WO2012005410A1 - Nouvelle souche ysl03 de chlamydomonas pitschmannii - Google Patents

Nouvelle souche ysl03 de chlamydomonas pitschmannii Download PDF

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WO2012005410A1
WO2012005410A1 PCT/KR2010/006561 KR2010006561W WO2012005410A1 WO 2012005410 A1 WO2012005410 A1 WO 2012005410A1 KR 2010006561 W KR2010006561 W KR 2010006561W WO 2012005410 A1 WO2012005410 A1 WO 2012005410A1
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ysl03
chlamydomonas
pitschmannii
strain
kctc
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PCT/KR2010/006561
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WO2012005410A9 (fr
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전병훈
샤납레다 에이 아이 아보우
황재훈
김영훈
오유관
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연세대학교 산학협력단
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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
    • C12N1/12—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
    • 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
    • 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
    • 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 Chlamydomonas pitschmanny high in wastewater resistance and lipid content YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain.
  • Biomass is a renewable energy source that does not affect the greenhouse effect [Widjaja, A., Chao-Chang Chien, Yi-Hsu Ju. 2009. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J. Taiwan Inst. Chem. Eng.40, 13-20.] It is continuously developed as it is used as energy in mass production.
  • Biodiesel is obtained from triglyceride oils and monohydric alcohols, and is a biomass energy source mainly produced from soybeans, sugar cane and animal fats [Lang, X., Dalai, AK. , Bakhshi, NN, Reaney, MJ, Hertz, PB, 2002. Preparation and characterization of biodiesels from various Bio-Oils. Biores. Technol. 80, 53-62.]. However, it is difficult to develop due to the food shortage theory.
  • algae which is an energy source capable of producing biodiesel in a stable and economical manner, are being developed due to the following characteristics of algae.
  • Algae are independent, heterotrophic photosynthetic bacteria that grow in the presence of solar energy, water, and carbon dioxide (sea, lake, river, etc.). 2) Better productivity than other materials (eg soybeans, corn, sugar cane, etc.) [Becker, 1994. Measurement of algal growth. In: Microalgae biotechnology and microbiology.
  • microalgae species having resistance to wastewater, growing rapidly in wastewater, and having a lipid content of 50% or more of biodiesel production material are separated from the natural environment, and thus, are useful for biodiesel production. .
  • the present invention is to solve the environmental pollution problem by using a novel microalgae in wastewater treatment, and to provide a method for solving the alternative energy by using the microalgae generated in the process as a raw material for biodiesel production.
  • Chlamydomonas Fitschmann YSL03 Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] strains and cultures thereof are characterized.
  • Chlamydomonas Fitschmann YSL03 Chlamydomonas pitschmannii YSL03
  • KCTC 11715BP Chlamydomonas Fitschmann YSL03
  • Chlamydomonas Fitschmann YSL03 Chlamydomonas pitschmannii YSL03
  • KCTC 11715BP Chlamydomonas pitschmannii YSL03
  • the present invention includes a method for culturing the new strain.
  • Chlamydomonas Fitschmann YSL03 Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] strain was used to examine wastewater resistance and lipid content as a biodiesel production source, thus securing the potential as a new strain for biodiesel production.
  • the new strain of the present invention has a strong wastewater resistance, and can directly apply N and P contained in the wastewater, thereby greatly improving the economics of the culture.
  • YSL03 Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] contains high content (more than 50%) lipids, which contributes to biodiesel production.
  • Chlamydomonas Fitschmann YSL03 Chlamydomonas pitschmannii YSL03
  • KCTC 11715BP Chlamydomonas Fitschmann YSL03
  • FIG. 1 Chlamydomonas Fitschmann The phylogenetic tree of YSL03 [KCTC 11715BP] is shown.
  • Fig. 4 Chlamydomonas Fitschmann Fatty acid distribution of YSL03 [KCTC 11715BP] [linolenic acid (C18: 3n6), linolenic acid (C18: 3n 3), oleic acid (C18: 1n9c), stearic acid (C18: 0), heptadecanoic acid (C17: 0 ), Palmitoleic acid (C16: 1), palmitic acid (C16: 0), myristic acid (C14: 0), lauryl acid (C12: 0)].
  • the present invention Chlamydomonas pitschmanny high in wastewater resistance and lipid content YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain.
  • the lipid content is at least 50% by weight of the dry cell weight, in particular, oleic acid and palmitic acid is characterized in that it contains at least 30% by weight of the total fatty acid.
  • Chlamydomonas pitschmanny a new strain of the present invention YSL03 was first identified and identified by the present inventors, and strains were isolated using BBM medium in sewage and wastewater treatment plants. As a result of analyzing the isolated strains by molecular genetic method by 28s rDNA sequencing sequence search, the standard strain was identified. Inn Klamidomonas Fitzmanny It was confirmed that the strain was 99% similar to AF183462.
  • the present inventors named the isolated and identified mycobacterium Klamidomonas Fitschmanny YSL03, and deposited on June 18, 2010 to the Korea Institute of Bioscience and Biotechnology Biological Resource Center was given accession number KCTC 11715BP.
  • Chlamydomonas Fitschmanny YSL03 can be cultured in wastewater.
  • the Chlamydomonas pitschmanny YSL03 is BBM medium (KH 2 PO 4 , CaCl 2 2H 2 O, MgSO 4 7H 2 O, NaNO 3 , K 2 HPO 4 , NaCl, H 3 BO 3 and trace amounts) Culture in an element containing an element).
  • the trace element may be at least one selected from the group consisting of ZnSO 4 .7H 2 O, MnCl 2 .4H 2 O, MoO 3 , CuSO 4 .5H 2 O, and Co (NO 3 ) 2 .6H 2 O.
  • Chlamydomonas pitschmanny YSL03 according to the present invention can be grown under high concentrations of nitrogen and phosphorus conditions, so that it can be cultured without significantly reducing the dilution ratio of the wastewater.
  • the high concentration means 500 to 1000 mg / L for nitrogen, 5 to 15 mg / L for phosphorus, and the experiment was performed under conditions of 800 mg / L nitrogen and 10 mg / L phosphorus in the examples.
  • the present invention is Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain or a culture medium for treating wastewater including a culture solution thereof can be provided.
  • the present invention is a method for removing nitrogen and phosphorus in the waste water by using an alga
  • the alga is Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain
  • Chlamydomonas pitschmanny according to the invention cultured using wastewater YSL03 provides fast growth rates and lipid-rich species ( Lipid content of 50% or more), which can be used as a component providing element capable of producing biodiesel by lipid extraction.
  • the present invention provides a method for separating lipids from algae or cultures thereof, wherein the algae is Chlamydomonas pitschmanny.
  • YSL03 Chlamydomonas pitschmannii YSL03
  • KCTC 11715BP KCTC 11715BP
  • the lipid is Chlamydomonas pitschmanny YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] biodiesel production method which is a lipid isolated from the strain is also included.
  • the lipid analysis of algae can be carried out using the Sulfophosphovanillin method, Kunkel method, and Brugdon method.
  • the new strain of the present invention was also analyzed for lipids by the conventional lipid analysis method as described above.
  • the biodiesel can be produced, and the present invention may also include a biodiesel production method using the lipid.
  • Samples were collected from the sewage and wastewater treatment plant in Wonju, Gangwon-do.
  • the collected sample was placed in a BBM liquid medium as shown in Table 1 in a 200 mL test tube and left to incubate for about 2 weeks.
  • Nitrogen-phosphorus was fed at 800 mg / L and 10 mg / L, respectively, and the concentration of wastewater was adjusted to about 24 watts on the surface of the culture tube using a fluorescent light source, followed by direct incubation.
  • 1 mL of the pre-cultured sample was placed in a 30 mL test tube containing 10 mL of sterile distilled water and mixed well to disperse the microalgal cells.
  • a 0.1 mL sample was taken using a micro pipette, and then mixed into a test tube containing sterile distilled water. After this procedure was repeated five times, about 0.1 mL of the sample was taken from each test tube, which was inoculated in a Petri dish containing BBM medium having a composition as shown in Table 2 below, followed by a temperature of 25 to 27 ° C. And stationary culture for 2-3 weeks in an incubator of roughness 50 ⁇ mol / m 2 -sec. When colonies of microalgae appeared, micron was used to check each microscope and each colony was transferred to a well cell culture plate using platinum teeth to start separation. Each hole was incubated for 10 to 14 days after administration of avian colonies and BBM medium 1: 1.
  • antibiotic streptomycin In order to inhibit the growth of some microorganisms, we used antibiotic streptomycin to grow only algae. The amount of antibiotic used was used 0.15 ⁇ m / mL per 1L of medium. After a period of time, the colonies cultured in each hole are examined under a microscope. At this time, most of the holes grow only the characteristic microalgae, the same shape was incubated in Petri dishes prepared with BBM medium. After a period of time, the cultivated algae colonies were predominantly cultured as one kind of colonies. The colonies were harvested and transplanted into 250 mL Erlenmeyer flasks containing 100 m of BBM medium.
  • YSL03 Chlamydomonas pitschmannii YSL03
  • BBM medium of Table 1 above YSL03 ( Chlamydomonas pitschmannii YSL03) strain was put in a 250 mL Erlenmeyer flask at 25 to 27 °C, pH 7.2 to 7.6 10 mL of the strain and incubated for about 14 days in a fluorescent stirred incubator. Stirring was maintained at 150 rpm and roughness 50 ⁇ mol / m 2 -sec.
  • Chlamydomonas Fitzmanny The sequencing of the strain YSL03 was analyzed by 28s rDNA sequencing. The state of the sample is Chlamydomonas Fitschmann. YSL03 colonies were plated, and as a request, PCR products by Extraction-gDNA and PCR Amplication were analyzed by sequencing reaction. Primers used for analysis are as follows.
  • Reverse primer 5'-TACTAGA-AGGTTCGATTAGTC-'3 (SEQ ID NO: 3)
  • the 28s rDNA sequencing results are described in the attached SEQ ID NO., And the phylogenetic results are shown in the phylogenetic tree results according to the neighbor-joining method using the Jukes-Cantor model. 2 is shown.
  • Chlamydomonas Fitzmanny The base sequence [SEQ ID NO: 1] of the strain YSL03 was classified as Chlamydomonas pitschmanny as shown in Table 3 below when the homology was examined in NCBI.
  • the microalgal bacteria isolated from the present invention when considering the characteristics of morphology, optimal temperature, pH, etc., except that it contains wastewater resistance and high content of lipids, Chlamydomonas Fitsch Manni Turned out to belong.
  • YSL03 Chlamydomonas pitschmannii In YSL03, algae were seeded according to different sources for consideration of nitrogen and phosphorus removal. Each reactor maintained a room temperature of 20-22 ° C., pH was maintained at 7.7-7.9 in nature and proceeded in the form of batch experiments. After the first planting, algae took about 10 days to acclimate, after which the removal of nitrogen and phosphorus occurred regardless of the initial concentrations of wastewater and BBM.
  • Algae are heterotrophic / independent nutrients composed of carbohydrates, proteins, and lipids that grow through photosynthesis.
  • a mixture of fatty acid methyl esters Mix RM3, Mix RM5, GLC50, GLC70 [Supelco, USA], heptadecanoic acid and gamma-linolenic acid [Supelco, USA] was used as a standard. Place a mass of microalgal lipid sample in a glass tube [11 mL, DH.GL28020, Daihan Scientific, Korea] with a Teflon stopper, and inject 2 mL of chloroform-methanol (2: 1, vol / vol) at room temperature. It was mixed for a minute with a vortex mixer (Vorex Genius 3. Ika, Italy).
  • 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].
  • Chlamydomonas pitschmanny according to the present invention YSL03 ( Chlamydomonas pitschmannii YSL03) strain was found to contain about 50% by weight of dry weight lipids in the cells [Table 4].
  • fatty acids palmitic acid (16: 0), stearic acid (C18: 0), oleic acid (C 18: 1n9c), and linolenic acid (C18: 3n3) are known as fatty acids for biodiesel production.
  • Chlamydomonas Fitschmann Palmitic acid (16: 0) is the most widely distributed fatty acid distribution of YSL03 strain.
  • the most stable fatty acids for biodiesel production of all microalgae reported to date are known as oleic acid (C 18: 1n9c) and palmitic acid (16: 0).
  • Chlamydomonas pitschmanny a new strain according to the present invention YSL03 is made up of 40% by weight of all fatty acids composed of oleic acid and palmitic acid for biodiesel production.

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Abstract

La présente invention concerne une nouvelle souche YSL03 [KCTC 11715BP] de Chlamydomonas pitschmannii et, plus particulièrement, la nouvelle souche YSL03 [KCTC 11715BP] de Chlamydomonas pitschmannii a une teneur élevée en lipides, est très efficace dans la production de biodiesel et est fortement résistante aux eaux usées et peut éliminer l'azote et le phosphore dans les eaux usées et permettre ainsi une production de bioénergie simultanément au traitement des eaux usées.
PCT/KR2010/006561 2010-07-05 2010-09-27 Nouvelle souche ysl03 de chlamydomonas pitschmannii WO2012005410A1 (fr)

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KR20100064306A KR101114426B1 (ko) 2010-07-05 2010-07-05 신균주 클라미도모나스 피트쉬만니 ｙｓｌ03

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Cited By (1)

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CN114606131A (zh) * 2022-03-30 2022-06-10 福建师范大学 一种绿藻菌株及其在稀土氨氮废水处理中的应用

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KR101394649B1 (ko) * 2012-02-08 2014-05-13 한국생명공학연구원 높은 이산화탄소 고정능 및 지질 생산능을 가지는 에틀리아 속 균주 및 이의 용도

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KR910006476A (ko) * 1989-09-20 1991-04-29 후미오 오오누끼 클라미도모나스속 단세포녹조 알사가 스트레인 95의 배양법 및 배양장치
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114606131A (zh) * 2022-03-30 2022-06-10 福建师范大学 一种绿藻菌株及其在稀土氨氮废水处理中的应用
CN114606131B (zh) * 2022-03-30 2023-07-25 福州文泽生物科技有限公司 一种绿藻菌株及其在稀土氨氮废水处理中的应用

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