WO2015122578A1 - Nouveau nephroselmis sp., nouvelle souche kge2 et procédé pour augmenter la teneur d'une nouvelle souche appropriée en acides gras - Google Patents

Nouveau nephroselmis sp., nouvelle souche kge2 et procédé pour augmenter la teneur d'une nouvelle souche appropriée en acides gras Download PDF

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WO2015122578A1
WO2015122578A1 PCT/KR2014/005699 KR2014005699W WO2015122578A1 WO 2015122578 A1 WO2015122578 A1 WO 2015122578A1 KR 2014005699 W KR2014005699 W KR 2014005699W WO 2015122578 A1 WO2015122578 A1 WO 2015122578A1
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strain
present
kge2
novel
nephroselmis
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PCT/KR2014/005699
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English (en)
Korean (ko)
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최재영
박영태
윤현식
송경근
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한국과학기술연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—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
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • 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
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • 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
    • 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
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/10—Nature of the water, waste water, sewage or sludge to be treated from quarries or from mining activities
    • 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
    • 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
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/30—Wastewater or sewage treatment systems using renewable energies
    • Y02W10/37—Wastewater or sewage treatment systems using renewable energies using solar energy

Definitions

  • the present invention relates to a novel strain of genus neproselmis and a method for increasing the fatty acid content in the strain using the same.
  • 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.
  • microalgae research for biofuel production has been limited to microalgae inhabiting the ocean, which may cause problems with the transport of large volumes of microalgae that are not dried from the ocean and must be injected with artificial minerals. There was a problem.
  • One aspect of the present invention to achieve the above object provides a Nephroselmis sp. KGE2 strain.
  • One aspect of the invention also provides a method of increasing the fatty acid content in a strain, comprising culturing the strain and wastewater together.
  • the novel strain of the present invention can be usefully used for biodiesel production, including fast growth rate and high content of lipids and fatty acids.
  • the novel strains of the present invention are isolated from acidic mines, and because of their resistance to heavy metals, they can maintain their activity even in wastewater.
  • the novel strain of the present invention has the advantage that it can grow using minerals in the wastewater to maintain its activity in harsh environments such as wastewater.
  • novel strains of the present invention can increase the content of fatty acids to the maximum when incubated with the mine drainage and wastewater, and can be used to produce alternative energy, which is available for biodiesel production.
  • novel strains of the present invention comprise a high content of lipids and thus high content of fatty acids such as palmitic acid, palmitoleic acid, oleic acid, linoleic acid (18: 2n6t), or g-linoleic acid. (18: 3n6) is contained in high content, and can be used usefully.
  • Figure 1 is a micrograph (1500 magnification) of the nephlocell miss genus KGE 2 (Nephroselmis sp.KGE2).
  • KGE 2 is a genus of Neplocell. The schematic of KGE 2 is shown.
  • Figure 5 is a table after measuring the amount of fatty acids in KGE 2 in neproselmis after treating mine drainage and livestock wastewater in various weight ratios.
  • the present invention relates to a four-cell Pro miss in KGE2 (Nephroselmis sp. KGE2) strain in one aspect.
  • Nephroselmis sp. KGE2 strain which is one aspect of the present invention, was isolated and identified for the first time by the present inventors.
  • the present inventors have for the separation of the identified new strains termed four pro-cell misses in KGE2 (Nephroselmis sp. KGE2) strains, July 30, the date in 2013 the deposit to deposit the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center number (KCTC 12456BP) Was granted.
  • KGE stands for KIST Gangneung Enviromental.
  • the strain of one aspect of the present invention may include the nucleotide sequence of SEQ ID NO: 3.
  • a strain in one aspect of the invention may comprise at least 30% by weight lipids relative to the total weight of the dry strain.
  • the content of lipids is difficult to exceed 20%.
  • the present invention contains 30% or more lipids, and a large amount of fatty acids can be actively utilized as biomass. Nephroselmis sp.
  • KGE2 strains of the present invention from the above point of view is 30.2% by weight, 30.4% by weight, 30.6% by weight, 30.8% by weight, 31% by weight, 31.2% by weight At least 31.4 wt%, at least 31.6 wt%, at least 31.8 wt%, 32 wt%, 32.2 wt%, 32.4 wt%, 32.6 wt%, 32.8 wt%, or at least 33 wt% lipid.
  • the strain may be Accession No. KCTC 12456BP.
  • a strain of one aspect of the present invention may include a high content of lipids and fatty acids as described above, and in particular, palmitoleic acid, heptadikenoic acid (Palmitoleic acid) Heptadecenoic acid, oleic acid (Oleic acid), may include one or more fatty acids selected from the group consisting of Linolelaidic acid (Linolelaidic acid).
  • palmitic acid is particularly suitable for fueling tropical climates because of its high oxidation stability and high cetane number (easily fuel ignition).
  • the present invention relates to a culture solution of the strain KGE2 of the genus Neproselmis.
  • the culture solution includes a product obtained by culturing the strain KGE2 of the genus Neproselmis, and has a constitution included in a conventional culture solution.
  • the present invention relates to a culturing method comprising the step of culturing the genus KGE2 strain of neproselmis in another aspect.
  • the culture method may include the step of culturing neproselmis genus KGE2 strain at 25 to 30 °C, pH 6 to 8 conditions, the growth rate of the new strain of the present invention may be high under the conditions.
  • 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 in a medium containing KH 2 PO 4 , CaCl 2 , MgSO 4 , NaNO 3 , K 2 HPO 4 , NaCl and H 3 BO 3 may comprise the step of culturing the genus KGE2 strain of neproselmis.
  • 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 present invention relates to a method of In another aspect, the four Pro cell misses in KGE2 (Nephroselmis sp. KGE2) increasing the fatty acid content in the, four Pro cell misses in KGE2 strain which comprises the culture with the strain and mine drainage and waste water .
  • KGE2 Nephroselmis sp. KGE2
  • KGE2 strain which comprises the culture with the strain and mine drainage and waste water .
  • the wastewater includes both wastewater or domestic wastewater including livestock manure.
  • the mine drainage may specifically include acid mine drainage, but is not limited thereto.
  • the livestock manure may mean manure of livestock containing a large amount of N, P.
  • the wastewater is at least one selected from the group consisting of wastewater and domestic wastewater including livestock manure.
  • the weight ratio between the acid mine drainage and the wastewater may be 0.5 to 2.5: 1. Within this range the production of fatty acids is the best. In view of the above, the weight ratio between the mine drainage and the wastewater may be 0.6 to 2.4: 1, 0.7 to 2.3: 1, 0.8 to 2.2: 1, 0.9 to 2.1: 1 or 1 to 2: 1.
  • the strain may be Accession No. KCTC 12456BP.
  • 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 isolated new strain sample was observed by magnification 1500 times through an optical microscope.
  • the new strain was placed in BBM liquid medium having the composition shown in Table 1 and cultured for 2 weeks.
  • the BBM liquid medium can provide an environment in which microalgae can be optimized for growth.
  • the microelement stock solution and the composition of solution 1 and solution 2 constituting the composition of the BBM liquid medium are shown in Table 2.
  • a 36-watt fluorescent lamp was used as a light source, and after two weeks of culture, an independent colony was collected and first spreaded evenly using platinum on an alar plate (15 g / L) containing BBM. After 8 days, the cultured colonies were second plated onto an alar plate containing BBM.
  • 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 a temperature 25 ⁇ 1 °C, the stirring speed 150rpm.
  • 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 '(SEQ ID NO: 2)
  • the present inventors named the isolated new strain KGE 2 ( Nephroselmis sp.KGE2) in neproselmis, deposited on August 19, 2013 at the Korea Biotechnology Research Institute microbial resource center and deposited the accession number KCTC 12456BP Granted.
  • MBR Primary treatment water that removes suspended solids by passing livestock wastewater through the membrane
  • 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) was transferred to a new container (with a centrifuged glass container and a Teflon liner inserted into the lid) using a paspice 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 lipid extract thus obtained, followed by centrifugation to transfer only the organic layer.
  • the organic layer was evaporated using a rotary vacuum evaporator to separate pure lipids. Thereafter, the dry weight of the pure lipid extract, the weight of the cultured microalgae and its dry weight were measured, and these were substituted into Equations 1 and 2 below to obtain Table 5 below.
  • Lipid content (%) dry weight of lipid extract (g) / dry weight of microalgae (g) X 100
  • Lipid productivity (g / L) microalgae production (g / L) X microalgal lipid content (%)
  • 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].
  • the present invention relates to a novel strain of genus neproselmis and a method for increasing the fatty acid content in the strain using the same.
  • the novel strain of the present invention can be usefully used for biodiesel production, including fast growth rate and high content of lipids and fatty acids.
  • the novel strains of the present invention are isolated from acidic mines, and because of their resistance to heavy metals, they can maintain their activity even in wastewater.
  • the novel strain of the present invention has the advantage that it can grow using minerals in the wastewater to maintain its activity in harsh environments such as wastewater.
  • novel strains of the present invention can increase the content of fatty acids to the maximum when incubated with the mine drainage and wastewater, and can be used to produce alternative energy, which is available for biodiesel production.
  • novel strains of the present invention comprise a high content of lipids and thus high content of fatty acids such as palmitic acid, palmitoleic acid, oleic acid, linoleic acid (18: 2n6t), or g-linoleic acid. (18: 3n6) is contained in high content, and can be used usefully.

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Abstract

La présente invention concerne une nouvelle souche de Nephroselmis sp. et un procédé pour augmenter la teneur d'une souche en acides gras, par son utilisation. La nouvelle souche de la présente invention présente un taux de croissance rapide et présente une teneur élevée en lipides et en acides gras, et peut donc être utile pour la production de biodiesel. De même, comme la nouvelle souche de la présente invention est séparée de la mine acide et a une résistance aux métaux lourds, son activité peut se conserver même dans les eaux résiduaires. En outre, la nouvelle souche de la présente invention présente l'avantage de pouvoir croître par utilisation des substances minérales se trouvant dans les eaux résiduaires, en conservant l'activité dans des environnements difficiles tels que les eaux résiduaires. En outre, dans le cas de la culture de la souche dans les eaux de mine et les eaux résiduaires, la nouvelle souche de la présente invention peut augmenter d'une manière maximale la teneur en l'acide gras, et est donc disponible pour la production d'un biodiesel et peut être utilisée pour produire une énergie alternative.
PCT/KR2014/005699 2014-02-11 2014-06-26 Nouveau nephroselmis sp., nouvelle souche kge2 et procédé pour augmenter la teneur d'une nouvelle souche appropriée en acides gras WO2015122578A1 (fr)

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KR10-2014-0015385 2014-02-11
KR1020140015385A KR101680048B1 (ko) 2014-02-11 2014-02-11 신규한 네프로셀미스 속 신균주 kge2 및 해당 신균주 내 지방산 함량을 증가시키는 방법

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8124400B2 (en) * 2008-12-10 2012-02-28 Synthetic Genomics, Inc. Production of branched-chain alcohols by photosynthetic microorganisms
WO2012087675A1 (fr) * 2010-12-23 2012-06-28 Exxonmobil Research And Engineering Company Culture d'un microorganisme dans un milieu contenant un niveau élevé d'une source de contre-ion carboxylate
KR101232279B1 (ko) * 2012-08-02 2013-02-12 한국과학기술연구원 고함량의 지질을 포함하는 신규한 균주

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101303946B1 (ko) * 2012-08-17 2013-09-05 한국과학기술연구원 광산배수를 이용하는 파이프 탈착형 미세조류 배양장치

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8124400B2 (en) * 2008-12-10 2012-02-28 Synthetic Genomics, Inc. Production of branched-chain alcohols by photosynthetic microorganisms
WO2012087675A1 (fr) * 2010-12-23 2012-06-28 Exxonmobil Research And Engineering Company Culture d'un microorganisme dans un milieu contenant un niveau élevé d'une source de contre-ion carboxylate
KR101232279B1 (ko) * 2012-08-02 2013-02-12 한국과학기술연구원 고함량의 지질을 포함하는 신규한 균주

Non-Patent Citations (2)

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Title
DATABASE GenBank [O] 15 July 2013 (2013-07-15), retrieved from ncbi Database accession no. HE861888.1 *
PARK, YOUNG-TAE ET AL.: "Removal of metal from acid mine drainage using a hybrid system including a pipes inserted microalgae reactor", BIORESOURCE TECHNOL., vol. 150, 10 October 2013 (2013-10-10), pages 242 - 248, XP028786650, ISSN: 0960-8524 *

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