WO2018043904A1 - Microalgae cell stimulating apparatus and method for increasing lipid production of microalgae using same - Google Patents

Abstract

The present invention relates to a microalgae cell stimulating apparatus and a method for increasing the lipid production of microalgae using the microalgae cell stimulating apparatus. When the microalgae cell stimulating apparatus according to the present invention is used to apply pressure to polyurethane beads in the microalgae cell stimulating apparatus, the restoring force of the polyurethane beads and the contact between adjacent polyurethane beads cause a chain of force dispersion events, thereby continuously applying a mechanical stimulus to the microalgae cells in the apparatus. Therefore, the present invention does not require any other external stimulus and thus is very efficient in view of energy, and does not give any chemical stimulus and thus acts stably on microalgae cells. Especially, the microalgae cell stimulating apparatus of the present invention has an effect of increasing the lipid production of the microalgae cells that have undergone a mechanical stimulus, and thus can be advantageously used in an environmentally friendly biodiesel production method.

Description

Microalgae cell stimulation device and method for increasing lipid production of microalgae using the same

The present invention relates to a microalgae cell stimulation device and a method for increasing lipid production of microalgae using the microalgae cell stimulation device.

Microalgae are single cell algae up to 50 μm in size. Because it is located at the lowest level in the natural food chain, it is an independent nutrient that produces organic matter through photosynthesis. Depending on the species, they can generally be divided into cell walls, which contain a lot of fiber, and internal extracts, which are filled with substances containing various sugars. The lipids contained in some species are very similar to vegetable oils and are very suitable for making them into biofuels, with some species reaching up to 80% of the dry matter. Microalgae can be mass cultured automatically in closed production systems such as culture facilities. In addition, in order to obtain lipids of the microalgae, culture conditions or systems are very important.

In conventional culture systems using large-capacity mechanical stress, a method using an impeller string has been used. Recently, large-scale mechanical stimulation systems have been developed in various ways such as compressive loading systems, longitudinal stretch systems and substrate distention systems. However, in the case of string, impeller rotational power consumption for fluid stress is high, which is inefficient in terms of energy, and the degree of stimulation (decreased stimulus intensity away from the impeller) according to the position inside the reactor is decreased. Because they are different, they have the limitation of not giving a spatially constant stress. In addition, in the case of the longitudinal elongation system or matrix expansion system, it has a limitation that it is applied only to adherent cells, and because it is a two-dimensional area unit stimulation method, the spatial inefficiency of high spatial consumption in order to stimulate a large amount of cells Have.

In order to solve the above problems, the present inventors while studying a method of increasing the lipid production of microalgae using a culture system using mechanical stimulation, microalgae to give mechanical stimulation to microalgae using polyurethane beads A cell stimulation device was constructed. When the pressure is applied to the elastomeric polymer beads and the compressed elastomeric polymer beads are restored by the apparatus, and mechanical stimulation is applied to the cells of the microalgae, it was confirmed that the lipid production of the microalgae is increased, thereby completing the present invention.

It is an object of the present invention to provide a microalgal cell stimulation device.

It is also an object of the present invention to provide a method for increasing lipid production of microalgae.

It is also an object of the present invention to provide a method for producing microalgae with increased lipid production.

It is also an object of the present invention to provide a microalgae in which lipid production is increased by the production method.

In order to solve the above problems, the present invention is an outlet is formed on one side, the stimulation tank having a filter formed to open and close the outlet; A stimulator having a stimulator head having a lateral movement in contact with an inner circumferential surface of the stimulation reaction tank to move up and down inside the stimulation reaction tank; A pump for injecting a solution into the stimulation tank or sucking and discharging the solution in the stimulation tank; And an elastomeric polymer bead accommodated in the stimulation reaction tank, and the stimulator head provides a microalgae cell stimulation device, characterized in that the nozzle is permeated with the solution.

In addition, the present invention, (a) compressing the elastomeric polymer beads using a stimulator of the microalgal cell stimulation apparatus; (b) injecting the microalgal cells into the inlet of the microalgal cell stimulation device; (c) synergizing the stimulator of the microalgal cell stimulation apparatus and then applying mechanical stimulation to the microalgal cells while restoring the elastomeric polymer beads; And (d) recovering the microalgae cells in a recovery tank after opening the filter of the microalgae cell stimulation device and lowering the stimulator, thereby providing a method for increasing lipid production of microalgae.

In addition, the present invention, (a) compressing the elastomeric polymer beads using a stimulator of the microalgal cell stimulation apparatus; (b) injecting the microalgal cells into the inlet of the microalgal cell stimulation device; (c) synergizing the stimulator of the microalgal cell stimulation apparatus and then applying mechanical stimulation to the microalgal cells while restoring the elastomeric polymer beads; And (d) recovering the microalgae cells in a recovery tank after opening the filter of the microalgae cell stimulation device and lowering the stimulator, thereby providing a microalgae with increased lipid production.

In another aspect, the present invention provides a microalgae with increased lipid production by the production method.

When pressure is applied to the elastomeric polymer beads in the microalgae cell stimulation apparatus using the microalgae cell stimulation apparatus according to the present invention, force dispersion occurs in series due to the restoring force of the elastomeric polymer beads and the contact between adjacent elastomeric polymer beads. It continuously exerts mechanical stimuli on the microalgal cells in the device. Therefore, the microalgae cell stimulation device according to the present invention does not require any external stimulation, and thus is very efficient in terms of energy, and does not give chemical stimulation, and thus has an advantage of stably acting on microalgal cells. In particular, the microalgal cell stimulation apparatus of the present invention has an effect of increasing the lipid production of microalgal cells subjected to mechanical stimulation, it can be usefully used in environmentally friendly biodiesel production method.

1 is a schematic diagram of a cell stimulation device according to a preferred embodiment of the present invention.

Figure 2a is a schematic of the process of increasing lipid production of stimulated Chlamydomonas Reinhardi. 2B is a diagram schematically illustrating a compression step, a de-compression step, and a recovery step of the cell stimulation device of the present invention.

Figure 3a is a view showing the shape of the polyurethane beads according to the polyurethane content (%) (w / v). 3 is a view showing the diameter of the beads according to the polyurethane content (%) (w / v). Figure 3c is a view showing the surface and cross-sectional shape of the 30% (w / v) polyurethane beads.

Figure 4a is a diagram confirming the degree of restoring force of the polyurethane beads according to the number of stimulation. Figure 4b is a diagram showing the result of confirming the absorbance of the liquid inside the beads to check whether the cells penetrated into the inside of the beads stimulated Chlamydomonas Reinharti.

5 a and b is a diagram confirming the calcium influx of the control and stimulated Chlamydomonas Reinharti. Figure 5c is a diagram confirming the cell size of Chlamydomonas Reinharti 12 hours and 24 hours of stimulation. 5d is a diagram confirming the mRNAs expression level of Mat3 and E2F1 of the control and stimulated Chlamydomonas Reinharti.

Figure 6 a and b is a diagram confirming the increase in fat granules of Chlamydomonas Reinharti with stimulation time and pressure.

Figure 7a is a diagram confirming the amount of intracellular lipid of a single cell with stimulation time. 7b is a diagram showing the degree of lipid granulation synthesis of total cells (black bar means control, gray bar means stimulated cells).

Figure 8 is a diagram showing the results of analyzing the lipid content of the control and stimulated Chlamydomonas Reinharti.

9 is a diagram confirming the mRNA expression level of ACCase, DGAT and LPAAT of Chlamydomonas Reinharti compressed and de-compressed over each time condition compared to the control group.

10A is a diagram schematically illustrating the conditions of direct stimulation of Chlamydomonas Reinharti cells. Figure 10b is a diagram showing the appearance of the cells by direct stimulation.

11 is a diagram showing the percentage of apoptosis of the compressed process and de-compressed Chlamydomonas Rainharti according to each time condition compared to the control.

In one aspect, the present invention is an outlet is formed on one side, the stimulation tank having a filter formed to open and close the outlet; A stimulator having a stimulator head having a lateral movement in contact with an inner circumferential surface of the stimulation reaction tank to move up and down inside the stimulation reaction tank; A pump for injecting a solution into the stimulation tank or sucking and discharging the solution in the stimulation tank; And an elastomeric polymer bead accommodated in the stimulation reaction tank, and the stimulator head provides a microalgal cell stimulation device, wherein a nozzle through which the solution is permeated is connected.

1 is a schematic diagram of a cell stimulation device according to a preferred embodiment of the present invention.

Referring to Figure 1, the microalgal cell stimulation apparatus 100 according to a preferred embodiment of the present invention will be described.

Microalgal cell stimulation apparatus 100 according to a preferred embodiment of the present invention is a stimulation reaction tank 110, stimulator 120, pump 130, supply tank 140, recovery tank 150 and elastomeric polymer beads ( 160).

Stimulus reaction tank 110 has an outlet is formed on one side, and has a filter 112 formed to open and close the outlet. The stimulation reaction tank 110 is formed in a straight tube shape, such as cylindrical.

The filter 112 is disposed at the outlet of the stimulation tank 110 to open and close the inside of the stimulation tank 110, the top of which is closed by the stimulator 120. The filter 112 may be implemented as a stainless steel membrane filter, the pore size of the filter 112 is formed to 800㎛ ~ 1200㎛ to filter the elastomeric polymer beads 160 accommodated inside the stimulation tank 110 Make it work.

Stimulator 120 is provided with a stimulator head 122 at one end thereof in contact with the inner circumferential surface of the stimulation reaction tank 110 to move up and down inside the stimulation reaction tank (110).

The stimulator head 122 is connected to a nozzle 132 through which a solution such as a culture solution for culturing microalgae cells C is performed to perform a filter role. The pore size of the nozzle 132 is 3 μm to 10 μm. It is formed to prevent the microalgae cells (C) to leak out of the stimulation reaction tank 110 together with the solution discharged during the discharge of the solution using the pump 130.

In addition, an inlet 126 for injecting and inoculating microalgae cells C into the stimulator head 122 in the inner space of the stimulation tank 110 and the stimulation tank 110 surrounded by the stimulator head 122. Is provided.

The pump 130 injects a solution such as a culture solution into the stimulation reaction tank 110 or inhales the solution in the stimulation reaction tank 110 and discharges it into the supply tank 140.

At this time, the pump 130 is connected to the tube 134 is formed so that the solution flows therein, the nozzle 132 is coupled to one end of the tube 134 facing the stimulation reaction tank 110.

At this time, the nozzle 132 is provided so that the other side that is not coupled to the tube 134 is connected to the stimulator head 122 in a state in which the stimulator head 122 is raised.

The supply tank 140 is discharged from the stimulation tank 110 by the pump 130, or the solution supplied to the stimulation tank 110 is stored.

The recovery tank 150 is a portion in which the microalgal cells C discharged through the filter 112 together with the solution in the opened state of the filter 112 are accommodated.

The elastomeric polymer bead 160 is formed of a material having elasticity and is accommodated inside the stimulation tank 110 to be deformed under pressure by the stimulator head 122 while being contained in the microalgae cells (110) It acts as a physical stimulus to C).

The elastomeric polymer beads may be prepared using a material having elasticity, and may preferably be microbeads, which are porous elastomeric polymers having porosity. In the case of using the porous elastomer polymer, the media is contained in the porous structure, and when the compression is carried out, the media is released by the intrinsic elastic properties, thereby inhibiting cell death. The elastic material usable in the present invention may be selected without limitation from elastic materials known in the art, and may be selected from nylon sponges, polyurethanes, porous synthetic resins, rubbers, and silicones. The porous elastomer polymer may be a biodegradable polymer having elasticity and may be poly (L-lactide-co-ε-caprolactone) (Poly (L-lactide-co-ε-caprolactone)), polylactic acid (PLA), or polyglycol. Acid (PGA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, L-lactide-coglycolide); PLGA), poly (caprolactone), diol / diacid based aliphatic polyester, polyester One or more synthetic polymers selected from the group consisting of -amide / polyester-urethane, polyurethane, polyethylene oxide, poly (valerolactone), poly (hydroxybutyrate) and poly (hydroxy valerate) Or chitosan, chitin, alginic acid, collagen, gelatin and hyaluronic acid can be prepared using any one or more polymers of at least one natural polymer selected from the group consisting of. In a preferred embodiment of the present invention discloses an example using polyurethane as an elastic material having a porosity.

In the present invention, the term "polyurethane" is a generic term for a high molecular compound bonded by a urethane bond made of a combination of an alcohol group and an isocyanic acid group. Polyurethane is elastic, firm and light because it has a foam structure. In addition, it has good resistance to chemicals and has good elasticity, and is also used as a substitute for rubber.

In one preferred embodiment of the present invention, the polyurethane beads may comprise polyurethane at a concentration of 10 to 50% (w / v), more preferably at a concentration of 25 to 30% (w / v) polyurethane. It is a bead.

2 is a view for explaining a process of applying a stimulation to the cell using a cell stimulation device according to a preferred embodiment of the present invention.

Hereinafter, a process of applying physical stimulation to a cell using the cell stimulation device 100 of the present invention described above with reference to FIG.

First, as shown in ① of FIG. 2, the stimulation reaction tank 110 is filled with a plurality of elastomer polymer beads 160 and a solution such as a culture solution, and the filter 112 is in a closed state (OFF). 110) Do not let the internal fillings leak to the outside.

Then, as shown in ② of FIG. 2, in the state in which the closed state (OFF) of the filter 112 is maintained, the stimulator 120 is lowered to compress the elastomeric polymer beads 160, and the inside of the stimulation tank 110 The solution is discharged to the upper space of the stimulator head 122 through the nozzle 132 is moved to the supply tank 140.

Thereafter, as shown in ③ of FIG. 2, the microalgae cells C are injected into the injection hole 126 while the closed state (OFF) of the filter 112 is maintained.

Then, as shown in ④ of FIG. 2, in the state in which the closed state (OFF) of the filter 112 is maintained, the stimulator 120 is raised to use the microalgae cells C by using the restoring force and elastic force of the elastomeric polymer bead 160. Mechanical stimulus). At this time, the solution of the supply tank 140 moves to the stimulation reaction tank 110 using the pump 130.

Thereafter, as shown in ⑤ of FIG. 2, while the open state (ON) of the filter 112 is maintained, the microalgal cells C subjected to mechanical stimulation are introduced into the recovery tank 150, and the elastomeric polymer beads ( The filters 160 are filtered by the filter 112 and remain inside the stimulation tank 110.

In addition, the present invention, (a) compressing the elastomeric polymer beads using a stimulator of the microalgal cell stimulation apparatus; (b) injecting the microalgal cells into the inlet of the microalgal cell stimulation device; (c) synergizing the stimulator of the microalgal cell stimulation apparatus and then applying mechanical stimulation to the microalgal cells while restoring the elastomeric polymer beads; And (d) recovering the microalgae cells in a recovery tank after opening the filter of the microalgae cell stimulation device and lowering the stimulator, thereby providing a method for increasing lipid production of microalgae.

The method of increasing lipid production of the microalgae includes a process of applying physical stimulation to cells using the stimulator of the cell stimulation device 100 of the present invention described above with reference to FIG. Description is omitted.

The term "mechanical stimulus" in the present invention means to cause a certain reaction by acting on the organism using a machine or a device, for example, in the present invention means a stimulus to restore the compressed elastomeric polymer to the cell.

The compression of (a) is 1 to 48 hours, but preferably 1 to 30 hours, more preferably 4 to 24 hours, but is not limited thereto.

The microalgae of the present invention may include without limitation various microalgae that can be induced metabolic changes through mechanical stimulation, specifically, when using the stimulation device of the present invention, the compression step, de-compression step and recovery In the case of mechanical stimulation through the step, membrane distortion may be induced, and thus, microalgae may be increased in the calcium inflow than non-stimulated cells. For example, the microalga is Chlamydomonas Reinharti(Chlamydomonas reinhardtii ), Chlamydomonas cardata (Chlamydomonas caudate), Chlamydomonas Moew( Chlamydomonas moewusii ), Chlamydomonas Nivaris(Chlamydomonas nivalis ), Chlamydomonas Oboys( Chlamydomonas ovoidae), Anastasis nidulans (Anacystis nidulans), Anchistrodesmus (Ankistrodesmus sp .), Bidulpa Aurita (Biddulpha aurita ), Boat Rio Cocous BrowniesBotryococcus braunii), Chitoceros sp., Chlamydomonas planta (Chlamydomonas applanata), Chlorella (Chlorella sp .), Chlorella Ellipsoide (Chlorella ellipsoidea), Chlorella EmulsonyChlorella emersonii ), Chlorella protosecoid (Chlorella protothecoides ), Chlorella Pyrenoidosa (Chlorella pyrenoidosa), Chlorella SorokiniChlorella sorokiniana), Chlorella Vulgaris (Chlorella vulgaris), Chlorella Minutishima (Chlorella minutissima), Chlorocoque litorrail (Chlororococcu littorale), Cyclotella cryptica( Cyclotella cryptica), Dunaliella Bardawil (Dunaliella bardawil), Dunaliella Salina (Dunaliella salina), Dunaliella Terthiolekta (Dunaliella tertiolecta ), Dunaliella Primorecta (Dunaliella primolecta), Gymnodium (Gymnodinum sp .), Hemeranomonas Catera (Hymenomonas carterae), Isocrisis Galvena (Isochrysis galbana), Isocrysis (Isochrysis sp .), Microcystis aeruginosa (Microcystis aeruginosa), Micromonas Fuscilla( Micromonas pusilla ), Monodus subterraneus (Monodus subterraneous), Nanochloris (Nannochloris sp .), Nanochloropsis (Nannochloropsis sp .), Nanochloropsis Atomus (Nannochloropsis atomus), Nanochloropsis Salina (Nannochloropsis salina), Navikula Pilikulosa (Navicula pelliculosa), Nitzia (Nitzschia sp .), The Nitzsia Closterium (Nitzscia closterium), Nitzia Palea (Nitzscia palea), Ossis polymorphia (Oocystis polymorpha), Aurocacus (Ourococcus sp .), Oscillatoria Rubeskens (Oscillatoria rubescens), Pavlova Lutheri (Pavlova lutheri), Paodaktrium Triconutum (Phaeodactylum tricornutum), Picnococcus probasoli (Pycnococcus provasolii), Pyramidonas cordata (Pyramimonas cordata), Spinula Platensis(Spirulina platensis), Stefanodiscus Minuturus (Stephanodiscus minutulus), Sticky note (Stichococcus sp .), Cinedraurna (Synedra ulna), Skenedesmus Obliques (Scenedesmus obliquus), Skeletonstrum grasire (Selenastrum gracile), Skeletonoma Costa Rum (Skeletonoma costalum), Tetraselmis Churro (Tetraselmis chui), Tetraselmis Makurata (Tetraselmis maculata), Tetraselmis (Tetraselmis sp .), Tetraselmis suesica (Tetraselmis suecica), Thalassio pseuudomona (Thalassiostra pseudomona), Anavana (Anabaena sp.), Carl Rodriguez (Calothrix sp .), Kamae Chiffon (Chaemisiphon sp .), Corokosidiopsis (Chroococcidiopsis sp.), Chanodese (Cyanothece sp .), Cylinder lossCylindrospermum sp .), Demo capella (Dermocarpella sp .), Fisherella (Fischerella sp.), Gloeocop company (Gloeocapsa sp .), Myxosina (Myxosarcina sp .), Northstock (Nostoc sp .), Ossilatoria (Oscillatoria sp .), Formium Corium (Phormidium corium), Pleurocap company (Pleurocapsa sp .), Procolococos (Prochlorococcus sp .), Peseudanavaena (Pseudanabaena sp .), Shineko Course (Synechococcus), Cinecosistis (Synechocystis sp .), Toliforrix (Tolypothrix sp .) And Genokocos (Xenococcus sp.It may be one or more selected from the group consisting of, preferably Chlamydomonas genus (Chlamydomonas sp .), And in one embodiment of the present invention Chlamydomonas Reinhardt (Chlamydomonas reinhardtiiExperimental example using the) is disclosed, but is not limited thereto.

The microalgae may be recovered as it is collected without culturing the collected in the sea, rivers, lakes, etc., may be microalgae cultured using a photobiological incubator.

The lipid may be a saturated or unsaturated fatty acid, and is a fatty acid having one or more chain lengths selected from the group consisting of C14, C16, C18, C20, C21, C24, C27, C28 and C29, but is not limited thereto.

In addition, the present invention, (a) compressing the elastomeric polymer beads using a stimulator of the microalgal cell stimulation apparatus; (b) injecting the microalgal cells into the inlet of the microalgal cell stimulation device; (c) synergizing the stimulator of the microalgal cell stimulation apparatus and then applying mechanical stimulation to the microalgal cells while restoring the elastomeric polymer beads; And (d) recovering the microalgae cells in a recovery tank after opening the filter of the microalgae cell stimulation device and lowering the stimulator, thereby providing a method for increasing lipid production of microalgae.

In addition, the present invention, (a) compressing the elastomeric polymer beads using a stimulator of the microalgal cell stimulation apparatus; (b) injecting the microalgal cells into the inlet of the microalgal cell stimulation device; (c) synergizing the stimulator of the microalgal cell stimulation apparatus and then applying mechanical stimulation to the microalgal cells while restoring the elastomeric polymer beads; And (d) recovering the microalgae cells in a recovery tank after opening the filter of the microalgae cell stimulation device and lowering the stimulator, thereby providing a microalgae with increased lipid production.

The microalgae may be deflagellation, but are not limited thereto.

In the present invention, "deflagellation" refers to a flagella which causes motility to the microalgae, and the flagella falls out.

In another aspect, the present invention provides a microalgae with increased lipid production by the production method.

In one embodiment of the present invention, using the stimulation device of the present invention, it was confirmed that the mechanical stimulation effectively applied to Chlamydomonas Reinharti cells through the compression step, de-compression step and recovery step. In addition, when using the 30% (w / v) polyurethane beads in the stimulation device, the beads have excellent elasticity, restoring force and toughness to effectively stimulate the cells, the stimulation time is 1 to 24 hours stimulation is optimal Confirmed.

Through the series of experiments, it was confirmed that the mechanical stimulation was effectively applied to Chlamydomonas Reinhardti cells through the compression, de-compression and recovery steps when using the stimulation device of the present invention. In addition, when using the 30% (w / v) polyurethane beads in the stimulation device, the beads have excellent elasticity, restoring force and toughness to effectively stimulate the cells, the stimulation time is 1 to 24 hours stimulation is optimal Confirmed.

In addition, it was confirmed that Chlamydomonas Reinharti cells stimulated using the stimulation device of the present invention induced membrane distortion, which increased calcium influx than control cells, resulting in deflagellation. In addition, it was confirmed that the expression of Mat3 mRNAs associated with physiological changes was increased, resulting in physiological changes, and that the size of the cells was larger than that of the unstimulated control group. In addition, mRNA expression of lipid related ACCase, DGAT and LPAAT is increased and stimulated Chlamydomonas reinhardti contains fatty acids with chain lengths of C14, C16, C18, C20, C21, C24, C27, C28 and C29 In comparison with the control group, polyunsaturated fatty acid (PUFA) was confirmed to occur, and accordingly, it was confirmed that the lipid composition was changed in the Chlamydomonas Reinharti cells by compressive stress.

Therefore, when using the microalgae cell stimulation apparatus according to the present invention, it is very efficient in terms of energy because it does not require other external stimulation, there is an advantage that can act stably on the microalgae cells without chemical stimulation. In particular, the microalgal cell stimulation apparatus of the present invention has an effect of increasing the lipid production of microalgal cells subjected to mechanical stimulation, it can be usefully used in environmentally friendly biodiesel production method.

Hereinafter, the present invention will be described in detail through examples. The following examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

Experimental Example 1. Preparation of microalgal cell stimulation device and experimental method

1-1. Manufacture of Microalgal Cell Stimulation Devices

The microalgal cell stimulation apparatus 100 of the present invention is shown in FIG. 1, and generally used a pumping principle of applying stimulation to cells such as a stimulation tank, and specifically, a stimulator 120, a pump 130, and a supply. It was manufactured to include a tank 140, a recovery tank 150 and the elastomeric polymer beads 160. More specifically, the stimulation reaction tank 110 is provided with an outlet formed on one side, the filter 112 formed to open and close the outlet. The stimulation reaction tank 110 is to be formed in a straight tube shape, such as cylindrical. The filter 112 is disposed at the outlet of the stimulation tank 110 so as to open and close the inside of the stimulation tank 110, the top of which is closed by the stimulator 120. The filter 112 may be implemented as a stainless steel membrane filter, the pore size of the filter 112 is formed to 800㎛ ~ 1200㎛ to filter the elastomeric polymer beads 160 accommodated inside the stimulation tank 110 Made to be produced.

In addition, the stimulator 120 is provided with a stimulator head 122 to move up and down inside the stimulation reaction tank 110 in contact with the inner peripheral surface of the stimulation reaction tank 110 at one end, the stimulator head 122 The nozzle 132 is connected to perform a filter role by passing through a solution such as a filter. The pore size of the nozzle 132 is 3 μm to 10 μm, and together with the solution discharged when the solution is discharged using the pump 130. Microalgae cells (C) was produced to prevent leakage to the outside of the stimulation tank (110). In addition, the stimulator head 122 is provided with an injection hole 126 for injecting and injecting the cells (C) into the inner space of the stimulation reaction tank 110 and the stimulation reaction tank 110 surrounded by the stimulation head (122). It was made to.

In addition, the pump 130 is a bidirectional pump for injecting a solution such as a culture solution into the stimulation tank 110, or to suck the solution inside the stimulation tank 110 to discharge to the supply tank 140. At this time, the pump 130 is connected to the tube 134 is formed so that the solution flows therein, the nozzle 132 is coupled to one end of the tube 134 facing the stimulation reaction tank (110). In addition, the nozzle 132 is manufactured so that the other side that is not coupled to the tube 134 is connected to the stimulator head 122 in a state in which the stimulator head 122 is raised.

In addition, the supply tank 140 is manufactured to be discharged from the stimulation tank 110 by the pump 130, or the solution supplied to the stimulation tank 110 is stored.

In addition, the recovery tank 150 is a portion in which the microalgal cells C discharged through the filter 112 together with the solution in the opened state of the filter 112 are accommodated.

In addition, the elastomeric polymer beads 160 are accommodated inside the stimulation tank 110 by using the elastomeric polymer beads having a concentration of 30% (w / v), and are deformed under pressure by the stimulator head 122. The microalgae cells (C) accommodated in the tank 110 were produced to act as a physical stimulus.

Using the microalgae cell stimulation device, the process of stimulating the Climidomonas Reinharti cells is shown in FIG. 2. Specifically, the stimulation tank 110 is filled with 30% (w / v) of the formulated elastomeric polymer beads 160, and the compression, de-compression and recovery steps were performed according to the process of FIG. .

In the case of the compression step, by applying a pressure to the stimulator 120 to compress the inside of the stimulation reaction tank 110 at the same time to discharge a certain amount of medium through the nozzle 132. After the elastomeric polymer beads 160 are completely compressed, the cells C are injected through the inlet 126. In this process, since cell loss may occur along with the medium discharge, cell injection was performed after the bead compression was completely completed. In addition, in the above process, the 1000 μm filter 112 at the lower end of the filter 112 was turned off so that the cells did not escape to the recovery tank 150 by compression. At this time, the elastomeric polymer bead 160 having a concentration of 30% (w / v) is in a compressed state and continuously exerts mechanical stimulation on surrounding cells due to the force to restore its original shape. In addition, dispersion of forces due to contact between beads occurs. This dispersion of forces occurs in series, causing the entire bead to compress equally, resulting in a spatially constant mechanical stimulus to the whole cell.

In the case of the de-compression step, the medium in the supply tank 140 was additionally injected into the stimulation tank 110 through the pump 130 while lifting the stimulator 120. At this time, the compressed elastomeric polymer beads 160 of 30% (w / v) concentration, due to the restoring force, again exerts mechanical stimulation on the whole cell (C).

In the recovery step, the elastomeric polymer beads 160, which are agglomerated by the supplied medium in a state where sufficient space is secured by the de-compression, are restored and completely released, and then the filter 112, which is in the off state of the lower end, is turned on, and the upper part is The cell C was recovered through the recovery tank 150 by applying pressure to the stimulator 120 while clogging the nozzle 132.

1-2. Polyurethane Bead (Polyurethane beed Production

Polyurethane (PU) was used as the material of the beads as an elastomeric polymer. The polyurethane template was dissolved in N, N-dimethylformamide (DMF) to form a 30% (w / v) polyurethane solution, then completely dissolved overnight and loaded into a plastic syringe to connect with the 22G needle. The syringe set was installed in a syringe pump (KD Scientific) and the flow rate was set to 3 ml / h. Polyurethane beads dropped into the sphere from the syringe were collected in the secondary distilled water contained in the beaker, and the drop height from the tip of the syringe to the distilled water was maintained at 9 cm, so that the polyurethane was 20% (w / v), 25% ( polyurethane beads containing 30% (w / v), 35% (w / v) and 40% (w / v) were formulated. Soak overnight in 95% ethanol (Samshun pure chemical) to remove residual DMF inside the formulated polyurethane beads. After washing three times with secondary distilled water, the beads were finally soaked overnight in TAP medium. In the process, the pressure was applied to the beads to discharge the solution absorbed therein, and the washing was performed to increase the elasticity of the beads and to have a restoring force against a predetermined pressure.

Scanning electron microscope (Hitachi S-4200) was used to observe the morphology of the formulated polyurethane beads. Each bead stored in a dessicator overnight was attached to the collector using a carbon tape. Prior to SEM, each bead was coated for 180 seconds using a Pt sputter. Finally, SEM images were obtained at a 10 kV acceleration voltage. In addition, in order to confirm the elastic force of each bead in the compression step or de-compression step, the degree of restoration of the beads of each concentration was observed at 10 minute intervals. In addition, the micrometer was used to measure the diameter change of the beads under each condition over time and the process of compressing the beads.

1-3. Chlamydomonas Reinhardty ( Chlamydomonas reinhardtii Culture

Wild-type Chlamydomonas Reinharti CC-620 was inoculated into a flask of 18 ml TAP media (Gibco, USA), pH 7.2, which was incubated in a shaker at 23 degrees and 120 rpm while maintaining 5% CO ₂ conditions. Light was provided by OSRAM DULUX L FPL36EX-D lamps and the light intensity was maintained at 300 μmm- ² s-1.

In order to confirm the synchronization of cell growth and differentiation, conditional changes (12 h / 12 h) of the light and cancer periods were performed. Synchronization of the cells was confirmed from the third cycle, and the synchronization of the cells was confirmed by drawing a growth curve curve by measuring the cell optical density per unit time using an X-ma 1000 spectrophotometer during the synchronized culture process. Growth curve changes were monitored during the third dark / light cycle. At the beginning of the fourth light period, an inoculum to be inoculated was prepared by adjusting the synchronous cell inoculation concentration (2 × 10 ⁶ cells / ml) through culture dilution. The inoculum was inoculated in a non-stress reactor and a fluid shear stress reactor, respectively.

1-4. Chlamydomonas Reinhardty ( Chlamydomonas reinhardtii ) Viability and apoptosis

Each sample obtained from the reactors 1-3 was stained at room temperature for 10 minutes through 1 mM sytox Green (Molecular Probes) containing TAP media. Each sample was taken using a confocal laser microscope (LSM 510 META), and live cells at 488 nm (Ar-laser) / 505-530 nm (excitation / emission) at 543 nm (HeNe-laser) / 560 nm (here / Dead cells were photographed. In addition, dual fluorescence images were merged through ZEN 2009 light Edition software (Carl Zeiss) and at least 500 cells were randomly set at × 100 magnification to determine the viability and degree of apoptosis of Chlamydomonas Reinhardti cells (%). Calculated.

1-5. Cell morphology

In order to compare and analyze the morphology of the compressed and de-compressed cells, the cells obtained in the compression and de-compression steps of 1-1 were centrifuged at 13,500 g for 20 seconds, which was 0.25% glutaraldehyde. Fixation). Subsequent microscopic cell membrane distribution and deflagellation The cell size was measured using a Cellometer Auto T4 Cell Counter (Nexcelom Bioscience) (n> 50).

1-6. Obtained from the stimulation device Chlamydomonas Reinhardty  cytoplasm Free calcium ions Confirmation of inflow

Obtained in the recovery tank of the stimulation device of the present invention. The supernatant was removed by centrifuging Chlamydomonas Reinharti (1 × 10 ⁵ cell / ml) at 13,500 g for 20 seconds. The remaining pellet was added to NMG + / K + buffer containing 1 mM sulfinpyrazone (containing 1 mM sulfinparazone, N-methyl-D-glucamine (NMG) was added to pH 5.6, 5 mM HEPES, 10 1 mM KCl, 200 μM K + BAPTA) and 3 μM Fura-2 dissolved in DMSO (Sigma-Aldrich). Each sample was incubated at 36 degrees for 2 hours and washed three times using a TAP medium. Thereafter, dye-free NMG + / K + buffer (pH 6.8) was added and incubated in dark at 10 ° C. for 10 minutes. Fura-2-Ca ²⁺ of cells was photographed using confocal microscopy.

1-7. Sulfo - Phospho Vanillin method Colorimetric ) ( Sulpho - phospho vanillin method (colorimetric method))

Obtained in the recovery tank of the stimulation device of the present invention. The supernatant was removed by centrifuging Chlamydomonas Reinharti (1 × 10 ⁵ cell / ml) at 13,500 g for 20 seconds. 95% ethanol (Samshun pure chemical) was added to the pellet of the cell, and then treated overnight, followed by centrifugation at 13,500 g for 10 minutes. The pellet is then removed and the supernatant (100% ethanol + lipid) transferred to a 96-well plate, evaporated in a dry oven at 90 degrees and 100 μl of sulfuric acid is added to each well for 20 minutes at 90 degrees. Reacted. Thereafter, 50 μl vanillin-phosphate (0.25 mg / ml vanillin in 17% phosphoric acid) was added to each well, reacted at room temperature for 10 minutes, and the OD value was measured at 540 nm by ELISA. .

1-8. GC -MS analysis

Obtained in the recovery tank of the stimulation device of the present invention. The supernatant was removed by centrifuging Chlamydomonas Reinharti (1 × 10 ⁵ cell / ml) at 13,500 g for 20 seconds. The pellet of cells was re-suspended by adding 1.0 ml of toluene and acetyl chloride in 10% (v / v) methanol. Thereafter, the sample was incubated at 70 ° C for 120 minutes, cooled, and then 0.2 ml of distilled water was added to stop the reaction. FAMEs were extracted by adding 1.25 ml of hexane-methyl tert-butyl ether (1: 1), gently shaken for 10 minutes, then centrifuged at 2500 × g for 6 minutes and the organic layer (top layer) was transferred to another new tube. To clean up the sample, 3 ml of cleaning reagent (10.8 g NaOH in 900 ml dH ₂ O) was added and shaken for 5 minutes. The organic layer (upper layer) was transferred to another new tube and evaporated at 40 ° C. Resuspension of FAMEs was performed using hexane, and the FAMEs sample included a J & W GC column DB-5ms (30 m × 0.25 mm × 0.25 μm), and was a LECO Pegasus III Time-of-Flight Mass Spectrometer (TOMS) as a detector. Agilent Tech) was installed in Agilent 6890N gas chromatography installed. A 1 μl injection volume was performed at 10: 1 split conditions at an inlet temperature of 280 ° C. The carrier gas was helium, and the fixed flow rate was 1 mL / min, 30 ° C for 10 minutes, and the lamp was 10 ° C. Conditions were maintained at 300 ° C./min and held for 10 minutes. GC analysis of FAMEs samples was analyzed by the Advanced Analysis Center (ACC) of the Korea Institute of Science and Technology (KIST).

1-9. Qualitative analysis of Chlamydomonas Reinhardti lipids by Nile red staining

Nile red staining (Sigma Aldrich) was performed to qualitatively confirm the change in lipid production of Chlamydomonas Reinharti mechanically stimulated using the stimulation device of the present invention. Obtained in the recovery tank of the stimulation device of the present invention. The culture of Chlamydomonas Reinharti was treated with 2.5% glutaraldehyde overnight to fix the cells. Then, the culture solution and Nile Red dye were mixed in a 3: 1 ratio and incubated in a dark condition at 40 degrees for 10 minutes. Images of the samples were confirmed by confocal microscopy with an excitation wavelength of 543 nm and an emission wavelength of 630 nm.

1-10. qRT - PCR Quantitative real-time PCR  Perform

Real time qPCR was performed to quantitatively analyze lipids by checking mRNA expression levels. Obtained in the recovery tank of the stimulation device of the present invention. Chlamydomonas Reinharti culture was fixed with gram iodine (10: 1 v / v) and cell number was calculated using a hemocytometer (Superior). In order to equalize the cell number condition of each sample, the cell number of each sample was diluted to 5-6 × 10 ⁶ cells / ml (reference value) based on the cell number calculation result. Each sample was washed with PBS and then centrifuged for 15 seconds at 10,000 rpm. Thereafter, the pellet from which the supernatant was removed was extracted with gDNA using RNAeasy Plant Mini Kit (Qiagen), and total RNA was extracted. To prevent RNA omission of each sample, cDNA was synthesized using Reverse Transcription Kit (Qiagen) and amplified by using QuantiFast SYBR Green PCR Kit (Qiagen) and Rotor-Gene Q (Qiagen). For PCR, a total of six primers (CBLP, Actin, ACCase, DGAT, LPAAT, and Mat3) shown in Table 1 were used, and the cycle time based on threshold 0.1 for each primer was determined using Rotor-Gene Q Series Software (Qiagen). The data was converted into delta-delta Ct value. Finally, delta-delta Ct was identified by generalizing the relative concentration of mRNA for each primer to the expression level of CBLP (housekeeping gene).

TABLE 1

Example  1.polyurethane Bead (Polyurethane beed ) Fabrication and its shape and diameter

According to the method of Experimental Example 1-2, the polyurethane was 20% (w / v), 25% (w / v), 30% (w / v), 35% (w / v) and 40% (w polyurethane beads contained in / v) were formulated, and their shape, diameter, surface and cross section were identified. The results are shown in Figure 3, Table 1 and Table 2.

As shown in FIG. 3 a and Table 1, each polyurethane concentration formulated in Example 1 was 20% (w / v), 25% (w / v), 30% (w / v), 35% ( The morphology of the polyurethane beads for w / v) and 40% (w / v) showed that 20% (w / v) beads showed very unstable formulations at low density, resulting in the shrinkage of beads after It was confirmed that a bead shrink phenomenon appeared. 35% (w / v) and 40% (w / v) beads were viscous and were formulated in tailed form to confirm instability. Formulations of polyurethane beads at concentrations of 25% (w / v) and 30% (w / v) were found to be spherical, resulting in a stable formulation.

In addition, as shown in b and Table 2 of FIG. 3, each of the polyurethane concentrations formulated in Example 1 20% (w / v), 25% (w / v), 30% (w / v), 35 Polyurethane beads at 20% (w / v) concentrations average 2.410 cm, 25% (w / v) concentrations as a result of checking the diameter of the polyurethane beads for% (w / v) and 40% (w / v) Polyurethane beads at an average of 2.507 cm, 30% (w / v) polyurethane beads at an average of 2.499 cm, 35% (w / v) polyurethane beads at an average of 2.483 cm and 40% (w / v) The concentration of polyurethane beads was found to be 2.353 cm on average.

In addition, as a result of checking c of FIG. 3, it was confirmed that 30% (w / v) of the surface of the polyurethane bead is a porous structure in which numerous fine pores exist, and an empty space having a specific size exists in the cross section.

In addition, the degree of restoration of polyurethane beads at each concentration in the compression or de-compression step was faster as the polyurethane concentration was lower, and all beads except 50% of the polyurethane beads were 40% (w / v). It was confirmed that it was restored within minutes.

Therefore, when used in the stimulation device of the present invention, it was confirmed that 30% (w / v) polyurethane beads having excellent elasticity and restoring force is the most optimal.

Example  2. Polyurethane under stimulation conditions using stimulation device of the present invention Bead  Robustness check

Robustness of the polyurethane beads in the stimulation conditions using the stimulation device of the present invention was confirmed.

Specifically, the physical properties change (size and elastic force) by repeatedly performing the polyurethane beads 1 to 10 times the compression step and the de-compression process to check the degree of restoration of 30% (w / v) of the polyurethane beads It was confirmed. In addition, as the bead is discolored to green under stimulation conditions using the stimulation device of the present invention, to determine whether the bead of Chlamydomonas Rheinharti infiltrate inside the bead that may affect the properties and recovery process of the bead In order to check the absorbance of Chlamydomonas Reinharti and the beads for more than a week, the surface of the beads (TAP media) (solution A) and the internal liquid (TAP media) solution (B) absorbed by the beads was confirmed. . The results are shown in FIG.

As shown in Figure 4a, it was confirmed that even in the compression step and the de-compression process, the size and elastic force of the polyurethane beads of the present invention remains unchanged, it was confirmed that the excellent toughness.

As shown in b of FIG. 4, it was confirmed that the released Chlamydomonas Reinhardt's pigment was adsorbed on the surface of the polyurethane beads, and the cells were not absorbed into the beads through the fine pores inside the beads. Therefore, the bead discoloration phenomenon was confirmed that does not affect the lipid production of Chlamydomonas Reinharti.

Example  3. Under stimulation conditions using the stimulation device of the present invention Chlamydomonas Reinhardty  Identify gene expression related to cell size, calcium influx-mediated deflagellation and physiological changes

The cell size, calcium influx-mediated deflagellation and morphological change-related gene expression of Chlamydomonas Reinharti were confirmed under stimulation conditions using the stimulation device of the present invention.

Specifically, according to the method of Experimental Example 1-5, Chlamydomonas Reinharti cells stimulated using the stimulation device of the present invention, according to the method of Experimental Example 1-5, for 12 hours or 24 hours And the size of control cells. According to the method of Experimental Example 1-6, it was confirmed for 2 hours to stimulate the Chlamydomonas line hareuti cells and cytoplasmic free (free) Ca ^{+ 2} influx of control cells. In addition, qRT-PCR (Quantitative real-time PCR) was performed according to the method of Experimental Example 1-10 to confirm the expression level of mRNAs of Mat3 and E2F1 associated with physiological changes.

As shown in a and b of Figure 5, Chlamydomonas Reinharti cells stimulated using the stimulation device of the present invention induced membrane distortion (membrane distortion) increased calcium influx than the control cells unbiased algae ( deflagellated).

As shown in Figure 5c, it was confirmed that the size of the Chlamydomonas Reinharti cells stimulated for 12 hours using the stimulation device of the present invention is up to 7.2 ㎛. The cells stimulated for 24 hours were 7.8 μm and the control was 6.7 μm. Therefore, Chlamydomonas Reinharti cells stimulated using the stimulation device of the present invention was confirmed that the size of the cells larger than the control group

As shown in Figure 5d, it was confirmed that the mRNAs of Mat3 associated with physiological changes in Chlamydomonas Reinharti cells stimulated using the stimulation device of the present invention significantly increased in 12 hours stimulation conditions. Since there is a significant change in the expression of Mat3, it was confirmed that physiological changes of the cell cycle of Chlamydomonas occurred by compressive stress.

Example  4. Under stimulation conditions using the stimulation device of the present invention Chlamydomonas Reinhardty  Confirmation of Lipid Generation

Lipid production of Chlamydomonas Reinharti was confirmed under stimulation conditions using the stimulation device of the present invention.

Specifically, the nile red staining method of Experimental Example 1-9 was used as compared to Chlamydomonas Reinhardti and its control cells stimulated using the stimulation device of the present invention, and the stimulation device of the present invention. Stimulation conditions were given using 0, 4, 8, 12 and 24 hours. In addition, each stimulation condition for the cells was varied by applying pressure to a volume of 0, 4 or 8 ml using a stimulator to the 50 ml stimulation tank of the present invention. Qualitative and quantitative analysis of lipid droplet formation of Chlamydomonas Reinharti cells or controls according to the above conditions was performed. The results are shown in FIG.

As shown in a and b of FIG. 6, it was confirmed that lipid granules inside the Chlamydomonas reinhardti cells stimulated using the stimulation device of the present invention increased with time or pressure.

Example  5. Under stimulation conditions using the stimulation device of the present invention Chlamydomonas Reinhardty  Confirmation of Lipid Production Efficiency

Lipid production efficiency of Chlamydomonas reinhardti was confirmed under stimulation conditions using the stimulation device of the present invention.

Specifically, the lipid production efficiency of Chlamydomonas Reinhardti cells or controls stimulated for 4, 8, 12 and 24 hours through a compression step or a de-compression step was determined by the sulfo-phospho-vanillin method of Experimental Example 1-7. It confirmed by (colorimetric method). The results are shown in FIG.

As shown in Figure 7a, as a result of confirming the degree of lipid granule synthesis of single cells, Chlamydomonas Reinharti cells subjected to 4 hours stimulation was confirmed that the high amount of lipid in the unit cell compared to the control. In addition, as the stimulation time increases every 4 hours, the analysis of the increase rate of lipids in the unit cell compared to the control cells showed that the increase rate of 65% (4 hours), 78% (8 hours), and 47% (12 hours) respectively. Confirmed.

As shown in b of FIG. 7, as a result of confirming the degree of lipid granule synthesis of the total cells, it was confirmed that the lipid particle synthesis of the total cells was increased compared to the control group.

Therefore, Chlamydomonas Reinharti stimulated using the stimulation device of the present invention was confirmed that the lipid production efficiency is superior to the control.

Example  6. Under stimulation conditions using the stimulation device of the present invention Chlamydomonas Reinhardty  Lipid content change

Changes in the lipid content of Chlamydomonas Reinharti under stimulation conditions using the stimulation device of the present invention were confirmed.

Specifically, 4, 8 and 12 hours of stimulation conditions were given using the stimulation device of the present invention. In addition, the 50 ml stimulation tank of the present invention using a stimulator to apply a pressure in a volume of 0 or 4ml to vary the respective stimulation conditions for the cells. Lipid content change of Chlamydomonas Reinharti cells stimulated according to the above conditions was analyzed according to the method of Experimental Example 1-8. The results are shown in Figure 8 and Table 2.

TABLE 2

It was found to contain fatty acids having chain lengths of C14, C16, C18, C20, C21, C24, C27, C28 and C29. In addition, it was confirmed that polyunsaturated fatty acid (PUFA) was generated in all Chlamydomonas Reinharti cells subjected to compressive stress compared to the control group. Therefore, it was found that lipid composition changes in Chlamydomonas Reinhardt cells due to compressive stress.

Example  7. Under stimulation conditions using the stimulation device of the present invention Chlamydomonas Reinhardty  Confirmation of lipid-related mRNA

The degree of lipid-related mRNA expression of Chlamydomonas reinhardti under stimulation conditions using the stimulation device of the present invention was confirmed.

Specifically, the degree of lipid-related mRNA expression of Chlamydomonas Reinharti cells stimulated according to 4, 8, 12 and 24 hour stimulation conditions was analyzed according to the method of Experimental Example 1-10. The results are shown in FIG.

As shown in Figure 9, Chlamydomonas Reinharti cells stimulated according to the stimulation device of the present invention compared with the control cells confirmed that the mRNA expression of ACCase, DGAT and LPAAT is up-regulated, the stimulation device of the present invention Thus stimulated Chlamydomonas Reinharti cells were found to produce lipids effectively.

Example  8. Under stimulation conditions using the stimulation device of the present invention Chlamydomonas Reinhardty  Confirmation of the degree of cell death

The degree of apoptosis of Chlamydomonas Reinharti cells was confirmed under stimulation conditions or direct stimulation conditions using the stimulation device of the present invention.

Specifically, stimulation conditions of the present invention were given 4, 8, 12 and 24 hour stimulation conditions. In addition, each stimulation condition for the cells was varied by applying pressure to a volume of 0 or 4 ml using a stimulator to the 50 ml stimulation tank of the present invention. In addition, as shown in Figure 10, the Petri dishes were filled with PDMS (polydimethylsiloxane) filled and hardened, and the conditions were applied directly to the cells using the flat surface of the hardened PDMS and Petri dishes.

The degree of apoptosis of Chlamydomonas Reinharti cells for each of the above conditions was confirmed according to the method of Experimental Example 1-4. The results are shown in FIG.

As shown in FIG. 11, it was confirmed that apoptosis rate (%) of 40% or more appeared under the condition of directly stimulating cells. In addition, it was confirmed that apoptosis was inhibited at 4, 8 and 12 hours of stimulation conditions and 4 ml volume of the pressure, except for 24 hours of stimulation conditions.

Through the series of experiments, it was confirmed that the mechanical stimulation was effectively applied to Chlamydomonas Reinhardti cells through the compression, de-compression and recovery steps when using the stimulation device of the present invention. In addition, when using the 30% (w / v) polyurethane beads in the stimulation device, the beads have excellent elasticity, restoring force and toughness to effectively stimulate the cells, the stimulation time is 1 to 24 hours stimulation is optimal Confirmed.

In addition, it was confirmed that Chlamydomonas Reinharti cells stimulated using the stimulation device of the present invention induced membrane distortion, which increased calcium influx than control cells, resulting in deflagellation. In addition, it was confirmed that the expression of Mat3 mRNAs associated with physiological changes was increased, resulting in physiological changes, and that the size of the cells was larger than that of the unstimulated control group. In addition, mRNA expression of lipid related ACCase, DGAT and LPAAT is increased and stimulated Chlamydomonas reinhardti contains fatty acids with chain lengths of C14, C16, C18, C20, C21, C24, C27, C28 and C29 In comparison with the control group, polyunsaturated fatty acid (PUFA) was confirmed to occur, and accordingly, it was confirmed that the lipid composition was changed in the Chlamydomonas Reinharti cells by compressive stress.

Claims (20)

1. An stimulation tank having an outlet formed at one side and having a filter formed to open and close the outlet;

   A stimulator having a stimulator head having a lateral movement in contact with an inner circumferential surface of the stimulation reaction tank to move up and down inside the stimulation reaction tank;

   A pump for injecting a solution into the stimulation tank or sucking and discharging the solution in the stimulation tank; And

   An elastic polymer bead accommodated in the stimulation tank;

   The stimulator head is a microalgae cell stimulation device, characterized in that the nozzle through which the solution is connected.
2. The method of claim 1,

   The stimulator head is a microalgae cell stimulation device, characterized in that the injection port is provided to inject the cells into the stimulation reaction tank.
3. The method of claim 1,

   A tube connected to the pump and in which the solution flows; And

   The nozzle is one side is coupled to one end of the tube facing the stimulation reaction tank, the other side that is not coupled to the tube is connected to the stimulator head, the microalgal cell stimulator, characterized in that the stimulator head is raised.
4. The method of claim 1,

   The microalgae cell stimulation apparatus further comprises a supply tank for storing a solution discharged from the stimulation reaction tank by the pump, or supplied to the stimulation reaction tank.
5. The method of claim 1,

   Cell stimulation apparatus, characterized in that the pore size of the filter is 800㎛ ~ 1200㎛.
6. The method of claim 3,

   The pore size of the nozzle is a cell stimulation device, characterized in that 3㎛ ~ 10㎛.
7. The method of claim 1,

   The elastomeric polymer beads may be poly (L-lactide-co-ε-caprolactone) (Poly (L-lactide-co-ε-caprolactone)), polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, L-lactide-coglycolide)), poly (caprolactone), diol / diacid based aliphatic polyester, polyester-amide / polyester-urethane, polyurethane, At least one member selected from the group consisting of polyethylene oxide, poly (valerolactone), poly (hydroxybutyrate), poly (hydroxy valerate), chitosan, chitin, alginic acid, collagen, gelatin and hyaluronic acid Microalgae cell stimulation apparatus.
8. The microalgal cell stimulation apparatus according to claim 7, wherein the polyurethane bead comprises polyurethane at a concentration of 10 to 50% (w / v).
9. The method of claim 1,

   The microalgae is a genus Chlamydomonas sp .
10. The method of claim 1,

    The genus Chlamydomonas Reinhardtii ( Chlamydomonas reinhardtii ), Chlamydomonas caudate , Chlamydomonas moewusii , Chlamydomonas nibaris ( Chlamydomonas nivalis ) and Chlamydomonas nivalis ( Chlamydomonas ovoidae ), characterized in that at least one selected from the group consisting of microalgae cell stimulation device.
11. (a) compressing the elastomeric polymer beads using the stimulator of the microalgal cell stimulation apparatus of claim 1;

    (b) injecting the microalgal cells into the inlet of the microalgal cell stimulation device;

    (c) synergizing the stimulator of the microalgal cell stimulation apparatus and then applying mechanical stimulation to the microalgal cells while restoring the elastomeric polymer beads; And

    (d) recovering the microalgae cells in a recovery tank after opening the filter of the microalgae cell stimulation device and lowering the stimulator.
12. The method of claim 11,

    The compression of (a) is characterized in that the addition of 1 to 48 hours, a method for increasing lipid production of microalgae.
13. The method of claim 11,

    The elastic polymer beads of (a) are polyurethane beads, characterized in that the lipid production of microalgae.
14. The method of claim 11,

    The microalgae is a genus Chlamydomonas sp. , Characterized in that the lipid production of microalgae.
15. The method of claim 14,

    The genus Chlamydomonas Reinhardtii ( Chlamydomonas reinhardtii ), Chlamydomonas caudate , Chlamydomonas moewusii , Chlamydomonas nibaris ( Chlamydomonas nivalis ) and Chlamydomonas nivalis ( Chlamydomonas ovoidae ) characterized in that at least one member selected from the group consisting of, a method for increasing lipid production of microalgae.
16. The method of claim 11,

    The lipid is a saturated or unsaturated fatty acid, characterized in that the lipid production of microalgae.
17. The method of claim 16,

    The saturated or unsaturated fatty acid is a fatty acid having at least one chain length selected from the group consisting of C14, C16, C18, C20, C21, C24, C27, C28 and C29, lipid production of microalgae.
18. (a) compressing the elastomeric polymer beads using the stimulator of the microalgal cell stimulation apparatus of claim 1;

    (b) injecting the microalgal cells into the inlet of the microalgal cell stimulation device;

    (c) synergizing the stimulator of the microalgal cell stimulation apparatus and then applying mechanical stimulation to the microalgal cells while restoring the elastomeric polymer beads; And

    and (d) recovering the microalgae cells in a recovery tank after opening the filter of the microalgae cell stimulation device and lowering the stimulator.
19. The method of claim 18,

    The microalgae is characterized in that the deflagellation (deflagellation), the production method of microalgae with increased lipid production.
20. Microalgae with increased lipid production by the method of claim 18.

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