KR20160056797A - Microfluidic device for capable of sequential operation from cell culture to lipid extraction and the method of extracting lipid using thereof - Google Patents

Microfluidic device for capable of sequential operation from cell culture to lipid extraction and the method of extracting lipid using thereof Download PDF

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KR20160056797A
KR20160056797A KR1020150154555A KR20150154555A KR20160056797A KR 20160056797 A KR20160056797 A KR 20160056797A KR 1020150154555 A KR1020150154555 A KR 1020150154555A KR 20150154555 A KR20150154555 A KR 20150154555A KR 20160056797 A KR20160056797 A KR 20160056797A
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culture
lipid
microalgae
chamber
organic solvent
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KR101756308B1 (en
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심상준
임현석
김영환
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고려대학교 산학협력단
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/12—Bioreactors or fermenters specially adapted for specific uses for producing fuels or solvents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/22—Transparent or translucent parts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M43/00—Combinations of bioreactors or fermenters with other apparatus
    • C12M43/02—Bioreactors or fermenters combined with devices for liquid fuel extraction; Biorefineries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Abstract

Disclosed are a microfluidic device enabling cell culture and lipid extraction, and a lipid extraction method using the same. According to an embodiment of the present invention, the microfluidic device of the present invention comprises: a microalgae culturing module having a plurality of separated culturing chambers for culturing the microalgae; and a lipid-dissolved solution filtration module coupled to an upper part of the microalgae culturing module by being stacked thereto, communicatively connected to the culturing chambers, and filtrating a lipid-dissolved solution containing dissolved lipid components derived from fractions of the microalgae pulverized by means of an organic solvent supplied to the culturing chambers.

Description

TECHNICAL FIELD [0001] The present invention relates to a microfluidic device capable of cell culture and lipid extraction, and a lipid extraction method using the microfluidic device and a lipid extraction method using the microfluidic device.

The present invention relates to a microfluidic device capable of cell culture and lipid extraction, and a lipid extraction method using the microfluidic device. More particularly, the present invention relates to a microfluidic device capable of performing a series of processes from microalgae culture to lipid component extraction A microfluidic device capable of culturing and extracting lipid, and a lipid extraction method using the microfluidic device.

Single-celled organisms, including bacteria, yeast, microalgae, etc., are used for a variety of purposes in agriculture, animal husbandry, fisheries, medicine and resources.

Here, microalgae refers to unicellular algae that contain various pigments such as chlorophyll, carotenoid, picobillins, etc., and can synthesize the cell growth and the organic materials necessary for photosynthesis, and most phytoplankton This belongs here.

Bacteria and yeast are used for the expression of medicinal proteins, and microalgae are attracting attention because they can produce various useful materials by using light energy, carbon dioxide and inorganic materials.

For example, microalgae are growing faster than plants and are rapidly emerging as alternatives to fossil fuels because they can produce large amounts of light energy and neutral lipids that can be converted from carbon dioxide and inorganic materials to biodiesel. In addition, It is attracting attention as a solution to greenhouse gas problem that warming is a problem.

Among the biomass produced from microalgae, the available oil content ranges from 30 to 70%, indicating higher fuel productivity than the oil produced in conventional plants.

Therefore, in order to utilize microalgae efficiently, various studies such as optimization of medium, optimum reactor design, metabolic process and product purification are required.

On the other hand, it is required to develop a system and a method for quickly and efficiently analyzing the microalgae, whose characteristics are not disclosed, in order to optimize the medium and to find out what kind of substances produced by the microalgae and what constituents there are.

At present, the analysis of components for the production of microalgae takes a long time, and a method of extracting lipid of microalgae uses a large amount of organic solvent such as chloroform, which is toxic substance.

Korean Patent Registration No. 10-0320786 discloses a method for extracting lipids from microalgae by dispersing microalgae collected by precipitating a microalgae culture solution and using a centrifugal separation method in a mixed solvent of chloroform and methanol to produce hydrocarbons A method for recovering a sample is disclosed.

The method of extracting lipids from microalgae according to the prior art can use only materials such as glass having high durability against an organic solvent, and there are restrictions on high cost and complicated manufacturing process.

In addition, the prior art has shown that the microalgae are destroyed in the dehydration process and extraction process, so that the useful carbohydrates, intracellular proteins, and useful biofuels, such as lipids, And it is difficult to reuse the biomass.

Therefore, in order to analyze efficiently the lipid produced from microalgae and its growth potential, it is necessary to study the culture of single-cell microalgae, lipid accumulation and lipid extraction rapidly and efficiently.

Korean Patent No. 10-0320786 (Announced on May 13, 2002)

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a method and apparatus for independently culturing at least one kind of microalgae, and a series of processes from the cultivation of microalgae to the extraction of lipid components can be performed quickly and efficiently A microfluidic device capable of cell culture and lipid extraction, and a lipid extraction method using the microfluidic device.

According to an aspect of the present invention, there is provided a microalga culture module having a plurality of independent culture chambers for culturing microalgae; And the microalgae culture module, wherein the lipid components of the microalgae in the microalgae lysed by the organic solvent, which is in communication with the plurality of culture chambers and supplied to the plurality of culture chambers, A microfluidic device capable of cell culture and lipid extraction including a lipid-solubilization filtration module for filtering a lipid-soluble solution can be provided.

The microalga culture module may further include a plurality of lipid solution containing chambers provided adjacent to the plurality of culture chambers and communicating with the lipid solution filtration module to receive the filtered lipid solution.

The lipid dissolution filtration module is configured to connect the adjacent culture chamber and the lipid solution reservoir chamber in a pair so as to communicate with each other so that the lipid dissolution solution is filtered and supplied from the culture chamber to the lipid solution reservoir chamber And may include a plurality of lipid solution filtration units.

Wherein each of the plurality of lipid solution filtration units includes: a lipid solution transfer passage through which the lipid solution is transferred from the adjacent culture chamber to the lipid solution storage chamber; And a plurality of filtration members, each of which is connected to the lipid solution transport passage and is connected to the culture chamber and the upper region of the lipid solution storage chamber, Wherein the lipolysis solution contained in the culture chamber is filtered by the plurality of filter members located in the upper region of the culture chamber and is moved to the lipolysis solution flow passage, And may be supplied to the lipid solution receiving chamber.

Wherein each of the plurality of filtration members is formed in a columnar shape and the plurality of filtration members positioned in an upper region of the culture chamber are connected to the one end of the filtration chamber so that one end thereof is immersed in the culture chamber and the other end is connected to the lipid- The plurality of filtration members positioned in the upper region of the lipid solution storage chamber may be connected so that one end thereof is immersed in the lipid solution storage chamber and the other end is connected to the lipid solution transport passage.

Wherein the microalgae culture module is connected to the plurality of culture chambers and includes a medium for supplying a medium for lipid accumulation in the microalgae and an organic solvent for crushing the microalgae in the plurality of culture chambers, And may further include a supply section.

The medium and the organic solvent supply unit include a medium and an organic solvent receiving chamber in which the medium and the organic solvent are accommodated; And a plurality of culture media and an organic solvent supply channel which connect the culture medium and the culture medium accommodating chamber to each other in a communicative manner, wherein the culture medium and the organic solvent supply channel are arranged in such a manner that the culture medium and the organic solvent supply channel, And may be formed in a zigzag shape from the solvent accommodating chamber to the incubation chamber.

The plurality of culture chambers are radially arranged around the medium and the organic solvent receiving chamber, and the plurality of lipid solution receiving chambers may be disposed adjacent to the outside of the plurality of culture chambers.

And a pump for supplying the medium and the organic solvent to the medium and the organic solvent-receiving chamber.

The microalgae culture module includes a plurality of microalgae supply units connected to the plurality of culture chambers to supply the microalgae to the plurality of culture chambers, respectively; And a lipid dissolution liquid discharge unit connected to the plurality of lipid solution storage chambers to discharge the lipid dissolution liquid contained in the plurality of lipid solution storage chambers, respectively.

The microalga culture module and the lipid solution filtration module may be formed of a transparent transparent material including polymethyl methacrylate (PMMA), polystyrene (PS), and polydimethylsiloxane (PDMS).

According to another aspect of the present invention, there is provided a microalgae culture module comprising: a micro-algae culture module; a micro-algae culture module; a micro-algae culture module; Accumulating lipids in the microalgae; Supplying an organic solvent to the culture chamber to crush the microalgae in the culture chamber; And filtering the lipolysis solution in which the lipid component of the microalgae is dissolved by using the lipolysis solution filtration module to extract lipids from the microalgae by using the microfluidic device.

The step of filtering and extracting the lipid-lysing solution may include a step of filtering the lipid-soluble solution by a plurality of filtration members of the lipid-solubilization filtration module, which is located in an upper region of the culture chamber and has a micro- Wherein the lipid dissolution liquid is located in an upper region of a lipid solution containing chamber provided adjacent to the culture chamber of the microalgae culture module and one end is immersed in the lipid solution containing chamber, To be accommodated in the lipid-dissolved-solution accommodating chamber.

The step of accumulating the lipid in the microalgae comprises supplying the nitrogen-deficient medium to the medium and the organic solvent supply unit provided adjacent to the culture chamber, exchanging the medium in the culture chamber with the nitrogen-deficient medium, and adding 40 mol photon m -2 it is possible to cultivate the micro-algae for 24 hours at a constant intensity of s -1 and for 4 days at 23 ° C.

The step of crushing the microalgae may be performed after the organic solvent is supplied to the culture chamber.

The organic solvent may include methanol, ethanol, and isopropanol.

The step of culturing the microalgae can be carried out by incubating the microalgae for 24 hours at 40 m photon m -2 s -1 intensity and for 3 days at 23 ° C.

The embodiment of the present invention can independently cultivate at least one kind of microalgae at the same time and can easily monitor the cultivation and lipid accumulation process of microalgae in real time.

In addition, the embodiment of the present invention can continuously and rapidly perform a series of processes from the cultivation of microalgae to the extraction of lipid components.

1 is a plan view showing a microfluidic device capable of cell culture and lipid extraction according to the present invention.
2 is an exploded perspective view showing a microfluidic device capable of cell culture and lipid extraction according to the present invention.
3 is a plan view showing a microalgae culture module according to the present invention.
4 is a plan view showing a lipid solution filtration module according to the present invention.
5 is an enlarged view of a portion A in Fig.
6 is an enlarged view of a portion B in Fig.
7 is an enlarged view of a portion C in Fig.
FIG. 8 is a flowchart illustrating a method for extracting lipids using a microfluidic device according to the present invention.
9 is a graph showing the results of culturing 8 different microalgae using the microalgae culture module according to the present invention.
10 is a graph showing the lipid extraction efficiency according to the present invention.
11 is a graph showing the lipid extraction efficiency according to the presence or absence of a plurality of filtration members of the present invention.
12 is a graph showing the lipid content of each of the eight microalgae extracted by the microfluidic device capable of cell culture and lipid extraction according to the present invention.

In order to fully understand the present invention, operational advantages of the present invention, and objects achieved by the practice of the present invention, reference should be made to the accompanying drawings and the accompanying drawings which illustrate preferred embodiments of the present invention.

Hereinafter, the present invention will be described in detail with reference to the preferred embodiments of the present invention with reference to the accompanying drawings. Like reference symbols in the drawings denote like elements.

"Microalgae" according to the present embodiment are collectively referred to as single-celled organisms that have photosynthetic activity with photosynthetic pigments. Microalgae according to the present embodiment include algae, diatoms, red algae, algae, algae, brown algae, Green algae, broad-leaved birds, or cyanobacteria.

In this embodiment, three kinds of micro-algae (Chloridomonas reinhardtii) (CC124, CC125, CC4348), three kinds of chlorella (Chlorella vulgaris, Chlorella protothecoides, Chlorella zofingiensis) (Neochloris oleoabundans) and Scenedesmus sp., Were used, but the present invention is not limited thereto.

In the present embodiment, "lipid" means a substance which constitutes a microalgae and does not dissolve in water but dissolves well in an organic solvent, and can be divided into a simple lipid and a complex lipid according to its component or chemical structure, and also includes triglyceride, Glycerol glycolipids, carotenoids and steroids. Especially, the lipid of microalgae contains a lot of triglyceride and has a molecular structure similar to that of petroleum oil, and its content is 10 times that of soybean, 7 ~ 8 times that of peanut.

FIG. 1 is a plan view showing a microfluidic device capable of cell culture and lipid extraction according to the present invention, FIG. 2 is an exploded perspective view showing a microfluidic device capable of cell culture and lipid extraction according to the present invention, FIG. 4 is a plan view showing a module for filtering a lipid solution according to the present invention, FIG. 5 is an enlarged view of a portion A of FIG. 3, and FIG. 6 is an enlarged view of a portion B of FIG. And Fig. 7 is an enlarged view of a portion C in Fig.

Referring to FIGS. 1 and 2, a microfluidic device 100 capable of cell culture and lipid extraction according to an embodiment of the present invention includes a microalgae culture module 200 for culturing microalgae, a microalgae culture module And a lipid solution filtration module 300 for filtering the lipid solution in which the lipid component of the microalgae cultured in the microalgae culture module 200 is dissolved.

The microalgae culturing module 200 and the lipid solution filtration module 300 according to the present embodiment are light transparent and transparent materials, and are not toxic to microalgae, can easily transfer the substances necessary for biological activity, Unbreakable materials or materials pretreated to have the above characteristics.

For example, the microalga culture module 200 and the lipid solu- tion filtration module 300 may be formed of a transparent transparent material including PMMA (polymethyl methacrylate), PS (polystyrene), and PDMS (polydimethylsiloxane) .

As described above, the microalgae culture module 200 and the lipid solution filtration module 300 are made of a transparent material, so that the cultivation of the microalgae and the lipid accumulation process can be easily and quickly monitored in real time using a microscope .

In the microfluidic device 100 capable of cell culture and lipid extraction according to an embodiment of the present invention, the microalgae culture module 200 is disposed at a lower portion, the microalgae culture module 200 is provided with a lipid solution The filtration module 300 is stacked and coupled.

A process of fabricating the microfluidic device 100 in which the microalga culture module 200 and the lipid solution filtration module 300 are combined will be described.

The microalgae culturing module 200 rotates the negative photoresist SU-8 on the silicon substrate and then forms a SU-8 mold by photo-lithography that covers the designed mask and exposes it to ultraviolet rays. SU-8 A polymer PDMS (Polydimethylsiloxane) and a hardener are mixed in a ratio of 10: 1 on the mold, and the culture chamber 210 and the lipid solution accommodating chamber 250 constituting the microalga culture module 200 to be described later are formed by photolithography do. Then, the lower part of the microalgae culture module 200 is bonded to the glass through a plasma treatment.

8, the SU-8 mold is fabricated by photolithography in which the designed mask is covered and exposed to ultraviolet rays, and the SU- A polymeric PDMS and a curing agent were mixed at a ratio of 20: 1, and a plurality of filter members 313 and the like constituting the lipid solution filtration module 300 to be described later were formed by photolithography, and they were hardened in an oven at 60 ° C for 10 minutes Is placed at a predetermined position on the upper part of the microalgae culture module 200, and further hardened in a 60 ° C oven for 30 minutes to be completely bonded to the microalgae culture module 200.

2 and 3, the microalgae culture module 200 according to the present embodiment includes a plurality of independent culture chambers 210 for culturing microalgae, and a plurality of culture chambers 210 provided adjacent to the plurality of culture chambers 210 A plurality of microalgae supply units 230 for supplying microalgae to the plurality of incubation chambers 210; a plurality of microalgae supply units 230 for supplying microalgae to the plurality of incubation chambers 210; A plurality of lipid solution receiving chambers 250 for receiving and storing the lipid dissolving solution in which the lipid components of the microalgae filtered in the lipid soluble solution filtration module 300 are stored, And a lipid solution discharging portion 270 provided adjacent to the plurality of lipid solution receiving chambers 250 and discharging the lipid solution contained in the plurality of lipid solution receiving chambers 250.

The culture chamber 210 serves to cultivate microalgae. In this embodiment, a plurality of culture chambers 210 are independently provided, so that one or more kinds of microalgae can be independently cultured at the same time.

Although eight culture chambers 210 are shown in this embodiment, the number of the culture chambers 210 is not limited thereto. In addition, although each culture chamber 210 according to the present embodiment is shown to have a diameter of 4 mm, the size of the culture chamber 210 is not limited thereto.

In this embodiment, three kinds of Chlamydomonas reinhardtii (CC124, CC125, CC4348), three kinds of Chlorella vulgaris (Chlorella vulgaris, Chlorella protothecoides, Chlorella zofingiensis), Neochloris oleoabundans and Scenedesmus sp. Were cultured in a total of 8 microalgae.

The microalgae supply unit 230 is provided in a number corresponding to the number of the plurality of culture chambers 210 and is disposed adjacent to and connected to each of the culture chambers 210 so as to be connected to the plurality of culture chambers 210 Supply microalgae. Although the microalgae supply unit 230 according to the present embodiment has a diameter of 2 mm, the size is not limited thereto.

On the other hand, the microalgae supply unit 230 may further supply a medium for culturing the microalgae. The medium for culturing the microalgae is a TAP medium and its constituents are shown in Table 1 below.

TAP Medium ingredient Content (in 1 L water) TAP salts (in 1 L water) 25 ml NH 4 Cl 15.0 g
MgSO 4揃 7H 2 O 4.0 g
CaCl 2 H 2 O 2.0 g Phosphate solution (in 100 ml water) 0.375 ml K 2 HPO 4 28.8 g
KH 2 PO 4 14.4 g Hutners trace elements 1.0 ml EDTA disodium salt 50 g (250 ml water)
ZnSO 4揃 7H 2 O (100 ml water)
H 3 BO 3 11. 4 g (200 ml water)
5.06 g (50 ml water) of MnCl 2 .4H 2 O)
1.61 g of CoCl 2 .6H 2 O (50 ml of water)
CuSO 4 · 5H 2 O 1.57g ( 50 ㎖ water)
(NH 4) 6 MO 7 O 24 · 4H 2 O 1.10g (50 ㎖ water)
FeSO 4 .7H 2 O 4.99 g (50 ml water) Tris base 2.42 g Glacial acetic acid 1.0 ml

The medium and the organic solvent supply unit 290 serve to supply a medium for accumulating lipid in the microalgae in the plurality of culture chambers 210 and an organic solvent for destroying microalgae in which lipid is accumulated.

Here, in this embodiment, the medium for lipid accumulation is TAP-N medium, which is a medium for inducing lipid accumulation in microalgae such as nitrogen nutrient deficient medium and saline-containing medium, and TAP-N medium is NH 4 Cl < / RTI > with the same molar amount of KCl.

Also, in this embodiment, the organic solvent dissolves the lipid component of the microalgae and the cell disruption in which the function of the cell membrane of the microalgae in which the lipid is accumulated is lost and the cell contents are dispersed or dissolved in water, and the organic solvent is microalgae culture Methanol, ethanol or isopropanol (IPA) may be used as long as it does not chemically react with the module 200 and the lipid solution filtration module 300 and does not cause deformation, but the claims of the present invention are not limited thereto.

3 and 5, in this embodiment, the medium and organic solvent supply unit 290 are located at the center of the microalgae culture module 200, wherein the plurality of culture chambers 210 are connected to the medium and the organic solvent supply unit 290 in the radial direction. This is so that the medium or the organic solvent can be supplied to the plurality of culture chambers 210 simultaneously or separately through one medium and the organic solvent supply unit 290. At this time, the plurality of microalgae supply units 230 may be disposed adjacent to the outside of the plurality of culture chambers 210.

Although one medium and an organic solvent supply unit 290 are illustrated in the present embodiment, the scope of the present invention is not limited thereto and a plurality of media and an organic solvent supply unit 290 may be provided.

In addition, the medium and the organic solvent supply unit 290 according to the present embodiment are shown to have a diameter of 1 mm, but the size is not limited thereto.

The culture medium and the organic solvent supply unit 290 are connected to the culture medium accommodating chamber 291 in which the TAP-N medium and the organic solvent are accommodated, the organic solvent accommodating chamber and the plurality of culture chambers 210, And a plurality of media and an organic solvent supply passage 293 for connection.

When the medium and the organic solvent are supplied to the plurality of culture chambers 210, the TAP-N medium or the organic solvent is supplied to the medium and the organic solvent accommodating chamber 291 disposed at the center of the microalga culture module 200, The TAP-N medium or organic solvent supplied to the culture medium and the organic solvent accommodating chamber 291 is supplied to each culture chamber 210 along the plurality of mediums and the organic solvent supply passage 293.

The microfluidic device 100 capable of cell culture and lipid extraction according to an embodiment of the present invention includes a pump (not shown) for supplying a culture medium and an organic solvent to the TAP-N medium and the organic solvent accommodation chamber 291 .

For example, in this embodiment, the pump may be a syringe pump, and the syringe pump may supply TAP-N medium or organic solvent at a constant rate to the medium and the organic solvent accommodating chamber 291, and the medium and the organic solvent accommodating chamber 291 ) And a plurality of culture chambers 210 to dissolve the lipid component of the microalgae in the microalgae lysate by applying pressure to the lipid solution filtration module 300 to the lipid solution reservoir chamber 250.

The culture medium according to the present embodiment and the organic solvent supply channel 293 serve to interconnect the culture chamber 210 and the culture medium and the organic solvent supply unit 290.

3 and 6, the TAP-N medium or the organic solvent supplied to the medium and the organic solvent supply unit 290 is supplied to the culture chamber 210 along the medium and the organic solvent supply path 293. The medium and the organic solvent supply passage 293 are provided to communicate with one medium and the organic solvent supply unit 290 and the plurality of culture chambers 210 in a communicative manner.

6, the medium and the organic solvent supply passage 293 are formed in a zigzag form in the medium and the organic solvent accommodating chamber 291 and the incubation chamber 210 in the medium and the organic solvent. And to prevent the TAP-N medium or the organic solvent from flowing back to the medium and the organic solvent supply unit 290 while the TAP-N medium or the organic solvent is supplied or supplied to the respective culture chambers 210 in the medium and the organic solvent supply unit 290.

In addition, the medium and the organic solvent supply passage 293 are shown to have a diameter of 3 mu m, but the size is not limited thereto.

The lipid solution receiving chamber 250 according to the present embodiment serves to receive the lipid dissolution solution in which the lipid component of the microalgae is dissolved in the microalgae lumps in the culture chamber 210.

That is, the lipid solution receiving chamber 250 is communicated with the culture chamber 210 by the lipid solution filtration module 300 to be described later, and the lipid solution dissolved by the organic solvent in the culture chamber 210 is lipid soluble And receives the lipid solution transferred through the liquid filtration module 300.

3, when a plurality of culture chambers 210 are radially disposed around the culture medium and the organic solvent supply unit 290, the plurality of lipid solution containing chambers 250 are divided into a plurality of culture chambers 210, And may be arranged radially around the medium and the organic solvent supply unit 290.

Each of the lipid solution containing chamber 250 and the culture chamber 210 may be arranged so as to be inline with respect to the medium and the organic solvent supply part 290. This is to form the shortest path in which the lipid solution in the culture chamber 210 is moved to the lipid solution containing chamber 250 by the lipid solution filtration part 310 of the lipid solution filtration module 300 to be described later .

The lipid solution discharging portion 270 according to the present embodiment is provided in a number corresponding to the number of the plurality of lipid solution receiving chambers 250 and adjacent to the respective lipid solution receiving chambers 250 And discharges the lipid solution contained in each of the lipid solution containing chambers 250. The lipid solution containing the lipid solution contained in each of the lipid solution containing chambers 250 is discharged.

The lipid solution discharging portion 270 according to the present embodiment is shown to have a diameter of 2 mm, but the size is not limited thereto.

The lipid solution filtration module 300 according to the present embodiment filters a lipid dissolving solution in which a lipid component of a microalgae in the culture chamber 210 is dissolved and supplies the lipid dissolution solution to the lipid solution receiving chamber 250 .

As described above, the lipid soluble solution filtration module 300 is a transparent transparent material that is free of toxicity to microalgae, facilitates the transfer of the substance necessary for biological activity, and does not interfere with the movement of microalgae. A material that does not have a chemical action or a material that is pretreated to have the above characteristics.

For example, the lipid solution filtration module 300 may be formed of a transparent transparent material including polymethyl methacrylate (PMMA), polystyrene (PS), and polydimethylsiloxane (PDMS).

The lipid solution filtration module 300 includes a plurality of lipid solution filtration parts 310 that connect the adjacent culture chambers 210 and the lipid solution storage chambers 250 in a pair.

The lipid solution filtration unit 310 interconnects the adjacent culture chamber 210 and the lipid solution containing chamber 250 in a pair and filters the lipid dissolution solution in the culture chamber 210, To the dissolving liquid accommodation chamber (250).

4, a plurality of lipid solution filtration units 310 are radially arranged with respect to a central portion of the lipid solution filtration module 300, and each of the lipid solution filtration units 310 is adjacent Are placed at the upper positions of the pair of culture chambers 210 and the lipid solution receiving chambers 250, respectively.

The lipid solution filtration unit 310 according to the present embodiment includes a lipid solution flow passage 311 through which the lipid solution is moved from the adjacent culture chamber 210 to the lipid solution containing chamber 250, A plurality of filtration members (not shown) are connected to the movement channel 311 and are disposed in the upper region of the culture chamber 210 and the lipid solution reservoir chamber 250, respectively, 313).

1, the lipid lysis solution contained in the culture chamber 210 is filtered by a plurality of filtration members 313 located in the upper region of the culture chamber 210 to be transported to the lipid solution transport passage 311, And is then moved to a plurality of filtration members 313 located in the upper region of the lipid solution reservoir chamber 250 and supplied to the lipid solution reservoir chamber 250. The lipid dissolution liquid supplied to the lipid solution reservoir chamber 250 is discharged to the outside through the lipid dissolution solution discharge unit 270.

The lipid solution transport passage 311 includes a plurality of filter members 313 located in the upper region of the culture chamber 210 and a plurality of filter members 313 located in the upper region of the lipid solution containing chamber 250. [ ).

The filtration member 313 is formed in a columnar shape, and a plurality of filtration members 313 are provided in the upper region of the culture chamber 210 and in the upper region of the lipid solution storage chamber 250, respectively.

Each of the plurality of filtration members 313 located in the upper region of the culture chamber 210 is immersed in the culture chamber 210 at one end and connected to the lipid dissolution liquid flow path 311 at the other end, Each of the plurality of filtration members 313 located in the upper region of the lipid solution transport passage 311 is immersed in the lipid solution reservoir chamber 250 at one end and connected to the lipid solution transport passage 311 at the other end.

As shown in FIG. 7, each of the plurality of filtration members 313 according to the present embodiment may be formed into a columnar shape having a minute-sized hollow through which the lipid-soluble solution is filtered to have a rectangular cross-section.

In this embodiment, the hollow section of the filtration member 313 may be formed to have a size of 10 μm × 10 μm in width and width, which may be changed depending on the size and type of microalgae.

Further, the height of the filtration member 313 may be 3 占 퐉, but it may be varied depending on the height difference between the culture chamber 210 and the lipid solution transport flow path 311.

When the organic solvent is introduced into the culture chamber 210 through the medium and the organic solvent supply unit 290 using the syringe pump, the microalgae are destroyed by the organic solvent and the lipids contained in the microalgae are dissolved in the organic solvent.

The lipid dissolving solution in which the lipid has been dissolved dissolves along the fine-sized hollow of the plurality of filter members 313 located in the upper region of the culture chamber 210 by the pressure applied by the syringe pump, And is moved to the lipid solution receiving chamber 250 along the micro-sized hollow of the plurality of filter members 313 located in the upper region of the lipid solution receiving chamber 250. [

At this time, the lysate of the microalgae except the lipid-soluble liquid among the microalgae of the microalgae in the culture chamber 210 remains between the plurality of columnar filter members 313, and only the lipolytic solution remains in the hollow of the filter member 313 And is then transferred to the lipid solution transport passage 311.

On the other hand, the columnar filter member 313 can be made of a material such as silicon, glass, and plastic polymer. In addition, the plastic material may be selected from the group consisting of a cycloolefin copolymer (COC), polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate (PC), polyamide (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyetheretherketone polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT) Which is selected from the group consisting of fluorinated ethylenepropylene (FEP) and perfluoralkoxyalkane (PFA) However, the present invention is not limited thereto.

A method of extracting lipid using the microfluidic device 100 according to an embodiment of the present invention will now be described.

FIG. 8 is a flow chart showing a method of extracting lipids using the microfluidic device according to the present invention, FIG. 9 is a graph showing the results of culturing 8 different microalgae using the microalgae culture module according to the present invention, 10 is a graph showing lipid extraction efficiency according to the present invention. FIG. 11 is a graph showing the lipid extraction efficiency according to the plurality of filter members of the present invention. FIG. 12 is a graph showing the lipid extraction efficiency according to the present invention. And the lipid content of each of the eight microalgae extracted by the microalgae.

Referring to FIG. 8, a method for extracting lipid using a microfluidic device 100 according to an embodiment of the present invention includes first supplying a TAP medium necessary for growth of microalgae and microalgae to a culture chamber 210, And cultured (S100). The microalgae and the TAP medium to be cultured are supplied to the respective culture chambers 210 through the microalgae supply unit 230 connected to each of the plurality of culture chambers 210.

In this embodiment, three kinds of Chlamydomonas reinhardtii (CC124, CC125, CC4348), three kinds of chlorella (Chlorella vulgaris, Chlorella protothecoides, Chlorella zofingiensis), neoprene Eight microalgae were cultivated in eight culture chambers 210 and eight different microalgae were cultured in each of eight culture chambers 210 at an initial concentration of 3.0 x 10 < 3 > cells ml < -1 & Diluted in TAP medium, and incubated for 24 hours at 40 m photon m -2 s -1 intensity and for 3 days at 23 ° C.

Then, the TAP-N medium is injected into the culture chamber 210 on the fourth day of culturing to induce the accumulation of lipid in the microalgae (S200). As shown in FIG. 9, lipid accumulation was confirmed by measuring the absorbance at a wavelength of 800 nm using a microplate reader (Infinite 200 Pro, Tecan, Middoshi) for the growth of microalgae.

The TAP medium in the culture chamber 210 is exchanged with the medium and the TAP-N medium supplied into the lipid and organic solvent-receiving chamber of the organic solvent supply unit 290. Lipid accumulation in microalgae is cultured for 24 hours at 40 m photon m -2 s -1 intensity and microalgae at 23 ° C for 4 days.

When lipid accumulation in the microalgae is induced, an organic solvent is supplied to the culture chamber 210 to break the microalgae in the culture chamber 210 (S300).

The organic solvent loses the function of the cell membrane of the microalgae in which the lipid is accumulated and dissolves the lipid component of the microalgae and cell disruption that the contents of the microalgae disperse or dissolve.

Organic solvents such as chloroform and hexane, which are commonly used for lipid extraction, have a problem of swelling and dissolving PDMS, which is a material of the microfluidic device 100 of the present invention.

Accordingly, in this embodiment, the 70% aqueous solution of methanol, ethanol or isopropanol (IPA) which does not chemically react with PDMS is supplied to the micro-algae in the culture chamber 210 to break the micro-algae. Then, in order to improve the extraction ability of the lipid-soluble solution, an organic solvent is supplied to the culture chamber 210 and then heated to a temperature of 60 ° C or higher.

Then, the lipid-soluble solution in which the lipid component of the microalgae is dissolved in the microalgae is extracted by filtration (S400).

The lipid solution filtration and extraction process is performed by using a syringe pump (PHD 2000 Advanced Syringe, Harvard Apparatus, USA), and the lipid solution in the micro-algae in the culture chamber 210 is placed in the upper region of the culture chamber 210 Is filtered through a plurality of filtration members 313 and moved along the lipid solution transport passage 311 and then along the plurality of filter members 313 located in the upper region of the lipid solution reservoir chamber 250, To the chamber 250.

10 shows a method of extracting Bligh-Dyer lipids using chloroform as a conventional organic solvent. The second method is a lipid extraction method using an aqueous solution of 70% isopurpanol (IPA) as an organic solvent in a conventional bulk state. The lipid extraction method using an aqueous solution of 70% isopropanol (IPA) as an organic solvent is compared and compared with the microfluidic device 100 of the present invention.

As shown in FIG. 10, the lipid extraction efficiency using the microfluidic device 100 of the present invention and the aqueous solution of 70% isopropanol (IPA) as an organic solvent was compared with the conventional lipid extraction method for the other eight microalgae As shown in FIG.

11 is a schematic sectional view of a microfluidic device 100 having a two-layer structure according to the present invention. In the microfluidic device 100, first, a lipid solution filtration unit 310 having a plurality of columnar filtration members 313 having micro- And the second case is a comparative analysis of a case in which a plurality of filter members 313 having a columnar shape and a fine size hollow are not provided.

As shown in FIG. 11, it can be seen that the lipid extraction efficiency is maintained at a high level when a plurality of columnar filter members 313 having micro-sized hollows are provided.

FIG. 12 is a graph showing the results obtained by using a 70% isopropanol (IPA) aqueous solution as an organic solvent in the microfluidic device 100 of the present invention, and simultaneously extracting lipid extracts from 8 different microalgae using a gas chromatograph, aglent technologies 7890A GC system ), And it was confirmed how many kinds of actual lipids and lipids were extracted from the other 8 microalgae.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Accordingly, such modifications or variations are intended to fall within the scope of the appended claims.

100: Microfluidic device 200: Microalgae culture module
210: culture chamber 230: microalgae supply unit
250: Lipid-containing solution receiving chamber 270: Lipid dissolving liquid discharging portion
290: medium and organic solvent supply part 291: medium and organic solvent reception chamber
293: medium and organic solvent supply channel 300: lipid solution filtration module
310: lipid solution filtration part 311: lipid solution liquid flow path
313: Filter element

Claims (17)

A microalga culture module having a plurality of independent culture chambers for culturing microalgae; And
Wherein the micro-algae culture module is stacked and bonded to the upper portion of the micro-algae culture module, wherein the micro-algae lysate, which is in contact with the plurality of culture chambers and is crushed by the organic solvent supplied to the plurality of culture chambers, A microfluidic device capable of cell culture and lipid extraction comprising a lipid solubilization filtration module for filtering lipid solubilization solution. The method according to claim 1,
In the microalgae culture module,
And a plurality of lipid solution containing chambers provided adjacent to the plurality of culture chambers and communicating with the lipid solution filtration module to receive the filtered lipid solution, . 3. The method of claim 2,
The lipid solution filtration module comprises:
And a plurality of lipid solution filtration units connecting the adjacent culture chambers and the lipid solution storage chamber in a pair so as to communicate with each other and supplying the lipid solution to the lipid solution storage chamber from the culture chamber by filtration Microfluidic device capable of cell culture and lipid extraction. The method of claim 3,
Wherein each of the plurality of lipid solution filtration units comprises:
A lipid solution transport passage through which the lipid dissolution solution moves from the adjacent culture chamber to the lipid solution storage chamber; And
A plurality of filtration members which are connected to the lipid solution transport passage and are connected to the lipid solution transport passage and disposed in the upper region of the culture chamber and the lipid solution storage chamber, ≪ / RTI &
The lipid solution contained in the culture chamber is filtered by the plurality of filter members located in the upper region of the culture chamber and is moved to the lipid solution transport passage, Wherein the microfluidic device is capable of performing cell culture and lipid extraction. 5. The method of claim 4,
Wherein each of the plurality of filter members is formed in a columnar shape,
The plurality of filtration members positioned in the upper region of the culture chamber are connected to the liposoluble solution transport channel in such a manner that one end thereof is immersed in the culture chamber and the other end is connected to the lipid-
Wherein the plurality of filtration members located in the upper region of the lipid solution storage chamber are connected to the lipid solution transport passage through one end thereof so as to be immersed in the lipid solution reservoir chamber and the other end to be connected to the lipid solution transport passage, Device. The method according to claim 1,
In the microalgae culture module,
A culture medium connected to the plurality of culture chambers to supply a medium for accumulating lipids in the microalgae and an organic solvent for crushing the microalgae in the plurality of culture chambers and an organic solvent supplier And a microfluidic device capable of lipid extraction. The method according to claim 6,
The culture medium and the organic solvent supply unit may include,
A medium and an organic solvent receiving chamber in which the medium and the organic solvent are accommodated; And
And a plurality of culture media and an organic solvent supply passage communicably connecting the culture medium, the culture medium accommodating chamber and the culture medium,
The medium and the organic solvent supply flow path are,
A microfluidic device capable of cell culture and lipid extraction in which a culture medium and an organic solvent are formed in a zigzag form from the culture medium and the organic solvent receiving chamber into the culture chamber. 8. The method of claim 7,
Wherein the plurality of culture chambers are arranged radially around the culture medium and the organic solvent receiving chamber,
Wherein the plurality of lipid solution containing chambers are disposed adjacent to the outside of the plurality of culture chambers. 8. The method of claim 7,
A microfluidic device capable of cell culture and lipid extraction, further comprising a pump for supplying a culture medium and an organic solvent to the culture medium and the organic solvent accommodation chamber. 3. The method of claim 2,
In the microalgae culture module,
A plurality of microalgae supply units connected to the plurality of culture chambers to supply the microalgae to the plurality of culture chambers, respectively; And
And a lipid dissolution liquid discharge unit connected to the plurality of lipid solution storage chambers to discharge the lipid solution contained in the plurality of lipid solution storage chambers. The method according to claim 1,
The microalgae culture module and the lipid solution filtration module,
A microfluidic device capable of cell culture and lipid extraction, which is formed of a transparent transparent material including polymethyl methacrylate (PMMA), polystyrene (PS), and polydimethylsiloxane (PDMS). Feeding a microalgae and a medium for growing the microalgae to a culture chamber of the microalgae culture module to cultivate the microalgae;
Accumulating lipids in the microalgae;
Supplying an organic solvent to the culture chamber to crush the microalgae in the culture chamber; And
A method for extracting lipids using a microfluidic device, comprising the steps of: filtering a lipid dissolving solution in which a lipid component of the microalgae is dissolved in a crushed product of the microalgae using a lipid solubilization filtration module. 13. The method of claim 12,
The step of filtering and extracting the lipid-
The lipid dissolution solution in the microalgae is filtered by a plurality of filtration members of the lipid solution filtration module, which is located in an upper region of the culture chamber and has a micro-sized hollow in which one end is immersed in the culture chamber, The lipid solution is moved to a plurality of filtration members, one end of which is located in an upper region of the lipid solution containing chamber provided adjacent to the culture chamber of the microalgae culture module, and immersed in the lipid solution containing chamber, A lipid extraction method using a microfluidic device accommodated in an accommodating chamber. 13. The method of claim 12,
The step of accumulating lipids in the microalgae comprises:
The culture medium in the culture chamber was replaced with a nitrogen-deficient medium, and a 24-hour continuous light condition of 40 m photon m -2 s -1 intensity and 23 Lt; RTI ID = 0.0 > C < / RTI > for 4 days. 13. The method of claim 12,
The step of crushing the microalgae comprises:
Wherein the organic solvent is supplied to the culture chamber and then heated. 13. The method of claim 12,
Wherein the organic solvent comprises methanol, ethanol, and isopropanol. 13. The method of claim 12,
The step of culturing the microalgae comprises:
40 m photon m -2 s -1 intensity for 24 hours and a microfluidic device for culturing the microalgae at 23 ° C for 3 days.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210093574A (en) 2020-01-20 2021-07-28 고려대학교 산학협력단 Microfluidic chip for the production of useful materials of microorganisms
WO2023168857A1 (en) * 2022-03-09 2023-09-14 苏州大学 Multi-organ-on-chip and application thereof in drug evaluation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210093574A (en) 2020-01-20 2021-07-28 고려대학교 산학협력단 Microfluidic chip for the production of useful materials of microorganisms
WO2023168857A1 (en) * 2022-03-09 2023-09-14 苏州大学 Multi-organ-on-chip and application thereof in drug evaluation

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