JPH0712303B2 - Continuous microorganism culture device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention relates to an improvement in a continuous microorganism culturing apparatus.

[Prior art] When continuously producing useful substances by utilizing the metabolic function of microorganisms, in order to improve the productivity, keep the bacterial cell concentration in the reaction tank high and increase the bacterial cell growth. It is necessary to remove the metabolites that are the inhibitory factors of recent years,
It has been attempted to use a filter in order to continuously separate cells and metabolites in a relatively short time.

In addition, if the lactic acid bacterium can be cultured at a high concentration by the method using the filter as described above, it can be used as a starter when producing many fermented dairy products such as yogurt and cheese. At present, a freeze-concentration starter is often used, but a concentration operation by centrifugation is required, and the activity of the starter decreases during centrifugation. On the other hand, the method using the filter does not cause such a problem.

By the way, conventionally, as the filter as described above, a tungsten sintered tube, an unfired ceramics support coated with diatomaceous earth, or a synthetic resin hollow fiber is used.

[Problems to be solved by the invention]

The problems with the filters for the above-mentioned applications are their durability and whether or not they can be stably used for a long time or reused. In particular, during use, whether or not backwashing and heat sterilization for avoiding clogging by bacterial cells can be a problem.

However, the tungsten sintered tube is easily immersed in acid and is not suitable for backwashing. Also, backwashing cannot be performed with a filter in which the ceramic support is coated with diatomaceous earth. Further, there are problems that synthetic resin hollow fibers cannot be backwashed and heat sterilized, and the amount of treatment is small.

Further, in practical use, it is of course desirable to solve the above problems and increase the processing amount.

[Means for solving problems]

The continuous microbial culture device of the present invention passes a culture solution in which a microbial reaction is carried out through a cylindrical filter, separates it into a filtrate containing metabolites and a concentrate containing cells, and culturing the concentrate. In a continuous microorganism culturing apparatus that circulates in a liquid, a ceramic filter that is an integral type having a plurality of passage holes and has a multilayer structure in which the pore diameter gradually increases around the passage holes is used as the filter. At the same time, a backwashing device is connected to the ceramic filter.

[Action]

In such a continuous microbial culture device, since the mechanical strength and heat resistance of the ceramic filter is good,
The filter can be backwashed and heat-sterilized, and it can be used stably for a long time and can be reused. Further, since the integrated ceramic filter having a plurality of passage holes is used, the filtration area can be increased and the flux of the filtration liquid can be increased.

It should be noted that passing the culture solution through the plurality of passage holes leads to a decrease in the flow flux due to a decrease in the liquid flow rate if the pump performance is the same. To this end, increase the filtration area by appropriately selecting the diameter of the passage holes (reducing the pore diameter to increase the liquid flow velocity) and the number (increasing the number to increase the filtration area). Since the contribution to the increase in the flux due to the above can be made to exceed the contribution to the decrease in the flux due to the decrease in the liquid flow velocity, the overall flux can be increased.

Further, if an attempt is made to obtain the same effect as that of the present invention by using a plurality of single filters each having a small pore size, the thickness of the ceramics must be increased in order to maintain the strength of each filter, which increases the cost. It becomes difficult to assemble the device. On the other hand, the integral ceramic filter having a plurality of through holes can maintain the strength even when the amount of the ceramic used is small, so that the cost can be reduced and the assembly of the device is easy.

〔Example〕

 Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a schematic configuration diagram of a continuous microorganism culturing apparatus according to the present invention. In FIG. 1, a culture tank 1 contains a culture solution 2 such as lactic acid bacteria. This culture solution 2 is in a constant temperature bath 3
It is maintained at a constant temperature by constant temperature water sent from. The culture solution 2 passes through a pipe 4, a pipe 7 having a pump 5 and a ball valve 6 interposed therebetween, a flow meter 8 and an inlet pipe 9 and then passes through a ceramic filter 11 provided in a filter case 10. The filtration liquid 12 containing the metabolites that has passed through the ceramic filter 11 is accommodated in the filtration liquid tank 15 through the filtration liquid pipe 14 having the solenoid valve 13 interposed therebetween. On the other hand, the concentrated liquid containing the bacterial cells is circulated in the culture tank 1 through an outlet pipe 17 having a ball valve 16 interposed. A bypass pipe 19 having a ball valve 18 is connected between the pipe 4 and the pipe 7 in parallel with the pump 5. Further, pressure gauges 20 and 21 are provided in the inlet pipe 9 and the outlet pipe 17, respectively. Further, a gas supply pipe 23 for backwashing which is provided with an electromagnetic valve 22 is connected to the filtration liquid pipe 14. Further, the culture tank 1 is provided with a liquid level gauge 24, and a new culture liquid containing lactose is supplied from the storage tank 26 to the culture tank 1 according to the amount of the liquid by a pump 25 linked with the liquid level gauge 24. It

The ceramic filter 11 is shown in FIGS.
The shape shown in the figure is used. The filter 31 shown in FIG. 2 is such that a ceramic seal 33 is attached to both ends of a hexagonal columnar filter body 32 made of alumina, and seven through holes 34 having a diameter of 4 mm are provided along the longitudinal direction thereof. Similarly, the filter 41 shown in FIG.
A ceramic seal 43 is attached to both ends of a prismatic alumina filter body 42, and 19 passing holes 44 having a diameter of 4 mm are provided along the longitudinal direction thereof. The total length of these filters is 750 mm. The filter bodies 32 and 42 have a multi-layer structure in which the pore diameters gradually increase around the passage holes 34 and 44 (Ceravert ceramic filter: trade name of Toshiba Ceramics Co., Ltd.).
Is. The pore diameter of alumina facing the passage hole of each filter is 0.2 μm.

Lactic acid bacteria (Streptcocc
us cremoris) was cultured. In addition, the experiment was carried out in the batch method in which the filter shown in FIG. 2 was used (Example 1) and the filter shown in FIG. 3 was used (Example 2), and filtration was not performed for comparison. (Reference example) When a ceramic filter having an inner diameter of 15 mm, an outer diameter of 19 mm and a total length of 750 mm is used (Comparative Example 1), a ceramic filter having an inner diameter of 15 mm, an outer diameter of 19 mm and a total length of 75
It was also carried out when two 0 mm ones were arranged in parallel (Comparative Example 2). Further, the filters used in Comparative Examples 1 and 2 are made of alumina having a multilayer structure in which the pore diameter gradually increases from the inside to the outside like the filters used in Examples 1 and 2 (Ceravale). Ceramic filter:
Product name of Toshiba Ceramics Co., Ltd.). The pore diameter of alumina facing the passage hole of each filter is 0.2 μm. Further, in Comparative Examples 1 and 2, a core was inserted in the passage hole to increase the liquid flux.

50g / lactose as a carbon source for lactic acid bacteria growth
And the amount of culture solution was 2. While pH control was performed using NH ₄ OH for the reference example and pH control was performed for Comparative Examples 1 and 2, and Examples 1 and 2, filtration was started 6 to 8 hours after the start of culture. Then, a culture solution containing an amount of lactose corresponding to the filtration flux was supplied to the culture tank to continue the filtration. However, in Comparative Example 2, the lactose concentration in the culture solution supplied halfway after the start of filtration was allowed to be 50 g / to 75 g /.

Then, the turbidity of the culture solution (OD ₅₇₀ ) and the lactose concentration (g / g
), The lactate concentration (mM) which is a metabolite was measured, and the flux was measured. These results are shown in FIGS. 4 to 8 in the order of Reference Example, Comparative Examples 1 and 2, and Examples 1 and 2. The arrows in FIG. 5 to FIG. 8 indicate the start of filtration. Further, in the following table, the liquid flow rate at the time of the experiment, the total amount of the filtration liquid until the growth of the lactic acid bacteria stopped (the time in parentheses until the growth stopped), the turbidity at the end of the experiment,
The dry cell amount and the cell number are collectively shown as the cell concentration measured separately from the turbidity.

As is clear from FIGS. 4 to 8, Comparative Examples 1, 2 and Examples in which the time for which the growth is stopped by the inhibition of lactate, which is a metabolite, is greater than that in Reference Example in which no filtration is performed. 1,
2 is longer. In Example 1, lactose was completely consumed 10 hours after the start of culture. However, even when the lactose concentration supplied after the start of filtration was 75 g /, the way of growth (turbidity curve) was Similarly, not lactose deficiency,
It was confirmed that the growth was stopped by being inhibited by the metabolite lactate. From this result, it is understood that lactate, which is a metabolite, can be removed by performing filtration, and lactic acid bacteria can be cultured at a high concentration.

Next, comparing FIGS. 5 and 6 (Comparative Examples 1 and 2) with FIGS. 7 and 8 (Examples 1 and 2), Comparative Examples 1 and 2
In contrast, the growth of lactic acid bacteria is temporarily stopped immediately after the start of filtration, whereas in Examples 1 and 2, such a phenomenon does not occur. From this, it can be seen that the concentration of lactic acid bacteria can be increased to a high concentration in a short time when the integrated filter having a plurality of passage holes is used as in Examples 1 and 2. Actually, Comparative Example 2 and Example 1 have substantially the same filtration area, and as shown in the table, the total filtration liquid amount of Comparative Example 2 is slightly larger than that of Example 1 ( Although the time is shorter in Example 1), the number of bacterial cells in Example 1 (1.92 × 10 ¹¹ , about 14 times that of the reference example) is in Comparative Example 2 (1.61 × 10 ¹¹ and about 13 times that of the reference example). ) Is more than.

Further, as is clear from FIG. 8 and the table, in the case of using the filter provided with 19 passage holes as in Example 2,
It can be seen that, although the liquid flow rate is small, both the total filtration liquid volume and the number of bacterial cells (3.5 × 10 ¹¹ , about 29 times that of the reference example) are extremely large.

In the above example, spherical lactic acid bacteria (Streptcoccus cr
The culturing of emoris) was described, but as shown in FIG. 9, the same results as in Example were obtained for rod-shaped lactic acid bacteria (Lactobaillus casei). Further, the continuous microorganism culturing apparatus of the present invention can be used not only for culturing lactic acid bacteria but also for all microbial reactions such as alcohol fermentation, acetone / butanol fermentation, cultivation and concentration of genetically modified bacteria using Escherichia coli and the like. It can also be used for production of useful substances by animal and plant cells and production of antibodies by cell fusion strains.

〔effect〕

As described in detail above, according to the continuous microorganism culturing apparatus of the present invention, stable use over a long period of time, re-use is possible, a large flux is large, and cells can be cultivated at a high concentration in a short time. It has a great effect.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a continuous microorganism culturing apparatus in an embodiment of the present invention, and FIGS. 2 and 3 show partially cutaway filters used in the apparatus of Examples 1 and 2 of the present invention, respectively. FIG. 9 is a perspective view, FIG. 4 to FIG. 8 are characteristic diagrams showing the results of culture experiments of the lactic acid bacteria obtained in Reference Example of the present invention, Comparative Example 1, Comparative Example 2, Example 1 and Example 2, respectively. FIG. 4 is a characteristic diagram showing the results of culture experiments of other lactic acid bacteria obtained in another example of the present invention. 1 ... Culture tank, 2 ... Culture solution, 3 ... Constant temperature tank, 4, 7 ...
… Piping, 5 …… Pump, 6,16,18 …… Ball valve,
8 ... Flowmeter, 9 ... Inlet piping, 10 ... Filter case, 11 ... Ceramic filter, 12 ... Filter liquid, 13,
22 …… solenoid valve, 14 …… boiler fluid piping, 15 …… boiler fluid tank, 17
...... Outlet piping, 19 …… Bypass piping, 20,21 …… Pressure gauge, 23 …… Gas supply piping, 31,41 …… Ceramic filter, 32,42 …… Filter body, 33,43 …… Ceramics seal, 34,44 …… Passing holes.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Masayuki Taniguchi 5-9 Kita-chikusa 1-chome, Chikusa-ku, Nagoya, Aichi Prefecture Nakada Housing RL-14 (72) Inventor Shunji Yasuda 1-26 Nishishinjuku, Shinjuku-ku, Tokyo No. 2 in Toshiba Ceramics Co., Ltd. (72) Inventor Takashi Ogawa 1 Minamitou, Ogakie-cho, Kariya city, Aichi Toshiba Ceramics Co., Ltd. in Kariya Plant (56) Reference JP-A-60-232084 (JP, A) JP-A 58-47485 (JP, A) Actually opened 60-144993 (JP, U)

Claims (1)

[Claims] 1. A continuous method in which a culture solution in which a microbial reaction is carried out is passed through a cylindrical filter to separate into a filtrate containing metabolites and a concentrate containing cells, and the concentrate is circulated in the culture solution. Type microorganism culturing apparatus, the filter is an integral type having a plurality of through holes, and a ceramic filter having a multilayer structure in which the pore diameter gradually increases around the through holes is used. A continuous-type microbial cultivating device characterized in that a backwash device is connected to the filter.