JP2010088335A - Bioreactor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bioreactor capable of holding specific microorganisms in high concentration. <P>SOLUTION: The bioreactor includes a cohesive container 100 for stirring a plurality of microorganisms and a raw material solution SS containing a raw material for bioreaction by rotating the mixture around a prescribed rotation axis L penetrating itself, and culturing the cohesive microorganisms, a detector 120 for detecting the kinds and the numbers of individuals of the microorganisms contained in the raw material solution SS, a controller 130 for controlling the rotating condition of the cohesive container 100 based on the detected result, and a discharging means 180A for discharging the cohesive bodies floating at a specific position in the depth direction in the raw material solution SS with the raw material solution SS in the cohesive container 100. The cohesive bodies AG having a plurality of sizes are formed according to the kinds of the microorganisms by the cohesion of the plurality of the microorganisms including the cohesive microorganisms attaching to the inner wall of the cohesive container 100 based on the rotation of the cohesive container 100, and the discharging means 180A discharges the cohesive bodies floating at the specific depth of the cohesive container 100 based on the size of the cohesive bodies. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a microorganism reaction apparatus.

  2. Description of the Related Art In recent years, technologies relating to so-called bioreactors, which are apparatuses for synthesizing and decomposing substances from raw materials in solution using enzyme reactions or biological reactions using microorganisms, have been actively developed. In general, biological reactions are slower than chemical reactions, but they do not require high temperatures and pressures unlike chemical reactions, so they have excellent energy savings, and the reaction equipment does not need to be heat and pressure resistant. ing. Furthermore, since it has the feature that a reaction system with few side reactions can be constructed from a specific raw material to a target product according to the species used in the reaction, it is industrially advantageous. Has attracted much attention (see, for example, Patent Document 1).

  In a bioreactor using microorganisms, in order to increase the reaction efficiency, a technique for retaining microorganisms that catalyze the reaction at a high concentration in the reaction apparatus is very important. As such a technique, conventionally, a method of supporting a microorganism with a structure (carrier) formed of a substance that is difficult to be decomposed by the microorganism has been widely used. Such a carrier can be roughly divided into a fixed type that is fixed inside the reaction vessel and a fluid type that freely flows in the reaction vessel, but the fluid type carrier is more suitable for the reaction vessel. Since it is possible to increase the contact efficiency between the carrier and the raw material, there is an advantage that the reaction efficiency is easily increased.

  The above-mentioned fluid type carrier is formed by encapsulating microorganisms when forming a carrier with a gel-like substance (for example, calcium alginate) having fine gaps (water channels), and has a high concentration of microorganisms inside the carrier. The embedding type | mold carrier implement | achieved can be mentioned. There is also known an attachment-type carrier that separately forms a carrier for attaching microorganisms to the surface and realizes a high concentration of microorganisms on the surface of the carrier (see, for example, Patent Document 2).

  Further, in a bioreactor using microorganisms, in order to perform a desired reaction, a technique for holding a specific type of microorganisms that catalyze the reaction in a high concentration in the reaction apparatus is very important.

As such a technique, conventionally, an embedding type carrier formed by encapsulating a specific microorganism in a high concentration is introduced into the reaction system. A method of planting only microorganisms has been used. Moreover, the technique which employ | adopts the control by selection pressure, maintaining the operating conditions suitable for a specific microorganism, the technique which employ | adopts the control by promoting or suppressing the proliferation of a certain microorganism by chemical | medical agent administration, etc. can be mentioned. On the other hand, in order to confirm that only specific types of microorganisms are propagated, various methods for measuring the types and number of microorganisms have been proposed (see, for example, Patent Documents 3 to 5).
JP 2006-325577 A JP 2001-292765 A JP 2003-254891 A JP-A-8-271447 JP-A-5-263411

  However, the above method for retaining a specific type of microorganism has the following problems.

  When an embedded carrier is used, the reaction efficiency is unstable due to the lifetime of the embedded microorganism, and the carrier needs to be replaced periodically, resulting in an increase in running cost. In the method of sterilizing the inflowing substance, the initial cost and running cost for sterilization increase, and if the sterilization is insufficient, the bacteria that are not used in the reaction are grown and the reaction system is contaminated. The Furthermore, control by selective pressure relies heavily on experience and is difficult to control. Control by drug administration increases running costs and is not applicable when the target product is a food or medicine.

  That is, in the conventional method, it is difficult to keep the concentration of a specific type of microorganisms, particularly when the operation is continuously performed. There is no suitable means to suppress the propagation of contaminated bacteria when the germs grow during continuous operation, so that the target microorganism concentration cannot be maintained and the reaction is interrupted. There is no other way but to sterilize and clean the inside.

  This invention is made | formed in view of such a situation, Comprising: It aims at providing the reaction apparatus which can hold | maintain a specific microorganisms at high concentration.

  As a result of various investigations, the inventor has come up with the idea that the reaction is controlled by a method using an aggregate in which aggregating microorganisms self-aggregate. Since the self-aggregates of microorganisms have different conditions such as agglutination force and agglutination rate depending on the type of aggregating microorganisms, the size of the aggregates varies depending on the type of microorganism used when the microorganisms are aggregated under the same conditions. Even if the germs started to grow, it was considered that the germs could be separated by using such a difference in the size of the aggregates.

  Therefore, in order to solve the above problems, the microorganism reaction apparatus of the present invention cultivates a plurality of microorganisms containing an aggregating microorganism to form an aggregate containing the aggregating microorganism, and for each of the plurality of microorganisms A microorganism reaction apparatus for controlling the number of individuals, wherein the plurality of microorganisms and a raw material solution containing a raw material for biological reaction using the plurality of microorganisms are rotated around a predetermined rotation axis penetrating the microorganisms. The aggregating vessel for agitation and culturing the aggregating microorganism, a detection device for detecting the type and number of the microorganisms contained in the raw material solution, and a rotation condition of the aggregation vessel based on the detection result in the detection device. A control device for controlling, and a discharge means for discharging the aggregate suspended in a position in a specific depth direction in the raw material solution together with the raw material solution in the aggregation container. A plurality of microorganisms including the aggregating microorganism attached to the inner wall of the container roll on the bottom surface of the aggregation container based on the rotation of the aggregation container and aggregate each other, so that the plurality of sizes according to the type of microorganism An aggregate is formed, and the discharge means is an aggregate having a specific size among the plurality of aggregates having different sizes, and the discharge unit is configured to store the aggregation container based on the size of the aggregate. It is characterized by discharging aggregates floating at a specific depth position.

  As the biological reaction progresses in the aggregation container, the aggregating microorganisms grow in the aggregation container, thereby forming minute aggregates in the raw material solution and attaching to the inner wall of the aggregation container. Here, since the raw material solution is agitated while the agglomeration container rotates, the fine aggregates formed in the raw material solution collide with each other due to convection generated by the agitation and become enlarged near the bottom of the agglomeration container. Alternatively, aggregating microorganisms (adherent microorganisms) adhering to the inner wall are partially separated from the inner wall due to frictional force between the raw material solution and the adhering microorganisms by stirring or gravity applied to the adhering microorganisms, and the raw material as aggregates of the aggregating microorganisms. Float in solution.

  Aggregates that have grown in solution in this way, or aggregates that have been peeled off from the wall surface, roll in the direction of rotation while taking in micro-aggregates in the raw material solution and adhering microorganisms adhering to the inner wall. It grows into large aggregates. Since such agglomeration of aggregates occurs simultaneously at a plurality of locations in the aggregation container, a plurality of aggregates are formed in the aggregation container. Thus, in the microorganism reaction apparatus provided with the configuration of the present invention, the aggregation of microorganisms is promoted to form an aggregate of microorganisms.

  Here, since the self-aggregate of microorganisms has different conditions such as agglutination force and agglutination speed depending on the type of aggregating microorganism, the size of the aggregate varies depending on the type of microorganism used when aggregating under the same conditions. . The difference in the size of the aggregates appears as a difference in the sedimentation speed in the raw material solution. Small aggregates are likely to float and large aggregates are likely to sink. For example, large agglomerates are not easily affected by the convection of the raw material solution due to agitation, and thus are easily settled. .

  Furthermore, the difference in the size of the aggregates is enlarged by changing the rotation conditions of the concentrator. Therefore, on the basis of the number of microorganisms detected by the detection device, the concentration device is designed so that the difference in the size of the aggregates between the microorganisms excluded from the raw material solution and the microorganisms to be retained in the raw material solution is enlarged. When the rotation is controlled, the difference in sedimentation speed is enlarged, and a difference in the position where the flocculation container stays in the depth direction is generated for each microorganism species. As a result, the aggregate stays at a specific position in the depth direction of the aggregation container based on the size of the aggregate due to the microorganism species.

  Then, after separating in the raw material solution according to the difference in the size of the aggregates, the amount of microorganisms (abundance ratio) in the raw material solution is controlled by discharging the aggregates of microorganisms to be excluded from the raw material solution. Can do. For example, when the microorganisms to be eliminated tend to form large aggregates, the microorganisms to be eliminated can be efficiently removed by extracting the aggregates that have settled near the bottom of the concentrator. In addition, when the microorganisms to be excluded are difficult to form aggregates, they float on the liquid surface of the raw material solution and stay at a high concentration near the liquid surface. Therefore, the raw material solution in the vicinity of the liquid surface is removed efficiently. be able to.

  Therefore, according to this structure, it can be set as the reaction apparatus which can hold | maintain a specific microorganisms at high concentration. In addition, since microorganisms form aggregates, highly efficient biological reactions can be realized by reactions using the aggregates.

  In the present invention, it is preferable that the detection apparatus uses a polynucleotide probe having a base sequence specific to the base sequence of a microorganism gene and counts the number of individuals for each type of microorganism contained in the raw material solution.

  According to this configuration, by using a polynucleotide probe having a base sequence specific to the base sequence of each microorganism gene, it is possible to quickly count for each type. Therefore, it is possible to quickly reflect the counting result in the rotational speed control of the concentrating device, and to control the microbial reaction device well.

  In the present invention, the term “polynucleotide probe having a base sequence specific to the base sequence of a microbial gene” can recognize a nucleic acid having a base sequence homologous or complementary to the gene of each microorganism. A nucleic acid having a base sequence homologous or complementary to a gene of this type of microorganism means a polynucleotide probe that is difficult to recognize.

In the present invention, it is preferable that the detection apparatus performs detection using a CARD-FISH method.
According to this configuration, since the fluorescent color and intensity differ depending on the type of microorganism and the type of fluorescent substance or enzyme used for labeling, the microorganism can be counted for each type. In addition, since it is possible to detect microorganisms with high sensitivity by increasing fluorescence intensity, it is possible to detect and count bacteria, for example, by observing under a fluorescence microscope with a lower magnification than before, thereby reducing the time required for counting. can do. Therefore, it is possible to quickly reflect the counting result in the rotational speed control of the concentrating device and to control the microbial reaction device satisfactorily.

In this invention, the said detection apparatus has an imaging means which images the image of the said microorganisms, and an analysis means which analyzes the image acquired by the said imaging means, The said detection means uses the said imaging means. The first step of taking an image of the microorganism and the image obtained in the first step using the analyzing means are binarized, and in the obtained binary image, a region where pixels are connected is represented by one individual. It is desirable to count the number of individuals of the microorganism by a counting method having a second step of recognizing as a microorganism and a third step of counting a region recognized as one individual microorganism.
According to this configuration, since the number of individuals can be counted semi-automatically from the acquired image, microorganisms can be counted quickly.

In the present invention, there is provided a reaction vessel to which the raw material solution containing the aggregate is supplied from the aggregation vessel, and in the reaction vessel, the reaction liquid containing the aggregate and the raw material solution is agitated, It is desirable to culture aggregating microorganisms.
According to this configuration, since the aggregate or the abundance ratio of the microorganisms supplied to the reaction vessel is controlled, it is possible to cause a good reaction without side reaction in the reaction vessel. Furthermore, when aggregating microorganisms are aggregated, if the raw material solution contains a solid, the solids inhibit aggregation between the aggregating microorganisms, so that no aggregates are generated. However, according to this configuration, the aggregation container that mainly forms aggregates and the reaction container that is mainly used as a field for biological reaction are separated, and therefore, the aggregates are first formed using a solution that does not contain solids. After forming the agglomerate, it is possible to supply the aggregate to the solution containing the solid and perform a good reaction.

In the present invention, the reaction vessel includes a detection device that detects the type and number of the microorganisms contained in the reaction solution, and a control device that controls the stirring condition of the reaction vessel based on the detection result of the detection device. A discharge means for discharging together with the reaction liquid in the reaction vessel, agglomerates floating at a position in a specific depth direction of the reaction liquid, and the aggregate contained in the reaction liquid comprises Agglomerating or disintegrating based on agitation of the reaction vessel to form the aggregates of a plurality of sizes according to the type of microorganisms, and the discharging means includes a plurality of the aggregates of different sizes. Among them, it is desirable to discharge the aggregate having a specific size, which is floating at a position in the specific depth direction of the reaction vessel based on the size of the aggregate.
According to this configuration, it is possible to provide a reaction apparatus that can maintain a specific microorganism at a high concentration by utilizing the difference in the size of aggregates that differ for each type of microorganism in the reaction vessel.

  According to the present invention, it is possible to separate mixed microorganisms by controlling the formation conditions of aggregates by utilizing the ease of formation of aggregates according to the type of microorganism and the difference in sedimentation speed depending on the size of aggregates. Thus, a reaction apparatus capable of maintaining the target microorganism at a high concentration can be obtained. For example, it is possible to eliminate contaminants growing in the reaction system, or to control the abundance ratio when two or more microbial species are used, and the microorganisms used form aggregates. In addition, a highly efficient biological reaction can be realized by the reaction using the aggregate.

[First Embodiment]
Hereinafter, the microorganism reaction apparatus according to the first embodiment of the present invention will be described with reference to FIGS. In all the drawings below, the film thicknesses and dimensional ratios of the constituent elements are appropriately changed in order to make the drawings easy to see.

  FIG. 1 is a schematic configuration diagram showing a microorganism reaction apparatus 1 of the present embodiment. As shown in the figure, the microorganism reaction apparatus 1 of the present embodiment includes an aggregation container 100 that rotates around the rotation axis L, and a rotation device 110 that rotates the aggregation container 100.

  In the aggregation container 100, a raw material solution SS containing a coagulable microorganism and a raw material for a biological reaction using the microorganism is stored. A plurality of aggregates AG of aggregating microorganisms are suspended in the raw material solution SS. The principle of promoting the formation of the aggregate AG will be described in detail later.

  The raw material solution SS has a solution S1 and a solution S2 in which different aggregates stay depending on the position in the depth direction. The solution S1 contains mainly an aggregate AG containing the target aggregating microorganism, and the solution S2 contains a lot of aggregate C containing a different type of germs different from the target aggregating microorganism. In the present embodiment, it is assumed that the contaminants are less likely to aggregate than the desired aggregating microorganism, and that the solution S1 stays near the bottom of the aggregation container 100 and the solution S2 stays near the liquid surface. To do. In the figure, the boundary between the solution S1 and the solution S2 is indicated by a wavy line for ease of explanation.

  The microorganism reaction apparatus 1 includes a tank 140 that stores the raw material solution SS, and is configured to supply the raw material solution SS to the inside of the aggregation container 100 via a pipe 150. The pipe 150 is provided with a pump 145 for supplying the raw material solution SS on the way.

  Furthermore, the microbial reaction apparatus 1 is provided with a pipe 170 for selectively discharging the solution S1 out of the raw material solution SS inside the aggregation container 100 to the outside of the container. Has been placed. At the time of discharge, a part of the aggregate AG is also discharged together with the raw material solution SS. When the aggregate AG is used at the discharge destination, the discharge pump 160 is not pulverized when the pump passes through the pump having a low shearing force in the pump such as a diaphragm pump. preferable.

  In the solution S1 discharged through the pipe 170, the reaction product generated by the biological reaction using the aggregating microorganism is also dissolved. Therefore, the target product (reaction product) can be obtained by recovering the solution S1 downstream of the pipe 170 and separating the reaction product.

  Moreover, the microorganism reaction apparatus 1 is provided with a discharge device (discharge means) 180A for selectively discharging the solution S2 out of the raw material solution SS in the aggregation container 100 to the outside of the container, and one end of the raw material solution SS. A pipe 180 located near the liquid level, and a discharge pump 185 provided in the middle of the pipe 180. When the discharge device 180 is used, it is possible to sort out and dispose of contaminating bacteria C that convect to the liquid surface at a high concentration.

  The pipe 170 is provided with a bypass 175 in part. A detection device 120 for detecting the type and number of microorganisms is provided in the middle of the bypass. Although the detection device 120 is connected to the bypass 175 of the pipe 170, the detection apparatus 120 is not connected to the pipe 170. If necessary, the raw material solution SS is taken out from a sampling port (not shown) and is not connected to the pipe 170. It is good also as detecting the kind and number of microorganisms using the apparatus 120.

  In the detection apparatus 120 of the present embodiment, a plurality of microorganisms contained in the raw material solution SS are counted for each type using a counting method using a polynucleotide probe. In the detection apparatus 120 of the present invention, the polynucleotide probe used for counting has a base sequence specific to the base sequence of the target microorganism gene to be counted, and the target microorganism and other types The polynucleotide probe is not particularly limited as long as it is a polynucleotide probe that can be detected separately from the microorganism.

  Here, “a polynucleotide probe having a base sequence specific to the base sequence of a microorganism gene” can recognize a nucleic acid having a base sequence homologous or complementary to the gene of each microorganism, but other types A nucleic acid having a base sequence that is homologous or complementary to a gene of a microorganism of the above means a polynucleotide probe that is difficult to recognize. Since it can be expected to be excellent in specificity, it is preferably a polynucleotide probe having a base sequence specific to the base sequence of the 16S rDNA gene of each microorganism. Such a polynucleotide probe can be designed and synthesized by a conventional method. Moreover, a well-known polynucleotide probe can also be used as a polynucleotide probe which can detect each microorganism.

  There is no particular limitation as long as it is a method of counting for each type of microorganism using a polynucleotide probe having a base sequence specific to the base sequence of the gene of each microorganism. Examples of the method include a hybridization method and a quantitative PCR (Polymerase Chain Reaction) method.

  Since it does not require a nucleic acid extraction operation and is simple, it is preferable to detect by an in situ hybridization (ISH) method. In particular, in order to be safe and sensitive, detection is preferably performed by a fluorescence in situ hybridization (FISH) method using a fluorescently labeled probe, and a CARD-FISH (Catalyzed Reporter Deposition-Fluorescence in situ Hybridization) method is used. (See Pernthaler et al., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2002, Vol. 68, pp. 3094-3101).

  The CARD-FISH method is an applied technology in which the fluorescence intensity is increased by activating a fluorescent dye using an enzyme reaction of HRP (Horse Radish Peroxidase). More specifically, after FISH is performed using an HPR labeled probe or the like, fluorescently labeled tyramide is used. Tyramide is radicalized by HPR, but the lifetime of this radical is so short that it is covalently bonded only to aromatic compounds such as tyrosine and tryptophan in the vicinity of HPR. As a result, since the radicalized fluorescently labeled tyramide accumulates in the microorganism labeled with the HPR labeled probe, the microorganism can be detected with higher sensitivity than in the conventional FISH method.

  For example, microorganisms that could not be detected without using an objective lens with a magnification of 60 to 100 in the conventional FISH method can be detected even with an objective lens with a magnification of 4 to 10 with the CARD-FISH method. That is, by using the CARD-FISH method, it is possible to detect and count bacteria by observation under a fluorescence microscope with a lower magnification than before, so that the time required for counting can be shortened. In this embodiment, this CARD-FISH method is used.

  The detection apparatus 120 according to the present embodiment includes an imaging unit that captures an image of a microorganism processed as described above, and simply detects the type and number of microorganisms by analyzing the captured image. Yes. Specifically, the detection device 120 binarizes the first step of capturing an image of the microorganism and the image obtained in the first step, and in the obtained binary image, the region where the pixels are connected is represented by one individual. Microorganisms are detected using a counting method that has a second step of recognizing as a microorganism and a third step of counting a region recognized as one individual microorganism in the second step. Hereinafter, it demonstrates for every process.

  First, as a first step, a digital image (image) of microorganisms contained in the raw material solution SS is acquired. A digital image of a microorganism can be obtained using a general-purpose microscope photographing apparatus having a microscope with an appropriate magnification and an imaging means such as a CCD camera connected to the microscope.

  Next, as a second step, the digital image obtained in the first step is binarized, and in the obtained binary image, a group of pixels connected to each other is recognized as one individual microorganism. For example, in a black-and-white binary image, a region surrounded by black and connected (continuously) in white is recognized as a single microorganism. Then, as the third step, the number of each region recognized as one individual microorganism is calculated.

  By binarization using an arbitrary threshold value, a digital image is identified with an extreme grayscale image. In the obtained binary image, the shape of the object (microorganism) separated from the background in the image is identified quickly and easily by recognizing a group of connected pixels as an integral microorganism. can do. The threshold used for binarization can be appropriately determined in consideration of the type and concentration of the fluorescent substance or enzyme used for labeling the microorganism.

  When such a treatment is performed, a microorganism having a gene that reacts in a homologous or complementary manner can be discriminated based on the type of polynucleotide probe used for each type of contained microorganism. In particular, since fluorescence of different colors and intensities is emitted according to the type and concentration of the fluorescent substance or enzyme used for labeling the microorganism, the type of microorganism can be determined according to the type of fluorescence. Furthermore, it is possible to quickly and easily count for each type of microorganism by imaging the fluorescent microorganism and performing image processing.

  In the detection apparatus 120 of the present embodiment, the number of microorganisms contained in the raw material solution SS is counted using the counting method as described above.

  The type and number of microorganisms detected by the detection device 120 are output to the control device 130 connected to the detection device 120. The control device 130 stores a predetermined value for the number of microbial individuals in the storage device, and compares the stored predetermined value with the number of detected microbial individuals using a comparison circuit to It is determined whether or not the operating condition is an operating condition for obtaining a desired microorganism.

  The predetermined value refers to, for example, the allowable amount of contaminants not used in the reaction of the microorganism reaction apparatus 1 of the present embodiment and the abundance ratio of a plurality of microorganism species, with respect to the total number of individuals of the microorganism species used. The ratio (namely, abundance ratio) of the number of individuals of each microorganism can be mentioned.

  A rotation device 110 is also connected to the control device 130 and controls the rotation speed of the aggregation container 100 based on the type and number of microorganisms detected by the detection device 120.

  In the microbial reaction apparatus 1 having such a configuration, the raw material solution SS is agitated by the rotation of the flocculation vessel 100 to promote a biological reaction using the flocculating microorganisms and to form an aggregate AG. FIG. 2 is a schematic cross-sectional view showing how the aggregate AG is formed in the aggregation container 100, and is a cross-sectional view corresponding to the line segment AA in FIG.

  First, as shown in FIG. 2A, when the biological reaction proceeds, the aggregating microorganisms grow and form minute aggregates ag in the raw material solution SS and adhere to the inner wall of the aggregation container 100. Since the agglomeration container 100 rotates and stirs the raw material solution SS, the aggregating microorganisms (adherent microorganisms MO) adhering to the inner wall cause frictional force between the raw material solution SS and the adhering microorganisms MO by stirring, gravity applied to the adhering microorganisms MO, and the like. As a result, a part is peeled off from the inner wall.

  Next, as shown in FIG. 2 (b), the detached attached microorganisms MO roll in the rotational direction while taking in the attached attached microorganisms MO and the minute aggregates ag in the raw material solution, and are enlarged into a snowball type. Aggregates AG are formed. Such an enlargement of the aggregate AG occurs simultaneously at a plurality of locations in the aggregation container 100, and a plurality of aggregates AG are formed in the aggregation container 100. In addition, the aggregate AG may collapse in the process of formation, but the collapsed aggregate becomes a nucleus, which is similarly enlarged to a snowball type and becomes an aggregate AG again. Thus, an aggregate AG of the aggregating microorganism is formed.

  The aggregating microorganism that can be used to form the aggregate AG is not particularly limited, and examples thereof include aggregating microorganisms such as Saccharomyces cerevisiae KF-7.

  The particle size of the aggregate AG formed in this way can be controlled by changing the rotation speed (the number of rotations per unit time) of the aggregation container 100. That is, when the rotation is slow, the aggregate AG rolls for a long time without collapsing, so that a large aggregate is formed. On the contrary, when the rotation is fast, the aggregate AG frequently collapses, resulting in a small aggregate. .

  The particle size of the aggregate AG varies depending on the type of the flocculating microorganism, such as the cohesive force and the aggregation speed. Therefore, when aggregated under the same conditions, the size of the aggregate AG varies depending on the microorganism species present. Occurs. Furthermore, by changing the rotation conditions of the aggregation container 100, the difference in size of the aggregate AG is expanded. Therefore, by controlling the rotation of the aggregation container 100, the difference in the ease of settling into the raw material solution SS (sedimentation speed) is expanded, utilizing the difference in the sedimentation speed due to the size of the aggregate AG, Microbial separation can be performed.

  In the microorganism reaction apparatus 1 of the present invention, the microorganism species in the aggregation container 100 can be controlled using the difference in the particle size of the aggregate AG generated in this way. For example, in the case where growth of contaminants that are not used in the target biological reaction is observed, the growth of contaminants can be suppressed and contamination in the aggregation container 100 can be prevented.

  FIG. 3 is a flowchart showing an example of an operation for controlling the microorganism species. In the following description of the flowchart, description will be made using the reference numerals shown in FIG.

  First, the inside of the aggregation container 100 is sterilized and washed, the raw material solution SS is stored, and desired microorganisms including aggregating microorganisms are inoculated. Then, the aggregation container 100 is rotated at a predetermined rotation speed to form an aggregate AG (step S11).

  Next, after a certain period of operation, the type and number of microorganisms contained in the raw material solution SS in the aggregation container 100 are detected using the detection device 120 to confirm the number of germs and the abundance ratio of the germs. Compare with tolerance. If the number of contaminating bacteria is less than the allowable value, the operation of the aggregation container 100 is continued as it is (step S12).

  If the number of germs detected by the detection device 120 is greater than or equal to the allowable value, the operating condition of the aggregation container 100 is changed (step S13). Since the self-aggregates of microorganisms have different conditions such as agglutination force and agglutination rate depending on the type of aggregating microorganisms, the size of the aggregates varies depending on the type of microorganism used when the microorganisms are aggregated under the same conditions. Utilizing this property, the operating conditions are changed depending on the ease of relative aggregation between the contaminating bacteria and the desired microorganism.

  That is, when the harmful bacteria are less likely to aggregate than the desired microorganism, the rotation speed of the aggregation container 100 is increased to control to inhibit the aggregation of the harmful bacteria. Then, the agglomerates containing the contaminating bacteria or the contaminating bacteria themselves are likely to float and stay near the liquid surface of the raw material solution SS in the aggregation container 100. Examples of such germs include filamentous bacteria.

  In addition, when the bacteria are more likely to aggregate than the desired microorganism, the rotation speed of the aggregation container 100 is decreased to control the aggregation of the bacteria. Then, the aggregate containing the contaminating bacteria easily settles and stays near the bottom of the aggregation container 100.

  After controlling the operating conditions in this way for a certain period, the aggregate C containing contaminants is extracted from the aggregation container 100. In the microorganism reaction apparatus 1 of the present embodiment, the solution S2 in which many foreign bacteria are retained is extracted from the aggregation container 100 using the discharge device 180A and discarded (step S14).

  After extracting the germs, the type and number of microorganisms contained in the raw material solution SS in the aggregation container 100 are detected again using the detection device 120. If the number of contaminating bacteria is less than the allowable value due to the change in the operating conditions of the aggregation container 100 and the subsequent extraction, the operation of the aggregation container 100 is continued. On the other hand, if the number of contaminating bacteria continues to be greater than or equal to the allowable value, the operating condition of the aggregation container 100 is changed again (S13) (step S15).

  For the circulation (loop) from step S13 to step S15, for example, the maximum number of continuous loops is set, and when a loop exceeding the set value of the number of continuous loops is performed, the apparatus is stopped and the cleaning / If sterilization is performed, it will not be unnecessarily operated for a long time.

  In addition, the maximum ratio of germs that can be controlled by the above flow is set in advance, and if the germs are contained above the maximum ratio, the device is stopped and washing and sterilization is performed. Even if it exists, it does not perform a driving | operation for a long time unnecessarily and is suitable.

  As described above, by controlling the operation of the microorganism reaction apparatus 1, the growth of contaminants is suppressed.

  In general, when the microorganisms adapt to the environment and gradually grow (the induction period, the lag period), the microorganisms that have grown to a certain level will reach an exponential growth period (exponential growth period, logarithmic growth period). For this reason, from the viewpoint of suppressing the growth of contaminating bacteria, it is preferable to change the operating conditions for suppressing the growth of contaminating bacteria before reaching the logarithmic growth phase or as early as possible after entering the logarithmic growth phase. Therefore, in the above flow, by confirming the number of contaminating bacteria at regular time intervals (S12) and assuming a period from the resulting growth rate of the contaminating bacteria to an allowable value, the measurement result of the contaminating bacteria is obtained. Even if it is less than the allowable value, the operating condition may be changed in S13.

  Moreover, when performing the biological reaction using 2 or more types of microorganisms containing an aggregating microorganism in the microorganism reaction apparatus 1 of this embodiment, it is good also as utilizing in order to maintain the suitable ratio of microorganisms. FIG. 4 is a flowchart showing an example of an operation for controlling the microbial species.

  That is, first, the inside of the aggregation container 100 is sterilized and washed, the raw material solution SS is stored, and a plurality of microorganisms including aggregating microorganisms are inoculated. Then, the aggregation container 100 is rotated at a predetermined rotation speed to form an aggregate AG (step S21).

  Next, after a certain period of operation, the type and number of microorganisms contained in the raw material solution SS in the aggregation container 100 are detected using the detection device 120, and the abundance ratio of the microorganisms is confirmed. If the abundance ratio of microorganisms is appropriate, the operation of the aggregation container 100 is continued as it is (step S22).

  If the abundance ratio of the microorganisms detected by the detection device 120 is inappropriate, the operation conditions of the aggregation container 100 are changed, and separation is performed using the ease of formation of aggregates due to microorganism species (step S23).

  After controlling the operating conditions in this way for a certain period of time, the aggregate AG is extracted from the aggregation container 100, and the abundance ratio of microorganisms is adjusted (step S24).

  After the extraction operation, the type and number of microorganisms contained in the raw material solution SS are detected again using the detection device 120, and the abundance ratio of the microorganisms is confirmed. If the abundance ratio of the microorganisms is an appropriate value by changing the operating conditions of the aggregation container 100 and the subsequent extraction, the operation of the aggregation container 100 is continued. On the other hand, if the abundance ratio of microorganisms continues to be unsuitable, the operation condition of the aggregation container 100 is changed again (S23) (step S25).

  For the loop from step S23 to step S25, the maximum number of continuous loops is set, and when a loop exceeding the set value of the number of continuous loops is performed, it is unnecessary to stop the apparatus. Do not drive for a long time. Also, if the maximum deviation of the controllable existence ratio is set in advance and the apparatus is stopped when it deviates greatly from the controllable deviation, it will not run unnecessarily for a long time. .

  As described above, the abundance ratio of microorganisms can be controlled by controlling the operation of the microorganism reaction apparatus 1.

  By combining the flows shown in FIG. 3 and FIG. 4, for example, when contaminants are generated in a reaction using two types of microorganisms, the contaminants are first removed from the aggregation vessel by the flow shown in FIG. After that, the abundance ratio can be controlled by the flow of FIG. Furthermore, when performing reaction using 3 or more types of microorganisms, the flow of FIG. 4 can be performed several times according to the kind of microorganisms to be used, and the abundance ratio of microorganisms can be controlled.

  According to the microorganism reaction apparatus 1 configured as described above, the formation conditions of the aggregate AG are determined by using the ease of formation of the aggregate AG according to the microorganism species and the difference in the sedimentation rate depending on the size of the aggregate AG. By controlling the above, it is possible to separate the microorganisms that are mixed, and the reaction apparatus can maintain the target microorganisms at a high concentration. When the microorganism reaction apparatus 1 is used, for example, it is possible to eliminate contaminants growing in the reaction system or to control the abundance ratio when two or more kinds of microorganisms are used. Since the aggregate AG is formed, a highly efficient biological reaction can be realized by the reaction using the aggregate AG.

  In the present embodiment, since it is determined that contaminants are less likely to aggregate than the desired microorganism, the solution S2 near the liquid surface is extracted using the discharge device 180A. The location varies depending on the nature of the germs. That is, when the contaminating bacteria are more likely to aggregate than the desired microorganism, the solution S2 stays in the vicinity of the bottom, so that the discharge device 180A extracts from the vicinity of the bottom. Moreover, when extracting from near the bottom, a separate outlet may be provided near the bottom of the aggregation container 100, and extraction may be performed by opening and closing the outlet.

[Second Embodiment]
FIG. 5 is a schematic configuration diagram showing a microorganism reaction device 2 according to the second embodiment of the present invention. The microorganism reaction apparatus 2 of the present embodiment is partially in common with the microorganism reaction apparatus 1 of the first embodiment. The difference is that a reaction vessel 200 is separately provided on the downstream side of the pipe 170 as a place for performing a biological reaction. Therefore, in this embodiment, the same code | symbol is attached | subjected about the component which is common in 1st Embodiment, and detailed description is abbreviate | omitted.

  The reaction vessel 200 is filled with a reaction solution RS containing a raw material solution SS supplied via a pipe 170. The composition of the reaction solution RS is not limited to the same composition as the raw material solution SS, and a solution containing various raw materials that perform a desired reaction using an aggregating microorganism that forms the aggregate AG can be used. Furthermore, the reaction solution RS may contain solids such as wood chips and leaves.

  The reaction solution RS has a solution S1 and a solution S2 in which different aggregates stay depending on the position in the depth direction, and the solution S1 contains an aggregate AG mainly containing a target aggregating microorganism. S2 contains a large amount of aggregate C containing contaminating bacteria. In the figure, the boundary between the solution S1 and the solution S2 is indicated by a wavy line for ease of explanation.

  In addition, the reaction vessel 200 has a stirring device 210 that stirs the reaction solution RS, a detection device 220 that detects the number of microorganisms contained in the reaction solution RS in the reaction vessel 200, and the reaction solution flows out of the reaction vessel 200. An outflow passage (discharge means) 240 is provided, and a solid-liquid separation tank 230 that separates the liquid and solid contained in the outflowing reaction solution RS is provided in the middle of the outflow passage 240. The microbial reaction apparatus 2 performs continuous operation control in which the raw material solution SS continuously flows into the reaction vessel 200 and the reaction solution RS continuously flows out from the reaction vessel 200 through the outflow path 240. However, batch operation control may be performed.

  The stirrer 210 has a stirrer 212 and a rotary motor 214 that operates the stirrer 212. In the present embodiment, the agitation method is performed with the agitator 212, but a fluidized bed type in which the reaction liquid RS is agitated with a gas blown into the reaction vessel 200 may be used. Moreover, you may provide both.

  A device similar to the detection device 120 can be used as the detection device 220. The number of microorganisms detected by the detection device 220 is output to the control device 130 connected to the detection device 220. A rotation motor 214 is connected to the control device 130, and the rotation speed with the stirrer 212 is controlled based on the input detection value to control the particle size of the aggregate AG in the reaction vessel 200.

  Aggregates AG in the reaction vessel 200 are pulverized by a shearing force generated by the stirring by the stirring device 210 and flow out of the reaction vessel 200 as small pieces SP of the aggregating microorganisms. Alternatively, due to adhesion of aggregating microorganisms that propagate in the reaction vessel 200, adhesion due to collision between the aggregates AG, and the like, the particle size increases and the fluid becomes poorly settled.

  The solid-liquid separation tank 230 has a function of causing solids contained in the reaction liquid RS flowing out of the reaction vessel 200 to settle and separate from the liquid. In the solid-liquid separation tank 230, since the small pieces SP included in the reaction liquid RS that flows out also settle, the settled small pieces SP are collected and put into the reaction vessel 200 again to keep the microorganism concentration in the reaction vessel 200 at a high concentration. I can do things.

This is because the microbial reaction apparatus 2 performs continuous operation control. From the outflow path 240, the reaction liquid RS flows out continuously. At that time, in the reaction liquid RS, the target aggregate AG tends to settle, so that it stays at the bottom of the reaction vessel 200 (solution S1), and the aggregate C containing contaminants stays near the liquid surface (solution). S <b> 2), the aggregate C containing contaminating bacteria is likely to flow out through the outflow path 240.
The microorganism reaction apparatus 2 of the present embodiment is configured as described above.

  Also in the microorganism reaction apparatus 2 of the present embodiment, it is possible to suppress the growth of contaminants in the reaction solution RS and to control the abundance ratio of microorganisms contained in the reaction solution RS. FIG. 6 is a flowchart showing an example of an operation for controlling the growth of contaminants in the microorganism reaction apparatus 2 of the present embodiment.

  First, after the inside of the reaction container 200 is sterilized and washed, the raw material solution SS supplied from the aggregation container 100 is stored. And the stirring apparatus 210 is drive | operated on predetermined stirring conditions, and stirring is performed (step S31).

  Next, after a certain period of operation, the type and number of microorganisms contained in the reaction solution RS are detected using the detection device 220 to confirm the number of germs and compare the presence ratio of the germs with an allowable value. . If the number of contaminating bacteria is less than the allowable value, the operation of the reaction vessel 200 is continued under the same stirring conditions (step S32).

  If the number of contaminating bacteria detected by the detection device 220 is greater than or equal to an allowable value, the stirring condition of the reaction vessel 200 is changed, and the size of the aggregate is mainly determined using the shearing force of stirring by the stirring device 210. Is controlled (step S33).

  After controlling the stirring conditions for a certain period of time, aggregates containing contaminating bacteria are extracted from the reaction vessel 200. In the case where contaminants are less likely to aggregate and easily float, the microorganism reaction apparatus 2 of the present embodiment flows out of the reaction vessel 200 due to overflow. When the contaminants are likely to aggregate and settle, the contaminants are extracted from the bottom of the reaction vessel 200 together with the reaction solution RS. In the present embodiment, since the aggregate C containing contaminating bacteria has the property of hardly aggregating and easily floating, the concentration of the aggregate C containing the contaminating bacteria in the solution S2 is increased by continuous operation control, and the reaction solution Along with the outflow of RS, more is discharged out of the system through the outflow path 240 (step S34).

  After extracting the germs, the type and number of microorganisms contained in the reaction solution RS are detected again using the detection device 220. If the number of germs is less than the allowable value, the operation of the reaction vessel 200 is performed. continue. On the other hand, if the number of contaminating bacteria continues to be greater than or equal to the allowable value, the stirring condition of the reaction vessel 200 is changed again (S33) (step S35).

  FIG. 7 is a flowchart showing an example of an operation for controlling the microorganism species in the microorganism reaction apparatus 2 of the present embodiment.

  That is, after the inside of the reaction container 200 is sterilized and washed, the raw material solution SS supplied from the aggregation container 100 is stored. Then, the apparatus is operated with stirring under predetermined stirring conditions (step S41).

  Next, after a certain period of operation, the type and number of microorganisms contained in the reaction liquid RS in the reaction vessel 200 are detected using the detection device 220, and the abundance ratio of the microorganisms is confirmed. If the abundance ratio of microorganisms is appropriate, the operation of the reaction vessel 200 is continued under the same stirring conditions (step S42).

  If the abundance ratio of microorganisms detected by the detection device 220 is inappropriate, the reaction container is changed so that a large amount of microorganisms having a small abundance ratio can be supplied by changing the operating conditions of the aggregation container 100. The abundance ratio of the microorganisms contained in the raw material solution SS supplied to 200 is changed (step S43). The operation condition of the aggregation container 100 is changed according to the flowchart shown in FIG.

  After supplying the raw material solution SS in which the abundance ratio of microorganisms is changed for a certain period, the type and number of microorganisms contained in the reaction solution RS are detected again using the detection device 220 to confirm the abundance ratio of microorganisms. If the abundance ratio of the microorganisms is an appropriate value, the operation of the aggregation container 100 is returned to the operation condition that provides the abundance ratio of the predetermined microorganism, and the subsequent operation is continued. On the other hand, if the abundance ratio of microorganisms continues to be unsuitable, the operating conditions of the aggregation container 100 are changed again (S43) (step S45).

  As described above, by controlling the operation of the microorganism reaction device 2, it is possible to suppress the growth of contaminants in the reaction solution RS and to control the abundance ratio of microorganisms contained in the reaction solution RS.

  According to the microorganism reaction apparatus 2 configured as described above, the aggregate AG supplied to the reaction vessel 200 is excellent in that there is no side reaction in the reaction vessel 200 because the number of microorganisms or the abundance ratio is controlled. Reaction can be performed. Furthermore, since the aggregation container 100 that mainly forms aggregates and the reaction container 200 that is mainly used as a place for biological reactions are separated, for example, even when the reaction liquid RS contains solids, After the aggregate AG is formed using a solution not containing the aggregate, the aggregate is supplied to the solution containing the solid, and a favorable reaction can be performed.

  In addition, since the reaction vessel 200 includes the detection device 220 and the control device 130 that controls the stirring condition of the stirring device 210, it is possible to maintain a specific microorganism at a high concentration in the reaction liquid RS in the reaction vessel 200. It can be a possible reactor.

  In the present embodiment, the number of aggregation containers 100 connected to the reaction container 200 is one, but a plurality of aggregation containers 100 may be connected. In the case where a plurality of aggregation containers 100 are used, it is preferable that each of the aggregation containers is similarly controlled to suppress contamination bacteria and to control the abundance ratio of microorganisms.

  Here, the abundance ratio of the microorganisms in the reaction vessel 200 is controlled by controlling the operating conditions of the agglutination vessel 100. It is good also as extracting microorganisms with many and controlling abundance ratio.

  The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, but it goes without saying that the present invention is not limited to such examples. Various shapes, combinations, and the like of the constituent members shown in the above-described examples are examples, and various modifications can be made based on design requirements and the like without departing from the gist of the present invention.

It is a schematic block diagram which shows the microorganisms reaction apparatus of 1st Embodiment of this invention. It is a schematic sectional drawing which shows the mode of formation of the aggregate in the aggregation container. It is a flowchart which shows an example of operation for controlling microbial species. It is a flowchart which shows an example of operation for controlling microbial species. It is a schematic block diagram which shows the microorganisms reaction apparatus 2 which concerns on 2nd Embodiment of this invention. It is a flowchart which shows an example of operation for controlling microbial species. It is a flowchart which shows an example of operation for controlling microbial species.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1,2 ... Microbe reaction apparatus, 100 ... Aggregation container, 120,220 ... Detection apparatus, 130 ... Control apparatus, 180 ... Side pipe (discharge port), 200 ... Reaction container, AG ... Aggregate, L ... Rotating shaft, RS ... reaction solution, SS ... raw material solution,

Claims (6)

A microorganism reaction apparatus for culturing a plurality of microorganisms containing an aggregating microorganism to form an aggregate containing the aggregating microorganism, and controlling the number of individuals for each of the plurality of microorganisms,
Aggregation container for agitating the plurality of microorganisms and a raw material solution containing a raw material for biological reaction using the plurality of microorganisms by rotating around a predetermined rotation axis penetrating the microorganisms and culturing the aggregating microorganisms When,
A detection device for detecting the type and number of the microorganisms contained in the raw material solution;
Based on the detection result in the detection device, a control device for controlling the rotation conditions of the aggregation container;
A discharge means for discharging the aggregate suspended at a position in a specific depth direction in the raw material solution together with the raw material solution in the aggregation container;
A plurality of microorganisms including the aggregating microorganisms adhering to the inner wall of the aggregation container roll on the bottom surface of the aggregation container based on rotation of the aggregation container to aggregate each other, thereby having a plurality of sizes according to the type of microorganism. Forming the aggregate of
The discharge means is an agglomerate having a specific size among the agglomerates having different sizes, and is arranged in a specific depth direction of the agglomeration container based on the size of the agglomerate. A microorganism reaction apparatus characterized by discharging aggregates floating at a position.   2. The detection apparatus according to claim 1, wherein a polynucleotide probe having a base sequence specific to a base sequence of a microbial gene is used to count the number of individuals for each type of microorganism contained in the raw material solution. The microorganism reaction apparatus as described.   The microorganism reaction apparatus according to claim 2, wherein the detection apparatus performs detection using a CARD-FISH method. The detection device has an imaging means for capturing an image of the microorganism,
A first step of taking an image of the microorganism using the imaging means;
A second step of binarizing the image obtained in the first step, and recognizing a region where pixels are connected as one individual microorganism in the obtained binary image;
The microorganism reaction apparatus according to claim 3, wherein the number of individuals of the microorganism is counted by a counting method including a third step of counting a region recognized as one individual microorganism. A reaction vessel to which the raw material solution containing the aggregate is supplied from the aggregation vessel,
The microorganism reaction apparatus according to any one of claims 1 to 4, wherein in the reaction vessel, a reaction liquid containing the aggregate and the raw material solution is stirred to culture the aggregating microorganism. The reaction vessel includes a detection device that detects the type and number of the microorganisms contained in the reaction solution;
A control device for controlling the stirring condition of the reaction vessel based on the detection result in the detection device;
A discharge means for discharging the agglomerates floating at a position in a specific depth direction of the reaction solution together with the reaction solution in the reaction vessel,
The aggregate contained in the reaction solution aggregates or disintegrates based on the stirring of the reaction vessel, thereby forming the aggregate of a plurality of sizes according to the type of microorganism,
The discharge means is an agglomerate having a specific size among the agglomerates having different sizes, and is arranged in a specific depth direction of the reaction vessel based on the size of the agglomerates. The microorganism reaction apparatus according to claim 5, wherein aggregates floating in the position are discharged.

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