JPS6188872A - Method and apparatus for continuous cultivation at high concentration - Google Patents

Abstract

PURPOSE:To enable the culture of microbial cells in high concentration, by supplying the culture medium to the second filtration membrane in the case of normal flow direction in the circulation circuit, and supplying to the first filtration membrane in the case of reverse flow and continuing the cultivation while removing the metabolic products from the medium. CONSTITUTION:A seed microorganism is inoculated in the culture tank 10, the cultivation is started, and when the concentration of the metabolic product reaches a specific level, the operation of membranes is started. In the case of the normal flow operation shown by the solid arrow, the cell liquid extracted from the culture tank 10 with the pump 37 is passed in the order of the first and the second filtration chambers 11, 12. The first filtration chamber 11 is operated under higher pressure, and the filtrate containing the metabolic product is introduced into the filtration chamber 27, the metabolic product is removed by the RO membrane, and the nutrient component having low molecular weight is returned to the medium supply tank 29. When the system is turned to the reverse-flow operation shown by the dotted arrow by the simultaneous change-over of the three-way solenoid valves 19, 20, 21, 23 and 32, the first filtration chamber is subjected to the back-washing, and is supplied with low-molecular nutrient component.

Description

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

(Prior Art) Generally, when culturing various types of bacterial cells, their growth is often inhibited by their metabolites.

Therefore, attempts have already been made at the laboratory level to remove metabolites produced during culture and achieve high concentration culture by combining a culture tank and a UF membrane filtration membrane.

In the one in Figure 2, a dialysis tank (3) is installed between the culture tank (1) and the holding tank (2), which are connected through a pump (4), and metabolites are removed through the dialysis membrane. This allows the bacterial fluid to circulate.

This is Journal the 5ociety Da
iry Technology Vol 30 NIL
Published in January 1979.

Also, The Au5tralian Journal o
f Dairy Technology-March 1
977 contains the following information: That is, in FIG. 3, the first RO film (5) and the second RO film (6)
) is connected to the raw material tank (9) through a pump (7) and a pressure control valve (8), and each RO membrane removes metabolites and recovers low-molecular nutrients, which are then returned to the raw material tank (9). It has become so.

(Problems to be solved by the invention) In the conventional method as described above, the low molecular weight nutrients removed from the dialysis membrane are directly supplied to the culture tank, the liquid level in the culture tank is maintained constant, and the culture is continued. It was continuing.

However, with this method, as the culture progresses, the liquid permeation rate rapidly decreases due to the increase in the viscosity of the culture solution and the clogging of the membrane surface with bacterial cells, and the metabolites produced in the culture tank cannot be removed sufficiently.
Therefore, there is a drawback that metabolites are accumulated in the tank and the growth of bacterial cells is suppressed.

(Means for Solving the Problems) Therefore, the technical problem of the present invention is a high-concentration continuous culture method capable of continuously cultivating bacterial cells at a high concentration by continuously culturing while removing metabolites. and for measuring equipment.

The technical means of the present invention to solve this technical problem is that when culturing various types of bacterial cells in a culture tank, the metabolites produced during the culture are collected in the first and second channels, which constitute a circulation circuit together with the culture tank.
In order to recover low-molecular active ingredients from the removed metabolites and supply them to the circulation circuit through the filtration membrane together with a fresh medium, in the forward direction of the circulation circuit, they are supplied to the second filtration membrane, and in the reverse flow direction. In this direction, the first filtration membrane is supplied with a highly concentrated bacterial solution, and the culture tank and the 1.2 filtration chamber, which uses a filtration membrane such as a UF membrane, are connected via piping equipment. The first and second filtration chambers are connected to form a reversible circulation circuit, and the first and second filtration chambers are switchably connected to a supply tank capable of supplying fresh culture medium and low-molecular active ingredients recovered from metabolites, and are also connected to a metabolite recovery device. However, in the forward flow direction of the circulation circuit, the second filtration chamber is connected to the supply tank,
In the reverse flow direction, piping is configured to connect the first filtration chamber to the supply tank.

(Effects of the Invention) In the present invention, metabolites produced during culture can be efficiently removed from the system using a filtration membrane such as a UF membrane, and the low-molecular active ingredients and fresh medium that flow out together It is possible to culture the bacteria while replenishing it through the membrane, and to continuously circulate the highly concentrated bacterial solution.

In other words, during normal flow operation, the bacterial cell liquid is transferred from the culture tank to the
The permeated liquid containing metabolites is discharged from the first filtration chamber on the high-pressure side to the outside of the thread through the filtration chambers in order, while low-molecular nutrients and fresh culture medium excluding metabolites are passed through the second filtration chamber on the low-pressure side. can be pushed in from outside the system to maintain a constant tank level inside the culture tank.

During reverse flow operation, the high-pressure side and low-pressure side of the first and second filtration chambers are reversed, and the first filtration chamber, which previously discharged the permeate from inside the system to the outside, is used to remove metabolites. Molecular nutrients and fresh culture medium are pushed into the system from outside the system, that is, enter a reverse '/'jc state, where bacterial bodies adhering to the membrane surface are removed and washed in preparation for the next forward flow operation. It can replenish low-molecular nutrients while also serving as a supplement.

In this way, by switching between the high pressure and low pressure sides of the 1.2 filtration chamber, different functions can be provided alternately, and the culture can be continued while continuously removing metabolites without reducing the permeation flow rate. The bacterial cells can be cultured.

Therefore, according to the present invention, culturing is carried out while removing growth-inhibiting substances produced during culturing, making it possible to achieve higher concentration culturing than the conventional batch method.

In addition, when producing a predetermined number of bacterial cells due to an increase in the concentration of bacterial cells, the volume of the culture tank can be greatly reduced, allowing for a small and compact design.

Furthermore, since the culture is carried out while effectively removing the metabolic products produced during the culture, the amount of culture medium required can be reduced to 115 or less, and since the medium to be replenished enters the system through the membrane, sterilization of the medium is not required, resulting in energy savings. It is possible.

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

In Figure 1, (10) is a culture tank, (11)
(12) is the first method using a follow fiber UF membrane.
．． 2 filtration chambers.

The culture tank (10) and the first overflow chamber (11) are connected to the pump (37).
) are connected by a pipe (13), and the first filter chamber (11) and the second filter 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).
The first filtration chamber (11) is connected to the culture tank (14) via a valve (20) from the pipe (13).
10).

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

From the above, during the forward flow operation indicated by the solid line, the bacterial liquid from the culture tank (10) flows from the tube (13) through the first filtration chamber (11) and the second filtration chamber (12). 15) to be returned to the culture tank (10), forming one circulation circuit.

Also, during reverse flow operation as indicated by the chain line arrow, the flow from the pipe (36) to the pipe (
15) to the second filtration chamber (12) and the first filtration chamber (1
1) and return to the culture tank (10) in the tube (14).

In the 1.2 filtration chamber, the culture medium is sent from the culture medium supply tank (29) to the pipe (30) through the pump (31), and the valve (
32) and into the 1.2 filtration chamber through any of the pipes (34) and (33).

In addition, low-molecular nutrients including metabolites removed from the UF membrane are transferred from the 1.2 filtration chambers (11) (12) to the pipes (22).
(40), valve (23), pipe (24) and pump (26) to the filtration chamber (27), where R
After the metabolites are removed through the O membrane, they are returned to the medium supply tank (29) through the pipe (28).

In addition, the concentrated bacterial liquid is collected from the pipe (13) through the pipe (16) and the pump (18) into the concentrated bacterial liquid recovery tank (17).

Now, to explain in detail according to the flow sheet shown in FIG. 1, first, inoculum is inoculated in a 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 line arrow, the pump (
The bacterial cell fluid drawn out in step 37) is passed through the 1.2 filtration chambers in this order.

The UF membrane in the 1.2 filtration chamber is the follow fiber U
In this case, the first filtration chamber (11) becomes the high pressure side, and the permeate containing metabolites flows through the tube (22), valve (23), tube (24), pump (26), tube (2).
5) to the filtration chamber (27), where metabolites are removed by an RO membrane, and the low molecular weight nutritional components from which the metabolites have been removed are passed through a tube (27).
28) and returned to the culture medium supply tank (29), so that the culture medium can be used effectively.

On the other hand, in the second filtration chamber (12) on the low pressure side, the pump (31
), low-molecular nutrients and fresh medium from which metabolites have been 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 (1o). be driven to do so.

If the above operating conditions are continued, the rate of permeate from the first filtration chamber will decrease due to increased viscosity due to the increase in the number of bacteria during the culture process and clogging of the membrane.
9) Switch (20), (21), (23), and (32) at the same time and enter reverse flow operation as indicated by the chain arrow.

By switching this 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, the tank (29 ), the pump (31), the pipe (30), the valve (32), and the pipe (34) enter the backwashing state, and bacteria adhering to the membrane surface are removed and prepared for the next forward flow operation. Replenishes low-molecular nutrients while also doing laundry.

In this way, by switching between the high pressure and low pressure sides of the 1.2 filtration chamber, different functions can be alternately provided, and the culture can be continued while continuously removing metabolites without reducing the permeation flow rate, thereby continuously increasing the concentration of bacteria. The body can be cultured.

In the embodiment, the first and second filtration chambers are arranged in series, but they may be arranged in parallel.

Further, although the examples show a continuous type operation, the operation may be completed in a batch type until the secondary growth phase.

An experiment of continuous high-concentration culture of Bacterium bifidus will be described below.

Yeast extract 1.0%, peptone 1.0%, vermeate If 13.0%, meat extract 0.5%, K2HP 040.5%, KH, PO40 in a 10ffi volumetric culture tank.
, 1% Na O, 1% ascorbate, and 93.8% water were prepared, and the medium 61 was sterilized at 120°C for 15 minutes, cooled, and precultured in advance.
ongum inoculum was inoculated and cultured at a temperature of 33°C±0.5°C.

Before inoculation, N gas is aerated into the culture medium to remove O2 gas dissolved in the culture medium, and N2 gas is sealed above the culture solution surface to prevent contact with O2 gas during culture. I declined. Figure 4 shows changes in the number of viable bacteria, lactose content, and concentration of lactic acid, a metabolite, during the culture process.

The initial pH is 6.8, but as the culture progresses, the pH decreases and growth stops when it exceeds a certain value. Therefore, in this experiment, a constant pH of 6.0 was controlled using 2N-NH,OH. went.

After 10 hours of culture, the concentration of lactose, which is the limiting substrate, was 2 g/
f or less, and the L-lactic acid concentration is 10 g/j! Since the growth stops beyond this point, the operation of the membrane system specified in the present invention is started from this period.

The membrane used in the experiment was a follow fiber membrane S E P4O10 (molecular weight cut off 6000,
(Membrane area: 0.2 rd, heat resistant temperature: 90°C). To sterilize the inside of the membrane and piping system, after circulating 80℃ hot water for 1 hour, immersing in 200 ppm hypochlorous acid solution for 3 hours, remove the hypochlorous acid solution using sterile water, and place in a 10jl' culture dunk. They were joined aseptically and used for experiments.

Ten hours after the start of culture, the peristaltic pump is activated to start membrane operation. The forward and reverse flow path switching was performed at 30 minute intervals using a timer.

The medium to be supplied is yeast extract 1.0% and peptone 1.0%.
, Vermeate flour 3.0%, Meat extract 0.5%, K2H
A medium having a composition of 0.5% PO, 0.1% KH, PO, 1% Na O ascorbate, and 93.8% water was previously passed through a 5EP1013 membrane.

In addition, the culture tank liquid level was kept constant at 6ρ.

As a result, the rate of liquid permeation through the membrane decreases as the bacteria multiply, as shown in Figure 5.6, but at a certain target concentration [1
When the bacterial liquid started to be discharged steadily after reaching 0'' (/mjri), no decrease in the liquid permeation rate was observed, and it was possible to continuously obtain the bacterial liquid.

The specific growth rate μ (Hr”) of the bacteria until steady operation is reached is the -order specific growth rate μ (when operating only 8 medium tanks)! is 0.23 CHr-'), and the second order specific growth rate μ (Hr'') is 0.23 CHr-') μ
2 (when performing culture tank operation while performing membrane operation) is 0.
10 (Hr).

By calculating the balance of substrates, viable bacteria, and products in the system using the above results, the seedling production rate, concentrated bacterial liquid extraction rate, substrate consumption rate, metabolite rate, etc. during continuous operation can be determined. I can do it.

In this way, after passing through the second growth period (10 hours) and the second growth period (8 hours), a solution with a bacterial cell concentration of 10' (-/mε), 0.6 J/HrX36Hr=21.6j! We were able to obtain it stably.

When the remaining liquid in the tank and the amount of liquid in the pipes are 6.81, it is 21.
6+6.8=28.1. The amount of medium used during this period was 1,001. The total operating time is 54 hours.

Conventionally, to obtain a high-concentration culture solution of this type of bacteria, a constant pH culture method was used to obtain a bacterial solution, and then the sludge-like bacteria were collected using a centrifugation method, and the dispersion was used to collect 1011 (/mjl). Comparing the results of this experiment with this conventional method, we find that: 1 1 Conventional method 1 Present method 1 Ratio 11 Culture tank volume 1 250 A l 10 / 10
．． 041 (Culture method 1 batch type 1 continuous type 1-
11 Culture time 116 hours 154 hours 1311
Amount of medium used l 200z 1100 e
10.5 11 Obtained bacterial liquid volume 1 1
1 1110” (-/ml) l 10j!
128.4112.8411 Conversion I
I 1 11 bacterial solution/Used medium 10.05 (-
) IO, 28415, 681111 (-) II As described above, when the present invention is used to obtain an equal volume of a highly concentrated solution containing a predetermined number of bacteria, the culture tank volume can be greatly reduced compared to the conventional method, and the culture medium used can be reduced. The amount can also be reduced.

Although the active ingredients in the permeate were not recovered in this experimental example, if this was done, it would be possible to further reduce the amount of medium used.

In addition, in this experimental example, a high-concentration culture example of Bifidus bacteria was shown, but it can also be fully used for culturing aerobic bacteria.

Furthermore, although a UF membrane with a molecular weight cutoff of 6000 was used in this experimental example, effective removal of metabolites is possible by using a microfilter with a size of about 0.1 to 0.45 μm, which has a sterilization effect.

[Brief explanation of the drawing]

Figure 1 is a flow sheet of the device of the present invention. Figure 2.3 is a flow sheet of a conventional culture device. Figure 4 is a culture time coat diagram. Figure 5 is a diagram of changes in permeation flow rate through the membrane. Figure 6 is a diagram of one membrane. Fig. 7 is a graph of the pressure distribution before and after the UF membrane during normal flow operation. (10)...Cultivation tank (11)...First filtration chamber (12)...Second filtration chamber (29)...Pa・Medium supply tank When culturing Figure 6 Culture promotion time (+-(r) Figure 7 (Inlet L1) (Exit) (Inlet) (Outlet D)
()Tr) Figure 5: 01 hours from the start of membrane operation

Claims (2)

[Claims] (1) When culturing various types of bacterial cells in a culture tank, metabolites produced during the culture are removed through the first and second filtration membranes, which together with the culture tank constitute a circulation circuit, and low-molecular active ingredients are extracted from the removed metabolites. When collecting and supplying the fresh medium to the circulation circuit through the filtration membrane, in the forward direction of the circulation circuit, it is supplied to the second filtration membrane, and in the reverse direction, it is supplied to the first filtration membrane to continuously maintain high concentration. A high-concentration continuous culture method characterized by making the bacterial solution moist. (2) The culture tank and the first and second filtration chambers using a filtration membrane such as a UF membrane are connected through a piping device to form a reversible circulation circuit, and the first and second filtration chambers are used for fresh culture medium and metabolic switchably connected to a supply tank capable of supplying low-molecular active ingredients recovered from the product, and connected to a metabolic product recovery device;
A high-concentration connected culture device, characterized in that the piping is configured such that the second filtration chamber is connected to the supply tank in the forward flow direction of the circulation circuit, and the first filtration chamber is connected to the supply tank in the reverse flow direction.