JPH03147778A - Sampling device of culture tank - Google Patents

Abstract

PURPOSE:To prevent the contamination of a sample when sampled, by disposing a switching means for switching between a process for filtering a culture solution from a culture tank with a filtration filter and a process for feeding steam into the filtration filter. CONSTITUTION:A valve 12, pipings 13, 14 and a container 35 receiving a filtration filter 15 in the order are connected to the downstream side of a piping 11 communicating with the interior of a culture tank 10. Pipings 16, 17 and a valve 18 in the order are connected to the filtrate-penetrating side of the container 35. A piping 19 is branched from the connection point of the pipings 13, 14 and a piping 21 is linked to the connection point of the pipings 16, 17. The piping 19 has a drain valve 20 and the upstream side of the piping 21 is connected with a steam-supplying source 23 through a valve 22.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a sampling device for collecting a culture fluid from a culture medium FTTFF, and more specifically, it is capable of aseptically collecting a sample directly from a culture tank that can separate bacterial cells and directly apply it to an analytical device such as chromatography. This relates to a sampling device that can be used. In addition, in this specification, the term "culture" shall be understood in the broadest sense, including "fermentation" which means the cultivation of microorganisms. Cell culture in which plant or animal cells are grown in a container is also included in the term "culture" in this specification. Therefore, in this specification, a culture tank, a fermentation tank, a culture solution, a fermentation solution, etc. are used interchangeably. Furthermore, in this specification, the terms "culture solution" and "medium" are treated as synonyms without any particular distinction. [Conventional technology]

Culture fluid (medium) containing metabolites, inhibitors, etc. in the culture tank - To understand the composition, judge the culture status, and control the culture, the culture fluid is often collected and analyzed during cultivation. A need arises. Various methods are used for analysis, but when using biosensors or liquid chromatography, gas chromatography, supercritical chromatography, etc. that can quantitatively analyze multiple components at once, solid-liquid analysis of the culture solution is used. Separation needs to be done. As a conventional culture tank sampling device capable of collecting samples that can be applied to these analytical devices, devices such as those shown in FIGS. 10 to 12, for example, have been proposed. The sampling device of FIG. 10 has a valve 5 downstream of the pipe 51.
A pipe 53 and a pipe 54 are arranged via a valve 55, and a pipe 56 is arranged downstream of the pipe 54 via a valve 55.
It has a piping system in which a pipe 58 with a valve 59 joins the pipe 58 at the connection point with the pipe 58. The upstream end of this piping 51 is connected to the culture tank 5
A filtration filter 60 with a pore diameter of 0.2μ or less is installed to prevent germs from passing through. In addition, the downstream of the piping 56 is connected to the sample tank 57,
Piping 58 is connected to a sterile air source 61 via a valve 59. Note that the piping 56 is provided with an extraction pump 62 if necessary. In such an apparatus, first, before the start of culture, steam is supplied into the culture tank 50 from a steam pipe (not shown) to sterilize the culture tank itself, the culture medium contained therein, and the filtration filter 60 by heating with steam. When sampling, all valves are closed, valves 59 are closed, valves 52 and 55 are opened, and the culture solution in the culture tank is pumped through the pipes 51, 53, 54, and 56 by the internal pressure of the culture tank.
The sample is collected into a sample tank 57 via the . When sampling is completed, valves 52 and 55 are closed. If the filter 60 becomes clogged during sampling, open the valve 59 while the valve 55 is closed, and supply sterile air to the pipe 58, pipe 53, and valve 5.
2. The water is supplied into the culture tank 50 via the piping 51 and the filter 60, and the filter 60 is backwashed. The reason why sterile air is used is that backwashing using steam is not possible because the culture solution in the culture tank contains bacterial cells, enzymes, etc. According to the sampling device shown in Fig. 10, sampling is carried out through a filter 60 with a pore size of 0.2μ or less provided in the culture medium, so the culture solution can be collected without contamination of bacteria into the culture tank. . In addition, since large solids and bacterial cells in the culture solution have been removed from the collected sample using a filtration filter, this sample can be used as it is. On the other hand, the sampling device shown in Fig. 11 and Fig. 12 (time open ■ f I 6l-5774) replaces the cross-flow type filtration filter installed in the culture tank as used in Fig. 10. A filtration filter is placed 1 m outside the culture medium, and a parallel flow type filtration filter that is relatively resistant to clogging is adopted. That is, the sampling device shown in FIG.
A culture solution circulation system consisting of a pipe 71, a circulation pump 72, and a pipe 73 is provided externally, and a cylindrical lξ-direction filter 74 with a pore diameter of 0.2μ or less that does not allow germs to pass is interposed in the pipe 73. The culture medium λ liquid flows inside the filtration filter 74 and the filtration ill comes out on the outer surface of the filtration filter. In such an apparatus, first, before the start of culture, the entire apparatus is placed in an autoclave, and the culture tank itself, the loaded medium, and the culture solution circulation system outside the culture tank are sterilized by heating and steam. When sampling, the circulation bomb 72 is started and the culture solution in the culture tank 70 is pumped through the piping 71, the bleaching ring pump 72,
The filtrate is circulated through the pipe 73, filter 74, and pipe 73 to pass through the outer surface of the filter 74, and the filtrate is collected as a sample 1175. In the sampling device of FIG. 12, instead of the pure cylindrical filter 74 shown in FIG. 11, a filter module 74' such as a ceramic filter module is used.
When the filtration filter becomes clogged, the filtrate and sterile air can be supplied into the culture tank through the filtration filter module 74' to backwash the filtration filter module 4'. This is how it was done. In this case as well, F? Since the culture solution in the i-culture tank contains bacterial cells, enzymes, etc., reverse 6T cannot be performed using steam. The above-mentioned filtration filter module 74' has a structure in which a number of cylindrical filtration filters or a plurality of cylindrical filtration filters are loaded in a storage container, and a culture solution circulates inside or outside the filtration filter cylinder. In the type where the culture medium flows inside the filter, the filtrate passes through the outside surface of the cylinder, and in the type where the culture medium flows outside the cylinder of the filtration filter, the filtrate permeates through the inside surface of the housing. Sample 1678 via connected piping 76 and valve 77
It is designed to flow. When backwashing the filter module 74', the filtrate from the sample tank 78 is supplied to the filter module 74' by the pump 79, or sterile air (not shown) is supplied to the filter module 74 through the piping 76. '(), and this filtrate or sterile air is passed through the culture tank side of the filtration filter. Also in the apparatuses shown in FIGS. 11 and 12 described above, the filtration filter 74 or Pore diameter 0.2
Since sampling is carried out through the microfiltration filter module 74', the culture medium milM can be collected without contaminating the culture medium 1itfl. In addition, large solids have been removed from the collected culture fluid using a filtration filter, and this sample can be directly applied to an analyzer.

[Problems to be Solved by the Invention] The conventional apparatus shown in Figures 10 to 12 is a sampling apparatus that can collect samples that can be directly applied to an analyzer such as chromatography. During backwashing, the filtrate and sterile air used for backwashing flow into the culture tank, so if bacteria grows in the downstream piping of the sampling device, these bacteria will be transferred to the filtrate and sterile air used for backwashing. This causes so-called contamination, which is entrained with the air and mixed into the culture tank. In order to prevent this, there is a restriction that a filter with a pore diameter of 0.2 μm or less must be used, which does not allow germs to pass through. Due to this constraint, on the other hand, when sampling culture fluids that contain a large amount of solids, large cells, cells with complex shapes, sticky cells, etc., the filtration filter can easily become clogged. Even in the devices shown in FIGS. 11 and 12, which employ parallel flow filters, the filters quickly become clogged. Therefore, the frequency of backwashing with filtrate or sterile air increases, and even with backwashing, it is difficult to completely recover from clogging, leading to problems in that the flow rate of filtrate passing through the filter is unstable. As a result, there is also the problem that it is difficult to connect the sampling device and an analyzer such as chromatography to automate sampling and analysis. Therefore, an object of the present invention is to provide a new and improved sampling device that is free from restrictions on the pore size of the filter, and free from the problem of contamination during sampling, and which can collect samples that can be directly applied to an analyzer. It is to make an offer. Another object of the present invention is that even if a filtration filter becomes viscous, it can be easily and sufficiently recovered by backwashing, and a sampling device and an analyzer such as a chromatography device can be An object of the present invention is to provide a new and improved sampling device that is easy to connect and automate sampling and analysis. A culture solution introduction pipe for introducing the culture solution from the culture tank is provided on one side of the filtration filter, and a filtrate extraction pipe for extracting the filtrate that has passed through the filtration filter is provided on the other side. A steam introduction pipe for introducing steam to the filtrate permeation side of the filtration filter, and a steam discharge pipe for discharging the steam that has passed through the filtration filter or its condensed water to the culture solution introduction side of the filtration filter, and from the culture tank. A step of introducing the culture solution into the culture solution introduction side of the filtration filter and extracting the filtrate that has passed through the filtrate permeation side, and introducing steam into the filtrate transmission side of the filtration filter and passing through the culture solution introduction side. It is characterized by providing a switching means for switching between the process of discharging steam or its condensed water.The above-mentioned filtration filter may be the above-mentioned direct flow type filter or parallel flow type filter. jj
In the flow method, the culture solution introduction pipe and the steam discharge pipe are placed on one side of the filtration filter of the filter storage container (culture solution introduction side), and the filtrate extraction pipe and the steam introduction pipe are placed on the side of the filter storage container. In most cases, it is directly connected to the other side of the filter (filtrate permeation side). On the other hand, in the parallel flow method, the culture solution flows inside the cylinder of the filtration filter.
In other words, the filtration ill extraction piping and the steam introduction piping may be connected to the filtration filter storage container (i.e., the filtration filter is provided on the other side of the filtration filter), and the filtration In the type where the culture solution flows outside the filter cylinder, the culture solution introduction pipe and the steam exhaust pipe are connected to the filtration filter storage container (
In other words, the filtrate discharge pipe and the steam introduction pipe may be directly connected to the filtration filter (that is, provided on the other surface of the filtration filter). As described above, there are various types of connection of each piping to the filter storage container and the filtration filter depending on the type of the filtration filter. In addition, the culture solution introduction pipe, filtrate extraction pipe, steam introduction pipe, and steam discharge pipe may each be provided independently in the filtration filter, and furthermore, the culture solution introduction pipe and the steam discharge pipe may be partially shared. Alternatively, the filtrate extraction piping and the steam introduction piping may be constructed from a part of common piping, and may be provided in the filter filter. Materials for filters include inorganic materials such as glass, ceramics, and silicone, organic polymer materials such as Teflon, nylon, polypropylene, polyvinyl alcohol, and cellulose resin, and metal materials such as porous stainless steel. However, other materials can be used as long as they can be sterilized by steam heating. A filtration filter that can be preferably used has a pore size of several tens of microns to 0.5 microns. t
Examples include μ ceramic filters and sintered metal filters, and macroporous membranes formed in a membrane shape with similar pore diameters and ultrafiltration membranes with a molecular weight of several thousand to several hundred thousand can also be used. . The pore size of the filtration filter is
It may be selected appropriately depending on the size of tlft+ cells, the components to be analyzed, etc. Preferably, one is selected that does not allow bacterial cells in the culture solution to pass through and has as large a pore size as possible. As the material for each of the above-mentioned pipes, stainless steel pipes can preferably be used, and flexible tubes made of organic polymeric materials such as silicone, Teflon, and polypropylene can be used. Further, it is preferable to make these piping as short as possible to facilitate steam heating sterilization. Further, as the element (4) of the storage container of the filter, stainless steel can be preferably used, and heat-resistant glass and the above-mentioned heat-resistant organic polymers and inorganic materials can also be used.

[Function] According to the sampling device of the present invention having the above-described configuration, there is a step of introducing the culture solution from the culture tank into the culture solution introduction side of the filtration filter and extracting the filtered waves that have passed through the filtrate permeation side. In other words, the switching means switches between the sampling process and the backwashing process in which steam is introduced into the filtrate permeation side of the filtration filter and the steam or condensed water that permeates into the culture medium inlet side is discharged. Even if steam is used in the washing step, this steam will not be introduced into the culture tank, and therefore will not affect the bacterial cells, enzymes, etc. in the culture tθ. In this way, in the device of the present invention, steam can be used in the backwashing process, so filter clogging can be removed much more easily and effectively than conventional backwashing of filtration filters using sterile air or filtrate. The filter can be sterilized by steam at the same time. In addition, in conventional backwashing using sterile air and filtrate, if bacteria grow in the downstream piping of the sampling device, these bacteria will be mixed into the culture tank along with the sterile air and filtrate used for cleaning. Because of the risk of contamination, there is a restriction that a filter with a pore diameter of 0.2 μm or less must be used, which does not allow germs to pass through. However, with the device of this invention, since steam can be used for backwashing, there is no risk of bacteria breeding in the piping, and therefore there is no longer a restriction that a filter with a pore diameter of 0.2 μm or smaller must be used to prevent bacteria from passing through. Ta. As a result, larger pore size filters can be used.
It is possible to sample culture fluids that would otherwise be clogged with conventional filters and cannot be sampled. As mentioned above, in this invention, Fn '1 +6
A filtration filter with as large a pore size as possible can be used without allowing the bacterial cells inside to pass through. As a result, although some solid content may be present in the filtrate, an analyzer using a biosensor can determine 7Ip+ without any problems, and even in high-performance liquid chromatography, the prefilter or precolumn is often clogged. This allows measurements to be taken without any problems, and therefore samples that can be directly applied to various analyzers can be collected. Embodiment FIG. 1 is an embodiment showing the basic configuration of a sampling device according to the present invention. This device includes a storage container 35 that houses a valve 12, a pipe 13, a pipe 14, and a filtration filter 15 on the downstream side of a pipe 11 that communicates with the inside of a culture tank 10.
are connected in this order, and the pipe 16, pipe 17, and valve 18 are connected in this order from the filtrate permeation side of the storage container 35, and further, one pipe branches from the connection point of the pipes 13 and 14,
A pipe 21 joins the connection point between the pipes 16 and 17. The pipe 19 has a drain valve 20 , and the upstream side of the pipe 21 is connected to a steam supply source 23 via a valve 22 . The operating procedure of the sampling device shown in FIG. 1 will be explained below. Sterilization of the filtration filter: Before starting the culture, open the valves 20 and 22, and turn on the steam supply source 23.
Piping 21, piping 16, filtration filter 15,
The filtration filter 15 is sterilized through the pipes 14 and 19. When the sterilization of the filter 15 is completed, the valve 12 is opened, the valve 20 is closed, and the steam is passed through the pipes 13 and 11.
Sterilize the piping system by passing it through. After the sterilization is completed, the valve 12 is closed, and the valves 22 and 2o are slightly opened to continue discharging steam little by little from the valve 20. Note that the steam may have a temperature that is used for heat sterilization in normal culture, and may contain some condensed water depending on the case. Next, the culture tank itself and the culture 7& charged therein are sterilized by heating and steam by supplying steam from a steam pipe (not shown) within θ1°. Sampling: Sampling of the culture solution in the culture tank 10 during cultivation. When performing this, the valve 22 and valve F20, which are slightly open, are closed, and then the valves 12 and 18 are opened, and the culture itl sent out from the plumber 1 due to the internal pressure of the culture tank 610 is filtered by the filtration filter 15. The filtrate is collected as a sample through pipes 16 and 17. Once the collection is complete, press valve 12.
Then, the valve 18 is closed, and then the valve 22 and the valve 20 are slightly opened again to continue discharging steam from the valve 20 little by little, and the steam seal is performed while backwashing the filter 15. Since sampling is performed through the filtration filter 15 in this way, the large solid content and cultured bacteria in the culture solution are removed from the collected sample, and the sample is directly transferred to an analyzer (not shown). can be applied. Furthermore, even if a filtration filter with a pore size exceeding 0.2μ is used, the sampling device including the filtration filter 15 on the upstream side of the valve 18 has already been sterilized, and furthermore, the filtration filter 15 acts like a check valve. So,
The filtrate (sample) does not backflow, and the culture solution can be collected without contaminating the culture tank 10 with bacteria. Backwashing of the filtration filter: As mentioned above, after the sampling operation is completed, a steam seal is performed, so this steam seal also serves as backwashing of the filtration filter, so it is usually necessary to backwash the filtration filter anew. There's no need. After the sampling operation is completed, if the valves 22 and 20 are not completely closed and steam sealing is not performed, the filtration filter 1
5, or as necessary after the completion of the above-mentioned sampling operation, valves 22 and 20 are opened while well 12 and valve 18 are closed, and the steam is passed through filter 15. The water is discharged through the valve 20 to perform backwashing of the filter. By steam sealing and steam backwashing as described above,
Compared to conventional backwashing using sterile air or filtrate, the clogging of the filter can be removed quickly and thoroughly, and the filter and the piping system connected to it can be sterilized at the same time. In addition, backwashing using steam thermally denatures the gel-like substance formed on the surface of the filter and improves its removability, making it far superior to conventional backwashing that cannot use steam. It is thought that this will produce an effect. Therefore, the steam backwashing made possible by the present invention can simultaneously recover from clogging of the filter and sterilize the filter, and has a synergistic effect. Furthermore, in the device of this invention, the filtration filter is
It is also possible to easily replace it aseptically without affecting the culture system in any way during culture. That is, in FIG. 1, with the valve 12 closed, the filtration filter 15 is removed from the storage container 35, or the storage container 35 is removed from the piping 14.16 and replaced. Thereafter, the valves 22 and 20 may be opened to pass steam through the newly replaced filter for heat sterilization. In addition, when replacing the filtration filter during culture is not considered, the storage container 35 may be configured integrally with the piping 14, 16, or furthermore, the storage container 35 itself may be configured with piping. Furthermore, in the apparatus of the present invention, samples containing bacterial cells and SS (suspended solid substances) can be collected from the drain port downstream of the valve 20, so various samples can be collected and a wide range of uses can be achieved. That is, valve 22 in FIG.
20 is opened and steam is passed through the pipes 14, 19, and 13 to heat and sterilize these pipe systems. Next, the valve 12 is slightly opened and the piping systems 13, 19 and 1.4 are cooled down while being washed with the culture solution. Next, the valve 22 is closed, and the culture solution is collected from the drain port downstream of the valve 20. When sampling is completed, valve 22 is opened while valve 12 is closed to re-sterilize the sampling device with steam, and the switching valves 20 and 22 are closed to return to the original state. This operation allows for aseptic sampling. Note that, as shown in FIG. 1, the downstream piping 24 of the valve 18
An extraction pump 25 may be provided in the culture tank 10, and filtration and sampling may be performed using the suction force of the pump 25 and the internal pressure of the culture tank 10. By providing the extraction pump 25, the sample can be easily extracted from the filtration filter 15, and the collected sample can be further transported to an analytical instrument. In the embodiment shown in Fig. 1, the valves 12, 18, and 20° 22 are all two-way valves, but if steam sealing is not performed,
Instead, the number of valves may be reduced by using T-boat type three-way valves at the connection points of the pipes 13° 1, 4, 19 and the pipes 16, 17° 21. In the embodiment shown in FIG. 1, the pipe 14 is used as a common pipe for introducing the culture solution into the filter and for discharging steam from the filter. Similarly,
The pipe 16 is used as a common pipe for introducing steam into the filter and for extracting the filter l& from the filter. However, as shown in FIG. 2, the culture l&introduction piping 13 and the steam discharge piping 19 are directly connected to the culture solution introduction side of the storage container 35 (filtration filter 15), and the steam introduction piping 21 and the filtrate extraction piping 17 may be directly connected to the filtrate permeation side of the storage container 35 (filtration filter 15). The filtration filter 15 in the embodiment shown in FIG. 1 can be used in either a direct flow type or a parallel flow type, but when a parallel flow type filtration filter is adopted,
It is preferable to provide a piping system as shown by broken lines in FIG. This piping system includes a pipe 26 connected to the culture wave introduction side of the filtration filter 15, a circulation pump (rotary pump in this example) 27, a valve 28, and a pipe 2 communicating with the culture wave introduction side 10.
9 are connected in this order. Further, a branch pipe 31 having a drain P30 is connected between the circulation pump 27 and the valve 28. As a result, the pipe 11,
A culture solution circulation system consisting of the valve 12, piping 13, piping 14, filtration filter 15, piping 26, circulation pump 27, valve 28, and piping 29 is formed, and this circulation system prevents clogging of the filtration filter 15. This can be prevented even more effectively. The operation of the apparatus shown in FIG. 1 equipped with the above-mentioned culture medium circulation system will be explained below. First, before starting culture, valve 12.28
is closed and steam is supplied into the culture tank 10 from a steam pipe (not shown) to sterilize the culture tank mortar and the medium charged therein, as well as the pipe 11 and the two pipes by heating with steam. On the other hand, with valve 18 closed, valves 20, 30, and 22 are opened to steam (
Steam is supplied from a 3% supply source 23, and piping 21, piping 16,
Filtration filter 15, piping 14, piping 13, piping 19,
The piping 26, the circulation pump 27, and the branch pipe 31 are sterilized by heating and steam. After sterilization, the valves 22, 20, and 30 are slightly opened, and steam sealing is performed in the same manner as described above. When sampling, close the valves 22, 20.30, then open the valve 12.28, start the circulation pump 27 to flow the culture solution in the culture tank 10 into the culture solution circulation system, and open the valve 18. Then, the filtered waves transmitted through the filter 15 are collected. When the sampling is completed, the circulation pump 27 is stopped, the valve 12.28.8 is closed, and then the valves 22 and 20.30 are slightly opened to perform the steam sealing as described above. This allows sterilization and backwashing to be performed at the same time. Note that if steam sealing is not performed, valves 12.28.
After completing the sampling operation by closing valve 18,
0.30.22 is opened and steam is supplied from the steam supply source 23 to the filtration filter 15 to backwash the filtration filter. Thereby, backwashing and sterilization can be performed simultaneously in this case as well. As a result of making the pore size of the filtration filter as large as possible without allowing the bacterial cells in the culture chamber to pass through, the filtrate (
Even if some solids remain in the sample), analyzers such as high-performance liquid chromatography systems have pre-filters and pre-columns, so even such filtered lfk can be directly applied to the analyzer. However, if you want to replace the prefilter or precolumn, or if you want to extend the cleaning interval, or if you want to replace the prefilter or precolumn
If it is desired to completely eliminate the pre-column, the filtration filters may be arranged in multiple stages as practically shown in FIG. In this case, a coarse filtration filter 1 is placed on the culture tank side.
5a, and a piercing filter 15 is used at the rear stage.
Use b. When filtration filters are arranged in multiple stages, the extraction pump 2 is placed after the latter stage filtration filter 15b.
5 must be installed for suction filtration. When a multistage filter as shown in FIG. 3 is used, the backwashing operation can be performed by the following procedure. For example, when the sampling operation is completed and all the valves in FIG. Steam from source 23 is passed through post-filter 15b. Next, in order to backwash the previous stage filter 158,
Valve 18a and drain valve 20a are opened while valve 22 remains open, and drain valve 20b is closed. As a result, the steam from the steam supply source 23 is introduced into the front filter 15H via the rear filter 15b. After these backwash operations are completed, either the valve 22 and the drain valve 20a are slightly opened to perform steam sealing, or all the valves are closed. FIG. 4 shows an example in which the sampling device of the present invention is directly connected to an analysis device to completely automate the analysis. In the figure, the parts incorporating the sampling device of the present invention shown in FIG. 1 are designated by the same reference numbers (12, 15, 18, 20
, 22°23 25.27, 28, 30.35), the explanation will be omitted. The sample liquid (a-filtrate) from the filtration filter 15 is sent by the pump 25 via the check valve 32 to a bubble removal sample pot, where air bubbles in the liquid are removed. A standard solution for calibrating the analyzer is sent to the bubble removal sample box 1 through a check valve 33 by a pump 41, or washing water for cleaning the piping system and each device is sent by a pump 42. It is sent through the check valve 34 and air bubbles are removed as well. The air bubble removal sample pot has a capacity of approximately 3 mg and consists of an F-shaped outer cylinder sealed by a top wall and a bottom wall, and a small diameter inner cylinder with an open top end, and an overflow pipe on the top wall of the outer cylinder. is connected, and the inner cylinder into which each liquid of sample liquid, standard liquid, and washing water flows is penetrated through the bottom wall of the outer cylinder. 11 The liquid overflowed from the top of the inner cylinder and the gas in the liquid was separated from the liquid.
One body flows down and stays between the outer circumference of the inner cylinder and the inner circumference of the outer cylinder, and is sucked and transported by the pump 43 from a pipe provided on the bottom wall of the outer cylinder. Therefore, in the bubble removal sample pot, the flow rates of the pumps 25, 41, 42 and the pump 43 are set so that the inflow amount is greater than the outflow amount, so that the liquid in the pot is exhausted and the pump 43 does not suck air. ing. The air (bubbles) separated from the liquid in the pot is sent from the top of the pot through an overflow pipe to a drain 1 and discharged to the outside. The sample liquid from which air bubbles have been removed in the air removal sample pot is sent via a sampling injector by a pump 43, a three-way valve 45, and a prefilter to an online automatic analyzer, where it is aliquoted. The sampling injector collects a predetermined volume of each of the sample liquid, floating QWI, or purified water sent from the pump 43, and transfers the predetermined amount of each liquid sampled into the injector to the eluent that is pumped from the pump 44. This sampling injector is a device that extrudes 1 m of each liquid to the device and discharges 1 m of excess liquid to the drain 2. This sampling injector is equipped with a six-way valve as shown in Fig. 5A and B. It consists of a sampling loop (tube), and the sampling loop holds a predetermined volume of each liquid to be sent to the analyzer.To explain an example of the operation of the sampling injector, in the state shown in Figure 5A, the pump The sample solution, washing water, or standard solution sent from the pump 43 passes through the sampling loop and is directly discharged to the drain 2. On the other hand, the eluent from the pump 44 passes through the six-way valve and flows to the analyzer. Next, when the six-way valve is rotated to the state shown in FIG. On the other hand, the sample liquid, washing water, or standard liquid continuously sent from the pump 43 passes through the six-way valve and is discharged as it is to the drain 2.The sample liquid from the sampling injector is passed through the three-way valve 45. The sample liquid is sent to the online automatic analyzer through a pre-filter.The pre-filter is provided to remove dust from the sample liquid and to prolong the replacement period of the pre-column. sample i (e.g. by time)
Switch the flow of & from prefilter A to B or from B to A. The online automatic analyzer consists of a PLC (high performance liquid chromatography) device, an analyzer, and a data computer. Cat 31 has UV (ultraviolet visible spectrometer) and R1 (differential spectrometer)
The data angle q analysis computer is equipped with each of them, and the analysis of the reciprocal term 1-1 is performed. In response to the signal received, the 11PI and C devices are controlled to start analysis, and the analysis results are sent to the computer. A sampling and analysis system for the gas in the culture tank is built into the system, and a gas mass spectrometer is used to determine the concentration of various gases in the culture tank, mainly oxygen concentration, carbon dioxide gas concentration, etc., and the measurement results are sent to a computer. The computer receives signals from various sensors and analyzers, interrupts the culture state, and instructs the process controller to take necessary measures.The process controller receives instructions from the computer and controls the culture. In addition to controlling the 11 controls of the sampling device of the present invention and the f of the analysis 11
Perform fiη control. Therefore, the supply of acid, alkali, and sterile air to the culture medium in the culture tank is controlled by the process controller via the computer based on information from the pH sensor, Do sensor, etc.
Nutrient sources such as sugars are controlled by a process controller via a computer based on the analysis results of samples by an 11 PLC device or the like. An example of the operation of the sampling device of the present invention will be described below with reference to the time chart of the sampling device shown in FIG. First, prior to the first sampling [1], the analyzer is calibrated using a standard solution. For this purpose, the pumps 41, 43 are activated to send the standard solution to the degassing sample pot and further to the sampling injector. Initially, this standard solution passes through the sampling loop in the sampling injector and is discharged to the drain 2, but by operating the sampling injector's main valve to bring it into the state shown in Figure 5B,
Specified capacitance 'l' included in the sampling loop
tR is pushed out by the melt M pump sent by the pump 44, which is constantly operating, and is sent through the pre-filter A by selection of the three-way valve 45 to the automatic needle one-loaf device, where it is calibrated. Go and adjust the analyzer. After the calibration of the analyzer with the standard solution is completed, the pump 41 is stopped and the pump 42 is activated. This allows the washing water to flow to the bubble removal sample pot and further to the pump 43.
and sent to the sampling injector. By operating the six-way valve in the sampling injector, a predetermined amount of wash water was sent to the analyzer through Prefilter-A in the same manner as above, and the standard solution was washed without being detected by the analyzer. Make sure that. Calibration using such a marker and cleaning with washing water may be performed about once a day. During the transport of the standard solution and wash water to the analyzer, the valves 30, 20 and 22 are kept slightly open to steam-seal the filter 15 and the piping system connected thereto. Then, in order to sample the first sample liquid from the mouth, the valves 30, 20 and 22 are closed;
Meanwhile, valves 28.12 and 18 are opened. As a result, the culture wave in the culture tank is filtered by the filtration filter 15, and the filtrate (sample liquid) is passed through the check valve 32 by the pump 25.
The sample is sent to the bubble removal sample pot via the pump 43, and further sent to the sampling injector by the pump 43. By operating the six-way valve in the sampling injector, a predetermined amount of sample liquid is sent to the analyzer through the prefilter A in the same manner as described above, and a predetermined analysis is performed. The surplus of the culture solution introduced from the culture tank into the filter 15 is circulated by the pump 27 through the valve 28 to the culture tank. After sampling, pumps 27.25 and 43 are stopped, valves 28.12 and 18 are closed, and valve 30.20 is closed.
and 22 are opened again to backwash the filtration filter 15 using steam from the steam supply source 23. After the first sampling (1) is completed, the valves 30, 20 and 22 are slightly opened to seal the filter 15 and the piping system connected thereto with steam. During this time, the three-way valve 45 is operated to switch from pre-filter A to B, and if necessary, pre-filter A is replaced. Part 2: “If you get excited about sampling 1 and after that,
Open valves 28.12 and 18, valves 30.20 and 22.
After closing, sampling is performed in the same manner as the first time, and after the sampling is completed, backwashing is performed in the same manner as in the first time 1. Experimental Example 1 Using the sampling device of the present invention shown in FIG. 1, which is equipped with a culture solution circulation system, an experiment was conducted to confirm the backwashing effect of the filter by steam. A ceramic filtration filter with a pore size of 0.2 μm was used as the filtration filter. While culturing baker's yeast in a medium containing molasses as a carbon source, the culture medium was continuously circulated through a filtration filter (membrane flow rate 0.16 a/5ec) every 20 minutes for 5 minutes (15
The filtrate was sampled at a sampling timing of 5 minutes after 5 minutes had elapsed, and the amount of culture solution permeated was measured. When the amount of transmission becomes 20 mJ7/ll1n or less,
The filtration filter was backwashed with steam, and the state of recovery of the amount of culture solution permeated after backwashing was investigated. The measured values of the amount of culture solution permeated were plotted and displayed as a continuous line, and the results are shown in FIG. As a comparative experiment, an experiment was conducted in which a conventional apparatus as shown in FIG. 12 was used, and filtrate was used instead of steam to backwash the filter. The specifications of the filter and other experimental conditions were the same as in the above experiment. The results of the comparative experiment are shown in the 8th
As shown in the figure. These experimental results show that backwashing using steam recovers the amount of permeation through the filter to the same level as the initial level, but when backwashing using filtrate, the amount of permeation does not recover much and the sample The result was that it was not possible to collect sufficient amount over a long period of time. Therefore, it can be seen that backwashing using steam is more effective. Experimental Example 2 Using the sampling device of the present invention shown in FIG.
Gllus nlger [Aspergillus niger (filamentous fungus)] was cultured, and a sampling experiment was conducted on a medium containing a large amount of bacterial cells and solid matter. For this experiment, the circulation pump was started at one point near the end of the culture, and the culture 11k was circulated through the filtration filter at a membrane surface flow rate of 0.16 m/sec, and the amount of culture solution permeated from the filtration filter was set to δP1. Established. Initially, a ceramic filter with a pore size of 0.2 μm was used as the filtration filter, but the filter became clogged within 10 minutes, making it impossible to collect a sample. After backwashing with steam, sampling was attempted again, but it soon became impossible to collect samples. Therefore, sampling was stopped, the ceramic filtration filter with a pore size of 0.2μ was replaced with a ceramic filtration filter with a pore diameter of 2.0μ, and after sterilization with steam, the circulation pump was started again and the culture solution was filtered at the same membrane surface flow rate. The amount of culture solution permeated was measured. The above results are shown in FIG. There was no contamination of the culture tank due to sampling operations, including filter replacement operations. From the results shown in Figure 9, it is possible to perform stable sampling with a filtration filter with a pore size of 2.0μ, and even more stable sterile continuous sampling is possible by using backwashing with steam. It was also found to be effective for sampling culture fluids of animals, etc.

【Effect of the invention】

According to the sampling device of the present invention having the above-described configuration, it is possible not only to separate bacterial cells etc. from the culture solution and collect a sample that can be directly applied to an analytical device such as chromatography, but also to heat the filtration filter using steam. Sterilization and backwashing in 11 times! You can (te) to j.As a result,
There is no problem of contamination with fresh sludge during sampling, and therefore there are no restrictions on the pore size of the filtration filter, and even if the filtration filter becomes clogged, the clogging can be prevented by backwashing, and it can be reversed by conventional filtrate etc. It can be removed much more effectively than washing and restore the filtration effect. The ability to backwash and heat sterilize the filtration filter with steam in this way, along with the ability to collect samples that can be applied directly to the analysis device, also makes it possible to perform sampling by connecting the sampling device to an analysis device such as chromatography. Facilitates the formation of automated analysis systems. Furthermore, since it is possible to collect samples containing bacterial cells and SS (suspended solid substances) from the drain port as necessary, various samples can be collected and a wide range of uses can be achieved.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of an embodiment showing the basic configuration of the sampling device of this invention; FIGS. 2 and 3 are explanatory diagrams showing different embodiments of the sampling device of this invention; FIG. An explanatory diagram showing an embodiment of a fully automated analysis system incorporating the sampling device of the invention; FIGS. 5A and B are explanatory diagrams showing the operation of the sampling injector used in the system of FIG. 4; FIG. A time chart of the sampling device of this invention in the system shown in the figure; FIG. 7 is a graph showing the backwashing effect of steam using the sampling device of this invention; FIG. 8 is a graph showing the backwashing effect of steam using the sampling device of this invention; Backwashing effect by filtered liquid 1
Figure 9 is a graph showing the difference in the backwashing effect of steam when filters with different pore sizes are used using the sampling device of the present invention; Figure 1O;
FIGS. 11 and 12 are explanatory diagrams each showing a conventional sampling device having an optical configuration. 10...Culture tank, 12.18.20.21...Valve (switching means), 13...Culture solution introduction piping, 15...
・Filtration filter 17...Filtrate extraction piping, 1
9...Steam exhaust pipe, 21...Steam introduction pipe,
35...Storage container.

Claims (1)

[Claims] 1. A culture solution introduction pipe for introducing the culture solution from the culture tank to one side of the filtration filter housed in a container, and a filtrate that has passed through the filtration filter to the other side. A filtrate extraction pipe is provided, and a steam introduction pipe for introducing steam to the filtrate permeation side of the filtration filter is provided, and a steam introduction pipe is provided to the culture solution introduction side of the filtration filter, and the steam that has passed through the filtration filter or its condensed water is provided. A step of providing a steam exhaust pipe for discharging steam, introducing the culture solution from the culture tank to the culture solution introduction side of the filtration filter, and extracting the filtrate that has permeated to the filtrate permeation side; 1. A sampling device for a culture tank, characterized in that a switching means is provided for switching between introducing steam into a permeation side and discharging permeated steam or condensed water to a culture solution introduction side. 2. The sampling device according to claim 1, wherein the culture solution introduction pipe, the filtrate extraction pipe, the steam introduction pipe, and the steam discharge pipe are each provided independently. 3. The sampling device according to claim 1, wherein the culture solution introduction pipe and the steam discharge pipe are partially constructed of a common pipe. 4. The sampling device according to claim 1, wherein the filtrate extraction piping and the steam introduction piping are partially constructed of a common piping. 5. The sampling device according to claim 1, wherein the switching means comprises a valve provided in each of the pipes.