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DEVICE FOR DETERMINING AND MONITORING THE PHYSIOLOGICAL STATES OF MICROBIAL CULTURES IN EACH INDIVIDUAL MICROBIOREACTOR OF A MICROTITER PLATE

20170313970 · 2017-11-02

    Inventors

    Cpc classification

    International classification

    Abstract

    A device for determining and monitoring the physiological state of microbial cultures in each individual microbioreactor of a microtiter plate, wherein a gas space of each microbioreactor of the microtiter plate is accessible via an inlet opening and outlet opening, includes means for shaking the microtiter plate and a gas supply system suitable for purging the gas space of each microbioreactor with a stream of purge gas in a purging phase. A shut-off device is arranged directly on each microbioreactor for interrupting the stream of purge gas. The flow resistances in the gas supply system and the flow resistance of each microbioreactor are configured so that the stream of purge gas in the purging phase is substantially equal in all of the microbioreactors. The device includes a measuring device configured to detect the physiological state of the microbial culture in each individual microbioreactor.

    Claims

    1. A device for determining and monitoring a physiological state of microbial cultures in each individual microbioreactor of a microtiter plate, comprising: a shaker device for shaking the microtiter plate, a gas supply system suitable for purging a gas space of each microbioreactor with a stream of purge gas through respective inlet openings and outlet openings for the each microbioreactor in a purging phase, a shut-off device arrangeable directly on each microbioreactor and suitable for interrupting the stream of purge gas, wherein flow resistances in the gas supply system and the flow resistance to the each microbioreactor are configured in such a way that the stream of purge gas in the purging phase is substantially equal in all of the microbioreactors, and a measuring device configured to detect the physiological state of the microbial culture in each individual microbioreactor in a measuring phase while the stream of purge gas is interrupted.

    2. The device according to claim 1, wherein the gas supply system comprises a purge gas feed-in and a gas distribution system including the inlet openings and the outlet openings, the gas distribution system being configured to deliver the fed-in purge gas and to remove the purge gas from the each individual microbioreactor.

    3. The device according to claim 2, wherein the gas distribution system further comprises a central delivery line, which extends from the gas feed-in to a subdistribution arranged on the microtiter plate for conducting the purge gas to and from the inlet openings and the outlet openings.

    4. The device according to claim 3, wherein the gas supply system includes a flow-controlling component arranged in the central delivery line.

    5. The device according to claim 3, wherein the gas supply system includes a wash bottle arranged in the central delivery line.

    6. The device according to claim 1, wherein the shut-off device includes an inlet valve with a valve seat surrounding the inlet opening and a pneumatically actuated shut-off membrane for opening and closing the inlet opening.

    7. The device according to claim 6, wherein the shut-off device further includes an outlet valve with a valve seat surrounding the outlet opening and also a pneumatically actuated shut-off membrane for opening and closing the outlet opening.

    8. The device according to claim 6, wherein a pressure chamber which can be acted upon by underpressure and/or overpressure, and which is configured for simultaneous pneumatic actuation of the shut-off membrane of several inlet valves, is arranged on the side of the shut-off membrane of several inlet valves that faces away from the valve seat.

    9. The device according to claim 7, wherein a pressure chamber which can be acted upon by underpressure and/or overpressure, and which is configured for simultaneous pneumatic actuation of the shut-off membrane of several outlet valves, is arranged on the side of the shut-off membrane of several outlet valves that faces away from the valve seat.

    10. The device according to claim 1, wherein the shut-off devices of all the microbioreactors are identical.

    11. The device according to claim 1, wherein the flow resistance of each microbioreactor in the purging phase is higher than the flow resistances of the gas distribution system as far as the respective microbioreactor by at least a factor of 50.

    12. The device according to claim 11, wherein the flow resistances of all the microbioreactors are substantially equal.

    13. The device according to claim 1, wherein the inlet opening determines the flow resistance of the each microbioreactor.

    14. The device according to claim 1, wherein the outlet opening determines the flow resistance of the each microbioreactor.

    15. The device according to claim 1, wherein a cross sectional area of the inlet opening of each microbioreactor is smaller than a cross sectional area of the outlet opening of each microbioreactor.

    16. The device according to claim 1, wherein at least a portion of the measuring device for detecting at least one parameter of the microbial culture representative of the respiration activity is arranged in each microbioreactor.

    17. The device according to claim 1, wherein the measuring device comprises: at least one passive measuring element arranged in each microbioreactor, a measurement signal of the at least one passive measuring element changing as a result of a change of respiration activity, transducers for converting the measurement signals to electrical signals, and transmission lines for transmitting the measurement signal between each passive measuring element and one of the transducers.

    18. The device according to claim 17, wherein each the at least one passive measuring element is an indicator layer arranged permanently on a transparent surface of the microbioreactor and reacts to changes of the gas concentration in the gas interior by changing the emitted electromagnetic radiation, and wherein each of the transducers is designed as an optoelectronic component.

    19. The device according to claim 17, further comprising an optical multiplexer with first ports and second ports, wherein the number of the passive measuring elements and the number of the transmission lines is greater than the number of the transducers by an integral multiple, and the transmission lines are connected to the first ports of the optical multiplexer and the transducers are connected to the second ports of the optical multiplexer, wherein measurement signals lying at different first ports can be switched through in succession to one of the second ports.

    20. The device according to claim 17, wherein each of the transducers comprises a modulatable light source and an optoelectronic sensor, and the light sources of all the transducers have different modulation frequencies.

    21. The device according to claim 21, wherein all of the shut-off devices are integrated in a cover that can be fitted onto the microtiter plate.

    22. The device according to claim 3, the subdistribution for the purge gas is integrated in a cover that can be fitted onto the microtiter plate.

    23. The device according to claim 8, wherein the pressure chamber which can be acted upon by underpressure and/or overpressure, and which are provided for pneumatic actuation of the shut-off membrane, is integrated in a cover that can be fitted onto the microtiter plate.

    24. The device according to claim 21, further comprising a sterile barrier arranged between the microtiter plate and the cover.

    25. The device according to claim 1, further comprising a barrier arranged between the inlet opening and outlet opening of each microbioreactor in such a way that a short circuit of the stream of purge gas between inlet and outlet is suppressed.

    26. The device according to claim 1, the shaking device for shaking the microtiter plate includes a shaker tray.

    27. The device according to claim 21, wherein the cover is composed of a plurality of interconnected plates.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0037] The invention is explained in more detail below with reference to the figures, in which:

    [0038] FIG. 1 shows a schematic view of a device according to the invention,

    [0039] FIG. 2 shows a schematic sectional view through a microbioreactor of the device according to the invention,

    [0040] FIGS. 3a and 3b show schematic views of pneumatically actuated valves of a microbioreactor according to FIG. 2, in different switching positions,

    [0041] FIGS. 4a and 4b show schematic sectional views along the lines 33, 34 according to FIG. 2,

    [0042] FIG. 5 shows a schematic sectional view of a cover for a microtiter plate according to the present invention,

    [0043] FIG. 6a shows an isometric view of an optical multiplexer, and

    [0044] FIG. 6b shows a schematic plan view of the multiplexer in order to illustrate the mode of operation.

    DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

    [0045] FIG. 1 shows a device for determining the physiological state of microbial cultures in each individual microbioreactor (2) of a microtiter plate (3), wherein the microtiter plate (3) is set in a shaking motion by a shaker device including a shaker tray (4). A controlled, uniform supply of a purge gas to each individual microbioreactor (2) is intended to be permitted according to the invention under the shaken conditions. With a device according to the invention, it is possible, for example, to detect an oxygen limitation in each individual microbioreactor (2), without directly measuring the dissolved oxygen concentration in the microbial culture.

    [0046] The oxygen transfer rate (OTR) is calculated as follows:

    [00001] O .Math. .Math. T .Math. .Math. R = d PO .Math. .Math. 2 dt .Math. Vg V f .Math. .Math. 1 .Math. R .Math. T formula .Math. .Math. 1 [0047] dpO.sub.2/dt: differential quotient for oxygen particle pressure over time [bar/min] [0048] V.sub.g: gas volume of the microbioreactor [ml] [0049] V.sub.fl: liquid volume of the microbioreactor [ml] [0050] T: temperature [K] [0051] R: gas constant [bar*l/mol/K]

    [0052] From the shape of the decay curve of the oxygen partial pressure in the measuring phase, it is possible to ascertain whether the oxygen transport is dependent on the oxygen consumption rate of the microorganisms (limited by reaction) or on the mass transfer (gas phase to liquid phase) (limited by mass transfer). In the first case, the oxygen consumption is independent of the driving partial pressure gradient, i.e., the differential quotient from the formula 1 can be replaced by a difference quotient:

    [00002] Δ PO .Math. .Math. 2 Δ .Math. .Math. t = O .Math. .Math. T .Math. .Math. R .Math. V fl .Math. R .Math. T Vg formula .Math. .Math. 2

    [0053] Formula 2 shows that, with a linear oxygen partial pressure drop in the measuring phase, there is no oxygen limitation of the culture.

    [0054] If there is an oxygen limitation (limited by mass transfer), the oxygen consumption is no longer independent of the driving partial pressure gradient, and the equation for the partial pressure drop in the measuring phase is as follows:

    [00003] ln .Math. P O .Math. .Math. 2 2 P O .Math. .Math. 2 1 Δ .Math. .Math. t = k L .Math. a .Math. V fl .Math. R .Math. T Vg .Math. He formula .Math. .Math. 3 [0055] k.sub.La: volumetric mass transfer coefficient [l/h] [0056] He: Henry's constant [bar*l/mol] [0057] P.sub.O21: oxygen partial pressure at the start of the measuring phase [bar] [0058] P.sub.O22: oxygen partial pressure at the end of the measuring phase [bar]

    [0059] This dependency on the driving partial pressure gradient leads to a non-linear curve shape.

    [0060] Each microbioreactor (2) has an inlet valve (5) and an outlet valve (6), which are closed in the measuring phase of the device. Both the inlet valve (5) and the outlet valve (6) are arranged directly on each microbioreactor, as can be seen from FIGS. 2-5. With the aid of measuring device (7), changes of the partial pressure of the purge gas are detected in each individual microbioreactor, and the transfer rates are calculated from these in a measurement computer (32). The measurement computer (32) moreover controls the inlet and outlet valves (5, 6), a mass flow regulator (8) and, if appropriate, an optional gas mixer unit (9).

    [0061] With a feed-in (10), the purge gas is delivered to the individual microbioreactors (2) via a gas distribution system comprising a central delivery line (11) and a subdistribution (12), which for reasons of clarity is not shown in FIG. 1, and said purge gas is removed from them again via the outlet valves (6). The subdistribution (12) is arranged in a cover (13) which is fitted onto the microtiter plate (3) and which has further functional elements for controlling the purge gas.

    [0062] A wash bottle (14) can additionally be arranged in the central delivery line (11) in order to compensate for loss of liquid caused by evaporation during cultivation. In order to ensure a substantially equal supply of purge gas in all of the microbioreactors (2), all of the inlet valves (5) and all of the outlet valves (6) of the microbioreactors (2) switch collectively in each case. Finally, the reference character (15) in FIG. 1 schematically indicates the flow resistance of each microbioreactor (2).

    [0063] The structure of the cover (13) is explained in more detail below with reference to the partial view in FIG. 2 in conjunction with FIG. 1. To illustrate the integration of the inlet and outlet valves (5, 6) and their pneumatic actuation, the view is not to scale. Between the cover (13) and the microtiter plate (3), a seal (16) is provided in order to prevent an escape of gas from the gas space (17) above the microbial culture (18) in the closed state of the two valves (5, 6). In structural terms, the cover (13) is composed of a transparent bottom plate (19), a middle plate (20) and a top plate (21). A switching membrane (22) is arranged between the top plate (21) and the middle plate (20).

    [0064] Inlet openings (23) and outlet openings (24) are located in the bottom plate (19), wherein the gas space (17) of each microbioreactor (2) is accessible via the inlet opening (23) and the outlet opening (24). Furthermore, the bottom plate (19) comprises a valve seat (25), which surrounds each inlet opening (23), and also a valve seat (26), which surrounds each outlet opening (24). The switching membrane (22) arranged between the middle plate (20) and the top plate (21) delimits first pressure chambers (27) and second pressure chambers (28). Depending on the controllable pressure prevailing inside the pressure chambers (27, 28), the elastic switching membrane (22) bears on the valve seats (25, 26) and closes the inlet and outlet valves (5, 6) collectively. The valve seats (25, 26) integrated in the bottom plate (19) form, together with the switching membrane (22) likewise integrated in the cover (13), all the shut-off means for interrupting the stream of purge gas into the microbioreactors (2).

    [0065] It will also be seen from FIG. 2 how the measuring device (7), which are shown merely schematically in FIG. 1, are partially integrated in the cover (13). A passive measuring element (29) is arranged on the surface of the transparent bottom plate (19) pointing toward the gas space (17) of each microbioreactor (2). The passive measuring element (29) is, for example, a fluorescence spot that is sensitive to partial pressure. The fluorescence spot reacts to changes of the gas concentration in the gas space (17) by changing the emitted electromagnetic radiation, which is transmitted contactlessly via a transmission line (30) from each microbioreactor (2) to a transducer (31) shown in FIGS. 6a and 6b. The transmission line (30), for example in the form of an optical fiber, is introduced, on the side lying opposite the passive measuring element (29), into a recess (30a) of the transparent cover (19).

    [0066] The reference character (35) finally indicates a part of the line structure of the subdistribution (12) delivering the purge gas, and the reference character (36) indicates a part of the line structure of the subdistribution (12) carrying off the purge gas. In addition, a flow-conducting barrier (37) is arranged on the underside of the bottom plate (19) between the inlet opening (23) and the outlet opening (24) of each microbioreactor (2), which barrier prevents a short circuit flow of the purge gas directly between the inlet and outlet openings (23, 24) when the valves are closed.

    [0067] With reference to FIGS. 3a and 3b, the actuation of the inlet valves (5), controlled by the measurement computer (32), is explained in more detail below on the basis of a sectional view of the cover (13). To generate an underpressure or overpressure in a first pressure chamber (27), an electrically driven pump (38) is provided whose suction side and pressure side are connected to the ports of a 5/2-way valve (39). In the switching position of the 5/2-way valve (39) shown in FIG. 3a, the suction side of the pump (38) is connected to the first pressure chamber (27) via a line (40). The elastic switching membrane (22) is pulled in the direction of the upper face of the first pressure chamber (27) by the underpressure. During the opened state of the inlet valves (5), the underpressure is maintained in order to ensure a controlled switching position. In the switching position of the 5/2-way valve (39) shown in FIG. 3b, the first pressure chamber (27) is connected fluidically to the pressure side of the pump (38). The overpressure now prevailing in the first pressure chamber (27) now presses the elastic switching membrane (22) onto the valve seat (25) of each inlet valve (5).

    [0068] Attached to the line (40) is a branch line with a throttle (53), which limits the underpressure in the switching state of the inlet valve (5) according to FIG. 3a and limits the overpressure in the switching state of the inlet valve (5) according to FIG. 3b.

    [0069] In order to switch the 5/2-way valve (39), the latter is connected to the measurement computer (32) via an actuator (39a). The actuation of the outlet valves (6) with the aid of the 2nd pressure chamber (28) takes place in the same way, and therefore a separate explanation of the valve actuation is unnecessary.

    [0070] The cross sections shown in FIGS. 4a and 4b, taken along the section lines 33, 34 according to FIG. 2, illustrate how several inlet valves (5) arranged in a row are actuated via a common first pressure chamber (27) and how several outlet valves (6) arranged in a row are actuated via a common second pressure chamber (28). For this purpose, each first pressure chamber (27) and each second pressure chamber (28), for actuation of a row of inlet and outlet valves (5, 6), is connected via a port (41) and (42), respectively, to the valve control system shown in FIG. 3.

    [0071] The partial section according to FIG. 5, corresponding to FIG. 2 in terms of the direction of sectioning, shows a total of three microbioreactors (2a, 2b, 2c) in order to illustrate the structure of the subdistribution (12) for the purge gas inside the cover (13). Each microbioreactor (2a, 2b, 2c) is a component part of a series of microbioreactors which extend perpendicularly with respect to the image plane and which are all separated from each other by partition walls (43). It will clearly be seen how the inlet valves (5) of the adjacent rows with microbioreactors (2a, 2b) are supplied with purge gas via the delivery line structure (35) in the opened state of the valves. It will moreover be seen how the outlet valves (6) of the adjacent rows with microbioreactors (2b, 2c) are fluidically connected to the removal line structure (36) for withdrawing the purge gas from the microbioreactors.

    [0072] The central delivery line (11), shown in FIG. 1, of the gas supply system is attached to the port (44) opening into the delivery line structure (35). By way of further line structures of the subdistribution (12) that are not visible in the sectional view according to FIG. 5, the purge gas delivered via the port (44) arrives at further rows (not shown in FIG. 5) of inlet valves (5).

    [0073] The length of the delivery and removal line structure (35, 36) to each individual microbioreactor (2a, 2b, 2c) is different. A uniform supply of purge gas to all of the microbioreactors is ensured in any case, since the cumulative flow resistances in the gas supply system as far as the respective microbioreactor (2a, 2b, 2c), i.e. in the delivery line (11) and subdistribution (12), are negligible compared to the flow resistance in the respective microbioreactor (2a, 2b, 2c). As a result, a single flow-controlling component, for example the mass flow regulator (8) shown in FIG. 1, is sufficient for a uniform supply of purge gas to all of the microbioreactors.

    [0074] The respiration activity of the microbial cultures leads to a change of the purge gas concentration in the closed gas space (17) of each microbioreactor (2) and is linked with a change of the electromagnetic radiation emitted by the fluorescence spot and coupled into the optical fiber. The optical fibers are routed from the microtiter plate (3) to an optical multiplexer (45), which is shown in FIG. 6a and which can be set up next to the shaker tray (4) shown in FIG. 1. The optical multiplexer (45) comprises a frame (46) with an annular body (47) which is mounted in a stationary position and on which a number of first ports (48) are arranged corresponding to the number of transmission lines (30) (optical fibers). Moreover, a rotary drive (49), for example in the form of a stepping motor, is secured on the frame (46), its output shaft being connected to a cylindrical mount (50). The axis of rotation of the mount (50) extends coaxially with respect to the axis of the annular body (47). Second ports (51) of the multiplexer are arranged on the mount (50) in such a way that they can be brought into communication with the first ports (48), by rotating the mount (50), for the measurement signals and the excitation light of the modulatable light source. One of the optoelectronic transducers (31) is attached to each second port (51). The number of the passive measuring elements (29) is greater than the number of the transducers (31) by an integral multiple.

    [0075] By actuation of the rotary drive, the electromagnetic signals at the first ports (48) can be switched through in succession to one of the second ports (51) and in this way delivered to one of the transducers (31), which converts the electromagnetic radiation delivered by the individual microbioreactors into electrical signals. Furthermore, the excitation light generated in the transducers (31) at the second ports (51) is switched through to one of the first ports (48) and in this way delivered to one of the passive measuring elements (29).

    LIST OF REFERENCE SIGNS

    [0076]

    TABLE-US-00001 No. Designation  1 device  2 a, b, c microbioreactors  3 microtiter plate  4 shaker tray  5 inlet valve  6 outlet valve  7 measuring device  8 mass flow regulator  9 gas mixer unit 10 feed-in (purge gas) 11 central delivery line 12 subdistribution 13 cover 14 wash bottle 15 flow resistance 16 seal 17 gas space 18 microbial structure 19 bottom plate 20 middle plate 21 top plate 22 switching membrane 23 inlet opening 24 outlet opening 25 valve seat (inlet valve) 26 valve seat (outlet valve) 27 1.sup.st pressure chamber 28 2.sup.nd pressure chamber 29 passive measuring element 30 transmission line 30a recess 31 transducer 32 measurement computer 33 section line 34 section line 35 delivery line structure 36 removal line structure 37 barrier 38 pump 39 5/2-way actuator 39a actuator 40 port 41 port of 1.sup.st pressure chamber 42 port of 2.sup.nd pressure chamber 43 partition walls 44 port 45 optical multiplexer 46 frame 47 annular body 48 first port 49 rotary drive 50 holder 51 second port 52 sterile barrier 53 throttle