JPS6363195B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a high-concentration continuous culture method and apparatus for culturing various bacterial cells. (Prior art) In the high-concentration culture method and device using the backwashing effect disclosed in Japanese Patent Application No. 59-211747, two membranes are used to discharge the permeated liquid from one membrane, and to discharge the permeate from the other membrane. A medium is supplied, and the flow path is switched at certain time intervals to alternately provide a filtration function. (Problems to be Solved by the Invention) In Japanese Patent Application No. 59-211747 (hereinafter referred to as the conventional method), when a certain membrane constituting the system moves from the medium supply side to the permeate discharge side, the membrane cartridge, that is, the filtration chamber The culture medium retained outside the membrane was discharged together with the permeate without being used. By the way, in the case of the Asahi Kasei Microfilter with a membrane area of ^6m2 , the raw water retention amount is 2.0 within the membrane surface and 2.5 outside the membrane surface, and the total amount is
It's now 4.5. Therefore, 2.5 volumes of medium outside the membrane surface will be lost in one change. This amount of loss varies depending on the time it takes to sequentially switch the solenoid valves, how the membrane system is assembled, operating conditions, etc., but it roughly reaches about 10% of the total amount of culture medium used. (Means for Solving the Problems) Therefore, it is an object of the present invention to provide a high-concentration continuous culture method and device that can prevent the outflow of the medium and increase its utilization efficiency. The technical means of the present invention to solve this technical problem is that, when culturing various bacterial cells in a culture tank, the metabolic products produced during the culture are circulated together with the culture tank. The fresh medium is removed through the plurality of filtration membranes constituting the filtration membrane, and the fresh medium is supplied to the circulation circuit through the filtration membranes, and the flow path is switched between the filtration membrane on the permeate discharge side and the filtration membrane on the medium supply side at regular intervals. When switching the filtration function, for example, an inert gas such as N ₂ gas is used to send the bacterial liquid remaining on the culture medium supply side to the other permeated liquid discharge side to remove the culture medium. A high-concentration continuous culture method characterized by effective utilization, and a culture tank and multiple filtration chambers using filtration membranes such as UF membranes are connected via piping equipment to form a reversible circulation circuit. Solenoid valves are arranged in the arrangement device so that some of the channels can be used as the permeate discharge side in forward flow operation, and the rest can be used as the medium supply side in reverse flow operation, and the flow paths can be selectively switched. A high-concentration system characterized by a structure in which a piping device for feeding is added, and the bacterial liquid stagnant on the culture medium supply side can be sent toward the other permeated liquid discharge side using gas when switching the flow path. It is a continuous culture device. (Effects of the Invention) According to this technical means, it is possible to send the medium remaining in the membrane cartridge to another membrane cartridge when switching the flow path, completely eliminating the amount of unused medium, and reducing the amount of medium used. You can save 10%. (Example) Hereinafter, an example shown in the drawings will be described. First, the conventional method will be explained. In FIG. 5, 10 is a culture tank, 11,
12 is the first using the FOLLOW EYEVER UF membrane,
2 filtration chambers. The culture tank 10 and the first filtration chamber 11 are connected by a pipe 13 having a pump 37, and the first filtration chamber 11 and the second filtration chamber 12 are connected by a pipe 35. The second filtration chamber 12 is also connected to the culture tank 1 via a pipe 15.
0, and the first filtration chamber 11 is connected to the culture tank 10 by a pipe 14 branched from a pipe 13 via a valve 20. In addition to the above, a pipe 36 branched from the valve 19 is connected to the pipe 15 through a valve 21 in the middle of the pipe 13 . From the above, during the forward flow operation indicated by the solid line, the bacterial fluid from the culture tank 10 is returned to the culture tank 10 through the pipe 13 through the first filtration chamber 11 and the second filtration chamber 12 through the pipe 15. They form a circular circuit. In addition, during the reverse flow operation as indicated by the arrow shown by the chain line, the second filtration chamber 12 and the first filtration chamber 1 are connected from the pipe 36 through the pipe 15.
1, and are returned to the culture tank 10 through a tube 14. The culture medium is sent to the first and second filtration chambers from a culture medium supply tank 29 through a pump 31 to a pipe 30, and is pushed into the first and second filtration chambers through either a pipe 34 or 33 via a valve 32. ing. In addition, low-molecular nutrients including metabolites removed from the UF membrane are transferred from the first and second filtration chambers 11 and 12 to the pipe 2.
2, 40, a valve 23, a pipe 24, and a pump 26 to a filtration chamber 27, where metabolites are removed through an RO membrane and then returned to a medium supply tank 29 through a pipe 28. In addition, concentrated bacterial liquid is collected into a concentrated bacterial liquid recovery tank 17 through a pipe 13, a pipe 16, and a pump 18. Now, to explain it in detail according to the flow sheet of FIG. 5, first, inoculum is inoculated in the culture tank 10 and culture is started. Metabolites begin to accumulate after a few hours, and when a certain concentration is reached, the membrane begins to operate. During forward flow operation as indicated by the solid arrow, the bacterial cell fluid drawn out from the culture tank 10 by the pump 37 is passed through the first and second filtration chambers in that order. The UF membranes in the first and second filtration chambers are composed of follow-up fiber UF membranes. In this case, the first filtration chamber 11 is on the high pressure side, and the permeate containing metabolites is passed through the pipe 22, valve 23, pipe 24, pump 26, tube 25
is led to a filtration chamber 27, where metabolites are removed by an RO membrane, and the low molecular weight nutrients from which metabolites have been removed are returned to a medium supply tank 29 through a pipe 28.
It is now possible to measure the effective use of culture media. On the other hand, in the second filtration chamber 12 on the low-pressure side, low-molecular nutrients and fresh culture medium from which metabolites have been removed are pumped by a pump 31 from a tank 29 to a pipe 30, a valve 32, and a pipe 3.
3 and is operated to maintain the tank level in the culture tank 10 at a constant level. If the above operating conditions are continued, the rate of permeate from the first filtration chamber will increase in viscosity as the number of bacteria increases during the culture process, and will decrease due to clogging of the membrane. ,20,21,
23 and 32 are switched simultaneously to enter a reverse flow operation as indicated by the chain arrow. By switching the valve, the high pressure side and the low pressure side are reversed, and the first filtration chamber, which had previously discharged the permeated liquid from inside the system to the outside, is now in a state where liquid is forced into the system from outside the system, that is, tank 29, Pump 31, pipe 3
0, enters a backwashing state via the valve 32 and pipe 34, and removes bacterial cells adhering to the membrane surface, and supplies low-molecular nutrients while also serving as washing in preparation for the next forward/reverse operation. In this way, by switching between the high pressure and low pressure sides of the first and second filtration chambers, different functions can be alternately provided, and the culture can be continued while continuously removing metabolites without reducing the permeation flow rate, resulting in continuous high concentration. can be cultured. As described above, the conventional method uses two membranes, discharges the permeate from one membrane, supplies the medium to the other membrane, and switches the flow path at certain time intervals to alternately perform the filtration function. It is designed to hold up. However, when a certain membrane moves from the medium supply side to the permeate discharge side when switching the flow path, the medium held outside the membrane surface in the membrane cartridge 35 is discharged together with the permeate without being used, as shown in FIG. . Reference numeral 36 in FIG. 1 shows a hollow fiber membrane filled with a culture solution (concentrated bacterial solution), and the space between the membranes 36 is filled with a supply medium or a permeated waste liquid. The amount of loss discharged as described above varies depending on the time of sequentially switching the solenoid valves, how the membrane system is assembled, operating conditions, etc., but it roughly reaches about 10% of the total amount of medium used. The present invention uses an inert gas such as N ₂ gas and a solenoid valve to prevent the medium from flowing out;
The aim is to increase the efficiency of its use. FIG. 2 shows a flowchart illustrating the principles of the invention. In the figure, MF-1 is on the medium supply side, MF-2
is on the permeate discharge side. In this case, the membrane cartridge of MF-1 is filled with a medium. Here, in the present invention, immediately before switching the flow path, _N2 gas is injected from the upper right side of the membrane of MF-2, which will be the medium supply side in the next step, to collect the permeate in the membrane cartridge. The permeate is discharged as shown in Figure 2 (A), and the flow path is switched at the same time as this permeate is discharged, so that the supply medium can be smoothly transferred from MF-1 to MF-2 as shown in Figure 2 (B). be. At this point, the MF-1 from which the medium has drained becomes the permeate discharge side and returns to normal operation. By performing the above-mentioned operation approximately 10 seconds before and after switching the main line in this way, the supply medium held in the membrane cartridge can be effectively used without loss. FIG. 3 shows the membrane arrangement system and piping lines, and FIG. 4 shows the operation of the solenoid valves at each step. The designations of the solenoid valves in FIG. 4 are the same as those shown in FIG. The solenoid valve newly added by this invention is 1G, 1
H, 1E, 2G, 2H... 12 in total, the sending side solenoid valve PF of the culture medium supply pump 37, and the return line side solenoid valve
There are a total of 14 PRs. This newly added solenoid valve was incorporated into the system so that the above-mentioned culture medium could be moved for 10 seconds before and after the 4 steps of switching the main line. When switching the main line, solenoid valves PR and PF are linked to temporarily stop the culture medium supply. The time before and after switching the main line is 10 in the example.
Although the flow rate is assumed to be seconds, the actual flow rate will vary depending on the membrane size, pipe diameter, etc., so preliminary experiments should be conducted to determine the actual flow rate. In any case, the operation of the electromagnetic valve shown in FIG. 4 is designed to smoothly discharge the permeate and move the culture medium when changing each step. The operation according to the present invention when switching the flow path from step 1 to step 2 on a trial basis will be explained as follows. That is, in step 1, the solenoid valve 1F is closed and the solenoid valves 2F, 3F, and 4F are open, so the bacterial liquid sent from the circulation pump 38 is
MF ₂ , MF ₃ , and MF ₄ are branched and ascend. In this case, the three membranes are on the high pressure side, and the permeate side solenoid valve 2
Since P, 3P, and 4P are opened in conjunction with each other, the permeate flows out from this line 55. The bacterial liquid that has passed through MF ₂ , MF ₃ , and MF ₄ is separated into MF 1 and return line 50 and returns to culture tank 10 .
Here, the MF ₁ becomes the low pressure side, the medium supply electromagnetic valve 1S opens, and the medium is supplied to this membrane and flows into the system with a backwashing effect. Next, MF ₂ has a backwashing effect and other MF ₁ ,
When switching MF ₃ and MF ₄ to the permeate discharge side, MF ₁
is on the medium supply side and MF ₂ is on the permeate discharge side.
In this case, the membrane cartridge of MF1 is filled with a medium. Therefore, in the next step, MF _2, which will be the medium supply side,
_N2 gas is pressurized from the tank 76 through the electromagnetic valves 2G and 2H from the upper stone side of the membrane, and the filtrate in the membrane cartridge is passed through the electromagnetic valve 2P into the pipe 5.
Let it be discharged from 5. At the same time as this permeate is discharged, the flow path is switched and the MF _1
N2 gas is injected into the top of the MF1, and the medium inside the MF1 is
Moves into MF ₂ via solenoid valves 1S and 2S. In this case, the gas in the MF ₂ is discharged via the electromagnetic valves 2H and 2E, and the MF ₁ from which the medium has drained becomes the permeate discharge side at this point and returns to normal operation. That is, MF ₂ undergoes a backwashing effect, and MF ₁ ,
MF ₃ and MF ₄ are on the permeate discharge side. The solenoid valves PF and PR are interlocked with each other. When PF is closed, PR is open, and when PF is open, PR is closed. When supplying culture medium, PR is open.
is closed and RF is open. However, from MF ₁ to MF ₂
No new medium is supplied when the medium is being moved. Therefore, the solenoid valve RF is closed and PR is opened, and the medium in the medium supply tank is circulated by the pump 37. Similarly, in steps 2, 3, and 4, MF ₂ , MF ₃ , and MF ₄ are sequentially subjected to backwashing by operating the solenoid valves shown in FIG. In seconds, permeate is drained from one membrane cartridge and feed medium is transferred from the other membrane. In the case of the example, anaerobic bacteria are cultured, but in the case of culturing aerobic bacteria, the culture tank 10 and the culture medium supply tank 29 are air-conditioned (ventilated) to increase the dissolved oxygen concentration, and the culture is carried out. Aseptic air is used instead of _N2 gas. The figure below shows the results obtained when operating according to the example shown in the conventional method for the amount of culture medium used according to the present invention and the conventional method.

[Table] By adding the operation according to the present invention as described above, it became possible to save about 10% of the medium.

[Brief explanation of drawings]

Figure 1 is a cross-sectional view of the hollow fiber membrane, Figure 2 (a) and (b) are flow diagrams of the culture medium and permeated waste liquid during flow path switching, Figure 3 is a membrane arrangement system and piping line diagram according to the present invention, and Figure 4 is a diagram of the membrane arrangement system and piping line according to the present invention. FIG. 5 is a diagram of the operation of the electromagnetic valve in each step, and a diagram of the membrane arrangement system and piping line according to the conventional method. 10... Culture tank, 29... Culture medium supply tank, 3
6... Hollow fiber membrane, 38... Circulation pump, 35, MF ₁ , MF ₂ , MF ₃ , MF ₄ ... Filtration chamber (membrane cartridge), 1F, 2F, 3F, 4F, 1
R, 2R, 3R, 4R, 1S, 2S, 3S, 4
S, 1P, 2P, 3P, 4P, 1G, 2G, 3
G, 4G, 1H, 2H, 3H, 4H, 1E, 2
E, 3E, 4E... Solenoid valve, 50... Return line pipe, 55... Permeate discharge pipe.

Claims (1)

[Scope of Claims] 1. When culturing various types of bacterial cells in a culture tank, metabolites produced during the culture are removed through a plurality of filtration membranes that constitute a circulation circuit together with the culture tank, and fresh culture medium is passed through the filtration membranes. The gas is supplied to the circulation circuit through the filter, and when switching the flow path between the filtration membrane on the permeate discharge side and the filtration membrane on the medium supply side at regular intervals to alternately provide the filtration function, the gas is A high-concentration continuous culture method characterized by using an inert gas such as N ₂ gas to send the bacterial liquid stagnant on the medium supply side toward another permeated liquid discharge side to effectively utilize the medium. 2. The culture tank and a plurality of filtration chambers using filtration membranes such as UF membranes are connected via piping equipment to form a reversible circulation circuit, and some of the filtration chambers are used as the permeate discharge side of normal flow operation, The remaining part is used as the culture medium supply side for reverse flow operation, and a solenoid valve is placed in the arrangement device so that the flow path can be selectively switched, and a piping device for gas feed is added to the flow path, so that the gas is supplied when switching the flow path. 1. A high-concentration continuous culture device characterized in that it is configured such that a bacterial solution staying on a culture medium supply side can be sent toward another permeated liquid discharge side using a.