Method for separating solid components, and solid-liquid separation system

Abstract

This technology provides ways to achieve energy savings in the production process of target substances through microbial fermentation. [Solution] A method for separating solid components from a liquid culture of organic acid-producing microorganisms, comprising: (a) adjusting the pH of the liquid culture to a predetermined value A different from the pH at the start of the culture; and (b) separating the solid components from the liquid culture whose pH has been adjusted to the predetermined value A, wherein the predetermined value A is a pH such that the value Z obtained by subjecting the liquid culture to zeta potential measurement by electrophoretic light scattering is in the range of -10mV to 10mV.

Description

Technical Field

[0001] The present invention relates to a method for separating a solid component containing microorganisms from a liquid culture of microorganisms, and a solid-liquid separation system.

Background Art

[0002] Conventionally, production of target substances by microbial fermentation has been widely performed. For example, microorganisms are mass-cultured in a tank, and the target substance is isolated from the liquid culture. The liquid culture of microorganisms contains a solid component containing microorganisms and a liquid component. The target substance may be contained in either the solid component or the liquid component.

[0003] From the recent trend of ensuring stable energy supply and preventing global warming, the need for energy conservation has been increasing further. In the culture process of microorganisms as well, it is preferable to perform the process with as little energy consumption as possible (for example, Patent Documents 1 and 2). However, in the production of target substances by mass-culturing microorganisms, various processes that consume a large amount of energy are included. The above-described separation of the solid component and the liquid component (solid-liquid separation) is an example of a process that may consume a large amount of energy. In addition, a large amount of liquid medium is required in the culture process of microorganisms, and this point can also be a cause of large energy consumption. This problem of energy consumption becomes more serious as the scale of culture is larger.

[0004] As means for separating the solid component and the liquid component of the liquid culture, for example, centrifugal separation, membrane separation, etc. are known. Here, generally, a centrifuge includes a rotating body such as a rotor and a motor that rotates the rotating body at high speed, and requires a large amount of energy.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] As mentioned above, the need for energy conservation is increasing globally, and even greater energy conservation is required in microbial culture processes. Therefore, the object of this invention is to provide a technology for achieving energy conservation in the production process of target substances by microbial fermentation. [Means for solving the problem]

[0007] The inventors identified energy-intensive processes in the production process of target substances by microbial fermentation in order to achieve energy savings. They focused on the process of separating solid components from a liquid microbial culture and investigated techniques to simplify this process. As a result, they found that adjusting the pH of the liquid culture simplifies the subsequent separation of solid and liquid components, thereby achieving energy savings.

[0008] One aspect of the present invention is a method for separating solid components, which includes organic acid-producing microorganisms, from a liquid culture of said microorganisms. (a) A step of adjusting the pH of the liquid culture to a predetermined value A that is different from the pH at the start of cultivation, and (b) A step of separating the solid components from the liquid culture whose pH has been adjusted to the predetermined value A, Includes, The predetermined value A is a pH such that the value Z obtained by measuring the zeta potential of the liquid culture by electrophoretic light scattering is in the range of -10mV to 10mV, which is a method for separating solid components.

[0009] This invention relates to a method for separating solid components containing microorganisms from a liquid culture of organic acid-producing microorganisms. In this invention, the pH of the liquid culture is adjusted to a predetermined value A, which is different from the pH at the start of cultivation, and then the solid components are separated from the liquid culture. In this invention, the predetermined value A is defined as "a pH at which the value Z obtained by measuring the zeta potential of the liquid culture by electrophoretic light scattering is in the range of -10mV to 10mV." As a result, in a liquid culture whose pH has been adjusted to the predetermined value A, at least some of the microorganisms contained in the solid components aggregate, making it easier to separate the solid components from the liquid components.

[0010] Preferably, the predetermined value A is 3.7 or more and 4.7 or less.

[0011] This aspect is particularly intended for cases where the target microorganism is bacteria.

[0012] Preferably, the organic acid is at least one selected from the group consisting of acetic acid, butyric acid, and lactic acid.

[0013] Preferably, the microorganism is an anaerobic bacterium.

[0014] Preferably, the anaerobic bacteria are Clostridium bacteria.

[0015] Preferably, the microorganism is further capable of producing amino acids.

[0016] Preferably, in step (a), the pH of the liquid culture is adjusted to the predetermined value A using the organic acid produced by the microorganism.

[0017] Preferably, in step (a), a pH adjusting agent is added to the liquid culture.

[0018] Preferably, in the step (b), the solid component is separated by natural sedimentation or centrifugation, and the amount of the solid component contained in the supernatant after the natural sedimentation or centrifugation is 0.3 g / L or less.

[0019] Preferably, the method further includes the step of providing a first container for culturing the microorganism and a second container for containing at least a part of the liquid culture of the microorganism cultured in the first container. The step (a) includes (a1) culturing the microorganism in the first container and adjusting the pH of the liquid culture to the predetermined value A; (a2) transferring part or all of the liquid culture with adjusted pH to the second container; and (a3) subjecting the liquid culture contained in the second container to the step (b).

[0020] Preferably, in the step (a2), the pH of the liquid culture is further adjusted.

[0021] Preferably, the step (b) includes returning the liquid component obtained by separating the solid component to the first container.

[0022] With such a configuration, the liquid component (mainly water) can be reused for the culture performed in the first container.

[0023] Another aspect of the present invention is a solid-liquid separation system for separating a solid component containing the microorganism from a liquid culture of a microorganism that produces an organic acid, a first container for culturing the microorganism; a second container for transferring and containing at least a part of the liquid culture of the microorganism cultured in the first container; a solid-liquid separation means for supplying the liquid culture contained in the second container and separating the solid component containing the microorganism from the liquid culture. The first container has a pH measurement means for measuring the pH of the liquid culture solution. ​During the cultivation of the microorganism, when the pH measured by the pH measuring means reaches a predetermined value A that is different from the pH at the start of cultivation, a portion or all of the liquid culture in the first container is automatically transferred to the second container. The predetermined value A is a solid-liquid separation system in which the pH is such that the value Z obtained by measuring the zeta potential of the liquid culture by electrophoretic light scattering is in the range of -10mV to 10mV.

[0024] This invention relates to a solid-liquid separation system for separating solid components containing organic acid-producing microorganisms from a liquid culture of said microorganisms. The system comprises a first container, a second container, and a solid-liquid separation means, and further comprises a pH measuring means for measuring the pH of the liquid culture medium in the first container. During the cultivation of microorganisms, when the pH measured by the pH measuring means reaches a predetermined value A, part or all of the liquid culture in the first container is automatically transferred to the second container. According to this invention, in the liquid culture transferred to the second container, at least a portion of the microorganisms contained in the solid components aggregate, making it easier to separate the solid components from the liquid components by the solid-liquid separation means.

[0025] Preferably, the predetermined value A is 3.7 or more and 4.7 or less.

[0026] Preferably, the second container has an additive means for adding a pH adjusting agent into the second container, and the pH of the liquid culture contained in the second container can be adjusted by adding the pH adjusting agent using the additive means.

[0027] Preferably, the solid-liquid separation means is a centrifugal separator.

[0028] Preferably, the liquid component separated by the solid-liquid separation means can be returned to the first container.

[0029] Preferably, the system further includes a third container, in which the liquid component is transferred to and contained, and in which the liquid component is transferred from the third container to the first container.

[0030] Preferably, the pH of the liquid component can be adjusted within the third container.

[0031] This configuration allows the pH of the reused liquid component (mainly water) to be pre-adjusted to a pH suitable for cultivation in the first container. [Effects of the Invention]

[0032] According to the present invention, the process of separating solid components containing microorganisms from a liquid culture of organic acid-producing microorganisms can be carried out more simply and with less energy. [Brief explanation of the drawing]

[0033] [Figure 1] This is an explanatory diagram showing the configuration of a solid-liquid separation system according to one embodiment of the present invention. [Figure 2] This graph shows the results of Example 1. [Figure 3] This is a photograph showing the results of Example 2. [Figure 4] This is a photograph showing the results of Example 3. [Figure 5] This graph shows the results of Example 4. [Figure 6] This is a photograph showing the results of Example 5. [Figure 7] This is a photograph showing the results of Example 6. [Modes for carrying out the invention]

[0034] The present invention relates to a method for separating solid components, including organic acid-producing microorganisms, from a liquid culture of said microorganisms. The method of the present invention is (a) A step of adjusting the pH of the liquid culture to a predetermined value A that is different from the pH at the start of cultivation, and (b) The process includes at least the step of separating the solid components from the liquid culture whose pH has been adjusted to the predetermined value A.

[0035] <Process (a)> In step (a), the pH of the liquid culture of the organic acid-producing microorganism is adjusted to a "predetermined value A" that is different from the pH at the start of the culture. The predetermined value A may be higher or lower than the pH at the start of the culture (hereinafter sometimes referred to as A0). In other words, "pH adjustment" includes both lowering the pH and raising the pH.

[0036] <Predetermined value A> The predetermined value A is the pH at which the value Z obtained by measuring the zeta potential of the liquid culture by electrophoretic light scattering is in the range of -10mV to 10mV (-10mV to 10mV). In other words, it is the pH at which the absolute value of Z is 0mV to 10mV (0mV to 10mV). That is, in this invention, by bringing the pH of the liquid culture closer to the predetermined value A, the charge state of the microbial surface changes, causing the microorganisms to aggregate. This facilitates the separation of the solid and liquid components in the subsequent step (b).

[0037] The absolute value of the Z value is preferably close to 0. The Z value is preferably between -8mV and 8mV (-8mV to 8mV), more preferably between -6mV and 6mV (-6mV to 6mV), even more preferably between -4mV and 4mV (-4mV to 4mV), and particularly preferably between -2mV and 2mV (-2mV to 2mV).

[0038] The charge state of a microbial surface is thought to change depending at least on the environment surrounding the microorganism, such as the composition of the culture medium, pH, ionic strength, etc. Furthermore, the charge state of a microbial surface is thought to differ depending on the type of microorganism. That is, since the structure of the cell membrane and the composition of membrane components may differ depending on the type of microorganism, the charge state of the microbial surface is also thought to differ depending on the type of microorganism. Therefore, the predetermined value A can be appropriately selected depending on the type of target microorganism, culture conditions, etc.

[0039] When the target microorganism is a common bacterium, the pH at the start of cultivation is around 7.0 to around 5.0 (from neutral to weakly acidic), and the pH often decreases as the cultivation progresses. And as the cultivation progresses, for example, when the pH drops to around 4.0 - 5.0, aggregation of the microorganisms begins to occur. In one preferred embodiment, the predetermined value A is 3.7 or more and 4.7 or less. In addition, the upper limit of the predetermined value A can be appropriately adjusted according to the type of the target microorganism to be 5.0 or less, 4.0 or less, 3.0 or less, etc. The lower limit of the predetermined value A can be appropriately adjusted according to the type of the target microorganism in the same way as the upper limit value, such as 3.0 or more, 4.0 or more, 5.0 or more, etc. For example, when the target microorganism is a bacterium belonging to the genus Clostridium, the predetermined value A is preferably 3.7 or more and 4.7 or less, more preferably 3.9 or more and 4.5 or less, and even more preferably 4.0 or more and 4.3 or less.

[0040] <pH adjustment> As a method for adjusting the pH of the liquid culture, it includes using organic acids produced by the microorganism itself during cultivation. For example, the pH of the liquid culture can be adjusted by organic acids excreted extracellularly by the microorganism during cultivation. In this case, the pH of the liquid culture decreases.

[0041] As other examples, it includes adding a pH adjuster to the liquid culture. For example, by adding an alkaline or acidic pH adjuster to the liquid culture, the pH of the liquid culture can be adjusted. When increasing the pH of the liquid culture, an alkaline pH adjuster is used. When decreasing the pH of the liquid culture, an acidic pH adjuster is used. Examples of the alkaline pH adjuster include aqueous ammonia; aqueous solutions of alkali metal hydroxides (such as sodium hydroxide), etc. Examples of the acidic pH adjuster include inorganic acids such as hydrochloric acid and phosphoric acid; organic acids such as acetic acid, butyric acid, lactic acid, and citric acid. It is also possible to use in combination using organic acids produced by the microorganism itself during cultivation and adding a pH adjuster.

[0042] <organic acid> There are no particular limitations on the organic acid produced by the target microorganism. In preferred embodiments, the organic acid is at least one selected from the group consisting of acetic acid, butyric acid, and lactic acid. For example, microorganisms generally called acetic acid-producing microorganisms, butyric acid-producing microorganisms, lactic acid-producing microorganisms, etc., are the subject of the present invention, and the pH adjustment described above is possible by the organic acid produced by these microorganisms.

[0043] <Microorganisms> The microorganisms covered by this invention are not particularly limited as long as they produce organic acids, and can include all microorganisms such as bacteria, yeasts, filamentous fungi, algae, and protozoa. There is no distinction between prokaryotes and eukaryotes, or between unicellular and multicellular organisms. Of these, bacteria are a representative example of microorganisms covered by this invention. There are no particular limitations on the bacteria covered by this invention, and there is no distinction between Gram-negative bacteria and Gram-positive bacteria, aerobic bacteria and anaerobic bacteria, eubacteria and archaea, etc.

[0044] In one embodiment, the target microorganism is an anaerobic bacterium such as a bacterium of the genus Clostridium. Examples of Clostridium bacteria include Clostridium ljungdahlii, Clostridium acetobutylicum, Clostridium aceticum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium magnum, Clostridium pasteurianum, Clostridium ragsdali, Clostridium scatologenes, Clostridium thermoaceticum, and Clostridium ultunense. These anaerobic bacteria are also examples of acetic acid-producing bacteria, butyrate-producing bacteria, and lactic acid-producing bacteria. In particular, when the microorganism is a bacterium of the genus Clostridium, it is preferable that the microorganism has the ability to produce at least one acetogene, and it is even more preferable that the microorganism has the ability to produce at least one of acetic acid or ethanol.

[0045] In one embodiment, the microorganism is capable of producing amino acids (amino acid-producing bacteria). Examples of such bacteria include oleic acid-requiring strains obtained from Brevibacrerium thiogenitalis, and threonine-requiring strains of the Corynebacterium genus and Brevibacterium flavum. Other amino acid-producing bacteria include intestinal bacteria belonging to the genera γ-proteobacteria such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Salmonella, and Morganella; so-called corynetype bacteria belonging to the genera Brevibacterium, Corynebacterium, and Microbacterium; and microorganisms belonging to genera such as Alicyclobacillus, Bacillus, and Saccharomyces. γ-proteobacteria can be used if they are classified as microorganisms according to the classification disclosed in the NCBI (National Center for Biotechnology Information Taxonomy Database). Examples of Escherichia bacteria include Escherichia coli. In particular, it is preferable that the microorganisms be Escherichia bacteria or corine-type bacteria. It is even more preferable that such microorganisms can produce aromatic compounds in the shikimic acid pathway from acetic acid or ethanol. Coumaric acid or caffeic acid are biosynthesized using tyrosine in the shikimic acid pathway as a precursor, and these are preferable because they can be used as raw materials for epoxy resins having a benzene ring. Such epoxy resins are preferable because they can be used as raw materials for resins requiring heat resistance.

[0046] <Solid component> Solid components are those components in a liquid culture other than the liquid components; in other words, they are water-insoluble components. The main components of solid components are microbial cells. Microbial cells include both living and dead cells. Furthermore, solid components may include cell debris (such as cell membrane components) larger than a certain size.

[0047] <Process (b)> In step (b), the solid component containing microorganisms is separated from the liquid culture whose pH has been adjusted to a predetermined value A. There are no particular limitations on the method for separating the solid component from the liquid component (solid-liquid separation method), and known methods such as centrifugation and membrane separation can be used.

[0048] In one embodiment, the solid components are separated by natural sedimentation or centrifugation. Here, the solid components subjected to step (b) after step (a) are in an aggregated state containing microorganisms (including living cells, dead cells, and cell debris), so they settle easily, and the solid components can be easily separated by natural sedimentation or centrifugation with low centrifugal force and / or short duration. In a preferred embodiment, it is preferable that the amount of the solid components contained in the supernatant after natural sedimentation or centrifugation is 0.3 g / L or less. It is more preferable that the amount of the solid components is 0.2 g / L or less. The value for the amount of solid components to be 0.2 g / L or less may be, for example, 0.1 g / L, 0.05 g / L, 0.02 g / L, or 0 g / L (below the detection limit). Furthermore, 0 g / L (below the detection limit) of the solid components does not mean that they are not contained at all, but rather that they are below the detectable value. By keeping the amount of solid components (mainly microorganisms) in the supernatant below the aforementioned value, the liquid components are less susceptible to the influence of microbial introduction when introduced into another process. The centrifugal force (relative centrifugal force) in the centrifugation is usually 10,000 G or less, preferably 5,000 G or less, and more preferably 3,000 G or less (G is the acceleration due to gravity).

[0049] <Solid-liquid separation system> A solid-liquid separation system according to one aspect of the present invention will be described with reference to the drawings. The solid-liquid separation system 1 shown in Figure 1 comprises a first container 2, a second container 3, a solid-liquid separation means 5, and a third container 6. The first container 2 and the second container 3 are connected by a first transfer line 7, the second container 3 and the solid-liquid separation means 5 are connected by a second transfer line 8, the solid-liquid separation means 5 and the third container 6 are connected by a third transfer line 11, and the third container 6 and the first container 2 are connected by a fourth transfer line 12.

[0050] The first container 2 is a container for liquid culture of microorganisms that produce organic acids, and is, for example, a culture tank. The first container 2 is equipped with a pH measuring means (not shown) for measuring the pH of the liquid culture. The pH measuring means is, for example, a pH sensor that can continuously measure the pH of the liquid culture.

[0051] The second container 3 is a container for temporarily holding the liquid culture. The second container 3 is connected to the first container 2 via the first transfer line 7. Part or all of the liquid culture in the first container 2 can be transferred to the second container 3 via the first transfer line 7. The second container 3 is equipped with an additive means (not shown) for adding a pH adjusting agent. This allows the pH of the liquid culture in the second container 3 to be adjusted. The second container 3 is also equipped with a turbidimeter (not shown) for measuring the turbidity of the liquid culture. A viewing window may be provided for visually checking the turbidity if necessary.

[0052] A valve (not shown) is provided in the first transfer line 7. This valve is an automatic valve that automatically opens and closes according to the pH of the liquid culture measured by the pH measuring means in the first container 2. When the automatic valve opens, the liquid culture in the second container 3 is transferred to the second container 3. The pH value at which the automatic valve opens can be freely set by the user of this system, and in this embodiment, it is set to the predetermined value A described above. The predetermined value A can be appropriately determined depending on the type of microorganism being targeted, etc.

[0053] The solid-liquid separation means 5 separates the solid and liquid components of the liquid culture. In this embodiment, the solid-liquid separation means 5 is a centrifuge. The solid-liquid separation means 5 is connected to the second container 3 via a second transfer line 8. The liquid culture in the second container 3 can be supplied to the solid-liquid separation means 5 through the second transfer line 8. A valve (not shown) is provided in the second transfer line 8. By opening this valve manually or automatically, the liquid culture in the second container 3 is transferred to the solid-liquid separation means 5.

[0054] The third container 6 is a container for temporarily holding the liquid component (centrifugal supernatant) separated by the solid-liquid separation means 5. The third container 6 is connected to the solid-liquid separation means 5 via the third transfer line 11. The liquid component (centrifugal supernatant) separated by the solid-liquid separation means 5 can be transferred to the third container 6 via the third transfer line 11. A valve (not shown) is provided in the third transfer line 11. By opening this valve manually or automatically, the liquid component separated by the solid-liquid separation means 5 is transferred to the third container 6. The third container 6 can be configured to allow adjustment of the pH of the liquid component contained therein as needed. For example, an additive means similar to that of the second container 3 may be provided, allowing a pH adjusting agent to be added to the third container 6.

[0055] The solid components separated by the solid-liquid separation means 5 can be recovered from the discharge line 10.

[0056] The third container 6 and the first container 2 are connected via the fourth transfer line 12. The liquid component (centrifugation supernatant) in the third container 6 can be transferred to the first container 2 via the fourth transfer line 12. A valve (not shown) is provided in the fourth transfer line 12. By opening this valve manually or automatically, the liquid component (centrifugation supernatant) in the third container 6 is transferred to the first container 2.

[0057] In other words, in the solid-liquid separation system 1, the first container 2, the first transfer line 7, the second container 3, the second transfer line 8, the solid-liquid separation means 5, the third transfer line 11, the third container 6, the fourth transfer line 12, and the first container 2 form a unidirectional annular flow path in this order.

[0058] An example of the operation and usage method of the solid-liquid separation system 1 will be described. First, small-scale preliminary experiments are conducted to determine a predetermined value A for the liquid culture of the target microorganism, that is, the pH at which the value Z obtained by measuring the zeta potential of the liquid culture of the microorganism using electrophoretic light scattering is in the range of -10mV to 10mV. Then, the predetermined value A is set in the solid-liquid separation system 1.

[0059] Liquid culture of microorganisms is started in the first container 2. The pH (A0) at the start of the culture is recorded. The pH of the liquid culture is continuously measured using a pH measuring device and monitored. If the target microorganism is bacteria, typically the pH (A0) at the start of the culture is around 7.0, and the pH decreases as the culture progresses. That is, the predetermined value A is lower than A0.

[0060] The pH of the liquid culture decreases and eventually reaches a predetermined value A. When the pH of the liquid culture reaches the predetermined value A, the microorganisms in the liquid culture aggregate, and the solid components are in a state where they are likely to precipitate. At this time, a valve provided in the first transfer line 7 automatically opens, and part or all of the liquid culture is transferred to the second container 3. In other words, in the solid-liquid separation system 1, when the pH of the liquid culture reaches a predetermined value A set in advance, the automatic valve opens, and part or all of the liquid culture is transferred to the second container 3. If you wish to continue culturing in the first container 2, you can transfer part of the liquid culture and leave part of it in the first container 2. On the other hand, if you wish to end culturing in the first container 2, you may transfer all of the liquid culture.

[0061] The liquid culture is temporarily stored in the second container 3. The pH of the liquid culture is adjusted as needed. The turbidity of the liquid culture is measured as needed to check its condition. Then, the valve of the second transfer line 8 is opened and the liquid culture is transferred to the solid-liquid separation means 5.

[0062] Solid-liquid separation is performed using the solid-liquid separation means 5 (centrifuge). In this embodiment, the microorganisms in the liquid culture aggregate, making it easy for the solid components to precipitate, so solid-liquid separation can be completed with low centrifugal force and / or short centrifugation time. After centrifugation, the solid components are recovered from the discharge line 10. The target substance can then be obtained from the recovered solid components. Meanwhile, the valve of the third transfer line 11 is opened, and the liquid components (centrifugation supernatant) are transferred to the third container 6. The solid-liquid separation means may contain additives to facilitate precipitation as needed. If such additives are added, it is preferable that they do not adversely affect microbial culture. Furthermore, the solid-liquid separation means may be a combination of multiple solid-liquid separation means. Examples of such combinations of solid-liquid separation means include two or more selected from, for example, heat transfer drying means and filtration means.

[0063] The liquid component (centrifugation supernatant) is temporarily stored in the third container 6. The pH of the liquid component is adjusted as needed. One way to adjust the pH is to set it to the same or close to the pH of A0 at the start of cultivation in the first container 2. Then, the valve of the fourth transfer line 12 is opened and the liquid component is transferred to the first container 2. This returns the liquid component separated by the solid-liquid separation means 5 to the first container 2.

[0064] By returning the liquid component separated by the solid-liquid separation means 5 to the first container 2, the liquid component can be reused for the next culture. That is, since a portion of the liquid culture remains in the first container 2, the culture can be restarted by adding the recovered liquid component to it. Here, it is preferable to adjust the pH of the liquid component in the third container 6 to the same or close value as A0, so that the pH at the start of the culture can be matched with the previous culture.

[0065] By repeating the cycle described above, cultivation can be repeated, and a large amount of solid components can be obtained. In this system, the liquid component (centrifugation supernatant) is reused for cultivation, which is particularly useful in terms of energy saving.

[0066] In the embodiment described above, there was one second container 3, but multiple second containers 3 may be provided. For example, multiple second containers 3 can be provided in parallel.

[0067] In this embodiment, the solid-liquid separation means 5 was a centrifuge, but other devices may be used. For example, the solid-liquid separation means 5 may be a solid-liquid separation tank that allows solid components to settle naturally. Alternatively, the solid-liquid separation means 5 may be a membrane separation device. [Examples]

[0068] [Example 1] Ten pH levels of 10 mM phosphate buffer were prepared. The pH levels were 3.0, 3.2, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, and 5.2.

[0069] Clostridium acetobutylicum (microorganism A) and Clostridium ljungdahlii (microorganism B) were cultured in liquid using a conventional method to obtain liquid cultures. The liquid cultures were subjected to centrifugation (13000G, 3 minutes), and the supernatant was removed to obtain the cells of each microorganism.

[0070] The obtained bacterial cells were suspended in each phosphate buffer so that the OD600 was 0.1. The resulting suspensions were used as samples, and their zeta potential was measured by electrophoretic light scattering. Zeta potential was measured using an Anton Paar Litesizer 500. Three measurements were performed, and the average value was calculated. The results are shown in Table 1 and Figure 1. In Figure 1, the vertical axis represents the average value of the zeta potential (mV), and the horizontal axis represents the pH of the suspension.

[0071] [Table 1]

[0072] A correlation was confirmed between the pH of the suspension and the zeta potential in both microorganisms A and B.

[0073] [Example 2] Clostridium ljungdahlii was cultured in liquid using a conventional method to obtain a liquid culture. The pH at the start of the culture was 7.0, and the pH at the end of the culture (pH of the liquid culture) was 4.9. The obtained liquid culture was uniformly suspended. Hydrochloric acid was added to this liquid culture (pH 4.9) to adjust the pH to "4.0" or "3.0". The cultures were left to stand in a 15 mL conical tube at room temperature for 16 hours, and the presence or absence of precipitate was visually confirmed. The results are shown in Figure 3. In Figure 3, from left to right, the results are for pH 4.9 (no pH adjustment), pH 4.0, and pH 3.0. Specifically, in the liquid culture adjusted to pH 4.0, a precipitate of solid components formed, and the supernatant was clear. On the other hand, in the liquid culture adjusted to pH 3.0, no precipitate formed, and it remained uniformly suspended.

[0074] [Example 3] Clostridium ljungdahlii was cultured in liquid using a conventional method to obtain a liquid culture. The pH at the start of the culture was 7.0, and the pH at the end of the culture (pH of the liquid culture) was 4.9. The obtained liquid culture was uniformly suspended. Sodium hydroxide was added to this liquid culture (pH 4.9) to adjust the pH to 5.3. Hydrochloric acid was also added to this liquid culture (pH 4.9) to adjust the pH to 4.5, 4.0, 3.5, or 3.0. The cultures were left to stand in 15 mL conical tubes at room temperature for 16 hours, and the presence or absence of precipitate was visually confirmed. The results are shown in Figure 4. In Figure 4, from left to right, the results are shown for pH 5.3, pH 4.9 (no pH adjustment), pH 4.5, pH 4.0, pH 3.5, and pH 3.0. Specifically, in the liquid cultures adjusted to pH 4.5 and pH 4.0, precipitate of solid components formed, and the supernatant was clear. On the other hand, in liquid cultures adjusted to other pH levels, no precipitate formed, and the mixture remained uniformly suspended.

[0075] [Example 4] Similar to Example 1, 10 mM phosphate buffers were prepared at various pH levels. The pH levels were 3.0, 3.2, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, and 5.2 (10 different pH levels).

[0076] Escherichia coli (BL21) (microorganism C) and Corynebacterium glutamicum (NBRC 12168) (microorganism D) were cultured in liquid according to a standard method to obtain liquid cultures. The liquid cultures were subjected to centrifugation (13000G, 3 minutes), and the supernatant was removed to obtain the cells of each microorganism.

[0077] The obtained bacterial cells were suspended in each phosphate buffer so that the OD600 was 0.02. The resulting suspensions were used as samples, and the zeta potential was measured by electrophoretic light scattering in the same manner as in Example 1. Three measurements were taken, and the average value was calculated. The results are shown in Table 2 and Figure 5. In Figure 5, the vertical axis represents the average value of the zeta potential (mV), and the horizontal axis represents the pH of the suspension.

[0078] [Table 2]

[0079] [Example 5] Escherichia coli was cultured in liquid using a conventional method to obtain a liquid culture. The culture was subjected to centrifugation (13000G, 3 minutes), and the supernatant was removed to obtain microbial cells. The cells were washed by suspending them in pH-adjusted phosphate buffer, centrifugation, and removing the supernatant. The cells were again suspended in pH-adjusted phosphate buffer and allowed to stand in a 3 mL tube for 6 hours, and the presence or absence of precipitate was visually confirmed. The results are shown in Figure 6. In Figure 6, from left to right, the results are shown for pH 2.0, pH 2.25, pH 2.5, pH 3.0, pH 3.5, and pH 4.0. Specifically, in suspensions adjusted to pH 2.0 to 2.5, precipitate of solid components formed, and the supernatant was clear. On the other hand, in suspensions adjusted to pH 3.5 or higher, no precipitate formed, and the suspension remained uniformly suspended.

[0080] [Example 6] The same procedure as in Example 5 was performed using Corynebacterium glutamicum instead of Escherichia coli. The results are shown in Figure 7. In Figure 7, the results are shown from left to right for pH 2.0, pH 2.25, pH 2.5, pH 3.0, pH 3.5, and pH 4.0. Specifically, in the suspension adjusted to pH 2.0 to 2.5, a precipitate of solid components formed, and the supernatant was clear. On the other hand, in the suspension adjusted to pH 3.5 or higher, no precipitate formed, and the suspension remained uniformly suspended. [Explanation of Symbols]

[0081] 1. Solid-liquid separation system 2 First container 3 Second container 5 Solid-liquid separation means 6 Third container

Claims

1. A method for separating solid components from a liquid culture of an organic acid-producing microorganism, wherein the solid component contains the said microorganism. (a) A step of adjusting the pH of the liquid culture to a predetermined value A that is different from the pH at the start of cultivation, and (b) A step of separating the solid components from the liquid culture whose pH has been adjusted to the predetermined value A, Includes, A method for separating solid components, wherein the predetermined value A is a pH such that the value Z obtained by measuring the zeta potential of the liquid culture by electrophoretic light scattering is in the range of -10 mV to 10 mV.

2. The method for separating solid components according to claim 1, wherein the predetermined value A is 3.7 or more and 4.7 or less.

3. The method for separating solid components according to claim 1, wherein the organic acid is at least one selected from the group consisting of acetic acid, butyric acid, and lactic acid.

4. The method for separating solid components according to claim 3, wherein the microorganism is an anaerobic bacterium.

5. The method for separating solid components according to claim 4, wherein the anaerobic bacteria are bacteria of the genus Clostridium.

6. The method for separating solid components according to claim 1, wherein the microorganism is capable of producing amino acids.

7. The method for separating solid components according to any one of claims 1 to 6, wherein in step (a), the pH of the liquid culture is adjusted to the predetermined value A using an organic acid produced by the microorganism.

8. The method for separating solid components according to any one of claims 1 to 6, wherein in step (a) above, a pH adjusting agent is added to the liquid culture.

9. The method for separating solid components according to claim 7, wherein in step (a), a pH adjusting agent is added to the liquid culture.

10. The method for separating a solid component according to any one of claims 1 to 6, wherein in step (b), the solid component is separated by natural sedimentation or centrifugation, and the amount of the solid component contained in the supernatant after natural sedimentation or centrifugation is 0.3 g / L or less.

11. The method further includes providing a first container for culturing the microorganism and a second container for containing at least a portion of the liquid culture of the microorganism cultivated in the first container, The above step (a) is, (a1) Culturing the microorganism in the first container and adjusting the pH of the liquid culture to the predetermined value A, (a2) Transferring part or all of the pH-adjusted liquid culture to the second container, (a3) The liquid culture contained in the second container is subjected to step (b), A method for separating solid components according to any one of claims 1 to 6, including the following:

12. The method for separating solid components according to claim 11, wherein in step (a2), the pH of the liquid culture is further adjusted.

13. The method for separating a solid component according to claim 11, further comprising, in step (b), returning the liquid component obtained by separating the solid component back to the first container.

14. A solid-liquid separation system for separating solid components containing organic acid-producing microorganisms from a liquid culture of said microorganisms, A first container for culturing the aforementioned microorganisms, A second container into which at least a portion of the liquid culture of microorganisms cultured in the first container is transferred and contained, The liquid culture contained in the second container is supplied, and the system includes a solid-liquid separation means for separating solid components, including the microorganisms, from the liquid culture. The first container has a pH measuring means for measuring the pH of the liquid culture solution, During the cultivation of the microorganism, when the pH measured by the pH measuring means reaches a predetermined value A that is different from the pH at the start of cultivation, a portion or all of the liquid culture in the first container is automatically transferred to the second container. The predetermined value A is a pH such that the value Z obtained by measuring the zeta potential of the liquid culture by electrophoretic light scattering is in the range of -10 mV to 10 mV, in a solid-liquid separation system.

15. The solid-liquid separation system according to claim 14, wherein the predetermined value A is 3.7 or more and 4.7 or less.

16. The second container has an additive means for adding a pH adjusting agent into the second container. The solid-liquid separation system according to claim 14, wherein the pH of the liquid culture contained in the second container can be adjusted by adding a pH adjusting agent using the aforementioned adding means.

17. The solid-liquid separation system according to claim 14, wherein the solid-liquid separation means is a centrifugal separator.

18. The solid-liquid separation system according to claim 14, wherein the liquid component separated by the solid-liquid separation means can be returned to the first container.

19. A third container is also provided, The liquid component is transferred to and contained in the third container. The solid-liquid separation system according to claim 18, wherein the liquid component is transferred from the third container to the first container.

20. The solid-liquid separation system according to claim 19, wherein the pH of the liquid component can be adjusted in the third container.

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