Method for producing microorganism carrier

Abstract

This method for producing a microorganism carrier comprises: a slurry preparation step for preparing a slurry containing a cement component that contains calcium phosphate and a microorganism-containing solution that contains active microorganisms, which are active, and water; and a curing step for curing the slurry. The active microorganisms may be one or more selected from fungi, bacteria, and archaea. The amount of the active microorganisms contained in the microorganism-containing solution may be 1.0 × 105 cells / mL to 1.0 × 109 cells / mL.

Description

Method for producing microbial carriers

[0001] This invention relates to a method for producing a microbial carrier. This application claims priority based on Japanese Patent Application No. 2024-193278, filed in Japan on November 1, 2024, the contents of which are incorporated herein by reference.

[0002] In recent years, the use of bioreactors has been explored in various fields, including water treatment, air purification, soil remediation, and biofuel production. A bioreactor is a biochemical reaction device that uses biocatalysts such as microorganisms to synthesize and decompose substances.

[0003] Some bioreactors utilize biocatalysts, such as microorganisms, supported on a carrier. Various carriers made from diverse materials, including polymers and inorganic materials, have been proposed in various shapes. For example, Non-Patent Document 1 describes solidified enzymes and solidified microorganisms. Non-Patent Document 1 also describes that methods similar to the carrier binding method, crosslinking method, and inclusion method used for enzyme immobilization are employed for immobilizing microbial cells.

[0004] Hydroxyapatite, a type of calcium phosphate, is the main inorganic component of bone. Due to its high affinity for bone, hydroxyapatite is used as an artificial bone material. For example, calcium phosphate cement (CPC), which is widely used as an artificial bone material, contains calcium phosphate powder as a binder, which hardens by forming hydroxyapatite through a reaction with water.

[0005] "On Immobilized Enzymes and Immobilized Microorganisms," Ichiro Senhata, Membrane 3(5), pp. 339-353 (1978)

[0006] However, conventional technology has made it difficult to produce microbial carriers on which a predetermined amount of microorganisms are supported. Therefore, there has been a need for a manufacturing method that can produce microbial carriers on which a predetermined amount of microorganisms are supported. The present invention has been made in view of the above problem, and aims to provide a method for producing microbial carriers on which a predetermined amount of microorganisms are supported.

[0007] The inventors focused on a method for supporting microorganisms inside a carrier and diligently investigated it as described below. As a result, they found that it is possible to prepare a slurry containing a cement component containing calcium phosphate and a microorganism-containing solution containing live microorganisms and water, and then harden this slurry.

[0008] A slurry containing cement components including calcium phosphate and water has little effect on microbial activity and can be hardened at low temperatures. Therefore, a microbial carrier made from the hardened slurry contains live microorganisms at a nearly uniform density within the carrier, and carries a predetermined amount of microorganisms corresponding to the amount of live microorganisms contained in the microbial solution.

[0009] Calcium phosphate cement has been widely used as an artificial bone material. Generally, when calcium phosphate cement is used as an artificial bone material, sufficient measures are taken during manufacturing to prevent the contamination of the hardened slurry containing calcium phosphate cement and water with microorganisms, from the perspective of preventing infection. Furthermore, it is undesirable for microorganisms to adhere to artificial bone manufactured using calcium phosphate cement, or for any attached microorganisms to survive in a highly active state. For this reason, the use of calcium phosphate cement, which has been conventionally used as an artificial bone material, as a carrier material for supporting microorganisms has not been considered.

[0010] Furthermore, conventional microbial carriers were manufactured by a method of supporting microorganisms on a carrier. Therefore, while it was possible to fix microorganisms to the surface of the carrier, it was difficult to adequately support microorganisms within the carrier itself. Consequently, conventional methods for manufacturing microbial carriers sometimes failed to produce carriers with a sufficient amount of supported microorganisms. Additionally, controlling the amount of microorganisms supported on the carrier was difficult with this method.

[0011] Another possible method for immobilizing microorganisms within a carrier is to incorporate live microorganisms during the manufacturing process of conventional carriers. However, when live microorganisms are incorporated during the manufacturing process of conventional carriers, it has been difficult to complete the carrier while maintaining the activity of the microorganisms. Specifically, for example, carriers made of inorganic materials are manufactured by mixing the inorganic materials that will become the carrier and then sintering them. Therefore, even if live microorganisms are incorporated into the inorganic materials at the stage of mixing the inorganic materials that will become the carrier, the activity of the microorganisms is lost during the sintering process.

[0012] Therefore, conventionally, the process of manufacturing carriers has not involved actively incorporating live microorganisms, making it difficult to manufacture microbial carriers that contain live microorganisms within them.

[0013] Furthermore, the inventors of the present invention conceived the present invention after confirming that the activity of microorganisms is maintained in a microbial carrier produced by preparing a slurry containing a cement component containing calcium phosphate and a microorganism-containing solution containing live microorganisms and water, and then hardening it. The present invention provides the following means.

[0014] [1] A method for producing a microbial carrier, comprising: a slurry preparation step of preparing a slurry containing a cement component containing calcium phosphate and a microorganism-containing solution containing active microorganisms and water; and a curing step of curing the slurry.

[0015] [2] The method for producing a microbial carrier according to [1], wherein the active microorganism is one or more selected from fungi, bacteria, and archaea. [3] The amount of the active microorganism contained in the microbial solution is 1.0 × 10 ５ cells / mL~1.0×10 ９ A method for producing a microbial carrier according to [1], wherein the amount is cells / mL.

[0016] [4] The method for producing a microbial carrier according to [1], wherein in the slurry preparation step, the active microorganism and the microbial culture medium are mixed to prepare the microorganism-containing solution. [5] The method for producing a microbial carrier according to [1], wherein in the slurry preparation step, the active microorganism, the microbial culture medium and sodium alginate are mixed to prepare the microorganism-containing solution.

[0017] [6] The method for producing a microbial carrier according to [4] or [5], wherein the active microorganism is yeast and the microbial medium is YPD medium. [7] The method for producing a microbial carrier according to [4] or [5], wherein the active microorganism is Escherichia coli and the microbial medium is LB medium.

[0018] [8] The method for producing a microbial carrier according to [1], wherein in the slurry preparation step, a slurry is prepared containing the cement component and the microorganism-containing solution in a mass ratio of 1:2 to 2.5:1.

[0019] [9] The method for producing a microbial carrier according to [1], wherein the cement component comprises (a) 0% to 100% by mass of α-tricalcium phosphate, (b) 0% to 80% by mass of tetracalcium phosphate, (c) 0% to 40% by mass of anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate, and (d) 0% to 10% by mass of hydroxyapatite, and contains 50% or more by mass of (a) α-tricalcium phosphate, or the total of (b) tetracalcium phosphate and (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate is 50% or more by mass.

[0020]

[10] A method for producing a microbial carrier according to [1], wherein the steps of preparing the microbial solution and curing the slurry are performed in an air atmosphere.

[0021] The present invention provides a method for producing a microbial carrier, comprising a slurry preparation step of preparing a slurry containing a cement component containing calcium phosphate and a microorganism-containing solution containing active microorganisms and water, and a curing step of curing the slurry. Therefore, according to the method for producing a microbial carrier of the present invention, active microorganisms are present in a substantially uniform density within the cured slurry, and a microbial carrier can be produced on which a predetermined amount of microorganisms corresponding to the amount of active microorganisms contained in the microorganism-containing solution is supported.

[0022] Figures 1A to 1D are photographs of microbial carriers and solutions in which the microbial carriers were immersed after a color reaction was observed by adding a microbial assay kit. Figure 1A is a photograph of the microbial carrier and solution in which the microbial carrier was immersed in Example 1. Figure 1B is a photograph of the microbial carrier and solution in which the microbial carrier was immersed in Example 2. Figure 1C is a photograph of the carrier and solution in which the carrier was immersed in Comparative Example 1. Figure 1D is a photograph of the carrier and solution in which the carrier was immersed in Comparative Example 2.

[0023] The method for producing a microbial carrier according to this embodiment will be described in detail below. The materials, dimensions, etc. exemplified in the following description are examples only, and the present invention is not limited to them. It can be implemented with appropriate modifications without changing the essence of the invention.

[0024] The method for producing a microbial carrier according to this embodiment includes a slurry preparation step and a hardening step. [Slurry Preparation Step] In the slurry preparation step according to this embodiment, a slurry is prepared that includes a cement component containing calcium phosphate and a microbial-containing solution containing active microorganisms and water.

[0025] (Cement component) In the method for producing a microbial carrier according to the present embodiment, the cement component used as a material contains calcium phosphate. The calcium phosphate contained in the cement component forms hydroxyapatite by reaction with water and hardens. The calcium phosphate and the reaction between calcium phosphate and water have little influence on the activity of microorganisms. Moreover, the reaction between calcium phosphate and water can proceed sufficiently at a low temperature of, for example, 30°C to 37°C. From these facts, according to the production method of the present embodiment, a microbial carrier in which active microorganisms having activity are present at a substantially uniform density inside the carrier can be produced.

[0026] The cement component used in the present embodiment preferably contains, as calcium phosphate, one or more compounds selected from, for example, α-tricalcium phosphate, tetracalcium phosphate, calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate, and more preferably contains α-tricalcium phosphate as a main component. The reason is that a slurry that hardens sufficiently can be easily obtained even if the content of the cement component forming the slurry is reduced.

[0027] The cement component used in the present embodiment is preferably (a) one having α-tricalcium phosphate as a main component, or (b) one having a mixture of tetracalcium phosphate and (c) calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate as a main component.

[0028] Specifically, the cement component used in the present embodiment contains (a) 0% by mass to 100% by mass of α-tricalcium phosphate, (b) 0% by mass to 80% by mass of tetracalcium phosphate, (c) 0% by mass to 40% by mass of calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate, (d) 0% by mass to 10% by mass of hydroxyapatite, and contains (a) 50% by mass or more of α-tricalcium phosphate, or (b) the total of tetracalcium phosphate and (c) calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate of 50% by mass or more.

[0029] When the content of (a) tricalcium phosphate contained in the cement component is 50% by mass or more, it is preferable because a slurry that easily hardens in the hardening process can be easily obtained. The content of (a) tricalcium phosphate is more preferably 70% by mass or more. Further, the cement component may be only (a) tricalcium phosphate (in other words, the content of (a) tricalcium phosphate is 100% by mass). However, when the content of (a) tricalcium phosphate contained in the cement component exceeds 90% by mass, the hardening reaction of the slurry tends to be slow. Therefore, the content of (a) tricalcium phosphate is preferably 90% by mass or less.

[0030] When the cement component contains 50% by mass or more of (a) tricalcium phosphate as the main component, it is preferable to contain 10% by mass or more of (b) tetracalcium phosphate together with (a) tricalcium phosphate. The reason is that when the content of (b) tetracalcium phosphate is 10% by mass or more, a slurry with an even faster hardening reaction can be obtained. The content of (b) tetracalcium phosphate is more preferably 15% by mass or more.

[0031] When the cement component contains 50% by mass or more of (a) tricalcium phosphate as the main component, it is preferable to contain 2% by mass or more of (c) calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate together with (a) tricalcium phosphate. The reason is that when the content of (c) calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate is 2% by mass or more, a slurry with an even faster hardening reaction can be obtained. The content of (c) calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate is more preferably 5% by mass or more.

[0032] Furthermore, if the cement component contains 50% by mass or more of a mixture of (b) tetracalcium phosphate and (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate as the main component, a slurry that hardens easily in the hardening process can be obtained even if (a) α-tricalcium phosphate is not included (in other words, even if the content of (a) α-tricalcium phosphate is 0% by mass). Therefore, the content of the above mixture in the cement component may be 100% by mass.

[0033] The ratio of (b) tetracalcium phosphate to (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate contained in the above mixture can be any ratio. For example, the ratio of (b) tetracalcium phosphate to (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate contained in the above mixture (component (b); component (c)) is preferably 50:50 to 90:10 by mass%, and more preferably 60:40 to 80:20.

[0034] When the cement components contain a total of 50% by mass or more of (b) tetracalcium phosphate and (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate, it is preferable that (b) tetracalcium phosphate is present in an amount of 60% by mass or more. This is because a slurry with a faster hardening reaction can be obtained. However, if the content of (b) tetracalcium phosphate in the cement components exceeds 80% by mass, the hardening reaction of the slurry tends to slow down, so it is preferable that the content of (b) tetracalcium phosphate be 80% by mass or less, and more preferably 75% by mass or less.

[0035] When the cement components contain a total of 50% by mass or more of (b) tetracalcium phosphate and (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate, it is preferable that (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate be present in an amount of 20% by mass or more. This is because a slurry with a faster hardening reaction can be obtained. However, if the content of (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate in the cement components exceeds 40% by mass, the hardening reaction of the slurry tends to slow down. Therefore, the content of (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate is preferably 40% by mass or less, and more preferably 35% by mass or less.

[0036] The cement components may optionally contain (d) hydroxyapatite. When (d) hydroxyapatite is included in the cement components, it functions as a seed crystal for hydroxyapatite particles in the slurry, thereby promoting the precipitation reaction of hydroxyapatite in the slurry. As a result, the reaction in which calcium phosphate contained in the cement components reacts with water to form hydroxyapatite is promoted, resulting in a slurry with an even faster hardening reaction, which is preferable.

[0037] The content of (d) hydroxyapatite in the cement component is preferably 1% by mass or more, and more preferably 2% by mass or more. Furthermore, the content of (d) hydroxyapatite in the cement component is preferably 10% by mass or less. This is because it makes it easier to ensure the content of components other than (d) hydroxyapatite, which is the main component that contributes to the hardening of the cement component.

[0038] The cement components used in this embodiment may further include, as needed, (a) α-tricalcium phosphate, (b) tetracalcium phosphate, (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate, and (d) other components other than hydroxyapatite. Other components may include conventionally known cement components, such as one or more components selected from calcium dihydrogen phosphate, magnesium phosphate, etc.

[0039] The composition of the cement components used in this embodiment can be appropriately determined depending on the type of active microorganism and the composition of the microorganism-containing solution. In this embodiment, commercially available calcium phosphate cement may be used as the cement component. Examples of commercially available calcium phosphate cements that can be used include Biopex (manufactured by HOYA Technosurgical Corporation) and Ceraharmo (manufactured by Integra Japan Corporation).

[0040] (Microbial Solution) The microbial solution used in the method for producing the microbial carrier of this embodiment contains active microorganisms and water. The type of active microorganism is not particularly limited and is determined as appropriate depending on the intended use of the microbial carrier of this embodiment. The microbial solution may contain only one type of active microorganism or two or more types.

[0041] Whether or not the microorganisms used in the method for producing the microbial carrier of this embodiment are alive (active or not) can be determined by known methods. For example, this can be determined by adding the microorganisms to water and stirring to form a microbial suspension, and then performing a color reaction on the resulting microbial suspension using a microbial assay kit (manufactured by Dojin Chemical Laboratories Co., Ltd.). Alternatively, it may be determined by placing the microorganisms in a liquid culture medium containing organic matter and examining whether or not the organic matter is decomposed. Another method is to culture the microorganisms in a liquid culture medium and measure the amount of gas or other substances produced by the biocatalytic action of the microorganisms.

[0042] Examples of active microorganisms include fungi, bacteria, and archaea. Examples of fungi include yeasts such as dry yeast and budding yeast, and filamentous fungi such as Aspergillus oryzae. Examples of bacteria include E. coli, rhizobia, Bacillus subtilis, and ammonia-oxidizing bacteria. Examples of archaea include methanogenic bacteria and ammonia-oxidizing archaea.

[0043] Microbial solutions may be prepared by mixing active microorganisms with a microbial culture medium. When a microbial solution contains a microbial culture medium, the activity of the active microorganisms in the microbial solution and the slurry containing the microbial solution increases. As a result, a microbial carrier with higher activity of the active microorganisms can be produced.

[0044] Alternatively, the microorganism-containing solution may be prepared by mixing active microorganisms, a microbial culture medium, and sodium alginate. When the microorganism-containing solution contains sodium alginate along with the active microorganisms and the microbial culture medium, the active microorganisms are protected by the sodium alginate. This increases the activity of the active microorganisms contained in the slurry containing the microorganism-containing solution, making it possible to produce a microbial carrier with higher activity of the active microorganisms.

[0045] Examples of microbial culture media that can be used in the method for producing the microbial carrier of this embodiment include known microbial culture media such as YPD medium and LB medium, and it is preferable to use a liquid microbial culture medium corresponding to the type of active microorganism. For example, when yeast is used as the active microorganism, it is preferable to use YPD medium as the microbial culture medium. When Escherichia coli is used as the active microorganism, it is preferable to use LB medium as the microbial culture medium. When methanogenic bacteria are used as the active microorganism, it is preferable to use a known medium that is recommended as a culture medium for methanogenic bacteria.

[0046] Alternatively, the microorganism-containing solution may be prepared by mixing active microorganisms with phosphate-buffered saline (PBS). When the microorganism-containing solution contains phosphate-buffered saline, the osmotic pressure of the microorganism-containing solution and the slurry containing the microorganism-containing solution for active microorganisms becomes stable. This, for example, increases the activity of the active microorganisms. As a result, a microbial carrier with higher activity of the active microorganisms can be produced.

[0047] Alternatively, the microorganism-containing solution may be prepared by mixing active microorganisms, phosphate-buffered saline, and sodium alginate. When the microorganism-containing solution contains sodium alginate along with phosphate-buffered saline, the active microorganisms are protected by the sodium alginate, and the phosphate-buffered saline stabilizes the osmotic pressure of the microorganism-containing solution and the slurry containing the microorganism-containing solution for the active microorganisms. This results in higher activity of the active microorganisms. Consequently, it becomes easier to obtain a microbial carrier with higher activity of the active microorganisms.

[0048] The microorganism-containing solution in this embodiment may be any solution containing active microorganisms and water, and is not limited to the above-described microorganism-containing solution. For example, the microorganism-containing solution in this embodiment may consist only of active microorganisms and water, consist of active microorganisms, water and sodium alginate, consist of active microorganisms, a microbial culture medium and phosphate-buffered saline, or consist of active microorganisms, a microbial culture medium, phosphate-buffered saline and sodium alginate.

[0049] Furthermore, the microorganism-containing solution may optionally contain other components besides active microorganisms, water, phosphate-buffered saline, microbial culture medium, and sodium alginate. Examples of other components include one or more selected from collagen, chitosan, chondroitin, and polyvinyl alcohol. The inclusion of these other components in the microorganism-containing solution may increase the activity of the active microorganisms contained in the solution and / or make it easier to maintain their activity.

[0050] In the slurry preparation process, a slurry containing the above-mentioned cement components and the above-mentioned microbial-containing solution is prepared. The lower the cement component content in the slurry, the more porous the microbial carrier tends to be. Microbial carriers with many pores are those in which gaseous components (or liquid components) can easily reach the active microorganisms present inside the carrier when the carrier is placed in a gas (or liquid) environment for use or storage. Furthermore, the higher the cement component content in the slurry, the more porous the microbial carrier tends to be, resulting in a carrier with fewer pores, higher strength, and greater density.

[0051] In this embodiment, it is preferable to prepare a slurry containing cement components and a microorganism-containing solution in a mass ratio (cement components:microorganism-containing solution) of 1:2 to 2.5:1. When the ratio of cement components to microorganism-containing solution is within the above range, the slurry contains sufficient cement components and there is no shortage of water in the microorganism-containing solution, so the reaction between calcium phosphate in the cement components and water proceeds sufficiently. As a result, a slurry is formed that is easy to harden and can form a microbial carrier with sufficient strength.

[0052] Furthermore, if the ratio of cement components to the microorganism-containing solution is within the above range, it is possible to prevent the microbial carrier from becoming too dense due to an excessive amount of cement components in the slurry. As a result, a slurry is formed that can create a microbial carrier in which the activity of the active microorganisms is less likely to decrease. It is more preferable that the ratio of cement components to the microorganism-containing solution in the slurry be 1:1 to 2:1 by mass ratio (cement components:microorganism-containing solution). The ratio of cement components to the microorganism-containing solution can be appropriately determined depending on the composition of the cement components and the microorganism-containing solution, the intended use of the microbial carrier, etc.

[0053] In this embodiment, a microbial carrier bearing a predetermined amount of microorganisms can be produced by adjusting the content of active microorganisms in the microbial-containing solution contained in the slurry. The content of active microorganisms in the microbial-containing solution is 1.0 × 10⁻⁶. ５ cells / mL~1.0×10 ９ It is preferable that the concentration is cells / mL. The amount of active microorganisms contained in the microbial solution is 1.0 × 10⁻⁶. ５ When the cells / mL or higher, the effect of containing active microorganisms in the microbial carrier obtained by curing the slurry becomes significant. Since it is possible to form a microbial carrier in which the effect of containing active microorganisms is more pronounced, the amount of active microorganisms contained in the microbial solution is 1.0 × 10⁻⁶. ６ A concentration of cells / mL or higher is more preferable.

[0054] Furthermore, the amount of active microorganisms contained in the microbial solution is 1.0 × 10９ If the cell content is less than or equal to 1.0 × 10⁻¹⁰, it is possible to prevent the microbial carrier obtained by curing the slurry from becoming brittle due to an excessive amount of active microorganisms in the microbial solution. ８ It is more preferable that the concentration be less than or equal to cells / mL. In this embodiment, the amount of active microorganisms contained in the microorganism-containing solution is a value calculated based on the amount of active microorganisms used (mass in a dry state) when producing the microorganism-containing solution used as a slurry material.

[0055] The slurry may contain not only the cement components and the microorganism-containing solution described above, but also other materials as needed, within a range that allows the effects of the present invention to be obtained. If the slurry contains other materials other than the cement components and the microorganism-containing solution described above, the content of the other materials in the slurry is preferably less than 90% by mass, and more preferably 80% by mass or less.

[0056] Other materials include, for example, hardened cement containing calcium phosphate and free of active microorganisms, conventionally known carriers free of active microorganisms, and naturally occurring granular structures. The material, shape, and size of the other materials are not particularly limited. For example, the shape of the other materials may be any particle shape such as columnar or spherical, or fibrous such as thread-like or cloth-like. The other materials are preferably porous materials, because hardening the slurry makes it easier to obtain a microbial carrier with higher activity of active microorganisms.

[0057] In the slurry preparation step of this embodiment, the method for mixing the cement components, the microorganism-containing solution, and other materials that may be included as needed can be a conventionally known method and is not particularly limited. Furthermore, the order in which the cement components, the microorganism-containing solution, and other materials that may be included as needed are mixed is not particularly limited.

[0058] [Curing Process] Next, in the method for producing the microbial carrier of this embodiment, a curing process is performed to harden the slurry. The slurry hardens by forming hydroxyapatite through a reaction between the cement components in the slurry and the water contained in the microbial solution.

[0059] The slurry can be cured by pouring it into a mold having a shape corresponding to the shape of the microbial carrier and holding it for 0.5 to 24 hours at a temperature of 30°C to 37°C and a humidity of 0% to 100%. The temperature for curing the slurry is more preferably in the range of 30°C to 35°C, as this more effectively suppresses the decrease in the activity of the active microorganisms caused by the curing of the slurry. The curing conditions for the slurry can be appropriately changed depending on the composition of the slurry, the type of active microorganisms contained in the slurry, the shape of the microbial carrier, etc.

[0060] In the method for producing a microbial carrier of this embodiment, a hardened cement product that does not contain active microorganisms, and / or a conventionally known carrier that does not contain active microorganisms, may be placed in a portion of the space within the mold corresponding to the shape of the microbial carrier, either before or after pouring the slurry. This makes it possible to produce a microbial carrier in which the hardened slurry product and the hardened cement product that does not contain active microorganisms, and / or a conventionally known carrier that does not contain active microorganisms, are integrated. The shape of the microbial carrier can be any shape, such as spherical, columnar, or plate-shaped, and can be appropriately determined according to the application of the microbial carrier.

[0061] In this embodiment, it is preferable to determine the atmosphere in which the steps of preparing the microorganism-containing solution in the slurry preparation step and curing the slurry in the curing step are performed according to the type of active microorganism. For example, if the active microorganism is an aerobic microorganism, it is preferable to perform the steps of preparing the microorganism-containing solution and curing the slurry in an air atmosphere. When the above steps are performed in an air atmosphere, the microbial carrier can be easily and efficiently manufactured without using special equipment. If the active microorganism is an anaerobic microorganism, the steps of preparing the microorganism-containing solution and curing the slurry can be performed in a carbon dioxide atmosphere, an argon atmosphere, or a nitrogen atmosphere, and it is preferable to perform them in a nitrogen atmosphere.

[0062] The microbial carrier obtained by the manufacturing method of the present embodiment can be installed and used in a bioreactor that performs, for example, water treatment, air purification, soil purification, production of biofuels, and the like. Further, it can also be suitably used for applications such as microbial soil improvement materials for agriculture and microbial carriers sprayed on soil to detoxify gases discharged from the soil.

[0063] The manufacturing method of the microbial carrier of the present embodiment includes a slurry preparation step of preparing a slurry containing a cement component containing calcium phosphate, a microbial-containing solution containing active microorganisms and water, and a curing step of curing the slurry. Therefore, according to the manufacturing method of the microbial carrier of the present embodiment, active microorganisms are present at a substantially uniform density in the cured product of the slurry, and a microbial carrier in which microorganisms are carried at a predetermined amount corresponding to the amount of active microorganisms contained in the microbial-containing solution can be manufactured.

[0064] As described above, the preferred embodiments of the present invention have been described in detail. However, the present invention is not limited to specific embodiments, and various modifications and changes are possible within the scope of the gist of the present invention described in the claims.

[0065] [Production of Microbial-Containing Solution] "Example 1" In an air atmosphere, dry yeast (manufactured by Nisshin Flour Milling Co., Ltd., Werner Co., Ltd.), which is an active microorganism, was put into 1 mL of a phosphate-buffered saline (PBS) solution containing sodium alginate at a concentration of 1.0% by mass and stirred. A microbial-containing solution of Example 1 having an active microorganism content of 1.7×10 ５ cells / mL was obtained.

[0066] "Example 2" In an air atmosphere, dry yeast (manufactured by Nisshin Flour Milling Co., Ltd., Werner Co., Ltd.), which is an active microorganism, was put into 1 mL of a phosphate-buffered saline (PBS) solution and stirred. A microbial-containing solution of Example 2 having an active microorganism content of 1.7×10 ５ cells / mL was obtained.

[0067] [Production of Microbial Carrier] "Example 1" A cement component was prepared containing 75% by mass of α-tricalcium phosphate, 18% by mass of tetracalcium phosphate, 5% by mass of calcium hydrogen phosphate dihydrate, and 2% by mass of hydroxyapatite.

[0068] Then, in an air atmosphere, the cement component and the microorganism-containing solution of Example 1 were mixed in a mass ratio (cement component:microorganism-containing solution) of 1:1, resulting in an active microorganism content of 1.7 × 10⁻⁶. ５ A slurry with cells / mL was prepared. Then, the slurry was placed in a mold made of polytetrafluoroethylene, having a diameter of 8 mm and a depth of 4 mm, with a roughly cylindrical inner surface, and cured by holding it in an air atmosphere at a temperature of 37°C and a humidity of 100% for 1 hour. This yielded the microbial carrier of Example 1.

[0069] "Example 2" Instead of the microorganism-containing solution of Example 1, the microorganism-containing solution of Example 2 was used, and the active microorganism content was 1.7 × 10 ５ The microbial carrier of Example 2 was obtained in the same manner as in Example 1, except that a slurry with cells / mL was prepared.

[0070] "Comparative Example 1" The carrier for Comparative Example 1 was obtained in the same manner as the microbial carrier for Example 1, except that the slurry was prepared using a phosphate-buffered saline (PBS) solution containing 1.0% by mass of sodium alginate instead of the microorganism-containing solution for Example 1.

[0071] "Comparative Example 2" The carrier for Comparative Example 2 was obtained in the same manner as the microbial carrier for Example 1, except that the slurry was prepared using a phosphate-buffered saline (PBS) solution instead of the microorganism-containing solution for Example 1.

[0072] Three microbial carriers each of the microbial carriers obtained in Example 1 and Example 2, and the carriers of Comparative Example 1 and Comparative Example 2 were prepared. These were placed in 1 mL of phosphate-buffered saline (PBS) in an air atmosphere, and 50 μL of a microbial assay kit (manufactured by Dojin Chemical Research Institute Co., Ltd.) was added to each. The samples were then incubated in an incubator (product name: IC402, manufactured by Yamato Scientific Co., Ltd.) at a temperature of 37°C and a humidity of 100% for 24 hours to allow the color reaction to occur. The results are shown in Figure 2.

[0073] If the microorganisms in the microbial carrier are active, adding the above-mentioned microbial assay kit and holding it under the above conditions for 24 hours will result in an orange color. Furthermore, the higher the energy metabolic activity of the microorganisms contained in the microbial carrier held under the above conditions for 24 hours, the darker the orange color will be.

[0074] Figure 2 shows photographs of the microbial carrier and the solution in which the microbial carrier was immersed after a color reaction was observed by adding the microbial assay kit. Figure 2(a) shows a photograph of the microbial carrier and the solution in which the microbial carrier was immersed in Example 1, and Figure 2(b) shows a photograph of the microbial carrier and the solution in which the microbial carrier was immersed in Example 2.

[0075] The microbial carriers of Example 1 and Example 2, and the carriers of Comparative Example 1 and Comparative Example 2 were immersed in the solution, and a microbial assay kit was added to induce a color reaction. The resulting solutions were then analyzed using a spectrophotometer (product name: V-730BIO, manufactured by JASCO Corporation) under fixed wavelength measurement conditions, and the peak intensity at a wavelength of 450 nm, which indicates the intensity of orange, was measured. The results are shown in Table 1.

[0076]

[0077] As shown in Figures 1A and 1B, the microbial carriers and the solutions in which the microbial carriers were immersed in the microbial carriers of Example 1 and Example 2 turned orange when the above-mentioned microbial assay kit was added and the solutions were held under the above conditions for 24 hours. In contrast, as shown in Figures 1C and 1D, the carriers and the solutions in which the carriers were immersed in the microbial carriers of Comparative Example 1 and Comparative Example 2 did not turn orange.

[0078] Furthermore, as shown in Table 1, the solutions in which the microbial carriers of Example 1 and Example 2 were immersed showed a stronger peak intensity at a wavelength of 450 nm, which indicates a darker orange color, compared to the solutions in which the carriers of Comparative Examples 1 and 2, which did not contain microorganisms, were immersed. From these findings, it was confirmed that the microorganisms in the microbial carriers of Example 1 and Example 2 were all active.

[0079] [Production of Microbial Solution] "Example 3" In an air atmosphere, budding yeast (Saccharomyces cerevisiae strain BY4741), an active microorganism, was added to 1 mL of YPD medium (Yeast Extract-Peptone-D(+)-Glucose medium) containing 1.0% by mass of sodium alginate and stirred until the active microorganism content reached 2.0 × 10⁶ ８ A microorganism-containing solution of Example 3 with cells / mL was obtained.

[0080] "Example 4" In an air atmosphere, 1 mL of YPD medium was mixed with the same budding yeast as in Example 3, which is an active microorganism, and the mixture was stirred until the active microorganism content reached 2.0 × 10⁶. ８ A microorganism-containing solution of Example 4 with cells / mL was obtained.

[0081] "Example 5" In an air atmosphere, budding yeast, the same as in Example 3, which is an active microorganism, was added to 1 mL of phosphate-buffered saline (PBS) solution containing 1.0% by mass of sodium alginate and stirred, and the active microorganism content was 2.0 × 10 ８ A microbial solution containing cells / mL was obtained for Example 5.

[0082] "Example 6" In an air atmosphere, budding yeast, which is an active microorganism, was added to 1 mL of phosphate-buffered saline (PBS) solution and stirred, and the active microorganism content was 2.0 × 10⁶. ８ A microbial solution containing cells / mL was obtained for Example 6.

[0083] "Example 7" In an air atmosphere, 1 mL of an aqueous solution containing 1.0% by mass of sodium alginate was mixed with the same budding yeast as in Example 3, which is an active microorganism, and stirred until the active microorganism content reached 2.0 × 10⁶ ８A microbial solution containing cells / mL was obtained for Example 7.

[0084] "Example 8" In an air atmosphere, 1 mL of distilled water was mixed with the same budding yeast as in Example 3, which is an active microorganism, and stirred until the active microorganism content reached 2.0 × 10⁶. ８ A microbial solution containing cells / mL was obtained for Example 8.

[0085] "Example 9" In an air atmosphere, 1 mL of LB medium containing 1.0% by mass of sodium alginate was mixed with the active microorganism Escherichia coli strain K-12, and the mixture was stirred until the active microorganism content reached 1.0 × 10⁶. ９ A microbial solution containing cells / mL was obtained according to Example 9.

[0086] For the LB medium (Luria-Bertani medium), we used commercially available LB medium (product name: Daigo, manufactured by Shioya MS Co., Ltd.) prepared by dissolving it in water. The prepared LB medium contained 10 g / L of peptone, 5 g / L of Yeast Extract, and 10 g / L of sodium chloride.

[0087] "Example 10" In an air atmosphere, the same LB medium as in Example 9 was mixed with the same active microorganism, E. coli, as in Example 9, and stirred until the active microorganism content reached 1.0 × 10⁶. ９ A microorganism-containing solution of Example 10 with cells / mL was obtained.

[0088] [Preparation of Microorganism-Containing Solution] "Example 11" In an air atmosphere, 1 mL of phosphate-buffered saline (PBS) solution containing 1.0% by mass of sodium alginate is mixed with the same active microorganism, Escherichia coli, as in Example 9, and stirred until the active microorganism content reaches 1.0 × 10⁶ ９ A microbial solution containing cells / mL was obtained for Example 11.

[0089] "Example 12" In an air atmosphere, 1 mL of phosphate-buffered saline (PBS) solution was mixed with the same active microorganism, E. coli as in Example 9, and the active microorganism content was 1.0 × 10⁶. ９ A microbial solution containing cells / mL was obtained for Example 12.

[0090] "Example 13" In an air atmosphere, 1 mL of an aqueous solution containing 1.0% by mass sodium alginate was mixed with the same active microorganism, E. coli as in Example 9, and stirred until the active microorganism content reached 1.0 × 10⁻⁶. ９ A microbial solution containing cells / mL was obtained for Example 13.

[0091] "Example 14" In an air atmosphere, 1 mL of distilled water was mixed with the same active microorganism, E. coli as in Example 9, and stirred until the active microorganism content reached 1.0 × 10⁶. ９ A microbial solution containing cells / mL was obtained for Example 14.

[0092] [Production of Microbial Carrier] "Example 3" A cement was prepared containing 75% by mass of α-tricalcium phosphate, 18% by mass of tetracalcium phosphate, 5% by mass of calcium hydrogen phosphate dihydrate, and 2% by mass of hydroxyapatite as the cement component.

[0093] Then, in an air atmosphere, the above cement component and the microorganism-containing solution of Example 3 were mixed in a mass ratio (cement component:microorganism-containing solution) of 1:1, and the active microorganism content was 1.0 × 10 ８ A slurry with cells / mL was prepared. Then, the slurry was placed in a mold made of polytetrafluoroethylene, having a diameter of 8 mm and a depth of 4 mm, with a roughly cylindrical inner surface, and cured by holding it in an air atmosphere at a temperature of 30°C and a humidity of 100% for 24 hours. This yielded the microbial carrier of Example 3.

[0094] "Example 4" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 4 was used, and the active microorganism content was 1.0 × 10 ８ The microbial carrier of Example 4 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0095] "Example 5" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 5 was used, and the active microorganism content was 1.0 × 10 ８ The microbial carrier of Example 5 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0096] "Example 6" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 6 was used, and the active microorganism content was 1.0 × 10 ８ The microbial carrier of Example 6 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0097] "Example 7" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 7 was used, and the active microorganism content was 1.0 × 10 ８ The microbial carrier of Example 7 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0098] "Example 8" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 8 was used, and the active microorganism content was 1.0 × 10 ８ The microbial carrier of Example 8 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0099] "Example 9" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 9 was used, and the active microorganism content was 5.0 × 10 ８ The microbial carrier of Example 9 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0100] "Example 10" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 10 was used, and the active microorganism content was 5.0 × 10 ８ The microbial carrier of Example 10 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0101] "Example 11" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 11 was used, and the active microorganism content was 5.0 × 10 ８ The microbial carrier of Example 11 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0102] "Example 12" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 12 was used, and the active microorganism content was 5.0 × 10 ８The microbial carrier of Example 12 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0103] "Example 13" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 13 was used, and the active microorganism content was 5.0 × 10 ８ The microbial carrier of Example 13 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0104] "Example 14" Instead of the microorganism-containing solution of Example 3, the microorganism-containing solution of Example 14 was used, and the active microorganism content was 5.0 × 10 ８ The microbial carrier of Example 14 was obtained in the same manner as in Example 3, except that a slurry with cells / mL was prepared.

[0105] Three microbial carriers were prepared for each of Examples 3 to 14, and each was placed in 1 mL of phosphate-buffered saline (PBS) in an air atmosphere. 50 μL of a microbial assay kit (manufactured by Dojin Chemical Research Institute Co., Ltd.) was added to each carrier, and the carriers were kept in an air atmosphere at 30°C and 100% humidity for 2 hours using an incubator (product name: IC-150MA, manufactured by AS ONE Corporation) to allow the color reaction to occur.

[0106] The microbial carriers of Examples 3 to 14 were immersed in the solution, and a microbial assay kit was added to induce a color reaction. The resulting solutions were then analyzed using a spectrophotometer (product name: V-730BIO, manufactured by JASCO Corporation) under fixed wavelength measurement conditions, and the peak intensity at a wavelength of 450 nm, which indicates the intensity of the orange color, was measured. The results are shown in Tables 2 and 3. The amount of active microorganisms per microbial carrier of Examples 3 to 14 is also shown in Tables 2 and 3.

[0107]

[0108]

[0109] As shown in Tables 2 and 3, the solutions obtained by immersing the microbial carriers of Examples 3 to 14 in the microbial assay kit for 2 hours showed a peak at a wavelength of 450 nm, indicating a deep orange color. This confirmed that the microorganisms in the microbial carriers of Examples 3 to 14 were all active.

[0110] Furthermore, as shown in Table 2, the solutions in which the microbial carriers of Examples 3 and 4 were immersed showed a stronger peak intensity at a wavelength of 450 nm, which indicates a darker orange color, compared to the solutions in which the microbial carriers of Examples 5 to 8 were immersed. From this, it was confirmed that a microbial carrier with high activity of active microorganisms can be obtained by using a slurry containing a microbial solution in which yeast is suspended in YPD medium.

[0111] In particular, Example 3 showed a particularly strong peak intensity at a wavelength of 450 nm. This indicates that using a slurry prepared with a microorganism-containing solution containing YPD medium and sodium alginate yields a microbial carrier with even higher activity of the active microorganisms.

[0112] Furthermore, as shown in Table 3, the solutions in which the microbial carriers of Examples 9 and 10 were immersed showed a stronger peak intensity at a wavelength of 450 nm, indicating a darker orange color, compared to the solutions in which the microbial carriers of Examples 11 to 14 were immersed. This confirms that a microbial carrier with high activity of active microorganisms can be obtained by using a slurry containing a microbial solution in which E. coli is suspended in LB medium. In particular, Example 9 showed a particularly strong peak intensity at a wavelength of 450 nm. This indicates that a microbial carrier with even higher activity of active microorganisms can be obtained by using a slurry prepared using a microbial solution containing LB medium and sodium alginate.

[0113] A microbial carrier can be manufactured that supports a predetermined amount of microorganisms corresponding to the amount of active microorganisms contained in a microbial solution.

Claims

1. A method for producing a microbial carrier, comprising: a slurry preparation step of preparing a slurry containing a cement component containing calcium phosphate and a microorganism-containing solution containing active microorganisms and water; and a curing step of curing the slurry.

2. The method for producing a microbial carrier according to claim 1, wherein the active microorganism is one or more selected from fungi, bacteria, and archaea.

3. The amount of active microorganisms contained in the microorganism-containing solution is 1.0 × 10 ５ cells / mL~1.0×10 ９ A method for producing a microbial carrier according to claim 1, wherein the concentration is cells / mL.

4. The method for producing a microbial carrier according to claim 1, wherein in the slurry preparation step, the active microorganism and the microbial culture medium are mixed to prepare the microorganism-containing solution.

5. The method for producing a microbial carrier according to claim 1, wherein in the slurry preparation step, the microbial-containing solution is prepared by mixing the active microorganism, the microbial culture medium, and sodium alginate.

6. The method for producing a microbial carrier according to claim 4 or 5, wherein the active microorganism is yeast and the microbial culture medium is YPD medium.

7. The method for producing a microbial carrier according to claim 4 or 5, wherein the active microorganism is Escherichia coli and the microbial culture medium is LB medium.

8. The method for producing a microbial carrier according to claim 1, wherein in the slurry preparation step, a slurry is prepared containing the cement component and the microorganism-containing solution in a mass ratio of 1:2 to 2.5:

1.

9. The method for producing a microbial carrier according to claim 1, wherein the cement component comprises (a) 0% to 100% by mass of α-tricalcium phosphate, (b) 0% to 80% by mass of tetracalcium phosphate, (c) 0% to 40% by mass of anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate, and (d) 0% to 10% by mass of hydroxyapatite, and (a) contains 50% or more by mass of α-tricalcium phosphate, or (b) contains 50% or more by mass of the total of tetracalcium phosphate and (c) anhydrous calcium hydrogen phosphate or calcium hydrogen phosphate dihydrate.

10. The method for producing a microbial carrier according to claim 1, wherein the steps of preparing the microbial solution and curing the slurry are performed in an air atmosphere.

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