KR102274119B1 - Novel Chlorella sp. strain having high oil productivity in fresh water, sea water and rackish water - Google Patents

Abstract

The present invention relates to a Chlorella sp. cell line, which is a microalgae isolated from rivers and capable of rapid growth in freshwater, seawater and brackish water, and excellent in biomass, pigment productivity and lipid productivity, and uses thereof, Unlike other microalgae and microorganisms, the isolated Chlorella HS2 can be cultured at a very fast growth rate in freshwater, seawater and brackish water, so it is free to use water for medium production, shows high biomass productivity, and has a high intracellular lipid content. It is excellent and can be used in the microalgal oil and biodiesel markets. Also, the pigment content is 1.8%, similar to or slightly higher than that of other microalgae, but due to the fast growth rate, the pigment productivity is higher than that of existing microalgae, and has potential as a resource for pigment production, which is very useful in related industries.

Description

A new strain of Chlorella with high oil productivity in freshwater, seawater and brackish water {Novel Chlorella sp. strain having high oil productivity in fresh water, sea water and rackish water}

The present invention relates to a Chlorella sp. HS2 cell line, which is a microalgae isolated from rivers and capable of rapid growth in freshwater, seawater and brackish water, and excellent in biomass, pigment productivity and lipid productivity, and uses thereof.

Recently, biodiesel has been receiving a lot of attention as an alternative fuel to diesel. Biodiesel is defined as methyl or ethyl ester compounds of fatty acids produced from vegetable oils or animal fats. Biodiesel is suitable as an eco-friendly vehicle fuel in that it can significantly reduce the emission of air pollutants such as carbon monoxide, fine dust, hydrocarbons, and toxic substances compared to conventional diesel. In addition, carbon dioxide from the combustion of biodiesel is absorbed and fixed again by the photosynthetic mechanism of plants, so there is almost no net carbon dioxide emission, so it is receiving great attention as a _{carbon dioxide neutral fuel (CO 2 -neutral fuel).}

In particular, the production of biofuels (bioethanol, biodiesel) is attracting attention, but the production of biofuels using crops causes problems such as destruction of the ecosystem and food shortage due to the expansion of arable land. However, the solar energy utilization efficiency of microalgae, a photosynthetic microorganism, is about 5%, about 25 times higher than that of terrestrial plants, and the carbon dioxide fixation rate is 15 times that of pine trees.

In addition, the biodiesel production per unit area of microalgae (when the oil content is 30%) is about 58,700 L/ha, which is 130 times higher than that of soybean 446 L/ha. Microalgae are 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 culture using idle arable land, ease of strain improvement, and irrelevance to 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 the natural material cycle, and the process is performed at room temperature and pressure, and the produced biomass is used as a useful material. There are advantages.

Meanwhile, Korean Patent No. 1625074 discloses 'microalgae with increased lipid content by using oxidative stress and a method for producing the same', and Korean Patent No. 1424852 discloses 'Chlorella vulgaris CV-16 producing biodiesel'. and 'a method for producing biodiesel using the same' are disclosed, but as in the present invention, a chlorella cell line, a microalgae with excellent pigment productivity and lipid productivity, isolated from rivers, and uses thereof, are not disclosed at all.

The present invention was derived from the above needs, and the present invention is a novel microalgae, Chlorella sp., HS2 cell line isolated from a river, when cultured in fresh water (BG11), excellent biomass and pigment productivity, and , it was confirmed that the lipid productivity was excellent when cultured in seawater (F/2).

In particular, it was confirmed that the growth rate of Chlorella HS2 was the fastest in freshwater (BG11) medium on average over the entire period, which was very fast compared to other microalgae, and exhibited the highest values in the maximum cell number and maximum cell dry weight. In addition, as cultured in medium with high salt concentration, total lipid content and FAME (fatty acid methyl esters) showed very high oil content, and fatty acid composition showed the highest content of oleic acid and linoleic acid, and pigment content including lutein. Also, the highest was confirmed.

As described above, the characteristic that the Chlorella HS2 cell line of the present invention can grow in freshwater, seawater and brackish water is a rare phenomenon not only in other microalgae but also in bacteria, confirming that it is a very specific phenomenon through the present invention.

Therefore, the Chlorella HS2 cell line of the present invention has very high water availability, high biomass productivity and high oil content, so it can be used as a microalgal oil and biodiesel production strain, and has potential as a lutein producing strain. , the present invention was completed.

In order to solve the above problems, the present invention provides a Chlorella sp. cell line, which is a microalgae separated from rivers and capable of rapid growth in freshwater, seawater and brackish water, and excellent in biomass, pigment productivity and lipid productivity.

In addition, the present invention provides a microbial preparation for lipid production comprising the cell line or a culture medium thereof as an active ingredient.

In addition, the present invention provides a method for producing a lipid, characterized in that the cell line is cultured and the lipid is separated from the culture solution.

In addition, the present invention is prepared by the above method, and contains oleic acid (C18:1) and linoleic acid (C18:2) as main fatty acids, and oleic acid (C18:1) and linoleic acid (C18:2) based on total lipid weight It provides lipids containing 45 to 55% of the polyunsaturated fatty acids and 10% or less based on the total weight of the lipids.

In addition, the present invention provides a method for producing biodiesel, comprising the steps of culturing the cell line, separating lipids from the culture medium, and transesterifying the lipids to produce fatty acid esters and glycerol.

In addition, the present invention provides a method for producing glycerol, comprising the steps of culturing the cell line, separating lipids from the culture medium, and transesterifying the lipids to produce fatty acid esters and glycerol.

Chlorella HS2 newly isolated in the present invention can be cultured at a very fast growth rate in freshwater, seawater, and brackish water, unlike other existing microalgae and microorganisms, so it is free to use water for medium production and shows high biomass productivity. Due to its excellent lipid content, it can be used in microalgal oil and biodiesel markets. In addition, the pigment content is similar to or slightly higher than that of other microalgae, but due to the rapid growth rate, the pigment productivity is higher than that of existing microalgae, and has potential as a resource for pigment production, which is very useful in related industries.

1 shows a phylogenetic diagram of Chlorella HS2 isolated in the present invention.
Figure 2 shows the results of confirming that Chlorella HS2 isolated in the present invention can grow in freshwater, brackish water and seawater.
3 is a result confirming the growth potential in fresh water, brackish water and seawater of Chlorella HS2 and other microalgae isolated in the present invention.
4 is a study of optimal conditions for culturing Chlorella HS2 isolated in the present invention.
5 is a culture result of Chlorella HS2 isolated in the present invention using fresh water, sea water and brackish water in a photobioreactor. A) Cell Number, B) Dry cell weight
6 shows the highest specific growth rate and generation time of Chlorella HS2 isolated in the present invention from freshwater, brackish water and seawater.
7 shows an optical microscope (A), a fluorescence microscope analysis (B) and a biomass color (C) of Chlorella HS2 isolated in the present invention.
8 is a result of analyzing the total lipid content (A), fatty acid composition ratio (B) and fatty acid content ratio (C) of Chlorella HS2 isolated in the present invention.
9 shows changes in cell components of each medium of Chlorella HS2 isolated in the present invention.
10 shows the change in the pigment content of each medium of Chlorella HS2 isolated in the present invention.
11 shows the pigment productivity (A) and lutein productivity (B) of Chlorella HS2 isolated in the present invention.

In order to achieve the object of the present invention, the present invention provides a Chlorella sp. cell line, which is a microalgae separated from rivers and capable of rapid growth in freshwater, seawater and brackish water, and has excellent biomass, pigment productivity and lipid productivity do. The cell line is preferably a Chlorella sp. HS2 cell line having an accession number of KCTC 13108BP.

The Chlorella HS2 cell line was identified through 18S rDNA sequencing of microalgae isolated from rivers, and it was confirmed that the Chlorella HS2 cell line was capable of rapid growth in freshwater, seawater and brackish water, and had excellent biomass, pigment productivity and lipid productivity. The Chlorella HS2 cell line capable of rapid growth in freshwater, seawater and brackish water and having excellent biomass, pigment productivity and lipid productivity was deposited with the Korea Research Institute of Biotechnology and Biotechnology (KCTC) on September 13, 2016 (Accession No.: KCTC 13108BP) ).

The Chlorella HS2 cell line of the present invention has excellent water availability, and, in particular, when cultured in fresh water, has excellent biomass and pigment productivity, and has excellent lipid productivity when cultured in seawater.

When the cell line according to an embodiment of the present invention is cultured in fresh water, the biomass production may be 600-700 mg/L/day, and the lutein pigment production may be 4-4.5 mg/L/day, but this not limited

The cell line according to an embodiment of the present invention preferably contains 60 to 70% by weight of the total lipid content based on cell components, and 60 to 70% by weight of fatty acid methyl ester (FAME) based on fatty acid when cultured in seawater. It may contain % by weight, and most preferably contains 60 to 65% by weight of the total lipid content based on cell components, and may contain 60 to 65% by weight of fatty acid methyl ester (FAME) based on fatty acid, It is not limited thereto.

In the chlorella cell line according to an embodiment of the present invention, the cell line can produce lipids including oleic acid (C18: 1) and linoleic acid (C18: 2) as major fatty acids, preferably the oleic acid (C18: 1) And the content of linoleic acid (C18:2) may be 45 to 55% based on the total lipid weight, but is not limited thereto.

The Chlorella HS2 cell line according to an embodiment of the present invention is the optimal culture condition under freshwater medium carbon dioxide condition, temperature 34~38℃, light 300~600μmol·m ^-2 ·s ^-1 , and temperature 34~36 under seawater medium carbon dioxide condition. ℃, light 450-600 μmol·m ^-2 ·s ^-1 Optimum culture conditions, but is not limited thereto.

In addition, the cell line polyhydric unsaturated fatty acid (PUFA) has three or more double bonds, the content of the polyunsaturated fatty acid may be 10% or less based on the total lipid weight, most preferably the total lipid weight It may be 6-7% based on, but is not limited thereto.

In addition, the present invention provides a microbial preparation for lipid production comprising the cell line or a culture medium thereof as an active ingredient. The microbial preparation may include the Chlorella HS2 cell line as an active ingredient, and can be effectively used for mass production of bio-oil. The mass-produced bio-oil may be used to produce biodiesel through a transesterification process.

In the microbial preparation according to an embodiment of the present invention, the lipid contains 45 to 55% of oleic acid (C18:1) and linoleic acid (C18:2) based on the total lipid weight, and contains polyunsaturated fatty acids based on the total lipid weight. It may contain 10% or less, preferably 45 to 50% of oleic acid (C18:1) and linoleic acid (C18:2) based on the total lipid weight, and 6-7 polyunsaturated fatty acids based on the total lipid weight %, but is not limited thereto.

In addition, the present invention provides a method for producing a lipid, characterized in that the cell line is cultured and the lipid is separated from the culture solution. The cell line may accumulate lipids at a high concentration at various culture temperatures, and any method known in the art may be used for the method of separating the lipids from the cell line culture medium.

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

Lipids according to an embodiment of the present invention include oleic acid (C18:1) and linoleic acid (C18:2) as major fatty acids, and oleic acid (C18:1) and linoleic acid (C18:2) are 45 based on total lipid weight. ~55%, polyunsaturated fatty acid may contain 10% or less based on the total lipid weight, preferably oleic acid (C18:1) and linoleic acid (C18:2) 45-50% based on the total lipid weight and may contain 6 to 7% of polyunsaturated fatty acids based on the total lipid weight, but is not limited thereto.

The present invention also comprises the steps of culturing the cell line of the present invention;

separating lipids from the culture solution; and

It provides a method for producing biodiesel comprising the step of transesterifying the lipid to produce a fatty acid ester and glycerol.

In the method of the present invention, culturing of the cell line may be preferably performed at a temperature of 34°C to 38°C. As the cell line culture medium, a medium commonly used in the art may be used. The fatty acid ester may preferably be a methyl ester or an ethyl ester, but is not limited thereto. The biodiesel may be produced in the form of a methyl ester or an ethyl ester by a transesterification process of fatty acids contained in biomass, and glycerol may 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 small, straight-chain molecules required by a regular diesel engine. As an emulsifier in the transesterification process, Triton X-100 or Tween 60 may be added, but is not limited thereto. The emulsifier promotes interfacial stability of the bio-oil and allows it to be mixed well, thereby increasing the reaction yield, thereby saving the cost of recovering biodiesel. In addition, sodium hydroxide or potassium hydroxide may be used as a reaction catalyst of the transesterification process, but is not limited thereto.

The biodiesel may have the following advantages as a biofuel; (1) Biofuels can be easily obtained from common biomass resources. (2) Biofuels represent the cycle of carbon dioxide in combustion. (3) Biofuels have the potential to be environmentally friendly. (4) There are many benefits to the environment, economy and consumers by using biofuels. (5) Biofuels can be broken down into harmless substances by microorganisms, contributing to sustainability.

The present invention also comprises the steps of culturing the cell line of the present invention;

separating lipids from the culture solution; and

It provides a method for producing glycerol comprising the step of transesterifying the lipid to produce a fatty acid ester and glycerol.

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

The glycerol is a lubricant, ointment or suppository, enema, desiccant or polishing agent for food or cosmetics, antifreeze, coolant, paint, pigment, printing ink, transparent soap production, laboratory analytical reagent, solvent, cellophane , may be used in the manufacture of adhesives, alkyd resins or dynamite, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Isolation and identification of microalgae

As a result of sequencing the 18S rDNA of microalgae isolated from domestic rivers and searching for it in NCBI blast, the isolated HS2 is Micratinium , Although homology with Chlorella was high (FIG. 1), there was a morphological difference from Micratinium, and Chlorella vulgaris ( Chlorella ) vulgaris ) or Chlorella sorokiniana ( Chlorella sorokiniana ), freshwater, seawater, and brackish water, all growthable characteristics ( FIG. 2 ) were judged to be different species from these and named as Chlorella sp. HS2. It was confirmed once again that E. coli, Bacillus, Pseudomonas, Ryzobium, which are bacteria widely used in laboratories, do not grow at all in seawater through the present invention (data not shown), and generally in seawater and freshwater through diversity analysis. Growable bacteria are clearly divided. However, it was confirmed that the characteristic of the Chlorella genus HS2 strain of the present invention, which can grow in freshwater, seawater and brackish water, is a rare phenomenon in not only microalgae but also in bacteria and is a very specific phenomenon (FIG. 3).

Example 2. Chlorella HS2 Analysis of optimal culture conditions of

Optimal culture conditions were analyzed through Photo-Biobox (Heo et al. 2015 Biochemical Engineering Journal, 103, 193-197). Photo-Biobox is a high throughput photo bioreactor that implements 96 different environments in one batch and is a useful device to quickly find the optimal conditions for microalgae by temperature, light, and presence of carbon dioxide. As a freshwater medium, BG11 medium, which is widely used as a green algae culture medium, was used, and as a seawater medium, F/2 medium was used. As the radix medium, a medium in which BG11 and F/2 were mixed at a ratio of 1:1 was used. As a result of checking the culture conditions of HS2 using this equipment, Chlorella HS2 is ^{the optimal culture condition of temperature 34~38℃ and light 300~600 μmol·m -2} ·s ^-1 under freshwater medium carbon dioxide condition, and in seawater, temperature 34~ It was confirmed as the optimal culture condition at 36°C and light 450-600 μmol·m ^-2 ·s ^{-1 ( FIG. 4 ).}

Example 3. in the photobioreactor Chlorella as an optimal condition HS2 culture of

Chlorella HS was cultured using fresh water (BG11), seawater (F/2) and brackish water (DHX) media, respectively, in a culture volume of 600 ml in a 1 L photobioreactor. The culture conditions were a temperature of 34° C., luminosity of 450 μmol·m ^-2 ·s ^-1 and 5% CO ₂ air was aerated at 0.2 vvm. After 6 days of culture, BG11 was 2.49 x 10 ⁸ cell/ml, F/2 was 5.04 x 10 ⁷ cell/ml, and DHX was 8.98 x 10 ⁷ cell/ml (Fig. 5A), and the dry cell weights were 4.25, 3.5 and 2.03, respectively. g/L was reached ( FIG. 5B ).

The specific growth rate (μ _Ave ) analyzed through culture was 0.85, the fastest in BG11 medium, on average over the entire period, and the generation time at this time was 0.81 days (FIG. 6). However, the short-term maximum specific growth rate (μ _Exp ) was the fastest growing at 1.63 in DHX medium between 0 and 2 days (Table 1). The growth rate of Chlorella HS2 was very fast compared to other microalgae, and it showed the highest values in the maximum cell number and maximum cell dry weight.

Example 4. Chlorella HS2 of oil accumulation

Microalgae cultured in PBR with Chlorella HS2 was stained with nile red and observed under a fluorescence microscope. As a result, the intracellular neutral lipid content increased as the salt concentration increased. Nile red by fluorescence microscopy It was confirmed by the strength of the dyeing reagent. In addition, as a result of freeze-drying the biomass obtained for oil extraction, it was confirmed that the color of the dried cells decreased in green and yellow increased as the salt concentration increased (FIG. 7). Oil extraction was performed using a solvent in which chloroform and methanol were mixed in a ratio of 2:1 after extraction, and the solvent was removed in a vacuum distiller. FAME content and composition analysis was performed using a DB-Wax column in GC-FID (Shimazu, Japan). FAME 37 mix (Sigma, USA) was used as a standard form for quantitative and qualitative analysis. As a result, the total lipid content was analyzed as BG11 26%, DHX 47% and F/2 62%, and the FAME content was 23%, 44% and 61%, respectively, indicating a very high oil content (FIG. 8A). And fatty acid composition occupied the highest content of oleic acid and linoleic acid (Figs. 8B and 8C). For C18:1 and C18:2 fatty acids, BG11 was 19% and 29%, DHX was 27% and 20%, and F/2 was 31% and 19%, and polyunsaturated fatty acids having three or more double bonds ( The content of PUFA, polyhydric unsaturated fatty acid) was very low at 5-7% in all of BG11, DHX and F/2 ( FIGS. 8B and 8C ). As such, the low content of PUFA and the high content of C18:1 and C18:2 fatty acids indicate that it is very good for use in biodiesel production.

Example 5. Cell composition analysis and pigment analysis

Cell components of biomass cultured in freshwater, seawater and brackish water were measured using the total lipid method, the total glucose measurement method, and the BioRed total protein assay kit. While there was no significant change in the protein content, it was confirmed that the carbohydrate content was high in freshwater with a low fat content (BG11), and the carbohydrate content decreased as the lipid increased ( FIG. 9 ). This means that intracellular carbohydrates are converted to fats for lipid accumulation.

Example 6. Color content and productivity

The pigment content was highest in the freshwater medium, and the pigment content decreased as the salt concentration increased. In particular, chlorophyll a was decreased the most. As carotenoid pigments, the lutein content was the highest at 0.6% of the total biomass, and in the case of lutein, the degree of reduction by the salt concentration was lower than that of other pigments (FIG. 10). As a result of confirming the productivity of pigment productivity and dominant carotenoid in each medium, the productivity of pigment and lutein in BG11 medium was 12.5 mg/L/day and 4.06 mg/L/day, showing the highest productivity (FIG. 11).

Example 7. Mixed culture ( Mixotrophic culture) and Heterotrophic culture) check the culture conditions

The HS2 strain of the genus Chlorella microalgae is cultured not only through photosynthesis, which is a general characteristic of microalgae, but also through a mixed culture that supplies light and organic carbon at the same time (Mixotorophic culture) and a heterotrophic culture that supplies organic carbon without supplying light. This is possible. Using Photobiobox, the optimal culture temperature and optimal light conditions were analyzed in a situation where air and carbon dioxide were supplied using BG11 medium containing 10,000 ppm of glucose. The condition for supplying the analysis air optimum temperature 37 ℃, light 200 ~ 250 μmol · m ^-2 · s ^-1 showed an optimum condition from, the carbon dioxide supply state temperature 35 ℃, light 100 ~ 300 μmol · m ^-2 Optimal conditions were confirmed at s ^{-1 .} Compared to BG11, the medium in which glucose was added to BG11 showed significantly higher absorbance values in both air and carbon dioxide conditions.

Example 8. Mixed culture ( Mixotrophic culture) and of heterotrophic culture Medium selection and optimal culture

It was confirmed that the HS2 strain of Chlorella genus could be cultured using organic carbon (glucose), and it was confirmed that the culture concentration (absorbance) was significantly increased in the medium containing 10,000 ppm of glucose in BG11 compared to the medium without glucose. did. In order to increase the efficiency of subculture and mixed culture, the medium used for subculture was selected and the growth of microalgae was confirmed by medium at 35°C. The medium used was confirmed by measuring the absorbance 48 hours after inoculation of LB (Difco), YM (Difco), TSB (Difco), and Marine broth (Difco). As a result, 10,000 ppm of glucose showed the highest absorbance in BG11 used as a control. , followed by TSB showed the highest growth. MB (Marine broth) showed a high growth rate considering that the cell density was relatively lower than that of other media, but considering that it was a seawater medium. The three media BG11-10,000 ppm glucose, TSB-5,000 ppm glucose and MB-5,000 ppm glucose (TSB and MB have low glucose content, about 2,500 ppm) were cultured in a 1 L baffle flask at 35°C in suspension. Cancer cultured.

As a result, after 5 days of culture, the TSB cell count ^{was the highest at 4.11×10 8} cell/ml, and the BG11 and MB medium were 2.78×10 ⁸ cell/ml and 2.07×10 ⁸ cell/ml, respectively. This result is considered to be due to the concentration of added glucose and the increase in the aeration efficiency by the baffle flask. In addition, as a result of analyzing the amount of glucose, nitrogen and phosphorus using BG11-glucose medium using an 8 L photoincubator, the cell number was 2.79×10 ⁸ cell/ml, and the weight was 6.8 g/L. During the process, a total of 17,500 ppm of glucose was used, and 350 ppm and 150 ppm of nitrogen and phosphorus were used, respectively. Based on these results, the specific growth rate, development time, cell number and cell weight of the mixed culture and subculture are summarized in Table 1.

Korea Institute of Bioscience and Biotechnology KCTC13108BP 20160913

Claims (12)

Chlorella HS2 (Chlorella sp. HS2) cell line with accession number KCTC 13108BP, a microalgae that is isolated from rivers and can grow in freshwater, seawater and brackish water, and produces biomass, pigments and lipids. delete The chlorella HS2 cell line according to claim 1, wherein, when the cell line is cultured in fresh water, the biomass production is 600-700 mg/L/day, and the lutein pigment production is 4-4.5 mg/L/day. According to claim 1, wherein when the cell line is cultured in seawater, the total lipid content contains 60 to 70% by weight based on cell components, and contains 60 to 70% by weight of fatty acid methyl ester (FAME) based on fatty acids. Chlorella HS2 cell line characterized. The method according to claim 1, wherein the cell line comprises oleic acid (C18:1) and linoleic acid (C18:2) as major fatty acids, and contains oleic acid (C18:1) and linoleic acid (C18:2) from 45 to based on the total lipid weight. Chlorella HS2 cell line, characterized in that it contains 55%. According to claim 1, wherein the cell line Chlorella HS2 cell line, characterized in that the content of polyunsaturated fatty acids (PUFA) is 10% or less based on the total lipid weight. A microbial preparation for lipid production comprising the cell line of any one of claims 1, 3 to 6 or a culture solution thereof as an active ingredient. The method of claim 7, wherein the lipid contains oleic acid (C18: 1) and linoleic acid (C18: 2) in an amount of 45 to 55% based on the total lipid weight, and contains polyunsaturated fatty acids in an amount of 10% or less based on the total lipid weight. Microbial preparation for lipid production, characterized in that. A method for producing a lipid, comprising culturing the cell line of any one of claims 1, 3 to 6, and separating the lipid from the culture solution. It is prepared by the method of claim 9, and contains oleic acid (C18:1) and linoleic acid (C18:2) as main fatty acids, and oleic acid (C18:1) and linoleic acid (C18:2) are 45- based on the total lipid weight. A lipid composition comprising 55%, polyunsaturated fatty acids in an amount of 10% or less based on the total lipid weight. Claims 1, 3 to 6, comprising the steps of culturing any one of the cell lines to obtain a culture solution;
separating lipids from the culture solution; and
A method for producing biodiesel comprising the step of transesterifying the lipid to produce a fatty acid ester and glycerol. Claims 1, 3 to 6, comprising the steps of culturing any one of the cell lines to obtain a culture solution;
separating lipids from the culture solution; and
A method for producing glycerol comprising the step of transesterifying the lipid to produce a fatty acid ester and glycerol.

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