JP2008125456A - Method of continuous culture and device of continuous culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and inexpensive method of continuous culture and device of continuous culture, performing the continuous culture, without much using sterilizing means such as complex high pressure steam sterilization requiring much labor and a high cost. <P>SOLUTION: This method for continuously culturing a medium supplied from a medium-storing vessel 1, in a fermentation reaction vessel 2 includes adding a neutralizing agent for neutralization-treating the medium in the fermentation reaction vessel 2 to the medium before being supplied into the fermentation reaction vessel 2 at the upstream side of the fermentation reaction vessel. The addition of the neutralizing agent is performed by a neutralizing agent-adding means 4 for adding the neutralizing agent to the medium-storing vessel 1. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a continuous culture method and a continuous culture apparatus effective for maintaining a sterilization environment of a culture medium and a culture system in a continuous fermentation culture system for producing an organic acid and the like.

  Fermentation methods, which are substance production methods or cell production methods that involve cultivation of microorganisms such as bacteria and fungi, and cultured cells such as animal cells, are largely divided into (1) batch fermentation methods (Batch fermentation methods) and fed-batch fermentation methods ( Fed-Batch fermentation method) and (2) continuous fermentation method. The batch and fed-batch fermentation method of (1) is simple in terms of equipment, and has an advantage that culture is completed in a short time and there is little damage due to contamination with various bacteria. However, the product concentration in the culture solution increases with time, and the productivity and yield decrease due to the influence of osmotic pressure or product inhibition. For this reason, it is difficult to stably maintain a high yield and high productivity over a long period of time.

  On the other hand, the continuous fermentation method (2) has a feature that a high yield and high productivity can be maintained over a long period of time by avoiding accumulation of the target substance at a high concentration in the fermenter. However, unlike the method of (1), in the continuous fermentation method, it is necessary to continuously supply a medium over a long period of time, so sterilization needs to be performed not only at the start of culture but also during the culture period. .

  As a method for sterilizing a culture medium or a culture apparatus, a method of sterilization using high-pressure steam is generally used. In this high-pressure steam sterilization method, it is necessary to install a large amount of boilers in order to produce and supply high-pressure steam, the utility cost is very high, the sterilization target container and piping need to be pressure-resistant containers, In order to completely sterilize the piping, extremely complicated operations are required. However, since it is indispensable to prevent contamination with microorganisms in microbial culture, it is impossible to avoid sterilization and development of a simpler and cheaper sterilization method is required.

Therefore, Patent Document 1 proposes a method in which a sterilization filter is provided in the middle of a pipe as a sterilization means in order to prevent contamination with bacteria during continuous culture. However, in this method, sterilization between the units tends to be insufficient, which may adversely affect the culture system, and a large number of sterilization filters are required.
JP-A-4-21357

  The present invention has been made in view of the above-mentioned problems in the prior art, and an object of the present invention is to provide a method that can be sterilized easily and inexpensively in a continuous fermentation method.

In order to solve the above problems, the present invention has the following configuration.
(1) In the method of continuously culturing the medium supplied from the medium storage tank in the fermentation reaction tank, a neutralizing agent for neutralizing the culture solution in the fermentation reaction tank is supplied into the fermentation reaction tank. A continuous culture method characterized by adding to the previous medium upstream of the fermentation reaction tank.
(2) The continuous culture method according to claim 1, wherein the position where the neutralizing agent is added is a medium reservoir.
(3) Neutralizing agent added to neutralize the culture solution in the fermentation reaction tank by the neutralizing agent added upstream of the fermentation reaction tank with respect to the medium before being supplied into the fermentation reaction tank The pH is adjusted by adding a neutralizing agent directly to the fermentation reaction tank according to the pH of the culture solution in the fermentation reaction tank. Continuous culture method.
(4) The continuous culture method according to any one of (1) to (3), wherein the continuous culture system is an organic acid fermentation continuous culture system, and the neutralizing agent is an alkali.

(5) In a continuous culture apparatus comprising a culture medium storage tank for storing a culture medium and a fermentation reaction tank for continuously fermenting a culture medium supplied from the culture medium storage tank, a pipe for supplying the culture medium to the fermentation reaction tank and / or A continuous culture apparatus characterized in that a neutralizing agent adding means for adding a neutralizing agent is disposed on the upstream side.
(6) The continuous culture apparatus according to (5), wherein a neutralizing agent addition means is attached to the culture medium storage tank.
(7) A neutralizing agent addition means for adding a neutralizing agent to the piping for supplying the medium to the fermentation reaction tank and / or to the medium storage tank on the upstream side of the pipe is provided. A second neutralizing agent adding means for adding a compatibilizer is arranged, a pH sensor for measuring the pH of the culture solution in the fermentation reaction tank is arranged, and the pH measured by the pH sensor The continuous culturing apparatus according to (5) or (6) above, wherein means for adjusting the amount of neutralizing agent added by the second neutralizing agent adding means according to the value is provided.

  According to the present invention, when continuously supplying the medium in the continuous fermentation method, a neutralizing agent for neutralizing the culture solution is preliminarily added to the medium on the upstream side, thereby performing sterilization. A sterilized medium can be supplied without frequently performing high-pressure steam sterilization with large utility costs and complicated operations.

  Hereinafter, the present invention will be specifically described.

  The method of the present invention can be carried out, for example, using a continuous culture apparatus as shown in FIG. The continuous culture apparatus includes a medium storage tank 1 for storing a medium and a fermentation reaction tank 2 for continuously fermenting the medium supplied from the medium storage tank 1. The medium storage tank 1 is provided with first neutralizing agent addition means 4 for adding a neutralizing agent. A second neutralizing agent is added to the fermentation reaction tank 2 to add a neutralizing agent in an amount corresponding to the pH measurement result and the pH measurement result in order to keep the pH of the culture solution in the fermentation reaction tank at a constant condition. Means 5 are provided. By this second neutralizing agent addition means 5, the pH of the fermentation reaction tank is finally kept constant. Furthermore, a medium supply pipe 8 and a medium supply pump 7 for supplying the medium from the medium storage tank 1 to the fermentation reaction tank 2 are provided, and a pipe and a pump 9 for extracting the culture solution from the fermentation reaction tank 2 are provided. Is provided. Moreover, the stirring means 6 for stirring the liquid in the fermentation reaction tank 2 is provided.

  In this continuous culture apparatus, first, when it is necessary to strictly maintain the sterilization environment, the medium storage tank 1, the medium supply pump 7, and the medium supply pipe 8 are sterilized by means such as steam sterilization once before use. However, in the present invention, this may be omitted depending on the medium and culture system used. The fermentation reaction tank 2, the devices attached thereto, and other solutions supplied to the fermentation reaction tank 2 are separately sterilized as necessary.

  Next, after performing batch culture at the time of start-up, when starting continuous fermentation, a medium is continuously or intermittently charged from the medium storage tank 1 to the fermentation reaction tank 2 by the medium supply pump 7. Here, a neutralizing agent is added in advance to the medium stored in the medium storage tank 1 by the first neutralizing agent adding means 4. That is, when the culture medium is put into the fermentation reaction tank 2, a neutralizing agent is also added at the same time. By adding a neutralizing agent to the culture medium in advance, the stored culture medium becomes a pH condition that greatly deviates from the pH conditions under which normal microorganisms can grow. The possibility of miscellaneous bacteria breeding in the system leading to can be suppressed. Furthermore, it is possible to suppress the propagation of various bacteria in the medium supply pipe 8 downstream from the medium storage tank 1. In addition, as a side effect of adding a neutralizing agent to the medium in advance and retaining it, depending on the type of medium and fermentation, hydrolysis of the raw material components in the medium partially proceeds, and the subsequent fermentation reaction tank In some cases, there is an effect that the utilization by microorganisms in the plant is improved.

  The amount of the neutralizing agent added to the culture medium storage tank 1 is the amount of neutralizing agent required in the fermentation reaction tank (calculated by preliminary examination) while the stored medium is introduced into the fermentation reaction tank 2. Do not exceed the amount. Preferably, the amount is slightly smaller than the amount of neutralizing agent required in the fermentation reaction tank, and the final pH adjustment of the culture solution is performed by a pH sensor and a second neutralizing agent addition means provided in the fermentation reaction tank. Preferably it is done. As a result, the pH of the culture solution can be maintained at a predetermined level while adding a neutralizing agent necessary for preventing the spoilage of the medium, and the intended goal can be achieved without waste of re-neutralization. .

  When the fermentation conditions of the fermentation production to be carried out are biased toward the liquid side (acid side or alkaline side) that changes with the progress of fermentation, the neutrality of the liquid is the opposite of the liquidity of the fermentation conditions. Use drugs that act as follows. By adding this neutralizing agent to the medium, it becomes an alkali side or an acid side, and the possibility of adversely affecting the fermentation production caused by the spoilage of the medium or the propagation of various bacteria can be reduced. For example, when pyruvic acid fermentation, which is a kind of organic acid fermentation, is performed, the pH tends to change to the acidic side as the pyruvic acid fermentation progresses, so an alkali is used as the neutralizing agent. Here, the fermentation conditions of pyruvic acid fermentation are biased toward the acidic side and have a pH of 5.5. However, since the alkali is supplied to the medium in advance, the medium exhibits alkalinity opposite to the fermentation conditions. In the unlikely event that contamination with alkali-resistant bacteria occurs in the culture medium storage tank or culture medium supply pipe, the adverse effects on fermentation production are limited because the bacteria are difficult to grow in the fermentation tank that is weakly acidic. . The same applies to other organic acid fermentations.

  If the reaction between the medium and the neutralizing agent results in an insoluble component or a component that adversely affects the culture, such as a component change, the component is individually sterilized and fermented separately. Can be added to the reaction vessel. Moreover, when the component which produces | generates an insoluble component cannot be divided, after removing an insoluble matter with a screen etc., it is good to send a culture medium to a fermentation reaction tank.

  The culture solution is discharged so that the amount of the solution in the fermentation reaction tank 2 can be maintained within a certain range. For example, the culture medium discharge pump 9 may discharge the liquid amount of the medium flowing into the fermentation reaction tank 2. As shown in FIG. 1, the culture solution may be discharged as it is, or as shown in FIG. 2, the culture solution taken out from the fermentation reaction tank is separated into a filtrate and an unfiltered solution by the membrane separation device 10. And the filtrate is taken out to recover the desired fermentation product, and the unfiltered liquid containing microorganisms and the like is refluxed to the fermentation reaction tank. As shown in FIG. The membrane separator 10 may be immersed in the tank 2 and the filtrate that has permeated the separation membrane of the membrane separator may be discharged.

  The membrane-separated continuous culture apparatus shown in FIGS. 2 and 3 is the same apparatus as in FIG. 1 except that the handling of the culture solution taken out from the fermentation reaction tank 2 is different as described above. As the separation membrane used in the membrane separation device 10 at this time, it is desirable to use a porous membrane that is less likely to be clogged by microorganisms and cultured cells used for fermentation and that can stably maintain filtration performance for a long period of time. . For example, a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm can be used. If the average pore size is within this range, it is possible to achieve both a high exclusion rate that does not cause leakage of bacterial cells and the like, and high water permeability. And can be implemented with reproducibility.

  The present invention can also be applied to the production of pharmaceuticals that require strict production quality where no contamination is allowed, but the method of the present invention is somewhat inferior in sterilization compared to conventional methods such as steam sterilization. There may be cases. For this reason, strict maintenance of pure culture is required, such as when there is no problem in production quality even if some bacteria are contaminated, or when the dominant risk of bacteria is low due to the culture solution environment such as product concentration and pH in the culture solution. Can be suitably applied in the case of fermentation production in which no is required. For example, as an example where strict production quality is not required, there can be mentioned large-scale fermentation production of industrial products called biorefinery exceeding 1000 tons / year. Moreover, organic acid fermentations, such as pyruvic acid fermentation, can be mentioned as an example with the low predominance risk of various bacteria. Since a high concentration of organic acid salt is present in the culture solution and the culture solution pH is low, the dominant risk of various bacteria is low, and the present invention can be suitably used.

  Hereinafter, in order to describe the present invention in more detail, an example in which the present invention is applied to a continuous production process of pyruvic acid will be described. The apparatus shown in FIG. 2 was used, and among the yeast Torulopsis glabrata, Torulopsis glabrata strain P120-5a (FERM P-16745) was used as a microorganism for producing pyruvic acid.

  First, the above P120-5a strain was tested in a test tube with 5 ml of pyruvate fermentation medium (glucose 100 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L as main components. And shaking culture overnight. The obtained culture solution was inoculated into 100 ml of a fresh pyruvate fermentation medium, and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 24 hours.

  The obtained culture broth was inoculated into a 1.5 L pyruvic acid fermentation medium of the membrane separation type continuous culture apparatus shown in FIG. 2, and the fermentation reaction tank 2 was stirred at 800 rpm by the attached stirrer 6. The amount of aeration and the temperature were adjusted in No. 2, and the culture was carried out for 48 hours. As for the sterilization of the medium, the fermentation reaction tank was sterilized by high-pressure steam sterilization (121 ° C., 15 minutes) only when the culture was started up. As the separation membrane of the membrane separation device, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) was used. The pH of the culture solution was adjusted to pH 5.5 by directly adding a 4N NaOH aqueous solution to the fermentation reaction tank 2. Thereafter, a pyruvic acid fermentation medium in which trace components such as vitamins and ammonium sulfate were removed was prepared in a medium storage tank. Sodium hydroxide was added thereto, and a pyruvic acid fermentation medium with a sodium hydroxide concentration of 5 g / L was prepared. This was continuously supplied to the fermentation reaction tank at a flow rate of 1.5 L / day, and a small amount of components such as vitamins sterilized separately and ammonium sulfate were also continuously supplied. Continuous culture was performed while controlling the amount of the membrane permeate in the membrane separator 10 so that the amount of the fermentation solution in the fermentation reaction tank 2 was 1.5 L, and pyruvic acid was produced by continuous fermentation. The final pH control of the culture solution in the fermentation reaction tank 2 was performed with a 4N NaOH aqueous solution added directly to the fermentation reaction tank 2. Even if the continuous fermentation test is conducted for 350 hours in this way, the membrane separation type continuous fermentation apparatus can stably produce pyruvic acid by continuous fermentation, and the culture solution can be observed with a microscope. In particular, no contamination was confirmed.

It is the apparatus schematic which shows one embodiment of the continuous culture apparatus by this invention. It is the apparatus schematic which shows another one embodiment of the continuous culture apparatus by this invention. It is the apparatus schematic which shows another one embodiment of the continuous culture apparatus by this invention.

Explanation of symbols

1: Medium storage tank 2: Fermentation reaction tank 3: pH sensor 4: Neutralizing agent addition means 5: Second neutralizing agent addition means 6: Stirring means 7: Medium supply pump 8: Medium supply pipe 9: Culture medium discharge pump 9 ': Filtrate discharge pump 10: Membrane separator 11: Circulation pump

Claims (7)

  In the method of continuously culturing the medium supplied from the medium storage tank in the fermentation reaction tank, the medium before neutralizing the culture solution in the fermentation reaction tank is supplied to the fermentation reaction tank. The continuous culture method characterized by adding in the upstream of a fermentation reaction tank.   The continuous culture method according to claim 1, wherein the neutralizing agent is added to a medium reservoir.   Part of the neutralizing agent required for the neutralizing agent added upstream of the fermentation reaction tank to the medium before being supplied into the fermentation reaction tank to neutralize the culture solution in the fermentation reaction tank And the neutralization agent is directly added to a fermentation reaction tank according to pH of the culture solution in a fermentation reaction tank, and pH adjustment is performed, The continuous culture method of Claim 1 or 2 characterized by the above-mentioned.   The continuous culture method according to any one of claims 1 to 3, wherein the continuous culture system is an organic acid fermentation continuous culture system, and the neutralizing agent is an alkali.   In a continuous culture apparatus comprising a culture medium storage tank for storing a culture medium and a fermentation reaction tank for continuously fermenting a culture medium supplied from the culture medium storage tank, and / or upstream of a pipe for supplying the culture medium to the fermentation reaction tank A continuous culture apparatus characterized in that a neutralizing agent adding means for adding a neutralizing agent is provided.   6. The continuous culture apparatus according to claim 5, wherein neutralizing agent addition means is attached to the culture medium storage tank.   In addition to the neutralizer addition means for adding the neutralizing agent to the pipe for supplying the culture medium to the fermentation reaction tank and / or the upstream medium storage tank, a neutralizing agent is added to the fermentation reaction tank. A second neutralizing agent adding means for adding, a pH sensor for measuring the pH of the culture solution in the fermentation reaction tank, and a pH value measured by the pH sensor The continuous culture apparatus according to claim 5 or 6, further comprising means for adjusting the amount of neutralizing agent added by the second neutralizing agent addition means.

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