JP7019144B2 - Method and device for separating the medium from the particle suspension - Google Patents

Description

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

Conventionally, there has been a wide range of industrial methods for producing useful substances by culturing microorganisms such as bacteria and yeast in a liquid culture, causing the microorganisms to produce and secrete useful substances, and recovering the useful substances secreted in the liquid medium. It is done. In order to carry out such a method, it is necessary to separate the culture medium and the microbial cells at the time of recovery of the useful substance. This separation has traditionally been performed by centrifugation or filtration.

However, centrifugation and filtration are batch-type, and it is not possible to continuously recover a liquid medium containing no microbial cells from a culture containing microbial cells.

On the other hand, Non-Patent Document 1 describes a method for separating particles in a particle suspension liquid containing particles of various sizes based on the size of the particles. In this method, a flow path device in which a narrow flow path and a wide flow path are connected is used to circulate the particle suspension liquid from the narrow flow path to the wide flow path, and in the wide flow path, large particles move around the center of the flow path. The particles are separated based on the particle size by utilizing the phenomenon that small particles flow through the outer part of the particles. Further, in order to arrange the particles on one wall surface of the narrow flow path, a liquid containing no particles is merged near the entrance of the narrow flow path.

However, it is neither described nor suggested that the method described in Non-Patent Document 1 separates a medium containing no particles, and depending on the apparatus described in Non-Patent Document 1, the medium containing no particles may be separated. Can not. Further, a method of separating plasma from whole blood by utilizing the axial concentration phenomenon of particles is also known (Patent Document 1 and Patent Document 2).

Japanese Unexamined Patent Publication No. 2010-237050 U.S. Patent Gazette 8,889,071 B2

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

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

As a result of diligent research, the inventors of the present application have found that when a particle suspension liquid is circulated from a narrow flow path to a wide flow path, the particles do not flow to the peripheral edge of the flow path in the wide flow path, and the particles are formed. By branching the flow path for collecting the medium from the peripheral portion of the flow path that does not flow and collecting the medium from this branch, it was conceived that the medium containing substantially no particles can be separated. The present invention was completed by experimental confirmation.

That is, the present invention is a method for recovering a medium from a particle suspension liquid in which particles are suspended in a liquid medium.
The first flow path, the 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 branching from the second flow path. The process of preparing a flow path device with a third flow path that is smaller in size than
A step of circulating the particle suspension liquid from the first flow path side to the second flow path side,
The step of collecting the medium from the third flow path is included, and the downstream end of the first flow path is connected to substantially the center of the upstream end of the second flow path, and the particles are formed. Provided is a method for recovering a medium of a particle suspension liquid , which is a microbial cell and the medium is a liquid medium, and the culture is circulated while supplementing the liquid medium .

Further, in the present invention, the first flow path, the 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 are branched from the second flow path. A particle having a third flow path that is smaller in size than the second flow path, the downstream end of the first flow path being connected to approximately the center of the upstream end of the second flow path. Provided is a medium recovery device for performing the above-mentioned method of the present invention, which recovers a medium from a suspended liquid.

INDUSTRIAL APPLICABILITY The present invention provides for the first time a method for separating a medium and an apparatus therefor, which can continuously recover a medium containing substantially no particles while circulating a suspended particle liquid. If the method of the present invention is applied to, for example, a method for recovering a medium from a culture of a microorganism producing a useful substance, the culture is circulated while being supplemented with a fresh liquid medium, and a medium containing the secreted useful substance is applied. Can be continuously recovered, so that the production efficiency of useful substances can be improved.

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

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

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 a size larger than that of the first flow path, and the second flow path. It branches from the flow path and includes a third flow path that is smaller in size than the second flow path. Here, "large size" means that the cross-sectional area of the flow path is large. Each flow path can also be formed in the substrate at the same depth. In this case, "large size" means "wide" and "small size" means "narrow". When the depth of each flow path is the same, the ratio of the width between the first flow path and the second flow path is such that if a particle-free region 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 widths of the second flow path and the third flow path is not particularly limited as long as the medium containing substantially no particles can be recovered from the third flow path, but is usually 100: 10 to 100: 10. It is about 100:30. Here, the medium containing substantially no particles means a medium in which the particle concentration is reduced to the extent that the target function is not hindered.

It is preferable that a fourth flow path having a size larger than that of the first flow path is connected to the upstream side of the first flow path. It is not necessary to provide the fourth flow path, but if the fourth flow path is present, the first flow path having a small size is small even when the particle suspension liquid is flowed through the fourth flow path with a relatively small pressure. Is preferable because a large pressure can be obtained. When the fourth flow path and the first flow path 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. Therefore, the depth of the flow path does not have to be the same. In this case, the flow path does not have to be the same. , The preferred width ratio between the first channel and the second channel described above, the preferred width ratio between the second channel and the third channel described above, and the fourth channel and the fourth channel described above. The ratio of the preferable width of the flow path of 1 is the ratio of the preferable cross-sectional area of each flow path.

The width and depth of the flow path in the above-mentioned flow path device are not particularly limited, but the flow path device is preferably in the form of a micro flow path chip. The microchannel chip is a flow in the substrate (chip) having a microchannel (that is, usually 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, particularly about 5 μm to 500 μm. The road) was formed. Since the microchannel chip has a small flow path, the amount of liquid flowing through the flow path is small, but because the amount of liquid is small, the efficiency of mixing and separation is good, and the liquid can be heated and cooled quickly. It has a favorable advantage. Although the flow rate of the liquid flowing through each flow path is small, the processing amount as a whole can be increased by using a large number of micro flow path chips at the same time. For this reason, it is being actively put into practical use in various industrial chemical processes in recent years. Also in the present invention, it is preferable to use the flow path device as a micro flow path chip because the recovery efficiency of the medium can be improved.

The microchannel chip can be easily manufactured, for example, by laminating and fixing a sheet (upper substrate) penetrating each channel on a flat plate (lower substrate) constituting the bottom surface of each channel. 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 microchannel chips, 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 the microchannel chip, the width of each channel is usually about 5 μm to 2 mm, particularly about 10 μm to 2 mm, and the depth is usually about 5 μm to 2 mm, particularly 5 μm to 500 μm. The width of the flow path is, for example, 10 μm to 200 μm, and the width of the second flow path is, for example, 200 μm to 2 mm. Further, the width of the third flow path and the fourth flow path is usually a width that is a ratio of these widths to the first flow path and the second flow path in the above range. Further, the length of the first flow path 12 may be arbitrary, for example, about 1 mm to 5 mm. The length of the other channels may be completely arbitrary.

FIG. 1 shows a schematic plan view of a preferred embodiment in which the flow path device is a micro flow path chip and a fourth flow path is also formed. The flow path device 10 shown in FIG. 1 has a first flow path 12, a second flow path connected to the first flow path 12, and wider than the first flow path 12, and the second flow path. It is provided with a third flow path that branches from the flow path of No. 1 and has a width smaller than that of 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 direction in which the liquid flows. First, the particle suspension liquid 20 is flowed from the upstream side of the fourth flow path 18 (the side opposite to the side to which the first flow path 12 is connected). This can be easily done using a pump (not shown). In FIG. 1, a large number of small circles indicated by reference number 22 schematically indicate particles suspended in the particle suspended liquid 20. The flow velocity 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 liquid 20 proceeds to the first flow path 12. In addition, from the fourth flow path 18, the downstream end of the fourth flow path 18 (the side closer to the first flow path 12) so that the particles 22 smoothly enter the first flow path 12. Is preferably provided with a tapered portion 18a.

Next, the particle suspension liquid 20 proceeds to the second flow path 14. At this time, the particle suspension liquid 20 has a pressure higher than the pressure in the fourth flow path 18 (generally, the pressure in the fourth flow path is adjusted to the pressure in the fourth flow path 18 and the first flow path 12). The pressure is multiplied by the ratio of the cross-sectional area) and is extruded into the second flow path 14.

The inventors of the present application have found that the particles 22 in the particle suspension liquid 20 extruded into the second flow path 14 proceed to the central portion of the second flow path 14 and do not advance to the peripheral portion. This situation is schematically shown in the lower figure of FIG. The lower figure of FIG. 1 is an enlarged schematic plan view in the vicinity of the first flow path 12. As schematically shown in the lower figure of FIG. 1, the particle 22 advances only to the region described in the figure as the “particle outflow region”, and the peripheral portion thereof has a region in which the particle 22 does not exist. Occurs. Therefore, by providing the third flow path 16 as a branch in this region, only the medium substantially free of 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. As shown in the figure, the third flow path 16 is preferably provided on the most upstream side of the second flow path. The particles 22 travel through the outflow region of the particles shown in the lower figure of FIG. 1, but diffuse again as they proceed to the downstream side and enter the peripheral edge of the second flow path 14, so that the particles 22 are at a site where diffusion has not yet occurred. It is preferable to branch the third flow path 16. The particle suspension liquid 20 containing the particles 22 proceeds as it is in the second flow path 14.

In one preferred embodiment described above, the depth of each flow path is the same and the particle outflow region is defined in the plane direction of the flow path, but the depth of each flow path is changed to allow the outflow of particles. It is also possible to define the region in any direction, for example, in the vertical direction. Channels with different depths can be produced by molding plastic, glass, paper, etc. using a mold, excavation using a microdrill, or the like.

In one preferred embodiment described above, the downstream end of the first flow path 12 is connected to approximately the center of the upstream end of the second flow path 14 .

In the apparatus described in Non-Patent Document 1, a further flow path is connected to the connection portion between the fourth flow path 18 and the first flow path 12, and a medium containing no particles is flowed to flow the particles to 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 the medium containing no particles without providing such a further flow path. No flow path is required.

The above-mentioned microchannel chip 10 is incorporated into a closed circuit connected to a particle suspension source tank (for example, a culture tank when applied to a microbial culture), and the particle suspension is continuously pumped into the circuit by a pump. By circulating the particles 22 continuously, the medium containing no particles 22 can be continuously recovered from the third flow path 16. In this case, by replenishing the same amount of fresh medium as the recovered medium, the medium containing no particles 22 can be continuously recovered for a long period of time. 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 for a long period of time without interrupting the culture of the cells. Since it is possible to recover the cells, it is not necessary to interrupt the culture at least partially and perform centrifugation or filtration as in the conventional method, and the recovery efficiency of useful substances can be significantly improved.

When the method of the present invention is industrially carried out, it is preferable to simultaneously use a large number of microchannel chips as described above because the amount of processing is small if 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. It can be connected in parallel (for example, a large number of entrances and exits are provided in the particle suspension tank to form a closed circuit, and the microchannel chip is incorporated in each circuit, etc.). It is possible, and both series connection and parallel connection can be used together. As a result, 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 supplied from the upstream side of the fourth channel 18 of the other microchannel chip, and the medium is supplied from the third channel 16 of the microchannel chip. By recovering the particles 22, the particles 22 that may be mixed in the medium recovered from the first microchannel chip can be reliably eliminated.

By using the particle suspension liquid having a reduced particle concentration obtained by the axial concentration of the particles as the particle suspension liquid supplied to the above-mentioned apparatus of the present invention, it is possible to more reliably recover the medium containing no particles. It is preferable because it becomes possible. In the phenomenon of "axial concentration of particles", when a particle suspension liquid is flowed in a linear microchannel, the particles are concentrated near the center of the channel, and as a result, the particle concentration is high near the center of the channel and the flow is high. It is a phenomenon that the particle concentration becomes low at the peripheral edge of the road. This phenomenon itself is known, and is described in, for example, Japanese Patent Application Laid-Open No. 2010-237050 and US Patent Publication No. 8,889,071 B2. That is, when the particle suspension is circulated in the 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 of the above, most of the particles flow from the center of the flow path within a range of about 80% of the diameter, and less particles flow to the periphery of the flow path by about 10% of the diameter. In order to cause the axial concentration phenomenon, it is necessary to accelerate the particle suspension until it reaches a predetermined flow velocity (hereinafter, may be referred to as "axis concentration acceleration" for convenience), and the predetermined flow velocity is the flow velocity of the flow path. It can be set as appropriate according to the width and depth. That is, since it can be easily known by microscopic observation whether or not the axis concentration occurs, the flow velocity at which the axis concentration phenomenon occurs can be easily set by a routine experiment. The photomicrograph of FIG. 2 shows a state when a particle suspension is flowed through a flow path 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 axial concentrated acceleration is 10 mm or more, and there is no particular upper limit, but it is useless if it is too long, so it is usually 10 mm to 50 mm, preferably 20 mm. It is about 30 mm.

To recover the suspended liquid having a reduced particle concentration from the suspended liquid in which the particles are axially concentrated, it is sufficient to simply provide a branch path (sixth flow path) in the flow path through which the suspended liquid in which the accelerated concentration of particles flows flows. A particle suspension whose concentration of particles has decreased due to the axial concentration phenomenon flows through this branch path, and therefore, when the first flow path or the fourth flow path is connected, a fourth flow path is connected. By supplying the medium to the flow path of the above, it becomes possible to more reliably recover the medium containing no particles. In this case, the width of the branch path is preferably about 5% to 30% of the width of the axis-concentrated acceleration flow path for axial-concentrated acceleration. Further, it is also possible to provide one branch path on each side when the flow path is viewed from above, and to connect the device of the present invention to each branch path.

Hereinafter, the present invention will be described in more detail based on Examples. 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 flow path 14 and the fourth flow path 18 was 2 mm, the width of the first flow path was 100 μm, the width of the third flow path was 500 μm, and the depth of each flow path was 100 μm. .. The length of the first flow path 12 was 5 mm. A pump, a flow meter and a pressure gauge were connected in series upstream of this microchannel tip.

A suspension of plastic beads having a diameter of 10 μm or 50 μm (water as a medium, particle concentration 2% by weight) was prepared. This bead suspension was pumped from upstream of the fourth flow path 18 of the microchannel tip. 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, the 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 flow path 12 was 5 mm. A pump, a flow meter and a pressure gauge were connected in series upstream of this microchannel tip.

A yeast culture solution having a diameter of about 5 μm was pumped from the upstream of the fourth channel 18 of the microchannel tip. 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 broth was prepared according to the method shown below. The Saccharomyces cerevisiae IFO 10217 strain was cultured on a YPD agar medium (10 g / l Yeast extract, 20 g / l Peptone, 20 g / l Extract, 2% Agar) at 30 ° C. overnight in a constant temperature layer. This culture was pre-inoculated into 50 ml of YPD liquid medium (10 g / l Yeast extract, 20 g / l Peptone, 20 g / l Extract) sterilized by autoclaving at 121 ° C for 20 minutes, and then inoculated to 30 ° C. It was sterilized for 24 hours. The obtained culture broth was used in subsequent experiments. As the Saccharomyces cerevisiae IFO 10217 strain, the one deposited at the National Institute of Advanced Industrial Science and Technology was used.

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

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 suspension liquid 22 Particles

Claims (5)

It is a method of recovering a medium from a particle suspension liquid in which particles are suspended in a liquid medium.
The first flow path, the 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 branching from the second flow path. The process of preparing a flow path device with a third flow path that is smaller in size than
A step of circulating the particle suspension liquid from the first flow path side to the second flow path side,
Including the step of collecting the medium from the third flow path, the downstream end of the first flow path is connected to substantially the center of the upstream end of the second flow path, and the particles are microorganisms. A method for recovering a medium of suspended particles, wherein the medium is a cell and the medium is a liquid medium, and the culture is circulated while supplementing the liquid medium. A fourth flow path having a size larger than that of the first flow path is connected to the upstream side of the first flow path, and the particle suspended liquid is transferred from the fourth flow path to the first flow path. The method according to claim 1, wherein the particles are distributed in the flow path. The method according to claim 1 or 2, wherein the flow path device is in the form of a micro flow path chip. The method according to any one of claims 1 to 3, wherein the microorganism is a bacterium or yeast. When a first flow path or a fourth flow path having a size larger than that of the first flow path is connected, the fourth flow path is obtained by axial concentration of particles. The method according to any one of claims 1 to 4, wherein a particle suspension liquid having a reduced particle concentration is supplied.

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