vvm or more and 0.5 vvm or less.
緑藻クラミドモナスとしては、特に限定されないが、例えば、Ｈｏｎｄａ ＤＲＥＡＭＯ株（受託番号ＦＥＲＭ ＢＰ－２２３０６）等が挙げられる。
The green alga Chlamydomonas is not particularly limited, but includes, for example, the Honda DREAMO strain (accession number FERM BP-22306).
ここで、Ｈｏｎｄａ ＤＲＥＡＭＯ株は、暗嫌気下において、エタノール産生能を有し、且つ、凝集しつつ、増殖する能力を獲得した変異株である（特許文献１参照）。
Here, the Honda DREAMO strain is a mutant strain that has the ability to produce ethanol under dark anaerobic conditions and has acquired the ability to grow while aggregating (see Patent Document 1).
緑藻クラミドモナスとして、Ｈｏｎｄａ ＤＲＥＡＭＯ株を用いる場合、本実施形態の緑藻クラミドモナスの培養方法は、暗嫌気下において、Ｈｏｎｄａ ＤＲＥＡＭＯ株を培養して、Ｈｏｎｄａ ＤＲＥＡＭＯ株にエタノールを産生させる工程をさらに含んでいてもよい。
When the Honda DREAMO strain is used as the green alga Chlamydomonas, the method for cultivating the green alga Chlamydomonas according to the present embodiment may further include a step of culturing the Honda DREAMO strain under dark anaerobic conditions to cause the Honda DREAMO strain to produce ethanol. good.
培養された緑藻クラミドモナスを回収する方法としては、特に限定されないが、例えば、遠心分離法、濾過法等が挙げられる。
The method for collecting the cultured green alga Chlamydomonas is not particularly limited, but examples include centrifugation and filtration.
回収された緑藻クラミドモナスから、デンプン、エタノール等を抽出することができる。
Starch, ethanol, etc. can be extracted from the collected Chlamydomonas algae.
緑藻クラミドモナスからエタノールを生成する方法としては、特に限定されないが、例えば、緑藻クラミドモナス固有のエタノール産生能によって自己発酵させる方法、緑藻クラミドモナスから抽出したデンプンを糖化発酵させる方法等が挙げられる。
The method of producing ethanol from the green alga Chlamydomonas is not particularly limited, but includes, for example, a method of self-fermentation using the inherent ethanol-producing ability of the green alga Chlamydomonas, and a method of saccharifying and fermenting starch extracted from the green alga Chlamydomonas.
なお、回収された緑藻クラミドモナスは、必要に応じて、乾燥させてもよい。
The collected green alga Chlamydomonas may be dried if necessary.
緑藻クラミドモナスを乾燥させる方法としては、特に限定されないが、例えば、凍結乾燥法等が挙げられる。
The method for drying the green alga Chlamydomonas is not particularly limited, but includes, for example, a freeze-drying method.
以下、本発明の実施例を説明するが、本発明は、実施例に限定されるものではない。
Examples of the present invention will be described below, but the present invention is not limited to the examples.
［比較例１］
人工気象器を用いて、所定の組成（表１参照）の液体培地中、以下に示す培養条件で緑藻クラミドモナス（Ｈｏｎｄａ ＤＲＥＡＭＯ株）を７日間通気培養した。ここで、液体培地は、窒素源（尿素）に含まれる窒素元素の含有量が１３０．９ｐｐｍであり、リン元素の含有量が４２０ｐｐｍである。 [Comparative Example 1]
Green alga Chlamydomonas (Honda DREAMO strain) was aerated for 7 days in a liquid medium having a predetermined composition (see Table 1) using an artificial climate chamber under the following culture conditions. Here, the liquid medium has a nitrogen element content of 130.9 ppm and a phosphorus element content of 420 ppm contained in the nitrogen source (urea).
人工気象器を用いて、所定の組成（表１参照）の液体培地中、以下に示す培養条件で緑藻クラミドモナス（Ｈｏｎｄａ ＤＲＥＡＭＯ株）を７日間通気培養した。ここで、液体培地は、窒素源（尿素）に含まれる窒素元素の含有量が１３０．９ｐｐｍであり、リン元素の含有量が４２０ｐｐｍである。 [Comparative Example 1]
Green alga Chlamydomonas (Honda DREAMO strain) was aerated for 7 days in a liquid medium having a predetermined composition (see Table 1) using an artificial climate chamber under the following culture conditions. Here, the liquid medium has a nitrogen element content of 130.9 ppm and a phosphorus element content of 420 ppm contained in the nitrogen source (urea).
温度：２５℃
明暗周期：明期１０時間、暗期１４時間
明期の光合成有効光量子束密度：４４０μｍｏｌ／ｍ２／ｓ
光源：白色ＬＥＤ
ガス組成物の通気量：０．１５ｖｖｍ
ガス組成物中の炭酸ガスの含有量：５体積％ Temperature: 25°C
Light-dark cycle: light period 10 hours, dark period 14 hours Photosynthetically effective photon flux density in light period: 440 μmol/m 2 /s
Light source: White LED
Aeration amount of gas composition: 0.15 vvm
Carbon dioxide content in gas composition: 5% by volume
明暗周期：明期１０時間、暗期１４時間
明期の光合成有効光量子束密度：４４０μｍｏｌ／ｍ２／ｓ
光源：白色ＬＥＤ
ガス組成物の通気量：０．１５ｖｖｍ
ガス組成物中の炭酸ガスの含有量：５体積％ Temperature: 25°C
Light-dark cycle: light period 10 hours, dark period 14 hours Photosynthetically effective photon flux density in light period: 440 μmol/m 2 /s
Light source: White LED
Aeration amount of gas composition: 0.15 vvm
Carbon dioxide content in gas composition: 5% by volume
［実施例１］
液体培地中の窒素源に含まれる窒素元素の含有量を１／３（４３．６ｐｐｍ；尿素９３．５ｐｐｍ）、リン元素の含有量を１．２倍（５０４ｐｐｍ；リン酸水素二カリウム１７２８ｐｐｍ、リン酸二水素カリウム８６４ｐｐｍ）とした以外は、比較例１と同様にして、緑藻クラミドモナスを培養した。 [Example 1]
The content of nitrogen element contained in the nitrogen source in the liquid medium is 1/3 (43.6 ppm; urea 93.5 ppm), and the content of phosphorus element is 1.2 times (504 ppm; dipotassium hydrogen phosphate 1728 ppm, phosphorus The green alga Chlamydomonas was cultured in the same manner as in Comparative Example 1, except that the amount of potassium dihydrogen acid was 864 ppm).
液体培地中の窒素源に含まれる窒素元素の含有量を１／３（４３．６ｐｐｍ；尿素９３．５ｐｐｍ）、リン元素の含有量を１．２倍（５０４ｐｐｍ；リン酸水素二カリウム１７２８ｐｐｍ、リン酸二水素カリウム８６４ｐｐｍ）とした以外は、比較例１と同様にして、緑藻クラミドモナスを培養した。 [Example 1]
The content of nitrogen element contained in the nitrogen source in the liquid medium is 1/3 (43.6 ppm; urea 93.5 ppm), and the content of phosphorus element is 1.2 times (504 ppm; dipotassium hydrogen phosphate 1728 ppm, phosphorus The green alga Chlamydomonas was cultured in the same manner as in Comparative Example 1, except that the amount of potassium dihydrogen acid was 864 ppm).
［実施例２］
液体培地中の窒素源に含まれる窒素元素の含有量を１／２（６５．５ｐｐｍ；尿素１４０．３ｐｐｍ）とした以外は、比較例１と同様にして、緑藻クラミドモナスを培養した。 [Example 2]
The green alga Chlamydomonas was cultured in the same manner as in Comparative Example 1, except that the nitrogen element content in the nitrogen source in the liquid medium was halved (65.5 ppm; urea: 140.3 ppm).
液体培地中の窒素源に含まれる窒素元素の含有量を１／２（６５．５ｐｐｍ；尿素１４０．３ｐｐｍ）とした以外は、比較例１と同様にして、緑藻クラミドモナスを培養した。 [Example 2]
The green alga Chlamydomonas was cultured in the same manner as in Comparative Example 1, except that the nitrogen element content in the nitrogen source in the liquid medium was halved (65.5 ppm; urea: 140.3 ppm).
［比較例２］
液体培地中の窒素源に含まれる窒素元素の含有量を１／４（３２．７ｐｐｍ；尿素７０．１ｐｐｍ）とした以外は、比較例１と同様にして、緑藻クラミドモナスを培養した。 [Comparative Example 2]
Green alga Chlamydomonas was cultured in the same manner as in Comparative Example 1, except that the nitrogen element content in the nitrogen source in the liquid medium was changed to 1/4 (32.7 ppm; urea: 70.1 ppm).
液体培地中の窒素源に含まれる窒素元素の含有量を１／４（３２．７ｐｐｍ；尿素７０．１ｐｐｍ）とした以外は、比較例１と同様にして、緑藻クラミドモナスを培養した。 [Comparative Example 2]
Green alga Chlamydomonas was cultured in the same manner as in Comparative Example 1, except that the nitrogen element content in the nitrogen source in the liquid medium was changed to 1/4 (32.7 ppm; urea: 70.1 ppm).
［デンプンの生産量］
回収した液体培地３ｍｌにα－アミラーゼ１ｍｌを添加した後、９５℃の温水バスに３０分間浸漬した。次に、空冷してからグルコアミラーゼ１ｍｌを添加した後、５０℃の温水バスに２時間浸漬した。次に、３ｍｌになるよう純水でメスアップした後、酵素電極法により、グルコース濃度を測定し、液体培地１Ｌあたりのデンプンの生産量［ｇ］を求めた。 [Starch production]
After adding 1 ml of α-amylase to 3 ml of the collected liquid medium, the mixture was immersed in a hot water bath at 95° C. for 30 minutes. Next, after cooling with air, 1 ml of glucoamylase was added, and the mixture was immersed in a warm water bath at 50°C for 2 hours. Next, after adding pure water to a volume of 3 ml, the glucose concentration was measured by the enzymatic electrode method, and the production amount [g] of starch per 1 L of liquid medium was determined.
回収した液体培地３ｍｌにα－アミラーゼ１ｍｌを添加した後、９５℃の温水バスに３０分間浸漬した。次に、空冷してからグルコアミラーゼ１ｍｌを添加した後、５０℃の温水バスに２時間浸漬した。次に、３ｍｌになるよう純水でメスアップした後、酵素電極法により、グルコース濃度を測定し、液体培地１Ｌあたりのデンプンの生産量［ｇ］を求めた。 [Starch production]
After adding 1 ml of α-amylase to 3 ml of the collected liquid medium, the mixture was immersed in a hot water bath at 95° C. for 30 minutes. Next, after cooling with air, 1 ml of glucoamylase was added, and the mixture was immersed in a warm water bath at 50°C for 2 hours. Next, after adding pure water to a volume of 3 ml, the glucose concentration was measured by the enzymatic electrode method, and the production amount [g] of starch per 1 L of liquid medium was determined.
［緑藻クラミドモナスの生産量］
回収した液体培地１５ｍｌを遠心分離した後、沈降物を純水で３回洗浄した。次に、Ｇ／Ｆろ紙でろ過した後、ろ紙を６０℃の乾燥器で１時間以上乾燥させ、ろ紙の重量変化量から、液体培地１Ｌあたりの緑藻クラミドモナスの生産量［ｇ／Ｌ］を求めた。 [Production volume of green alga Chlamydomonas]
After centrifuging 15 ml of the collected liquid medium, the sediment was washed with pure water three times. Next, after filtering with a G/F filter paper, the filter paper is dried in a dryer at 60°C for 1 hour or more, and the production amount [g/L] of the green alga Chlamydomonas per 1 L of the liquid medium is obtained from the weight change of the filter paper. rice field.
回収した液体培地１５ｍｌを遠心分離した後、沈降物を純水で３回洗浄した。次に、Ｇ／Ｆろ紙でろ過した後、ろ紙を６０℃の乾燥器で１時間以上乾燥させ、ろ紙の重量変化量から、液体培地１Ｌあたりの緑藻クラミドモナスの生産量［ｇ／Ｌ］を求めた。 [Production volume of green alga Chlamydomonas]
After centrifuging 15 ml of the collected liquid medium, the sediment was washed with pure water three times. Next, after filtering with a G/F filter paper, the filter paper is dried in a dryer at 60°C for 1 hour or more, and the production amount [g/L] of the green alga Chlamydomonas per 1 L of the liquid medium is obtained from the weight change of the filter paper. rice field.
［緑藻クラミドモナス中のデンプンの蓄積量］
式
（液体培地１Ｌあたりのデンプンの生産量［ｇ／Ｌ］）／（液体培地１Ｌあたりの緑藻クラミドモナスの生産量［ｇ／Ｌ］）×１００
により、緑藻クラミドモナス中のデンプンの蓄積量［質量％］を算出した。 [Amount of accumulated starch in green alga Chlamydomonas]
Formula (Production amount of starch per 1 L of liquid medium [g/L])/(Production amount of green alga Chlamydomonas per 1 L of liquid medium [g/L]) x 100
The accumulation amount [% by mass] of starch in the green alga Chlamydomonas was calculated.
式
（液体培地１Ｌあたりのデンプンの生産量［ｇ／Ｌ］）／（液体培地１Ｌあたりの緑藻クラミドモナスの生産量［ｇ／Ｌ］）×１００
により、緑藻クラミドモナス中のデンプンの蓄積量［質量％］を算出した。 [Amount of accumulated starch in green alga Chlamydomonas]
Formula (Production amount of starch per 1 L of liquid medium [g/L])/(Production amount of green alga Chlamydomonas per 1 L of liquid medium [g/L]) x 100
The accumulation amount [% by mass] of starch in the green alga Chlamydomonas was calculated.
［デンプンの生産量に及ぼす液体培地中の尿素に含まれる窒素元素の含有量の影響］
図１に、リン元素の含有量が４２０ｐｐｍに設定された液体培地中の尿素の含有量に対するデンプンの生産量の関係を示す。 [Influence of content of nitrogen element contained in urea in liquid medium on starch production]
FIG. 1 shows the relationship between the amount of starch produced and the content of urea in a liquid medium in which the elemental phosphorus content was set to 420 ppm.
図１に、リン元素の含有量が４２０ｐｐｍに設定された液体培地中の尿素の含有量に対するデンプンの生産量の関係を示す。 [Influence of content of nitrogen element contained in urea in liquid medium on starch production]
FIG. 1 shows the relationship between the amount of starch produced and the content of urea in a liquid medium in which the elemental phosphorus content was set to 420 ppm.
図１から、液体培地中の窒素源である尿素に含まれる窒素元素の含有量を、３２．７ｐｐｍ（尿素７０．１ｐｐｍ）、４３．６ｐｐｍ（尿素９３．５ｐｐｍ）、６５．５ｐｐｍ（尿素１４０．３ｐｐｍ）、１３０．９ｐｐｍ（尿素２８０．５ｐｐｍ）と変化させると、窒素元素の含有量が４３．６ｐｐｍのときに、デンプンの生産量が最大となることがわかる。また、窒素元素の含有量が６５．５ｐｐｍのときに、デンプンの生産量が２番目に大きくなり、窒素元素の含有量が３２．７ｐｐｍおよび１３０．９ｐｐｍのときに、デンプンの生産量が減少していることがわかる。
From FIG. 1, the contents of nitrogen elements contained in urea, which is the nitrogen source in the liquid medium, are 32.7 ppm (70.1 ppm of urea), 43.6 ppm (93.5 ppm of urea), 65.5 ppm (140 ppm of urea). 3 ppm) and 130.9 ppm (urea 280.5 ppm), it can be seen that when the nitrogen element content is 43.6 ppm, the amount of starch production is maximized. In addition, when the nitrogen element content is 65.5 ppm, the starch production amount is the second largest, and when the nitrogen element content is 32.7 ppm and 130.9 ppm, the starch production amount decreases. It can be seen that
［緑藻クラミドモナスの生産量および緑藻クラミドモナス中のデンプンの蓄積量に及ぼす液体培地中の尿素に含まれる窒素元素の含有量の影響］
図２に、リン元素の含有量が４２０ｐｐｍに設定された液体培地中の尿素の含有量に対する緑藻クラミドモナスの生産量および緑藻クラミドモナス中のデンプンの蓄積量の関係を示す。 [Influence of content of nitrogen element contained in urea in liquid medium on production amount of green alga Chlamydomonas and accumulation of starch in green alga Chlamydomonas]
FIG. 2 shows the relationship between the production amount of green alga Chlamydomonas and the amount of starch accumulated in Chlamydomonas green algae with respect to the content of urea in a liquid medium in which the elemental phosphorus content was set to 420 ppm.
図２に、リン元素の含有量が４２０ｐｐｍに設定された液体培地中の尿素の含有量に対する緑藻クラミドモナスの生産量および緑藻クラミドモナス中のデンプンの蓄積量の関係を示す。 [Influence of content of nitrogen element contained in urea in liquid medium on production amount of green alga Chlamydomonas and accumulation of starch in green alga Chlamydomonas]
FIG. 2 shows the relationship between the production amount of green alga Chlamydomonas and the amount of starch accumulated in Chlamydomonas green algae with respect to the content of urea in a liquid medium in which the elemental phosphorus content was set to 420 ppm.
図２から、液体培地中の窒素源である尿素に含まれる窒素元素の含有量を３２．７ｐｐｍ（尿素７０．１ｐｐｍ）、４３．６ｐｐｍ（尿素９３．５ｐｐｍ）、６５．５ｐｐｍ（尿素１４０．３ｐｐｍ）、１３０．９ｐｐｍ（尿素２８０．５ｐｐｍ）と変化させると、緑藻クラミドモナスの生産量は、窒素元素の含有量が減少するのに伴い、減少していることがわかる。一方で、デンプンの蓄積量は、窒素元素の含有量が減少するのに伴い、増加していることがわかる。
From FIG. 2, the contents of nitrogen elements contained in urea, which is the nitrogen source in the liquid medium, are 32.7 ppm (urea 70.1 ppm), 43.6 ppm (urea 93.5 ppm), 65.5 ppm (urea 140.3 ppm). ), 130.9 ppm (urea 280.5 ppm), the production of the green alga Chlamydomonas decreased as the nitrogen element content decreased. On the other hand, it can be seen that the accumulated amount of starch increases as the nitrogen element content decreases.
［緑藻クラミドモナスの生産量に及ぼす液体培地中のリン元素の含有量の影響］
液体培地中のリン酸塩の含有量を変更した以外は、実施例１と同様にして、緑藻クラミドモナスを培養した。 [Effect of elemental phosphorus content in liquid medium on production of green alga Chlamydomonas]
The green alga Chlamydomonas was cultured in the same manner as in Example 1, except that the phosphate content in the liquid medium was changed.
液体培地中のリン酸塩の含有量を変更した以外は、実施例１と同様にして、緑藻クラミドモナスを培養した。 [Effect of elemental phosphorus content in liquid medium on production of green alga Chlamydomonas]
The green alga Chlamydomonas was cultured in the same manner as in Example 1, except that the phosphate content in the liquid medium was changed.
図３に、液体培地中のリン酸塩の含有量に対する緑藻クラミドモナスの生産量の関係を示す。
Fig. 3 shows the relationship between the production amount of the green alga Chlamydomonas and the phosphate content in the liquid medium.
図３から、液体培地中のリン酸塩の含有量が２１６０ｐｐｍ未満である、すなわち、液体培地中のリン元素の含有量が４２０ｐｐｍ未満であると、緑藻クラミドモナスの生産量が低下することがわかる。
From FIG. 3, it can be seen that when the phosphate content in the liquid medium is less than 2160 ppm, that is, when the elemental phosphorus content in the liquid medium is less than 420 ppm, the production of the green alga Chlamydomonas decreases.
［デンプンの生産量］
図４に、実施例１、２および比較例１、２のデンプンの生産量を示す。 [Starch production]
FIG. 4 shows the production amounts of starch in Examples 1 and 2 and Comparative Examples 1 and 2.
図４に、実施例１、２および比較例１、２のデンプンの生産量を示す。 [Starch production]
FIG. 4 shows the production amounts of starch in Examples 1 and 2 and Comparative Examples 1 and 2.
図４から、実施例２は、比較例１および比較例２と対比して、デンプンの生産量が多いことがわかる。また、液体培地中の尿素に含まれるリン元素の含有量を１．２倍に調整した実施例１では、デンプンの生産量が最も多いことがわかる。
From FIG. 4, it can be seen that Example 2 produced a large amount of starch compared to Comparative Examples 1 and 2. Moreover, it can be seen that in Example 1 in which the content of elemental phosphorus contained in urea in the liquid medium was adjusted to 1.2 times, the amount of starch produced was the highest.
Claims (6)
- 液体培地中で緑藻クラミドモナスを培養して、前記緑藻クラミドモナスにデンプンを蓄積させる工程を含み、
前記液体培地は、窒素源に含まれる窒素元素の含有量が３５ｐｐｍ以上７０ｐｐｍ以下であり、リン元素の含有量が４２０ｐｐｍ以上である、緑藻クラミドモナスの培養方法。 culturing the green alga Chlamydomonas in a liquid medium to accumulate starch in the green alga Chlamydomonas;
A method for cultivating green alga Chlamydomonas, wherein the liquid medium contains 35 ppm or more and 70 ppm or less of elemental nitrogen and a content of 420 ppm or more of elemental phosphorus contained in the nitrogen source. - 前記窒素源は、尿素である、請求項１に記載の緑藻クラミドモナスの培養方法。 The method for culturing the green alga Chlamydomonas according to claim 1, wherein the nitrogen source is urea.
- 前記リン元素は、リン酸塩に含まれる、請求項１または２に記載の緑藻クラミドモナスの培養方法。 The method for culturing the green alga Chlamydomonas according to claim 1 or 2, wherein the elemental phosphorus is contained in a phosphate.
- 前記リン酸塩は、リン酸水素二カリウムおよびリン酸二水素カリウムである、請求項３に記載の緑藻クラミドモナスの培養方法。 The method for cultivating the green alga Chlamydomonas according to claim 3, wherein the phosphates are dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
- 前記液体培地は、リン酸水素二カリウムの含有量が１４４０ｐｐｍ以上であり、リン酸二水素カリウムの含有量が７２０ｐｐｍ以上である、請求項４に記載の緑藻クラミドモナスの培養方法。 The method for cultivating the green alga Chlamydomonas according to claim 4, wherein the liquid medium has a content of dipotassium hydrogen phosphate of 1440 ppm or more and a content of potassium dihydrogen phosphate of 720 ppm or more.
- 前記緑藻クラミドモナスは、Ｈｏｎｄａ ＤＲＥＡＭＯ株（受託番号ＦＥＲＭ ＢＰ－２２３０６）である、請求項１から５のいずれか一項に記載の緑藻クラミドモナスの培養方法。 The method for culturing the green alga Chlamydomonas according to any one of claims 1 to 5, wherein the green alga Chlamydomonas is Honda DREAMO strain (accession number FERM BP-22306).
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015025552A1 (en) * | 2013-08-23 | 2015-02-26 | 国立大学法人神戸大学 | Method for generating oil/fat component and method for producing higher unsaturated fatty acid |
| WO2017217116A1 (en) * | 2016-06-17 | 2017-12-21 | 本田技研工業株式会社 | Novel microalga having aggregation ability |
| JP2019527052A (en) * | 2016-06-30 | 2019-09-26 | クーンル アグロシステムズ，インコーポレイテッド | Improved heterotrophic production method and bioproducts for microbial biomass |
-
2022
- 2022-09-27 WO PCT/JP2022/035883 patent/WO2023074226A1/en unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015025552A1 (en) * | 2013-08-23 | 2015-02-26 | 国立大学法人神戸大学 | Method for generating oil/fat component and method for producing higher unsaturated fatty acid |
| WO2015025553A1 (en) * | 2013-08-23 | 2015-02-26 | 国立大学法人神戸大学 | Method for generating oil/fat component, method for producing higher unsaturated fatty acid, and chlamydomonas species strain jsc4 |
| WO2017217116A1 (en) * | 2016-06-17 | 2017-12-21 | 本田技研工業株式会社 | Novel microalga having aggregation ability |
| JP2019527052A (en) * | 2016-06-30 | 2019-09-26 | クーンル アグロシステムズ，インコーポレイテッド | Improved heterotrophic production method and bioproducts for microbial biomass |
Non-Patent Citations (5)
| Title |
|---|
| BARRETT MICHAEL R., KOCH ARTHUR R.: "Effects of Ammonium Sulfate and Urea on the Growth of Chlorophycean Algae From Rice Fields", JOURNAL OF ENVIRONMENTAL QUALITY, AMERICAN SOCIETY OF AGRONOMY, CROP SCIENCE SOCIETY OF AMERICA AND SOIL SCIENCE SOCIETY OF AMERICA, US, vol. 11, no. 2, 1 April 1982 (1982-04-01), US , pages 187 - 191, XP093060961, ISSN: 0047-2425, DOI: 10.2134/jeq1982.00472425001100020009x * |
| BATISTA ALINE D.; ROSA RINAMARA M.; MACHADO MARIANA; MAGALHãES ALAN S.; SHALAGUTI BáRBARA A.; GOMES PRISCILLA F.; COVELL: "Increased urea availability promotes adjustments in C/N metabolism and lipid content without impacting growth inChlamydomonas reinhardtii", METABOLOMICS, SPRINGER US, NEW YORK, vol. 15, no. 3, 28 February 2019 (2019-02-28), New York, pages 1 - 14, XP036762665, ISSN: 1573-3882, DOI: 10.1007/s11306-019-1496-3 * |
| GOPAL NIKHIL, SUDHAKAR KUMARASWAMY: "Ultraviolet modification of Chlamydomonas reinhardtii for carbon capture", RESEARCH AND REPORTS IN BIOLOGY, vol. 7, 1 April 2016 (2016-04-01), pages 41 - 46, XP093060955, DOI: 10.2147/RRB.S98536 * |
| YANG LEI, CHEN JUN, QIN SHAN, ZENG MIN, JIANG YONGGUANG, HU LANG, XIAO PENG, HAO WENLONG, HU ZHANGLI, LEI ANPING, WANG JIANGXIN: "Growth and lipid accumulation by different nutrients in the microalga Chlamydomonas reinhardtii", BIOTECHNOLOGY FOR BIOFUELS, vol. 11, no. 1, 1 December 2018 (2018-12-01), pages 40, XP093060960, DOI: 10.1186/s13068-018-1041-z * |
| YU QIAN, HE JIAYI, ZHAO QIANQIAN, WANG XIUFENG, ZHI YANNA, LI XIAONAN, LI XIANJUN, LI LONGJIAN, GE BAOSHENG: "Regulation of nitrogen source for enhanced photobiological H2 production by co-culture of Chlamydomonas reinhardtii and Mesorhizobium sangaii", ALGAL RESEARCH, ELSEVIER BV, NL, vol. 58, 1 October 2021 (2021-10-01), NL , pages 102422, XP093060962, ISSN: 2211-9264, DOI: 10.1016/j.algal.2021.102422 * |
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