JP2015053892A - Semicontinuous culture apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a semicontinuous culture apparatus which has a simple structure, can separate a fungus body from a culture solution, and can maintain permeability performance over a long period of time.SOLUTION: A semicontinuous culture apparatus 1 includes: a culture tank 3 for holding a culture solution 2 and culturing a fungus; first filtering means 5 for filtering the culture solution 2 extracted from the culture tank 3 and collecting the product produced by the fungus as filtered matter; and filtrate circulating means 8 for circulating the filtrate produced by the first filtering means 5 to the culture tank 3. The semicontinuous culture apparatus also includes: solid-liquid separating means 4 which is disposed inside the culture tank 3 to separate the fungus body from the culture solution 2; and culture solution supplying means 6 for supplying the culture solution 2 from which the fungus body is separated by the solid-liquid separating means 4 to the first filtering means 5.

Description

  The present invention relates to a semi-continuous culture apparatus for culturing a bacterium to recover a product such as an enzyme produced by the bacterium.

  Conventionally, a culture solution is extracted from a culture tank for cultivating bacteria, the culture solution is filtered, and a product such as an enzyme produced by the bacteria is collected as a filtrate, and the culture solution that has not been filtered is cultured in the culture medium. A semi-continuous culture apparatus that circulates in a tank is known (see, for example, Patent Document 1).

  In the conventional semi-continuous culture apparatus, by using a porous membrane having an average pore diameter in the range of 0.01 to 2.0 μm for filtration of the culture solution, the bacteria are separated from the culture solution and generated. It is said that things can be recovered. In addition, the porous film is less likely to be clogged and can maintain high permeation performance for a long time. However, in the conventional semi-continuous culture apparatus, when culturing a fungus that forms a bacterial mass such as a filamentous fungus, the porous membrane is easily clogged by the bacterial body such as the bacterial mass, There is a problem that it is difficult to maintain the performance of recovering the product.

  In order to solve the above problem, a solid-liquid separation device such as a filter press for separating the bacterial cells from the culture solution extracted from the culture vessel is provided outside the culture vessel, and the bacterial cells are separated by the solid-liquid separation device. It is conceivable to collect the product as a filtrate by filtering the culture solution from which the product has been separated.

JP 2009-296921 A

  However, when the solid-liquid separation device is provided outside the culture tank, a mechanism for cleaning or sterilizing the solid-liquid separation device is also required, which not only increases the complexity and size of the device but also increases the complexity of the device. There is a disadvantage that the risk of contamination increases.

  The present invention eliminates such inconvenience, can separate the cells from the culture solution with a simple configuration, and can maintain the ability to recover the product over a long period of time. An object is to provide an apparatus.

  In order to achieve such an object, the present invention includes a culture vessel that contains a culture solution and cultures bacteria in the culture solution, and the culture solution extracted from the culture vessel is filtered to obtain the fungus as a filtrate. A semi-continuous culture apparatus comprising a first filtration means for recovering a produced product and a filtrate circulation means for circulating a filtrate of the first filtration means to the culture tank. A solid-liquid separation means for separating the cells from the culture solution by causing the culture solution to flow in parallel to the filter by rotating a hollow cylindrical filter around the axis, and performing cross-flow filtration; A culture liquid supply means for supplying the culture liquid from which the cells have been separated by the solid-liquid separation means to the first filtration means.

  In the semi-continuous culture apparatus of the present invention, a culture solution containing the product is obtained by culturing the bacterium in a culture solution accommodated in the culture tank and causing the bacterium to produce a product such as an enzyme. Can do. Therefore, the culture solution extracted from the culture tank is filtered by the first filtration means to collect the product as a filtrate, while the culture solution as a filtrate is collected by the filtrate circulation means. Semi-continuous culture is performed by circulating in a tank.

  At this time, there is a concern that the first filtering means may be clogged, for example, because the bacterial cells form a bacterial mass. Therefore, the semi-continuous culture apparatus of the present invention comprises the solid-liquid separation means in the culture tank, and the culture liquid from which the cells have been separated by the solid-liquid separation means is 1 filtration means.

  The solid-liquid separation means forms a flow in which the culture solution flows in parallel to the filter in the culture tank by rotating the hollow cylindrical filter about its axis. As a result, the bacterial cells contained in the culture solution do not substantially permeate the filter, and the culture solution containing the product permeates the filter.

  Therefore, the solid-liquid separation means can separate the cells from the culture without clogging itself, and the culture liquid from which the cells have been substantially separated by the solid-liquid separation means By supplying the first filtration means, clogging of the first filtration means can be prevented.

  Therefore, the semi-continuous culture apparatus of the present invention can maintain the performance of recovering the product for a long time.

  Further, according to the semi-continuous culture apparatus of the present invention, since the solid-liquid separation means is disposed in the culture tank, the solid-liquid separation means can be washed and sterilized simultaneously with the culture tank, There is no need to provide an independent cleaning or sterilization mechanism in the solid-liquid separation means. Therefore, the semi-continuous culture apparatus of the present invention can separate the cells from the culture solution with a simple configuration, and can reduce the risk of contamination.

  In the semi-continuous culture apparatus of the present invention, the filter preferably includes pores having a pore diameter in the range of 0.001 to 1.0 mm. If the pore diameter of the filter exceeds 1.0 mm, the cells cannot be sufficiently separated. Further, it is technically difficult to make the pore diameter of the filter smaller than 0.001 mm including the cost.

  The semi-continuous culture apparatus of the present invention is characterized in that the first filtration means includes an ultrafiltration membrane, and the second filtration means includes a microfiltration membrane disposed in the middle of the culture solution supply means, It is preferable to include a third filtration means provided in the middle of the filtrate circulation means and provided with a microfiltration membrane.

  The semi-continuous culture apparatus of the present invention further completely separates the cells from the culture solution by disposing a second filtration unit having a microfiltration membrane in the middle of the culture solution supply unit. Can do.

  As a result, the culture solution substantially free of the cells can be supplied to the first filtration unit by the culture solution supply unit. At this time, since the first filtration means includes the ultrafiltration membrane, the culture solution substantially free of the cells is filtered, and the product is efficiently recovered as a filtrate. be able to.

  Furthermore, the semi-continuous culture apparatus of the present invention can separate and remove germs and the like mixed in the filtrate by providing a third filtration means having a microfiltration membrane in the middle of the filtrate circulation means. It is possible to reliably prevent contamination in the culture tank.

The system block diagram which shows the structure of the semi-continuous culture apparatus of this invention. FIG. 2 is a perspective view showing a partially broken configuration of the spin filter shown in FIG. 1.

  Next, embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

  As shown in FIG. 1, the semi-continuous culture apparatus 1 of the present embodiment contains a culture solution 2 and a culture vessel 3 for culturing bacteria in the culture solution 2, and is disposed in the culture vessel 3. A spin filter 4 as a solid-liquid separation means for separating bacterial cells from the culture medium, and an ultrafiltration membrane device 5 as a first filtration means for recovering a product produced by the bacteria from the culture solution 2. The spin filter 4 is connected to a culture fluid conduit 6 as a culture fluid supply means for supplying the culture fluid 2 to the ultrafiltration membrane device 5, and the culture fluid conduit 6 serves as a second filtration means disposed in the middle. Is connected to the ultrafiltration membrane device 5 through the microfiltration membrane device 7.

  The ultrafiltration membrane device 5 includes a filtrate conduit 8 as a filtrate circulation means for circulating the filtrate in the culture tank 3, and the filtrate conduit 8 is a microfiltration membrane device as a third filtration means disposed in the middle. 9 is connected to the culture tank 3 through 9.

  The culture tank 3 can stir the culture solution 2 by a shaft 11 suspended from a motor 10 provided at an upper portion outside the tank and a stirring blade 12 attached to the shaft 11.

  As shown in FIG. 2, the spin filter 4 includes a hollow shaft 13, and a plurality of circular frames 4 a that are externally fitted to the hollow shaft 13 at intervals, and along the outer peripheral edge of the frame 4 a. And a filter 4b formed in a hollow cylindrical shape. The upper and lower sides of the filter 4b are sealed by lids 4c and 4d. The filter 4b is formed of, for example, punching metal, ceramics, resin, or the like, and includes pores having a pore diameter in the range of 0.001 to 1.0 mm.

  The hollow shaft 13 is connected to the culture medium conduit 6 via the rotary joint 14 at the upper part outside the culture tank 3, and a pulley 15 provided below the rotary joint 14 is connected to the rotating shaft of the motor 16.

  The ultrafiltration membrane device 5 includes an ultrafiltration membrane 17. The culture solution 2 is filtered by the ultrafiltration membrane 17, and the product produced by the bacterium as a filtrate, for example, a concentrated solution in which an enzyme is concentrated. Get. For example, the ultrafiltration membrane 17 has a molecular weight cut-off in the range of 5000 to 30000, and constitutes a hollow fiber membrane module. The concentrated liquid is supplied to a subsequent process through a concentrated liquid conduit 18 provided in the ultrafiltration membrane device 5. On the other hand, the filtrate of the ultrafiltration membrane 17 is discharged from the ultrafiltration membrane device 5 through the filtrate conduit 8.

  The microfiltration membrane device 7 includes a microfiltration membrane 19. The culture solution 2 is filtered by the microfiltration membrane 19, so that the cells that have not been separated by the spin filter 4 are separated and discharged from the discharge conduit 20. The microfiltration membrane 19 has a pore diameter of 0.2 μm, for example, and constitutes a hollow fiber membrane module. The microfiltration membrane device 7 is connected to a medium conduit 21 for supplying a new medium.

  The microfiltration membrane device 9 includes a microfiltration membrane 22, which separates germs and the like in the filtrate circulated to the culture tank 3 by the filtrate conduit 8 and discharges it from the discharge conduit 23. The microfiltration membrane 22 has a pore size of 0.2 μm, for example, and constitutes a flat membrane module.

  Next, operation | movement of the semi-continuous culture apparatus 1 of this embodiment is demonstrated.

  First, the culture tank 3 contains a predetermined culture solution 2, and the culture solution 2 is stirred by a stirring blade 12 that is rotated by a motor 10 via a shaft 11, thereby cultivating bacteria with the culture solution 2. The bacterium is not particularly limited as long as it is a bacterium that produces a useful product such as an enzyme, but is a fungus such as a filamentous fungus, and the microbial cells are entangled with each other to form a bacterial mass. Can be suitably used.

  Examples of the filamentous fungus include bacteria such as zygomycetes and ascomycetous fungi, and the filamentous fungus may contain incomplete bacteria. Specific examples of the filamentous fungi include Aspergillus, Trichoderma, Penicillium, Acremonium, and Fusarium.

  In the semi-continuous culture apparatus 1, a part of the culture solution 2 is taken out through the spin filter 4 and the culture solution conduit 6 while culturing the bacteria in the culture tank 3. At this time, the spin filter 4 separates the cells contained in the culture solution 2 by rotating the hollow cylindrical filter 4 b via the motor 16 and the pulley 15.

  The spin filter 4 forms a flow in which the culture solution 2 flows parallel to the surface of the filter 4b by rotating the filter 4b as described above. As a result, according to the spin filter 4, cross flow filtration can be performed, and the culture solution 2 substantially free of the bacterial cells and containing a product such as an enzyme produced by the fungus is a hollow cylindrical filter 4 b. Flows into the interior.

  The culture solution 2 that has flowed into the filter 4 b flows into the inside of a hole (not shown) provided in the hollow shaft 13 and is introduced into the culture solution conduit 6 through the hollow shaft 13. Therefore, according to the spin filter 4, even when the culture solution 2 contains microbial cells such as the fungal mass of the filamentous fungus, the microbial cells can be separated without being clogged by the microbial cells.

  Next, the culture solution 2 from which the cells have been separated by the spin filter 4 is supplied to the microfiltration membrane device 7 through the culture solution conduit 6. In the microfiltration membrane device 7, the culture solution 2 is filtered by the microfiltration membrane 19, and the microbial cells slightly mixed in the culture solution 2 without being separated by the spin filter 4 are also separated and discharged from the discharge conduit 20. . Further, in the microfiltration membrane device 7, a new culture medium supplied from the culture medium conduit 21 is dissolved in the culture solution 2, and germs and the like contained in the culture medium are separated by the microfiltration membrane 19. The miscellaneous bacteria and the like are discarded together with the cells by the discharge conduit 20.

  The culture solution 2 filtered by the microfiltration membrane device 7 is then supplied to the ultrafiltration membrane device 5 via the culture solution conduit 6 and filtered by the ultrafiltration membrane 17. The culture solution 2 at this stage does not contain bacterial cells, but contains products such as enzymes produced by the bacteria. Thus, by filtering through the ultrafiltration membrane 17, a concentrated solution in which the product such as the enzyme is concentrated as a filtrate is obtained. Supplied to the process.

  On the other hand, since the filtrate of the ultrafiltration membrane device 5 is only the culture solution 2 that does not contain the cells and the products produced by the bacteria, it is circulated to the culture tank 3 via the microfiltration membrane device 9 by the filtrate conduit 8. The In the microfiltration membrane device 9, the culture solution 2 as the filtrate is filtered through the microfiltration membrane 22, and various germs and the like are separated as filtrate. Filtrate such as bacteria is discarded through the discharge conduit 23.

  According to the semi-continuous culture apparatus 1 of the present embodiment, the filter 4b was produced by the bacteria without clogging even in the case of bacteria forming a bacterial mass such as filamentous fungi, as described above. Products such as enzymes can be recovered. Moreover, since the culture solution 2 taken out from the culture tank 3 is circulated to the culture tank 3 by adding a new medium, semi-continuous culture of the bacteria can be performed.

  Furthermore, since the spin filter 4 is disposed in the culture tank 3 in the semi-continuous culture apparatus 1 of the present embodiment, the culture tank 3 and the spin filter are used when the apparatus is cleaned and sterilized when the operation is stopped. 4 can be washed and sterilized together.

  DESCRIPTION OF SYMBOLS 1 ... Semi-continuous culture apparatus, 2 ... Culture solution, 3 ... Culture tank, 4 ... Spin filter, 5 ... Ultrafiltration membrane apparatus, 6 ... Culture solution conduit, 7 ... Microfiltration membrane device, 8 ... Filtrate conduit, 9 ... Microfiltration membrane device.

Claims (3)

A culture tank for containing the culture solution and culturing the fungus in the culture solution, and a first filtering means for collecting the product produced by the fungus by filtering the culture solution extracted from the culture vessel And a semi-continuous culture apparatus comprising a filtrate circulation means for circulating the filtrate of the first filtration means to the culture tank,
By rotating a hollow cylindrical filter disposed in the culture tank around the axis, the culture fluid is caused to flow in parallel to the filter, and cross-flow filtration is performed, thereby producing bacterial cells from the culture fluid. A semi-continuous culture, comprising: a solid-liquid separation means for separating the culture medium; and a culture liquid supply means for supplying the culture liquid from which the cells have been separated by the solid-liquid separation means to the first filtration means. apparatus.   The semi-continuous culture apparatus according to claim 1, wherein the filter includes pores having a pore diameter in the range of 0.001 to 1.0 mm.   The semi-continuous culture apparatus according to claim 1 or 2, wherein the first filtration means includes an ultrafiltration membrane, and the second filtration includes a microfiltration membrane disposed in the middle of the culture solution supply means. A semi-continuous culture apparatus comprising: means; and third filtration means provided in the middle of the filtrate circulation means and provided with a microfiltration membrane.