JP2000287672A - Method and apparatus for continuously culturing microorganisms in a tubular culture tank - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase the effective surface area of a carrier in a culture solution for continuous culture and to prevent microorganisms having optimal activity by culturing while preventing substrate inhibition and competition inhibition. Provide a way to harvest without. SOLUTION: A culture solution is continuously supplied from one end,
A method and apparatus for continuously culturing microorganisms, wherein the microorganisms are cultured in a tube-shaped culture tank discharged from the other end.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for continuously culturing microorganisms.

[0002]

2. Description of the Related Art In continuously culturing microorganisms, a carrier for adsorbing the microorganisms has been conventionally introduced into a culture tank. The main factor is that a large amount of microorganisms can be produced by continuous culture because the concentration of microorganisms in the culture tank increases as the microorganisms are adsorbed and retained on the carrier, and as a result, the amount of microorganisms that grow as seeds increases. That is the reason. In addition, the advantage of the introduction of the carrier is that the growth rate of the microorganism is increased and the activity of the microorganism is improved by the adsorption of the microorganism on the carrier.

[0003] As an apparatus for continuous culture using a carrier, a stirred tank type, a fixed bed type and a fluidized bed type are generally used. In each case, a microorganism to be cultured and a carrier are administered into a culture tank, a culture solution of the microorganism is introduced from one side, and a culture solution containing the cultured microorganism is taken out from the other side.

[0004] In the stirring tank type, stirring blades are installed in the culture tank,
The carrier is suspended in the culture solution by stirring the culture solution by rotation of the stirring blade.

[0005] In the fixed-bed type, the carrier is densely filled in a culture tank, and the culture solution flows between the carriers by introducing the culture solution, whereby the carrier and the culture solution are contacted.

[0006] In the fluidized bed type, a carrier is administered in a state of low filling in a culture tank, and the carrier floats in the culture tank by introducing the culture solution or flowing the culture solution by introducing a gas such as air. .

[0007] In addition, a basket is installed at the center of a culture tank of a stirring tank type or a fluidized bed type, and a carrier is held there.

In a stirred tank type or a fluidized bed type, the carrier needs to be suspended in the culture solution by stirring or flowing the culture solution. When a carrier having a specific gravity larger than that of the culture solution is used, the stirring speed or the flow rate is increased. The carrier cannot be suspended in the culture solution unless the speed is increased accordingly. However, when the stirring speed or the flow speed is increased, the degree of damage to the microorganisms and the carrier itself in the culture solution due to the shearing force increases, and the microorganisms adsorbed on the carrier are peeled off. In addition, there is also a problem that an increase in power required for stirring and flowing increases operating costs.

On the other hand, when a carrier is used for continuous culture, it is the surface of the carrier that adsorbs microorganisms, and it is important to maximize the total surface area of the carrier with respect to the culture solution in order to exert the effect of the carrier. Come. For this purpose, it is required to use a carrier having a large surface area per volume, that is, a carrier having a relatively small particle size or a porous carrier.

[0010] On the other hand, when culturing microorganisms, when the substrate concentration of the culture solution increases, the growth and activity of the microorganisms may be inhibited, and this is generally called substrate inhibition. In the case of batch culture, fed-batch culture in which a substrate that acts as an inhibitor is added little by little so that the concentration in the culture solution does not increase is used as a method for preventing substrate inhibition. In this case, if the number of additions is increased, the concentration of the finally produced microorganism will increase.

On the other hand, in continuous culture, the culture solution is supplied to the culture tank little by little continuously, so that the substrate concentration in the culture solution does not increase as in fed-batch culture, and continuous culture avoids substrate inhibition. This is considered to be a suitable culture method.

There is also a phenomenon called competition inhibition in which the substrate of the culture solution competes for the enzyme of the microorganism with the target substance which is to act on the microorganism, and inhibits the action of the target microorganism.

A specific example is the decomposition of an organochlorine compound by a microorganism. In order for microorganisms to decompose organochlorine compounds, the presence of a chemical substance called an inducer is required. That is, the target organic chlorine compound can be decomposed by the enzyme expressed to decompose the inducer.

[0014] The inducers known at present include aromatic compounds such as phenol, cresol and toluene, methane and the like, and most of them can be used as a nutrient source for culturing degrading bacteria.

In the case of decomposing an organic chlorine compound using this inducer, if an inducer is present in a reaction field where the decomposition is carried out, the decomposing enzyme is used only for decomposing the inducer, and the decomposition of the organic chlorine compound is essential. Stalls, that is, competitive inhibition occurs.

[0016] Competition inhibition by the inducer occurs in a form in which the inducer competes for an organochlorine compound and a degrading enzyme. A dramatic result, such as a complete disappearance of the decomposition activity of organochlorine compounds by only a few ppm increase, would be obtained.

In the case of batch culture, the inducer is eventually consumed and is hardly a problem, but in the case of a continuous culture reactor, a culture solution containing the inducer is supplied to the culture tank one after another. It is necessary not to bring the inducer into a place where organic chlorine compounds are decomposed, that is, to set the feed rate of the inducer so that the inducer is almost completely consumed during the culture.

[0018]

[Problem to be Solved by the Invention] [First Problem] When a carrier having a small particle size and a low specific gravity is used, the culture can be efficiently performed by a stirring tank type or fluidized bed type culture apparatus. However, when a carrier having a small particle size and a large specific gravity is used, it becomes difficult to culture in a stirred tank type or fluidized bed type as described above.

Even when a carrier having a small particle size and a large specific gravity is used, when a basket for holding the carrier is installed at the center of the culture tank with a stirring tank type or fluidized bed type culture apparatus, or when a fixed bed type is used. Does not cause the problem of suspending the carrier in the culture solution.

However, when the cage is filled with a carrier having a small particle size, the gap between the carriers is small, and it is difficult for the culture solution to flow into the cage. Since the pressure loss received when the culture solution flows between the filled carriers is proportional to the reciprocal of the square of the particle size of the carrier, the pressure loss is quadrupled when the particle size is reduced by half. Therefore, when the particle size is small, the growth of microorganisms on the surface of the carrier present inside the basket becomes inactive, and the utilization efficiency of the carrier is reduced.

For example, a cylindrical cage (inner diameter 30 mm, mesh hole diameter 0.5) containing an anion exchange resin having a particle diameter of 0.6 mm.
mm) is immersed in a microorganism culture solution containing a culture solution, and even when the culture solution is flown, it has been experimentally confirmed that microorganisms hardly grow within an inner radius of 15 mm of the basket ( Comparative Example 1-1).

In the case of the fixed bed type, a channel in which the culture solution easily flows is often formed in the gap between the filled carriers, and a state in which the culture solution is in contact only with the surface of a specific carrier is likely to occur. .

In any type of culture apparatus, when the carrier is filled vertically, the packing density increases, so that the contact area between the carriers increases, and the contact area between the carrier and the culture solution necessarily decreases. . In particular, when the carrier is flexible, when the carrier is filled and laminated, the lower carrier is compressed, and the contact area between the carriers becomes larger. In addition, the gap between the carriers becomes smaller, making it more difficult for the culture solution to flow into the inside of the cage, and the channels through which the culture solution easily flows are more easily formed.

When a porous carrier is used, the smaller the pore size, the larger the surface area of the carrier. However, as the pore diameter becomes smaller, the culture solution becomes less likely to flow into the pores due to the surface tension of the culture solution. In particular, in the case of a carrier made of a hydrophobic material, it becomes difficult to flow the culture solution even with a pore diameter of about several mm.

Even if the microorganisms can enter the pores, it is difficult to maintain the growth rate of the microorganisms in the pores at the same level as the outside of the carrier because the exchange rate of the culture solution is low inside the pores. Become. Therefore, it is considered that the surface area of the carrier increases as the pore diameter is reduced, but the surface area effectively used for the adsorption of microorganisms reaches a peak at a certain pore diameter, and thereafter decreases.

From the above, when aiming to increase the effective carrier surface area in the culture solution of continuous culture, when using a conventional culture apparatus, when using a non-porous carrier, the particle size is small. A carrier with a low specific gravity must be used in a stirred tank type or fluidized bed type culture device, and the type of carrier used is very limited.Even when using a porous carrier, the inner surface of the pores can absorb microorganisms. There was a problem that it was difficult to use it effectively.

[Second problem] In a continuous culture, when the supply rate of the culture solution is set, the maximum concentration of the culture solution to be supplied is limited to a level at which the substrate is not inhibited. Accordingly, there is a problem that it is not possible to produce a high concentration of microorganisms.

For example, when the substrate to be decomposed is an organochlorine compound, the inducer is previously induced with an inducer as described above. On the other hand, in order to avoid competition inhibition, do not bring the inducer into a place where the organochlorine compound is decomposed. In other words, it is necessary to set the feed rate of the inducer so that the inducer is almost completely consumed during the culture. However, the culture conditions and the growth rate of the microorganism to be cultured,
The concentration of the residual inducer in the culture solution tends to change due to the change in the inducer consumption rate, and it is very difficult to control the concentration to the optimum concentration.

If the supply rate of the culture solution is reduced more than necessary to avoid the residual of the inducer, the time required for the enzyme produced by the inducer to be used becomes longer, and the activity of the enzyme decreases during that time. Might be.

When the inducer concentration in the culture solution is set low, the inducer for the degrading bacteria is weakened, and the production of the degrading enzyme is reduced. In particular, when the inducer also serves as a growth substrate for the microorganism, the concentration of the produced microorganism is reduced, which also reduces the decomposition activity.

On the other hand, in the continuous culture, the supplied culture solution is almost instantaneously diluted by the culture solution in the culture tank. However, since the culture solution diluted with the culture solution is discharged from the culture tank as it is, production from the culture device is performed. This means that the culture medium to be prepared always contains the same components as the supply culture medium having a constant concentration. Therefore, when the supply rate of the culture solution containing the inducer is increased, the concentration of the inducer remaining in the culture solution discharged from the culture tank also increases, and the competitive inhibition appears remarkably.

The fact that a certain amount of unused culture solution is always contained in the culture solution discharged from the culture tank means that a certain amount of culture solution is always wasted and wasted. When the speed is increased, the culture cost is not negligible.

Furthermore, when an inducer such as phenol or toluene is used, the degrading bacteria may be damaged by the increase in the concentration of the inducer itself, and the above-mentioned substrate inhibition may occur. If the concentration of the inducer decreases, the active bacteria may not be cultured even if the inducer concentration is reduced.

As described above, in continuous culture, particularly in the case of continuously culturing microorganisms that decompose organochlorine compounds using an inducer, substrate inhibition and competition inhibition are prevented while preventing
A major issue is how to harvest microorganisms having optimal activity by culturing without deterioration.

An object of the present invention is to solve the above-mentioned problems, and is a continuous culture apparatus using a tubular culture tank and a continuous culture method using the same.

[0036]

That is, the present invention provides a continuous microorganism culture wherein a culture solution is continuously supplied from one end and the microorganism is cultured in a tube-shaped culture tank discharged from the other end. This is a culture method.

It is preferable that the culture tank is filled with a carrier, and further, it is preferable that the packing density of the carrier in the culture tank can be arbitrarily adjusted.

Alternatively, it is preferable that at least the inside of the carrier is porous and the inside can be impregnated with a liquid, and the pores opened on the surface of the carrier communicate with the pores inside the carrier.

In addition, one or more branch pipes that can communicate between the inside and outside of the culture tank are provided besides both ends of the tubular culture tank, or an extension tube is continuously provided at the discharge end of the culture solution in the culture tank. By doing so, it is preferable that the length of the tube for culturing can be arbitrarily extended, and that the extension tube is provided with one or more branch tubes capable of communicating the inside and the outside of the extension tube.

In this case, by supplying the culture solution from the branch tube, the substrate in the culture solution in the tube is suppressed to a certain concentration or less, and the amount of substrate added in the whole culture step is increased without causing substrate inhibition. And discharging and collecting the culture solution from the branch pipe, or controlling the residence time of the culture solution by the length of the tube.

Controlling the concentration of residual substrate in the culture solution;
In particular, the concentration of the residual substrate is preferably a concentration that does not cause competition inhibition for microorganisms.

Further, it is preferable to continuously culture the microorganisms decomposing organochlorine compounds using an inducer in the culture tank, and discharge and collect the culture liquid from the branch pipe to further discharge the culture liquid from the culture tank. By extending the tube length by continuing the tube from the end, it is preferable to control the residual inducer concentration in the discharged and collected culture solution to a concentration suitable for decomposing the organochlorine compound.

Further, it is preferable that the residual inducer concentration is a concentration that does not cause competitive inhibition against the decomposition of the organic chlorine compound by the microorganisms that decompose the organic chlorine compound.

Further, it is preferable to monitor the culture solution from the branch tube, and to determine the branch tube from which the culture solution is supplied or the culture solution is recovered.

Further, a tube-shaped culture tank is provided as a means for culturing microorganisms, and the culture tank has means for continuously supplying a culture solution from one end and means for discharging the culture solution from the other end. This is a continuous culture device for microorganisms.

In addition, the culture vessel is filled with a carrier, and one or more branch pipes that allow communication between the inside and the outside of the culture vessel are provided other than at both ends of the culture vessel, or It is preferable to have a connection portion for an extension tube at the culture solution discharge end of the culture tank.

In particular, it is preferable that the extension tube has at least one branch pipe capable of communicating the inside with the outside.

[0048]

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, for the first problem, it is necessary to fill a tube-shaped culture tank with a carrier. Hereinafter, the continuous culture method using the present apparatus will be specifically described by distinguishing between a non-porous carrier (one not impregnated with a liquid) and a porous carrier (one impregnated with a liquid). (The structure and properties of the carrier will be described later).

First, the non-porous carrier 102 having no liquid impregnated therein will be described with reference to FIG. Non-porous carrier 102
When a pre-cultured microorganism is charged into a tube 101 filled with, and a culture solution 103 is continuously introduced from one end of the tube 101, a flow 104 of the culture solution is generated in the tube.
After the culture solution 105 fills the tube, the tube 101
Is discharged from the other end.

Under the flowing state of the culture solution in such a tube, when the culture solution 3 first comes into contact with the microorganism, the growth of the microorganism starts using the nutrients in the culture solution. As the concentration of bacteria in the culture solution in the tube increases, the amount of microorganisms adsorbed on the carrier also increases, and the growth of microorganisms on the carrier becomes active. When the density of the microorganisms on the carrier increases to some extent, the microorganisms proliferated from the microorganisms on the carrier are released into the culture solution 105.

The microorganisms grown in the culture solution 105 and the microorganisms grown on the carrier and desorbed into the culture solution 1
The microbial concentration in 05 becomes higher. Microorganisms cultured at a high concentration by such a mechanism are discharged from the tube and provided for use.

Next, the porous carrier 111 impregnated with a liquid therein will be described with reference to FIG. A pre-cultured microorganism is charged into a tube filled with the porous carrier 111, and the tube 1
When the culture solution 103 is continuously introduced from one end of 01, the culture solution 104 flows in the tube.

Here, when the portion of the porous carrier 111 is viewed in detail in FIG. 12, the flowing culture solution 5 flows from the carrier surface 112 through the pores 113 through the pores 113 due to the pressurization.
, And further released to the outside of the carrier by pressurization,
Then, the adjacent carrier 111 is similarly impregnated. The above steps are repeated, and the culture solution 105 is discharged from the other end of the tube 1 after filling the inside of the tube.

Under the flow state of the culture solution 105 passing through the inside 114 of the carrier in the tube, when the culture solution 103 first comes into contact with the microorganism, the growth of the microorganism starts using the nutrients in the culture solution. . As the concentration of bacteria in the culture solution in the tube increases, the amount of microorganisms adsorbed on the carrier also increases, and the pore surface 1 outside the carrier or inside the carrier 1
Even at 15, the growth of microorganisms becomes active.

When the density of the microorganisms on the inner and outer surfaces of the carrier is increased to some extent, the microorganisms grown from the microorganisms on the inner and outer surfaces of the carrier are detached into the culture solution 105. The concentration of microorganisms in the culture solution 105 increases due to the microorganisms that have grown in the culture solution 105 and the microorganisms that have grown on the inner and outer surfaces of the carrier and have been eliminated in the culture solution. Microorganisms cultured at a high concentration by such a mechanism
Emitted from 1 and provided for use.

For the second problem, it is not particularly necessary to fill the tube-shaped culture tank with the carrier. Even when the carrier is not filled, continuous culture may be performed in the same manner as described above, except that the carrier is not filled in the culture tank. This is shown in FIG.

In addition to the presence or absence of the carrier, a branch pipe 12 capable of supplying a new culture medium or discharging a flowing culture medium at an appropriate interval in the middle of a tubular culture tank as shown in FIG.
1 or a new tube 131 extending from the tip 132 of the tube-shaped culture tank to extend the residence time of the culture solution.

The present continuous culture apparatus is effective in solving the first problem for the following reasons.

That is, when a small-sized carrier is used, even if the size of the carrier is small and the packing density is high and the speed of diffusion of the culture solution in the gap between the carriers is reduced, the carrier is filled in a tube having a small inner diameter. As long as it is performed, the carrier is present in the vicinity of the flow of the culture solution, and the distance required for diffusion is shortened, so that the influence of the decrease in the diffusion rate can be reduced. This effect can be realized regardless of the specific gravity of the carrier.

If the axial direction of the tube is not vertical, the direction of filling of the carrier in the tube will not be vertical, so that it is possible to prevent the packing from becoming closest. Therefore, it is possible to set the filling density relatively slowly,
The diffusion rate of the culture solution in the gap between the carriers can be increased.

Furthermore, when a carrier impregnated with a liquid is used, the culture solution can be supplied to the inside of the carrier by pressurizing the culture solution flowing in the tube. It becomes possible to grow the microorganisms by giving the culture solution components to the microorganisms, and it is possible to collect the microorganisms that have grown on the inner surface of the carrier and detached into the culture solution inside the carrier outside the carrier. Become. Therefore, since the inner surface of the carrier can also contribute to the growth of microorganisms, the surface area per carrier volume can be increased.

As described above, since the surface area of a small-sized carrier or a porous carrier in which a liquid is impregnated therein can be used with high efficiency, the effective surface area per carrier volume can be increased, and therefore, the effective volume per culture medium can be increased. It is possible to improve the effect of the carrier, that is, the ability of the culture device to grow microorganisms, thereby making the device scale compact. Furthermore, since the residence time of the culture solution in the culture tank can be shortened, it becomes possible to supply freshly active microorganisms to the outside.

Regarding the second problem, first, regarding the substrate inhibition, unlike a normal culture tank having a large cross-sectional area in the flow direction of the culture liquid, the culture liquid flows almost completely in a laminar flow in a tubular culture tank. Therefore, the concentration of the inhibitory substrate in the culture solution supplied from the tube end can be reduced as the medium is flowed through the tube-shaped culture tank. Since the culture medium can be supplied gradually little by little from the branch pipe installed in the middle of the tank, the branch pipe installed in the middle of the tubular culture tank at the site where the substrate concentration in the culture medium supplied from the tube end has decreased to some extent It is possible to add a new substrate from, and to add the substrate during continuous culture while maintaining the substrate concentration at a level not inhibited by another branch tube. It is possible to increase the added substrate concentration barrel.

In addition, competition inhibition, particularly competition inhibition by an inducer for decomposing organochlorine compounds, can be almost completely caused to flow in a laminar flow in a tubular culture tank. Even when the feed rate is increased, the culture solution diluted by mixing with the culture solution is discharged from the culture tank as it is, and there is no danger that a competitive inhibition substrate such as an inducer remains in the reaction field, thereby preventing competition inhibition. It becomes possible. The culture solution supplied by adjusting the tube length can be completely used in the tubular culture tank, so that the cost of the culture solution can be reduced.

In addition, since microorganisms in the culture solution are induced as the microorganisms flow in the tube-shaped culture tank, if the residence time of the culture solution is adjusted as in a general culture tank, the microorganism after contact with the inducer can be obtained. Recovering young microorganisms that have a short duration and a low level of inducement can also be caused by cultivating old microorganisms that have already completed induction due to a long time from contact with the inducer and enzyme activity has begun to decrease. Only those microorganisms having optimal activity that have just been induced can be harvested at a high concentration without being contained in the liquid.

Further, when the inducer consumption rate of the microorganism flowing in the culture tank increases due to a change in the culture condition, the culture solution is sampled from a branch pipe provided in the middle of the culture tank, and the microorganism is analyzed. Since the point having the optimum activity can be known, the microorganism having the optimum activity can be harvested again by collecting the culture solution from the point. In addition, when the inducer consumption rate is reduced even when the entire length of the culture tank is used, it is possible to similarly harvest microorganisms having optimal activity by newly extending the tube from the tip of the tube.

In addition, sampling and analysis of the culture solution
(Monitoring) can also be applied to determine additional supply points for the culture.

Next, the configuration of the continuous culture apparatus of the present invention will be specifically described.

First, the tube can be made of any material as long as the culture solution can flow therethrough and does not adsorb microorganisms. However, it is preferable that the tube is flexible because it can be compactly stored at the time of use. Particularly, a fluororesin made of PFA, which is a fluororesin which has little adsorption of microorganisms and is flexible, is useful.

The size of the tube may be any. However, in order to sufficiently realize the effects of the present invention, the inner diameter of the tube is preferably 10 mm or less, more preferably 5 mm or less.
mm or less. This depends on the material of the tube and the supply speed of the contaminated gas and the culture solution, but if the tube diameter is small, the culture solution fills the inner cross section of the tube due to surface tension, so that the culture solution infiltrates the entire carrier, This is because the inside of the carrier can be further transmitted.

On the other hand, the inner diameter of the tube may be set so as to prevent the flow of the culture solution from being clogged in the middle and to obtain a stable flow. For example, the inner diameter of the tube is 1 mm or more, more preferably 3 mm or more. be able to.

The method of filling the tube with the carrier is as follows.
Any method and packing density is possible unless the tube is filled with the tube axis vertical, but in the case of a carrier that does not impregnate liquid inside, the carrier size is very small, etc.
When the gap between the carriers is particularly small, it is preferable to lower the packing density and increase the diffusion rate of the culture solution into the gap. In this case, it is preferable to take measures to prevent the carrier from flowing out, such as providing filters at both ends of the filling section.

On the other hand, in the case of a carrier impregnated with a liquid therein, it is desirable to adopt a method in which the carrier is densely packed and the gap is reduced when the cross section of the tube is viewed. This is to prevent the portion not filled with the filler from becoming a tap water and the culture solution passing therethrough preferentially, and preventing the culture solution from permeating the inside of the carrier.

The material of the carrier must be one that is not harmful to the growth of the microorganism to be cultured, that is capable of adsorbing the microorganism and increasing the concentration of the microorganism in the culture solution, and that allows the grown microorganism to be easily detached from the carrier. is there. Further, it is more desirable that the growth ability and activity of the microorganisms be increased by adsorption to the carrier.

The size of the carrier may be smaller than the inner diameter of the tube. If the size is smaller than several tens of microns, the gap between the fillers is too small, which makes it difficult to transmit the culture solution.

As for the carrier impregnating the liquid inside, if the material is flexible and the inner wall of the tube and the carrier or the carrier are in close contact with each other and the gap between the tube and the carrier or the carrier can be eliminated, the inside of the carrier is impregnated. Since the permeation of the culture solution can be sufficiently performed, a high effect can be expected.

The shape of the pores inside the carrier is inappropriate if it is closed inside the carrier, and it is necessary that the pores opened on the surface of the carrier always communicate with a plurality of other pores. . The pore size is desirably several tens of microns, which facilitates the passage of the culture solution and does not make the transfer of microorganisms difficult.

Specific examples of the carrier having no liquid impregnated therein include anion exchange resin, glass beads, alumina beads, silica sand and the like.

Examples of the carrier in which the liquid is impregnated include chitopearl (manufactured by Fuji Boseki Co., Ltd.), zeolite, cellulose, Teflon, porous glass, polyurethane and the like.

The conditions for culturing the microorganisms are not particularly different from those in the case of culturing in a usual culturing apparatus. However, since the concentration of the cultivated microorganisms is high, the concentration of nutrients in the culture solution, the supply speed of the culture solution, There is a possibility that the culture can be performed more efficiently by making changes from the culture state of the microorganism, such as making the oxygen concentration in the culture solution higher than usual.

In the case of aerobic cultivation, the oxygen required for cultivation is dissolved in the culture solution in advance by aeration or the like, an aeration tank is provided immediately before the culture tank, or the culture solution is added to the culture tank. By supplying air or the like in the interval between the above. In particular, when a carrier is used, the culture solution is prevented from being discharged from the culture tank at the same speed as the aeration speed because the culture solution adheres to the surface of the carrier even if the amount of aeration increases.

For example, when a predetermined amount of the culture solution and a predetermined amount of the gas are alternately introduced from one opening of the tubular bioreactor, the decomposition microorganism is retained in an airtight tank. A liquid phase of the culture solution and a gas phase in contact with the liquid phase are formed, and the culture solution and gas of the microorganism are further flowed into the liquid phase. A method can be employed in which a culture solution containing the microorganism and the gas are alternately discharged from a discharge pipe where an opening is located, and are introduced into a tubular bioreactor.

As a method of installing a branch pipe in the tube of the culture tank, any method may be used as long as it is suitable for the material of the tube. To prevent liquid transfer,
It is desirable to attach a cock near the part connected to the culture tank.

Also, when a new tube is continued at the end of the tube of the culture tank to extend the tube length, any method may be used as a method of continuing the tube as long as it is suitable for the material of the tube.

[0085]

The present invention will be described more specifically with reference to the following examples.

[Example 1] Continuous culture in a tubular culture tank using a liquid impervious carrier Pseudomonas cepacia KK01 strain, a trichloroethylene-degrading bacterium (Biotechnological Research Institute, Ministry of International Trade and Industry, Ministry of International Trade and Industry) Place, accession number: FER
(MBP-4235, commission date: March 11, 1992), a continuous culture was carried out in a tubular culture tank filled with a carrier having no liquid permeability. The KK01 strain requires an inducer to express a trichlorethylene-degrading enzyme, and in this example, phenol was used also as a growth substrate.

The KK01 strain was inoculated into 200 mL of an M9 medium containing 200 ppm of phenol and minerals in a Sakaguchi flask, and pre-cultured by shaking at 23.5 ° C. for 24 hours. This culture was used with a particle size of 0.6 mm as shown in FIG.
Anion exchange resin (Amberlite IRA41)
0, specific gravity 1.25) 100 g of a carrier 102 as a tube-shaped culture tank 101 (manufactured by PFA, 4 mmΦ × 36
The culture solution was injected into a culture medium (700 mm, stored culture volume: 380 mL), the culture solution was supplied at 10 mL / hr from one end, and continuous culture was performed at 23.5 ° C. while the culture solution was discharged from the other end. FIG. 14 shows the entire apparatus.

As the culture solution 3, an M9 medium containing 1000 ppm of phenol and minerals was used.
And bubbled with diffuser 16 at 20 mL / min.

After culturing for 3 days and acclimatization,
10 mL of the discharged culture solution was sampled,
The solution was added to a vial having a volume of 0 mL and sealed with a Teflon-coated stopper. The trichlorethylene gas was added to the vial so that the liquid phase concentration in the vial became about 40 ppm.
5 mL was injected with a syringe and shaken at 23.5 ° C.

The concentration of trichlorethylene in the gas phase of the vial was measured over time by gas chromatography, and the initial rate of decomposition of trichlorethylene was determined from the rate of decrease of trichlorethylene. The concentration of residual phenol in the culture was measured by the aminoantipyrine method.

Further, the culture solution supply speed was set to 20, 30, 4
Increase gradually to 0, 50, 60, 70 mL / hr, 3 for each
After acclimatization for ４4 days, the above steps were performed in the same manner to determine the initial decomposition rate of trichloroethylene and the concentration of residual phenol in the culture at each culture solution supply rate.

The above results are shown in FIGS.

[Comparative Example 1-1] The KK01 strain was pre-cultured in the same manner as in Example 1, and the culture solution was used in a bubble column type stirring type culture tank 41 (46 mmΦ × 400 mm, stored culture solution 38) shown in FIG.
0 mL). A cylindrical basket 140 (inner diameter 30 mm, mesh hole diameter 0.5 mm) was installed at the center of the culture tank, and was filled with 100 g of the same carrier as in Example 1.

[0094] From the lower part of the culture tank,
And M9 medium containing minerals at 10 mL / hr,
Bubble with diffuser at 20mL / min,
The culture solution was discharged from the upper surface of the culture solution and continuously cultured at 23.5 ° C.

After culturing for 3 days and acclimatization,
The initial decomposition rate of trichlorethylene was determined in the same manner as in Example 1. The concentration of residual phenol in the culture was measured by the aminoantipyrine method.

Further, the culture solution supply rate was set to 15, 20, 2
After gradually adjusting to 3, 30, and 35 mL / hr, the above steps were similarly performed after acclimation for 3 to 4 days, and the initial rate of trichloroethylene decomposition and the concentration of residual phenol in the culture solution at each culture solution supply rate were determined. Was.

The above measurement results are shown in FIGS.

[Comparative Example 1-2] The KK01 strain was pre-cultured in the same manner as in Example 1, and the culture solution was filled with 100 g of the same carrier as in Example 1;
Φ × 400 mm, 380 mL of a stored culture solution). A culture solution was supplied at a rate of 10 mL / hr from the lower portion of the culture tank, the culture solution was discharged from the upper surface of the culture solution, and continuous culture was performed at 23.5 ° C. The culture solution was stored in a 5 L reservoir using an M9 medium containing phenol 1000 ppm and minerals.
Bubbling was performed with a diffuser at 20 mL / min.

After continuing the culture for 3 days and performing the adaptation,
The initial decomposition rate of trichlorethylene was determined in the same manner as in Example 1. The concentration of residual phenol in the culture was measured by the aminoantipyrine method.

Further, the culture solution supply speed was set to 15, 20, 2
After gradually adjusting to 3, 30, and 35 mL / hr, the above steps were similarly performed after acclimation for 3 to 4 days, and the initial rate of trichloroethylene decomposition and the concentration of residual phenol in the culture solution at each culture solution supply rate were determined. Was.

The above measurement results are shown in FIGS.

In Example 1, even if the supply amount of the culture solution was increased, the initial rate of decomposition of trichlorethylene was high, and Comparative Examples 1-1 and 1-
As compared with 2, the amount of culture solution that can be produced even with the same amount of stored culture solution is high, indicating that the culture tank has a high culture efficiency.

[Example 2] Continuous culture in a tubular culture tank using a liquid-permeable carrier The trichloroethylene-degrading bacterium was used as the J1 strain (Biotech Industrial Research Institute, Ministry of International Trade and Industry, accession number: FERM BP-
5102, Contract date: May 25, 1994), phenol as an inducer / proliferation substrate, 24 g of liquid-permeable polyurethane as a carrier, PFA in a tubular culture tank, 4 m
mΦ × 38300 mm, and using 380 mL of the stored culture solution, the culture solution supply speed was 10, 15, 20, 25,
The same procedure as in Example 1 was performed except that the flow rate was changed to 30, 35, and 40 mL / hr, and the initial rate of decomposition of trichlorethylene and the concentration of residual phenol in the culture solution were determined.

The results are shown in FIGS.

[Comparative Example 2-1] The J1 strain was used as a trichloroethylene-degrading bacterium, phenol was used as an inducer / growth substrate, and 24 g of a liquid-permeable polyurethane was used as a carrier. The same procedure as in Comparative Example 1-1 was carried out except that it was not used, and the initial decomposition rate of trichloroethylene and the concentration of residual phenol in the culture solution were determined.

The results are shown in FIGS.

[Comparative Example 2-2] The same procedure as in Comparative Example 1-2 was carried out except that the J1 strain was used as a trichloroethylene-decomposing bacterium, phenol was used as an inducer and growth substrate, and 24 g of a liquid-permeable polyurethane was used as a carrier. , The initial rate of decomposition of trichlorethylene and the concentration of residual phenol in the culture solution were determined.

The results are shown in FIGS.

In Example 2, even if the supply amount of the culture solution was increased, the initial rate of decomposition of trichlorethylene was high, and Comparative Examples 2-1 and 2-
As compared with 2, the amount of culture solution that can be produced even with the same amount of stored culture solution is high, indicating that the culture tank has a high culture efficiency.

[Example 3] Continuous culture in a tubular culture tank for recovering a culture solution from a branch tube and an extension tube In a case where competition is inhibited by a substrate, it is assumed that the concentration of the remaining substrate changes due to a change in culture conditions. Then, an experiment was conducted in which a change in the concentration of the remaining substrate was simulated by changing the concentration of the substrate in the culture solution.

A culture solution of the KK01 strain, which had been pre-cultured in the same manner as in Example 1, was fed into a tube-shaped culture tank having branch pipes at 5000 mm intervals (4 mmΦ × 30000 mm, stored culture solution volume of 3).
60 mL), a culture was supplied at 20 mL / hr from one end, and continuous culture was performed at 23.5 ° C. while discharging the culture from the other end. The culture solution was stored in a 5 L reservoir using an M9 medium containing 800 ppm of phenol and minerals, and bubbled at 20 mL / min with a diffuser.

The components of the medium are as shown below.

After culturing for 3 days and acclimatization,
The culture solution discharged from each branch pipe and tube end placed in the middle of the culture tank was sampled. Further, a similar tube having a similar branch tube was continued to the end where the culture solution was discharged, and the culture solution discharged from each branch tube and a new tube end was sampled. Then, the initial rate of trichlorethylene decomposition of each culture solution was measured in the same manner as in Example 1,
The highest decomposition initial rate was determined. The residual phenol concentration in the culture was measured by the aminoantipyrine method.

Furthermore, the phenol concentration in the culture was adjusted to 90
0, 1000, 1100, 1200, and 1300 ppm, and the above steps were repeated after acclimation for 3 to 4 days. The highest initial trichloroethylene decomposition rate at each phenol concentration and the residual phenol concentration in the culture solution I asked.

The above results are shown in FIGS.

[Comparative Example 3] A tube-shaped culture tank without branch pipe (4 mmΦ × 30000 mm, stored culture liquid amount 360 m)
L) was carried out in the same manner as in Example 3 except that the tube was not extended, and the initial rate of trichlorethylene decomposition at each phenol concentration and the residual phenol concentration in the culture solution were determined.

The above measurement results are shown in FIGS.

In Example 3, even when the culture conditions were changed and the phenol concentration remaining in the culture solution was increased, the residence time was extended by extending the tube, and the inhibition of competition by phenol could be prevented. Was. When the concentration of phenol remaining in the culture solution became low, the culture solution having the highest residence time could be harvested by harvesting the culture solution from the branch tube.

[Example 4] Continuous culture in a tubular culture tank in which a culture solution is added from a branch tube In a case where substrate inhibition occurs, an experiment to increase the base mass to be added in all the culturing steps was performed using a tubular culture tank having a branch tube. Was performed using

A culture solution of the KK01 strain, which had been pre-cultured in the same manner as in Example 1, was placed in a tube-shaped culture tank having a branch tube at the center part (4 mmΦ × 30000 mm, stored culture solution volume of 3).
60 ml), and supply M9 medium containing phenol (800 ppm) and minerals at 15 ml / hr from one end, and supply the same culture solution at 5 ml / hr from the branch tube, and discharge the culture solution from the other end. Continuous culture was performed at 23.5 ° C. The culture was stored in a 5 L reservoir and bubbled at 20 mL / min with a diffuser.

After culturing for 3 days and acclimatization,
10 mL of the discharged culture solution was sampled,
The solution was added to a vial having a volume of 0 mL and sealed with a Teflon-coated stopper. The trichlorethylene gas was added to the vial so that the liquid phase concentration in the vial became about 40 ppm.
5 mL was injected with a syringe and shaken at 23.5 ° C. The concentration of trichlorethylene in the gas phase portion of this vial was measured over time by gas chromatography, and the initial rate of decomposition of trichlorethylene was determined from the rate of decrease of trichlorethylene. The concentration of residual phenol in the culture was measured by the aminoantipyrine method.

Further, the phenol concentration in the culture was adjusted to 90
0, 1000, 1100, 1200, and 1300 ppm, and the above steps were repeated after acclimation for 3 to 4 days. The highest initial trichloroethylene decomposition rate at each phenol concentration and the residual phenol concentration in the culture solution I asked.

The above results are shown in FIGS.

[Comparative Example 4] Since the experiment was the same as that of Comparative Example 3, data on the initial decomposition rate of trichloroethylene and the concentration of residual phenol in the culture solution of Comparative Example 3 were used.

The above results are shown in FIGS.

In Example 4 and Comparative Example 4, the amount of phenol provided in all the culturing steps was the same, but in Example 4, phenol as a substrate was used with a difference in the residence time of the culture solution.
It can be seen that, even when the phenol concentration in the culture solution was increased, the increase in the residual phenol concentration was suppressed, and substrate inhibition was unlikely to occur because the phenol concentration in the culture solution was increased.

[0127]

According to the present invention, a carrier having a large specific gravity or a carrier having a small particle size can be effectively used. In addition, since the inner surface of the pores of the porous carrier can be effectively used, the effective surface area of the carrier in the culture solution can be increased, and the efficiency of continuous culture can be improved.

Further, in continuous culture, in particular, in the case of continuously culturing organochlorine-decomposing microorganisms using an inducer, it is possible to prevent substrate inhibition or competition inhibition and selectively harvest a microorganism culture solution having a high activity. It becomes possible.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the culture solution supply rate and the initial decomposition rate of trichlorethylene for each type of culture tank when a carrier having no liquid permeability is used.

FIG. 2 is a graph showing the relationship between the culture solution supply rate and the concentration of residual phenol in the culture solution in the case of using a carrier having no liquid permeability, for each type of culture tank.

FIG. 3 is a graph showing a relationship between a culture solution supply rate and a trichloroethylene decomposition initial rate in the case of using a liquid-permeable carrier, for each type of culture tank.

FIG. 4 is a graph showing the relationship between the culture solution supply rate and the concentration of residual phenol in the culture solution in the case of using a liquid-permeable carrier for each type of culture tank.

FIG. 5 is a graph showing the relationship between the phenol concentration in the feed culture solution and the initial rate of trichlorethylene decomposition in a tubular culture tank for collecting the culture solution from the branch tube and the extension tube, in comparison with the case where no collection is performed. It is.

FIG. 6 is a graph showing the relationship between the phenol concentration and the residual phenol concentration in a feed culture solution in a tubular culture tank for collecting a culture solution from a branch pipe and an extension tube in comparison with a case where no recovery is performed. is there.

FIG. 7 is a graph showing the relationship between the phenol concentration in the feed culture solution and the initial rate of trichlorethylene decomposition in a tubular culture tank to which the culture solution is added from the branch tube, in comparison with the case where no addition is performed.

FIG. 8 is a graph showing a relationship between a phenol concentration and a residual phenol concentration in a feed culture solution in a tubular culture tank to which a culture solution is added from a branch tube, in comparison with a case where addition is not performed.

FIG. 9 is a view showing a tubular culture tank when no carrier is filled.

FIG. 10 is a view showing a tubular culture tank in which a carrier having no liquid permeability is filled.

FIG. 11 is a diagram showing a tubular culture tank in which a liquid-permeable carrier is filled.

FIG. 12 is a structural view of a liquid-permeable carrier.

FIG. 13 is a view showing a case where a branch pipe and an extension tube are attached to a tubular culture tank.

FIG. 14 is an overall view of an apparatus according to the first and second embodiments.

FIG. 15 is an overall view of the apparatus of Comparative Example 1-1 (when a bubble column type stirred tank type culture tank is used).

FIG. 16 is an overall view of the apparatus of Comparative Example 1-2 (in the case of using a fixed-layer type culture tank).

[Explanation of symbols]

 3: Culture solution (supply) 10: Pump / motor 11: Reservoir 12, 22: Culture solution 14: Air 15: Exhaust 16, 26: Diffuser 17: Bubbles 18, 38: Discharge pipe 19, 39: Discharge pipe opening 35 : Exhaust 41: Bubble tower type stirred tank type culture tank 51: Fixed bed type culture tank 53: Carrier 101: Tubular culture tank 102: Non-porous carrier 103: Culture medium (inflow) 104: Culture medium (internal flow) 105 : Culture solution (outflow) 111: porous carrier 112: carrier surface 113: pore 114: inside carrier 115: pore surface 121: branch tube 131: (extension) tube 132: end of tubular culture tank (extension connection)

Claims (27)

[Claims] 1. A method for continuously culturing microorganisms, comprising culturing microorganisms in a tube-shaped culture tank in which a culture solution is continuously supplied from one end and discharged from the other end. 2. The method according to claim 1, wherein the culture tank is filled with a carrier. 3. The method according to claim 2, wherein the packing density of the carrier in the culture tank is arbitrarily adjusted. 4. The method according to claim 2, wherein the carrier has a particle size of 0.6 mm or less. 5. The method according to claim 2, wherein the specific gravity of the carrier is higher than the culture solution. 6. The method according to claim 2, wherein at least the inside of the carrier is porous and can be impregnated with a liquid therein, and the pores opened on the surface of the carrier communicate with the pores inside the carrier. 7. The method according to claim 1, wherein one or more branch pipes capable of communicating between the inside of the culture tank and the outside are provided in addition to both ends of the tubular culture tank. 8. The method according to claim 1, wherein the length of the tube for culturing can be arbitrarily extended by continuing the extension tube at the discharge end of the culture solution in the culture tank. 9. The method according to claim 8, wherein the extension tube is provided with one or more branch pipes capable of communicating the inside and the outside of the extension tube. 10. Supplying a culture solution from the branch tube suppresses the substrate in the culture solution in the tube to a certain concentration or less,
10. The method according to claim 7 or 9, wherein the amount of substrate added in all the culture steps is increased without causing substrate inhibition. 11. The method according to claim 7, wherein the residence time of the culture solution is controlled by discharging and collecting the culture solution from the branch pipe. 12. The method according to claim 8, wherein the residence time of the culture solution is controlled by the length of the tube. 13. The method according to claim 1, wherein continuous culture of an organochlorine compound-degrading microorganism is performed by using the inducer in the culture tank. 14. The method according to claim 13, wherein the concentration of the residual substrate in the discharged and recovered culture is controlled by discharging and recovering the culture from the branch pipe. 15. The method according to claim 13, wherein the concentration of the residual substrate in the discharged and collected culture solution is controlled by the length of the tube. 16. The method according to claim 14, wherein the concentration of the residual substrate is a concentration that does not cause competitive inhibition of the microorganism. 17. The method according to claim 13, wherein the concentration of the residual inducer in the discharged and collected culture solution is controlled to a concentration suitable for decomposing the organochlorine compound by discharging and collecting the culture solution from the branch pipe. 18. The length of the tube is extended by continuing the tube from the culture solution discharge end of the culture tank, whereby the concentration of the residual inducer in the culture solution discharged and recovered is adjusted to a concentration suitable for the decomposition of the organochlorine compound. 14. The method according to claim 13, wherein the control is performed. 19. The method according to claim 17, wherein said residual inducer concentration is a concentration that does not cause competitive inhibition of organochlorine compound decomposing microorganisms. 20. The method according to claim 7, wherein the culture solution is monitored from the branch tube. 21. A branch tube for supplying a culture solution by monitoring the culture solution from the branch tube.
The described method. 22. A branch tube for collecting a culture solution by monitoring the culture solution from the branch tube.
20. The method of any one of claims 19 to 19. 23. A method for culturing microorganisms, comprising a tubular culture tank, wherein the culture tank has means for continuously supplying a culture solution from one end and means for discharging the culture liquid from the other end. Continuous culture equipment for microorganisms. 24. The apparatus according to claim 23, wherein the culture tank is filled with a carrier. 25. The apparatus according to claim 23, wherein one or more branch pipes that allow communication between the inside and the outside of the culture tank are provided in addition to both ends of the culture tank. 26. The apparatus according to claim 23, further comprising a connection portion of an extension tube at a culture solution discharge end of said culture tank. 27. The apparatus according to claim 26, wherein the extension tube has one or more branch pipes that can communicate between the inside and the outside.