Apparatus for material production and method for material production

Abstract

To improve the production efficiency of a target product in microbe-based substance production techniques.SOLUTION: A substance production device 1 comprises: a reaction tank 2 that stores an aqueous layer 12 comprising a hydrophilic medium, an organic layer 14 comprising a hydrophobic organic solvent, and a microbial layer 16 comprising microbes growing at an interface between the aqueous layer 12 and organic layer 14 and that facilitates the leaching of substances produced by the microbes into an organic solvent; and an adsorption tank 4 that allows the organic solvent from the reaction tank 2 to flow thereinto and comprises an adsorbent 18 for adsorbing the substances within the organic solvent.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a substance production apparatus and a substance production method using microorganisms. Background Art

[0002] Patent Document 1 discloses a bioreactor that uses microorganisms growing at the interface between a hydrophilic carrier and an organic liquid layer to convert a substrate into a useful substance, and accumulates the useful substance in the organic liquid layer.

[0003] Also known is a biofermenter that utilizes the metabolic pathways of the aforementioned microorganisms to accumulate metabolites produced by the microorganisms, for example, primary metabolites such as fatty acids, and secondary metabolites such as antibiotics and anticancer active substances, as useful substances in an organic liquid layer. Prior Art Literature Patent Literature

[0004] Patent Literature 1 Japanese Unexamined Patent Publication No. Hei 5-91878 Summary of the Invention Problem to be Solved by the Invention

[0005] The present inventors have conducted intensive studies on the aforementioned substance production technology using microorganisms such as bioreactors and biofermenters, and have found a technology for improving the production efficiency of a target product.

[0006] The present invention has been made in view of such circumstances, and one of the objects thereof is to provide a technique for improving the production efficiency of a target product in a substance production technique using microorganisms. Means for Solving the Problem

[0007] One aspect of the present invention is a substance production apparatus. This apparatus comprises a reaction vessel containing an aqueous layer containing a hydrophilic medium, an organic layer containing a hydrophobic organic solvent, and a microbial layer containing microorganisms that grow at the interface between the aqueous and organic layers, and for eluting substances produced by the microorganisms into the organic solvent; and an adsorption vessel containing an adsorbent that receives the organic solvent from the reaction vessel and adsorbs substances in the organic solvent.

[0008] Another aspect of the present invention is a method for producing a substance. This method involves producing a substance using microorganisms that grow at the interface between an aqueous layer containing a hydrophilic medium and an organic layer containing a hydrophobic organic solvent, eluting the substance into an organic solvent, transferring the organic solvent to an adsorption tank containing an adsorbent, and adsorbing the substance in the organic solvent onto the adsorbent.

[0009] Furthermore, any combination of the above components, or any substitution of the components or expressions of the present invention between methods, apparatus, systems, etc., are also valid embodiments of the present invention. [Effects of the Invention]

[0010] According to the present invention, the production efficiency of target products can be improved in substance production technology using microorganisms. [Brief explanation of the drawing]

[0011] [Figure 1] This is a schematic diagram of a substance production apparatus according to an embodiment. [Figure 2] This figure shows the recovery results of the target product. [Modes for carrying out the invention]

[0012] The present invention will be described below with reference to the drawings, based on preferred embodiments. The embodiments are illustrative and not limiting, and not all features or combinations thereof described in the embodiments are necessarily essential to the invention. The same or equivalent components, members, and processes shown in each drawing are denoted by the same reference numerals, and redundant explanations are omitted as appropriate. Furthermore, the scale and shape of each part shown in each drawing are set for convenience to facilitate explanation and are not to be interpreted restrictively unless otherwise specified. In addition, when terms such as "first," "second," etc. are used in this specification or claims, these terms do not indicate any order or importance, but are used to distinguish one configuration from another. Furthermore, some components that are not important for explaining the embodiments are omitted in each drawing.

[0013] Figure 1 is a schematic diagram of a substance production apparatus 1 according to an embodiment. The substance production apparatus 1 comprises a reaction vessel 2, an adsorption vessel 4, a circulation channel 6, a circulation pump 8, and a control unit 10. The substance production apparatus 1 shown in Figure 1 comprises six reaction vessels 2 and one adsorption vessel 4, but is not limited to this configuration. There may be one reaction vessel 2 or more than six. Alternatively, multiple reaction vessels 2 may be stacked to form a reaction vessel tower, and multiple reaction vessel towers may be installed in parallel. This makes it possible to scale up the substance production apparatus 1. There may also be multiple adsorption vessels 4.

[0014] Each reactor 2 functions as a bioreactor or biofermenter. Each reactor 2 contains an aqueous layer 12, an organic layer 14, and a microbial layer 16. The aqueous layer 12 contains a hydrophilic medium. There are no particular restrictions on the hydrophilic medium that can be used, as long as it is suitable for use in known interfacial bioreactors or interfacial biofermenters; water is an example. A nutrient source for microorganisms is also added to the aqueous layer 12. This nutrient source can be appropriately selected depending on the microorganisms used.

[0015] The microbial layer 16 contains microorganisms that grow at the interface between the aqueous layer 12 and the organic layer 14. There are no particular restrictions on the microorganisms that can be used, as long as they are usable in known interface bioreactors and interface biofermenters. Examples include filamentous fungi such as Aspergillus, Penicillium, Trichoderma, and Talaromyces; yeasts such as Saccharomyces, Rhodotorula, and Pichia; actinomycetes such as Streptomyces and Rhodococcus; and bacteria such as Bacillus, Pseudomonas, and Lactobacillus.

[0016] The organic layer 14 contains a hydrophobic organic solvent. The organic solvent is less polar than the substances produced by the microorganisms contained in the microbial layer 16 (hereinafter also referred to as products). Examples of such organic solvents include n-alkanes such as n-decane, n-dodecane, and n-tridecane; low-viscosity silicone oils such as KF-96L-1CS (manufactured by Shin-Etsu Chemical Co., Ltd.) and KF-96L-1.5CS (manufactured by Shin-Etsu Chemical Co., Ltd.); medium-chain aliphatic ethers such as di-n-hexyl ether and diisoamyl ether; and low-toxicity terpenes such as limonene and α-pinene, which are non-polar to slightly polar organic solvents. Note that limonene and α-pinene can also be used as substrates for microbial transformation. When reaction vessel 2 is used as a bioreactor, the substrates for microbial transformation will be dissolved in the organic layer 14 at high concentrations. In this case, based on the toxicity mitigation phenomenon at the interface, it is possible to set extremely high substrate concentrations while avoiding the toxicity of the substrate being introduced (Oda S and Ohta H, Biosci. Biotech. Biochem., 56, 1515-1517 (1992)) (Oda S and Ohta H, Biosci. Biotech. Biochem., 56, 2041-2045 (1992)).

[0017] Reactor 2 elutes substances produced by microorganisms in the microbial layer 16 into the organic solvent in the organic layer 14. When reaction vessel 2 functions as a bioreactor, microorganisms convert substrates into useful substances and release them into the organic solvent. When reaction vessel 2 functions as a biofermenter, microorganisms produce metabolites and release them into the organic solvent. As a result, microbial conversion products and microbial metabolites, which are the target products, accumulate in the organic solvent. Preferably, the products of the microorganisms are substances that are more soluble in the organic solvent of the organic layer 14 than in the hydrophilic environment within the microorganisms or the hydrophilic medium of the aqueous layer 12.

[0018] Furthermore, the products of this embodiment are more polar than the organic solvents in the organic layer 14. The products are also, for example, slightly polar to moderately polar. "Slightly polar to moderately polar" means that the LogP value, an indicator of hydrophobicity, is between 1.0 and 7.0. The LogP value is the common logarithm of the partition coefficient between water and 1-octanol. When the reactor 2 functions as a bioreactor, examples of target products include saturated lactones such as δ-lactones and γ-lactones; terpenes such as verbenone, α-pinene oxide, limonene oxide, and β-caryophyllene oxide; hydroxysteroids such as 11β-hydroxyprogesterone; and androstenediones such as 1-androsten-3,17-dione. When the reactor 2 functions as a biofermenter, examples of target products include unsaturated lactones such as 6-pentyl-α-pyrone (6PP) and massoialactone; and biologically active secondary metabolites such as sclerothioline and monascus pigment. Examples of monascus pigments include monacin and rubropunctatin.

[0019] The above-mentioned saturated lactones, terpenes, and unsaturated lactones are usable as perfume raw materials. The above-mentioned hydroxysteroids and androstenediones are usable as pharmaceutical raw materials. The above-mentioned biologically active secondary metabolites are usable as pharmaceutically useful biologically active substances. For example, the above-mentioned Monascus pigments are usable as anticancer active substances, therapeutic substances for hyperlipidemia, antibacterial active substances, antiviral active substances, anti-obesity active substances, anti-inflammatory active substances, anti-Alzheimer's active substances, therapeutic substances for hypertension, immunosuppressive substances, and the like.

[0020] Note that the reaction tank 2 may be of a solid / liquid interface type, or may be of a liquid / liquid interface type. The solid / liquid interface type reaction tank 2 comprises an aqueous layer 12 formed of a hydrophilic gel such as agar, a microbial layer 16 formed of microorganisms grown on the hydrophilic gel, and an organic layer 14 formed of a hydrophobic organic solvent that covers the microbial layer 16. Further, the liquid / liquid interface type reaction tank 2 comprises an aqueous layer 12 formed of a liquid medium, a microbial layer 16 formed of hollow microparticles that float on the surface of the liquid medium and carry microorganisms, and an organic layer 14 formed of a hydrophobic organic solvent that covers the microbial layer 16.

[0021] The adsorption tank 4 is connected to the reaction tank 2 via a circulation flow path 6 that connects the reaction tank 2 and the adsorption tank 4. An organic solvent flows into the adsorption tank 4 from the reaction tank 2. The circulation flow path 6 includes a forward path 6a and a return path 6b. One end of each of the forward path 6a and the return path 6b is connected to the reaction tank 2, and the other end of each is connected to the adsorption tank 4. The circulation flow path 6 can be formed of a known flow path forming structure such as a tube or a pipe. A circulation pump 8 is connected to the circulation flow path 6. As an example, the circulation pump 8 is connected midway along the return path 6b. A known pump such as a peristaltic pump or a diaphragm pump can be used as the circulation pump 8. Driving of the circulation pump 8 generates a flow of the organic solvent from the reaction tank 2 to the adsorption tank 4 via the forward path 6a, and a flow of the organic solvent from the adsorption tank 4 back to the reaction tank 2 via the return path 6b.

[0022] In the present embodiment, as described above, six reaction tanks 2 are stacked to form a reaction tank column. The outward path 6a connects two vertically adjacent reaction tanks 2 to each other. That is, the outward path 6a connects the uppermost reaction tank 2 and the second reaction tank 2 from the top. It also connects the second reaction tank 2 from the top and the third reaction tank 2 from the top. It further connects the third reaction tank 2 from the top and the fourth reaction tank 2 from the top. It also connects the fourth reaction tank 2 from the top and the fifth reaction tank 2 from the top. In addition, it connects the fifth reaction tank 2 from the top and the lowermost reaction tank 2. Moreover, the outward path 6a connects the lowermost reaction tank 2 and the adsorption tank 4. The outward path 6a connecting each pair of reaction tanks 2 constitutes a so-called overflow line. The return path 6b connects the adsorption tank 4 and the uppermost reaction tank 2. This forms an organic solvent circulation line in which each reaction tank 2 and the adsorption tank 4 are connected in series via the circulation flow path 6.

[0023] The organic solvent in the uppermost reaction tank 2 sequentially moves to the lower reaction tanks 2 by natural flow down through the outward path 6a (overflow line). In this process, the product in each reaction tank 2 moves to the lower reaction tank 2 together with the organic solvent. The organic solvent that has reached the lowermost reaction tank 2 flows into the adsorption tank 4 by natural flow down through the outward path 6a. This causes the organic solvent in each reaction tank 2 to move to the adsorption tank 4.

[0024] The adsorption tank 4 contains an adsorbent 18 that adsorbs the product in the organic solvent. The adsorbent 18 of the present embodiment selectively adsorbs the product based on the difference in polarity between the organic solvent and the product. Examples of usable adsorbents 18 include silica gel having a particle diameter of 200 µm to 2000 µm, preferably 1000 µm to 2000 µm; synthetic adsorbents such as Amberlite (registered trademark) XAD-7 (manufactured by Organo Corporation) and Amberlyst (registered trademark) 200CT Na (manufactured by Organo Corporation). In the present embodiment, the polarity of the organic solvent is relatively low, the polarity of the product is relatively high, and the adsorbent 18 has a polarity as high as that of the product. Accordingly, the product with relatively high polarity is adsorbed to the adsorbent 18, while the organic solvent with relatively low polarity flows out of the adsorption tank 4 without being adsorbed to the adsorbent 18.

[0025] Organic solvent flows continuously from reaction vessel 2 to adsorption vessel 4, and the adsorbent 18 sequentially adsorbs the products in the organic solvent. This allows for the recovery of products from the organic solvent. As the adsorbent 18 recovers the products from the organic solvent, the concentration of products in the organic solvent decreases. The organic solvent with reduced product concentration is returned from adsorption vessel 4 to the uppermost reaction vessel 2 via the return path 6b by the circulation pump 8. The organic solvent returned to the uppermost reaction vessel 2 then circulates through each of the reaction vessels 2 again.

[0026] In this embodiment, the flow of organic solvent from the reaction vessel 2 to the adsorption vessel 4 via the forward passage 6a is generated by gravity. However, this gravity flow is generated by the circulation pump 8 pumping organic solvent from the adsorption vessel 4 to the uppermost reaction vessel 2. Therefore, the flow of organic solvent from the reaction vessel 2 to the adsorption vessel 4 via the forward passage 6a can also be interpreted as being generated by the operation of the circulation pump 8.

[0027] The inflow rate of the organic solvent from the adsorption tank 4 to the uppermost reaction tank 2, that is, the circulation rate of the organic solvent in the material production apparatus 1, is not particularly limited as long as it is below the limit of the outflow rate of the organic solvent in the overflow line, and can be set appropriately according to the production rate of the product by microorganisms, the operational stability of the material production apparatus 1, etc.

[0028] By introducing an organic solvent into the adsorption tank 4 for a predetermined time and then replacing it with a new adsorption tank 4, the target product can be continuously recovered over a long period of time. The target product adsorbed by the adsorbent 18 can be easily recovered by known methods, such as circulating a polar solvent such as ethyl acetate through the adsorption tank 4. This recovery process corresponds to the purification process of the target product. Therefore, according to the substance production apparatus 1 of this embodiment, the production and purification of the target product can be easily carried out.

[0029] The control unit 10 controls the drive of the circulation pump 8. The control unit 10 is implemented as a hardware component consisting of elements and circuits such as the CPU and memory of a computer, and as a software component consisting of a computer program, etc., but in Figure 1 it is depicted as a functional block realized through the cooperation of these components. It will be obvious to those skilled in the art that this functional block can be realized in various forms through combinations of hardware and software.

[0030] For example, the substance production apparatus 1 is equipped with a concentration sensor (not shown) for measuring the concentration of the product in the organic solvent. Known concentration measuring methods such as a high-performance liquid chromatograph, a gas chromatograph, or a spectrophotometer (e.g., one capable of measuring absorbance in the ultraviolet to visible region) can be used as the concentration sensor. The measurement results from the concentration sensor are sent to the control unit 10. Based on the measurement results from the concentration sensor, the control unit 10 drives the circulation pump 8 to circulate the organic solvent. For example, the control unit 10 has pre-stored information regarding the concentration threshold of the product in the organic solvent. The concentration threshold can be set appropriately based on experiments or simulations conducted by the designer.

[0031] When the control unit 10 detects from the measurement results of the concentration sensor that the concentration of the product in the organic solvent has reached a concentration threshold, it starts driving the circulation pump 8. This prevents the organic solvent from being wasted when the concentration of the product in the organic solvent is low, such as during the initial operation of the material production device 1. Therefore, the power consumption of the material production device 1 can be reduced and the production efficiency of the product can be improved.

[0032] Furthermore, the control unit 10 can also control the circulation pump 8 based on a pre-set fixed operation program, rather than using feedback control based on the measurement results of the concentration sensor. Alternatively, the circulation pump 8 may be controlled manually by an operator. In this case, the control unit 10 can be omitted.

[0033] As described above, in the substance production apparatus 1 of this embodiment, the adsorption tank 4, which is attached to the reaction tank 2, selectively adsorbs and recovers the products of microorganisms. This suppresses the excessive accumulation of products in the reaction tank 2. Therefore, it is possible to suppress product inhibition, in which the growth and metabolic activity of microorganisms are inhibited by the products. Furthermore, if the products are toxic, it is possible to suppress the death of microorganisms due to the products. It is also possible to suppress the decomposition of products by microorganisms. Therefore, the target product can be produced efficiently. In addition, the target product can be recovered at low cost and with high purity.

[0034] Furthermore, in the substance production apparatus 1 of this embodiment, products that are poorly soluble or insoluble in water can be eluted into an organic solvent. Therefore, accumulation of products in microorganisms can be avoided, and feedback inhibition that would otherwise occur to avoid excessive accumulation can be suppressed. In addition, the adsorption tank 4 can reduce the concentration of products in the organic solvent. This promotes the elution of products from microorganisms into the organic solvent. Therefore, the production efficiency of the target product can be increased.

[0035] Furthermore, in general microbial culture, it is known that when a large amount of easily metabolizable carbon source, such as glucose, is present in the culture medium, the production of the target fermentation product is suppressed at the genetic level, a phenomenon known as catabolite suppression. To avoid catabolite suppression, the concentration of fermentation raw materials must usually be reduced. In contrast, the substance production apparatus 1 of this embodiment employs an interfacial culture method that produces useful substances using microorganisms that grow at the interface between the aqueous layer 12 and the organic layer 14. According to the interfacial culture method, the occurrence of catabolite suppression can be effectively avoided (Oda S., et al., J. Biosci. Bioeng., 113, 742-745 (2012)).

[0036] The embodiments of the present invention have been described in detail above. The embodiments described above are merely examples of how to implement the present invention. The content of the embodiments does not limit the technical scope of the present invention, and many design changes, such as changes, additions, and deletions of components, are possible as long as they do not depart from the spirit of the invention as defined in the claims. The new embodiments to which design changes have been made combine the effects of the respective embodiments and modifications. In the embodiments described above, the content in which such design changes are possible is emphasized with notations such as "in this embodiment" or "in this embodiment," but design changes are also permitted even if there are no such notations. Any combination of the above components is also valid as an embodiment of the present invention.

[0037] As a variation of the above-described embodiment, a dehydration tank may be provided downstream of the reaction tank 2 and upstream of the adsorption tank 4. The dehydration tank is filled with anhydrous inorganic salts such as calcium chloride, sodium sulfate, and magnesium sulfate as a desiccant. When the organic solvent is a slightly polar solvent such as isoamyl ether, trace amounts of water from the culture medium and water droplets adhering to the inner walls of each reaction tank 2 may be mixed into the organic solvent. When the organic solvent contaminated with water flows into the adsorption tank 4, the water is preferentially adsorbed by the adsorbent 18 rather than the product. As a result, the recovery of the product from the organic solvent by the adsorbent 18, and the extraction and recovery of the product from the adsorbent 18, may be hindered. In contrast, by providing a dehydration tank upstream of the adsorption tank 4, the above-mentioned problems caused by water can be suppressed, thereby increasing the production efficiency of the target product.

[0038] The embodiments may be specified by the items described below. (1st item) A reaction vessel (2) containing an aqueous layer (12) containing a hydrophilic medium, an organic layer (14) containing a hydrophobic organic solvent, and a microbial layer (16) containing microorganisms that grow at the interface between the aqueous layer (12) and the organic layer (14), and for eluting substances produced by the microorganisms into the organic solvent, The system includes an adsorption tank (4) into which an organic solvent flows from a reaction tank (2) and which contains an adsorbent (18) that adsorbs substances in the organic solvent, Material production apparatus (1). (Second item) Organic solvents are less polar than substances. The adsorbent (18) selectively adsorbs substances based on differences in polarity. The material production apparatus (1) described in item 1. (3rd item) A circulation channel (6) including a forward path (6a) and a return path (6b) connecting the reaction vessel (2) and the adsorption vessel (4), The system includes a circulation pump (8) connected to a circulation channel (6) that generates a flow of organic solvent from the reaction vessel (2) to the adsorption vessel (4) via a forward path (6a), and a flow of organic solvent from the adsorption vessel (4) to the reaction vessel (2) via a return path (6b), A substance production apparatus (1) as described in item 1 or item 2. (Item 4) The method involves producing a substance using microorganisms that grow at the interface between an aqueous layer (12) containing a hydrophilic medium and an organic layer (14) containing a hydrophobic organic solvent, eluting the substance into an organic solvent, transferring the organic solvent to an adsorption tank (4) containing an adsorbent (18), and adsorbing the substance in the organic solvent onto the adsorbent (18). Methods of producing substances. [Examples]

[0039] The following describes embodiments of the present invention, but these embodiments are merely illustrative examples for suitably illustrating the present invention and do not limit the present invention in any way.

[0040] (Example 1) Three polypropylene trays with lids, measuring 115 mm in length, 155 mm in width, and 65 mm in height, were prepared as fermenter units (reaction vessels). Inlets and outlets for organic solvents were provided on the sides of each tray. Each unit was sterilized with formaldehyde (formalin) gas and then degassed. A mixture was prepared by vigorously mixing 600 ml of liquid culture medium, 5 g of hollow microparticles MFL-80GTA (manufactured by Matsumoto Oil & Fat Pharmaceutical Co., Ltd.), and 2 ml of seed culture solution of Trichoderma atroviride NBRC 100586. The liquid culture medium used consisted of 100 g of glucose, 10 g of peptone, trace amounts of ferrous sulfate, trace amounts of manganese sulfate, and trace amounts of calcium chloride (pH 6.0). The mixture was dispensed into each unit, and a mold / hollow microparticle layer was formed on the surface of the liquid culture medium. By allowing the culture to stand still for 3 days at 25°C, a physically robust mold-hollow particle composite mat (microbial layer) was formed on the surface of the liquid culture medium (aqueous layer).

[0041] Subsequently, three units were stacked, and the outlets of each upper unit were connected to the inlets of each lower unit with solvent-resistant rubber tubing. The outlet of the bottom unit was also connected to the inlet of the top unit with solvent-resistant rubber tubing. An adsorption tank and a circulation pump were installed in the middle of the tubing connecting the bottom and top units. 40g of silica gel 60N (manufactured by Kanto Chemical Co., Ltd.) was packed into the adsorption tank as an adsorbent. This formed an organic solvent circulation line consisting of the three units, the adsorption tank, and the tubing (circulation channel). Then, 2.5L of hydrophobic low-viscosity dimethyl silicone oil KF-96L-1CS (manufactured by Shin-Etsu Chemical Co., Ltd.) was injected into the circulation line, and a batch operation of 1 week was repeated four times at a circulation rate of 2mL / min and 25°C. After the fourth batch operation, one 12-day batch operation was performed.

[0042] As a result, it was confirmed that 6PP, a fungicidal secondary metabolite, was recovered into the adsorption tank. Furthermore, it was confirmed that the concentration of 6PP in the organic solvent was consistently maintained at a low level of 200 mg / L or less during operation. From the above, it can be understood that 6PP produced by the bacterial cells and secreted into the organic solvent can be efficiently recovered by the adsorbent.

[0043] Furthermore, the 6PP adsorbed on the adsorbent was eluted with ethyl acetate and quantified. The weight of 6PP recovered in each batch is shown in Figure 2. Figure 2 shows the recovery results of the target product. As shown in Figure 2, it was confirmed that 6PP could be repeatedly produced and recovered at a yield of 200-400 mg / 40 g silica gel by batch operation for 1 week to 12 days. It was also confirmed that the yield of 6PP increased as the number of batch operations increased. Therefore, it was confirmed that the metabolic capacity of the bacterial cells could be maintained and even improved for more than one month, and that 6PP could be produced stably.

[0044] 6PP has strong mycogenic properties. Therefore, in conventional liquid culture using flasks, the maximum production is only about 200 mg / L. Furthermore, biodegradation of 6PP progresses towards the end of the culture period. For this reason, it has been confirmed that the concentration of 6PP gradually decreases after the 10th day of culture. In contrast, in the substance production apparatus 1 according to this embodiment, 6PP secreted into the organic solvent is adsorbed by an adsorbent and recovered from the organic solvent. This suppresses both the killing of mold by 6PP and the biodegradation of 6PP by the mold, allowing for the efficient production and recovery of 6PP.

[0045] (Example 2) Two stainless steel trays, measuring 135 mm in length, 180 mm in width, and 55 mm in height, were prepared as fermenter units (reaction vessels). Inlets and outlets for organic solvents were provided on the sides of the trays. Each unit was sterilized with formaldehyde (formalin) gas, followed by degassing. 400 ml of agar medium (1% agar concentration) with the same composition as the liquid medium in Example 1 was dispensed into each unit and allowed to solidify. 4 mL of a 2-fold diluted solution of Trichoderma atroviride NBRC 100586 seed culture solution was spread over the surface of the agar medium in each unit. A mold mat (microbial layer) was formed on the surface of the agar medium (aqueous layer) by static incubation at 25°C for 3 days.

[0046] Subsequently, in each unit, the outlet and inlet were connected with solvent-resistant rubber tubing, forming independent circulation channels for each unit. An adsorption tank and circulation pump were installed in the middle of the tubing connected to one unit. The adsorption tank was filled with 15 g of silica gel 60N as the adsorbent. Only a circulation pump was installed in the middle of the tubing connected to the other unit. This resulted in two circulation lines: one with an adsorption tank and one without. Then, 600 mL of n-decane was injected into each circulation line, and batch operation was repeated for one week at a circulation rate of 2 mL / min and 25°C.

[0047] As a result, in the circulation line without an adsorption tank, the concentration of 6PP in the organic solvent remained low, at 110 mg / L in the first batch and 150 mg / L in the second batch. Furthermore, a decrease in the accumulation rate of 6PP was observed in the second batch. This suggests that the decomposition of 6PP by mold had already begun.

[0048] On the other hand, in the circulation line equipped with an adsorption tank, the concentration of 6PP in the organic solvent was maintained below the detection limit for two weeks. Furthermore, 6PP was recovered into the adsorption tank at a yield of 240 mg / 15g silica gel in the first batch and 260 mg / 15g silica gel in the second batch. Therefore, it was confirmed that the target product can be efficiently produced and recovered by installing an adsorption tank. [Explanation of symbols]

[0049] 1. Material production apparatus, 2. Reaction vessel, 4. Adsorption vessel, 6. Circulation channel, 6a. Forward path, 6b. Return path, 8. Circulation pump, 12. Aqueous layer, 14. Organic layer, 16. Microbial layer, 18. Adsorbent.

Claims

1. A reaction vessel containing an aqueous layer containing a hydrophilic medium, an organic layer containing a hydrophobic organic solvent, and a microbial layer containing microorganisms that grow at the interface between the aqueous layer and the organic layer, and for eluting substances produced by the microorganisms into the organic solvent, A substance production apparatus comprising: an adsorption tank into which the organic solvent flows from the reaction tank and which contains an adsorbent for adsorbing the substance in the organic solvent, The polarity of the organic solvent is lower than the polarity of the substance. The adsorbent is silica gel, whose polarity is closer to that of the product than to that of the organic solvent, and based on the difference in polarity, it allows the relatively low-polarity organic solvent to flow out of the adsorption tank without adsorbing it, and selectively adsorbs the relatively high-polarity substance. The aforementioned substance production apparatus is A circulation channel including a forward and return path connecting the reaction tank and the adsorption tank, The system further comprises a circulation pump connected to the circulation channel, which generates a flow of the organic solvent from the reaction vessel to the adsorption vessel via the forward path, and a flow of the organic solvent from the adsorption vessel to the reaction vessel via the return path, By driving the circulation pump, the organic solvent is circulated between the reaction vessel and the adsorption vessel, thereby enabling the production of the substance by the microorganisms and the recovery of the substance by the adsorbent to be carried out simultaneously and continuously. Material production device.

2. The particle size of the silica gel is 1000 μm or more and 2000 μm or less. The material production apparatus according to claim 1.

3. By using microorganisms that grow at the interface between an aqueous layer containing a hydrophilic medium and an organic layer containing a hydrophobic organic solvent, a substance is produced. The substance is eluted into the aforementioned organic solvent, This includes transferring the organic solvent to an adsorption tank containing an adsorbent, and allowing the adsorbent to adsorb the substance in the organic solvent, The polarity of the organic solvent is lower than the polarity of the substance. The adsorbent is silica gel, whose polarity is closer to that of the product than to that of the organic solvent, and based on the difference in polarity, it allows the relatively low-polarity organic solvent to flow out of the adsorption tank without adsorbing it, and selectively adsorbs the relatively high-polarity substance. This method involves circulating the organic solvent between the reaction vessel and the adsorption vessel via a circulation channel connecting the reaction vessel containing the aqueous layer, the organic layer, and the microbial layer containing the microorganisms, thereby simultaneously and continuously carrying out the production of the substance by the microorganisms and the recovery of the substance by the adsorbent. Methods of producing substances.

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