KR20120057321A - Chlorella vulgaris CV-18 producing biodiesel, and method for producing biodiesel using the strain - Google Patents

Abstract

PURPOSE: A method for producing biodiesel using chlorella vulgaris CV-18 is provided to enhance lipid content and biodiesel productivity. CONSTITUTION: A method for producing biodiesel comprises: a step of culturing chlorella vulgaris AG10032 and treating with ethyl methanesulfonate(EMS) to induce mutation; and a step of culturing mutated chlorella vulgaris and extracting lipid and fatty acid from culture liquid. The chlorella vulgaris is chlorella CV-18(KCTC 11776BP). The fatty acid has oleic acid(C18:1) and linoleic acid(C18:2).

Description

Chlorella vulgaris CV-18 producing biodiesel, and method for producing biodiesel using the strain

The present invention relates to Chlorella vulgaris CV-18 for producing biodiesel and a method for producing biodiesel using the same.

The recent increase in the use of fossil fuels raises the concentration of carbon dioxide in the atmosphere, causing global warming and other serious problems worldwide. Therefore, researches to reduce carbon dioxide in the atmosphere are actively conducted, and recently, forest composition is recommended on land to strengthen carbon dioxide absorption sources of forests. However, the land area for forming forests is limited, and the photosynthetic metabolism of terrestrial plants is so slow that it is not sufficient to reduce carbon dioxide. Therefore, attention has been drawn to the use of microalgae 10 times higher utilization of solar energy and carbon dioxide than land plants.

Methods for reducing carbon dioxide increase in the atmosphere due to industrialization include physical / chemical control methods, biological fixation methods, and marine storage methods. Among them, the biological fixation method is the most environmentally friendly method using the natural carbon cycle, and there is an advantage that no other secondary pollution occurs at all. In particular, the carbon fixing method using the microalgae has the advantage that the photosynthetic efficiency is superior to that of higher plants, and the reaction proceeds at room temperature and atmospheric pressure and thus consumes less energy.

Recently, due to the sharp rise in crude oil prices, biofuel (bioethanol, biodiesel) technology using biomass as an alternative fuel source of petroleum has attracted attention. However, the development of biofuels using edible crops causes problems such as ecosystem destruction and food shortage due to the expansion of arable land. Therefore, in order to solve these problems, technology that utilizes microalgae as a raw material instead of edible crops has attracted much attention as the next generation biodiesel technology.

Microalgae are phytoplankton that do photosynthesis in the ocean, which occupies 71% of the earth's surface. The microalgae collectively refers to unicellular photosynthetic microorganisms capable of photosynthetic growth using water, carbon dioxide, and sunlight. Microalgae can be cultured anywhere in the wasteland, on the coast, and in the sea if only photosynthesis is possible, which has the advantage of being able to cultivate using idle farmland. It also inhabits the entire ocean, producing 50% of the world's oxygen production. Therefore, microalgae can be cultivated in large quantities in idle cropland, and there is an advantage that can be harvested every day. In addition, since the microalgae can directly grow by absorbing high concentration carbon dioxide (15% level) in by-product gas such as a thermal power plant, the carbon dioxide reduction effect is also very large. Photovoltaic microalgae use about 5% solar energy efficiency, about 25 times higher than 0.2% of land plants, and carbon dioxide immobilization rate is 15 times higher than that of pine (Matsumoto, H., N. Shioji, A Hamasaki, Y. Ikuta, Y. Fukuda, M. Sato, N. Endo, and T. Tsukamoto. 1995. Carbon dioxide fixation by microalgae photosynthesis using actual flue gas discharged from a boiler.Appl.Biochem.Biotech.51 / 52. : 681-692.). In addition, biodiesel production per unit area of microalgae (when oil content is 30%) is about 58,700 L / ha, which is 130 times higher than 446 L / ha of soybeans (Chisti, Y. 2007. Biodiesel from microalgae.Biotechnol Adv. 25: 294-306.). In addition, microalgae can produce biodiesel to replace diesel produced from fossil fuels due to various advantages such as cultivation using idle cropland, ease of strain improvement, irrelevant to food problems, and dramatic increase in carbon dioxide fixation capacity. It is rated as the only resource. That is, biofuels based on microalgae (third generation biofuels), which have higher unit productivity than cellulose or lignin, biological waste resources, and herbs of plants classified as so-called second generation biofuels, are considered as renewable energy resources. In addition, the biological conversion and treatment of carbon dioxide is an environmentally friendly method using photosynthesis, which is the basic principle of natural material circulation, and the process is performed at room temperature and atmospheric pressure, and has the advantage of using biomass produced as a useful material.

On the other hand, Chlorella (Chlorella) is byunryu as green algae, a classification system is present in a single cell of a round or oval, the diameter 10㎛ than a fresh water algae belonging to Chlorophyta and neck, Trebouxiophyceae. There is no flagella, no movement, and the cell has one nucleus and one cup-shaped chloroplast. About 10 species of chlorella are known and have been commonly used in the study of plants photosynthesis because of their excellent photosynthetic capacity and easy cultivation. In particular, chlorella increases about 10 times a day under moderate conditions, so the annual organic production is about 8 times that of rice. In addition, depending on the culture conditions can increase the content of fat 20 ~ 80%, protein 90%, and carbohydrates 37%, has been studied to solve the food problem of humans by culturing in large quantities. However, if the individual is too small and eats as it is, it is not digested well, and it is not developed as a food because it has no taste, but health supplement foods and dairy products have been developed that increase digestion absorption by sterile pure water culture. Chlorella is widely distributed in fresh water all over the world and contains 15-25% lipids in the body. The composition of chlorella is palmitic acid (C16: 0), oleic acid (C18: 1) and linoleic acid (18: 2). It consists of active ingredients (Yoo, C., Jun, SY., Lee, JY., Ahn, CY., And Oh, HM. 2010. Selection of microalgae for lipid production under high levels carbon dioxide.Biores.Technol. 101: S71-S74). In addition, chlorella-derived lipids are easily converted to biodiesel and have similar properties to petroleum-based diesel.

In order to produce biodiesel from microalgae, growth and lipid content of microalgae are important factors. However, the growth of microalgae and the accumulation of lipids are opposite results, that is, when the growth of microalgae is fast, the lipid content is low, and when the lipid content of the microalgae is high, growth is slow.

Therefore, there is an urgent need for the development of microalgae with fast growth and high lipid content in order to improve the productivity of biodiesel.

The present inventors studied to develop microalgae that grow fast and have high lipid content, and induce mutants by culturing chlorella vulgaris and treating with ethyl methanesulfonate (EMS). Bulgari's cell growth rate, biomass productivity, lipid productivity and fatty acid productivity was improved and the lipid content was confirmed, the present invention was completed.

The present invention is to provide a chlorella vulgaris CV-18 for producing biodiesel and a method for producing biodiesel using the same.

1 is a view of chlorella vulgaris CV-18 of the present invention ( Chlorella vulgaris CV-18, KCTC 11776BP) observed with an optical microscope.
Figure 2 shows the growth curves of Chlorella vulgaris AG10032 and Chlorella vulgaris CV-18.
FIG. 3 shows biomass productivity, lipid productivity and fatty acid productivity of Chlorella vulgaris AG10032 and Chlorella vulgaris CV-18.
4 is a diagram comparing the fatty acid composition of Chlorella vulgaris AG10032 and Chlorella vulgaris CV-18.

The present invention provides a Chlorella vulgaris CV-18 to produce the biodiesel (Chlorella vulgaris CV-18, KCTC 11776BP).

In addition,

1) incubating Chlorella vulgaris AG10032 and inducing mutations by treating with ethyl methanesulfonate (EMS), and

2) culturing the mutation-induced chlorella vulgaris, and provides a method for producing biodiesel comprising the step of extracting and separating lipids and fatty acids from the culture.

Hereinafter, the present invention will be described in detail.

Chlorella vulgaris CV-18 according to the present invention ( Chlorella vulgaris CV-18, KCTC 11776BP) is characterized by inducing mutation by treating with Chlorella vulgaris AG10032, treated with ethyl methanesulfonate (EMS).

As a result of observing the mutant-induced chlorella vulgaris under an optical microscope, it was present as a single cell and showed no morphological difference with the parent strain chlorella vulgaris AG10032, and the base of the 18S rDNA of the mutant chlorella vulgaris had the same base sequence as the chlorella vulgaris AG10032. Have Therefore, it was confirmed that the mutant-induced chlorella vulgaris was a chlorella genus, and the mutant-induced chlorella vulgaris was named Chlorella vulgaris CV-18 (KCTC 11776BP).

Cell growth rate, biomass productivity, lipid productivity and fatty acid productivity of Chlorella vulgaris CV-18 of the present invention are improved by 1.9, 1.4, 3.0 and 2.4 times, respectively, compared to Chlorella vulgaris AG10032. In addition, the total lipid content of chlorella vulgaris CV-18 is more than doubled compared to chlorella vulgaris AG10032, and the fatty acid composition of chlorella vulgaris CV-18 is oleic acid (C18: 1) and linoleic acid (C18: 2). ), And the content of oleic acid (C18: 1) in Chlorella vulgaris CV-18 is slightly increased compared to Chlorella vulgaris AG10032.

As described above, the chlorella vulgaris CV-18 according to the present invention induces mutations by treating the chlorella vulgaris strain with ethyl methanesulfonate (EMS), thereby increasing cell growth rate and resulting cell division, metabolism and High CO2 fixation ability results in high biomass productivity, high lipid content, high lipid productivity and fatty acid productivity. Therefore, Chlorella Bulgari CV-18 can increase the productivity of biodiesel through mass cultivation, which can be usefully used as a source of bioenergy, and can greatly contribute to carbon dioxide reduction, eco-friendly fuel development and new green industry creation. I think there will be.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1  : Preparation of Mutant Chlorella Bulgari

Chlorella vulgaris AG10032 used in this experiment was used by the Korea Institute of Bioscience and Biotechnology. As culture conditions, shaking culture was performed by irradiating light at a light incubator at a temperature of 120 ± 5 μmol / photons / ㎡ / s and a temperature of 26 ± 0.2 ° C. for 24 hours. The composition of the BG11 medium used for the culture is shown in Table 1 below.

Composition Components of BG11 Medium Content (g / L) NaNO ₃ 1.500 K ₂ HPO ₄ 0.039 MgSO _4? 7H ₂ O 0.075 Na ₂ CO ₃ 0.021 CaCl ₂ 0.027 Na ₂ SiO _3? 9H ₂ O 0.058 Ferric citrate 0.006 Citric acid 0.006 EDTA 0.001 Trace ingredients
H ₃ BO ₃ 2.86 MnCl _2? 4H ₂ O 1.86 ZnSO _4? 7H ₂ O 0.22 Na ₂ MoO _4? 2H ₂ O 0.391 CuSO _4? 5H ₂ O 0.079 Co (NO ₃ ) _2? 6H ₂ O 0.0494

1. Mutation Induction of Chlorella vulgaris

After incubating Chlorella vulgaris AG10032 for 10 days in BG11 medium, the culture was centrifuged at 5,000 rpm for 10 minutes, washed three times with distilled water and then suspended in BG11 medium. To the suspension was added ethyl methanesulfonate (EMS), a mutation inducer, to a final concentration of 0.24% (v / v). The reaction solution was aliquoted into a micro tube (2 ml) by 1 ml and reacted at room temperature for 10 minutes, 20 minutes and 30 minutes respectively to induce mutations. After the reaction, the supernatant was removed by centrifugation at 5,000 rpm for 10 minutes, and the reaction was stopped by adding 1 ml of 5% sodium thiosulfate. After completion of the reaction, the supernatant was removed by centrifugation at 5,000 rpm for 10 minutes, washed three times with 1 ml of BG11 medium to remove residual ethyl methanesulfonate and thiosulfate, and then suspended with BG11 medium. A portion of the suspension (100 μl) is plated on a BG11 agar solid plate medium and shaken for 2 weeks in a light incubator for 24 hours under light conditions of 120 ± 5 μmol / photons / m2 / s and a temperature of 26 ± 0.2 ° C. It was. Colonies formed after the culture were transferred to BG11 liquid medium using a Pasteur pipette and shaken under the same conditions.

2. Identification of Mutant Chlorella Bulgari

The mutant-induced chlorella vulgaris in 1 was cultured in BG11 medium and observed with an optical microscope, Microphot FX-A (Nikon, JAPAN), and bird illustration (Prescott, Algae of the western great lakes area, 1973; Canter-Lund and Lund, Freshwater algae: Their microscopic world explored, 1995). In addition, parental and mutant strains were compared using 18S rDNA sequences used for molecular biological identification.

The results of observing the mutant-induced chlorella vulgaris with an optical microscope are shown in FIG. 1, and the 18S rDNA nucleotide sequences of the mutant-induced chlorella vulgaris are shown in Table 2.

Mutant Chlorella Bulgari
18S rDNA sequence Blast Search Results gctcgtagtt ggatttcgga tggggcctgc cggtccgccg tttcggtgtg cactggcagg gcccaccttg ttgccgggga cgggctcctg ggcttcactg tccgggactc ggagtcggcg
ctgttacttt gagtaaatta gagtgttcaa agcaggccta cgctctgaat acattagcat ggaataacac gataggactc tggcctatcc tgttggtctg taggaccgga gtaatgatta
aaagggacag tcgggggcat tcgtatttca ttgtcaaagg tgaaattctt ggatttatga aagacaaact act gi | 283896766 | emb | FM205862.1 | Chlorella sp. UTEX 938 18S rRNA gene (partial), ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene (partial), strain UTEX 938

Length = 2822
Score = 547 bits (606),
Expect = 1e ^-152
Identities = 309/313 (99%),
Gaps = 0/313 (0%)

As shown in FIG. 1, the mutant-induced chlorella vulgaris existed as single cells and did not show morphological differences with the parent strain chlorella vulgaris AG10032.

In addition, as shown in Table 2, as a result of confirming the presence or absence of a base phase change by ethyl methanesulfonate (EMS) of mutant Chlorella vulgaris, it was confirmed that the base of 18S rDNA of mutant Chlorella vulgaris has the same base sequence as Chlorella vulgaris AG10032. . Of course, the whole DNA was not tested, but the length of the assay was too short, and no variation in the nucleotide sequence was observed.

Therefore, it was confirmed that the mutant-induced chlorella vulgaris was a chlorella genus, and the mutant-induced chlorella vulgaris was named Chlorella vulgaris CV-18. Deposited on date (KCTC 11776BP).

Experimental Example 1  : Growth rate measurement of Chlorella vulgaris CV-18

To determine the growth rate of chlorella vulgaris, chlorella vulgaris AG10032 and chlorella vulgaris CV-18 were incubated for 12 days in BG11 medium. Cultivation was carried out using a light incubator supplied with a light amount of 120 μmol / photons / ㎡ / s using a fluorescent lamp, the culture conditions were maintained at 26 ℃ constant temperature and stirred at 120rpm. After inoculation with Chlorella vulgaris, the samples were measured for absorbance at 680 nm using a spectrophotometer, and the cell growth rate (specific growth rate, μ) was calculated by Equation 1 below.

[Equation 1]

μ = Ln (N ₁ / N ₀ ) / (t ₁ -t ₀ )

* N ₀ : absorbance at initial 680 nm, N ₁ : absorbance at final 680 nm,

t ₀ : initial culture day, t ₁ : final culture day.

The growth curves of chlorella vulgaris AG10032 and chlorella vulgaris CV-18 are shown in FIG. 2, and cell growth rates and drainage of chlorella vulgaris AG10032 and chlorella vulgaris CV-18 are shown in Table 3. In addition, the biomass productivity of chlorella vulgaris AG10032 and chlorella vulgaris CV-18 is shown in FIG.

Microalgae Cell growth rate (/ d) Drainer (day) Chlorella vulgaris AG10032 0.116 6.00 Chlorella vulgaris CV-18 0.219 3.17

As shown in FIG. 2 and Table 3, the cell growth rate of Chlorella vulgaris AG10032 was 0.116 / d, while the cell growth rate of Chlorella vulgaris CV-18 was 0.219 / d, about 1.9 times faster. In addition, in the case of doubling time indicating a doubling time, Chlorella vulgaris AG10032 was 6.0 days, while Chlorella vulgaris CV-18 was 3.2 days, which was able to multiply more than twice at the same time. Therefore, it can be seen that when incubated with Chlorella vulgaris CV-18, the period for producing the same biomass can be shortened.

In addition, as shown in FIG. 3, the biomass productivity of Chlorella vulgaris AG10032 was 20.0 mg / L / d, whereas the Chlorella vulgaris CV-18 was approximately 1.4-fold improved to 28.3 mg / L / d.

Experimental Example 2  : Quantitative Analysis of Lipids and Fatty Acids in Chlorella Bulgari CV-18

Total lipid analysis was performed according to Bligh and Dyer's method. Total lipids were eluted from the microalgal solids using chloroform-methanol solution (2: 1, v / v). After elution, water was added to separate the chloroform and methanol layers. The chloroform layer was washed with 5% sodium chloride (NaCl) solution and then the solvent was evaporated using an evaporator. The weight remaining after evaporation was measured to obtain the total amount of lipid. Fatty acid analysis was performed by gas chromatography after pretreatment according to the method of Lepage and Roy.

The total lipid and fatty acid contents of chlorella vulgaris AG10032 and chlorella vulgaris CV-18 are shown in Table 4 and Table 5, respectively. The lipid and fatty acid productivity of chlorella vulgaris AG10032 and chlorella vulgaris CV-18 are shown in FIG. The result of comparing the fatty acid composition of Bulgari AG10032 and Chlorella Bulgari CV-18 is shown in FIG. 4.

Microalgae Total lipid content (%) Chlorella vulgaris AG10032 15.0 Chlorella vulgaris CV-18 32.3

Microalgae Fatty acid content (%) C16: 0 C16: 1 C18: 0 C18: 1 C18: 2 Chlorella vulgaris AG10032 11.7 2.2 5.1 45.8 27.2 Chlorella vulgaris CV-18 12.0 1.9 5.2 48.3 22.1

As shown in Table 4, the total lipid content of Chlorella vulgaris CV-18 was more than doubled compared to Chlorella vulgaris AG10032.

In addition, as shown in Table 5 and FIG. 4, the fatty acid compositions of chlorella vulgaris AG10032 and chlorella vulgaris CV-18 are oleic acid (C18: 1) (45.8%, 48.3%, respectively) and linoleic acid (C18: 2). (27.2% and 22.1%, respectively), and the content of oleic acid (C18: 1) in Chlorella vulgaris CV-18 was slightly increased compared to Chlorella vulgaris AG10032.

In addition, as shown in Figure 3, the lipid productivity and fatty acid productivity of Chlorella vulgaris CV-18 increased by 3.0 and 2.4 times, respectively, compared to Chlorella vulgaris AG10032.

Chlorella vulgaris CV-18 according to the present invention induces mutations by treating chlorella vulgaris strain with ethyl methanesulfonate (EMS), thereby increasing cell growth rate and fast cell growth resulting in increased cell division, metabolism and carbon dioxide fixation capacity High productivity, high lipid content, high lipid productivity and fatty acid productivity. Therefore, Chlorella Bulgari CV-18 can increase the productivity of biodiesel through mass cultivation, which can be usefully used as a source of bioenergy, and can greatly contribute to carbon dioxide reduction, eco-friendly fuel development and new green industry creation. I think there will be.

Korea Biotechnology Research Institute KCTC11776BP 20101005

Claims (3)

Chlorella vulgaris CV-18 to produce the biodiesel (Chlorella vulgaris CV-18, KCTC 11776BP). 1) incubating Chlorella vulgaris AG10032 and inducing mutations by treating with ethyl methanesulfonate (EMS), and
2) culturing the mutation-induced chlorella vulgaris, and extracting and separating lipids and fatty acids from the culture, biodiesel production method. Claim 2 wherein said mutation induced Chlorella vulgaris is Chlorella vulgaris CV-18 (Chlorella vulgaris CV- 18, KCTC 11776BP) of the method of the production of biodiesel, characterized in.

Images