KR102024456B1 - Effects of on the Mass culture of the Prasinophyte Tetraselmis suecica and T. tetrathele using Light Emitting Diodes (LEDs) - Google Patents

Abstract

The LED (Light-Emitting Diodes; LEDs) in the bulk culture method using the genus Tetraselmis the amount of light and wavelength of, Tetraselmis The genus is Tetraselmis . suecica and Tetraselmis . Tetrathele , the light emitting diode light source is a red light having a wavelength of 620nm to 680nm or a yellow light having a wavelength of 590nm , the luminous intensity of the light emitting diode light source is 10 to 300μmol / m ² / s Light-Emitting Diodes; Green Tetraselmis using Light and Wavelength of LEDs) suecica and Tetraselmis . Tetraselmis by providing a method for mass culturing tetrathele It is possible to mass cultivate through improving the growth of the genus.

Description

Effect of on the Mass culture of the Prasinophyte Tetraselmis suecica and T. tetrathele using Light Emitting Diodes (LEDs)}

The present invention relates to a method for cultivating bile green algae in large quantities, and more particularly, by identifying and investigating the optimum light quantity and wavelength of species-specific light emitting diodes of microalgae, high carbohydrate, protein, and lipid content Green algae Tetraselmis using light emitting diodes that may include It relates to a mass culture method of suecica and Tetraselmis tetrathele .

Marine ecosystems are made up of a variety of biological groups, and the microorganisms occupy the lowest level. Microalgae are independent nutrients that synthesize inorganic matter into organic matter through photosynthesis through light energy. It is also the primary producer of oxygen, one of the important biogroups on which bioproduction is based in ecosystems.

Microalgae are widely used in various industrial fields, such as agriculture, fisheries, alternative energy, food, medicine, feed additives and environmental purification. It is used as feed organisms such as rotifers and shellfish, or it is provided as an animal feed source through processing. In addition, it is possible to extract microalgae nutrients from microalgae and use them as raw materials for food and pharmaceuticals, or intracellular lipids can be converted into fatty acid methyl esters, which are biodiesel, and are attracting attention as energy to replace fossil fuels. There is also the use of domestic and industrial wastewater and sludge purification utilizing nutrient availability and photosynthetic oxygen supply.

As such, for the commercial use of microalgae, mass culture is essential because high concentration of cell density is required within a short period of time. Currently, there are two methods for culturing microalgae, open ponds and closed culture systems (Photo-Bioreactors). Open mass cultivation is a method of cultivating outdoors through sunlight to be used for non-purity applications.

Closed culture is used for cultivation requiring high purity and stability, such as pharmaceuticals, food and feed, but operating costs are high, in the case of halogen and fluorescent lamps used as lighting, it consumes a lot of power consumption. Among them, light conditions are easier to control than other environments, and thus may change nutrients or growth.

Biochemical and physical environmental conditions (water temperature, salinity, dissolved gas, pH, advection, nutrients, and environmental acceptability) affect the growth of microalgae, but most of them are photosynthetic organisms. It is an important factor for growth.

In particular, since the change of the light wavelength changes the biochemical composition and content of the cells according to the reactivity of the photosynthetic pigments of the microalgae, it is expected to be utilized in industrial fields such as increasing useful materials.

On the other hand, a light emitting diode (LED) is a semiconductor device that can irradiate light by supplying a current through a compound such as gallium phosphide or gallium arsenide, and is environmentally friendly, long life and low consumption due to no mercury. Due to the advantages of power, it has developed rapidly in the industry.

LED can be adjusted to short wavelength or plural wavelength, and light irradiation time can be irradiated only minute or second wavelengths to enhance the growth of microalgae or to enhance the nutrient reinforcement. For mass cultivation, studies to find the optimum culture conditions of microalgae are being actively conducted.

Chlorella sp. Showed higher growth rate as the amount of light increased in the red wavelength compared to the blue and yellow wavelengths, and the diatom Chaetoceros calcitrans In the case of, red growth showed high growth rate and biomass. Diatom Thalassiosira Pseudonana showed high growth in white fluorescent lamps, and protein content was high in blue-green wavelength, and it is considered that there is a specificity of light wavelength depending on species.

Tetraselmis Is It is known to be a species of green algae, and it is a species that is photochlorinated. It is easily replaced by chlorella, which is vulnerable to high temperature in summer. It is also nutritionally excellent and widely used as a food organism.

In addition, like other microalgae, it is possible to promote useful substances in cells through changes in environmental conditions such as water temperature, salinity, and light wavelength, and is known to have high nutrient absorption. However, wavelength studies for ocean culture in Tetraselmis through the irradiation of light emitting diodes are less than other microalgae.

In Korean Patent No. 10-1382955, a microalgae using a light emitting diode in which an optical fiber having a light emitting diode (LED) as a light source is installed inside the culture tank so that the light source can be evenly transmitted to the entire culture tank. It relates to a culture apparatus. A culture tank containing microalgae and culture solution therein and a light source unit installed inside the culture tank for irradiating light and a carbon dioxide supply unit connected to the inside of the culture tank to supply carbon dioxide and connected to the light source unit and supplied through the carbon dioxide supply unit Disclosed is a microalgae culture apparatus using a light emitting diode including a carbon dioxide discharge unit for discharging the carbon dioxide into the culture tank. In Korean Patent No. 10-1447386, the amount of photosynthetic pigment and photosynthesis generated by the growth of marine bacteria and Synechococcus sp. In order to determine the effect of the photosynthetic dye content and gene expression was prepared a primer for detecting the amount of the photosynthetic pigment content and gene expression after illumination for each LED wavelength and intensity. As a result, the short wavelength green and blue LED light source increases the content of photosynthetic pigments of Synechococcus sp., Induces the mRNA expression of photosystem related genes and delivers the energy generated in the cells. Disclosed is a method of culturing using an increase in photosynthesis of marine bacterium through irradiation of a light emitting diode light source. In Korean Patent No. 10-0415150, maximization of light efficiency by dynamic internal light source using a dynamic internal light source using a light emitting diode (LED) or a rectangular fluorescent light source and a conventional turbine agitated bioreactor during high concentration culture by microalgae And agitated light emitting turbine device for mass culture of microalgae, which can provide agitated photobioreactor equipped with a light emitting turbine device for mass culture of microalgae, which can simultaneously obtain very high mixing efficiency of the turbine and agitated reactor. A fluorescent bioreactor is disclosed. Korean Patent Publication No. 10-2015-0057350 maximizes the utilization of light by using LED light source and reflector plate when culturing microalgae, and has an inclined plate to enhance the agitation effect of microalgae with the power of bubbler without any external power device. The efficiency of minimizing the device can be increased, and the convenience of cultivation can be achieved by providing equipment using transparent soft materials such as polyethylene and urethane, which are highly accessible in place of expensive glass and other containers when culturing microalgae. Disclosed is an LED microalgae chlorella incubator having a reflecting device inclined inside thereof, which has an effect of increasing the efficiency. However, the prior document includes the step of culturing the microalgae consisting of Tetraselmis suecica or Tetraselmis tetrathele by irradiating light using light-emitting diodes (LEDs) of the present invention, the induction phase during the growth of the microalgae LED light source irradiation of at least one stage selected from the early stage, algebraic growth stage, etc. to increase the carbohydrate, protein, lipid content in the microalgae by irradiating the yellow light wavelength of 590 nm, mid-term algebraic stage during the growth phase of the microalgae Is irradiated with a light emitting diode light source of a red light wavelength of 620nm to 680nm, the light emitting diode light source Tetraselmis suecica using light quantity and wavelength of light-emitting diodes (LEDs) of 10 to 300 μmol / m2 / s And Tetraselmis. The method of mass culturing of tetrathele is not disclosed and shows a difference.

The present invention is the Tetraselmis through irradiation of light emitting diodes in the microalgae industrialization To a method for culturing in the ocean other than solving the above problems, insufficient algae, a high industrial value Tetraselmis and T. suecica thetrathele that to identify the effects of LED intensity and wavelength on the Growth applying this light-emitting diode (Light-Emitting Diodes; LEDs) Tetraselmis using the amount of light and the wavelength of the Provides a method of mass culture of the genus.

Tetraselmis by irradiating light using light-emitting diodes (LEDs) as a means to solve the above problems Including the step of culturing the microalgae consisting of suecica or Tetraselmis tetrathele , the light emitting diode light source irradiation step of at least one selected from the induction period, early stage, algebraic growth stage, etc. of the growth phase of the microalgae is irradiated with a wavelength of 590 nm Increasing the carbohydrate, protein, lipid content in the microalgae, during the growth stage of the microalgae irradiated light emitting diode light source of red light wavelength of 620nm to 680nm, the light emitting diode light source is 10 to 300 μmol / m Light green steel Tetraselmis using light quantity and wavelength of ² / s Light-Emitting Diodes (LEDs) suecica and Tetraselmis . To provide a method for mass culturing tetrathele .

As a result of culturing the microalgae by the method according to the present invention, the culture light source can be expected to save electricity by irradiating with a light emitting diode, and the yellow light wavelength of the induction phase, early stage, logarithmic growth phase during the growth phase of the microalgae Increasing the accumulation of carbohydrates, proteins, and lipids in the cells of microalgae biochemical composition can be cultured to facilitate food and food nutrition, biofuel production and water or sediment environment.

In addition, during the growth phase of the microalgae, the medium-term logarithmic growth period is irradiated with red wavelengths, thereby promoting growth.

1 is Tetraselmis used in the experiment used in the experiment . suecica (LIMS-PS-0007) and Tetraselmis. Picture of tetrathele (LIMS-PS-0053).
2 shows wavelength conditions of a light source.
3 shows T. suecica The change in cell density according to the amount of light for each wavelength is shown.
Figure 4 shows the change in cell density according to the amount of light for each wavelength of T. tetrathele .
FIG. 5 shows the results of the relation between light quantity and growth rate of wavelengths of T. suecica and T. tetrathele through an improved equation of Lederman and Tett's model.
Figure 6 shows a graph showing the change of the pigment for each wavelength of T. suecica and T. tetrathele .
Figure 7 shows the change in carbohydrate content of each T. suecica and T. tetrathele wavelength.
Figure 8 shows the protein changes by wavelength of T. suecica and T. tetrathele .
Figure 9 shows the lipid content of the T. suecica and T. tetrathele by wavelength.
Figure 10 shows the change in the concentration of ammonia nitrogen of the culture medium according to the wavelength of T. suecica and T. tetrathele .
11 shows the phosphate concentration of the culture medium according to the wavelength by the time of T. suecica and T. tetrathele.
12 shows a schematic diagram of ammonia nitrogen absorption process of microalgae.
Figure 13 shows the concentration change of ammonia nitrogen in the culture medium according to the wavelength of T. suecica and T. tetrathele depleted nitrogen source.
Figure 14 shows the change in the concentration of phosphorus phosphate in the culture medium according to the wavelength of T. suecica and T. tetrathele depleted by personnel.
15 is an example of the light-emitting diode Tetraselmis suecica and Tetraselmis using light quantity and wavelength of the light emitting diode proposed in the present invention. The method of mass culturing tetrathele is shown.

Light green steel Tetraselmis using the light quantity and wavelength of light-emitting diodes (LEDs) of the present invention suecica and Tetraselmis. When explaining the experimental example specific to the mass culture method of tetrathele as follows.

1. Experiment Method

1-1 Tetraselmis in the growth rate

1 is Tetraselmis used in the experiment used in the experiment . suecica (LIMS-PS-0007) and Tetraselmis. Picture of tetrathele (LIMS-PS-0053). Tetraselmis . suecica (LIMS-PS-0007) and Tetraselmis . tetrathele (LIMS-PS-0053) was distributed by the Korea Maritime Institute of Maritime Science Library. The medium used for the culture was f / 2, and the final concentration of selenium (H ₂ SeO ₃ ) was added to 0.001 μM.

Water temperature and salinity for the maintenance were 20 ℃, 30 psu, the light amount was controlled by 100 μmol / m ² / s (L: D = 12L: 12D; cool-white fluorescent lamp). The instrument used in the experiments was used after autoclaving (121 ° C, 20 min) to block secondary biological contamination, and all work was performed at aseptic mass.

AM9 antibiotics were used for aseptic treatment of the experimental species, and the apparatus used in the experiment was used after high temperature and high pressure sterilization (121 ℃, 202 kpa, 20min) to prevent secondary biological contamination. Was performed.

Tetraselmis according to wavelength to know the increase or decrease of cell density The growth rate of suecica and T. tetrathele were confirmed by increasing and decreasing cell density. The increase and decrease of cell density was measured by in vivo chlorophyll fluorescence, not by direct microscopic examination. According to the increase and decrease of cell density, the growth stage could be divided into the following three stages.

Induction phase: When microalgal cells are inoculated in fresh medium, the number of cells does not increase before logarithmic growth.

-Exponential phase (algebraic growth period): the period in which cell proliferation increases at a constant rate during cell growth.

-Normal phase (nutrient enrichment phase): When cell proliferation and death have a certain ratio, there is no increase in cell number

This is because the use of a culture tube that can be measured directly through the fluorescence spectrometer is not necessary to open and close directly to prevent contamination of the cells, and to be able to process a large amount of samples quickly.

2 shows wavelength conditions of a light source. As a light source for the growth experiment according to the amount of light for each wavelength, a fluorescence lamp (three-wavelength lamp, Namyoung Bulb Co., Ltd.) was used, and the single wavelength was a blue LED ( λ _max = 450 nm; LUMILEDS), a yellow LED ( λ _max = 590 nm; LUMILEDS), red LEDs ( λ _max = 630 nm; LUMILEDS).

Two species of Tetraselmis were grown to algebraic growth period, and 5 mL of f / 2 medium was injected into 9 mL culture tube (13 mm, disposable culture tubes, KIMBLE CHASE), and the final cell density was inoculated to about 1.0 ² cells / mL. .

Light conditions were adjusted in 7 steps of 10, 30, 50, 70, 100, 200 and 300 μmol / m ² / s (QSL-2100, Biospherical Instrument Inc.) using a visible light shielding film. Thereafter, the fluorescence value was measured at 10 am at 10 am with a fluorophotometer (10-AU-Fluorometer, Turner Designs,).

Growth rate was calculated by substituting the following equation using the cell density during the period of logarithmic growth. Each light condition experiment was carried out by triplicate, and the growth rate was expressed as their average value by the following formula (except when the value obviously found out of triplicate was not averaged).

μ: specific growth rate (day)

N ₀ , N _t : Fluorescence value at the beginning and after t time (day) in algebra

t: 대수생장기의 기간(day)

t: period of logarithmic growth (day)

The relationship between growth rate and light intensity was calculated using the following equation, which was modified from Lederman and Tett's (1981) model.

μ: specific growth rate (day)

μ _max : maximum specific growth rate (/ day)

I: amount of radiation (μmol / m ² / s)

I ₀ : compensation PFD (μmol / m ² / s)

K _S : half-saturation light intensity (μmol / m ² / s)

1-2. Biochemical Measurement of the Genus Tetraselmis

In order to confirm the cell count, the water temperature and salinity for the preculture were 20 ° C. and 30 psu, and the light amount was adjusted to 100 μmol / m ² / s (L: D = 12L: 12D). 3 L of f / 2 medium was injected into a 4 L PE bottle to inoculate a final cell density of about 1.0 ² cells / mL. Cell counts were directly examined under a microscope at 10 am once every two days.

Table 1 shows the measured pigment items. Chl a, b, c, phaeophytin a , chlorophyllide a and 13 subsidiary pigments were measured. Samples for analysis were incubated to algebraic growth period, and then frozen (-20 ° C) immediately after filtration and stored in cows.

Pigment UPLC (Ultra Performance Liquid Chromatography) was measured Chl a, b, c, phaeophytin a, chlorophyllide a, auxiliary dye 13 species System (ACQUITY UPLC H-Class, Waters , USA).

Measured pigment item Peak no. Pigment Retention One Chlorophyllide a 2.1 2 Chlorophyll c ₂ 2.3 3 Peridinin 2.6 4 19'-butanoyloxyfucoxanthin 2.8 5 Fucoxanthin 2.9 6 Neoxanthin 3.0 7 Prasinoxanthin 3.1 8 Violaxanthin 3.2 9 19'-hexanoyloxyfucoxanthin 3.2 10  Diadinoxanthin 3.6 11 Alloxanthin 4.0 12 Diatoxanthin 4.3 13 Lutein 4.6 14 Zeaxanthin 4.7 15 Chlorophyll b 8.0 16 Chlororoll a 8.3 17 Pheophytin a 8.7 18 β-carotene 9.0

For photosynthetic pigment analysis, 100 mL of the biological samples grown until the logarithmic growth period was filtered through GF / F filter paper and stored at -20 ° C.

The dye was extracted by the following method. 5 mL of 100% acetone was added and 100 μL of canthaxanthin, an internal standard, was added, followed by ultrasonic grinding for about 5 minutes. Then extracted at -20 ℃ cold dark 24 hours. Filter paper was crushed using a grinder during the extraction period of 24 hours, 1 mL of the supernatant centrifuged at 2000 rpm for 10 minutes and 300 μL of UPLC grade water were added to the vial, mixed, and injected into the UPLC loop for analysis.

Table 2 shows the composition ratio of the solvent for color analysis. The composition ratio of the solvent for the dye analysis was set according to the conditions of the column using the ACQUITY UPLC Columns Calculator program.

Composition of Solvent for Pigment Analysis UPLC: Waters ACQUITY UPLC H-Class
Detector: Absorbance (Waters PDA) at 436 nm
Column: Waters HSS C18 (1.8 μm, 2.1mm)
Solvent A: MeOH 80%, Ammonium Acetate (0.5 M) 20%
Solvent B: Acetonitrile 87.5%, H ₂ O 12.5%
Solvent C: Ethyl Acetate 100%
Flow rate: 0.35 mL / min

Carbohydrates were analyzed by Phenol-sulphuric acid method. Allowed to stand 40 minutes after the phenol was added 1 mL of 5% over a standard solution of glucose, and the cells are added to extract the in carbohydrate, H ₂ SO ₄ 5 mL. Allow to cool for 10 minutes after cooling. Mix homogeneously and centrifuge for 15 minutes at 2000 rpm. Afterwards, the absorbance was measured at 490 nm and 750 nm using UV / Vis spectrophotometer (X-ma, 3000PC) to determine the concentration.

Protein was analyzed by the following method. A standard solution was prepared using Protein standard solution, and 5 mL of alkaline copper solution, a mixed reagent, was added and mixed homogeneously. After leaving for 10 minutes, the protein was extracted and left for 1 hour and 30 minutes with folin ciocalteu and mixed homogeneously. After centrifugation at 3000 rpm for 10 minutes, the absorbance was measured at 750 nm using a UV / Vis spectrophotometer to determine the concentration.

Lipids were analyzed using the following method to extract lipids using chloroform and methanol. glycerintripalmitat (tripalmitin) was used as a standard solution, and the lipid extraction process was as follows.

Ultrapure water is added to a supernatant containing lipids extracted by chloroform and methanol. After distillation into methanol, ultrapure water, chloroform and lipid layers, the methanol and ultrapure water of the upper layer is removed and stored in a dryer at 40 ° C for 48 hours. When all samples were dried, H ₂ SO ₄ and ultrapure water were added, and the absorbance was measured at 360 nm and 750 nm by UV / Vis spectrophotometer to determine the concentration.

1-3 Measurement of Nutrients Absorption Capacity in Tetraselmis

T. suecica and T. tetrathele were aseptically treated with AM 9. AM 9 mixture contains dihydrostreptreptomycin sulfute (5 mg / mL), potassium penicillin G (3.5 mg / mL), polymyxin B sulfate (0.194 mg / mL), tetracycline (0.5 mg / mL), chloramphenicol (0.05 mg / mL) and neomycin A mixture of six antibiotics, sulfate (0.25 mg / mL), was prepared and used.

The cultured cell solution and the AM 9 mixture were mixed in a ratio of 0.9: 0.1, 0.8: 0.2, 0.7: 0.3, and 0.6: 0.4, respectively, and adjusted to a total of 1 mL. 24 hours of 0.6 the ratio of the mixture was high AM 9: 0.4 were both species cell death, T. suecica on standing 0.8: confirmed the viability of the cells in 0.3: 0.2, T. tetrathele in 0.7. The viable cell solution was cultured under f / 2 medium.

The cultured cell solution was mixed to a final concentration of glutaaldehyde to 1% and stored in a cool dark place. Thereafter, DAPI (4,6-diamidino-2-phenylindole) was mixed at a final concentration of 1 μg / mL, and then checked for sterility by fluorescence microscopy. In addition, to prevent secondary contamination, all experiments were performed on a clean bench, and the experimental apparatus was used by high temperature and high pressure sterilization (121 ° C., 202 kpa, 20 min).

In order to understand the relationship between the microalgae absorption rate and nutrient concentration, surge uptake after nutrient addition should be performed and the experiment should be performed within the time when there is no change of intracellular nutrients. Therefore, before the nutrient absorption experiment, the constant velocity absorption was identified through a preliminary experiment.

First, T. suecica and T. tetrathele were pre-cultured in L1 media based on AK artificial seawater without nitrogen or phosphorus to deplete intracellular nitrogen and phosphorus. Thereafter, the cells were cultured until the growth of cells was stopped. Intracellular nitrogen and phosphorus-depleted cell solution were inoculated into 700 mL of L1 medium containing 20 μM of ammonia nitrogen and 3 μM of phosphoric acid.

At the start of the culture (0 minutes), 10, 20, 30, 45, 60, 90, 120, 180, 240 minutes each of the culture medium was taken and filtered with a 0.45 μm filter and the ammonia nitrogen and phosphorus phosphate were measured respectively. The same experiment was conducted in the fluorescent lamp, red wavelength (630 nm), yellow wavelength (590 nm), and blue wavelength (450 nm) to determine the change according to the wavelength. It was selected as the experiment time.

The nutrient absorption rate is 7 steps of 1.0, 5.0, 10, 20, 50, 70, 100 μM of ammonia nitrogen, and 7 steps of 0.1, 0.5, 1.0, 3.0, 5.0, 10, 20 μM of phosphorus phosphate in a 300 mL culture vessel. Nitrogen and phosphorus-depleted cell solution (about 1 × 10 ⁴ cells / mL) were inoculated into the L1 medium prepared with each.

Incubation time was set to the time when the constant absorption was shown in the preliminary experiment. In particular, the addition of the culture medium containing the nitrogen and phosphorus depleted cells is lower than the concentration of the nutrients provided in the absorption experiment, so that the concentration was checked immediately after inoculation. Experiments were performed under 100 μmol / m ² / s at all wavelengths to determine the nutrient absorption rate according to the wavelength.

The Michaelis-Menten equation was used to determine the relationship between nutrient absorption rates and nutrients. The variable value Ks was calculated by using the nonlinear least-squares method by substituting the measured experimental value into Equation (1).

(1)

(One)

ρ _max : Maximum absorption rate of nutrients (pmol / cell / hr)

Ks: half saturation constant (μM) S: nutrient concentration (μM)

All assays for nutrient measurements were tested according to the Marine Environmental Process Testing Standard (Ministry of Land, Transport and Maritime Affairs, 2014), and the analysis items included ammonia nitrogen and phosphorus phosphate, as described below.

Ammonia nitrogen is oxidized with a basic hypochlorous acid solution to produce a monoamine. Monoamines produce blue indophenol by phenol, the nitroprosheets, and hypochlorous acid. After a 1 hour reaction time in a thermostat 35 ℃, the absorbance of the indophenol color developed at 640 nm, the maximum absorption wavelength was measured by UV / Vis Spectrophotometer. The concentration of ammonia was calculated using the calibration curve.

The molybden method is most commonly used to measure phosphorus in seawater. Phosphorous phosphate reacts with ammonium phosphomolymoldenate and potassium antimonyl tartrate under acidic conditions to form a phosphorus-molybdenic acid complex. This complex is reduced by ascorbic acid to produce a blue solution. Since the intensity of this blue color is proportional to the concentration of phosphorus phosphate, the solution was measured for absorbance at 885 nm, the maximum absorption wavelength by UV / Vis Spectrophotometer (X-ma, 3000PC). The concentration of phosphorus phosphate was calculated using a calibration curve.

2. Experimental Results

2-1 Growth Results of the Genus Tetraselmis According to Wavelength and Light Amount

The microalgae photochemical device is composed of a reaction center consisting of chlorophyll a main pigment and an antenna complex consisting of some chlorophyll a (chl- a ) and sub-pigment carotenoid and phycobilin.

Photosynthetic pigment is an important classification key because the composition is different at the microalgal level, and the absorption wavelength is determined by the photosynthetic pigment. It seems to affect until.

3 shows T. suecica The change in cell density according to the amount of light for each wavelength is shown. The growth curves for the fluorescent lamp, the blue wavelength, the yellow wavelength, and the red wavelength are shown for each light amount. As a result, it was confirmed that the maximum cell density increased with increasing the amount of light at all wavelengths, and was similar at a certain amount of light. The maximum cell density increased with increasing the amount of light up to 100 μmol / m ² / s for fluorescent lamps, blue and red wavelengths, and 300 μmol / m ² / s for yellow wavelengths.

Figure 4 shows the change in cell density according to the amount of light for each wavelength of T. tetrathele and the growth curve for the fluorescent lamp, blue wavelength, yellow wavelength, red wavelength is shown for each light amount. As a result, it was confirmed that the maximum cell density increased with increasing the amount of light at all wavelengths, and was similar at a certain amount of light. In the case of fluorescent lamps and red wavelengths, the maximum cell density increased up to 200 μmol / m ² / s and blue and yellow wavelengths up to 300 μmol / m ² / s.

The results showed no photoinhibition of the growth rate reported in some microalgae, and showed the maximum cell density at relatively low light intensity when fluorescent lamps and red wavelengths were light sources. It can be expected that the electricity saving effect will be expected.

Relationship between Light Amount and Growth Rate of T. suecica and T. tetrathele by Wavelength Species Wavelength Hyperbobolic equation μ _max
(/ day) Ks
(μmol / m ² / s) I ₀
(μmol / m ² / s) T. suecica Fluorescent lamp μ = 0.91 (I-4.73) / (I + 37.6) 0.91 47.1 4.73 Blue LED μ = 0.94 (I-1.50) / (I + 17.8) 0.94 20.8 1.50 Yellow LED μ = 0.89 (I-5.45) / (I + 32.9) 0.89 22.0 5.45 Red LED μ = 1.14 (I-9.01) / (I + 46.2) 1.14 64.2 9.01 T. tetrathele Fluorescent lamp μ = 0.96 (I-10.7) / (I + 44.1) 0.96 65.6 10.7 Blue LED μ = 0.83 (I-12.7) / (I + 49.7) 0.83 75.2 12.7 Yellow LED μ = 0.63 (I-3.74) / (I + 19.8) 0.63 12.4 3.74 Red LED μ = 0.95 (I-10.4) / (I + 51.6) 0.95 72.5 10.4

5 and Table 3 show the results of the relationship between the light quantity and growth rate for each wavelength of T. suecica and T. tetrathele through an improved equation of Lederman and Tett's model. I ₀ is the minimum amount of light that can be grown by cells with the same amount of respiration and photosynthesis, and Ks is the affinity index of light.

The growth rate also increased as the amount of light increased at each wavelength condition, and showed similar growth rate above a certain amount of light. Μ _max of T. suecica appeared in the order of red wavelength, blue wavelength, fluorescent lamp, and yellow wavelength. Therefore, the growth rate of T. suecica by wavelength was the highest in red wavelength and the lowest in yellow wavelength.

Ks and I ₀ had the lowest blue wavelength and were relatively high in the slow red growth. Ks was similarly the lowest in blue and yellow, and the highest in red.

As a result, considering the blue wavelengths I ₀ and Ks, it is low in T. suecica , and it is possible to grow at low light quantity and to grow to a certain number of cells quickly.

In the case of T. tetrathele , the growth rate also increased with increasing amount of light at all wavelengths. μ _max was similar in fluorescent lamp and red wavelength, followed by blue wavelength and yellow wavelength. Therefore, the growth rate of T. tetrathele was the highest in fluorescent lamp and red wavelength, and the lowest in yellow wavelength.

Ks was similar at 71.1 ± 5.00 μmol / m ² / s in all light conditions except yellow wavelength and showed the lowest value in yellow wavelength. I ₀ was similar in the fluorescent, blue, and red wavelengths with an average of 11.2 ± 1.25 μmol / m ² / s, which was the lowest in the yellow wavelength.

When confirming the above results showed the highest growth rate in the red wavelength LED light source in both species, T. tetrathele (0.95 / day) a higher growth rate in the T. suecica (1.12 / day) than was found.

In the case of T. tetrathele, the growth rate was similar to the red wavelength in the fluorescent lamp. However, when looking at the power consumption compared to the fluorescent lamp, the LED can realize a high photosynthetic effect with a low power consumption compared to the normal fluorescent lamp of 40W.

To find out the economic effect of this, the electricity bill was calculated. Based on KEPCO's electricity bill, we saw that the average industrial electricity rate (kWh) was about 73.2 won, and based on this, the fluorescent lamp (Samyoung Bulb, 40 W, life time 8000 h) and LED (Smart LED, 20 W, life time) 25000 h) was compared. As a result, based on 25,000 hours of use, fluorescent lamps cost electricity of 79,450 won and LEDs cost 49,100 won, saving about 45%. Therefore, even though LEDs show the growth of microalgae at a level similar to fluorescent lamps, they can expect about half the electricity savings in the long term.

Microalgae have different levels of absorption spectrum for each species, and green algae show high growth at blue or red wavelengths due to the effect of chlorophyll a .

According to the experimental example of the present invention, T. suecica and T. tetrathele showed a high growth rate in red wavelength, which is judged to be an effect on the absorption spectrum of photosynthetic pigment. Green Algae There is a unique and containing a second pigment chlorophyll b (chl- b), the maximum absorption wavelength of chl- b as well as a blue 455 nm and 642 nm, to absorb red wavelength pass optical energy into a chl- Done.

The center wavelength of the red LED used in the above example was 630 nm, which was similar to chl- b and the maximum absorption wavelength. As a result, the growth rate is high at a specific wavelength range, and the high growth rate at the red wavelength is mostly green algae and cyanobacteria.

Green algae are C. pyrenodosa , C. vulgaris , Haematococcus pluvialis, blue-green algae is the Spirulina platensis, Synechococcus sp. have been reported. Red algae Porphyra has a higher light absorption rate in red light than blue light and is 1.2 to 1.5 times more efficient in transferring to the next stage for photosynthesis. Therefore, photostimulation is thought to show high growth rate by activating metabolism including enzyme activity as well as high carbon accumulation rate.

In the case of yellow wavelength, the growth rate was thought to be low, but both species showed 70% of the maximum growth rate and showed relatively lower Ks and I ₀ than other wavelengths. Secondary pigment which generally has a hollow Tetraselmis is chl- b and a neoxanthin, violaxanthin, lutein, and carotenoids account for the highest number of rate including the zeaxanthin, the absorption wavelength of the carotenoids is 400 ~ 550 nm (green in purple).

However, since the absorption spectrum (absorption spectrum) the genus Tetraselmis is chl- a is and is able to absorb most of the wavelengths except the wavelength of green, chl- b is able to absorb most of the wavelengths including the yellow wavelength, the present invention According to the experimental example of the growth in the yellow wavelength range is considered possible.

As a result of the embodiment of the present invention, the growth rate according to the amount of light by wavelength of T. suecica and T. tetrathele showed the highest growth rate in the red wavelength, which is a complex including high light absorption transmission and high carbon accumulation rate in the red wavelength. It may be due to the factor.

However, Ks and I ₀ were rather low at yellow wavelength. Therefore, the use of yellow wavelength in induction and early algebraic growth period and red wavelength in mid-term algebraic growth phase of Tetraselmis is effective in promoting growth.

2-2 Biochemical Composition of Tetraselmis Species According to Wavelength and Light Amount

Carbohydrates, proteins, lipids, and pigments, which are intracellular components of microalgae, are commercially available. When microalgae are used as food organisms, high biochemicals are required because they affect the body components of the feeder that feeds on the food organisms.

Thus, the biochemical composition and content of algae can be changed according to the light conditions, and in the embodiment of the present invention is a report that an enrichment in the environmental conditions under which growth is inhibited Tetraselmis in the LED wavelengths photosynthetic pigments, carbohydrates, Protein and lipid content changes were measured.

Microalgae pigments accumulate through photosynthesis and contain not only the main chlorophyll but also several subsidiary pigments. Its composition varies depending on the species of microalgae, and the types of auxiliary pigments also vary.

Among the commercially available auxiliary pigments are carotenoids, such as β-carotene, astaxanthin, and lutein. As such, the pigment content and accumulation rate may vary according to the culture environment, so that the T. suecica and T. tetrathele Changes to photosynthetic pigments and auxiliary pigments were measured by UPLC.

Changes in the Pigments of T. suecica and T. tetrathele by Wavelength Species Wavelength Chlild a Chl c2 Neo Viol + hex Allo Dtx Lut
+
Zea Chl b Chl a Pht a β-caro T. suecica Fluorescent lamp 0.021 0.000 0.046 0.058 0.005 0.002 0.059 1.350 1.371 0.028 0.040 Blue LED 0.038 0.000 0.075 0.132 0.005 0.006 0.106 1.677 1.827 0.155 0.136 Yellow LED 0.055 0.000 0.156 0.176 0.010 0.007 0.143 4.206 4.077 0.158 0.220 Red LED 0.041 0.000 0.099 0.130 0.010 0.007 0.111 2.193 2.212 0.172 0.115 T. tetrathele Fluorescent lamp 0.015 0.002 0.021 0.024 0.001 0.001 0.030 0.590 0.636 0.006 0.013 Blue LED 0.020 0.000 0.030 0.050 0.002 0.002 0.042 0.696 0.801 0.049 0.041 Yellow LED 0.018 0.000 0.066 0.077 0.002 0.002 0.059 1.624 1.856 0.053 0.098 Red LED 0.010 0.000 0.020 0.032 0.001 0.001 0.027 0.582 0.711 0.024 0.039

Table 4 and FIG. 6 are graphs showing changes in pigments for each wavelength of T. suecica and T. tetrathele, and the pigment concentrations for fluorescent lamps, blue wavelengths, yellow wavelengths, and red wavelengths are indicated by items.

Figure 6a shows the change of the pigment for each wavelength of T. suecica . Most of the items were found to be high in the yellow wavelength. The main pigments, chlorophyll a (chl- a ) and chlorophyll b (chl- b ), were about 2.97 and 3.11 times higher in yellow wavelengths, respectively, compared with fluorescent lamps, which are the light sources used for mass culture.

The yellow wavelength was about 1.84 and 1.91 times higher than the red wavelength which showed rapid growth. As a result of the concentration of the sub-pigment, neoxanthin, violaxanthin + 19-hexanoyloxyfucoxanthin, lutein + zeaxanthin, and β-carotene had the highest yellow wavelength. Compared with the fluorescent lamps, the concentrations were 3.1, 3.1, 2.4, and 5.6 times higher. Yellow wavelengths were 1.6, 1.4, 1.3, and 1.9 times higher than red wavelengths, respectively.

However, chlorophyllide a , alloxanthin and diatoxanthin showed similar concentrations at all wavelengths. Chlorophyll c2 was not detected in this experiment. The total pigment concentration of each wavelength was also the highest in yellow wavelength. The main pigment concentration was 8.38 pg / cell and the total auxiliary pigment concentration was 0.93 pg / cell. In fluorescent lamp, red wavelength, and blue wavelength, 2.7 ～ 4.4 pg / cell, respectively. , 0.26 to 0.69 pg / cell.

Figure 6b is a graph showing the change of the pigment for each wavelength of T. tetrathele . Most of the items were found to be high in the yellow wavelength. The main colors of chl a and chl b were about 2.9 and 2.8 times higher in yellow wavelengths, respectively, compared with fluorescent lamps, which are light sources used in conventional mass culture. Compared with the red wavelength which showed rapid growth, it was about 2.6 and 2.8 times higher. As a result of the wavelengths of the secondary pigments, neoxanthin, violaxanthin + 19-hexanoyloxyfucoxanthin, lutein + zeaxanthin, and β-carotene showed the highest concentrations in yellow wavelength.

The yellow wavelengths were 3.2, 3.2, 2.0 and 7.8 times higher than the fluorescent lamps, respectively. Yellow wavelengths were 3.4, 2.4, 2.2, and 2.5 times higher than those of red, respectively. However chlorophyllide a, alloxanthin, diatoxanthin showed similar levels in all wavelengths.

In addition, chlorophyll c2 does not appear at all wavelengths, so T. tetrathele does not seem to contain chlorophyll c2 . The total pigment concentration of each wavelength was also highest in yellow wavelength, with main pigment concentration of 3.5 pg / cell and total auxiliary pigment concentration of 0.38 pg / cell. In fluorescent lamp, red wavelength, and blue wavelength, 1.2 ~ 1.5 pg / cell, respectively. , 0.11 to 0.24 pg / cell. When the two species were compared, both the main and secondary pigments were 2.5 and 2.9 times higher in T. suecica , respectively.

The composition and content of the pigment contained in each microalgae was species-specific and the pigment content of the Tetraselmis genus of the present invention was found to show a cumulative effect due to optical stress at a yellow wavelength.

During cell division under stress environment, a defense mechanism is applied to maximize the amount of light absorption, resulting in higher accumulation of pigments per cell and higher chl a concentration at green wavelengths with slower growth than the faster red wavelengths. Because the reason is not the wavelength absorption region of the main pigment, the mismatch of the light conditions for growth and pigment content is known to lead to a decrease in the growth rate with a decrease in the number of cells when subjected to high stress by light.

In the above results, β-carotene increased up to 7.8 times in yellow wavelength. Green algae generally report high growth at red or blue wavelengths and nutrient accumulation due to low growth at green wavelengths. However, because the yellow wavelength does not absorb the carotenoid family in the absorption spectrum, it can increase the light stress. Therefore, it is judged that the yellow LED is suitable as a light source for pigment accumulation in Tetraselmis .

The change of pigment with wavelength of T. suecica and T. tetrathele showed high pigment accumulation at yellow wavelength, which was the slowest growth. The reason is that, as described above, the yellow wavelength is not the absorption wavelength region of the main pigment, so Tetraselmis Because it stressed the growth of the genus. Yellow wavelength is not absorbed spectrum wavelength of carotenoid series, so it is considered that it can give higher optical stress than red, blue or green wavelength. In addition, the reason for the low pigment concentration in the red wavelength is thought to be due to the fast cleavage rate, which cannot afford to accumulate the pigment.

Thus Tetraselmis The content of photosynthetic pigments in the mass culture of the genus is expected to be higher in the yellow wavelength than the red wavelength, which showed rapid growth with the fluorescent lamp which is the existing mass culture light source.

Carbohydrates are polymers linked to monosaccharides and are used as important energy sources for animals and plants. Also, microalgae are photosynthetic by light energy to convert chlorophyll into carbohydrates, so light conditions are an important factor in carbohydrate content in microalgae. The microalgae have different carbohydrate accumulation patterns depending on the culture stress of each species, and thus experiments were carried out on the carbohydrate change in the Tetraselmis genera according to the wavelength.

Changes in Carbohydrate Content of T. suecica (a) and T. tetrathele (b) by Wavelength Species Wavelength Carbohydrate content
(pg / cell) T. suecica Fluorescent lamp 4.63 ± 0.8 Blue LED 3.80 ± 3.0 Yellow LED 6.15 ± 2.8 Red LED 1.70 ± 2.0 T. tetrathele Fluorescent lamp 1.84 ± 0.2 Blue LED 2.43 ± 0.1 Yellow LED 2.35 ± 0.0 Red LED 1.73 ± 0.5

7 and Table 5 show the carbohydrate content of each wavelength of T. suecica (a) and T. tetrathele (b) and the carbohydrate content for fluorescent lamps, blue wavelengths, yellow wavelengths, and red wavelengths.

Referring to (a) of FIG. 7, T. suecica showed the highest concentration at the yellow wavelength, followed by fluorescent lamps, blue wavelengths, and red wavelengths. This confirmed that the carbohydrates showed the lowest growth rate in the red wavelength.

The yellow wavelength showed about 1.3 times higher carbohydrate content than the red wavelength which led to the growth of 1.3 times faster than the fluorescent lamp which is the existing mass culture light source. In addition, T. of suecica In order to know the carbohydrate ratio in the cell, it was obtained by referring to the contents of protein and lipid. As a result, the content of carbohydrates in the cells of T. suecica accounted for the lowest proportion compared to protein and lipid 4-9%, there were no significant differences across wavelengths.

Looking at Figure 7 (b) of T. tetrathele Carbohydrate content by wavelength showed the highest concentration at blue and yellow wavelengths, followed by fluorescent lamps and red wavelengths. This confirmed that both species of Tetraselmis had the lowest carbohydrates in the fast growing red wavelength. When comparing the carbohydrate content of the fluorescent lamp, which is a light source used in the existing mass culture, with the yellow wavelength, the yellow wavelength showed 1.3 times higher content.

When compared with the red wavelength, which showed the fastest growth, the yellow wavelength showed 1.4 times higher content. In addition, the carbohydrate ratio in the cell was obtained by referring to the protein and lipid content. As a result, the carbohydrate content was about 3-6% in the cell, and there was no significant difference by wavelength. Comparing the two experimental species, both the fluorescent lamp, which is a mass culture light source, and the yellow wavelength with high accumulation showed high contents in T. suecica .

According to the experimental results, T. suecica and T. tetrathele showed the highest carbohydrate content in the slow yellow wavelengths and the lowest concentration in the red wavelengths.

This is because the decrease in the rate of cell division alters the chemical composition and enzyme activity of the cell along with protein synthesis, increasing lipid and carbohydrate content, resulting in a slow growth rate in yellow wavelengths and a high rate of carbohydrate content in yellow wavelengths. Judging.

In the fast growing red wavelength, the short time to accumulate carbohydrate is useful in promoting the growth of the Tetraselmis genus, but the synthesis of carbohydrates is derived efficiently from the yellow wavelength.

In the Tetraselmis genus according to the present experimental example, carbohydrates accounted for 3-9 % of the total biochemical ratio. The microalgae used for carbohydrate extraction had the highest percentage of carbohydrates in biochemical composition. The relatively low proportion of carbohydrates in Tetraselmis seems to be difficult to commercially use.

However, when used as a food organism, it showed higher carbohydrate accumulation than the fluorescent lamp which is a conventional mass culture light source. This is thought to be helpful for those who use carbohydrates in microalgae as energy sources.

Microalgae usually contain the highest percentage of protein in the cell. Most of the protein is in amino acid form. In order for microalgae to be used as a food organism, biochemical composition such as fat, carbohydrates and proteins is important, and the nutrition of the food has a great influence on the growth and survival rate of floating larvae.

In addition, because protein and fat are used for biosynthesis in living organisms, it is important to understand the content of microalgae. The biochemical composition of the microalgae may vary depending on light conditions, and research on this has been actively conducted.

Wavelength-specific protein changes in the fluorescent lamp, the blue wavelength and yellow wavelength, a red wavelength of the T. suecica and T. tetrathele Species Wavelength Protein content
(pg / cell) T. suecica Fluorescent lamp 35.7 ± 0.2 Blue LED 24.4 ± 0.0 Yellow LED 42.8 ± 0.0 Red LED 28.6 ± 0.8 T. tetrathele Fluorescent lamp 17.1 ± 1.5 Blue LED 18.2 ± 0.2 Yellow LED 38.5 ± 3.8 Red LED 15.4 ± 1.3

FIG. 8 and Table 6 show changes in proteins for wavelengths of fluorescent lamps, blue wavelengths, yellow wavelengths, and red wavelengths of T. suecica and T. tetrathele .

Referring to (a) of FIG. 8, T. suecica showed the highest concentration at the yellow wavelength, followed by fluorescent lamps, red wavelengths, and blue wavelengths. Through this, it was confirmed that the highest protein content was observed in the yellow wavelength, which was the slowest growth.

The yellow wavelength showed about 1.2 times higher protein content than the red wavelength which led to the rapid growth, which is 1.2 times higher than that of the fluorescent lamp, which is a mass culture light source. In addition, T. of suecica In order to know the protein ratio in the cell, it was obtained by referring to the carbohydrate and lipid content. As a result, the protein content of T. suecica was the highest at 48 ～ 69%. Comparing the two experimental species, both the fluorescent lamp, which is a mass culture light source, and the yellow wavelength with high accumulation showed high contents in T. suecica .

Looking at (b) of Table 8, T. tetrathele Protein content of each wavelength was highest in blue and yellow wavelengths, followed by blue, fluorescent and red wavelengths.

It was confirmed that both Tetraselmis two kinds of growth is likely the highest protein content in Cannes noticed the yellow wavelengths. When comparing the protein content of the fluorescent lamp, which is a light source used in the existing mass culture, and the yellow wavelength, the yellow wavelength showed 2.3 times higher content.

When compared with the red wavelength, which showed the fastest growth, the yellow wavelength was 2.5 times higher. In addition, it was obtained by referring to the carbohydrate and lipid content in order to know the protein ratio in the cell. As a result, the protein content in the cell was about 42-56%, which was high.

The change of protein with wavelength of T. suecica and T. tetrathele showed high protein accumulation at yellow wavelength, which was the slowest growth. The reason for this is that, as described above, the decrease of cell division in the yellow wavelength is caused by Tetraselmis. This may be due to the high protein content induced by stress in the stomach.

Carbohydrate experiments also used several wavelengths to accumulate, but as mentioned in the pigment results, the yellowing gave a high stress due to the light stress. In addition, it is thought that the time for inducing protein synthesis was short in red wavelengths with high cell division rate. Thus Tetraselmis The red wavelength is useful for the growth of the genus, but the protein content is thought to be efficiently accumulated in the yellow wavelength.

The fat content of the food organism plays an important role in the growth of marine fish. Intracellular lipids of microalgae do not always have a constant content, but because they can be changed by the culture environment, such as water temperature, salinity, nutrient status and light, research on this is being actively conducted.

Of T. suecica and T. tetrathele Lipid content by wavelength Species Wavelength Lipid content
(pg / cell) T. suecica Fluorescent lamp 11.3 ± 1.3 Blue LED 20.3 ± 1.3 Yellow LED 39.4 ± 0.4 Red LED 13.0 ± 2.6 T. tetrathele Fluorescent lamp 15.6 ± 0.4 Blue LED 22.7 ± 4.3 Yellow LED 28.0 ± 1.4 Red LED 15.9 ± 0.2

9 and Table 7 show T. suecica and T. tetrathele. It shows the lipid content of each wavelength for fluorescent lamp, blue wavelength, yellow wavelength and red wavelength.

Referring to (a) of FIG. 9, T. suecica showed the highest concentration at yellow wavelength, followed by blue wavelength, red wavelength, and fluorescent lamps. Through this, it was confirmed that the highest lipid content was observed in the yellow wavelength, which was the slowest growth.

When comparing the lipid content of the fluorescent lamp, which is a light source used in the existing mass culture, with the yellow wavelength, the yellow wavelength showed about 3.5 times higher content. The yellow wavelength was about 3.0 times higher than the red wavelength, which was the fastest growing. In addition, T. of suecica In order to know the ratio of lipids in the cells, the carbohydrate and protein contents were referred to. As a result, the lipid content in the cells of T. suecica was 22-45 %, which was the second highest after protein.

Looking at Figure 9 (b) of T. tetrathele The protein content of each wavelength showed the highest concentration at yellow wavelength, followed by blue wavelength, red wavelength, and fluorescent lamp. It was confirmed that both species of Tetraselmis had the highest lipid content at the slow yellow wavelength.

When comparing the lipid content of the fluorescent lamp, which is a light source used in the existing mass culture, with the yellow wavelength, the yellow wavelength showed about 1.8 times higher content. Compared to the red wavelength, which was the fastest growing, it was about 1.8 times higher in the yellow wavelength. In addition, it was obtained by referring to the carbohydrate and protein content to know the ratio of intracellular lipids. As a result, the lipid content in the cells accounted for a high rate of about 42-56%.

As a result of this experiment, it was confirmed that T. suecica and T. tetrathele showed high lipid content in yellow wavelength, which was the slowest growth. Similar results were found in Chlorella sp., Which showed high growth in red and slow growth in blue.

Reduction of cell division rate is accompanied by protein synthesis and changes in cell chemical composition and enzyme activity to increase lipid and carbohydrate content, and the enhancement of lipid to microalgae is induced by salinity and nitrogen deficiency.

Experimental Example of the present invention was confirmed that the lipid accumulates due to stress even in the light wavelength that can be easily adjusted in place of environmental factors such as salts difficult to change the conditions. In addition, more effective lipid accumulation may be possible by applying a combination of stressing environmental conditions to the microalgae.

The lipid change according to the wavelength of T. suecica and T. tetrathele according to the embodiment of the present invention was confirmed to have a high lipid accumulation in the yellow wavelength of the slowest growth. The reason for this is that, as mentioned above, the decrease of cell division in the yellow wavelength increases the lipid and carbohydrate content by changing the chemical composition and enzyme activity of the cell along with protein synthesis.

In addition, because of the rapid cell division in the red wavelength, relatively low lipid accumulation was induced. Thus Tetraselmis When using the red wavelength as a light source, high growth rate can be expected. The lipid content is similar to the fluorescent lamp, which is a conventional mass culture light source.

In the mass culture of Tetraselmis genus, it was confirmed that there is a difference between a light source for promoting growth and a light source for enhancing the biochemical composition, and it is appropriate that the culture step for promoting growth and the step for cultivating biochemical composition are performed step by step.

The growth rate according to the amount of light according to the experimental example of the present invention was confirmed that the growth rate is fast in the red wavelength, but the light affinity is high in the yellow wavelength. This means that yellow wavelength may be advantageous for growth induction. In other words, it is appropriate to incubate growth in the induction phase in the early stages of algebraic growth during the induction phase, and to promote growth through red wavelengths from the late phase of the algebraic growth, and to intensify nutrients using the yellow wavelength in the stop phase.

2-3 Results of nutrient absorption in the genus Tetraselmis

Among the nutrients essential for the growth of microalgae, phosphorus and nitrogen can be limited by the environment. In general, the use of nutrients of microalgae varies with light conditions, but the rate of absorption increases with the increase of the amount of light, but it is known that the absorption of nutrients also increases under conditions where the growth rate is enhanced and the absorption rate is similar. have.

However, as the growth rate increases, the rate of nutrient absorption decreases or species-specific growth rate and absorption rate are unrelated, and there is a range of nutrient concentrations showing optimum growth for each nutrient. For example, the nutrient absorption rate according to the wavelength was confirmed by using Michaelis-Menten equation to determine the nutrient absorption capacity of Tetraselmis .

Figure 10 shows the change in ammonia nitrogen concentration of the culture medium according to the fluorescent lamp, blue wavelength, yellow wavelength, red wavelength of T. suecica and T. tetrathele . As a result, the absorption rate of ammonia over time for each wavelength is started culture (10 min.) From was a tendency that the absorption rate significantly lower after showing a constant-speed-absorption to a certain time (t -test, p <0.05) .

T. suecica was the longest at 30 minutes in yellow wavelength and 20 minutes in fluorescent, red, and blue wavelengths. T. tetrathele showed the same 10 minutes at all wavelengths except yellow wavelength and 20 minutes at yellow wavelength. The rate of constant absorption by wavelength was the highest at 2.75 ～ 3.49 pmol / cell / hr in red wavelength, followed by fluorescent lamp, blue wavelength and yellow wavelength.

Constant Velocity Absorption Time of T. suecica and T. tetrathele by Wavelength Species Wavelength Nutrient Incubation time (min) Uptake rate
(pmol / cell / hr) Surge uptake time (min) T. suecica Fluorescent lamp Ammonium 0 to 20 2.17 ～ 3.17 20 20 to 240 0.0 to 0.40 Blue LED 0 to 20 1.74 ～ 2.23 20 20 to 240 0.01 to 0.36 Yellow LED 0 to 30 1.74 ～ 2.40 30 30 to 240 0.00 ～ 0.36 Red LED 0 to 20 2.75 ～ 3.49 20 20 to 240 0.03 to 0.54 T. tetrathele Fluorescent lamp Ammonium 0 to 10 4.88 10 10 to 240 0.00 ～ 1.03 Blue LED 0 to 10 3.72 10 10 to 240 0.02 to 0.80 Yellow LED 0 to 10 1.34 ～ 3.05 20 10 to 240 0.03 ～ 0.71 Red LED 0 to 10 4.50 10 10 to 240 0.03 to 0.46

Table 8 shows the times of constant speed absorption of T. suecica and T. tetrathele by wavelength. Based on the results, the constant speed absorption time was selected as the culture time for the ammonia absorption experiment, and the absorption rate experiment was performed according to the concentration of ammonia nitrogen for each wavelength to be described later.

In this experiment, constant speed absorption time was determined in ammonia nitrogen of T. suecica and T. tetrathele . As a result, T. suecica showed 20 minutes except yellow wavelength of 30 minutes, and T. tetrathele was 20 minutes at yellow wavelength and 10 minutes at the remaining wavelength. In the constant absorption time, the absorption rate of T. suecica was higher in red wavelength and T. tetrathele in fluorescent lamp and red wavelength.

Absorption rate within the constant speed absorption time according to the experimental example of the present invention was consistent with the results of the growth rate according to the wavelength of the previous field, it was confirmed that the absorption of ammonia nitrogen also increased at a faster wavelength growth rate.

Figure 11 shows the phosphate concentration change in the culture medium according to the fluorescent lamp, blue wavelength, yellow wavelength, red wavelength of T. suecica and T. tetrathele . As a result, the absorption rate of phosphorus phosphate with time for each wavelength showed a constant rate of absorption from the start of the culture (0 min) to a certain time, and then the absorption rate was significantly decreased ( t -test, p <0.05).

T. suecica showed 20 minutes of constant absorption time at all wavelengths. T. tetrathele was the shortest for 10 minutes in fluorescent and red wavelengths, and 20 minutes in blue and yellow wavelengths. As a result of the experimental example according to the present invention, it was confirmed that T. suecica and T. tetrathele were also short in the constant absorption of nutrients, which was faster at faster wavelengths.

Constant Velocity Absorption of Phosphoric Acid by Wavelength Species Wavelength Nutrient Incubation time (min) Uptake rate
(pmol / cell / hr) Surge uptake time (min) T. suecica Fluorescent lamp Phosphate 0 to 20 0.12 to 0.13 20 20 to 240 0.01 to 0.08 Blue LED 0 to 20 0.12 20 20 to 240 0.01 to 0.06 Yellow LED 0 to 30 0.12 20 30 to 240 0.01 to 0.04 Red LED 0 to 20 0.12 to 0.14 20 20 to 240 0.01 to 0.06 T. tetrathele Fluorescent lamp Phosphate 0 to 10 0.20 10 10 to 240 0.01 to 0.08 Blue LED 0 to 20 0.14 to 0.18 20 20 to 240 0.00 ～ 0.07 Yellow LED 0 to 20 0.15 to 0.17 20 20 to 240 0.01 to 0.08 Red LED 0 to 10 0.23 10 10 to 240 0.02 to 0.12

Table 9 shows the constant speed absorption of each phosphorus phosphate. Based on these results, the time of constant absorption was selected as the incubation time for phosphate absorption experiment.

In the experimental example of the present invention, the constant speed absorption time was determined in the phosphorus phosphate of T. suecica and T. tetrathele . As a result, all of T. suecica showed 20 minutes, and T. tetrathele showed 10 minutes in fluorescent and red wavelengths, and 20 minutes in blue and yellow wavelengths. Absorption rate within the constant absorption time was higher in T. suecica with red wavelength and T. tetrathele with fluorescent lamp and red wavelength.

Looking at the experimental example, the absorption rate in the constant speed absorption time was consistent with the results of the growth rate according to the wavelength of the preceding field, it was confirmed that the absorption to the phosphorus phosphate also increased at a faster wavelength growth rate. This means rapid growth and absorption of nutrients in mass culture, which can increase efficiency in mass culture in a short period of time.

The nutrient absorption rate of microalgae varies according to the concentration of nutrients. In general, the rate of absorption increases with the increase of nutrient concentration, as the growth is promoted by the increase of light quantity. However, as the high light quantity causes photoinhibition, the absorption limit may be rather high at a high concentration of nutrients or the absorption rate may be similar at a certain concentration or higher.

In addition, the nutrient absorption rate for the microalgae should be understood to know the nutrient conditions required for microalgae culture. This can minimize the addition of nutrients for cultivation, thereby reducing the cost of mass culture.

Therefore, in the following experimental example, the absorption rate of the T. suecica and T. tetrathele was measured by using ammonia nitrogen and phosphorus phosphate to determine the nutrient absorption capacity. Incubation time was set to the time when the constant absorption was shown in the laboratory example.

12 shows the ammonia nitrogen absorption process of the microalgae. Microalgae are known to have a "preference" mechanism that consumes ammonia nitrogen from a nitrogen source and then consumes nitrogen nitrate. Since this 'preference' mechanism is advantageous in terms of energy use compared to nitrate nitrogen, microalgae can use a source of nitrogen in the form of ammonium ion in the body. The use of nitrogen nitrate requires a lot of energy while undergoing a reduction process through two stages of enzymatic reactions, so microalgae prefer ammonia nitrogen as the nitrogen source.

Therefore, the following experimental example was determined to absorb the nitrogen source with ammonia nitrogen instead of nitrate nitrogen. In the concentration section, nitrogen and phosphorus depleted cell solution (about 1 × 10 ⁴ cells / mL) was inoculated into L1 medium prepared in 7 steps of 1, 5, 10, 20, 50, 70, and 100 μM, respectively.

P _max (maximum absorption rate) and Ks (half saturation constant) for the wavelengths of T. suecica and T. tetrathele derived from Michaelis-Menten equation Species Wavelength Ks (μM) p _max
(pmol / cell / hr) T. suecica Fluorescent lamp 11.5 5.65 Blue LED 16.6 5.78 Yellow LED 15.5 5.15 Red LED 9.43 6.35 T. tetrathele Fluorescent lamp 26.1 9.88 Blue LED 26.3 6.46 Yellow LED 32.2 5.71 Red LED 21.2 9.85

Table 10 shows p _max (maximum absorption rate) and Ks ( half saturation constant) for each wavelength of T. suecica and T. tetrathele derived from the Michaelis-Menten equation. The p _max was the highest in the red wavelength, followed by the blue, fluorescent and yellow wavelengths. Figure 13 shows the concentration change of ammonia nitrogen in the culture medium according to the wavelength of T. suecica and T. tetrathele depleted nitrogen source.

Figure 13 (a) is the concentration change of ammonia nitrogen in the culture medium according to the time of wavelength of T. suecica depleted nitrogen source. As a result, the absorption rate also increased as the ammonia nitrogen concentration increased to 50 μM at all wavelengths.

Looking at p _max (maximum absorption rate) and Ks ( half saturation constant) of T. suecica derived from Michaelis-Menten equation of Table 10, Ks is a measure of affinity for nutrients. It shows favorable characteristics for growth under nutrient condition. Ks had a red wavelength of about 1.5 times lower than the average of the other wavelengths, and the other wavelengths ranged from 11.5 to 15.5 μM (average 14.5 ± 2.68 μM).

Figure 13 (b) is a change in the concentration of ammonia nitrogen in the culture medium according to the time of wavelength of T. tetrathele depleted nitrogen source. As a result, the absorption rate also increased as the ammonia nitrogen concentration increased to 70 μM at all wavelengths, and showed a similar tendency at higher concentrations.

Table 10 of the Michaelis-Menten equation derived T. tetrathele the wavelengths of p _max (maximum absorption rate), Ks Referring to (half picture number), in the case of p _max indicates the maximum absorption rate was highest in the fluorescent lamp and the red wavelength , Blue wavelength, yellow wavelength. Ks, the affinity index for limiting nutrients, was lower in order of red wavelength, fluorescent lamp, blue wavelength, and yellow wavelength.

When comparing the two kinds of experiments, p _max is shown as the average 5.73 ± 0.49 pmol / cell / hr , T. tetrathele average of 7.98 ± 2.20 pmol / cell / hr in T. suecica, was about 1.4 times the T. tetrathele. For Ks, show an average 13.3 ± 3.36 μM, T. average in tetrathele 26.5 ± 4.50 μM at T. suecica, T. tetrathele is about 2.0 times higher.

As a result of this experiment, T. suecica and T. tetrathele also had high absorption rate of ammonia nitrogen in red wavelengths which grew rapidly. Thus Tetraselmis The genus is thought to be effective in promoting nutrient absorption and growth growth when mass culture in red wavelength.

P _max , Ks by wavelength of T. suecica and T. tetrathele derived from Michaelis - Menten equation Species Wavelength Ks (μM) p _max
(pmol / cell / hr) T. suecica Fluorescent lamp 0.89 0.28 Blue LED 0.94 0.22 Yellow LED 1.27 0.18 Red LED 0.98 0.29 T. tetrathele Fluorescent lamp 1.00 0.30 Blue LED 1.15 0.23 Yellow LED 1.11 0.11 Red LED 1.07 0.33

In water, phosphorus is divided into dissolved inorganic, dissolved organic phosphorus and suspended particulate organic phosphorus. Microalgae use a variety of nitrogen sources, but in the case of phosphorus it only absorbs phosphorus. If the concentration of phosphorus phosphate in the water is sufficient, the microalgae are absorbed (luxury consumption) and stored and used in the cell as a polyphosphate compound.

Therefore, phosphoric phosphate was selected for the absorption rate experiment for the personnel in this experimental example. In the concentration section, nitrogen and phosphorus depleted cell solution (about 1 × 10 ⁴ cells / mL) were inoculated into L1 medium prepared in seven steps of 0.1, 0.5, 1.0, 3.0, 5.0, 10, and 20 μM, respectively.

Table 11 shows p _max and Ks of wavelengths of T. suecica and T. tetrathele derived from Michaelis-Menten equation. Figure 14 shows the change in the concentration of phosphorus phosphate in the culture medium according to the wavelength of T. suecica and T. tetrathele depleted by personnel.

Figure 14 (a) is the concentration change of phosphorus phosphate in the culture medium according to the time of wavelength of T. suecica depleted by personnel. As a result, the absorption rate also increased as phosphorus phosphate concentration increased up to 5 μM at all wavelengths.

Table 11 of the Michaelis-Menten equation deriving the T. suecica wavelengths of p _max, referring to Ks, for p _max indicates the maximum absorption rate was the highest at red wavelength, appeared as a fluorescent lamp, a blue wavelength and yellow wavelength order. Ks was the highest at 1.27 μM in yellow wavelength and 0.89∼ 0.98 μM (average 0.94 ± 0.05 μM) at the remaining wavelength.

Figure 14 (b) is a change in the concentration of phosphorus phosphate in the culture medium according to the wavelength of the depleted T. tetrathele time. As a result, the absorption rate also increased as phosphorus phosphate concentration increased up to 5 μM at all wavelengths.

Table 11 of the Michaelis-Menten equation derived T. tetrathele the wavelengths of p _max, referring to Ks, for p _max indicates the maximum absorption rate was the highest at red wavelength, appeared as a fluorescent lamp, a blue wavelength and yellow wavelength order.

Ks, the affinity index for limiting nutrients, was lower in order of fluorescent lamp, red wavelength, yellow wavelength, and blue wavelength. When comparing the two kinds of experiments, p _max is showed on average 0.24 ± 0.05 pmol / cell / hr , average 0.24 ± 0.10 pmol / cell / hr in T. tetrathele in T. suecica, Ks average at 1.02 T. suecica The average of 1.08 ± 0.06 μM in ± 0.17 μM and T. tetrathele showed similar results.

As a result of this experimental example, it was confirmed that T. suecica and T. tetrathele also had high absorption rate of phosphorus phosphate in red wavelengths which grew rapidly. P _max in the genus Tetraselmis , compared to other species, Halampora veneta with Achnanthes It was higher than lanceolata . In addition, the benthic microalgae Navicula minima and Nitzschia It has a higher affinity for phosphate than microcephala . Thus Tetraselmis The genus is thought to be able to grow well even under low phosphate conditions in mass culture.

The half saturation constant Ks provides the nutrient concentrations required for microalgae cultivation. In other words, the culture of Tetraselmis genus requires 11.5 to 21.2 μM of nitrogen source and 0.89 to 1.00 μM of phosphorus based on fluorescent lamps. This needs to be taken into account as the required nutrient concentrations vary depending on the LED wavelength.

In the red wavelengths, Ks value for fast absorption rate and the required nutrient concentration was low, but the relatively low absorption rate and high Ks value in yellow wavelengths could be used to identify the nutrients needed for mass cultivation in Tetraselmis .

Fig. 15 shows the Tetraselmis-tipped green steel Tetraselmis using the light quantity and wavelength of the light emitting diode proposed from the present invention. suecica and Tetraselmis The method of mass culturing tetrathele is shown. From the results of the present invention, green algae Tetraselmis suecica and Tetraselmis For the efficient mass cultivation of tetrathele , cultivation should be carried out step by step to promote growth as an initial culture and to enhance nutrition by a later culture. In the existing studies, two-stage cultures have been proposed that do not consider growth according to the amount of light, but in this study, three-stage cultures were found to be effective. That is, the induction phase, which is the early stage of cultivation, should be irradiated with yellow wavelength, which showed low compensation and semi-saturated intensity, and the red wavelength should be used to promote growth in algebraic growth period, and then the yellow wavelength should be irradiated to enhance nutritional substances.

Among the microalgae that are widely used in various fields, microalgae can be grown in a short period of time by controlling the wavelength and quantity of light-emitting diodes in the Tetraselmis, which are easy to mass-cultivate, and controlling light. There is industrial applicability in relation to this.

Claims (9)

The growth stages of the microalga Tetraselmis tetrathele are classified into induction stage, algebraic growth stage, and nutrient reinforcement, and the wavelengths of the light emitting diodes are changed at each stage.
During the growth phase of the microalgae, the inducer irradiates a light emitting diode light source having a wavelength of 590 nm yellow light, the logarithmic organ irradiates a light emitting diode light source having a red light wavelength of 620 nm to 680 nm, and the nutrient enhancer has a yellow light wavelength of 590 nm. Incubated by irradiating the light emitting diode light source of
The algebraic growth unit adds a nitrogen source at a concentration of 21.2 μM and a personnel of 1.07 μM under light source conditions of red light wavelength;
The nutritional substance enhancer is a large-scale cultivation method of Tetraselmis tetrathele of light green algae microorganisms using a light emitting diode, characterized in that the cultivation by adding a nitrogen source of 32.2 μM, personnel in a concentration of 1.27 μM under light source conditions of the yellow light wavelength. delete delete delete delete delete delete delete delete

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