KR101576622B1 - Method of increasing oil accumulation from microalgae - Google Patents
Method of increasing oil accumulation from microalgae Download PDFInfo
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- KR101576622B1 KR101576622B1 KR1020150060808A KR20150060808A KR101576622B1 KR 101576622 B1 KR101576622 B1 KR 101576622B1 KR 1020150060808 A KR1020150060808 A KR 1020150060808A KR 20150060808 A KR20150060808 A KR 20150060808A KR 101576622 B1 KR101576622 B1 KR 101576622B1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
Abstract
Conventional nutrient inhibition and two-stage processes are complex,
Oil accumulation technology using light wavelength with high effect is needed. The present invention relates to a method for culturing microalgae in a two-phase, and it relates to a method for growing a microalgae in a first phase (for example, blue wavelength) or a second phase (for example, a red wavelength) To increase the oil accumulation amount.
Description
Many studies have been conducted on biodiesel, and biodiesel is being produced from waste cooking oil, rice bran, and soybean oil as raw materials in Korea. Commercialization of biodiesel started in July 2006 through a two-year pilot project from 2004 .
At present, the oil accumulation technique using microalgae is stressed through the substrate inhibition (nitrate, phosphate, silica, salinity) of the growth medium to increase the oil content. The microalgae growth and oil accumulation process are performed in each medium.
Conventional nutrient inhibition and two-stage processes have been complicated by the use of separate media for microalgae growth and oil accumulation processes, and a simple and effective oil accumulation technique has been demanded.
The conventional two-stage culture process as shown in FIG. 10 produces the greatest amount of biomass in the first stage culture, and when cells reach the stationary stage, cells are recovered using a centrifuge. The recovered cells are resuspended in a fresh medium from which a nutrient source such as nitrogen source or phosphorus is removed and re-inoculated for the second step culture. Two-step cultivation with nutrient source removed increases the oil accumulation rate. After 3 days of cultivation, cells are recovered by centrifugation. The recovered cells are separated through an oil extraction process.
The present invention relates to a method for culturing a microalgae in a two-phase, wherein a first phage (for example, Blue wavelength, Red wavelength) and an oil accumulation increase (second wavelength) phase; for example, Red wavelength, Blue wavelength, Green wavelength).
The present invention relates to a method for accumulating high-concentration oil in a microalgae, wherein the microalgae contains a chlorophyll a substance, β-carotene and phycocyanin, and comprises one section for increasing biomass and two sections for increasing oil accumulation rate , The same medium is continuously used in the first section and the second section, and the wavelength and intensity of the irradiation light can be controlled.
In the present invention, the wavelength range of the LED light irradiated in the first section differs from the wavelength range of the LED light irradiated in the second section.
The present invention relates to a method for accumulating high-concentration oil in microalgae, comprising the steps of: increasing biomass; and increasing the oil accumulation rate, wherein the same medium is continuously used in the first section and the second section, The wavelength range of the LED light irradiated in the first section is either 465 nm (blue) or 660 nm (red), and the wavelength range of the LED light irradiated in the second section is either 465 nm (blue), 520 nm (green), or 660 nm Lt; / RTI >
In this regard, when a wavelength of 465 nm (blue) was used as a light energy source in the first section, Isochrysis galbana, Pheodactylum tricornutum, Nannochloropsis oculata, Nannochloropsis oceanica, Nanochloropsis salina, Dunaliella salina and Dunaliella tertiolecta were 0.97, 1.08, , 0.84, 0.79, 0.75 g / L, and Nannochloris atomus produced 0.87 g / L of maximum biomass at a wavelength of 660 nm (red), producing more than two times biomass than when cultured in a fluorescent lamp (FIG. 5) . The oil content (%) based on dry cell weight also increased significantly (Fig. 6).
The maximum oil content was accumulated on the 1st to 3rd day of culture when the wavelength was changed to 456, 520, or 660 nm (blue, red, green) in the 2-step culture. The oil content of I. galbana was red 60% oil content was measured at 56% and green (520 nm) at 660 nm (FIG. 8A)
P. tricornutum was measured at 58% at red (660 nm) and 62% at green (520 nm) (Figure 8b), and N. oculata was 50% at red (660 nm) (Figure 8c). N. oceanica was measured 47% at red (660nm) and 53% at green (520nm) on day 2 (Figure 8d) N. salina was measured 47% in red (660 nm) and 52% in green (520 nm) on day 3 (FIG. 8f) D. tertiolecta was measured at 46% green (520) and red (660 nm) at
The maximum oil content at two wavelengths of Green 520 nm was 30% for I. galbana, 26% for P. tricornutum, and 2.60% for N. oculata 28% for N. oceanica, 30% for N. oceanica, 8a, 8b, 8c, 8d, 8e, 8e, 8e, and 8e show an increase in oil content of 28% (based on 660 nm) for Atomus, 27% for N. salina, 27% for D. salina, and 28% for D. tertiolecta , Figs. 8F, 8G, 8H).
In the present invention, in order to cultivate at least one species selected from the group consisting of Isochrysis galbana, Pheodactylum triocornutum, Nannochloropsis oculata, Nannochloropsis oceanica, Nannochloropsis salina, Dunaliella salina and Dunaliella tertiolecta, the wavelength of the LED light irradiated in one section is set to 465 nm (red, green), and the light intensities were 40, 70, 100, and 130 mol m - 2 s - 2 , respectively, 1 < / RTI >
1 was used as a source of light energy at a wavelength of 465 nm (blue), and at a luminous intensity of 100 mol m -2 s -1 , Isochrysis galbana, Pheodactylum tricornutum, Nannochloropsis oculata, Nannochloropsis oceanica, Nannochloropsis salina, Dunaliella salina, Dunaliella tertiolecta (Figs. 3a, 3b, 3c, 3d, 3f, 3g and 3h), the biomass was produced at a concentration of 0.58, 0.65, 0.69, 0.61, 0.59, 0.55,
In order to cultivate Nannochloris atomus, the wavelength of the LED light irradiated in the 1 section can be set to 660 nm (red), and the light intensities can be set to 40, 70, 100, and 130 mol m -2 s -1 . Nannochloris atomus produced 0.52 g / L biomass at 100 mol m -2 s -1 . (Fig. 3E)
In the present invention, the light / dark cycle is 12h: 12h in the case where the wavelength of the LED light of the first section is either 465 nm (blue) or 660 nm (red), and the wavelength of the LED light of the two sections The light / dark cycle is 12h: 12h in any of the ranges of 465, 520, and 660 nm (blue, green, red).
In the present invention, nitrate concentrations of 16, 24 and 36 mg / L were applied as the culture conditions at a temperature of 201, a pH of 7.0 to pH 9.0, and a nitrogen source.
Referring to FIGS. 4a, 4b, 4c, 4d, 4e, 4f, 4g and 4h, I. galbana and P. tricornutum were cultured in a medium containing 2 mg of a nitrogen source at a concentration of 8 mg / N. oocyte, N. atomica, N. salina, D. salina, and D. tertiolecta produced three times the basal nitrogen source concentration of 24 (mg / L) At the concentration of mg / L, maximum biomass was produced at 1.01, 0.91, 0.87, 0.84, 0.79, 0.75 g / L.
The high concentration oil accumulation system of the microalgae of the present invention comprises a transparent incubator; An LED illumination surrounding the incubator and illuminating the interior; An LED illumination inserted into the incubator; And a controller for controlling the wavelength, the intensity and the time of the LED light emitted from the inside and the outside of the incubator for each of the
In the present invention, the wavelengths absorbed by the material are respectively 465 nm, 520 nm, and 660 nm.
In the present invention, the illumination inserted into the incubator is installed through a transparent tube.
In the present invention, LED light surrounding the incubator and LED light inserted into the incubator may include chips having wavelengths arranged in at least one of dot, line or plane.
In the high concentration oil accumulation system of the microalgae of the present invention, a transparent incubator; An LED illumination surrounding the incubator and illuminating the interior; An LED illumination inserted into the incubator; And a controller for controlling the wavelength, intensity and time of the LED light emitted from the inside and the outside of the incubator for each of the
In the present invention, the wavelength is at least one of 465 nm, 520 nm, and 660 nm.
In the high concentration oil accumulation system of the microalgae of the present invention, a transparent incubator; An LED illumination surrounding the incubator and illuminating the interior; An LED illumination inserted into the incubator; And a controller for controlling the wavelength, intensity, and time of the LED light emitted from the inside and the outside of the incubator for each of the first and second sections, wherein the incubator includes a first section for increasing the biomass to the micro- ; And two sections to increase the oil accumulation rate; And the LED illumination selectively illuminates one or more of the red, green, and blue wavelengths.
In the present invention, the wavelength is at least one of 465 nm, 520 nm, and 660 nm.
The present invention is simpler than the complex process of the two-stage method using the conventional nutrient inhibition method and the different media, and is a method of using a single medium as an oil accumulation technique using light wavelength instead of the oil accumulation method due to nutritional inhibition Method.
The present invention is simpler than the existing oil accumulation method and uses the same medium to shorten the time required for the process and ensures an oil content higher than the oil accumulation amount by the conventional technique, thus providing high technical efficiency and excellent economy.
Figures 1a, 1b, 1c, 1d, 1e, 1f, 1g and 1h show the growth curves of microalgae at each wavelength
Figure 2 shows the wavelength absorption of Chlorophyll a, β-carotene, and Phycocycianin
Figures 3a, 3b, 3c, 3d, 3e, 3f, 3g and 3h show the microalgal growth curves
Figs. 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H show microbial growth curves at various nitrogen source concentrations
Figure 5 compares production of biomass under fluorescent and LED conditions (single wavelength application in 1 and 2 sections)
Fig. 6 shows the comparison of the oil content at the optimum conditions of fluorescent lamps and LEDs (single wavelength application in 1 and 2 sections)
FIG. 7 is a graph showing changes in the oil content of the two-
8A, 8B, 8C, 8D, 8E, 8F, 8G and 8H show changes in oil content in the two-phase process using wavelength conversion
Figs. 9A, 9B and 9C are views showing a micro-algae culture apparatus according to the present invention
Figure 10 shows a two-stage culture process
FIG. 11 shows the results of a two-phase culture process
In the present invention, microalgae can be used interchangeably. The algae refers to a group of eukaryotes belonging to the protozoan system. Preferably, the algae may be any one selected from the group consisting of diatoms, brown algae, cyanobacteria, euglena, and green algae. Most preferably Isochrysis galbana, Pheodactylum tricornutum, Nannochloropsis oculata, Nannochloropsis oceanica, Nannochloropsis salina, Dunaliella salina, Dunaliella tertiolecta and Nannochloris atomus. To find the optimum wavelength for each of the eight kinds of microalgae using Red (660 nm), Orange ( 640 nm), 70mol m -2 in a wavelength Green (520 nm), Blue ( 465 nm), Violet (405 nm) s -1 . < / RTI >
1 a, 1 b, 1 c, 1 d, 1 e, 1 f, 1 g and 1 h show the results of cultivating each wavelength of each microalgae in order to find the optimum wavelength to be used. As a result, Isochrysis galbana, Pheodactylum tricornutum, Nannochloropsis Nannochlorisis oceanica, Nannochloropsis salina, Dunaliella salina and Dunaliella tertiolecta produced maximal biomass of 0.44, 0.54, 0.42, 0.41, 0.42, 0.40 and 0.40 g / L in Blue (465 nm) (660 nm) produced the maximum biomass of 0.40 g / L.
The eight microalgae contain light-absorbing Chlorophyll a substance to utilize photosynthesis and contain auxiliary pigments of β-carotene and phycocyanin. Chlorophylla mainly absorbs the blue (400 ~ 500 nm) and red (600 ~ 700 nm) wavelengths. The auxiliary pigment β-carotene absorbs the blue-green (600-700 nm) region to be used for photosynthesis. Wave scanning was performed using a UVspectrophotometer, and the results are shown in FIG. 1a-1f showing the results of experiments on these wavelengths, microalgae I. galbana, P. tricornutum, N. oculata and N. oceanica, N. salina, D. salina, D. tertiolecta The wavelength is Blue (465 nm) and Red (660 nm) (Fig. 1e) for N atomus.
Light intensity optimization experiments were performed to determine the biomass growth rates of microalgae I. galbana, P. tricornutum, N. oculata, N. oceanica, N. salina, D. salina, D. tertiolecta and N.atomus. Microalgae were cultured at various light intensities of 40, 70, 100, and 130 mol m -2 s -1 in order to find the optimal light intensity at the optimal wavelengths identified for each microalgae. Figures 3a, 3b, 3c, 3d, 3e, 3f, 3g and 3h are the result graphs. I. galbana, P. tricornutum, N. oculata , N. oceanica, N. atomus, N. salina, D. maximum biomass eight species of salina and D. tertiolecta is in mol m -2 s -1
4a, 4b, 4c, 4d, 4e, 4f, 4g and 4h, the nitrogen source concentration was set to 16, 24 and 32 mg / L in order to produce high concentration biomass Results. I.galbana and P. tricornutum produced maximal biomass of 0.97 and 1.08 g / L at a concentration of 16 mg / L, which was twice the concentration of 8 mg / L of nitrogen source used in the basic culture (FIGS. 4A and 4B) N. oocata, N. oceanica, N. atomus, N. salina, D. salina, and D. tertiolecta showed maximum biomass concentrations of 1.01, 0.91, 0.87, 0.84, 0.79, 0.75 g / L. In order to confirm the effect of the present invention, the maximum biomass production was compared with that of the control fluorescent lamp culture at the optimum LED wavelength (Blue, 465 nm, Red, 660 nm) and luminous intensity (100 mol -2 s -1 ). FIG. 5 is a graph comparing the maximum biomass measured after culturing using an LED wavelength and a fluorescent lamp at an optimum light intensity for each microalgae. Eight kinds of microalgae showed an increase of biomass production more than 2 times in the optimum LED wavelength and light intensity compared with the fluorescent light of the control group. The oil content was measured by solvent extraction method. Solvent extraction was carried out by using a homogenizer, and then chloroform and methanol were mixed at a ratio of 1: 2, and the amount of 18 ml per 10 mg was added. The remaining cell debris was removed and chloroform and distilled water were mixed at a ratio of 1: 1 12 ml was added.
The layers were separated by centrifugation at 2000 rpm for 5 minutes, and the oil content was measured by removing the upper layer and evaporating the lower layer with oil at 30. The results of comparative analysis of the fluorescent content and the oil content in the optimum LED wavelength culture are shown in FIG. The microalgae I. galbana, P. tricornutum, N. oculata, N. oceanica, N. atomus, N. salina, D. salina and D. tertiolecta accumulated about 1.5 times more oil than the control fluorescent lamp. In the present invention, two-phase processes were carried out in the same medium through wavelength conversion in order to improve the oil accumulation amount of microalgae based on the experimental results. In the case of microalgae, biomass is increased but the amount of oil accumulation is decreased under high growth conditions. In the case of growth restriction, biomass is decreased but oil accumulation is increased. A schematic diagram thereof is shown in Fig. Blue (465 nm), Red (660 nm), and Green (520 nm) were observed when the microalgae cultured at a light intensity of 100 mol m -2 s -1 at Blue (465 nm) nm). < / RTI >
I. galbana measured 56% of the oil content in red (660 nm) and 60% in green (520 nm) on day 2 (FIG. 8a) and P. tricornutum was 58% in red (660 nm) N. oculata was measured at 50% at red (660 nm) and 56% at green (520 nm) (FIG. 8c), and N. oceanica was measured at
In this experiment, the error range of each wavelength is 5 nm, the error range of light intensity is 5 mol m -2 s -1 , the error of nitrate is 10%, the error of DCW is 5%, and the error of oil is 5%. The 5% is the experimental concentration value and 5% of the resulting value. For example, the error of nitrate of 8g / L is 0.4g / L which is 5% of 8. The experimental data to which each error applies are DCW of FIG. 1, DCW of FIG. 3, DCW and nitrate of FIG. 4, DCW of FIG. 5, Oil of FIG.
FIGS. 9A, 9B and 9C are diagrams showing a high concentration oil accumulation system for microalgae according to the present invention, in which a hollow cylinder in which LEDs are installed is inserted into a transparent tubular incubator capable of doubly transmitting LED light inside and outside . 9A shows a container into which a tube for supplying air to a transparent container (incubator) and a container into which a bubbler is inserted, and a lighting device (
The two-phase culture process of the present invention as shown in FIG. 11 produces the maximum amount of biomass at the wavelengths of blue (465 nm) and red (660 nm) in the one-section culture and when the cells reach the stationary phase, And the wavelength was changed to blue (465 nm), red (660 nm) or green (520 nm) to improve the oil accumulation rate. After cultivation, the cells are recovered by centrifugation and separated by an oil extraction process.
The difference between the conventional two-stage culture process and the two-phase culture process of the present invention and the advantages of the present invention are summarized as follows. In the two-stage culture process, the maximum amount of biomass is produced in the first stage culture, and only the whole cell is recovered by centrifugation. After that, it is resuspended in a new medium in which the nitrogen source has been removed as a stressor, and is subjected to a process of re-inoculation for the second stage culture. However, in the two-phase culture process, when the cell reaches the stationary phase where growth ceases, it is necessary to convert only the wavelength to increase the oil accumulation rate Culturing is carried out in two sections. Therefore, the two-phase culture process is more efficient than the two-stage culture process because the oil can be accumulated easily without changing the medium.
In addition, general fluorescent lamps contain various wavelengths. Chlorophyll a contained in microalgae absorbs only blue (465 nm) and red (660 nm), which are specific wavelengths of various wavelengths, to utilize photosynthesis. However, in the case of LED, the efficiency of photosynthesis is increased by irradiating only the blue (465 nm), which is the wavelength absorbed by chlorophyll a, with the same amount of the fluorescent lamp, and the energy consumption is lower than that of the fluorescent lamp, .
Claims (15)
1 section to increase biomass;
And the two sections that increase the oil accumulation rate are the same,
The wavelength of the LED light to be irradiated in the first section is Blue (460 to 470 nm) and the wavelength of the LED light to be irradiated in the second section is the high concentration oil accumulation of microalgae, which is either Green (515-525 nm) or Red (655-665 nm) Way.
Wherein the incubation period of the one section is 20 days and the incubation period of the two sections is 3 days under the culture conditions of 100 mol m -2 s -1 and the light / dark cycle of 12h: 12 h in the section 1, Method for accumulating high concentration oil of microalgae.
1 section to increase biomass;
And the two sections that increase the oil accumulation rate are the same,
Wherein the wavelength of the LED light irradiated in the first section is 655-665 nm and the wavelength of the LED light irradiated in the second section is any one of 460-470 nm or 515-525 nm wavelength.
Wherein the incubation period of the one section is 20 days and the incubation period of the two sections is 3 days under the culturing conditions of the light intensity of the wavelength of 1 in the section 1 and the light / dark cycle of 12h: 12h in the light intensity of 100 mol m -2 s -1 Method for accumulating high concentration oil of microalgae.
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| KR101287384B1 (en) * | 2013-04-17 | 2013-07-23 | 이수연 | Method for increasing growht and lipid content of microalgae using mixture of led light |
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| KR102039824B1 (en) * | 2018-01-17 | 2019-11-01 | 부경대학교 산학협력단 | Production method of high unsaturated fatty acid using marine microalgae |
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