KR20180037520A - 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 microalgae Chlorella sp. cell strain that is a microalgae which is separated from rivers, can be rapidly grown in fresh water, seawater, and brackish water, and is excellent in biomass, pigment productivity and lipid productivity, and a use thereof. Chlorella sp. HS2 newly separated in the present invention can be cultivated at a very rapid growth rate in the fresh water, the seawater, and the brackish water unlike conventional other microalgae and microorganisms, thereby being free in the use of water for producing a culture medium, can show high biomass productivity, and can be used in microalgae oil and biodiesel markets by high biomass productivity and excellent lipid contents in a cell. Also, a pigment content is 1.8% which is similar to or slightly higher than other microalgae, but the pigment productivity is higher than conventional microalgae due to the rapid growth rate thereof rate and thus has potential as a resource for pigment production, thereby being very useful for related industries.

Description

A novel chlorella strain having high oil productivity in fresh water, sea water, and nose water {Novel Chlorella sp. strain high oil productivity in fresh water, sea water and rackish water}

The present invention relates to a microalgae Chlorella sp. HS2 cell line which is isolated from a river and is capable of rapid growth in fresh water, seawater and nose, and is excellent in biomass, pigment productivity and lipid productivity, and its use.

Recently, biodiesel has attracted much attention as an alternative to diesel. Biodiesel is defined as methyl or ethyl ester compounds of fatty acids produced from vegetable oils or animal fats. Biodiesel is more suitable for environmentally friendly vehicles because it can reduce the emission of air pollutants such as carbon monoxide, fine dust, hydrocarbons and toxic substances more than conventional diesel. In addition, the carbon dioxide from the combustion of biodiesel is absorbed and fixed by the plant's photosynthesis mechanism, so there is little net carbon dioxide emission, and carbon dioxide neutral fuel (CO ₂ -neutral fuel) is attracting much attention.

Particularly, biofuel (bioethanol, biodiesel) production is attracting attention, but production of biofuel using crops causes problems such as destruction of ecosystem and food shortage due to enlargement of cultivated land. 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.

Korean Patent No. 1625074 discloses microalgae having increased lipid content by using oxidative stress and a method for producing the microalgae. Korean Patent No. 1424852 discloses a microalgae containing chlorella bruvirus CV-16 And a method for producing biodiesel using the same. 'However, as in the present invention, the chlorella cell line, which is a microalga having excellent biomass, pigment productivity and lipid productivity, isolated from a river, and its use have never been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and it is an object of the present invention to provide a novel microalgae, Chlorella sp. HS2 cell line, isolated from a river, which is excellent in biomass and pigment productivity when cultured in fresh water (BG11) , And sea water (F / 2), respectively.

In particular, the growth rate of chlorella HS2 was the fastest in the freshwater (BG11) medium as averaged over the whole period, which was much faster than other microalgae and highest in the highest cell number and maximum cell dry weight. The lipid contents of oleic acid and linoleic acid showed the highest contents of total lipid and FAME (fatty acid methyl esters), and the content of lutein The highest was also confirmed.

As described above, the characteristic feature that the chlorella HS2 cell line of the present invention can be grown in both fresh water, seawater and nadir is a rare phenomenon not only in other microalgae but also in bacteria, and has been confirmed to be a very unusual phenomenon through the present invention.

Accordingly, the chlorella HS2 cell line of the present invention has high water availability, high biomass productivity and high oil content, and thus can be used as microalgae oil and biodiesel production strains, and has potential as a lutein producing strain , Thereby completing the present invention.

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

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 the main fatty acids, and oleic acid (C18: 1) and linoleic acid And 45 to 55% by weight of polyunsaturated fatty acids and 10% or less by weight of total lipids.

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 freshly isolated chlorella HS2 of the present invention can be cultivated at a very high growth rate in fresh water, seawater and nordic water, unlike other microalgae and microorganisms, and is free to use water for producing medium, exhibits high biomass productivity, It has excellent lipid content and can be used in micro-algae oil and biodiesel market. Also, the pigment content is similar to or slightly higher than other microalgae, but its pigmentation productivity is higher than conventional microalgae due to its rapid growth rate, and thus has potential as a resource for producing pigment, which is very useful for related industries.

Figure 1 shows a schematic diagram of chlorella HS2 isolated in the present invention.
FIG. 2 shows the results of confirming that chlorella HS2 isolated in the present invention can be grown in fresh water, brackish water and sea water.
FIG. 3 shows the results of confirming the extent of growth of chlorella HS2 and other microalgae isolated in the present invention in fresh water, sea water, and seawater.
FIG. 4 is a graph showing the optimal culture conditions of chlorella HS2 isolated in the present invention.
FIG. 5 shows the results of culturing of chlorella HS2 isolated from the present invention using fresh water, sea water and nadir in a photobioreactor. A) Cell number, B) Dry cell weight
Figure 6 shows the highest specific growth rate and generation time of chlorella HS2 isolated in the present invention in fresh water, nose water and seawater.
Fig. 7 shows an optical microscope (A), a fluorescence microscope analysis (B) and a biomass color (C) of chlorella HS2 isolated in the present invention.
FIG. 8 shows the results of analysis of total lipid content (A), fatty acid composition ratio (B) and fatty acid content ratio (C) of chlorella HS2 isolated in the present invention.
FIG. 9 shows changes in cell components of the medium of chlorella HS2 isolated in the present invention. FIG.
FIG. 10 shows changes in the content of pigment in the medium of chlorella HS2 isolated in the present invention. FIG.
Fig. 11 shows the pigment productivity (A) and the 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 a river and capable of rapid growth in fresh water, seawater and nursery, and excellent in biomass, pigment productivity and lipid productivity do. The cell line is preferably a Chlorella sp. HS2 cell line with the accession number KCTC 13108BP.

The chlorella HS2 cell line was identified through 18S rDNA sequencing of microalgae isolated from the river, and chlorella HS2 cell line capable of rapid growth in fresh water, seawater and nadir, and excellent in biomass, pigment productivity and lipid productivity were identified. A chlorella HS2 cell line capable of rapid growth in the fresh water, seawater and nursery and having excellent biomass, pigment productivity, and lipid productivity was deposited at KCTC on Sep. 13, 2016 (Accession No .: KCTC 13108BP ).

The chlorella HS2 cell line of the present invention is excellent in water utility, and particularly excellent in biomass and pigment productivity when cultured in fresh water, and excellent in lipid productivity when cultured in seawater.

The cell line according to an embodiment of the present invention preferably has a biomass production of 600 to 700 mg / L / day and a lutein pigment production of 4 to 4.5 mg / L / day when cultured in fresh water, It is not limited.

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

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 the major fatty acids, preferably oleic acid (C18: And linoleic acid (C18: 2) may be 45 to 55% based on the total lipid weight, but are not limited thereto.

According to one embodiment of the present invention, the chlorella HS2 cell line is cultured under fresh water medium carbon dioxide at a temperature of 34 to 38 ° C. and light of 300 to 600 μmol · m ^-2 · s ^-1 under optimal conditions, ° C, light 450 to 600 μmol · m ^-2 · s ^-1 , but is not limited thereto.

In addition, the cell line may have a polyhydric unsaturated fatty acid (PUFA) having three or more double bonds. The content of the polyunsaturated fatty acid may be 10% or less based on the total lipid weight, and most preferably, But it 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 HS2 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 a microbial formulation according to one embodiment of the invention, the lipid comprises oleic acid (C18: 1) and linoleic acid (C18: 2) in an amount of 45-55% by weight of total lipids, polyunsaturated fatty acid (C18: 1) and linoleic acid (C18: 2) in an amount of 45 to 50% by weight of the total lipids, polyunsaturated fatty acids in an amount of 6 to 7 %, But 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 lipid at a high concentration at various culture temperatures, and 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 comprises oleic acid (C18: 1) and linoleic acid (C18: 2) as major fatty acids and oleic acid (C18: 1) and linoleic acid (C18: (C18: 1) and linoleic acid (C18: 2) in an amount of 45 to 50% by weight, based on the total lipid content, of polyunsaturated fatty acids, And polyunsaturated fatty acids may be contained in an amount of 6 to 7% by weight of the total lipids, but are not limited thereto.

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 34 ° C to 38 ° 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 by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Isolation and identification of microalgae

The microalgae isolated from domestic rivers were sequenced by 18S rDNA and searched in NCBI blast. As a result, isolated HS2 was identified as Micratinium , It was highly homologous with Chlorella (Fig. 1), but it was morphologically different from Micrathium, and Chlorella vulgaris) or determined in different species Chlorella Thoreau Kearney Ana (Chlorella sorokiniana) than the growth potential characteristic (Fig. 2) at all, unlike fresh water, sea water and riders and these were named as the Chlorella genus (Chlorella sp.) HS2. Bacteria such as Escherichia coli, Bacillus, Pseudomonas, Rizobium, which are widely used in laboratories, have been confirmed by the present invention that no growth occurs in seawater (data not yet available) Growthable bacteria are clearly divided. However, it was confirmed that the characteristic feature of the genus Chlorella HS2 of the present invention, which can grow both in fresh water, seawater and nose, is a very unusual phenomenon (Fig. 3), which is a rare phenomenon not only in microalgae but also in bacteria.

Example  2. Chlorella HS2 Analysis of Optimal Culture Conditions

Optimal culture conditions were analyzed using Photo-Biobox (Heo et al., 2015 Biochemical Engineering Journal, 103, 193-197). Photo-Biobox is a high-throughput photo bioreactor that can be used to find micro-algae optimum conditions due to rapid temperature, light, and carbon dioxide by implementing 96 different environments in one batch. As the fresh water medium, BG11 medium, which is widely used as a green algae culture medium, was used, and F / 2 medium was used as a seawater medium. As the radionuclide medium, 1: 1 mixture of BG11 and F / 2 was used. As a result of confirming the culturing conditions of HS2 using this equipment, chlorella HS2 is the optimal culture condition of 34 ~ 38 ℃ and light 300 ~ 600 ㎛ · m ^-2 · s ^-1 under fresh water medium carbon dioxide condition, 36 ° C, and light 450 to 600 μmol · m ^-2 · s ^-1 (FIG. 4).

Example  3. In the photobioreactor  Optimal conditions include chlorella HS2 Cultivation of

Chlorella HS was cultured in a 1 L photobioreactor using fresh water (BG11), seawater (F / 2), and radionuclide (DHX) medium in a 600 ml culture volume. The culture conditions were a temperature of 34 ° C, a luminous intensity of 450 μmol · m ^-2 · s ^-1 and a 5% CO ₂ air aeration of 0.2 vvm. After 6 days of culture, the cell dry weights were 2.49 × 10 ⁸ cells / ml for BG11, 5.04 × 10 ⁷ cells / ml for F / 2 and 8.98 × 10 ⁷ cells / ml for DHX (Figure 5A) g / L (Fig. 5B).

The non-growth rate (μ _Ave ) analyzed through culture was the fastest 0.85 in the BG11 medium and the generation time was 0.81 days (FIG. 6). However, the shortest maximum growth rate (μ _Exp ) was the fastest growth from 0 to 2 days in DHX medium to 1.63 (Table 1). The growth rate of chlorella HS2 was much faster than other microalgae, and the highest value was obtained even at the highest cell number and maximum cell dry weight.

Example  4. Chlorella HS2 Oil accumulation

The microalgae cultured on PBR of chlorella HS2 were stained with neutral lipids by nile red and observed by fluorescence microscope. The results showed that the intracellular neutral lipid content was increased with increasing salt concentration, It was confirmed by the intensity of the dyeing reagent. As a result of lyophilization of the biomass obtained for oil extraction, it was confirmed that the color of dried cells decreased in green color and increased in yellow color toward a medium having high salt concentration (FIG. 7). The oil extraction was carried out using a solvent mixture of chloroform and methanol 2: 1 and then the solvent was removed in a vacuum distillation apparatus. The FAME content and composition were analyzed using a DB-Wax column in GC-FID (Shimazu, Japan) FAME 37 mix (Sigma, USA) as a standard foam. As a result, total lipid contents were analyzed as BG11 26%, DHX 47% and F / 2 62%, and the FAME contents were 23%, 44% and 61%, respectively. And oleic acid and linoleic acid had the highest fatty acid composition (Fig. 8B and Fig. 8C). The content of C18: 1 and C18: 2 fatty acids was very high, 19% and 29% for BG11, 27% and 20% for DHX and 31% and 19% for F / 2, respectively. Polyvalent unsaturated fatty acids with three or more double bonds PUFA, Polyhydric unsaturated fatty acid) was very low (5 to 7% in both BG11, DHX and F / 2) (Fig. 8B and Fig. 8C). 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.

Example  5. Cell component analysis and pigment analysis

The cellular constituents of biomass cultured in freshwater, seawater and nephew were measured using total lipid method, total sugar method and Bio Red total protein assay kit. The content of protein was not significantly changed, but the carbohydrate content was high in fresh water (BG11) having low fat content, and the carbohydrate content was decreased as lipid was increased (FIG. 9). This means that intracellular carbohydrates have been converted to fat for lipid accumulation.

Example  6. Pigment content and productivity

Pigment content was highest in freshwater medium and decreased in pigment concentration. In particular, chlorophyll-a decreased the greatest. Lutein content was the highest at 0.6% of total biomass in the carotenoid pigment, while lutein was lower in salt concentration than other pigments (Fig. 10). The productivity of pigment and lutein in the BG11 medium was highest at 12.5 mg / L / day and 4.06 mg / L / day, respectively (FIG. 11).

Example  7. Mixed culture ( Mixotrophic  culture) and Heterotrophic culture)

The microalgae Chlorella HS2 strain can be cultivated through a heterotrophic culture which does not supply light but also supplies organic carbon as well as photosynthesis, which is a general characteristic of microalgae, as well as light and organic carbon at the same time This is possible. Optimum culture temperature and optimal light condition were analyzed using BG11 medium containing glucose at 10,000 ppm by using Photobiobox in the condition of supplying air and carbon dioxide. As a result of the analysis, optimum conditions were shown at the optimum temperature of 37 ° C and light of 200 to 250 μmol · m ^-2 · s ^-1 under the condition of air supply. In the case of the carbon dioxide supply condition, the temperature was 35 ° C. and the light was 100 to 300 μmol · m ^-2 · S ^-1 . Compared with BG11, the medium containing glucose in BG11 showed much higher absorbance values in both air and carbon dioxide conditions.

Example  8. Mixed Culture ( Mixotrophic  culture) and Heterotrophic culture  Selection of medium and cultivation of optimal conditions

It was confirmed that the strain HS2 of the genus Chlorella could be cultured using organic carbon (glucose), and it was confirmed that the culture concentration (absorbance) was greatly increased in the medium containing 10,000 ppm glucose in BG11 as compared with the medium not containing glucose Respectively. In order to increase the efficiency of the subculture and mixed cultivation, microbial growth was observed at 35 ℃ in the medium used for the subculture. The absorbance was measured 48 hours after inoculation of LB (Difco), YM (Difco), TSB (Difco) and Marine broth (Difco) and the highest absorbance of glucose was found in BG11 , Followed by TSB with high growth. The MB (Marine broth) had a lower cell density than the other media but showed a high growth rate considering that it was a seawater medium. The three mediums BG11-10,000 ppm glucose, TSB-5,000 ppm glucose, and MB-5,000 ppm glucose (TSB and MB are glucose-poor at about 2,500 ppm) were cultured in a 1 L baffle flask at 35 ° C in a suspension culture Lt; / RTI >

As a result, the number of TSB cells was the highest at 4.11 × 10 ⁸ cells / ml after 5 days of culture, and the BG11 and MB media were 2.78 × 10 ⁸ cells / ml and 2.07 × 10 ⁸ cells / ml, respectively. These results are attributed to the increase in the concentration of added glucose and aeration efficiency by baffle flask. The amount of glucose, nitrogen and phosphorus was measured using a BG11-glucose medium using a mixotrophic culture method using an 8 L light incubator. The cells were cultured at a cell number of 2.79 × 10 ⁸ cells / ml and a weight of 6.8 g / L While 17,500 ppm of glucose was used, and 350 ppm and 150 ppm of nitrogen and phosphorus were used, respectively. Based on these results, non-growth rate, development time, cell number and cell weight of the mixed culture and subculture are summarized in Table 1.

Korea Biotechnology Research Institute KCTC13108BP 20160913

Claims (12)

Chlorella sp., A microalgae that is separated from rivers and capable of rapid growth in fresh water, seawater, and nose, and has excellent biomass, pigment yield and lipid productivity. The cell line according to claim 1, wherein the cell line is a Chlorella sp. HS2 cell line having the accession number KCTC 13108BP. The cell line according to claim 1, wherein the cell line has a biomass production of 600 to 700 mg / L / day and a lutein pigment production of 4 to 4.5 mg / L / day when cultured in fresh water. The cell line according to claim 1, wherein the cell line contains 60 to 70% by weight of the total lipid content based on the cell component and 60 to 70% by weight of the fatty acid methyl ester (FAME) based on fatty acid when cultured in seawater Cell lines characterized. 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 and oleic acid (C18: 1) and linoleic acid (C18: 55%. The cell line according to claim 1, wherein the cell line has a polyunsaturated fatty acid (PUFA) content of 10% or less based on the total lipid weight. A microorganism preparation for producing a lipid comprising the cell line of any one of claims 1 to 6 or a culture thereof as an active ingredient. 8. The method of claim 7, wherein the lipid comprises oleic acid (C18: 1) and linoleic acid (C18: 2) in an amount of 45-55% by weight of total lipids, and polyunsaturated fatty acids Wherein the microorganism is a microorganism. A method for producing a lipid characterized by culturing the cell line according to any one of claims 1 to 6 and separating the lipid from the culture. (C18: 1) and linoleic acid (C18: 2) in an amount of 45 to 80% by weight, based on the total lipid weight, of oleic acid (C18: 1) and linoleic acid 55%, and contains polyunsaturated fatty acids in an amount of 10% or less based on the total lipid weight. Culturing the cell line of any one of claims 1 to 6;
Separating the lipid from the culture liquid; And
And transesterifying the lipid to produce a fatty acid ester and glycerol. Culturing the cell line of any one of claims 1 to 6;
Separating the lipid from the culture liquid; And
Transesterifying the lipid to produce a fatty acid ester and glycerol.

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