JPS61254184A - Method of continuous culture in high concentration and device therefor - Google Patents

Abstract

PURPOSE:To utilize effectively a membrane area and to cultivate continuously a mold in high concentration, by discharging a filtered solution from some filter membranes among plural filter membranes, and changing channels at fixed intervals. CONSTITUTION:The solenoid value 1F is closed, the solenoid valves 2F-4F are opened, the solenoid valves 2P-4P at the filtered solution side are opened, a mold solution in the culture tank 10 is passed through the pipe 66, the circulating pump 38 and the pipes 42-44, sent to the filter changers MF2-4, filtered, the filtered solution is sent through the pipes 57-59 and the pipe 55 and discharged. The mold solution is separated into the filter chamber MF1 and the pipe 49, blended with a medium sent through the solenoid value 1S, provided with a reverse washing effect and circulated through the culture tank 10 (Step 1). Then, after a fixed time is over, the mold solution is circulated while being provided with reverse washing effect by the operation of the solenoid valves shown in the Steps 2-3. through the filter chambers MF2-4.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a high-concentration continuous culture device and device for culturing various types of bacterial cells.

(Prior art) In the high-concentration culture method and device using the backwashing effect disclosed in Japanese Patent Application No. 59-21-1747, two wooden membranes are used to discharge the permeated liquid from one membrane and drain the permeated liquid from the other membrane. A culture medium is supplied to the filter, and the flow path is switched at certain time intervals to alternately provide a filtration function.

(Problems to be Solved by the Invention) When scaling up the above-described apparatus, 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, 1
Since the membrane area of a book is limited, it is safe to combine a large number of membranes to make effective use of the area.

Regarding the discrepancies in patent application No. 59-211.747, lr<
Both the oil supply side and the permeated liquid 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 culture medium supply example hardly decreases during the course of culture.

Therefore, in the later stages of culture, there is an imbalance between the supply side and discharge side capacities, and as a result, the membrane area on the discharge side becomes
1 hika will be decided.

(Means for Solving the Problem) 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, and to solve this technical problem. The technical means of the present invention, when culturing various types of bacterial cells in a culture tank, removes metabolites produced during the culture through a plurality of filtration membranes that together with the culture tank constitute a circulation circuit, and also allows fresh culture medium to pass through the filtration membranes. When supplying to the circulation circuit, some of the filtration membranes are used on the permeate discharge side in forward flow operation, and the rest are used on the medium supply side in reverse flow operation, so that the membrane area ratio of the discharge full to the supply example is at least 1 on the supply side. A high-concentration continuous culture method characterized by selectively switching the flow path so that the number of discharge sides (iη) is reached to maximize the effective use of the membrane area;
A culture tank and a plurality of filtration chambers using filtration membranes such as OF membranes are connected through a piping device to form a reversible circulation circuit 111.
! Some of the I, II & 2 passage chambers are used as the permeate discharge side for normal flow operation, and the rest are used as the culture medium for J-flow operation! ! The piping device is configured such that the flow path can be selectively switched so that the membrane area ratio of the discharge side to the supply side is at least several times that of the supply side on the supply side. This is a high concentration continuous culture method.

(Effects of the invention) This technical means not only achieves the effects of the patent application No. 59-211747 (hereinafter referred to as the conventional method), but also achieves the effects described below. It is.

In other words, in the fj'ff conventional method, when four membranes are used to arrange the membranes in the same area on the supply and discharge sides, the effective filtration area is 0.1 μ, assuming 0.2 m/i of microfibers. However, according to the present invention, if one crotch is arranged on the supply side and three crotches on the discharge side, then 0.2X 3 =0.6 m', and the present invention Compared to conventional methods, the effective filtration area is 50% larger.

According to the present invention, if one membrane is arranged on the supply side and three membranes are arranged on the discharge side, and the cycle time is set to the same time as the conventional method, the time required for backwashing of one membrane is the same as that of the conventional method. /2
becomes.

In this way, the time of backwashing g is reduced by half in the present invention, 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 Figure 7, (]0) is a culture tank, (]1)(
12) is No. 1.2 using follow fiber UF membrane.
It is a filtration chamber.

The culture tank (10) and the first i1!6 filtration chamber (11) are connected by a pipe (13) with a pump (37), and the first filtration chamber (
11) and the second filtration chamber (12>) are connected through a pipe (35).

The second filtration chamber (12) is also connected to the culture tank (
10), and the first filtration chamber (11) is connected to the culture tank (10) by a pipe (14) branched from the pipe (13) via a valve (20).

In addition to the above, in the middle of the pipe (13), a pipe (36) branched from the valve (19) connects to the pipe (15) via the valve (21).
is connected to.

From the above, in the forward flow operation indicated by the solid line, the bacterial fluid from the culture tank (10) passes through the tube (13), the first filtration chamber (11), and the second filtration chamber (12). The water is returned to the culture tank (10) through a pipe (5), forming one circulation circuit.

Also, during reverse flow operation as indicated by the ifj line -, 'i:i(
3G) through the tube (15) to the second σV overchamber (12)
, the first filtration chamber (11) in this order, and is returned to the culture tank (10) through a pipe (14).

1.211&, the culture medium is sent from the culture medium supply tank (29) to the pipe (30) through the pump (31), and to either of Ai' (34) (33) through the valve (32). Through the 1.2 rr = pushed into the excess chamber a) yo).

In addition, low-molecular nutritional components including metabolites removed from the UF tube are passed from the 1.2 filtration chamber (11,) (12) to the tube (22).
) (40), valve (23), pipe (24>,
1 no. through the pump (26)! The medium is led to a chamber (27) where, after removing metabolites through an RO membrane, it is returned to a medium supply tank (29) through a pipe (28).

Others: pipe (13) to pipe (16), pump (18)
The concentrated bacterial liquid is collected into a concentrated bacterial liquid recovery tank (17) through the tank.

Now, to explain it in detail according to the flow sheet in Figure 7, first, inoculate the inoculum in the culture tank (10).
L. Start culturing.

After a few hours, metabolites begin to accumulate, and when they reach a certain concentration, they begin to drive.

During forward flow operation as indicated by the solid line arrow, the pump (
The bacterial cell liquid drawn out in step 37) is passed through the 1st and 2nd filtration chambers in this order.

The UF crotch in the 1.2 filtration chamber is a follow fiber U
In this case, the first filtration chamber (11) is on the high pressure side, and the permeate containing metabolites is passed through the tube (22), valve (23), tube (24), pump (26), tube ( 25) to the filtration chamber (27), where metabolites are removed by an RO membrane. This allows for effective use of the culture medium.

On the other hand, in the second filtration chamber (12) on the low pressure side, the pump (3
1), low-molecular nutrients and Shinmachi culture medium with metabolites removed are pushed from the tank (29) through the pipe (30), valve (32), and pipe (33) to maintain a constant tank level in the culture tank (10). (It is operated so that λj is maintained.

If the above operating conditions are continued, the permeate rate from the first filtration chamber will increase in viscosity as the number of bacteria increases during the culture process.
At this time, the three-way solenoid valve (19) (20) (21) (23) (
32) at the same time and enters reverse flow operation as indicated by the chain arrow.

By switching this valve, the high-pressure side and low-pressure side are reversed, and the permeated liquid is forced into the system from the first filtration chamber, which had previously been discharged from the system to the tank. (29), the pump (3]), the pipe (30), the valve (32), and the pipe (34) enter the backwashing Hg state, which removes the bacterial cells on the membrane surface, and then the next forward flow. In preparation for operation, it also serves as a washing machine and replenishes low-molecular nutrients.

In this way, by energizing the high pressure and low pressure sides of the 1.2 filtration chamber, the light function is alternately provided, and the culture is continued while continuously removing substances without reducing the permeation flow rate. It is possible to culture a high concentration of bacterial cells.

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 perform the filtration function alternately. It is designed to hold up.

In contrast, the present invention utilizes the installed membrane area as effectively as possible.

That is, the one shown in Figure 1 uses four membranes to
This is a combination of several solenoid valves.

MFz, MF2. M7. MFz and MuJ filtration chambers are shown, 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 (66).
) to each filtration chamber by a circulation pump (38).

Pipe (39) (40) is pipe (41) (4
2) Connected to each filtration chamber through (43) and (44), and pipes (45) (46) (47) (48)
) constitutes a return line through the pipe (49) to the i1'' nutrient tank (10).

And the permeate is piped (56) (57) (5
8) Collected from (59) to pipe (55), pipe (
64) to branch pipe (60) (61,) (62
) The culture medium is supplied to either of the filtration chambers through (63). IF, 2F, 3F,
,IF.

], R, 2R, 3R, 4R, 11], 2P, 3P.

4, P, Is, 2S, 3S, 4.3 indicate each electromagnetic valve in the piping.

Therefore, we used the sequencer shown in Figure 3 to set up one solenoid valve.
Switching operation can be performed at 0 minute intervals, 5 steps
], --3tep 2-→5tep 3-tStep
By cutting out the channels in the membrane in the order of 4-→5Lep 1-5tep2..., each membrane is backwashed and can fully exhibit its obscuring ability.

To explain step 1, the solenoid valve IF is closed and the solenoid valves 2F, 3F, and 4F are open, so the bacterial fluid sent from the circulation pump (38) flows through the pipe (42) (
43) Through (44), MF2. MF3゜M Fψ
It branches out in the middle of the river and flows up and down.

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

MF2, MF, the bacterial fluid that has passed through MF is MF
/ Divided into return line and pipe (49)
At (50), return to the culture tank (10).

Here, MF7 is on the low pressure side, and the solenoid valve IS for culture medium supply is
When the membrane opens, medium is supplied to this membrane, creating a backwashing effect.
It flows into the system. This is shown in Figure 3 (b).

Below are 5 steps 2. 3. 4 are respectively MF2.4 by operating the solenoid valves shown in FIG. MF3. MF4 can perform saikling while receiving backwashing.

FIG. 3(C) shows 5tep2.

In order to compare the method of the present invention with the conventional method as described above, the sostem line of silk cloth made by the conventional method is shown in FIG. The same parts as in Figure 1 are marked with the same symbols.

The operation of opening and closing the solenoid valve using the sequencer shown in Fig. 4 will be explained starting from 5 steps.
is open and solenoid valve 2F is closed, and furthermore, solenoid valve IR is closed and solenoid valve 2R is open, so the bacterial fluid sent from the circulation pump (38) flows through the pipes (39) (74) (72)
(78), branches into MF, MF2° and rises. The bacterial fluid flowing out from MF/, MF2 is transferred from pipes (75) (77) (76) to MF3.
MF≠ enter pipe (79) (73) (50)
MF/
, MF2 are on the high pressure side, and M Fj and MF bodies are on the low pressure side.

The permeated liquid of MF, MF2 is discharged from the pipe (68) and supplied to MF and MFψ through the pipe clover 4). Therefore, the low pressure side has a backwashing effect. This is shown in Figure 4 (b).

According to 5tep2, MF, , MFμ are on the high pressure side, and MF, , MF2 are on the low pressure side. This is shown in FIG. 4(c).

According to the above, both the culture medium supply side and the permeate discharge side have the same membrane area, and the number of solenoid valves is smaller compared to the present invention, so the line is simplified, but the permeate side membrane The area is densely small, 0.2 x 2 = 0.4 m.

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.

In a 10ff volumetric culture tank, yeast extract 1.0%, peptone 1.0%, permini-1 to powder 3.0%, meat extract 0°5%, K11 (POge 0.5%, KH2PO, IO,
1% Na ascorbic acid, 0.1% Na ascorbate, and 93.8% water were prepared, and after sterilization at 120°C for 15 minutes, the medium was cooled and precultured.
After inoculating um inoculum and culturing at a temperature of 35±0.5°C, 1'g of membrane was grown. I started driving Le.

Note that P■ decreases as the operation progresses, so 2N N
11. A constant I'l+ culture with l111=6.0 was carried out by foll.

Other pretreatment conditions are the same as in the conventional method.

The membrane used in the experiment was Asahi Kasei! It is a 0.1μ microfiber (membrane surface & J 1 piece 0.2 m).

(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 the operation was carried out to make effective use of the medium by supplying a medium commensurate with the growth of bacterial cells.

The feeding rate of this medium (this value is equal to the amount of permeate) was determined by optimization from the values of the bacterial cell concentration, the lactose concentration in the permeate, 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 did not increase much with the conventional method (and the amount of permeate did not increase much after 7 to 8 hours). (The positive force was sustained by 9.

On the other hand, in the present invention, an optimized permeation flow rate was obtained for 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 thought to be 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. The situation was finally there.

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.

In addition, referring to the effect of the ratio Sp/Ss of the membrane area on the permeate side Sp and the membrane area Ss on the medium supply side from the present experimental results, in the conventional method, this value is 1.
．． While it is 0, it is 3.0 in the method of the present invention. The supply side membrane area Ss is 0.4 in the conventional method, whereas it is 0.4 in the method of the present invention. 2m and 172, and the flow rate through the membrane area during backwashing is twice as high in the latter as in the former.

As described above, in the present invention, there are four 5-step steps in which the solenoid valve is sequentially switched, whereas in the conventional method, the 5-step steps are fewer than two times. Therefore, if the time of one cycle is divided into seven times for both parties, then
In the present invention, the time required for backwashing one membrane is 1 in the conventional method.
/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 the drawing]

FIG. 1 shows a membrane arrangement system according to the present invention. FIG. 2 shows a membrane arrangement system according to a conventional method. FIG. Figure 4 (a), (b), and (c) are flow diagrams of the liquid at each step according to the conventional method. Figure 5 is a diagram of the change in permeate flow rate over time. Figure 6 is a bacterial cell growth curve diagram. Figure 7 is a diagram showing the conventional method. It is. MF, , MF, , MFJ, MF Me...Filtration chamber (10)...Culture tank (29)...Medium supply tank (38)...Circulation pump

Claims (2)

[Claims] (1) When culturing various types of bacterial cells in a culture tank, metabolites produced during cultivation are removed through multiple filter membranes that together with the culture tank constitute a circulation circuit, and fresh culture medium is supplied to the circulation circuit through the filter membranes. , 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 several times that of the supply side. A high-concentration continuous culture method characterized by selectively switching flow channels to make the most effective use of membrane area. (2) The culture tank and multiple 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 connected to the permeate discharge side for forward flow operation. The piping system is designed so that the flow path can be selectively switched so that the membrane area ratio of the discharge side to the supply side is at least several times that of the supply side, with the remainder being used as the culture medium supply side in reverse flow operation. A high-temperature continuous culture device characterized by the following configuration.