WO2015088127A1 - Method for preparing microalgae with increased lipid content - Google Patents

Abstract

The present invention relates to a method for preparing microalgae with an increased microalgae lipid content, the method comprising a step of culturing microalgae in stress conditions; to microalgae with an increased microalgae lipid content, prepared by the method; to a biomass comprising the microalgae; to a method for producing biodiesel using the biomass; to biodiesel produced by the method; and a use of microalgae with an increased lipid content for the production of the biodiesel. The use of the method of the present invention can prepare microalgae exhibiting high productivity per hour while increasing the lipid content in cells, and the prepared microalgae with an increased microalgae lipid content can be used as a biomass for the production of biodiesel, and thus can be widely utilized in the economical production of biodiesel.

Description

Manufacturing method of microalgae with increased lipid content

The present invention relates to a method for producing microalgae with an increased lipid content, and more particularly, the present invention relates to a method for producing a microalgae with increased lipid content in cells, comprising culturing the microalgae under stress conditions. Prepared by the microalgae increased the lipid content in the cell, biomass containing the microalgae, a method for producing biodiesel using the biomass, the biodiesel produced by the method and for the production of the biodiesel It relates to the use of microalgae with increased lipid content.

In the face of depletion of fossil energy and global warming all over the world in recent years, the development of new and renewable energy such as eco-friendly bio, hydrogen, solar, etc. Promoting supply and use. Although many efforts are being made to develop new and renewable energy in Korea, it is still in the research stage. Bioenergy is renewable and has the advantage of reducing global warming acceleration by fixing carbon dioxide. Currently, research on bioethanol, biobutanol, biodiesel, etc. has been actively conducted.

Among the bioenergy, the most active research is biodiesel. Biodiesel is produced by conversion into methyl ester or ethyl ester form by the transesterification process of fatty acid contained in biomass, and has a flash point of 150 ℃, which is less than diesel. It does not stick well and is classified as a non-flammable liquid because it is more stable than gasoline (45 ° C.), which is highly volatile at low temperatures, and is more stable because it catches fire at high temperatures. In addition, unlike diesel, biodiesel is known to have low emissions of carcinogens and mutations when burned, and other emissions are known to have significantly lower nontoxic energy.

Such biodiesel is known to be biologically produced using microalgae. Microalgae have 25 times higher efficiency of solar energy than plants, so they are used for feed or fertilizer. Especially, Spirulina sp., Chlorella sp. And Dunaliella sp. Nostoc sp. Is also used as a health food. Since the microalgae have excellent solar energy utilization efficiency, the microalgae having a high lipid content can be used as biomass for the production of biodiesel. Such microalgae with excellent lipid content are known as Botryococcus sp. And Schiochytrium sp. The microalgae do not have high lipid content in normal living conditions, but nutrient supply is stopped. After a certain period of time, the lipids accumulate in the cells. The microalgae may be used as a biomass for the production of biodiesel using such characteristics, but in order to increase the lipid content of the microalgae, the first step of culturing and growing the microalgae and the expanded microalgae In order to stop the supply of nutrients and incubate for a certain period of time to increase the lipid content in the cells, a second step is required. Since the second step requires excessive time, it is not yet used for industrial production of biodiesel. Development of microalgae with excellent lipid content for the production of biodiesel is urgently needed.

The present inventors have diligently researched to develop microalgae having a high lipid content, and thus, when microalgae are cultured under stress conditions, their lipid content is increased, and thus, the microalgae with increased lipid content are produced for biodiesel production. It was confirmed that it can be used as biomass, the present invention was completed.

One object of the present invention is to provide a method for producing microalgae with increased lipid content in cells.

Another object of the present invention is to provide a microalgae with increased lipid content in the cells produced by the above method.

Still another object of the present invention is to provide a biomass including microalgae with increased lipid content in cells.

Still another object of the present invention is to provide a method for preparing biodiesel using the biomass.

Another object of the present invention is to provide a biodiesel produced by the above method.

Still another object of the present invention is to provide a microalgae having an increased lipid content for producing the biodiesel.

By using the method of the present invention, it is possible to produce microalgae with increased lipid content in cells while showing high productivity per hour, and the prepared microalgae with increased lipid content in cells are biomass for the production of biodiesel. As it can be used, it can be widely used for economic production of biodiesel.

FIG. 1A is a TEM photograph showing the inside of cells of chlorella soroquinia or HS strain cultured in BG11 medium containing no salt, and FIG. 1B is a chlorella soroquinia or HS strain cultured in BG11 medium containing salt. TEM image showing the inside of the cell.

Figure 2 is a graph showing the change in lipid content of the mycelia of Chlorella Sorokonia or HS strain according to the concentration of salts contained in the medium.

Figure 3 is a graph showing the composition of lipids in cells of chlorella sorokinia or HS strain cultured under various salt stress conditions.

4 is incubated for 6, 12, 24 or 30 hours in BG11 medium containing 0, 7, 10 or 15% (w / v) of NaCl, Chlorella sorokinina HS strain, the cultured Chlorella sorokiniana It is a graph showing the results of analyzing the lipid content in these cells in HS strains.

FIG. 5 shows chlorella sorokiniana or HS strains cultured in a salt-free medium, 0, 6, in BG11 medium containing 0, 1, 3, 5, 7, 10 or 15% (w / v) NaCl. After culturing for 24 or 30 hours, it is a fluorescence micrograph showing the result of performing Nile red staining on the cultured Chlorella Sorokonia or HS strain.

FIG. 6 is a graph showing the change in lipid content in cells over time after the addition of hydrogen peroxide to chlorella vulgaris OW-01 cultured in BG11 medium not containing hydrogen peroxide as an oxidizing agent.

FIG. 7 is a micrograph showing changes in lipid content in cells over time after oxidative stress was added by adding hydrogen peroxide to chlorella vulgaris OW-01 cultured in BG11 medium without hydrogen peroxide as an oxidizing agent.

8 is incubated for 0, 6, or 24 hours in BG11 medium containing 0, 1, 5, 10 or 20 mM hydrogen peroxide for chlorella sorokinina or HS strains, It is a graph showing the result of analyzing lipid content in cells.

9 is a chlorella sorokinina HS strain incubated for 0, 6, 24 or 30 hours in BG11 medium containing 0, 1, 3, 5, 10, 15 or 20 mM hydrogen peroxide, the cultured chlorella sorokinina Fluorescence micrograph showing the result of performing Nile red staining on HS strain.

FIG. 10 shows HS strains of the genus Stizelonium incubated for 6, 12 or 24 hours in BG11 medium containing 0, 1, 3, 5, 10, 15 or 20 mM hydrogen peroxide, It is a graph showing the results of analyzing the lipid content in these cells in HS strains.

FIG. 11 is incubated with 0, 1, 3, 5, 10, 15, or 20 mM BG11 medium containing 0, 1, 3, 5, 10, 15, or 20 mM hydrogen peroxide for 0, 6, 12, or 24 hours, and the cultured Stizeoclo Fluorescence micrograph showing the result of performing Nile red staining on the HS strain of the genus.

The present inventors have been paying attention to a method of culturing the microalgae under stress conditions while performing various studies to develop microalgae having a high lipid content for the production of biodiesel.

Among the microalgae, the microalgae that can survive in various stress conditions such as salt stress or oxidative stress are given a stress condition, while reducing the number of cells by inhibiting the growth of the cells, increasing the volume of the cells themselves, external stress Increasing the resistance to the condition ultimately increases the viability, which is expected to increase the content of lipids in the cells as a means of causing an increase in the volume of such cells. In order to confirm this, the microalgae were cultured under the conditions of salt stress or oxidative stress, and the lipid content contained in the cultured cells was measured. It was confirmed that the lipid content in the cells was significantly increased than that of the microalgae cultured in the environment.

Conventionally, in order to increase the lipid content of microalgae in the microbial cells, a method of culturing the microalgae for a long time under the condition that the nutrient supply is stopped, the method is low productivity per hour because it takes a long time incubation time There was a problem. Unlike the prior art, the method of culturing the microalgae under the stress conditions provided by the present invention does not require a long incubation time, and thus has an advantage of better productivity per hour than the prior art.

Therefore, the microalgae excellent in the lipid content of the microbial cells prepared by the method of culturing the microalgae under stress conditions provided by the present invention can be used not only as a biomass for the production of biodiesel, but also for the economic production of biodiesel. Could be utilized.

As one embodiment for achieving the above object, the present invention provides a method for producing a microalgae with increased lipid content, comprising culturing the microalgae under stress conditions.

The term "stress condition" of the present invention means a condition that can inhibit the growth of microalgae by deteriorating the culture environment of the microalgae. The stress condition is not particularly limited as long as it can increase the lipid content in the cell by inhibiting the growth of microalgae, but may preferably be salt stress, oxidative stress and the like.

As used herein, the term "salt stress" refers to stress applied to microalgae because salt, which is a combined product of acid and base, is excessively present in the microalgae culture environment. When the salts increase in the culture environment of the microalgae, the osmotic pressure in the microalgae decreases, so that the water inside the microalgae flows out into the cultured environment, and the growth of the microalgae decreases as the concentration of the salts in the cultured environment increases. If it is above a certain level, the microalgae will die.

In the present invention, the salt is added to the culture medium of the microalgae is used as a means for increasing the lipid content in the microalgal cells, the salt is particularly limited to this as long as it can increase the content of lipids in the microalgae cells But preferably sodium chloride (NaCl), magnesium chloride (MgCl ₂ ), magnesium sulfate (MgSO ₄ ), calcium sulfate (CaSO ₄ ), potassium sulfate (K ₂ SO ₄ ), calcium carbonate (CaCO ₃ ), brominated Magnesium (MgBr ₂ ) and the like may be used alone or in combination. The addition amount of the salts is not particularly limited, but it is preferably 1 to 30% (w / v), more preferably 5 to 20% ( w / v), most preferably 10 to 15% (w / v).

As used herein, the term "oxidative stress" refers to a stress in which a high content of an oxidizing substance is present in a cell's surroundings, thereby increasing the possibility of oxidizing components such as lipids contained in the cell membrane due to the oxidizing substance. it means. In vivo, the oxidative stress can be reduced by releasing an antioxidant which can alleviate the oxidative stress, but in vitro conditions, the antioxidant should be added artificially. In particular, when oxidative stress is added to the culture environment of microalgae such as chlorella strains, oxidative stress in the microalgae cell membranes inhibits various metabolisms such as growth and proliferation of microalgae. As the level of is increased, the growth of the microalgae decreases, and when the level is above a certain level, the microalgae are killed.

In the present invention, the oxidative stress may be formed by adding an oxidizing agent to the culture environment of the microalgae, and may be interpreted as a cause for providing a herb for increasing the content of lipids in the microalgae, the oxidizing agent. Is not particularly limited as long as it can increase the content of lipids in microalgae cells, preferably hydrogen peroxide (H ₂ O ₂ ), superoxide ion (0 ^2- ), hydroxyl radical, OH ⁻ ), hypochlorous acid (HOCl) and the like may be used alone or in combination, and the addition amount of the oxidizing agent is not particularly limited thereto, but is preferably 5 to 100 mM, more preferably 5 to 50 mM, Most preferably, it can be added to the medium of the strain of the genus Chlorella at a concentration of 5-20 mM.

The term "microalgae" of the present invention, also referred to as "phytoplankton", refers to a unicellular prokaryote that survives in an aquatic environment, reproduces with spores, and photosynthesizes with photosynthetic pigments.

In the present invention, the microalgae is not particularly limited as long as it can accumulate lipids in the cells while living in a stress environment, but preferably may be a strain of the genus Chlorella, the strain of the genus Stizelonium, and the like. Advantageously chlorella Thoreau Kearney Ana HS strain (chlorella sorokiniana HS) (KCTC 12171BP ), chlorella vulgaris OW-01 strain (chlorella vulgaris OW-01) ( KCTC 12553BP), styryl fifth claw nium in HS strain (Stigeoclonium sp. HS) ( KCTC 12676BP).

Chlorella sorochiniana HS strain provided by the present invention means a strain deposited with the accession number KCTC 12171BP to the Korea Biotechnology Center (Korean Collection for Type Culture) as of March 21, 2012.

Chlorella vulgaris OW-01 strain provided by the present invention means a strain deposited with the accession number KCTC 12553BP to the Korea Collection for Type Culture (Korea Biotechnology Research Institute) on February 11, 2014.

HS strain of the genus styeoclonium provided by the present invention means a strain deposited with the accession number KCTC 12676BP to the Korea Biotechnology Center (Korean Collection for Type Culture) on September 12, 2014.

As used herein, the term "culture" refers to a series of actions for growing microorganisms under appropriately artificially controlled environmental conditions.

In the present invention, the culturing may be interpreted to mean a method of culturing microalgae, the culturing method may be performed using a method well known in the art. Specifically, the culturing may be performed continuously in a batch process or in a fed batch or repeated fed batch process.

In order to culture the microalgae, it is necessary to meet the survival requirements of specific strains in an appropriate manner while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like. Carbon sources that can be used are mainly CO ₂ and carbonate, and mixed sugars of glucose and xylose may be used as the carbon source, and sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose, and soybean oil Oils such as sunflower oil, castor oil, coconut oil and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used. For example, microalgae as a culture medium for the _{NaNO 3, K 2 HPO 4,} MgSO 4 .7H 2 O, CaCl 2 .2H 2 O, citric acid, Na ₂ EDTA, ammonium ferric citrate, green (Ammonium ferric citrate green) , Na ₂ CO _3, and trace metals solution _{(H 3 BO 3, MnCl 2} .4H 2 O, ZnSO 4 .7H 2 O, Na 2 MoO 4 .2H 2 O, CuSO 4 .5H 2 O, Co (NO 3) BG11 medium containing ₂ .6H ₂ O) can be used.

In addition, antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation. Gas (eg, air) can be injected to maintain aerobic conditions. The temperature of the culture can usually be maintained at 20 ° C to 35 ° C, preferably 25 ° C to 30 ° C.

In addition, the nature of the microalgae to perform photosynthesis is preferable to provide light during the cultivation, the amount of light provided and the time provided can be appropriately adjusted by those skilled in the art as needed, luminous conditions of about 50 to 100μmole / ㎡ / s Can provide light.

According to one embodiment of the present invention, BG11 medium containing no salt and BG11 medium containing 3% NaCl were inoculated with Chlorella sorokinia or HS strain, cultured, and then in cells using an electron microscope (TEM). As a result of confirming the change, when chlorella sorkinonia or HS strains were cultured in a salt-containing medium, the contents of starch and lipids in the cells were increased to increase the size of the cells as a whole (FIG. 1). As a result of measuring the content of lipids contained in the cells of Chlorella soroquinia or HS strains cultured in BG11 medium containing 1, 2, 3 or 4% of salt, the strains cultured in BG11 medium containing no salts were measured. The lipid content in the cells was about 10%, the lipid content in the cells of the strain cultured in BG11 medium containing 1% of the salt was about 15%, and cultured in BG11 medium containing 2% of the salt Strain The lipid content in the sieve is about 25%, the lipid content in the cells of the strain cultured in BG11 medium containing 3% of the salt is about 34%, and cultured in BG11 medium containing 4% of the salt Lipid content contained in the cells of the strain was found to be about 32% (Fig. 2). In addition, the strain was cultured in a salt-free medium, and it was confirmed that the lipid content contained in the cells was increased even when the salt shock was added to the strain by adding salts (FIG. 4, FIG. 5 and Table 2).

Therefore, it was found that the microalgae cultured in a medium containing salts increased lipid content in cells.

According to another embodiment of the present invention, the chlorella vulgaris OW-01 strain, a strain of the genus Chlorella, is cultured in a medium to which hydrogen peroxide, an oxidizing agent of various concentrations, is added, and the change of lipid content in the strain is measured with the cultivation time. As a result, it was confirmed that the lipid content in the strain was increased when the oxidizing agent was 5mM or more, and the lipid content in the strain was further increased when the oxidizing agent was 10mM or more, and thus the increased lipid content was continuously maintained. It was confirmed that the maximum lipid content was about 1.7 times the lipid content of the strain cultured in the medium without oxidative stress (FIGS. 6 and 7). In addition, chlorella soroquinia or HS strains, which are strains of other chlorella species, were cultured in a medium to which hydrogen peroxide, which is an oxidizing agent of various concentrations, was measured, and the change in lipid content in the strain was measured as a result of culturing time. When the lipid content in the strain was increased, the lipid content in the strain was further increased when the oxidizing agent was treated more than 15mM. The increased lipid content was continuously maintained, and the maximum lipid content was given to the oxidative stress. It was confirmed that about 1.6 times the lipid content of the strain cultured in the medium (Fig. 8 and 9). In addition, it was confirmed that the lipid content in the microbial cells was increased even when cultured under the condition that oxidative stress of HS strain of Stizeoclonium, which is another microalgae (FIG. 10, 11 and Table 3).

Therefore, it was found that the microalgal cultured in the medium to which oxidative stress was added increased lipid content in cells.

As another embodiment for achieving the above object, the present invention provides a microalgae prepared using the above method, the lipid content in the cells is increased compared to the strain cultured without stress conditions.

The microalgae prepared by the above method is characterized in that the lipid content in the cells is increased compared to the cells cultured in a medium containing no salts. For example, when culturing microalgae chlorella sorokinia or HS is cultured in a salt stressed environment, it is about 3 to 4 times, preferably about 3.4 than when cultured in a salt stressed environment. Contains three times the enhanced content of lipids. At this time, the lipid contained in the Chlorella Sorokini or HS is palmitate (palmitate, C16: 0), palmitate (palmitoleate, C16: 1), stearate (stearate, C18: 0), oleate (oleate, C18) : 1), linoleate (C18: 2), linolenate (C18: 3) and the like. In particular, palmitoleate (C16: 1) is not included in chlorella sorokinia or HS cultured in a medium containing no salts. Thus, palmitolate can be used as the main marker to distinguish the microalgae prepared by the method of the present invention from microalgae prepared by the conventional method.

According to one embodiment of the present invention, as a result of analyzing the composition of the lipid contained in Chlorella sorokinya or HS cultured in BG11 medium containing 0, 1, 2, 3 or 4% of salt, palmitate (C16) : 0), palmitoleate (C16: 1), stearate (C18: 0), oleate (C18: 1), linoleate (C18: 2), linolenate , C18: 3) and the like. In particular, palmitate is not included in chlorella sorokinia or HS cultured in a medium that does not contain salts, and is contained only in chlorella sorokinia or HS cultured in a medium containing salts. As the concentration was increased, it was confirmed that the content of the fatty acid tended to increase (FIG. 3).

As another embodiment for achieving the above object, the present invention provides a biomass comprising microalgae with increased lipid content in cells and a method for producing biodiesel using the biomass and the bio-diesel produced by the method Provide diesel.

As used herein, the term "biomass" refers to a variety of algae and plant resources produced by photosynthesis, such as trees, grasses, branches of crops, leaves, roots, fruits and the like.

In the present invention, the biomass may be interpreted as microalgae, cultures containing the microalgae, culture fractions, etc., in which the lipid content in cells is increased by culturing in a medium under stress conditions, and the biomass is biodiesel It can be used as a raw material for the preparation.

On the other hand, the method for producing a biodiesel of the present invention (a) obtaining a lipid component from the biomass; And (b) adding methanol to the obtained lipids and reacting under an alkali catalyst to obtain FAME (fatty acid methyl), which is biodiesel. At this time, in order to increase the yield of FAME may further comprise the step of removing the reaction by-product glycerol.

In the production method of the biodiesel of the present invention, in order to obtain a lipid component from the biomass, various known methods can be used. For example, the microalgae contained in the biomass may be dried and obtained by physically crushing the microalgae, or lipid components may be extracted from the microalgae by adding an organic solvent to the biomass. In this case, a non-polar solvent may be used as the solvent, and preferably hexane, dimethyl sulfoxide (DMSO), dimethyl carnonate (DMC), or the like may be used.

In addition, since the lipid component obtained from the biomass is derived from the microalgae with increased lipid content in the cells provided by the present invention, palmitate (palmitate, C16: 0), palmitoleate (palmitoleate, C16: 1) , Stearate (C18: 0), oleate (C18: 1), linoleate (C18: 2), linolenate (C18: 3), etc., alone or in combination Can be.

The term "bio-diesel" of the present invention broadly refers to the whole of pollution-free fuel made from vegetable oils, and narrowly to fatty acid methyl esters prepared using vegetable oils such as soybean oil as raw materials. ). The biodiesel can be prepared by various methods, and is usually prepared by treating methanol with an alkaline catalyst in triglycerides.

As another embodiment for achieving the above object, the present invention provides the use of microalgae with increased lipid content for the production of biodiesel.

As described above, since the microalgae increased in the lipid content produced by the method of the present invention contain a large amount of lipids containing various fatty acids in the microalgal cells, it is used as a raw material (biomass) in the production of biodiesel. Can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Effect of Salt Stress on Microalgae

When culturing Chlorella sorokiniana HS strain (KCTC 12171BP) isolated from livestock wastewater, the inventors found that the increase in the concentration of salt in the medium reduced the number of cells, but increased the volume of the cells themselves. It was confirmed that it exhibits a characteristic of increasing the dry weight, and a patent application (Korean Patent Application No. 2012-0073061, July 4, 2012). In the following, it was intended to analyze the effect of salt stress on the microalgae.

Example 1-1 Cultivation of Strains Under Salt Stress Conditions

In order to analyze the effect of salt stress conditions using a culture solution containing a high concentration of salt on chlorella soroquinia or HS strain, the chlorella in BG11 medium containing no salt and BG11 medium containing 3% of salt (NaCl) was not included. Sorokonia or HS strains were inoculated, incubated and the cultures collected. At this time, the BG11 medium was prepared by mixing Stock No. 1 to Stock No. 9 disclosed in Table 1 below, specifically, per 1 L of medium, 100 ml of Stock No. 1, 10 ml of Stock No. 2, and Stock No. 3 10 ml, 10 ml of Stock No. 4, 10 ml of Stock No. 5, 10 ml of Stock No. 7, 10 ml of Stock No. 8, 10 ml of Stock No. 8 and 10 ml of excess distilled water. After that, 1 ml of Stock No. 6 was finally added to prepare.

Table 1 Composition of BG11 Medium

Stock no.	Medium	Volume	Addition per liter
One	NaNO 3	15 g / ℓ	100 ml
2	K 2 HPO 4	4.0 g / 500 ml	10 ml
3	MgSO 4 .7H 2 O	3.75 g / 500 ml	10 ml
4	CaCl 2 .2H 2 O	1.80 g / 500 ml	10 ml
5	citric acid	0.30 g / 500 ml	10 ml
6	EDTA Na 2	0.05 g / 500ml	1 ml
7	Ammonium ferric citrate green	0.30 g / 500 ml	10 ml
8	Na 2 CO 3	1.0 g / 500 ml	10 ml
9	Trace metal solution		10 ml
	H 3 BO 3	2.86 g / ℓ
	MnCl 2 4H 2 O	1.81 g / ℓ
	ZnSO 4 7H 2 O	0.22 g / ℓ
	Na 2 MoO 4 2H 2 O	0.39 g / ℓ
	CuSO 4 5H 2 O	0.08 g / ℓ
	Co (NO 3 ) 2 6H 2 O	0.05 g / ℓ

Example 1-2 Morphological Characterization of Strains Cultured Under Salt Stress Conditions

The culture collected in Example 1-1 was applied to an electron microscope (TEM) to confirm the intracellular changes contained in the culture (FIG. 1).

FIG. 1A is a TEM photograph showing the inside of cells of chlorella soroquinia or HS strain cultured in BG11 medium containing no salt, and FIG. 1B is a chlorella soroquinia or HS strain cultured in BG11 medium containing salt. TEM image showing the inside of the cell. As shown in Figure 1A, Chlorella Sorokonia or HS strains cultured in BG11 medium containing no salts have a size of about 2㎛, and in the cells, thylakoids, chloroplasts, pyrenoids, starch (S ) And lipids (L) were found. On the contrary, as shown in FIG. 1B, the Chlorella sorokiniana or HS strain cultured in BG11 medium containing salts has a size of about 6 μm, and thylakoids, chloroplasts, pyrenoids, and a plurality of strains are Starch (S) and a large number of lipids (L) was found to be included. From this, when chlorella Sorokonia or HS strains were cultured in a medium containing salts, it was found that the content of starch and lipids in the cells increased, thereby increasing the cell size as a whole.

Example 1-3 Lipid Content Analysis

From the results of Example 1-2, it was confirmed that the chlorella soroquinia or HS strain cultured under salt stress conditions increased the size of the cells as a result of increasing the content of starch and lipids in the cells. The change in lipid content was measured.

Specifically, inoculating BG11 medium containing 0, 1, 2, 3 or 4% of the salt (NaCl) inoculated with the chlorella soro kina or HS strain culture to collect the cultures, each cell was obtained from the culture solution. . To each cell obtained, a mixed solvent containing chloroform and methanol in a ratio of 2: 1 (v / v) was added thereto, followed by stirring. Then, distilled water was further added. Finally, the ratio of chloroform, methanol, and distilled water was 1: 1: 0.9. (v / v / v). Then, the stirring was stopped, the chloroform layer was fractionated, and the content of lipid derived from each cell dissolved in the fractionated chloroform was measured by a known method (Bioresource Technology 101: S75-S77) (FIG. 2).

Figure 2 is a graph showing the change in lipid content of the mycelia of Chlorella Sorokonia or HS strain according to the concentration of salts contained in the medium. As shown in Figure 2, the lipid content contained in the cells of the strain cultured in BG11 medium containing no salt is about 10%, lipid content contained in the cells of the strain cultured in BG11 medium containing 1% salt Is about 15% and the lipid content contained in the cells of the strain cultured in BG11 medium containing 2% of the salt is about 25%, contained in the cells of the strain cultured in BG11 medium containing 3% of the salt Lipid content is about 34%, it was confirmed that the lipid content contained in the cells of the strain cultured in BG11 medium containing 4% salt of about 32%. From this, the cells cultured in a medium containing 3% salt showed the highest intracellular lipid content, and the lipid content in the cells was increased by about 3.4 times as compared to the cells cultured in the medium containing no salt. Cells cultured in a medium containing salts showed a tendency to decrease the lipid content in the cells slightly.

Example 1-4 Analysis of the Composition of Lipids in Cells of Chlorella Sorokini and HS Strains

The composition of each cell-derived lipid obtained in Example 1-3 was confirmed by GC analysis.

Specifically, 1 mL of KOH-CH ₃ OH was added to 50 mg of the culture cell obtained in Example 1-3 and reacted at 75 ° C. for 10 minutes, and 5% HCl and methanol were added thereto and then again at 75 ° C. The reaction was carried out for 10 minutes. Subsequently, hexane and (CH ₃ ) ₃ COCH ₃ were added to the reactants, and the mixture was mixed vigorously after distilled water was added. The mixture was left at room temperature and fractionated to obtain an organic solvent layer containing lipid components. The obtained organic solvent layer was concentrated under reduced pressure and lyophilized to obtain a lipid component, and GC analysis was performed using the obtained lipid component under the following conditions: Gas chromatograph Shimadzu GC-2010, Japan), DB-WAX (30 m, 0.25 mm) was used as the GC column, helium (30 ml / min) was used as the carrier gas, and the injection volume was 1 μl. The split ratio was 20: 1, and the oven condition was raised to 5 ° C. per minute up to 250 ° C. for 1 minute at 170 ° C. and maintained at 250 ° C. for 12 minutes (FIG. 3). .

Figure 3 is a graph showing the composition of lipids in cells of chlorella sorokinia or HS strain cultured under various salt stress conditions. As shown in Fig. 3, the intracellular lipids of chlorella soroquinia or HS strain are palmitate (C16: 0), palmitoleate (C16: 1), stearate (Stearate, C18: 0) as major fatty acids. , Oleate (C18: 1), linoleate (C18: 2), linolenate (C18: 3) and the like. In particular, palmitate is not included in chlorella sorokinia or HS cultured in a medium that does not contain salts, and is contained only in chlorella sorokinia or HS cultured in a medium containing salts. As the concentration was increased, it was confirmed that the content of the fatty acid tended to increase.

Example 1-5 Nile red Staining Analysis of Chlorella Sorokini or HS Strains Cultured Under Stress Conditions by Salt Shock

From the results of Examples 1-3, when chlorella Sorokonia or HS strains were cultured in a medium containing salts, it was confirmed that the content of lipids in the cells increased, the strains were cultured in a salt-free medium, The addition of the salt was to determine whether the same result can be obtained even if the salt shock to the strain.

Example 1-5-1: Cultivation of Strains Under Salt Stress Conditions

Cells were obtained by culturing the Chlorella Sorokiana HS strain in BG11 medium without salts, and then the cells were obtained at various concentrations (0, 1, 3, 5, 7, 10 or 15% (w / v). Each culture was collected at 6, 12, 24 or 30 hours after inoculation into BG11 medium containing NaCl and further incubation for 30 hours.

Example 1-5-2: Lipid Content Analysis

Chlorella sorokini cultured for 6, 12, 24 or 30 hours in BG11 medium containing 0, 7, 10 or 15% (w / v) of NaCl in the culture collected in Example 1-5-1 Except for using the culture of Ana HS strain, the method of Example 1-3 was carried out to analyze the content of lipids contained in the cells of Chlorella sorokinia or HS strain cultured under salt stress conditions ( 4 and Table 2).

4 is incubated for 6, 12, 24 or 30 hours in BG11 medium containing 0, 7, 10 or 15% (w / v) of NaCl, Chlorella sorokinina HS strain, the cultured Chlorella sorokiniana It is a graph showing the results of analyzing the lipid content in these cells in HS strains.

TABLE 2 Intracellular Lipid Contents of Chlorella Sorokini and HS Strains Cultured in Salt Stress Conditions

Incubation time	NaCl concentration in the medium
	0%	7%	10%	15%
6 hours	20.94%	26.57%	26.72%	27.00%
12 hours	20.94%	33.52%	34.09%	37.26%
24 hours	24.52%	36.58%	38.63%	38.70%
30 hours	24.79%	25.00%	38.35%	38.90%

As shown in Figure 4 and Table 2, Chlorella Sorokonia or HS strain increased the lipid content in the cells in proportion to the concentration and the culture time of NaCl contained in the medium, but contains more than 10% (w / v) NaCl Incubation in BG11 medium for 24 hours or more did not increase the lipid content in the cells even after the incubation time. The highest value of lipid content in cells obtained by culturing the Chlorella Sorokonia or HS strain in a medium containing NaCl was 38.90 obtained in a cell cultured for 30 hours in BG11 medium containing 15% (w / v) NaCl. It was found to be%.

Example 1-5-3: Nile red staining analysis

In cultures collected in Examples 1-5-1, 0, 6, 24 or 30 hours in BG11 medium containing 0, 1, 3, 5, 7, 10 or 15% (w / v) of NaCl Cells were obtained from each of the cultures of Chlorella Sorokonia or HS strains cultured during the period, and Nile red staining was performed by lipids in the cells, and the results were observed with a fluorescence microscope (FIG. 5). At this time, Nile red staining, the dye solution in which Nile red was dissolved in acetone at a concentration of 0.25mg / ㎖ was added to each of the cell samples and reacted for 5 minutes while stirring, confocal fluorescence microscope (ZEISS LSM510Meta, Germany) was used to measure excitation at 490 nm and emission value at 585 nm.

FIG. 5 shows chlorella sorokiniana or HS strains cultured in a salt-free medium, 0, 6, in BG11 medium containing 0, 1, 3, 5, 7, 10 or 15% (w / v) NaCl. After culturing for 24 or 30 hours, it is a fluorescence micrograph showing the result of performing Nile red staining on the cultured Chlorella Sorokonia or HS strain. As shown in Figure 5, when the content of NaCl contained in the medium was increased, the lipid content in the cells was confirmed that the Nile red staining level is increased.

From the results of Examples 1-5-1 to 1-5-3, it was found that increasing the content of salts in the medium increased the lipid content in the microalgae cells.

Example 2 Effect of Oxidative Stress on Microalgae

Microalgae Chlorella vulgaris OW-01 strain (Chlorella vulgaris OW-01) ( KCTC 12553BP), Chlorella Thoreau Kearney Ana HS strain (Chlorella sorokiniana HS) (KCTC 12171BP ) or styryl fifth claw nium in HS strain (Stigeoclonium sp. HS) We tried to analyze the effect of oxidative stress on (KCTC 12676BP).

Example 2-1 Effect of Oxidative Stress on Chlorella vulgaris OW-01 Strain

The oxidative stress was added to the medium of Chlorella vulgaris OW-01 strain, and then the level of change in lipid content was measured.

Example 2-1-1: Cultivation of Strains Under Oxidative Stress Conditions

Chlorella vulgaris was treated with OW-01 strain (Chlorella vulgaris OW-01) ( KCTC 12553BP) , the concentration of hydrogen peroxide of inoculation, thereby oxidizing agent in BG11 medium of 0, 1, 5, 10, or 20 mM was the oxidative stress Each culture was collected at 6 and 24 hours after incubation.

Example 2-1-2: Lipid Content Analysis

Except for using the culture collected in Example 2-1-1, by performing the method of Example 1-3, contained in the cells of Chlorella vulgaris OW-01 strain cultured under oxidative stress conditions The content of lipids was analyzed (FIG. 6).

FIG. 6 is a graph showing the change in lipid content in cells over time after the addition of hydrogen peroxide to chlorella vulgaris OW-01 cultured in BG11 medium not containing hydrogen peroxide as an oxidizing agent. As shown in Figure 6, the content of the lipid contained in the strain of the control group (0mM) without addition of the oxidant is about 18.22%, the content of lipids contained in the strain of the experimental group incubated for 24 hours with the addition of 1mM oxidant There was no difference as about 18.93%. However, the content of lipid in the strain of the experimental group incubated for 24 hours with 5 mM oxidant was about 24.23%, and the content of lipid in the strain of the experimental group incubated for 24 hours with 10 mM oxidant was 31.67%, the content of lipid contained in the strain of the experimental group incubated for 24 hours with the addition of 20mM oxidant was confirmed that about 31.25%.

Example 2-1-3: Nile red staining analysis

Except for using the culture collected in Example 2-1-1, by performing the method of Example 1-5-3, cells of Chlorella vulgaris OW-01 strain cultured under oxidative stress conditions Nile red staining analysis was performed on the subject (FIG. 7).

FIG. 7 is a micrograph showing changes in lipid content in cells over time after oxidative stress was added by adding hydrogen peroxide to chlorella vulgaris OW-01 cultured in BG11 medium without hydrogen peroxide as an oxidizing agent. As shown in Figure 7, in the control group without addition of the oxidizing agent (0mM) was not observed in the yellow strain, but as the concentration of the oxidizing agent and the incubation time was confirmed that the number of strains displayed in yellow increases It was. In particular, in the experimental group treated with the oxidant at a concentration of 10mM or more, it was confirmed that most strains are displayed in yellow.

From the results of Examples 2-1-1 to 2-1-3, when oxidizing agent is treated with Chlorella vulgaris OW-01 strain at a concentration of 5 mM or more, the lipid content in the strain is increased, and when 10 mM oxidant is treated. , The lipid content in the strain showed a maximum value, it was found that the increased lipid content is continuously maintained when the oxidant is treated at a concentration of 10mM or more. The maximum value of the lipid content in the cells increased by oxidative stress was about 1.7 times compared to the control.

Example 2-2 Effect of Oxidative Stress on Chlorella Sorokini and HS Strains

An oxidative stress was added to chlorella soroquinia or HS strain, and then the level of change in lipid content was measured.

Example 2-2-1: Culture of Strains under Oxidative Stress Conditions

Chlorella sorokiniana HS strain (KCTC 12171BP) was cultured in BG11 medium without salts to obtain the cells, and then the cells were obtained at various concentrations (0, 1, 3, 5, 10). , 15 or 20 mM) was inoculated in BG11 medium containing hydrogen peroxide and further incubated for 30 hours, with each culture collected at time points 0, 6, 12, 24 or 30 hours had elapsed.

Example 2-2-2: lipid content analysis

Among the cultures collected in Example 2-2-1, cultures of Chlorella sorokiniana or HS strains cultured for 0, 6 or 24 hours in BG11 medium containing 0, 1, 5, 10 or 20 mM hydrogen peroxide Except for using, the method of Example 1-3 was carried out to analyze the content of lipids contained in the cells of Chlorella Sorokonia or HS strain cultured under oxidative stress conditions (Fig. 8).

8 is incubated for 0, 6, or 24 hours in BG11 medium containing 0, 1, 5, 10 or 20 mM hydrogen peroxide for chlorella sorokinina or HS strains, It is a graph showing the result of analyzing lipid content in cells. As shown in FIG. 8, the chlorella soroquinia or HS strain increased the lipid content in the cells in proportion to the concentration of hydrogen peroxide in the medium and the culture time, but the culture time when incubated in BG11 medium containing more than 10 mM hydrogen peroxide Even after this time, it was found that the lipid content in the cells was no longer increased. The maximum value of lipid content in cells obtained by culturing the Chlorella Sorokonia or HS strain in a medium containing hydrogen peroxide was 31.67% obtained in cells cultured in BG11 medium containing 10 mM hydrogen peroxide for 24 hours.

Example 2-2-3: Nile red staining analysis

Chlorella sorokini cultured for 0, 6, 24 or 30 hours in BG11 medium containing 0, 1, 3, 5, 10, 15 or 20 mM hydrogen peroxide in the culture collected in Example 2-2-1 Except for using the culture of the Ana HS strain, by performing the method of Example 1-5-3, Nile red staining analysis was performed on the cells of Chlorella Sorokonia or HS strain cultured under oxidative stress conditions Was performed (FIG. 9).

9 is a chlorella sorokinina HS strain incubated for 0, 6, 24 or 30 hours in BG11 medium containing 0, 1, 3, 5, 10, 15 or 20 mM hydrogen peroxide, the cultured chlorella sorokinina Fluorescence micrograph showing the result of performing Nile red staining on HS strain. As shown in Figure 9, when the content of hydrogen peroxide contained in the medium was increased, the lipid content in the cells was confirmed that the Nile red staining level is increased. Specifically, an increase in Nile red staining level due to an increase in lipid content was observed in a medium containing more than 5 mM hydrogen peroxide in the case of Chlorella Sorokonia or HS strain, and furthermore Nile red staining level in a medium containing more than 10 mM hydrogen peroxide. This did not increase. In addition, even when cultured in a medium containing a high concentration of hydrogen peroxide, the difference in the Nile red staining level according to the culture time was observed.

From the results of Examples 2-2-1 to 2-2-3, as well as Chlorella vulgaris OW-01 strain, Chlorella sorokinia or HS strain also increased the lipid content in the cells when the oxidant content was increased in the medium. Could know.

Example 2-3: Effect of Oxidative Stress on HS Strains of Stimeoclonium

When the chlorella vulgaris OW-01 strain or chlorella Sorokiana HS strain, which is a chlorella strain under conditions subjected to oxidative stress, was observed from the results of Examples 2-1 and 2-2, it was confirmed that the lipid content in the cells was increased. , To determine whether the same effect in other strains than the strain of the genus Chlorella.

Example 2-3-1: Culture of Strains under Oxidative Stress Conditions

Stigeoclonium sp. HS (KCTC 12676BP), which belongs to the fungus, was cultured in BG11 medium without salts to obtain the cells, and then the cells were obtained at various concentrations (0, 1, Each culture was collected at 0, 6, 12, 24 or 30 hours, inoculating BG11 medium containing 3, 5, 10, 15 or 20 mM hydrogen peroxide and further incubating for 30 hours. .

Example 2-3-2: Lipid Content Analysis

Among the cultures collected in Example 2-3-1, the genus Stizeoclonium cultured for 6, 12 or 24 hours in BG11 medium containing 0, 1, 3, 5, 10, 15 or 20 mM hydrogen peroxide Except for using the culture of the HS strain, the method of Example 1-3 was carried out to analyze the content of lipids contained in the cells of the HS strain of the genus Styreclonium cultured under oxidative stress conditions ( 10 and Table 3).

FIG. 10 shows HS strains of the genus Stizelonium incubated for 6, 12 or 24 hours in BG11 medium containing 0, 1, 3, 5, 10, 15 or 20 mM hydrogen peroxide, It is a graph showing the results of analyzing the lipid content in these cells in HS strains.

TABLE 3 Intracellular Lipid Contents of HS Strains of Stimeoclonium Cultured in Various Conditions

Incubation time	Hydrogen Peroxide Concentration in Medium
	0mM
	1 mM	3mM	5 mM	10 mM	15 mM	20mM
6 hours	21.95%	26.72%	27.12%	29.62%	28.53%	28.00%	29.08%
12 hours	23.58%	29.65%	30.25%	33.73%	37.09%	38.21%	38.24%
24 hours	24.52%	34.52%	35.17%	36.84%	38.51%	39.51%	37.51%

As shown in FIG. 10 and Table 3, HS strains of the genus Stizeoclonium increased the lipid content in the cells in proportion to the concentration of hydrogen peroxide contained in the medium and incubation time, to be cultured in BG11 medium containing more than 10 mM hydrogen peroxide In this case, it was found that the lipid content in the cells did not increase any more even after the incubation time. It can be seen that the highest value of lipid content in cells obtained by culturing the HS strain of S. zeoclonium in a medium containing hydrogen peroxide was 39.51% obtained in cells cultured in BG11 medium containing 15 mM hydrogen peroxide for 24 hours. .

Example 2-3-3: Nile red staining analysis

Among the cultures collected in Example 2-3-1, Stizeoclo incubated for 0, 6, 12 or 24 hours in BG11 medium containing 0, 1, 3, 5, 10, 15 or 20 mM hydrogen peroxide Nile red staining was performed on the cells of HS strain of S. zeoclonium cultured under oxidative stress conditions by performing the method of Example 1-5-3, except that a culture of the strain of S. nilium was used. Analysis was performed (FIG. 11).

FIG. 11 is incubated with 0, 1, 3, 5, 10, 15, or 20 mM BG11 medium containing 0, 1, 3, 5, 10, 15, or 20 mM hydrogen peroxide for 0, 6, 12, or 24 hours, and the cultured Stizeoclo Fluorescence micrograph showing the result of performing Nile red staining on the HS strain of the genus. As shown in Figure 11, when the content of hydrogen peroxide contained in the medium was increased, the lipid content in the cells was confirmed that the Nile red staining level is increased. Specifically, an increase in Nile red staining level was observed due to an increase in lipid content in a medium containing more than 1 mM hydrogen peroxide, and when incubated in a medium containing more than 10 mM hydrogen peroxide for 12 hours, Nile red was no longer present. No difference in staining levels was observed.

From the results of the above Examples 2-3-1 to 2-3-3, increasing the content of the oxidant in the medium in the microalgae other than the genus Chlorella provided by the present invention, the lipid content in the microalgae is increased Could know.

[Correction under Article 91 of the Rule 27.11.2014]

[Correction under Article 91 of the Rule 27.11.2014]

[Correction under Article 91 of the Rule 27.11.2014]

Claims (9)

1. Comprising a step of culturing the microalgae under stress conditions, a method of producing a microalgae with increased lipid content.
2. The method of claim 1,

   Wherein said stress condition is caused by salt stress or oxidative stress.
3. The method of claim 2,

   The salt stress is sodium chloride (NaCl), magnesium chloride (MgCl ₂ ), magnesium sulfate (MgSO ₄ ), calcium sulfate (CaSO ₄ ), potassium sulfate (K ₂ SO ₄ ), calcium carbonate (CaCO ₃ ), magnesium bromide ( MgBr ₂ ) and a salt selected from the group consisting of a combination thereof are formed by adding to the medium of microalgae.
4. The method of claim 3,

   The concentration of the salt contained in the medium is 1 to 30% (w / v).
5. The method of claim 2,

   The oxidative stress is hydrogen peroxide (H ₂ O _2), excess oxidizing ion (superoxide ion, 0 ^2-), hydroxyl radicals (hydroxyl radical, OH ^-), tea acid (hypochlorous acid, HOCl), and from the group consisting of a combination of Wherein the oxidant of choice is added to the medium of microalgae.
6. The method of claim 5,

   The concentration of the oxidant contained in the medium is 5 to 100mM.
7. The method of claim 1,

   Wherein said microalgae is a strain selected from the group consisting of chlorella genus strain, stigeoclonium strain and combinations thereof.
8. A microalgae prepared using the method of any one of claims 1 to 7, wherein the lipid content in the cells is increased compared to the strain cultured without stress conditions.
9. A biomass comprising microalgae having an increased lipid content in cells of claim 8.

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