JPS6363194B2 - - 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.

(Problem to be solved by the invention) When scaling up the above-mentioned device,
Not only the capacity of the culture tank must be increased, but also the membrane area must be increased. However, in the case of the hollow fiber type membrane used, there is a limit to the area of one membrane, so it is necessary to combine a large number of membranes to make effective use of the membrane.

According to Japanese Patent Application No. 59-211747, both the culture medium supply side and the permeate discharge side are arranged with the same area. In this method, only 50% of the installed membrane area can be used as an effective filtration area.

In actual experiments, the number of bacteria in the system increased as the culture progressed, and although the effect of backwashing using the culture medium was observed, the flow rate of permeate on the discharge side decreased. On the other hand, the capacity of the medium supply side hardly decreases during the course of culture.

Therefore, in the latter stages of culture, an imbalance occurs between the capacity on the supply side and the capacity on the discharge side, and as a result, the membrane area on the discharge side determines the capacity of the device.

(Means for Solving the Problems) Therefore, the technical problem of the present invention is to provide a method and device for effectively utilizing the membrane area in a membrane culture device. The technical means of the present invention to solve the problem is that when culturing various types of bacterial cells in a culture tank, metabolites produced during the culture are removed through a plurality of filter membranes that constitute a circulation circuit together with the culture tank, and fresh When the culture medium is supplied to the circulation circuit through the filtration membrane, some of the filtration membranes are used as the permeate discharge side in forward flow operation, and the rest are used as the medium supply side in reverse flow operation, so that the membrane area ratio of the discharge side to the supply side is at least the supply side. A high-concentration continuous culture method characterized by selectively switching the flow path so that the discharge side is several times larger than that of the previous one to make the most effective use of the membrane area, and a culture tank and a filtration membrane such as a UF membrane. The plurality of filtration chambers used are connected via piping equipment to form a reversible circulation circuit, with some of the filtration chambers serving as the permeate discharge side for forward flow operation, and the rest serving as the medium supply side for reverse flow operation. A high-concentration continuous culture device characterized in that the piping device is configured such that the flow path can be selectively switched so that the membrane area ratio on the supply side is at least several times that on the discharge side as compared to the supply side. It is.

(Effects of the invention) According to this technical means, not only can the functions and effects of Japanese Patent Application No. 59-211747 (hereinafter referred to as the conventional method) be achieved as they are, but also the functions and effects described below can be achieved. It is.

In other words, in the conventional method, when four membranes are used to arrange the membranes on the supply side and the discharge side with equal area,
Effective filtration area is 0.1μ microfiber 0.2m ² /
If one membrane is used, it will be 0.2 x 2 = 0.4 ^m2 , but according to the present invention, if one membrane is placed on the supply side and three membranes are placed on the discharge side, it will be 0.2 x 3 = 0.6 ^m2 . can obtain 50% more effective filtration area than conventional methods.

According to the invention, there is one on the supply side and three on the discharge side.
If two membranes are arranged and the time for one cycle is set to the same time as the conventional method, the time required for backwashing one membrane will be 1/2 that of the conventional method.

As described above, in the present invention, the backwashing time is reduced by half, but since the membrane surface flow velocity can take a high value, the effect is large and can be sufficiently put to practical use.

In any case, according to the present invention, there is no imbalance between the supply side and discharge side capacities even in the later stages of culture, and the membrane culture apparatus can be scaled up.

(Example) The embodiments shown in the drawings will be described below.

First, the conventional method will be explained.

In FIG. 7, 10 is a culture tank, 11,
12 is the first one 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 in detail according to the flow sheet of FIG. 7, first, seed bacteria are 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 velocity of the 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 membrane clogging. 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, reinforcing low-molecular nutrients while also serving as washing in preparation for the next forward flow 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. The bacterial cells 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 was designed to hold up.

In contrast, the present invention utilizes the installed membrane area as effectively as possible. In other words, the one shown in Figure 1 uses four membranes to
This is a combination of several solenoid valves.

MF ₁ , MF ₂ , MF ₃ , and MF ₄ indicate filtration chambers, and these and the culture tank 10 are connected by a circulation pump 38 to form a circulation circuit.

That is, the bacterial fluid from the culture tank 10 is transferred to the pipe 6.
6 and sent to each filtration chamber by a circulation pump 38.

Pipes 39, 40 are pipes 41, 42, 43,
Connected to each filtration chamber through 44, pipe 45,
46, 47, and 48 constitute a return line to the culture tank 10 through a pipe 49.

And the permeate is pipe 56, 57, 58, 5
The culture medium is collected from pipe 9 into pipe 55 and supplied from pipe 64 to one of the filtration chambers through branch pipes 60, 61, 62, and 63. 1
F, 2F, 3F, 4F, 1R, 2R, 3R, 4
R, 1P, 2P, 3P, 4P, 1S, 2S, 3
S and 4S indicate each electromagnetic valve in the piping.

Therefore, using the sequencer shown in Figure 3, the solenoid valve can be switched and operated at 10 minute intervals, Step1→Step2→Step3→Step4→Step1→
Step 2: Switching the flow path in the membrane in the following order:
Each membrane can fully perform its filtration function by undergoing backwashing.

To explain Step 1, since the solenoid valve 1F is closed and the solenoid valves 2F, 3F, and 4F are open, the bacterial fluid sent from the circulation pump 38 is transferred to the pipe 4.
It branches through MF ₂ , MF ₃ , and MF ₄ through MF 2 , 43 , and 44 and flows upward.

In this case, the three membranes MF ₂ , MF ₃ , MF ₄ are on the high pressure side, and the permeate side solenoid valves 2P, 3P, 4P are opened in conjunction with each other, so the permeate flows out from this line to the pipe 55 .

The bacterial fluid that has passed through MF ₂ , MF ₃ , and MF ₄ is divided into MF ₁ and a return line, and returns to the culture tank 10 through pipes 49 and 50 .

Here, the MF ₁ becomes the low pressure side, the medium supply electromagnetic valve 1S opens, and the medium is supplied to this membrane, causing it to flow into the system with a backwashing effect. This is shown in Figure 3B.

In steps 2, 3, and 4, MF ₂ , MF ₃ , and MF ₄ can be cycled while receiving backwashing by operating the solenoid valves shown in FIG. 3, respectively. Figure 3C shows Step 2.

In order to compare the present invention as described above with the conventional method, a system line assembled by the conventional method is shown in FIG. The same parts as in FIG. 1 are given the same reference numerals.

The operation of opening and closing the solenoid valve using the sequencer shown in Figure 4 will be explained from Step 1.
Since the solenoid valve 1F is open and the solenoid valve 2F is closed, and the solenoid valve 1R is closed and the solenoid valve 2R is open, the bacterial fluid sent from the circulation pump 38 flows into the pipes 39 and 7.
4, 72, and 78, branching into MF ₁ and MF ₂ and ascending. The bacterial fluid flowing out from MF ₁ and MF ₂ enters MF ₃ and MF ₄ through pipes 75, 77, and 76 and returns to the culture tank 10 through pipes 79, 73, and 50, and MF ₁ and MF ₂ are on the high pressure side. in
MF ₃ and MF ₄ are on the low pressure side.

The permeated liquids of MF ₁ and MF ₂ are discharged from pipe 68 and supplied to MF ₃ and MF ₄ through pipe 64. Therefore, the low pressure side has a backwashing effect. This is shown in Figure 4B.

According to Step 2, MF ₃ and MF ₄ are on the high pressure side,
MF ₁ and MF ₂ are on the low pressure side. This is shown in Figure 4C.

According to the above, both the culture medium supply side and the permeated liquid discharge side have the same membrane area, and the number of solenoid valves is smaller than that of the present invention, so the line is simplified, but the permeated liquid Lateral membrane area is always 0.2×
2 = 0.4m ² , which is small.

In any case, the two membranes on the supply side and the two membranes on the discharge side are switched alternately, and the solenoid valve on the permeate discharge side, which is the high pressure side, opens in conjunction with the solenoid valve to allow the permeate to flow out, while the medium supply, which is the low pressure side. The solenoid valve on the side opens in conjunction with the solenoid valve to supply the culture medium into the membrane.

(Experimental Example) The present invention as described above and the conventional method will be compared using the following experimental method.

Yeast extract 1.0%, peptone 1.0%, permeate flour 3.0%, meat extract 0.5 in a 10 volume culture tank
%, K ₂ HPO ₄ 0.5%, KH ₂ PO ₄ 0.1%, Na ascorbic acid 0.1%, water 93.8% Medium 6
After sterilizing at 120°C for 15 minutes, cool and inoculate with Blongum seed that had been pre-cultured.
After culturing at a temperature of 35°C ± 0.5°C, membrane culture operation was started.

In addition, as the pH decreases as the operation progresses, 2N
-NH ₄ OH was used to perform constant pH culture at PH = 6.0.
Other pretreatment conditions are the same as in the conventional method.

The membrane used in the experiment was Asahi Kasei's 0.1 μ microfiber (one membrane area: 0.2 m ² ).

(Experimental Results) FIG. 5 shows the change in the permeate flow rate after the start of membrane operation. At the start of membrane operation, the bacteria concentration was low and the permeate discharge capacity was sufficient, but operation was carried out to ensure effective utilization of the culture medium by supplying a culture medium commensurate with the growth of bacterial cells. The feeding rate of this medium (this value is approximately equal to the amount of permeated liquid) was determined by optimization from the values of the bacterial cell concentration, the lactose concentration in the permeated liquid, and the specific growth rate of the bacterial cells.

It was possible to operate according to this optimal supply rate until about 5 hours after the start of operation, but after this period, the amount of permeate stopped increasing with the conventional method, and decreased after 7 to 8 hours. A tendency to do so was observed.

In contrast, in the present invention, an optimized amount of permeate was obtained up to about 10 hours after the start of culture, and the difference between the two is clearly considered to be due to the difference in the membrane area on the permeate side.

Figure 6 shows a comparison of the growth rates of bacterial cells when experimenting with both devices. In the present invention, the bacteria grew at a constant specific growth rate from 0 to 12 hours, whereas in the conventional method, the cells grew at a constant specific growth rate. This is probably because the increase in the permeate flow rate slowed down after 5 hours, but the specific growth rate began to decline, and in the latter stages of culture, it was difficult to maintain the number of bacteria despite supplying the medium. It was finally in a good condition.

Therefore, there is a large difference in the final concentrations obtained by both methods after 12 hours of culture.

The results of this experiment show that how the membrane area is used is a major factor in determining the device capacity.

Furthermore, based on the results of this experiment, referring to the influence of the ratio Sp/Ss of the permeate side membrane area Sp to the medium supply side membrane area Ss on the backwashing effect, it is 1.0 in the conventional method, whereas in this type, In the method of the present invention, it is 3.0. The supply side membrane area Ss is 0.4 m ² in the conventional method, but it is 0.2 m ² in the method of the present invention, and the flow rate through the membrane area during backwashing is twice as high in the former as in the latter. shows. As mentioned above, the present invention sequentially switches the solenoid valve.
There are four steps, whereas in the conventional method, there are four steps.
There are only 2 steps. Therefore, if one cycle time is set to the same time for both, the time required for backwashing of one membrane is 1/1/2 of the conventional method in the present invention.
It becomes 2.

In this way, the backwashing time is halved in the present invention, but since the membrane surface flow velocity can take a high value as described above, the effect is large and can be sufficiently put to practical use.

[Brief explanation of drawings]

Fig. 1 is a diagram showing a membrane arrangement system according to the present invention, Fig. 2 is a diagram showing a membrane arrangement system according to a conventional method, Fig. 3 is a flow chart of liquid at each step according to the invention, Fig. 4 A, B, and C are flow diagrams of the liquid at each step according to the conventional method, FIG. 5 is a diagram showing changes in permeate flow rate over time, FIG. 6 is a bacterial cell growth curve diagram, and FIG. 7 is a diagram showing the conventional method. MF ₁ , MF ₂ , MF ₃ , MF ₄ ...filtration chamber, 10...
...Culture tank, 29...Medium supply tank, 38...Circulation pump, 55...Permeate discharge pipe, 64...
Medium supply pipe, {1F, 2F, 3F, 4F, 1
R, 2R, 3R, 4R, 1P, 2P, 3P, 4
P, 1S, 2S, 3S, 4S}... Solenoid valve.

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. When supplying to the circulation circuit through the filter, some of the filtration membranes are used as the permeate discharge side for forward flow operation, and the rest are used as the medium supply side for reverse flow operation, so that the membrane area ratio of the discharge side to the supply side is at least as high as the discharge side with respect to the supply side. A high-concentration continuous culture method characterized by selectively switching the flow path so that the side is several times as large as possible to make the most effective use of the membrane area. 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 piping device was configured so that the flow path could be selectively switched so that the remaining membrane area was on the culture medium supply side in reverse flow operation and the membrane area ratio of the discharge side to the supply side was at least several times that of the supply side. A high-concentration continuous culture device characterized by: