KR20120002101A - Method and device for producing cell and fat solubles material by culturing cell - Google Patents

Method and device for producing cell and fat solubles material by culturing cell Download PDF

Info

Publication number
KR20120002101A
KR20120002101A KR1020100062817A KR20100062817A KR20120002101A KR 20120002101 A KR20120002101 A KR 20120002101A KR 1020100062817 A KR1020100062817 A KR 1020100062817A KR 20100062817 A KR20100062817 A KR 20100062817A KR 20120002101 A KR20120002101 A KR 20120002101A
Authority
KR
South Korea
Prior art keywords
fat
cells
soluble
solvent
tank
Prior art date
Application number
KR1020100062817A
Other languages
Korean (ko)
Inventor
김성천
Original Assignee
김성천
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 김성천 filed Critical 김성천
Priority to KR1020100062817A priority Critical patent/KR20120002101A/en
Publication of KR20120002101A publication Critical patent/KR20120002101A/en

Links

Images

Classifications

    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material

Abstract

The present invention relates to a method and apparatus for producing intact cells and fat-soluble substances from cell culture fluids at low cost and high efficiency. Apparatus (1) for producing cells and fat-soluble substances through cell culture according to the present invention includes a culture tank (10) for culturing cells containing fat-soluble substances; A solvent bath 20 for storing a fat soluble material extraction solvent in which the fat soluble material is dissolved; A mixing tank 30 for mixing the culture solution of the cells from the culture tank 10 and the fat-soluble substance extraction solvent from the solvent tank 20; An agglomeration tank (40) having an ultrasonic resonance field generating device (41) and applying an ultrasonic resonance field to the mixture from the mixing tank (30) to aggregate the cells; A fractionation tank (50) for dividing the mixture from the flocculation tank (40) into a fat-soluble substance-solvent in which the fat-soluble substance of the cell culture solution is dissolved in the fat-soluble substance extracting solvent; A cell-receiving device 80 for receiving or processing the cells fractionated from the fractionation tank 50; And a fat-soluble substance-receiving device 90 for receiving or treating the fat-soluble substance-solvent fractionated from the fractionation tank 50; It includes.

Description

Method and device for producing cell and fat soluble substance through cell culture {Method and Device for Producing Cell and Fat Solubles Material by Culturing Cell}

The present invention relates to a method and apparatus for producing cells and fat-soluble substances through cell culture, and more particularly, to a method and apparatus for producing intact cells and fat-soluble substances from cell culture medium at low cost and high efficiency.

Various biomasses obtained from the cultured cells are used in various ways, such as raw materials for health functional foods and pharmaceutical products, and their usability is being expanded to produce feeds, alternative energy raw materials, and biochemicals.

Separation of cells or microorganisms cultured in a bioreactor or fermentor uses an ultrasound resonance field, a filtration membrane and a centrifuge. Separation apparatus using the ultrasonic resonance field used for the separation of cells or microorganisms is a device that can perform almost permanent filtration function while consuming very little power with only a simple device, the biggest advantage is that without the addition of other mechanical devices The cells can be separated while remaining intact.

Application of an ultrasonic resonance field can overcome many of the problems caused when using a conventional filter membrane. For example, the conventional filtration membrane method has a need to replace the membrane due to the blockage of the membrane when used for a long time, the ultrasonic resonance field can not only reduce this inconvenience, but also prevent the contamination of the membrane has a long-term stability. Centrifugal separation, which is one of the existing cell recovery methods, is difficult to apply to on-line systems of fermenters, while ultrasonic resonance fields can be applied to on-line systems. It can be applied to on-line clarification and perfusion culture of fermenter. Therefore, if the ultrasonic resonance field separator is applied to the recovery of cells or extracellular products, the conventional ultrafiltration and microfiltration processes may be replaced.

Separation of cells or microorganisms by using an ultrasonic resonance field is known to have little effect on microorganisms or the life and death of cells because they use very little power, and in perfusion culture for high concentration culture of plant cells, insect cells and animal cells, etc. It is used as a device for maintaining cells without killing, and is also used for continuous perfusion culture for production of monoclonal antibodies.

Various cells contain not only lipids, proteins and carbohydrates as primary metabolites but also bioactive substances as secondary metabolites. The most abundant substance that can be obtained from cells is protein, but protein is a component that should be considered mainly in terms of using cell itself, and in terms of using substances produced by cells, lipids, carbohydrates, pigments, vitamins, minerals And special ingredients. In addition, carbohydrates, proteins, nucleic acids, and lipids, intermediate metabolites that occur during the synthesis and degradation of metabolism and metabolic regulators are also essential compounds of the cell, these compounds can be obtained from the cell.

Plants also contain a number of compounds that are not essential for survival (primarily secondary metabolites), and more than 100,000 species have been known to date. These compounds often have one compound distributed only on several or several plants. . Secondary metabolites can be broadly divided into alkaloids, phenolic compounds, terpenes, and other compounds depending on the structure and synthesis process.

The conventional extraction method for obtaining various useful materials from cell biomass is to remove water as much as possible through centrifugation, filtration, and drying processes that inhibit the growth of cells, and then separate and purify useful materials through cell crushing process. .

However, the conventional extraction method has difficulty in culturing or reusing cells due to the destruction of cells in the dehydration and extraction processes and the release and loss of other useful substances. Have.

In order to solve the problem of the conventional method for extracting useful substances from cells (microalgae), the present inventors of the Republic of Korea Patent Application No. 10-2010-0043955 (2010. 05. 11 application) 'Biofuel extraction by microalgal culture Method and Apparatus' and Korean Patent Application No. 10-2010-0059100 (filed Jun. 22, 2010) have proposed a method and apparatus for producing high density microalgae and concentrated fat-soluble substances by culturing microalgae.

An object of the present invention is to mix a cell culture medium containing a fat-soluble material with a solvent-soluble material extraction solvent so that the fat-soluble material of the cell is dissolved in the fat-soluble material extraction solvent, agglomerate the cells by applying an ultrasonic resonance field to the mixture, and then the mixed solution Is fractionated into cells and a fat soluble substance-solvent (a solution in which the fat soluble substance is dissolved in a fat soluble substance extracting solvent), and then, the cultured cells are cultivated, and the cultured cells are re-fractionated into fractionated fat soluble substance-solvents. By repeating the process as many times as necessary, the final cell obtains a fat-soluble substance-solvent containing a fat-soluble substance that has been concentrated to the desired level with the cells having the desired density. It can be used for the production of functional foods, biofuels and hydrolysates, and from the latter fat-soluble solvents Submitted a fat-soluble substance is to provide a method and apparatus for producing cells and fat-soluble substance without cell damage at a low cost through a cell culture that enables the production of pharmaceuticals, health functional food, bio-diesel in high yield.

According to the present invention, a method for producing cells and fat-soluble substances through cell culture is provided.

Method for producing cells and fat-soluble substances according to the present invention, the process of culturing cells containing fat-soluble substances; Mixing the fat soluble material extraction solvent in which the fat soluble material is dissolved with the culture medium of the cell to contact the fat soluble material extraction solvent with the cell culture solution to dissolve the fat soluble material of the cell in the fat soluble material extraction solvent; Applying an ultrasonic resonance field to the mixture to aggregate the cells in the mixture; Fractionating the cells in which the fat-soluble material is dissolved in the fat-soluble material extraction solvent and the cells are free of cell damage; Obtaining the fractionated cells and the fat-soluble substance-solvent, respectively; .

Preferably, the method for producing cells and fat-soluble substances according to the present invention comprises the steps of culturing the fractionated cells between the fractionation process and the process of obtaining the cells and the fat-soluble substance; Re-splitting said cells cultured using said fat soluble material extraction solvent or fractionated fat soluble material-solvent; And finally performing the culturing of the cells and the re-fractionation process once or twice or more times, and finally fractionating the fat-soluble substance-solvent containing the concentrated fat-soluble substance and the cells of high density. It further comprises, so that after the final fractionation process, it is possible to obtain the fat-solvent-solvent containing the cells of high density and the concentrated fat-soluble material.

Preferably, when the cell culture medium and the fat soluble material extraction solvent are mixed, at least one of a process of vibrating and pulverizing the cell culture solution and agitating the mixture, the cell culture medium and the fat soluble material extraction solvent Can increase contact.

According to the present invention, an apparatus for producing cells and fat-soluble substances through cell culture is provided.

An apparatus for producing cells and fat soluble substances according to the present invention includes a culture tank 10 for culturing cells containing fat soluble substances; A solvent bath 20 for storing a fat soluble material extraction solvent in which the fat soluble material is dissolved; A mixing tank 30 for mixing the culture solution of the cells from the culture tank 10 and the fat-soluble substance extraction solvent from the solvent tank 20; An agglomeration tank (40) having an ultrasonic resonance field generating device (41) and applying an ultrasonic resonance field to the mixture from the mixing tank (30) to aggregate the cells; A fractionation tank (50) for dividing the mixture from the flocculation tank (40) into a fat-soluble substance-solvent in which the fat-soluble substance of the cell culture solution is dissolved in the fat-soluble substance extracting solvent; A cell-receiving device 80 for receiving or processing the cells fractionated from the fractionation tank 50; And a fat-soluble substance-receiving device 90 for receiving or treating the fat-soluble substance-solvent fractionated from the fractionation tank 50; It includes.

Preferably, the apparatus for producing cells and fat-soluble substances according to the present invention, the cell-circulation line (60) for circulating the cells fractionated from the fractionation tank (50) to the culture tank (10); And a solvent-circulation line 70 for circulating the fractionated fat-soluble substance-solvent from the fractionation tank 50 into the solvent bath 20. It includes more.

The cell-receiving device 80 is the high-density cells finally fractionated from the fractionation tank 50 after one or two or more cultures in the culture vessel 10 through the cell-circulation line 60 Accept or process; The fat-soluble substance-receiving device 90 is the concentrated fraction obtained by re-fractionation once or twice or more in the fractionation tank 50 through the solvent-circulation line 70 and finally fractionated from the fractionation tank 50. Fat-Soluble-Receive or process solvent.

Preferably, the cell and the fat-soluble material production apparatus according to the present invention, the cell culture medium of the culture tank 10 and the agent for supplying a fixed amount of the fat-soluble material extraction solvent of the solvent tank 20 to the mixing tank 30 1 peristaltic pump 2; A second to transfer the cells fractionated in the fractionation tank 50 to the culture tank 10 or to the cell-receptor 80 selectively through the cell-circulation line 60 according to the density thereof Peristaltic pump 3; And a third peristaltic pump 4 which transfers the fat-soluble material-solvent fractionated in the fractionation tank 50 to the solvent tank 20 or to the fat-soluble material-receiving device 90 according to its concentration. It further includes.

Preferably, the ultrasonic resonance field generating device 41 is an acoustic cell filter 41 including an acoustic chamber 42, an ultrasonic oscillator 43, an ultrasonic vibrator 44 and a reflecting film 45; The first traveling wave propagated from the ultrasonic vibrator 44 by the ultrasonic oscillator 43 and the second traveling wave reflected by the reflecting film 45 and propagated in the opposite direction to the first traveling wave collide with each other. By generating standing waves in the acoustic chamber 42 between the ultrasonic vibrator 44 and the reflecting film 45, the ultrasonic resonance field caused by the standing waves aggregates the cells to form cell aggregates.

Preferably, the mixing tank further comprises at least one of a vibratory grinding device for vibrating and pulverizing the cell culture and an agitating device for stirring the mixture so as to increase the contact between the cell culture and the fat-soluble substance extraction solvent. do.

Preferably, the soluble solvent extraction solvent is a hydrocarbon solvent.

In the present invention, 'lipophilic substance' is an oil-soluble substance, phospholipids, free fatty acids, esters of fatty acids, triacylglycerols, sterols and sterol esters, carotenoids, xanthophylls (for example, oxycarotenoids), hydrocarbons, alkaloids, It includes phenolic compounds, terpenes, isoprenoid-derived compounds, and other substances that are soluble in oil.

In the present invention, the cells include animal cells, plant cells, fungi, diatoms, coarse imitation steels, flaky imitation birds, red algae, red algae, green algae, and prokaryotes.

Method and apparatus for producing cells and fat-soluble substances through cell culture according to the present invention, by mixing the culture medium of the cells containing the fat-soluble substance with the solvent-soluble material extraction solvent to dissolve the fat-soluble material of the cell in the fat-soluble material extraction solvent, After the ultrasonic resonance field is applied to the cells, the cells are aggregated, and the mixed solution is fractionated into the cells and the fat-soluble material-solvent, and then the cultured cells are cultivated, and the cultured cells are extracted from the fat-soluble material or the fat-soluble material-solvent. As a method and apparatus for acquiring fat-soluble solvents containing fat-soluble substances together with cells without cell damage, at high density and high concentration without the need for separate pretreatment, by repeating the process of re-fractionation as necessary. With the high production yield at cost, it is effective to obtain cells and fat-soluble substances without cell damage, This will improve the productivity of cells (biomass) that can be used for the production of bio-compounds, pharmaceuticals, health functional foods, biofuels and proteolytic products, and fat-soluble substances that can be used for the production of pharmaceuticals, health functional foods, and biodiesel. It can be maximized.

Unlike the conventional culture, harvesting and extraction methods that require dehydration, the method and apparatus according to the present invention can be used at low cost through high-density cells and concentrated fat-soluble substances through repeated culture and refraction using peristaltic pumps. Can produce.

Since the method and apparatus according to the present invention extract the fat soluble substance without destroying the cells, it is possible to prevent the loss of other useful components in the cell, thereby improving the usefulness of the cells from which the fat soluble substance is extracted, Maximize cell productivity.

The method and apparatus according to the present invention can further improve the production yield of fat-soluble substances and cells through effective contact between the cell culture solution and the fat-soluble substance extraction solvent.

1 is a block diagram of an exemplary apparatus to which a method for producing cells and fat-soluble substances through cell culture according to the present invention is applied;
2 is a graph showing the effect of the fat-soluble substance extraction solvent on the survival of cells,
Figure 3 is a graph showing the effect of the fat-soluble material extraction solvent (alkanes) and vibration grinding on the extraction of fat-soluble material from the cells,
Figure 4 is a graph showing the effect of the fat-soluble material extraction solvent (alkanes) and the ultrasonic resonance field on the extraction of fat-soluble material from the cells,
Figure 5 is a graph of the GC-TOF-MS analysis results for biodiesel extracted from microalgae by the present invention.

Hereinafter, a method and apparatus for producing cells and fat-soluble substances through cell culture according to the present invention will be described in detail. The following examples are illustrative of the present invention but are not intended to limit the scope of the present invention.

First, with reference to Figure 1 will be described an apparatus 1 for producing cells and fat-soluble substances through cell culture according to the present invention.

As illustrated in FIG. 1, the apparatus 1 according to the present invention basically includes a culture tank 10, a solvent tank 20, a mixing tank 30, a flocculation tank 40, a fractionation tank 50, Cell-receiving device 80 and fat-soluble material-receiving device 90, preferably further comprises a cell-circulation line 60 and a solvent-circulation line 70.

The culture tank 10 is a device for culturing cells and supplying cell culture fluid. The culture tank 10 can use a wide range of systems suitable for culturing cells. The culture tank 10 includes ponds, artificial open field culture facilities, bioreactors, plastic bags, tubes, fermenters, shake flasks, and pneumatic lift columns. lift columns), and there are no restrictions on the type of cells that can be cultured. The culture method may be independent nutrition or heterotrophic culture alone, or independent culture and independent culture after heterotrophic culture or heterotrophic culture.

The solvent bath 20 is a device for storing and supplying a fat-soluble material extraction solvent for extracting the fat-soluble material contained in the cells of the cell culture solution.

The mixing tank 30 is a fat-soluble material contained in the cells of the cell culture solution by uniformly mixing the cell culture medium from the culture tank 10 and the fat-soluble material extraction solvent from the solvent tank 20 to contact them. It is a device that is dissolved in a material extraction solvent to produce a fat-soluble material-solvent. Preferably, the mixing tank 30 mixes the cell culture solution and the fat-soluble substance extraction solvent in an approximately 5: 1 ratio.

Preferably, the mixing tank 30 may be equipped with a vibration grinding device 31 to vibrate the cell culture solution. The vibration grinding device 31 preferably treats the culture solution by ultrasonic waves. Vibratory grinding breaks up molecular aggregates to separate or permeate them. Vibration pulverization improves the extraction efficiency of fat-soluble substances by making the cell culture medium into small particles so that the cells are exposed (contacted) with more solvent-soluble solvents.

In addition to or in place of the vibration grinding device 31, a stirring device 32 for agitating the mixture of the cell culture liquid and the fat-soluble material extraction solvent may be provided, and the stirring device 32 may also be used with the cell culture solution. Improve the extraction efficiency of fat-soluble substances by increasing the contact of the fat-soluble substance extraction solvent.

Preferably, the cell culture solution of the culture tank 10 and the fat-soluble substance extraction solvent of the solvent tank 20 are transferred to the mixing tank 30 at a predetermined flow rate through the first peristaltic pump 2.

To this end, the line 11 from the culture vessel 10 and the line 21 from the solvent vessel 20 are each connected to the first peristaltic pump 2, respectively, and the line from the first peristaltic pump 2. 33 is connected to the mixing tank 30.

The agglomeration tank 40 applies an ultrasonic resonance field to the mixture transferred from the mixing tank 30 through the line 34 from the mixing tank 30 so that the cells of the cell culture liquid from which the fat-soluble substance is extracted are aggregated with each other. It is a device to promote the precipitation of cells in the fractionation process of.

In the flocculation tank 40, an ultrasonic resonance field generating device 41, which can be represented by, for example, an acoustic cell filter 41 (Nature Biotechnology 12, 281-284 (1994)), is provided. Ultrasonic Resonance Field is applied to allow cells to aggregate.

The illustrated acoustic cell filter 41 includes an acoustic chamber 42, an ultrasonic generator 43, an ultrasonic transducer 44, and a reflector 45, and the cells of the mixture. An ultrasonic resonance field is applied to the cells so that the cells aggregate in the ultrasonic resonance field to form aggregates.

As the ultrasonic resonance field generating device 41 such as the acoustic cell filter 41 is installed in the agglomeration tank 40, the ultrasonic oscillator 44 generates the first traveling wave by the action of the ultrasonic oscillator 43, and the ultrasonic vibrator. The first traveling wave applied from the 44 to the reflective film 45 is reflected by the reflective film 45 in the reverse direction to generate the second traveling wave, which is reflected by the first traveling wave generated by the ultrasonic vibrator 44 and the reflective film 45 and is reversed. The second traveling wave traveling in the direction collides in the acoustic chamber 42 to generate a standing wave between the ultrasonic vibrator 44 and the reflective film 45.

In the specific example shown in FIG. 1, an example in which the mixing tank 30 and the coagulation tank 40 are constructed in separate configurations is illustrated, but the mixing tank 30 and the coagulation tank 40 are indicated by dotted lines. ) Can also be integrated into a single reactor (100) in which the function is integrated.

The fractionation tank 50 transfers the mixture transferred from the flocculation tank 40 through the line 51 from the flocculation tank 40 to the fat-soluble substance-solvent (layer) (the fat-soluble substance in the cell culture is a fat-soluble substance extraction solvent). Solution) and cells (layers). That is, in the fractionation tank 50, the mixture from the flocculation tank 40 is fractionated into the upper fat-soluble substance-solvent (layer), the middle water (layer), and the lower cell (layer).

As will be described later, the lower cells (layers) fractionated in the fractionation tank (50) are transferred to the cell-receptor (80) for later use in the intended use, and the upper fat-soluble material-solvent (layer) is The fat-soluble material-receiving device 90 is then used for subsequent desired use.

The cell-circulation line 60, which can be optionally added, is a line for selectively circulating the cells fractionated in the lower layer in the fractionation tank 50 back to the culture tank 10. That is, in the preferred embodiment, the cells of the fractionation tank 50 are not directly transferred to the cell-receptor 80, but are re-supplied to the culture tank 10 through the cell-circulation line 60.

The solvent-circulation line 70 which can be optionally added is a line for selectively circulating the oil-soluble substance-solvent fractionated in the upper layer in the fractionation tank 50 back to the solvent bath 20. That is, in the preferred embodiment, the fat-soluble material-solvent of the fractionation tank 50 is not directly transferred to the fat-soluble material-receiving device 90, but is re-supplied to the solvent tank 20 through the solvent-circulation line 70. do.

Thereafter, the circulated cells are cultivated in the culture tank 10, and the cultivated cells and the fat-soluble material extraction solvent or the circulated fat-soluble material-solvent of the solvent tank 20 are supplied and mixed again to the mixing tank 30. After further dissolving the fat-soluble substance from the cells, the mixture is supplied to the flocculation tank 40 and the fractionation tank 50 again, and the process of aggregation and fractionation is repeated.

Preferably, the culturing and re-fractionation of such cells can be repeated one or more times until the cells are densified to the desired density and the fat-soluble material dissolved in the fat-soluble material-solvent is concentrated to the desired degree. .

The cell-receiving device 80 is densified to the desired density of cells cultivated one or more times while being directly transferred to the cells once fractionated in the fractionation tank 50 or circulating the cell-circulation line 60. When the high-density cells are transferred from the fractionation tank 50, the device is temporarily accommodated or subjected to a desired treatment for subsequent treatment.

For example, in the cell-receiving device 80, various kinds of useful substances such as bio-compounds, medicines, health foods, biofuels, and protein hydrolysates are processed from the cells by treating the high-density cells with various treatments such as ethanol fermentation, butanol fermentation, and organic acid fermentation. Temporarily receive prior to production or delivery to processing equipment to produce such useful materials.

For example, if the cell-receptor 80 performs various fermentations such as ethanol fermentation, the cell-receptor 80 becomes a fermenter, and the cell-receptor 80 is temporarily transferred before being sent to a separate fermenter. In the case of storing high density cells, the cell-receiving device 80 becomes a storage tank.

The fat-soluble material-receiving device 90 receives the fat-soluble material-solvent fractionated once in the fractionation tank 50 or re-fractions one or more times in the fractionation tank 50 while circulating the solvent-circulation line 70. When the fat-soluble substance of the solvent-soluble fat-soluble substance is concentrated to the desired concentration, the fat-soluble substance-solvent is transferred from the fractionation tank 50, and then temporarily received for the desired treatment or the apparatus for the desired treatment.

For example, the fat-soluble substance-receiving device 90 extracts the fat-soluble substance from the fat-soluble substance-solvent and performs various treatments to produce various useful substances such as medicines, health functional foods, biodiesel, etc. Temporarily receive prior to sending to processing equipment for production.

For example, if the fat-soluble substance-receptor 90 extracts the fat-soluble substance from the fat-soluble substance-solvent and produces biodiesel from the extracted fat-soluble substance, the fat-soluble substance-receptor 90 is a distillation-biodiesel production tank. When the fat-soluble material-storage is temporarily stored before sending the fat-soluble material-solvent to a separate distillation-biodiesel production tank, the fat-soluble material-receiving device 90 becomes a storage tank.

Preferably, the device 1 of the present invention, according to the density of the cells fractionated in the fractionation tank 50 is selectively transferred to the culture tank 10 through the cell-circulation line 60 or the cell-receiving device ( And a second peristaltic pump 3 which is transferred to 80. To this end, a second peristaltic pump 3 is installed in the cell-circulating line 60 between the fractionation tank 50 and the culture vessel 10, and an additional line 81 from the second peristaltic pump 3 is provided. Is connected to the cell-receiving device 80.

In a preferred embodiment, if the density of the cells of the fractionation tank 50 does not reach the desired density by the action of the second peristaltic pump 3, the cells are repeatedly circulated to the culture tank 10 to be repeated one or more times Cultivation, fat soluble lysis of cells, aggregation and fractionation of cells and transfer to cell-receptor 80 at desired density.

Preferably, the apparatus 1 of the present invention is a third interlocking unit for transferring the fat-soluble substance-solvent fractionated in the fractionation tank 50 to the solvent tank 20 or the fat-soluble substance-receiving apparatus 90 according to the concentration thereof. A pump 4. To this end, a third peristaltic pump 4 is installed in the solvent-circulation line 70 between the fractionation tank 50 and the solvent bath 20, and an additional line 91 from the third peristaltic pump 4 is provided. Fat-soluble material-receiving device (90).

In a preferred embodiment, if the concentration of the fat-soluble material of the fraction tank 50-solvent of the fat-soluble material of the fraction tank 50 does not reach the desired concentration by the action of the third peristaltic pump 4, the fat-soluble material-solvent again the solvent tank 20 It is recycled to and used for refraction once or more, and when it reaches the desired concentration it is transferred to the fat-soluble substance-receptor 90.

Hereinafter, a method of producing cells and fat-soluble substances through cell culture according to the present invention will be described with reference to FIGS. 1 to 5.

1. Cell (High Density) Culture

Cells cultured in the culture tank 10 include animal cells, plant cells, fungi, diatoms, coarse wool, complex green algae, cyanobacteria, red algae, green algae, prokaryotes, and the like.

For example, Chlorella protothecoides can be used in the present invention. C. protothecoides can be cultured at 10 times higher cell density than most microalgae, which is very suitable for securing biomass. C. protothecoides can harvest biomass at yields up to 35 gfw / L under ideal conditions in heterotrophic conditions, and store 55% of the biomass as fat-soluble.

Given the relatively constant rate of production of fat-soluble substances by cells, it is natural that higher biomass densities will result in higher total amounts of useful substances produced per volume. Current conventional fermentation methods for growing cells produce a biomass density of about 50 to about 80 g / L or less.

The inventors have found that by applying the method of the present invention, biomass density significantly higher than currently known biomass density can be achieved.

Preferably, the process of the invention is at least about 100 g / L, more preferably at least about 130 g / L, more preferably at least about 150 g / L, even more preferably at least about 170 g / L, most preferably A biomass density of cells in excess of 200 g / L is achieved.

Thus, at such high cell biomass densities, although the rate of production of useful materials in the cells is slightly reduced, the total usefulness production rate per volume is significantly higher than currently known processes.

In a preferred embodiment, the method of the present invention, after culturing the cells, after primary mixing of the cell culture medium and the fat-soluble material extraction solvent, agglomeration and fractionation of the cells, cultivated the fractionated cells and the cultured cells again The process of mixing, coagulating, and re-fractionation with a fat-soluble substance extraction solvent or a fat-soluble substance-solvent (a solution in which the fat-soluble substance of the cell culture is dissolved in the fat-soluble substance extraction solvent) is repeated as many times as necessary. High productivity is ensured by densely culturing the cells to the desired density.

C. protothecoides can be heterotrophically grown on glucose or corn sweetener hydrolysates (CSH). Heterotrophic growth can increase fat-soluble content and reduce direct dependence on solar energy. The energy density of biodiesel produced from C. protothecoides is substantially equivalent to that of petroleum-based diesel. Chlorella is easy to engineer by molecular biological methods and can be cultured in large-scale photobiotors with enhanced CO 2 .

2. Extraction of fat-soluble substances from cells

A major cost associated with the production of biomass and useful materials using cells arises from the harvesting of cells from large volumes of culture. In the conventional fat-soluble material production method, which involves harvesting cells from cell culture, drying and destroying them to extract fat-soluble substances, the cost of this process accounts for 40-60% of the total cost.

Conventional methods are difficult to harvest other biomolecules other than fat-soluble substances due to cell breakdown in the extraction of fat-soluble substances, but the present invention alleviates and overcomes these conventional problems by low-cost, non-destructive recycle culture.

The fat-soluble substance extraction solvent used in the present invention is a solvent which is highly selective for the fat-soluble substance and is biologically suitable and can be contacted with the cell without a significant loss in cell activity. In general, the number of octanols (log Poct, octanol water Log of partition coefficient) is greater than or equal to 5 (Dodecanone is an exception to this rule). Hexane and heptane are toxic to cells in a solvent having an octanol number of 4-5, and decanol and dipentyl ether are harmless to cells.

Exemplary fat soluble extracting solvents applicable to the present invention include 1,12-dodecanedioic acid diethyl ether, n-hexane, n-heptane (n-heptane), n-octane, n-dodecane, n-dodecane, dodecyl acetate, decane, dihexyl ether, isopar ), 1-dodecanol, 1-octanol, butyoxyethoxyehteane, 3-octanone, cyclic paraffins, varsol, isoparaffin ( isoparaffins, branched alkane, oleyl alcohol, dihecylether, 2-dodecane and the like.

The fat-soluble substance extraction solvent used in the present invention may include one or more C4-C16 hydrocarbons, and may include C10, C11, C12, C13, C14, C15 or C16 hydrocarbons.

Ultrasonic irradiation to the microorganisms without cell damage by the vibration crushing device 31 is dose-dependent at low frequencies. As the frequency increases, the microorganisms survive long irradiation times. Various frequency and intensity studies have been conducted to determine the appropriately subdivided frequency range and intensity over different exposure times at a frequency regime (20 kHz to 1 MHz) that has no effect on cell activity and can optimize the extraction of fat-soluble substances. And the exposure time influenced the extraction efficiency.

It can be used to optimize the extraction of fat-soluble substances without damaging the cells with ideal intensity over different exposure times in the appropriate frequency range (20 kHz to 60 kHz). In other words, frequency, intensity and exposure time affect the extraction efficiency of fat-soluble substances. Since cell size, cell morphology, cell wall composition and physiological state are complex effects on the interaction between cells and ultrasound, 20 kHz and 1 MHz, 20-100 kHz, 20-60 kHz, 30 The ideal frequency can be determined from various frequencies such as -50 kHz or 40 kHz. With the proper combination of solvent-soluble extraction solvent and vibration pulverization, extraction efficiency of oil-soluble substances (10% of total cell fatty acids) can be achieved by almost 100%.

Even when agitating the cell culture and the fat soluble material extraction solution mixture with the agitator 32 instead of the vibration pulverization, the contact between the cell culture and the fat soluble material extraction solvent is increased (improved), thereby improving the extraction efficiency of the fat soluble material like vibration grinding. Can be. It is of course also possible to carry out vibration grinding and stirring together.

3. Cell Aggregation Without Cell Damage Using Ultrasonic Resonance Field

Ultrasonic resonance field is applied to the agglomeration tank 40 so as to perform cell movement, aggregation, and separation accurately and efficiently without damaging the cells. To this end, the agglomeration tank 40 has an ultrasonic resonance field generator 41. Is installed.

As an example of the ultrasonic resonance field generating device 41, the aggregation of the cells by the acoustic cell filter 41 is made by ultrasonic standing waves generated by the ultrasonic vibrator 44. The standing waves come from opposite directions. Two independent traveling waves combine to form.

As described above with reference to FIG. 1, the standing waves of the ultrasonic waves are structured by the ultrasonic vibrators (for example, piezoelectric transducers) and the reflective film 45 which face each other at a predetermined distance, or at a predetermined distance. It can be generated from two independent ultrasonic vibrators installed facing each other.

Ultrasonic standing waves have nodes and antinodes. The pressure amplitudes of these ultrasonic standing waves are the largest in the abdomen and have a minimum value at the node and appear twice at a wavelength. Due to the discontinuity of the particles, cells or droplets in the ultrasonic resonance field thus formed, the ultrasonic resonance field forms position-dependent acoustic potential energy. By this phenomenon, cells move to the lowest acoustic potential energy and are trapped in the standing waves of ultrasonic waves. This allows cells to be trapped at pressure nodes that exist at half of the wavelengths. These collected particles aggregate inside the standing wave to form aggregates.

Therefore, the extraction efficiency of fat-soluble substances (10% of the total fatty acids of the cell) can be achieved by a proper combination of the fat-soluble substance extraction solvent and the ultrasonic frequency of the ultrasonic resonance field, the distance between the ultrasonic vibrator 44 and the reflective film 45, and the like. Can be.

As another ultrasonic resonance field generating device 41, a piezoelectric transducer connected to both ends of the upper glass substrate and converting electrical input from the outside into mechanical vibration and applied to the upper glass substrate, and a lower portion parallel to the upper glass substrate. And one or N electrodes arranged on a substrate, wherein the upper glass substrate and the lower substrate are filled with a fluid mixed with cells, and each electrode is perpendicular to the length direction of the piezoelectric transducer. The N electrodes can be applied to a cell separation device arranged at regular intervals along the longitudinal direction of the piezoelectric transducer.

According to the cell separation device, the vibration applied to the upper glass substrate by the piezoelectric transducer generates a surface wave traveling through the upper glass substrate, and collides with another surface wave traveling in a direction opposite to the traveling direction of the surface wave, thereby causing a fluid. Particles, such as cells within, are generated perpendicular to the longitudinal direction of the piezoelectric transducer while moving in a direction perpendicular to the upper glass substrate. The surface wave generates an acoustic wave propagating from the upper glass substrate to the lower substrate in the fluid, and the acoustic wave is reflected from the lower substrate and reflected onto the upper glass substrate, thereby generating an ultrasonic resonance field in the fluid. Create

As a cell separation apparatus applicable to the ultrasonic resonance field generating apparatus as described above, reference may be made to the 'Mutilayered plezoelectric resonator for the separation of suspended particles' of US Patent 5711888 (registered on Jan. 27, 1998).

Hereinafter, a method for producing cells and fat-soluble substances through cell culture according to the present invention by specific examples.

Example 1. Cultivation of Cells

Chlorella protothecoides were used in the present invention while maintaining in proteose agar slant.

The basic medium consisted of KH 2 P0 4 (0.7g), K 2 HPO 4 (0.3g), MgSO 4 · 7H 2 O (0.3g), FeSO 4 · 7H 2 O (3mg) per liter. Urea (1 g), Arnon's A Solution (1 ml), thiamine hvdrochloride (10 μg), pH 6.3. The culture was carried out in 5% CO 2 , 20 degrees, 15,000 lux fluorescent lamp.

The composition of the Arnon's A5 solution consists of H 3 BOS 3 (2.9 g), MnCl 2 · 4H 2 O (1.8 g), ZnSO 4 · 7H 2 O (0.22 g), CuSO 4 · 5H 2 O (0.08 g), MoO 3 (0.018 g).

Heterotrophic culture of Chlorella protothecoides was performed in basal medium with 0.01% urea and 4.0% glucose instead of 0.1% urea.

Example 2 Effect of Fat-Soluble Extracting Medium on Cells

After treatment with C. protothecoides culture solution for 5 minutes using a C10 to C16 alkanes fat-soluble extracting solvent at a 5: 1 ratio, 1 ml of the fractionated C. protothecoides solution was diluted 1 / 100,000-fold and smeared on a 1.5% agar plate. The colony formed was counted to evaluate the effect of soluble extracting solvent on the survival of C. protothecoides. The results are shown in FIG. 2, and the soluble extracting solvent did not affect cell survival. .

Example 3 Effects of Fat-Soluble Extraction Solvent and Ultrasonic Vibration Grinding on Extraction of Fat-Soluble Compounds from Cells

C. The growth rate of protothecoides in the log section was treated with hexane and decane solvent (oil soluble extraction solvent) for 5 min at 5: 1 ratio, and then vibrated at 40 kHz for 2 seconds in a water bath. .

The fat-soluble substance extracted with the fat-soluble substance extraction solvent was saponified, and the free fatty acid was measured by LC-MS analysis using C17 as a standard. The results are shown in Figure 3, 10% total cell fatty acids were extracted by mixing the solvent-soluble solvent extraction for 5 minutes and vibration grinding for an additional 2 seconds. Short vibration grinding increased the extraction of fat-soluble substances by 75%.

Example 4. Effects of fat-soluble substance extraction solvent and ultrasonic resonance field on extraction of fat-soluble substance from cells

C. The growth rate of protothecoides is treated with the culture solution in the log section and the hexane or decane solvent (lipophilic material extraction solvent) for 5 minutes at 5: 1 ratio, and then the acoustic cell filter 41 operates. Transfer to tank 40 and successively fractionate in fractionation tank 50.

As the acoustic cell filter 41, as shown in FIG. 1, an acoustic chamber 42, a 3 MHz ultrasonic oscillator 43, an ultrasonic vibrator 44, and a reflective film 45 were used. The acoustic chamber 42 was produced using an acrylic tube, and glass was used as the reflective film 45. In the acoustic chamber 42, it was confirmed that cell aggregation by the ultrasonic resonance field occurs.

The fat-soluble substance extracted with the fat-soluble substance extraction solvent was saponified, and the free fatty acid was measured by LC-MS analysis using C17 as a standard. The results are shown in Figure 4, 10% of total cell fatty acids were extracted by mixing the solvent-soluble extracting solvent for 5 minutes and vibration grinding for an additional 2 seconds. The acoustic cell filter 41 increased the extraction of fat-soluble substances by about 75%.

Example 5 (High Density) Culture of Cells and Fractionation of Fat-Soluble-Solvent Mixtures

C. protothecoides were incubated in a 5 L culture tank (10) under agitation speed, 150 rpm, roughness 15,000 lux or under a dark reaction to a log section.

Transfer the cell culture solution of the culture tank 10 and the decane solvent (lipophilic material extraction solvent) of the solvent tank 20 to the mixing tank 30, and transfer them at a 5: 1 ratio, and mix them to form an acoustic cell filter ( 41) is transferred to the coagulation tank 40 in operation and the mixture is continuously transferred to the fractionation tank 50 and fractionated, so that the fat-soluble substance-solvent (layer), water (layer) and cells (layer) are divided up and down. Confirmed.

Then, the cells (layers) were transferred to the culture tank 10 through the cell-circulation line 60 and cultured by adding the culture solution, and the fat-soluble material-solvent (layer) was the solvent tank through the solvent-circulation line 70. Transfer to 20.

As a result of analyzing the cell culture after extraction of fat-soluble material from the cells, that is, the cell culture fluid inhibited by the fat-soluble material extraction solvent by the extraction of fat-soluble material was transferred to the culture tank 10 and cultured. 0.028 g / h.

In the case of general fermenter culture in the culture tank, there was no problem in culturing the strain after the simultaneous extraction compared to the growth rate of about 0.033 g / h.

The culture, mixing, aggregation and fractionation process is carried out once a day, or once a day 2 to 20 times to cultivate the cells to a density of 50 ~ 200 gdw / L and fat-soluble substance-solvent containing a concentrated fat-soluble substance Secured. As the fraction and the culture process was repeated, it was confirmed that the microalgae density and fat-soluble substance increased by 2 to 3 times at each step and saturated.

Example 6 Extraction of Fatty Acids from Fractionated Fat-Soluble-Solvents

Extraction of fat-soluble fatty acids was carried out using a Buchi 210/215 rotovapor (Buchi, Switzerland) with a round bottom flask corresponding to an example of a fat-soluble substance-receptor 90. The fractionated fat-soluble substance-solvent was transferred to an evaporator and placed in a round bottom flask. Cold raw water flowed into the condenser and the oil bath of the distillation flask was set at 174 ° C.

When distillation started, gas decane passed through the instrument into the condenser and collected in the receiving flask in liquid form. At the end of the distillation, the volume of decane recovered in the receiving flask and the volume of cell fat solubles left in the distillation flask were measured and measured by LC-MS analysis using C17 as a standard. The recovered decane solvent was transferred to the solvent bath 20 and reused.

Example 7. Biodiesel Extraction from Cellular Fatty Acids

Methanol and caustic soda were stirred to prepare methoxide, and the prepared methoxide was added to a stirrer and stirred to react the extracted cell fat soluble substance with methoxide to form biodiesel, glycerin, and soap solid component. . These products were fed to a centrifuge and centrifuged, and the top and bottom were separated so that the biodiesel was positioned at the top and the heavy glycerine and soap components were located at the bottom by the difference in specific gravity.

The separated glycerin and soap components separated in this way were discharged into separate glycerin storage tanks, and the biodiesel contained in the biodiesel was added to the stirrer by stirring with water of about twice the amount of biodiesel and biodiesel placed at the top. The glycerin, soap component and methanol component dissolved in water were dissolved in water, and the stirred solution was put into a centrifuge again to separate miscellaneous components such as glycerin dissolved in water, and the wastewater solution containing these miscellaneous components was The product was discharged to a glycerin storage tank through a separate discharge line. Finally, a biodiesel was harvested by performing a distillation process by evaporating 1 ~ 2% of water remaining in the biodiesel by operating a distiller, which is a heater. It was.

GC-TOF-MS (Gas Chromatography / Time of Flight / Mass spectrometry; GC-6890N, Agilent Technologies, USA) analysis of the harvested biodiesel is shown in FIG.

Example 8 Analysis of Beta-Carotene in Fractionated Fat-Soluble-Solvents

The content of beta-carotene in the fractionated fat-soluble material-solvent was measured using HPLC (Hewlett Packard Series model 1100) equipped with a Waters Spherisorb S5 ODS2 cartridge column (4.6x250 mm).

Solvent was flowed at a rate of 1.0 ml / min to separate the dye, 90% acetonitrile, 9.99% distilled water and 0.01% triethylamine in 0 to 1 minute, 86% acetonitrile in 2 to 14 minutes, distilled water 8.99 %, Triethylamine 0.01% and ethyl acetate 5%, 100% ethyl acetate was used for 15 to 21 minutes. Post-runs were run for 9 minutes with the first solvent. When the reference (Jin et al., 2001 Biochim Biophys Acta 1506: 244-2597) was set at 550 nm, the beta-carotene pigment was detected at 445 nm, and a standard curve for quantifying beta-carotene (DHI water and environment, Denmark) was used. The amount of beta-carotene was measured on a basis of 8.72 × 10 −10 μM.

Example 9. Ethanol Fermentation of Fractionated Cells

A fermenter, an example of the cell-receiving device 80, was constructed using a microbial fermenter (INNO 200603, Innobio, Korea). Clostridium phytofermentans were grown in culture tubes containing GS-2 medium containing the indicated amounts of the fractionated cells, respectively.

GS-2 medium contained yeast extract 6.0, urea 2.1, K 2 HPO 4 2.9, KH 2 PO 4 1.5, MOPS 10.0, trisodium citrate dihydrate 3.0, cysteine hydrochloride 2.0 (represented in g / L, respectively) . The initial pH of the medium was 7.5, the initial Clostridium phytofermentans concentration was 0.8-1.1 × 10 7 cells / mL, and the cells were cultured under N 2 atmosphere at 30 ° C.

Ethanol concentration was determined upon completion of fermentation of the fractionated cells. Clostridium phytofermentans generated hydrogen simultaneously with ethanol fermentation. Ethanol concentration was analyzed using HPLC equipped with RI detector (Breeze HPLC system, Waters Co., USA) and the column was Aminex HPX-87H (3007.8 mm, Bio-rad).

When the fractionated cell density was 10 g / L, the concentration of ethanol was 0.23 to 0.26% (v / v). When the fractionated cell density was 20 g / L, the concentration of ethanol was 0.42 to 0.54% (v / v). When the fractionated cell density was 40 g / L, the concentration of ethanol was 0.92-1.20% (v / v). Ethanol distillation was performed using a distillation tank.

These results indicate that higher density of fractionated cells does not inhibit the action of Clostridium phytofermentans because the concentration of ethanol increases as the density of fractionated cells increases. As a result, it can be seen that Clostridium phytofermentans ferment these cellulose feedstocks with ethanol without chemical pretreatment of fractionated cell feedstocks and addition of cellulase or other enzymes.

Example 10 Extraction of Ethanol

Fermentation products produced by ethanol fermentation were transferred to a distiller to distill ethanol. At this time, the oil bath was operated to evaporate ethanol and heated above the evaporation temperature of ethanol. If the water is higher than the evaporation temperature during heating, water may evaporate and be mixed with ethanol to reduce the concentration of ethanol. Therefore, the vaporization temperature is formed between 78.3∼85 ℃ and heated for a certain time, and the high concentration of ethanol is vaporized separately. Condensation was carried out in an ethanol storage tank.

Example 11 Butanol Fermentation of Fractionated Cells

A fermenter, an example of the cell-receiving device 80, was constructed using a microbial fermenter (INNO 200603, Innobio, Korea).

Clostridium thermocellum was anaerobicly cultured with DSM medium at a temperature of 60 ° C., anaerobic conditions and 150 rpm. The fractionated cells were transferred to a 5 L fermenter 50 and inoculated with the C. thermocellum culture solution (5%, v / v), and then cultured for 3 days with stirring at a temperature of 60 ° C., anaerobic conditions and 150 rpm for 3 days. Biotechnology Letters Vol 7 No 7 509-514 (1985). After inoculation, the culture solution was injected with nitrogen gas to maintain anaerobic conditions.

For butanol fermentation, Clostridium acetobutylicum, a spore suspension, was heated for 10 minutes at 80 degrees Celsius. Thereafter, the cells were anaerobicly cultured at a temperature of 37 ° C.

After inoculating the C. acetobutylicum culture solution (5%, v / v) to the fermenter, anaerobic butanol fermentation was carried out while stirring at a temperature of 37 ° C., anaerobic conditions and 180 rpm. During the fermentation process, samples were taken periodically to analyze the growth and butanol concentration of the microorganisms.

The DSM medium used was 1.3 g (NH 4 ) 2 SO 4 , 2.6 g MgCl 2 · 6H 2 O, 1.43 g KH 2 PO 4 , 7.2 g K 2 HPO 4 · 3H 2 O, 0.13 g CaCl 2 · 6H 2 O, 1.1 mg of FeSO 4 · 7H 2 O, 6.0 g of sodium β-glycerophosphate, 4.5 g of yeast extract, 10 g of carbon source (filter paper, cellulose processed mass or cellobiose ), 0.25 g of reduced glutathione and 1 mg of resazurin. pH was adjusted from 5.0 to 8.0 with 1 M HCl or 1 M NaOH.

C. acetobutylicum culture medium is 0.75 g of KH 2 PO 4, 0.75 g of K 2 HPO 4, 0.4 g of MgSO 4 · H 2 O, 0.01 g MnSO 4 · H 2 O, 0.01 g FeSO 4 · 7H 2 of 0, 0.5 g of cysteine; 5 g of yeast extract, 2 g of asparagine.H 2 O, 2 g of (NH 4 ) 2 SO 4 were included.

Acetone, butanol and ethanol produced by the microorganisms after the continuous process were quantified using gas chromatography (Agilent technology 6890N Network GC system) equipped with Flame Ionization Detector (FID), and the column was HP-INNOWAX (30 cm x 250). Agilent technology) was used. The temperature of the sample injection part and the detection part was set to 250 degreeC, and the oven was raised to 50 degreeC from 50 degreeC to 10 degreeC / min. The fermentation broth contained 13 g / L butanol, 8 g / L acetone and 0.5 g / L ethanol.

Example 12 Extraction of Butanol

Ionic liquid BMIM-TFSI imide [1-butyl-3-methyl imidazolium bis (trifluoromethylsulfonyl)] [1-butyl-3-methyl imidazolium bis (trifluoromethylsulfonyl) imide], and BMIM-PF6 Butanol was extracted using (1-butyl-3-methyl imidazolium hexafluorophosphate) (1-butyl-3-methyl imidazolium hexafluorophosphate). Butanol was extracted by vortexing a mixed solution obtained by mixing the same amount of BMIM-TFSI (Sigma Aldrich, USA) into the fermentation broth in the fermentor, and a mixed solution of BMIM-PF6 (Sigma Aldrich, USA) in the same amount as the fermentation broth. It is also possible to use what was manufactured using the general ionic liquid manufacturing method at this time. As a result, 60 ± 1% butanol was extracted using BMIM-TFSI, and 64 ± 1% butanol was extracted using BMIM-PF6.

Example 13. Organic Acid Fermentation in Fractionated Cells

A fermenter, an example of the cell-receiving device 80, was constructed using a microbial fermenter (INNO 200603, Innobio, Korea). Lactobacillus brevis subsp. brevis ) is a PYG culture medium (20.0 g of peptone per liter, medium 5.0 g, glucose, yeast powder 10.0 g, NaCl 0.08 g, Cysteine hydrochloride 0.5 g, Calcium chloride 0.008 g, MgSO 4 0.008 g, K 2 HPO 4 0.04 g, 0.04 g of KH 2 PO 4 , 0.4 g of sodium bicarbonate, pH 7.1-7.3), and then grown by centrifugation and washed with 0.085% saline. It was inoculated into a fermenter 50 with the transferred cells. The initial pH of the medium was 7.2, the initial lactic acid fermentation strain was 0.8-1.1 × 10 8 cells / mL, and cultured with injection of N 2 gas at 30 ° C. Upon completion of the fermentation of the fractionated cells, the concentrations of organic acids, such as malate, lactate, acetate, citrate and butyrate were determined.

The concentrations of the organic acids, malic acid, lactic acid, acetic acid, and guic acid, were determined by HPLC (HP placard, Japan) with a Platinum EPS C18 organic acid analysis column (250 mm x 4.6 mm, 5 μm) and set at 0.05 M KH 2 PO 4 at pH 2.4. Butyric acid was analyzed by gas chromatograph (HP placard, Japan) using a CP 58 Wax (FFAP) (30 cm × 0.25 mm ID, 0.25 μm) column.

The results showed that malic acid, lactic acid, acetic acid, guic acid and butyric acid were at concentrations of 870.30 + 13.15, 746.16 + 8.91, 4,233.23 + 76.06, 318.04 + 47.75 and 1.99 + 1.99 mM, respectively.

These results show that the lactic acid fermentation strains ferment these cellulose feedstocks with lactic acid without chemical pretreatment of the fractionated cell feedstock and the addition of cellulase or other enzymes.

Example 14 Extraction of Lactic Acid

Ca (OH) 2 was added to the fermentation broth to adjust the pH to 10 and then heated to increase the solubility of calcium lactate, kill the lactic acid bacteria, and coagulate the protein. This was filtered at high temperature, recovered with calcium lactate and cooled to precipitate calcium lactate. After dissolving calcium lactate at high temperature, sulfuric acid was treated to precipitate CaSO 4 to recover lactic acid.

1: Device of the invention 2: First peristaltic pump 3: Second peristaltic pump
4: 3rd peristaltic pump
10: culture tank 11, 21, 33, 34, 51, 81, 91: line
20: solvent bath 30: mixing tank 31: vibration grinding device
32: stirring device 40: coagulation tank
41: ultrasonic resonance field generator (acoustic cell filter)
42: acoustic chamber 43: ultrasonic oscillator 44: ultrasonic oscillator
45: reflective membrane 50: fractionation tank 60: cell-circulation line
70: solvent-circulating line 80: cell-receiving device 90: fat-soluble material-receiving device

Claims (8)

Culturing cells containing fat-soluble substances;
Mixing the fat soluble material extraction solvent in which the fat soluble material is dissolved with the culture medium of the cell to contact the fat soluble material extraction solvent with the cell culture solution to dissolve the fat soluble material of the cell in the fat soluble material extraction solvent;
Applying an ultrasonic resonance field to the mixture to aggregate the cells in the mixture;
Fractionating the cells in which the fat-soluble material is dissolved in the fat-soluble material extraction solvent and the cells are free of cell damage; And
Obtaining the fractionated cells and the fat-soluble substance-solvent, respectively;
Characterized in that it comprises a cell and a fat-soluble substance through cell culture. The method of claim 1,
Between the fractionation process and the process of obtaining the cells and the fat-soluble solvent,
Culturing the fractionated cells;
Re-splitting said cells cultured using said fat soluble material extraction solvent or fractionated fat soluble material-solvent; And
A step of finally fractionating the soluble-solvent and the high-density cells containing the concentrated soluble material after the culturing of the cells and the re-fractionation process once or twice or more times; More,
Thus, after the final fractionation process, the fat-soluble material-solvent comprising the cells of high density and the concentrated fat-soluble material is obtained, the method of producing cells and fat-soluble material through cell culture. The method according to claim 1 or 2,
When the cell culture medium and the fat soluble material extraction solvent are mixed, the contact between the cell culture medium and the fat soluble material extraction solvent is increased by performing at least one of the steps of vibrating the cell culture solution and stirring the mixture. Method for producing cells and fat-soluble substances through cell culture, characterized in that. A culture tank 10 for culturing cells containing fat-soluble substances;
A solvent bath 20 for storing a fat soluble material extraction solvent in which the fat soluble material is dissolved;
A mixing tank 30 for mixing the culture solution of the cells from the culture tank 10 and the fat-soluble substance extraction solvent from the solvent tank 20;
An agglomeration tank (40) having an ultrasonic resonance field generating device (41) and applying an ultrasonic resonance field to the mixture from the mixing tank (30) to aggregate the cells;
A fractionation tank (50) for dividing the mixture from the flocculation tank (40) into a fat-soluble substance-solvent in which the fat-soluble substance of the cell culture solution is dissolved in the fat-soluble substance extracting solvent;
A cell-receiving device 80 for receiving or processing the cells fractionated from the fractionation tank 50; And
A fat-soluble material-receiving device 90 for receiving or treating the fat-soluble material-solvent fractionated from the fractionation tank 50;
Characterized in that it comprises a device for producing cells and fat-soluble materials through cell culture. The method of claim 4, wherein
A cell-circulating line circulating the cells fractionated from the fractionation tank 50 to the culture tank 10; And
A solvent-circulating line (70) for circulating the fractionated fat-soluble substance-solvent from the fractionation tank (50) to the solvent tank (20); More,
The cell-receiving device 80 is the high-density cells finally fractionated from the fractionation tank 50 after one or two or more cultures in the culture vessel 10 through the cell-circulation line 60 Accept or process;
The fat-soluble substance-receiving device 90 is the concentrated fraction obtained by re-fractionation once or twice or more in the fractionation tank 50 through the solvent-circulation line 70 and finally fractionated from the fractionation tank 50. Apparatus for producing cells and fat-soluble substances through cell culture, characterized in that for receiving or processing a fat-soluble substance-solvent. The method of claim 5,
A first peristaltic pump (2) for supplying a predetermined amount of the cell culture solution of the culture tank (10) and the fat-soluble substance extraction solvent of the solvent tank (20) to the mixing tank (30);
A second to transfer the cells fractionated in the fractionation tank 50 to the culture tank 10 or to the cell-receptor 80 selectively through the cell-circulation line 60 according to the density thereof Peristaltic pump 3; And
A third peristaltic pump 4 transferring the fat-soluble material-solvent fractionated in the fractionation tank 50 to the solvent tank 20 or to the fat-soluble material-receiving device 90 according to its concentration;
Characterized in that it comprises a device for producing cells and fat-soluble materials through cell culture. The method according to any one of claims 4 to 6,
The ultrasonic resonance field generating device 41 is an acoustic cell filter 41 including an acoustic chamber 42, an ultrasonic oscillator 43, an ultrasonic vibrator 44 and a reflecting film 45;
The first traveling wave propagated from the ultrasonic vibrator 44 by the ultrasonic oscillator 43 and the second traveling wave reflected by the reflecting film 45 and propagated in the opposite direction to the first traveling wave collide with each other. By generating a standing wave in the acoustic chamber 42 between the ultrasonic vibrator 44 and the reflecting film 45, the ultrasonic resonance field by the standing wave aggregates the cells to form a cell aggregate, Apparatus for producing cells and fat-soluble substances through cell culture. The method of claim 7, wherein
In the mixing tank 30, at least one of a vibration pulverizing device 31 for vibrating and pulverizing the cell culture and a stirring device 32 for stirring the mixture so as to increase the contact between the cell culture and the fat-soluble substance extraction solvent. The apparatus for producing cells and fat-soluble substances through cell culture, characterized in that it further comprises one device.
KR1020100062817A 2010-06-30 2010-06-30 Method and device for producing cell and fat solubles material by culturing cell KR20120002101A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020100062817A KR20120002101A (en) 2010-06-30 2010-06-30 Method and device for producing cell and fat solubles material by culturing cell

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020100062817A KR20120002101A (en) 2010-06-30 2010-06-30 Method and device for producing cell and fat solubles material by culturing cell

Publications (1)

Publication Number Publication Date
KR20120002101A true KR20120002101A (en) 2012-01-05

Family

ID=45609570

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020100062817A KR20120002101A (en) 2010-06-30 2010-06-30 Method and device for producing cell and fat solubles material by culturing cell

Country Status (1)

Country Link
KR (1) KR20120002101A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110075566A (en) * 2019-05-07 2019-08-02 河南康谱生物科技有限公司 A kind of cell extraction mixing arrangement
KR20210155968A (en) * 2019-06-18 2021-12-24 가천대학교 산학협력단 Spheroid Producing Device in Liquid Phase Medium and Method Thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110075566A (en) * 2019-05-07 2019-08-02 河南康谱生物科技有限公司 A kind of cell extraction mixing arrangement
KR20210155968A (en) * 2019-06-18 2021-12-24 가천대학교 산학협력단 Spheroid Producing Device in Liquid Phase Medium and Method Thereof

Similar Documents

Publication Publication Date Title
US9605288B2 (en) Processing biomass
KR101194942B1 (en) Method and System for Producing Biogas, Fat Solubles Material and Microalgae by Microalgae Culture of Organic Waste
US8377687B2 (en) System for harvesting algae in continuous fermentation
US20130309757A1 (en) Method and apparatus for producing cells and fat soluble materials by cell culture
Khan et al. Insights into diatom microalgal farming for treatment of wastewater and pretreatment of algal cells by ultrasonication for value creation
US20120094355A1 (en) Processing biomass
CN113308387B (en) Bacterial strain for co-production of unsaturated fatty acid and carotenoid and application thereof
Salinas-Salazar et al. Methods for extraction of valuable products from microalgae biomass
Zheng et al. Progress in ultrasound-assisted extraction of the value-added products from microorganisms
US10961497B2 (en) Method for concentrating a cell suspension comprising a mucilaginous biomass of oleaginous yeasts
CN103497171A (en) Device and method for extracting V<E> (vitamin E) in enhanced manner by means of ultrasonically pre-treating deodorized and distilled products of plant oil
CN108004015A (en) A kind of Suaeda salsa seed oil supercritical carbon dioxide extraction process
KR101244469B1 (en) Method and Device for Producing Bio-Diesel and Fermentation Material by Culturing Microalgae
JP2022519874A (en) Methods for Producing Microbial Lipids
KR20120002101A (en) Method and device for producing cell and fat solubles material by culturing cell
KR101251191B1 (en) Method and Device for Producing Cell and Fat Solubles Material by Culturing Cell
AU2015200713A1 (en) Processing biomass
Mat Husin et al. A review on pretreatment methods for lipid extraction from microalgae biomass
AU2013202834B2 (en) Processing biomass
KR20110138922A (en) Method and device for producing high-density microalgae and high-concentrate fat solubles material by culturing microalgae
KR101446392B1 (en) Method for Producing Cell and Fat Solubles Material by Culturing Cell
KR102143001B1 (en) The shredding process of oleaginous microorganism using supersonic disperser and manufacturing method of bio-oil using it
CN107937474A (en) A kind of Suaeda salsa seed oil fermenting extraction process
Antezana Zbinden Investigation of pulsed electric field (PEF) as an intensification pretreatment for solvent lipid extraction from microalgae, utilizing ethyl acetate as a greener substitute to chloroform-based extraction
CN203558992U (en) Ultrasonic pretreatment vegetable oil deodorizer distillate-enhanced VE extraction device

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E601 Decision to refuse application