KR101575208B1 - Microalgae Chlorella strain high-producing starch and lipid isolated from arctic ocean and uses thereof - Google Patents

Abstract

The present invention relates to a novel microalgae Chlorella sp. Which accumulates functional starch and lipid at a high concentration. The chlorella ArM29B cell line according to the present invention has been identified as a cell line that accumulates starch and lipid at high concentration during culture, And can be used as a material for the production of biodiesel and functional lipids by confirming accumulation of high concentration of lipid by nile red assay capable of specifically staining droplets of neutral oil in the cell . In addition, the present cell line Chlorella ArM29B cell line is suitable for microalgae for biodiesel because it accumulates lipids at a high concentration during cultivation. Since it grows well above the freezing temperature, it is not necessary to set the temperature condition during cultivation. Therefore, cultivation and production are possible throughout the year.

Description

Microalgae Chlorella strain high-producing starch and lipid isolated from arctic ocean and uses thereof,

The present invention relates to a microalgae Chlorella cell line for accumulating functional starch and lipid at a high concentration and a use thereof, and more particularly to a microalgae Chlorella cell line which can be used for production of biodiesel and functional lipids. The chlorella ArM29B cell line according to the present invention has been identified as a cell line that accumulates starch and lipid at a high concentration during culturing and can be cultured under various temperature conditions and can be used for nile red which can specifically stain neutral oil droplets ) Method, it is an industrially very useful microalgae.

Recently, the rapid rise in crude oil prices has attracted attention to the development of alternative energy using biomass, especially biofuel (bioethanol, biodiesel) production. However, the production of biofuel using crops has been affected by the destruction of ecosystems due to the expansion of cultivated land, And the like. However, microalgae, a photosynthetic microorganism, has a solar energy utilization efficiency of about 5%, about 25 times higher than that of 0.2% of land plants, and the carbon dioxide immobilization rate is 15 times higher than that of pine trees.

In addition, biodiesel production (with 30% oil content) per unit area of microalgae is about 58,700 L / ha, which is 130 times higher than 446 L / ha of soybean. The microalgae have been evaluated as the only resource capable of producing biodiesel to replace diesel produced from fossil fuels in the long term due to various advantages such as cultivation using idle cultivated land, ease of strain improvement, and irrespective of food problems. In addition, the biological conversion and treatment of carbon dioxide is an environmentally friendly method by using photosynthesis, which is the basic principle of natural material circulation. The process is carried out at room temperature and atmospheric pressure, and biomass produced is used as a useful substance There are advantages.

In recent years, bio-energy production materials (biomaterials) have been demanded as a result of the surge in demand for bio-energy. Most of the studies of micro-algae biodiesel have been conducted for selecting species capable of producing lipids and producing lipids , And mass production of lipid-producing microalgae chlorella and clamidomonas cell lines through cell non-destructive extraction method and cell high-density cultivation method enables biocomponents such as bio-fuels such as renewable energy, food additives and medical materials, A project to produce large quantities of antioxidants such as carotenoids is underway.

At present, it is possible to apply to large-scale cultivation of microalgae for biodiesel, since mass cultivation and utilization techniques of microalgae (Chlorella, Dunalliela, Haematococcus, Spirilluna, etc.) which can be used as antioxidants and food additives feeds have been developed. In the case of microalgae (especially, Botryococcus, Schiochytrium, etc.), which has been the most noted in the production of biodiesel due to high lipid content, R & D is being concentrated mainly on finding optimal culture conditions for increasing the lipid content. In addition, microalgae accumulating lipids at high concentrations typically accumulate lipid bodies in the cells. According to the results thus far, there are many differences in optimum production conditions of lipids depending on the species. In general, at a temperature of about 23 ° C, limiting the supply of nitrogen source and increasing the concentration of carbon dioxide, .

However, microalgae accumulating lipid at a high concentration is limited to a part, and it is general to accumulate lipid under optimum temperature and specific culture conditions.

Korean Patent Laid-Open Publication No. 2011-0125576 discloses a lipid-producing microalgae clamidomonas cell line isolated from Arctic Ocean and its use. Japanese Patent Registration No. 3004510 discloses a process for producing ethanol from microalgae However, starch and lipid-producing microalgae Chlorella cell lines isolated from Arctic Ocean as in the present invention and their uses have not been disclosed.

It is an object of the present invention to provide a material for producing biodiesel and functional lipids by accumulating starch and lipids at a high concentration while being able to grow under various temperature conditions.

Accordingly, the present inventors screened chlorella cell lines of polar microalgae by GC analysis and nile red staining method to select microalgae that can be developed for high fatty acid production and bio energy because of their high lipid content, The present inventors completed the present invention by selecting a chlorella ArM29B cell line which accumulates high concentrations of starch and lipid and grows well.

In order to solve the above problems, the present invention provides a Chlorella sp. ArM29B cell line, which is a microalgae produced from Arctic Ocean.

The present invention also provides a microorganism preparation for preparing a lipid comprising the cell line or a culture solution thereof as an active ingredient.

The present invention also provides a method for producing a lipid characterized in that the cell line is cultured and lipids are separated from the culture.

(C18: 1) and linoleic acid (C18: 2) as main fatty acids and oleic acid (C18: 1) and linoleic acid ) In a total lipid weight basis of 50 to 60% and a polyunsaturated fatty acid content of 10% or less based on the total lipid weight.

The present invention also provides a method for producing biodiesel comprising culturing the cell line, separating lipids from the culture, and transesterifying the lipids to produce fatty acid esters and glycerol.

The present invention also provides a method for producing glycerol comprising culturing the cell line, separating lipid from the culture, and transesterifying the lipid to produce a fatty acid ester and glycerol.

The chlorella ArM29B cell line according to the present invention has been identified as a cell line that accumulates starch and lipid at a high concentration during culturing and can be cultured under various temperature conditions and can be used for nile red which can specifically stain neutral oil droplets ) Method, it can be used as a material for biodiesel and functional lipid production.

In addition, since the chlorella ArM29B cell line accumulates lipids at a high concentration during cultivation, it is suitable for microalgae for biodiesel. Since it grows well above the freezing temperature, it is not necessary to set the temperature condition during cultivation. Cultivation and production are economically feasible.

Fig. 1 shows the result of identification with Chlorella ArM29B cell line ( Chlorella sp. ArM29B). ArM29B and Chlorella sp. Chlorella Thoreau Kearney Ana (Chlorella sorokiniana, HM101339), Chlorella guru Pierre Noi (Chlorella pyrenoidosa , AB240145), Auxenochlorella protothecoides , EU038285), Chlorella variabilis, shows a sequence comparison of the rbc L gene of AB260903) and Chlorella vulgaris (Chlorella vulgaris, AB260909).
Figure 2 shows the results of analysis of the growth rate of chlorella ArM29B cell line of the present invention at various temperatures. Chlorella vulgaris was used as a control. Data are presented as means ± SD (n = 3).
Fig. 3 shows that the temperature at the highest temperature as a result of the outdoor culture of the chlorella ArM29B cell line grows at a temperature of 36 캜.
FIG. 4 shows the result of observing starch granules and lipplets in electron microscopic observation of cells cultured under the nitrogen-deficient condition of chlorella ArM29B cell line. (a) is a result of a confocal microscopic observation of ArM29B cells, and bar indicates 5 mu m. (B) is a result of TEM analysis of ArM29B cells, and bar indicates 1 mu m; S is starch granule, L is lipid remnant. (C) is the result of observing the lipid remnants of ArM29B with Nile red staining. Yellow and red represent lipid remnants and chloroplasts, respectively.
FIG. 5 shows the result of analysis of the fatty acid composition in the chlorella ArM29B cell line. Each fatty acid and total fatty acid was expressed in mg / cell dry weight.
FIG. 6 shows changes in intracellular starch and fatty acid content in the chlorella ArM29B cell line according to the culture period.

In order to accomplish the object of the present invention, the present invention provides a Chlorella sp. Cell line, which is an arctic marine starch and lipid-producing microalgae. The cell line is preferably a Chlorella sp. ArM29B cell line (KCTC 12331BP).

The chlorella ArM29B cell line was sampled from sea ice of the Arctic Ocean, and single colonies formed by streaking on an agar medium were selected and identified through 18S rDNA and rbcL gene sequencing analysis. Starch and lipid-producing chlorella ArM29B cell lines Respectively. The above starch and lipid-producing chlorella ArM29B cell line was deposited at KCTC on Dec. 3, 2012 (Accession No .: KCTC 12331BP).

In a chlorella cell line according to one embodiment of the present invention, the cell line can accumulate starch and lipid at a high concentration, and preferably the starch and lipid yields of the cell line are 25-35% and 35-45% And more preferably the starch and lipid yields of the cell line may be 30% and 39%, respectively, per cell line dry weight, but are not limited thereto.

In a chlorella cell line according to an embodiment of the present invention, the cell line can accumulate starch and lipid at a high concentration at various culture temperatures, and preferably the culture temperature may be 4 to 36 ° C, Do not.

In a chlorella cell line according to an embodiment of the present invention, the cell line can produce lipids containing oleic acid (C18: 1) and linoleic acid (C18: 2) as major fatty acids, preferably oleic acid C18: 1) and linoleic acid (C18: 2) may be 50 to 60%, and most preferably 53 to 55%, based on the total lipid weight. In addition, the cell line may have a content of polyhydric unsaturated fatty acid (PUFA) of 10% or less based on the total lipid weight, and most preferably 6 to 7% of the total lipid weight, Do not.

In a chlorella cell line according to one embodiment of the present invention, the cell line may grow well in both fresh water and seawater, but is not limited thereto.

The present invention also provides a microorganism preparation for preparing a lipid comprising the cell line or a culture solution thereof as an active ingredient. The microorganism preparation may contain chlorella ArM29B cell line as an active ingredient, and can be effectively used for mass production of bio oil. The mass produced bio-oil can be used to produce biodiesel through a trans-esterification process.

In the microbial formulation according to one embodiment of the present invention, the lipid comprises oleic acid (C18: 1) and linoleic acid (C18: 2) in an amount of 50 to 60% by weight of the total lipid, polyunsaturated fatty acid (C18: 1) and linoleic acid (C18: 2) in an amount of 53 to 55% based on the total lipid weight, and the polyunsaturated fatty acid is used as a whole lipid But it is not limited thereto.

The present invention also provides a method for producing a lipid characterized in that the cell line is cultured and lipids are separated from the culture. The cell line can accumulate lipids at high concentrations at various culturing temperatures, and the culturing temperature may preferably be 4 ° C to 36 ° C. Any method known in the art can be used as a method for separating lipids from the cell culture medium.

The present invention also provides a lipid produced by the method for producing the lipid.

The lipid according to one embodiment of the present invention contains oleic acid (C18: 1) and linoleic acid (C18: 2) as major fatty acids, oleic acid (C18: 1) and linoleic acid (C18: 1) and linoleic acid (C18: 2) can be contained in an amount of 50 to 60%, based on the total lipid weight, of polyunsaturated fatty acids, 53 to 55% based on the total lipid weight, and may contain, but not limited to, polyunsaturated fatty acids by 6 to 7% by weight of total lipids.

The present invention also provides a method for producing a cell line, comprising: culturing the cell line of the present invention;

Separating the lipid from the culture liquid; And

And transesterifying the lipid to produce a fatty acid ester and glycerol.

In the method of the present invention, the cultivation of the cell line is preferably carried out at a temperature of 4 ° C to 36 ° C. The cell culture medium may be a medium commonly used in the art. The fatty acid ester is preferably, but not limited to, a methyl ester or an ethyl ester. The biodiesel can be produced in the form of methyl ester or ethyl ester by transesterification of the fatty acid contained in the biomass, and glycerol can be produced as a by-product of the transesterification process.

The transesterification process refers to a method of transforming a large branched molecular structure of fat contained in biomass into a small, linear chain molecule required by a regular diesel engine. As the emulsifier in the transesterification process, Triton X-100 or Tween 60 may be added, but the present invention is not limited thereto. The emulsifier can be used to stabilize the interface of the bio-oil and mix well, thereby increasing the reaction yield and thus saving the biodiesel recovery cost. In addition, sodium hydroxide or potassium hydroxide may be used as a reaction catalyst in the transesterification process, but the present invention is not limited thereto.

The biodiesel may have the following advantages as a biofuel: (1) Biofuels can easily be obtained from common biomass resources. (2) Biofuels indicate the circulation of carbon dioxide in combustion. (3) Biofuels have the potential to be environmentally friendly. (4) By using biofuels, environmental, economic and consumer benefits are gained. (5) Biofuels can be decomposed into harmless substances by microorganisms, contributing to sustainability.

The present invention also provides a method for producing a cell line, comprising: culturing the cell line of the present invention;

Separating the lipid from the culture liquid; And

And transesterifying the lipid to produce a fatty acid ester and glycerol.

In the method of the present invention, the culture temperature and culture medium of the cell line and the method of lipid separation are as described above.

The glycerol can be used as a lubricant, a preparation of medicine such as an ointment or a suppository, an antioxidant, an antiseptic agent for food or cosmetics or an antiseptic agent, antifreeze, coolant, paint, pigment, printing ink, transparent soap, , An adhesive, an alkyd resin or a dynamite, but the present invention is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Method

Isolation of Arctic microalgae ArM29B

Samples collected on sea ice of Dasan Station were cultured on agar medium and serially diluted to separate microalgae. The isolated green microalgae were then cultured in Tris-acetate-phosphate (TAP) medium (Harris 1989, Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use, Academic Press, San Diego, CA, 780) The aseptic line was analyzed under a microscope and named ArM29B. Then, the genomic DNA was isolated and the rbcL gene was amplified by PCR amplification according to the method of Hoham et al. (Hoham et al. 2002 J. Phycol. 38, 1051-1064) and the base sequence was determined. BLASTN search ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) and ClustalW2-Multiple Sequence Alignment ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ) in the NCBI database. The rbcL nucleotide sequences of ArM29B and related species were compared by the analytical method.

Culture conditions

In the present invention ArM29B and Chlorella vulgaris UTEX 395 (Chlorella vulgaris UTEX 395) was used. ArM29B cells were able to grow on TAP medium supplemented with 300 mM NaCl, but grew better when NaCl was not added. To analyze the growth rate, about 2 × 10 ⁵ cells were inoculated into 50 ml TAP liquid medium and cultured under continuous light condition of ⁴⁰ μmolm ^-2 s ^-1 while suspending at 200 rpm at 4, 15, 25 ° C temperature condition. The degree of cell proliferation was measured every 24 hours for 9 days by absorbance (OD ₇₅₀ ). For fatty acid analysis, about 2 x 10 ⁵ cells were inoculated in 50 ml of nitrogen-deficient TAP (NH ₄ Cl-removed from TAP salt) medium and cultured at 25 ° C. The cells were harvested at day 12 and 15 days after harvesting.

Fatty acid analysis

20 mg of the lyophilized cells were separated according to the method of Sasser (1990 Identification of bacteria by gas chromatography of cellular fatty acids. Microbial ID Inc., Newark, Del.). Hexane / methyl tert-butyl ether (1: 1) was then used to extract fatty acids. The fatty acid analysis was performed using GC (YL 6100 GC, Young Lin, Korea). The detector was a flame ionization detector (FID) and the components of each fatty acid (each FAME) were identified and quantified using the Supelco 37 Component FAME Mix (Sigma, USA).

Electron Microscopy Analysis / TEM ( Transmission electron microscopy )

Cells cultured for 7 days in a nitrogen source-deficient medium were washed twice with 250 mM phosphate buffer (pH 7.0) and washed with 2.5% (v / v) glutaraldehyde / 1% (v / v) osmium tetraoxide solution . A concentration gradient of ethanol (50-100%) was used for dehydration. Samples were embedding in Spurr's resin and the slices were made using ultramicrotome (Ultracut UCT, LEICA, Germany) and stained with uranyl acetate. The prepared samples were observed with a TEM (transmission electron microscope, Tecnai, FEI, Netherlands).

Nile Red  Lipid Remains by Dyeing droplet ) observe

Cells were stained with acetone containing 1 μg / ml of 9-diethylamino-5H-benzo [a] phenoxazine-5-one (Sigma-Aldrich, USA) for 30 min. The stained intracellular lipids were observed with a confocal microscope (Zeiss LSM 5 PASCAL confocal microscope system with PASCAL version 4.0 software (Jena, Germany)).

Example  1. Chlorella ArM29B Isolation and identification

The rbcL nucleotide sequence of ArM29B showed very high homology with chlorella species; Chlorella pyrenoidosa , 96%), Chlorella vulgaris , 94%) (Fig. 1). However, it showed 90% or less homology with other microalgae including Chlamydomonas and Senedesmus . Therefore, ArM29B was classified as Chlorella sp. Growth rates at various temperatures were compared with those of the control, chlorella bulgaris, at all the temperatures examined (FIG. 2). In particular, the natural habitat of ArM29B was rapidly growing even at 25 ° C (Fig. 2) in spite of polarity, and grown at 36 ° C in outdoor culture (Fig. 3).

Example  2. Analysis of relative lipid content of chlorella ArM29B cell line

ArM29B cells were spherical with a diameter of 4-8 탆 and had no flagella. Cells cultured under nitrogen depletion conditions were filled with starch granules and lipplets upon electron microscopy (Fig. 4). Analysis of fatty acids by GC analysis showed that the total fatty acid content was 39% of the dry weight and the major fatty acids were C16: 0, C18: 1 and C18: 2 (FIG. 5). The contents of C18: 1 and C18: 2 fatty acids were 54% and the content of polyhydric unsaturated fatty acids (PUFAs) with 3 or more double bonds was very low at 7%. The low content of PUFA and the high contents of C18: 1 and C18: 2 fatty acids indicate that they are excellent for use in the production of biodiesel.

In addition, the starch content in the chlorella ArM29B cell line was increased to 30% of the starch content per dry weight (Fig. 6), and the starch content decreased after 7 days of culture. On the other hand, It grew slowly at the early stage of culture and increased rapidly after 7 days of culture, and the fatty acid content per dry weight increased to 39%.

Korea Biotechnology Research Institute KCTC12331BP 20121203

Claims (15)

The Chlorella sp. ArM29B cell line, an Arctic marine starch and lipid production microalgae, characterized by the deposit number KCTC 12331BP. delete The cell line according to claim 1, wherein the starch and lipid production amount of the cell line is 25 to 35% and 35 to 45%, respectively, per cell weight. The cell line according to claim 1, wherein the cell line grows well at a culturing temperature of 4 ° C to 36 ° C. The cell line according to claim 1, wherein the cell line comprises oleic acid (C18: 1) and linoleic acid (C18: 2) as major fatty acids. 6. The cell line according to claim 5, wherein the content of oleic acid (C18: 1) and linoleic acid (C18: 2) is 50 to 60% based on the total lipid weight. The cell line according to claim 1, wherein the cell line grows well in both fresh water and seawater. The cell line according to claim 1, wherein the cell line has a polyunsaturated fatty acid content of 10% or less based on the total lipid weight. 9. A microorganism preparation for producing a lipid comprising the cell line of any one of claims 1 to 8 or a culture thereof as an active ingredient. 10. The method of claim 9, wherein the lipid comprises oleic acid (C18: 1) and linoleic acid (C18: 2) in an amount of 50 to 60% by weight of total lipids, polyunsaturated fatty acids in an amount of 10% Wherein the microorganism is a microorganism. 9. A method for producing a lipid comprising culturing the cell line according to any one of claims 1 to 8 and separating lipids from the culture. 12. The method of claim 11, wherein the culturing of the cell line is performed at a temperature of from 4 캜 to 36 캜. A process for preparing oleic acid (C18: 1) and linoleic acid (C18: 2), which comprises oleic acid (C18: 1) and linoleic acid (C18: 2) Wherein the lipid comprises 50 to 60% by weight of lipid, and the polyunsaturated fatty acid contains 10% or less by weight of the total lipid. 9. A method for producing a cell line, comprising culturing the cell line of any one of claims 1 to 8 to obtain a culture solution;
Separating the lipid from the culture liquid; And
And transesterifying the lipid to produce a fatty acid ester and glycerol. 9. A method for producing a cell line, comprising culturing the cell line of any one of claims 1 to 8 to obtain a culture solution;
Separating the lipid from the culture liquid; And
Transesterifying the lipid to produce a fatty acid ester and glycerol.

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