WO2014021508A1 - Nouvelle souche à haute teneur en lipides - Google Patents

Nouvelle souche à haute teneur en lipides Download PDF

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Publication number
WO2014021508A1
WO2014021508A1 PCT/KR2012/009801 KR2012009801W WO2014021508A1 WO 2014021508 A1 WO2014021508 A1 WO 2014021508A1 KR 2012009801 W KR2012009801 W KR 2012009801W WO 2014021508 A1 WO2014021508 A1 WO 2014021508A1
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WIPO (PCT)
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strain
neproselmis
kge8
present
nephroselmis
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PCT/KR2012/009801
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English (en)
Korean (ko)
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최재영
윤현식
박영태
지은도
송경근
이우람
지민규
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한국과학기술연구원
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Publication of WO2014021508A1 publication Critical patent/WO2014021508A1/fr

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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
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/02—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only
    • C10L1/026—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only for compression ignition
    • 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
    • C12N1/125—Unicellular algae isolates
    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae
    • 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 novel strain comprising a high content of lipids.
  • bioenergy sources are mainly beans, sugar cane and the like.
  • the production efficiency of the biodiesel for the vast area required for cultivation thereof is low, and thus faces difficulties. Therefore, the study of microalgae as biomass is needed for stable and economic production of biodiesel.
  • the present inventors have made diligent efforts to solve the above problems, and have confirmed that the microalgae inhabiting acid mine drainage are novel strains containing lipids with high content and completed the present invention.
  • the present invention provides a Nephroselmis sp. Strain, culture medium of the strain and a method for culturing the strain, comprising at least 35% by weight of lipids relative to the total weight of the dry strain. to provide.
  • the novel strains of the present invention contain high amounts of lipids, high amounts of fatty acids, in particular palmitic acid, and can be usefully used for biodiesel production.
  • the novel strains of the present invention are also isolated from acid mines and have heavy metal resistance.
  • the novel strains of the present invention are high in lipid high fatty acids, especially palmitic acid, palmitoleic acid, oleic acid, linoleic acid (18: 2n6t), or g-linoleic acid (18: 3n6). In addition to containing a high content, the growth rate is fast, the productivity is excellent.
  • KCTC 12235BP KCTC 12235BP
  • Figure 2 is neproselmis sp. Of the present invention in BBM (Bold's Basal Medium). Strain ( Nephroselmis sp.KGE8, accession number KCTC 12235BP) Growth and nitrogen, phosphorus removal effect is shown.
  • neproselmis sp shows the lipid content of the strain ( Nephroselmis sp. KGE8, Accession No. KCTC 12235BP).
  • neproselmis sp Fatty acid distribution of strain ( Nephroselmis sp. KGE8, Accession No. KCTC 12235BP) is shown.
  • the strain was originally identified and identified by the inventors.
  • the present inventors identified the isolated new strain of neproselmis sp. It was named KGE8 ( Nephroselmis sp.KGE8, Accession No. KCTC 12235BP), and was deposited on July 3, 2012 at the Korea Research Institute of Bioscience and Biotechnology.
  • neproselmis sp. KGE8 (Nephroselmis sp.KGE8, Accession No. KCTC 12235BP) relates to a strain.
  • the invention in another aspect, relates to a Nephroselmis sp. Strain comprising at least 35% by weight of lipids relative to the total weight of the dry strain.
  • KGE stands for KIST Guengnung Enviromental.
  • the content of lipids is difficult to exceed mid 20%.
  • the present invention contains more than 35% of the lipids, and a large amount of fatty acids can be utilized as a biomass.
  • the nephroselmis sp. Strain of the present invention is at least 35.5 wt%, at least 36 wt%, at least 36.5 wt%, at least 37 wt%, at least 37.5 wt%, at least 38 wt% , At least 38.5 wt%, at least 39 wt%, at least 39.5 wt%, at least 40 wt%, at least 40.5 wt% or at least 41 wt% lipid.
  • the nephroselmis sp. Strain of the present invention is 90% by weight, 88% by weight, 86% by weight, 84% by weight, 82% by weight, 80% by weight or less , 78% by weight, 76% by weight, 74% by weight, 72% by weight, 70% by weight, 68% by weight, 66% by weight, 64% by weight, 62% by weight, 60% by weight Or up to 58% by weight of lipids.
  • the strain may be Nephroselmis pyriformis .
  • the strain may comprise the DNA sequence of SEQ ID NO: 1.
  • the strain may comprise the DNA sequence of SEQ ID NO: 1.
  • the strain may have an access number of KCTC 12235BP.
  • the present invention in another aspect, the present invention, the neproselmis sp. It relates to a culture medium in which the strain is cultured.
  • the culture solution was neproselmis sp.
  • the product obtained by culturing a strain is contained, and it has a structure contained in a normal culture liquid.
  • the present invention in another aspect, the present invention, the neproselmis sp. It is a culturing method of strains, and relates to a culturing method of culturing the strains under conditions of 25 to 30 °C, pH 6-8. Under these conditions, the growth rate of the new strain of the present invention is the highest.
  • the strain may be incubated at a temperature of 25.5 to 29.5 ° C, 26 to 29 ° C or 26.5 to 28.5 ° C, and may be cultured at pH 6.2 to 7.8, pH 6.4 to 7.6 or pH 6.4 to 7.4. have.
  • the culture method KH 2 PO 4 , CaCl 2 , MgSO 4 , NaNO 3 , K 2 HPO 4 , NaCl and H 3 BO 3
  • the neprocell Miss sp. Strains can be cultured.
  • the medium may further include one or more elements selected from the group consisting of ZnSO 4 , MnCl 2 , MoO 3 , CuSO 4 and Co (NO 3 ) 2 .
  • the samples were collected from the acid mine drainage area of Heungil Taebong, Mojeon-myeon, Gangneung-si, Gangwon-do. Sampling was carried out in the sterilized water sample pack (1L), the soil, water quality and cold storage using an ice box to the laboratory. The collected sample was partially separated and washed and centrifuged three times using sterilized DIW to obtain a new strain.
  • the new strain was placed in BBM liquid medium having the composition shown in Table 5 and allowed to stand for 2 weeks.
  • the composition of the solution constituting the BBM liquid medium is shown in Tables 1 to 4.
  • a 36 watt fluorescent lamp was used as the light source.
  • an independent colony was taken and first spread evenly spread using a platinum plate on an alar plate (15 g / L) containing BBM. After 8 days, the cultured colonies were second plated onto an alar plate containing BBM. After 12 days, the cultured colonies were harvested and the microalgae were dispersed in a Erlenmeyer flask containing a liquid medium containing BBM. And microalgae are incubated using a constant temperature shaker with a fluorescent lamp. At this time, the culture conditions were the temperature 25 °C, stirring speed 150rpm.
  • KGE 8 Nephroselmis sp. KGE8, Accession No. KCTC 12235BP was analyzed by 28 s rRNA sequencing.
  • microalgae A portion of the microalgae was taken during the cultivation and centrifuged to use the sample for analysis.
  • the microalgae were extracted using a DNA extraction kit (SolGent, Korea), and the extracted DNA was amplified by PCR. Primer 28S D1D2 was used.
  • Reverse primer 5'-TACTAGA-AGGTTCGATTAGTC-3 '
  • the resulting nucleotide sequence is described in SEQ ID NO: 1, using the base sequence was prepared by referring to the NCBI information (Fig. 1).
  • the new strain of the present invention was identified to have a homology of 89% with Nephroselmis pyriformis (HE561886).
  • the present inventors have identified the strain of the present invention neproselmis sp. It was named KGE8 ( Nephroselmis sp.KGE8, Accession No. KCTC 12235BP), and was deposited on July 3, 2012 at the Korea Research Institute of Bioscience and Biotechnology.
  • the strain was incubated with BBM medium. After filling 300ml medium, a portion of the strain (OD 680nm 0.01) was inoculated and used. During the incubation, a fluorescent light source of 100umol / m 2 ⁇ s was used and incubated at 28 ° C.
  • the concentration of nitrogen and phosphorus with initial concentration of 50mg / L was measured using an analysis kit (C-Mac, Korea) with an initial concentration of 50mg / L of nitrogen and phosphorus, and absorbance at 680nm with UV meter (Hach, USA). (Optical Density) was measured to determine the growth rate.
  • the sample to which the solvent was added to the microalgae was mixed well with a high speed stirrer for 5 minutes and crushed using a sonicator for 30 minutes to start extraction.
  • the crushed microalgae were extracted with stirring (30 ° C., 150 rpm) for one day with a constant temperature water shaker under conditions of 150 rpm and 30 ° C.
  • 1.25ml of chloro forum was added to the extracted sample using a glass pipette, and further stirred in a constant temperature shaker for 2 hours.
  • 1.25ml of distilled water was added to the extracted sample by using a micropipette and centrifuged to separate an organic layer and a water layer.
  • the organic layer (primary extract) is transferred to a new container (with a Teflon liner inserted into a centrifuged glass container and lid) using a Paspan pipette and centrifuged once more by adding 1.25 ml of distilled water to the remaining material.
  • the organic layer (secondary extract) and the water layer were separated, and the organic layer was separated using a Paspan pipette, and the primary and secondary extracts were combined to obtain a lipid extract. 5 ml of NaCl (5%) was added to the thus obtained lipid extract, followed by centrifugation to transfer only the organic layer.
  • the organic layer was evaporated using a rotary vacuum evaporator to separate 207 mg of the dried lipid extract.
  • Total lipid content dry weight of lipid extract (mg) / dry weight of microalgae (mg) X 100
  • the growth rate of the microalgae was measured during the period from 7 days to 17 days after the start of microalgae culture. During the above period, the culture solution containing the microalgae was taken 1 L daily, centrifuged and lyophilized, and the difference in weight from the previous day's dry weight was calculated. As a result, the growth rate of the microalgae of the present invention was 560 mg / L day.
  • the lipid productivity of the microalgae of the present invention was calculated by substituting the growth rate and the total lipid content obtained in Equation 1 into Equation 2, and the results shown in Table 7 were obtained.
  • the lower layer (organic phase) was extracted with a disposable PP syringe (Norm-ject, Germany) and filtered with a disposable 0.22 ⁇ m PVDF syringe filter (Millex-Gv, Millipore, USA), followed by gas chromatography with an automatic injector [Model 7890 , Agilent, USA].
  • palmitic acid (16: 0) 27.3 wt%, palmitoleic acid (16: 1) 2.9 wt%, oleic acid (18: 1 n9) 13.4 wt%, linole Iksan (18: 2n6) 5.9 wt% and g-linoleic acid (18: 3n6) 43.7 wt% were detected. Therefore, it may be provided as an index for producing high quality materials in the production of biodiesel in the future.
  • the novel strains of the present invention contain high amounts of lipids, high amounts of fatty acids, in particular palmitic acid, and can be usefully used for biodiesel production.
  • the novel strains of the present invention are also isolated from acid mines and have heavy metal resistance.
  • the novel strains of the present invention are high in lipid high fatty acids, especially palmitic acid, palmitoleic acid, oleic acid, linoleic acid (18: 2n6t), or g-linoleic acid (18: 3n6). In addition to containing a high content, the growth rate is fast, the productivity is excellent.

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Abstract

Cette invention concerne une nouvelle souche à haute teneur en lipides. La nouvelle souche est riche en lipides, en acides gras et renferme, en particulier, de l'acide palmitique, elle peut donc être utilisée avec efficacité dans la production de biocarburant. La nouvelle souche est par ailleurs séparée d'un effluent minier acide et présente une résistance aux métaux lourds. Outre sa richesse en lipides, en acides gras et, en particulier, sa teneur élevée en acide palmitique, la souche de l'invention présente une excellente vitesse de croissance et donc une excellente productivité.
PCT/KR2012/009801 2012-08-02 2012-11-19 Nouvelle souche à haute teneur en lipides WO2014021508A1 (fr)

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KR10-2012-0084922 2012-08-02
KR1020120084922A KR101232279B1 (ko) 2012-08-02 2012-08-02 고함량의 지질을 포함하는 신규한 균주

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101767948B1 (ko) 2016-01-07 2017-08-17 한국과학기술연구원 네프로셀미스 속 kge 8 미세조류를 사용하여 유용물질을 생산하는 방법

Families Citing this family (1)

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KR101680048B1 (ko) * 2014-02-11 2016-11-28 한국과학기술연구원 신규한 네프로셀미스 속 신균주 kge2 및 해당 신균주 내 지방산 함량을 증가시키는 방법

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US20110014665A1 (en) * 2007-06-01 2011-01-20 Solazyme, Inc. Production of Oil in Microorganisms
US20110020914A1 (en) * 2009-07-24 2011-01-27 Novus International Inc Methods for enhancing growth of organisms in an aqueous growth medium
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KR101147450B1 (ko) * 2010-05-04 2012-05-21 한국생명공학연구원 신규 유지성 미세조류 ｋｒｓ１０１ 균주 및 이를 이용한 바이오오일의 제조방법

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US20110020914A1 (en) * 2009-07-24 2011-01-27 Novus International Inc Methods for enhancing growth of organisms in an aqueous growth medium
KR101147450B1 (ko) * 2010-05-04 2012-05-21 한국생명공학연구원 신규 유지성 미세조류 ｋｒｓ１０１ 균주 및 이를 이용한 바이오오일의 제조방법
KR20110125576A (ko) * 2010-05-13 2011-11-21 한국생명공학연구원 북극 해양에서 분리한 지질 고생산 미세조류 클라미도모나스 세포주 및 이의 용도

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KR101767948B1 (ko) 2016-01-07 2017-08-17 한국과학기술연구원 네프로셀미스 속 kge 8 미세조류를 사용하여 유용물질을 생산하는 방법

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