JPWO2017104812A1 - Method and apparatus for separating media from particle suspension - Google Patents

Abstract

SUMMARY A method and apparatus for recovering a medium from a particle suspension in which particles are dispersed in a liquid medium is disclosed that can recover the medium continuously from the particle suspension. A method of recovering a medium from a particle suspension in which particles are dispersed in a liquid medium includes a first channel and a second channel connected to the first channel and having a size larger than that of the first channel. A step of preparing a flow channel device including a flow channel and a third flow channel branched from the second flow channel and having a smaller size than the second flow channel; and the particle floating from the first flow channel side A step of circulating a liquid to the second flow path side, and a step of recovering the medium from the third flow path.

Description

  The present invention relates to a method and an apparatus for separating a medium from a particle suspension in which particles are suspended in a liquid medium.

  Conventionally, a method for producing useful substances by liquid culture of microorganisms such as bacteria and yeast to produce and secrete useful substances to the microorganisms and recovering the useful substances secreted in the liquid medium has been widely used industrially. Has been done. In order to carry out such a method, it is necessary to separate the culture solution and the microbial cells when the useful substance is collected. This separation is conventionally performed by centrifugation or filtration.

  However, centrifugation and filtration are batch-type, and it is not possible to continuously recover a liquid medium that does not contain microbial cells from a culture that contains microbial cells.

  On the other hand, Non-Patent Document 1 describes a method of separating particles in a particle suspension containing particles of various sizes based on the size of the particles. This method uses a flow channel device in which a narrow flow channel and a wide flow channel are connected. The particle suspension is circulated from the narrow flow channel to the wide flow channel, and large particles move around the center of the flow channel in the wide flow channel. The particles are separated based on the particle size by utilizing the phenomenon that small particles flow through the outer part. Furthermore, in order to arrange the particles on one wall surface of the narrow channel, a liquid not containing particles is joined in the vicinity of the inlet of the narrow channel.

  However, in the method described in Non-Patent Document 1, it is neither described nor suggested that the medium containing no particles is separated, and depending on the apparatus described in Non-Patent Document 1, it is not possible to separate the medium containing no particles. Can not. Also known is a method of separating plasma from whole blood using the phenomenon of axial concentration of particles (Patent Document 1, Patent Document 2).

JP 2010-237050 U.S. Patent Publication 8,889,071 B2

Masumi Yamada et al., Anal. Chem. 2004, 76, 5465-5471

  An object of the present invention is a method and apparatus for separating a particle-free medium from a particle suspension liquid in which particles are dispersed in a liquid medium, wherein the medium can be separated from the particle suspension liquid continuously. Is to provide.

  As a result of diligent research, the inventors of the present application have found that when a particle suspension is circulated from a narrow channel to a wide channel, the particle does not flow to the periphery of the channel in the wide channel. It is conceived that a medium substantially free of particles can be separated by branching a flow path for collecting the medium from the peripheral edge of the flow path that does not flow, and collecting the medium from this branch, and This invention was completed experimentally.

That is, the present invention is a method for recovering a medium from a particle suspension in which particles are dispersed in a liquid medium,
A first flow channel, a second flow channel connected to the first flow channel and having a size larger than the first flow channel, and the second flow channel branched from the second flow channel. Preparing a flow path device comprising a third flow path having a smaller size than
Circulating the particle suspension from the first channel side to the second channel side;
Recovering the medium from the third flow path.

  The present invention also includes a first flow channel, a second flow channel connected to the first flow channel and having a size larger than the first flow channel, and a branch from the second flow channel, Provided is a medium recovery device for recovering a medium from a particle suspension, which includes a third flow path having a size smaller than that of the second flow path.

  According to the present invention, for the first time, a method for separating a medium and an apparatus therefor are provided, which can continuously recover a medium substantially free of particles while circulating a particle suspension. If the method of the present invention is applied to, for example, a method for recovering a medium from a culture of microorganisms that produce useful substances, the culture medium is circulated while supplementing a fresh liquid medium, and a medium containing secreted useful substances Can be recovered continuously, so that the production efficiency of useful substances can be improved.

The upper diagram of FIG. 1 is a schematic plan view of a separation apparatus according to a preferred embodiment of the present invention, and the lower diagram is an enlarged view of the main part of the upper diagram. FIG. 2 is a photomicrograph showing the axial concentration phenomenon that can be used in the method of the present invention.

  The method of the present invention is a method for separating and recovering a medium from a particle suspension in which particles are dispersed in a liquid medium. Here, the “particle” is a particle having a particle size (diameter, but not a spherical shape, but a major axis when a major axis and a minor axis are present) is usually about 0.5 μm to 50 μm, preferably about 1 μm to 10 μm. It is. Examples of particles include bacteria such as Escherichia coli and microorganisms such as yeast; cells of multicellular organisms such as mammals, but are not limited to these, and are produced industrially. Latex particles, plastic particles, and the like may be used. The “liquid medium” is a medium in which the above-described particles are suspended (suspended), and is not limited as long as the liquid can float the particles. In the case of cell culture, it is a liquid medium or the like, and in the case of industrial particles, it is a solvent or a dispersion medium used for the production of the particles. The medium may be a solution in which an arbitrary component is dissolved. “Particle suspension” is a solution in which the above particles are suspended in the above-mentioned medium. As typical examples, a culture of cells of microorganisms or multicellular organisms, or industrially produced particles are dispersed. And the like.

  The flow path device used in the method of the present invention includes a first flow path, a second flow path connected to the first flow path and having a size larger than that of the first flow path, and the second flow path. A third channel branched from the channel and smaller in size than the second channel is provided. Here, “large size” means that the cross-sectional area of the flow path is large. Each flow path can also be formed at the same depth in the substrate. In this case, “large size” means “wide” and “small size” means “narrow”. If the depth of each flow path is the same, the ratio of the widths of the first flow path and the second flow path is such that a region that does not include particles is formed in the peripheral portion of the second flow path. Although not particularly limited, it is usually about 5: 100 to 30: 100. Further, the ratio of the width of the second flow path to the third flow path is not particularly limited as long as a medium substantially free of particles can be recovered from the third flow path. It is around 100: 30. Here, the medium substantially not containing particles means a medium in which the particle concentration is reduced to such an extent that the intended function is not hindered.

  It is preferable that a fourth channel having a size larger than that of the first channel is connected to the upstream side of the first channel. The fourth flow path may not be provided, but if the fourth flow path exists, the first flow path having a small size can be obtained even when the particle suspension is caused to flow through the fourth flow path with a relatively small pressure. Is preferable because a large pressure can be obtained. When the fourth channel and the first channel have the same depth, the width ratio is not particularly limited, but is usually about 100: 5 to 100: 30. If the flow path is formed by molding plastic, glass, paper, etc., the depth of the flow path can be easily changed, so the depth of the flow path need not be the same. The preferred ratio of the width of the first channel and the second channel, the preferred ratio of the width of the second channel and the third channel, and the fourth channel and the second channel. A preferred width ratio of one flow path is a preferred cross-sectional area ratio of each flow path.

  The width and depth of the channel in the above-described channel device are not particularly limited, but the channel device is preferably in the form of a microchannel chip. A microchannel chip is a microchannel (that is, a flow having a width of about 5 μm to 2 mm, particularly about 10 μm to 2 mm, and a depth of about 5 μm to 2 mm, especially about 5 μm to 500 μm in a substrate (chip). Road). The micro-channel chip has a small channel, so the amount of liquid flowing through the channel is small, but because the amount of liquid is small, the efficiency of mixing and separation is high, and the liquid can be heated and cooled quickly. Has advantageous advantages. Although the flow rate of the liquid flowing through each flow path is small, if a large number of micro flow path chips are used together, the overall processing amount can be increased. For this reason, it has been actively put into practical use in various industrial chemical processes in recent years. Also in the present invention, it is preferable to use a microchannel chip as the channel device because the medium recovery efficiency can be increased.

  The microchannel chip can be easily manufactured by, for example, stacking and fixing a sheet (upper substrate) penetrating each channel on a flat plate (lower substrate) constituting the bottom surface of each channel. it can. In this case, since the thickness of the sheet is constant, the depth of each flow path is the same. Therefore, the size of the flow path is defined by the width of the flow path. However, as described above, even with a microchannel chip, the depth of each channel can be easily changed by molding plastic, glass, paper, etc. using a mold, so the depth is the same. That is not essential.

  In the case of a microchannel chip, the width of each channel is usually about 5 μm to 2 mm, especially about 10 μm to 2 mm, and the depth is usually about 5 μm to 2 mm, especially 5 μm to 500 μm. The width of the channel is, for example, 10 μm to 200 μm, and the width of the second channel is, for example, 200 μm to 2 mm. In addition, the widths of the third flow path and the fourth flow path are usually widths that are the ratios of the above ranges with respect to the first flow path and the second flow path having these widths. Moreover, the length of the 1st flow path 12 may be arbitrary, for example, is about 1 mm-5 mm. The length of the other channel may be completely arbitrary.

  A schematic plan view of a preferred embodiment in which the channel device is a microchannel chip and a fourth channel is also formed is shown in FIG. The flow channel device 10 shown in FIG. 1 includes a first flow channel 12, a second flow channel that is connected to the first flow channel 12 and has a width larger than the first flow channel 12, and the second flow channel device 10. The third flow path is branched from the first flow path and has a smaller width than the second flow path. Further, a fourth flow path 18 having a width larger than that of the first flow path 12 is connected to the upstream side of the first flow path 12.

  Next, an operation method and an operation principle for carrying out the method of the present invention using the microchannel chip shown in FIG. 1 will be described. The three white arrows in FIG. 1 indicate the directions in which the liquid flows. First, the particle suspension 20 is flowed from the upstream side of the fourth channel 18 (the side opposite to the side to which the first channel 12 is connected). This can be easily performed using a pump (not shown). In FIG. 1, many small circles indicated by reference numeral 22 schematically indicate particles suspended in the particle suspension 20. The flow rate at this time is not particularly limited, but is usually about 10 μL / min to 200 μL / min. The pressure is not particularly limited, but is usually about 50 hectopascals to 500 hectopascals.

  Then, the particle suspension 20 proceeds to the first flow path 12. It should be noted that the downstream end of the fourth channel 18 (on the side close to the first channel 12) so that the particles 22 smoothly enter the first channel 12 from the fourth channel 18. Is preferably provided with a tapered portion 18a.

  Next, the particle suspension 20 proceeds to the second flow path 14. At this time, the particle suspension liquid 20 is larger in pressure than the pressure in the fourth flow path 18 (generally, the pressure in the fourth flow path 18 and the first flow path 12 is increased to the pressure in the fourth flow path). The pressure is multiplied by the ratio of the cross-sectional areas).

  The inventors of the present application have found that the particles 22 in the particle suspension 20 pushed out to the second flow path 14 travel through the central portion of the second flow path 14 and do not travel to the peripheral portion. This is schematically shown in the lower diagram of FIG. The lower view of FIG. 1 is a schematic plan view in which the vicinity of the first flow path 12 is enlarged. As schematically shown in the lower diagram of FIG. 1, the particle 22 does not advance only to the region described in the drawing as “particle outflow region”, and a region where the particle 22 does not exist is present at the peripheral portion. Arise. Therefore, by providing the third flow path 16 as a branch in this region, only the medium that does not substantially contain the particles 22 enters the third flow path 16. Therefore, only the medium substantially free of the particles 22 can be recovered from the third flow path 16. The third channel 16 is preferably provided on the most upstream side of the second channel, as shown. The particles 22 proceed through the particle outflow region shown in the lower diagram of FIG. 1, but diffuse again as they proceed downstream, and enter the peripheral portion of the second flow path 14. It is preferable to branch the third flow path 16. Note that the particle suspension 20 containing the particles 22 travels in the second flow path 14 as it is.

  In the above-described preferred embodiment, the depth of each flow path is the same, and the outflow region of the particles is defined in the planar direction of the flow path. It is also possible to define the region in an arbitrary direction such as a vertical direction. Channels with different depths can be produced by molding plastic, glass, paper or the like using a mold, drilling using a micro drill, or the like.

  In the above-described preferred embodiment, the downstream end of the first flow path 12 is connected to substantially the center of the upstream end of the second flow path 14, but at a position shifted from the center (a peripheral edge is also acceptable). It is also possible to connect. In this case, it is preferable to provide the third flow path 16 at the peripheral edge on the side far from the first flow path 12 in order to avoid mixing of the particles 22.

  In the apparatus described in Non-Patent Document 1, a further flow path is connected to the connection portion of the fourth flow path 18 and the first flow path 12, and a medium not containing particles is flowed to cause the particles to flow through the first flow path. Although it is in contact with one wall surface in the flow path, in the present invention, it is possible to recover a medium containing no particles without providing such a further flow path. A flow path is not required.

  The microchannel chip 10 described above is incorporated into a closed circuit connected to a particle suspension supply tank (for example, a culture tank when applied to a culture of microorganisms), and the particle suspension is continuously supplied to the circuit by a pump. Therefore, the medium that does not contain the particles 22 can be continuously recovered from the third flow path 16. In this case, the medium containing no particles 22 can be recovered continuously over a long period of time by replenishing the same amount of fresh medium as the medium to be recovered. Therefore, when the method of the present invention is applied to a culture of cells such as microorganisms, a liquid medium containing useful substances produced by the cells is continuously applied over a long period without interrupting the culture of the cells. Therefore, unlike the conventional method, there is no need to interrupt the culture at least partially and perform centrifugation or filtration, and the collection efficiency of useful substances can be greatly improved.

  When the method of the present invention is industrially implemented, it is preferable to use a large number of the above-described microchannel chips at the same time because the amount of processing is small when only one microchannel chip is used. In this case, a plurality of microchannel chips are connected in series (that is, the downstream end of the second channel 14 of the microchannel is connected to the upstream end of the fourth channel 18 of the next microchannel chip). Can be connected in parallel (for example, a closed circuit can be formed by providing a large number of inlets / outlets in the particle suspension supply source tank, and the microchannel chip is incorporated in each circuit). It is possible to use both serial connection and parallel connection. Thus, it is possible to achieve a processing amount that can be industrially put into practical use. This has also been confirmed in various other chemical processes. Further, the medium recovered from the third channel 16 is further supplied from the upstream side of the fourth channel 18 of another microchannel chip, and the medium is supplied from the third channel 16 of the microchannel chip. By collecting the particles, the particles 22 that may be mixed into the medium collected from the first microchannel chip can be surely excluded.

  As a particle suspension supplied to the apparatus of the present invention described above, a particle suspension with reduced particle concentration obtained by axial concentration of particles can be used to more reliably recover a medium that does not contain particles. This is preferable because it becomes possible. The “particle axial concentration” phenomenon is caused by flowing a particle suspension in a linear microchannel, and the particles concentrate near the center of the channel, resulting in a high concentration of particles near the center of the channel. This is a phenomenon in which the particle concentration is lowered at the periphery of the road. This phenomenon is known per se and is described in, for example, Japanese Patent Application Laid-Open No. 2010-237050 and US Pat. No. 8,889,071 B2. That is, when the particle suspension is circulated through a microchannel having a width of 30 μm to 500 μm, preferably 100 μm to 400 μm and a depth of 10 μm to 500 μm, preferably 50 μm to 400 μm, for example, FIG. As shown in the photomicrograph, most of the particles flow within a range of about 80% of the diameter from the center of the flow path, and less particles flow to the periphery of the flow path by about 10% of the diameter. In order to cause an axial concentration phenomenon, it is necessary to accelerate the particle suspension until a predetermined flow velocity is reached (hereinafter, sometimes referred to as “axial concentration acceleration” for convenience). It can be set as appropriate according to the width and depth. That is, since whether or not axial concentration occurs can be easily determined by microscopic observation, the flow velocity at which the axial concentration phenomenon occurs can be easily set through routine experiments. The micrograph in FIG. 2 shows a state when a particle suspension is flowed through a flow channel having a width of 100 μm and a depth of 40 μm at a flow rate of 30 μL / min. The length of the flow path (fifth flow path) for performing concentrated acceleration is 10 mm or more, and there is no upper limit in particular. However, since it is useless if it is too long, it is usually 10 mm to 50 mm, preferably 20 mm. About 30 mm.

  In order to recover the suspended liquid having a reduced particle concentration from the suspended particle liquid in which the particles are axially concentrated, a branch path (sixth flow path) may be provided in the flow path in which the suspended liquid having the axial concentration accelerated. Since the particle suspension liquid whose particle concentration is reduced due to the axial concentration phenomenon flows in this branch path, this is the fourth flow path when the first flow path or the fourth flow path is connected. By supplying to the flow path, it is possible to more reliably recover a medium that does not contain particles. In this case, the width of the branch path is preferably about 5% to 30% of the width of the axial concentrated acceleration channel that performs axial concentrated acceleration. It is also possible to provide one branch path on each of the left and right sides of the flow path when viewed from above, and connect the apparatus of the present invention to each branch path.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1
The microchannel chip shown in FIG. 1 was produced. The width of the second channel 14 and the fourth channel 18 was 2 mm, the width of the first channel was 100 μm, the width of the third channel was 500 μm, and the depth of each channel was 100 μm. . The length of the first channel 12 was 5 mm. A pump, a flow meter, and a pressure gauge were connected in series upstream of the microchannel chip.

  A suspension of plastic beads having a diameter of 10 μm or 50 μm (medium is water, particle concentration is 2% by weight) was prepared. This bead suspension was allowed to flow from the upstream side of the fourth channel 18 of the microchannel chip by a pump. The pressure at this time was 125 hectopascals or 150 hectopascals, and the flow rate was 30 μL / min to 50 μL / min.

  As a result, a medium containing no particles could be continuously recovered from the third flow path 16. The recovery rate was 1.75 μL / min (125 hectopascals or 3.5 μL / min (250 hectopascals).

Example 2
The microchannel chip shown in FIG. 1 was produced. The width of the second flow path 14 and the fourth flow path 18 was 300 μm, the width of the first flow path was 100 μm, the width of the third flow path was 100 μm, and the depth of each flow path was 100 μm. . The length of the first channel 12 was 5 mm. A pump, a flow meter, and a pressure gauge were connected in series upstream of the microchannel chip.

  A yeast culture solution having a diameter of about 5 μm was allowed to flow from the upstream side of the fourth channel 18 of the microchannel chip by a pump. The pressure at this time was 100 hectopascals or 300 hectopascals, and the flow rate was 30 μL / min to 90 μL / min.

  The yeast culture solution was prepared according to the following procedure. Saccharomyces cerevisiae IFO 10217 strain was cultured on a YPD agar medium (10 g / l Yeast extract, 20 g / l Peptone, 20 g / l Dextrose, 2% Agar) at 30 ° C. overnight in a constant temperature layer. This culture was inoculated in an amount of one platinum loop in 50 ml of YPD liquid medium (10 g / l Yeast extract, 20 g / l Peptone, 20 g / l Dextrose) previously autoclaved at 121 ° C. for 20 minutes. For 24 hours. The obtained culture broth was used for subsequent experiments. The Saccharomyces cerevisiae IFO 10217 strain used was deposited with the National Institute of Advanced Industrial Science and Technology.

  As a result, it was possible to continuously recover the liquid medium containing no yeast from the third flow path 16. The recovery rate was 0.7 μL / min (100 hectopascals) or 4.0 μL / min (300 hectopascals).

DESCRIPTION OF SYMBOLS 10 Micro flow path chip 12 1st flow path 14 2nd flow path 16 3rd flow path 18 4th flow path 18a Tapered part of 4th flow path 20 Particle floating liquid 22 Particles

Claims (11)

A method of recovering a medium from a particle suspension in which particles are suspended in a liquid medium,
A first flow channel, a second flow channel connected to the first flow channel and having a size larger than the first flow channel, and the second flow channel branched from the second flow channel. Preparing a flow path device comprising a third flow path having a smaller size than
Circulating the particle suspension from the first channel side to the second channel side;
Collecting the medium of the particle suspension from the third flow path.   A fourth flow channel having a size larger than that of the first flow channel is connected to the upstream side of the first flow channel, and the particle suspension is transferred from the fourth flow channel to the first flow channel. The method according to claim 1, wherein the method is circulated in a flow path.   The method according to claim 1 or 2, wherein the flow channel device is in the form of a microchannel chip.   The method according to claim 1, wherein the particles are cells, and the medium is a liquid medium.   The method of claim 4, wherein the cell is a microbial cell.   The method according to claim 5, wherein the microorganism is a bacterium or a yeast.   When the first flow path or a fourth flow path having a size larger than the first flow path is connected, the fourth flow path is obtained by the axial concentration of particles. The method according to any one of claims 1 to 6, wherein a particle suspension having a reduced particle concentration is supplied.   A first flow channel, a second flow channel connected to the first flow channel and having a size larger than the first flow channel, and the second flow channel branched from the second flow channel. The medium collection | recovery apparatus which collects a medium from a particle | grain suspension liquid which comprises the 3rd flow path smaller than size.   The apparatus according to claim 8, wherein a fourth channel having a size larger than that of the first channel is connected to the upstream side of the first channel.   The apparatus according to claim 8 or 9, wherein the flow channel device is in the form of a microchannel chip.   The apparatus includes a fifth flow path for axial concentration of particles, and a sixth flow path for collecting the particle suspension liquid branched from the fifth flow path and generated by axial concentration and having a reduced particle concentration. And the sixth channel is connected to the upstream side of the first channel or a fourth channel having a size larger than the first channel. The apparatus of any one of Claims 8-10 connected to the upstream of 4 flow paths.