FR2781391A1 - Homogenizing two liquids in bioreactor operation using two vessels, one for e.g. sampling, measurement and nutrient introduction, one for culturing, with flow alternating between them through filter maintaining sterility - Google Patents

Abstract

A process for homogenizing two liquids containing solids, solutes, and/or microorganisms comprises two hermetically sealed vessels (1, 2) connected by a tube (5) discharging into their upper volumes, and extended by a filter (6) in either or both of them. Liquid is transferred back and forth through the filter(s). A process for homogenizing two liquids containing solid and/or dissolved components and/or microorganisms comprises two hermetically sealed vessels (1, 2) connected by a tube (5) discharging into their upper volumes, and extended by a filter (6) in either or both of them. The filter comprises hollow fiber membranes connected at one end to the tube by a fitting (7), and sealed at the other. The filter retains a required minimum particle size. Each vessel is filled with one of the liquids, such that the tube is immersed at each end, covering the filter(s). Liquid is transferred back and forth through the filter(s). Measurements and/or samples are taken periodically from the reservoir(s). An Independent claim is also included for the corresponding equipment.

Description

METHOD AND APPARATUS FOR HOMOGENEATION

  TWO LIQUID MEDIA CONTAINING COMPONENTS

  SOLIDS OR DISSOLVED AND / OR MICROORGANISMS

  The invention relates to a method and an apparatus for the homogenization of two liquid media containing solid or

dissolved and / or microorganisms.

  Fermentation processes involving micro-

  organisms, such as yeasts or bacteria, are commonly used in many areas such as the food industry, oenology, the pharmaceutical industry, cosmetics, etc. However, the implementation of these techniques is is currently encountering difficulties encountered, on the one hand in the laboratory phase for obtaining reliable kinetic data intended for the optimization and modeling of fermentation processes, and on the other hand, in the exploitation phase industrial for monitoring and control of different parameters. Regarding the laboratory phase, the fermentation processes involving a single species of microorganisms, such as yeasts or bacteria, are currently fairly well controlled. Indeed, there are different models for predicting changes over time in biomass,

  substrates and products. On the other hand, when several species of micro-

  organisms are involved, an increasingly frequent case in an industrial environment, modeling, essential for the conduct of the process, of the various interactions that should be encouraged or inhibited depending on the goal to be achieved, is currently difficult or even nonexistent due to the lack or imprecision of experimental data, in particular on the distinct cell concentrations of each family involved, resulting from the heaviness of the analytical methods

discriminating to implement.

  The current techniques used in the analytical phase of the study of mixed populations of microorganisms are indeed long and

  tedious, or turn out to lead to very random results.

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  Thus, for example, observation under the microscope carried out in the case where the species are morphologically different, provides results of the growth of each species with a minimum percentage of error of the order of 7% which can reach 25% depending on the culture age, when microorganisms tend to have similar contours. In the most frequent cases of morphologically similar species, the current technique consists in resorting to DNA extractions and analyzes by electroporesis which prove to be cumbersome and expensive methods. Finally, in the case of amensalism-type interaction, o0 tests on solid or liquid culture media have been developed. However, their implementation requires a new fermentation, that is to say about twenty additional hours of experimentation, and this to provide a single point to

the main experiment.

  In an attempt to overcome these drawbacks, an apparatus has been designed, described in US Pat. No. 3,893,891, aiming to make it possible to use only the most conventional methods in microbiology recognized as reliable and

  rapid, in order to master the study of mixed populations of microorganisms.

  This device comprises for this purpose several culture chambers separated by filter membranes in each of which a culture can evolve, while allowing free diffusion between said chambers of metabolic products.

  of each of said cultures. So in the case of solids or micro-

  organisms, the particles or strains being pure in each chamber, the samples and analyzes of dry matter or biomass are carried out exactly as in the case of suspensions or pure cultures, techniques which are easy and perfectly mastered. However, in practice, such an apparatus designed to ensure, by natural diffusion, the transfer through the membranes of the dissolved compounds, has most often been found to be incapable of allowing the monitoring of the biomass concentration, because of the slowness from this transfer and from the presence of faster biological reactions, concentration gradients and the resulting osmotic pressure differences lead to problems of maintaining volumes in the chambers. Therefore, such a device is only intended for use with growing cells

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  slow such as more particularly plant cells, and not with microorganisms, such as yeasts or bacteria, more particularly intended

  to fermentations of industrial interest.

  Regarding the industrial exploitation phase of fermentation techniques, current conventional fermenters include covers with an abundance of tappings intended for the measurement of various operating parameters or for the introduction of multiple probes. However, such a design leads to numerous drawbacks. Indeed, and in the first place, the different tappings constitute as many weak points both from the point of

  l0 view of the tightness as the pressure resistance and sterility.

  In addition, the various measurement probes used must be sterilized and therefore made of a sterilizable material, the cost price of which obstructs the cost of such probes. In addition, any defection of a probe requiring its replacement or even any verification of the state of a probe, leads to

  the obligation to completely stop production of the bioreactor.

  Furthermore, when monitoring the process requires taking samples, the samples taken cannot be re-injected into the reactor afterwards and are therefore lost. It should be noted regarding this last point that when the measurements require only a simple contact with the solution, it has been

  designed measuring instruments that do not require sampling.

  However, such instruments appear to retain microorganisms, and by

  Consequently, they become clogged and quickly become unusable.

  The present invention aims to overcome all of the above-described drawbacks of current fermentation techniques and has as its first objective to provide a process making it possible to master the study of

  mixed populations of solid and / or dissolved components and / or micro-

  organisms by implementing conventional methods recognized as reliable and

rapid in microbiology.

  Another objective of the invention is to provide an apparatus which overcomes the drawbacks inherent in the concepts of current fermenters in

  matter of measurement and sample taking.

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  To this end, the invention relates to a process for the homogenization of two liquid media containing solid and / or dissolved components and / or microorganisms, characterized in that: two hermetically sealed reservoirs are used communicating via '' a pipe opening into the upper volume of said tanks, and extended in at least one of these tanks by a filtration module consisting of hollow fiber membranes sealed at one of their ends in a nozzle mounted on said pipe and closed at from their opposite end, said hollow fiber membranes having

  a cutoff threshold lower than the minimum size of the components and / or micro-

  organisms that we wish to confine in each tank, we fill each tank with one of the liquid media so that the communication line plunges into each of the liquid media and that each filtration module is submerged, 15. we control cyclically and alternatively the transfer of a volume of the liquid phase contained in one of the tanks to the other tank, through the communication line and each filtration module, and measurement measurements are made periodically.

  and / or taking samples from at least one of the tanks.

  The method according to the invention therefore consisted in isolating two liquid media on either side of a filtration module consisting of hollow fiber membranes so as to separate two species of components and / or micro-organisms while retaining a composition identity of the liquid phase 2s thanks to rapid transfer through the membrane ensuring mixing

  and the homogenization of the liquid phases of the two reservoirs.

  In addition, the mixing and homogenization of the liquid phases of the two reservoirs is obtained according to this process thanks to an alternating transfer of the latter between the two reservoirs through a single pipe, which also leads advantageously, to a regeneration of the

  filtration module and therefore gives this module a self-cleaning character.

2781391

  It should be noted, moreover, that the filtration module is technologically easy to produce and can be sterilized at the same time as the apparatus. In addition, given its positioning, this filtration module is easily accessible. Finally, by its design, this filtration module has a high filling rate (filtration area per unit volume of the module) and leads to a limitation of the dead volume from a point of view

charge transfers.

  According to a first application, such a method can be implemented for the study in the laboratory of mixed cultures of solid and / or dissolved components or / and of microorganisms. In this case, the particles or the strains being pure in each reservoir, the samples and analyzes of dry matter or biomass can be carried out as in the case of suspensions or pure cultures, techniques which are easy and perfectly controlled. According to a second application, this method can be implemented for the production of a sterile sampling chamber associated with a bioreactor, this sampling chamber then constituting one of the reservoirs, and the bioreactor the other reservoir. In this case, the measurement probes do not need to be sterilized, and the risks of contamination are reduced by reducing the tapping in the cover of the bioreactor. In addition, the probes are isolated from the bioreactor, and their verification or replacement does not require

no production stops.

  In addition, the method according to the invention has the advantage of making it possible to carry out all of the measurements and samples in the sampling chamber and to reintroduce the excess of sample into the bioreactor, and this without worrying about sterility due to the sterilizing nature of

  hollow fiber membranes used.

  According to this application, in addition, the filtration module

  can be placed either in the bioreactor or in the sampling chamber.

  The first solution has the advantage of eliminating all sterilization problems, apart from the bioreactor itself. The second solution presents

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  the advantage of allowing checks and possible replacements

easy from the filtration module.

  According to a preferred embodiment of the method according to the invention, each tank is kept under pressure by means of a pneumatic source, and the alternating transfer of the liquid phases is controlled by increasing the pneumatic pressure in one of the tanks, and by putting

  simultaneously the other tank at atmospheric pressure.

  This preferred mode of implementation firstly has the advantage of leading to an optimal reduction in dead volume from the point of view of charge transfer. In addition, the choice of gas allowing overpressure of the tanks and transfer remains wide. One can indeed operate with inert gases (strict anaerobic) or in aerobic condition. Finally, putting in the cyclic atmosphere of each of the reservoirs makes it possible to naturally evacuate the carbon dioxide produced by the

fermentation.

  According to another characteristic of the invention, the direction of flow of the liquid phases is reversed after transfer of a predetermined volume of each of said liquid phases. This mode of implementation makes it possible to maintain substantially stable liquid levels

in the tanks.

  In addition, in order to adjust the transferred volume, and advantageously, the level of liquid is detected in at least one of the reservoirs, and the reversal of the flow direction is controlled when a level is obtained. of

  predetermined liquid in said tank.

  The invention extends to an apparatus for the homogenization of two liquid media containing solid and / or dissolved components and / or microorganisms, characterized in that it comprises in combination: - two hermetically sealed reservoirs intended to contain each a liquid medium, a line for placing the two reservoirs in communication, having end sections each extending in one of said reservoirs so as to immerse in the liquid medium contained in the latter,

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  at least one filtration module mounted in the extension of one of the end sections of the tank connection pipe so as to be immersed in the liquid medium contained in said tank, said filtration module consisting of a bundle of hollow fiber membranes having a cutoff threshold lower than the minimum size of the solids and / or microorganisms to be confined in the reservoirs, said hollow fiber membranes being sealed at one of their ends, called the top, in a nozzle mounted on the end section of the pipe, and being closed at their opposite end, called the lower end, - circulation means adapted to allow transfers of liquid in both directions between the tanks, through of the communication line and of each filtration module, - means for controlling the circulation means adapted for comma cyclically and alternatively the

  transfer of a volume of the liquid contained in one reservoir to the other reservoir.

  According to another characteristic of the invention, the means for circulating comprise pneumatic means for pressurizing each tank, and means for venting each of said tanks, and the servo means are suitable for alternatively control the pressurization of one of the tanks and simultaneously the

  venting the other tank.

  In this case, moreover, and according to another characteristic of the invention: - the pneumatic means comprise a pneumatic source connected to each of the reservoirs by channeling means comprising, for each of said reservoirs, a closure member, - the air release members consist, for each tank, of a shutter member installed on the upper wall of said tank, - the servo means are connected to the shutter members of pressurization and setting atmosphere of the reservoirs, and

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  adapted for sequentially controlling the opening and closing cycles

of said organs.

  Furthermore, each tank is preferably equipped with a level detector, and the servo means are connected to said level detectors and programmed to control cycle inversions.

  for predetermined levels of liquid in said tanks.

  Other characteristics, aims and advantages of the invention

  will emerge from the detailed description which follows with reference to the appended drawings

  which represent by way of nonlimiting examples two embodiments

  preferential. On these drawings which form an integral part of this description:

  - Figure 1 is a schematic view of a first embodiment of an apparatus according to the invention, - Figure 2 is a curve representing the evolution in biomass and glucose for fermentation with Kluyveromices marxianus, - the Figure 3 is a curve representing the evolution in biomass and glucose for a fermentation with Saccharomyces cerevisiae, - Figure 4 is a curve representing the evolution in biomass and glucose for a fermentation with Kluyveromices and Saccharomyces, with separate strains according to the invention, - Figure 5 is a curve representing the evolution in biomass and glucose for fermentation with Kluyveromices and Saccharomyces, with strains mixed in a conventional fermenter, Figure 6 is a curve representing the evolution of the time of mixing depending on the volume moved, - and Figure 7 is a schematic view of a second

  embodiment of an apparatus according to the invention.

  The apparatus represented in FIG. 1 is an apparatus for studying in the laboratory mixed cultures of microorganisms intended to allow

  optimize industrial processes.

  It consists of two identical tanks 1, 2 with a maximum capacity of two liters each, closed by covers 3, 4

  airtight holes pierced with tapping holes.

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  These two reservoirs 1, 2 are placed in communication via a pipe 5 extending through orifices provided in the

  covers 3, 4 so as to open into the upper volume of said tanks.

  In addition, one end of the supply line 5 located in one of the tanks 2 is extended by a filtration module 6 consisting of hollow fiber membranes which can be sterilized (for example by steam), folded in a U shape and sealed together, at the level from their open end, in a molded resin end piece 7 designed to be mounted around said pipe by

  a system of the type known per se of ring and cable gland.

  The hollow fiber membranes of this filtration module 6 are microfiltration fibers (cut-off threshold of approximately 0.2 μm) and have, for example, respective internal and external diameters of 0.3 mm and 0.45 mm. The filtration module is made up of around a thousand fibers and thus has a filtration area of around a thousand square centimeters. In addition, the

  average fiber permeability is 2 ml / min / bar / cm2.

  This device also comprises a first pair of solenoid valves 8, 9, each mounted on the cover 3, 4 of a reservoir 1, 2, the feed inlets of which are provided with a sterilization membrane at their inlet. supply and are powered by an air source

  tablet (not shown) common to said solenoid valves.

  This device further comprises a second pair of solenoid valves 10, 11, each also mounted on the cover 3, 4 of a tank 1, 2, intended to act as vents for said tanks and provided

  a sterilization membrane at their outlet.

  The opening and closing cycle of these solenoid valves 8, 11, intended to allow an alternative transfer of a given volume of liquid from a reservoir 1, 2 to the other, is managed by an electronic unit 12 to which are connected two level detectors 13, 14 made of stainless steel arranged

each in a tank 1, 2.

  The operating conditions intended for monitoring the growth of mixed populations are as follows: firstly, 2781391 first, each reservoir 1, 2 is partially filled by means of the same volume of one of the liquid media containing one of the species considered, adapted so that the ends of the communication line 5 plunge into said liquid media and that the filtration module 6 is immersed. Prior to the actual start of the experiment, the volume of liquid to be transferred during each alternation of

  cycle is determined. It turns out that there is an ideal exchange volume,

  beyond which no gain in terms of quality of mixture can be obtained. This phenomenon emerges from the analysis of FIG. 6 which represents the experimental evolution of the mixing time as a function of the volume moved during each transfer. It emerges, indeed, from the examination of this figure, that for exchange volumes greater than or equal to 100 ml, the curves obtained are almost identical, and that consequently, it is useless to exchange

  s5 volumes greater than 100 ml within the framework of an apparatus as described above

  above. Once this exchange volume has been determined, the operating phase is effectively launched, and samples are taken periodically from a tap 15, 16 in each of the

covers 3, 4.

  Figures 2, 3 and 5 show evolution curves obtained during experiments carried out with a view to validating the device according to the invention. Firstly, FIGS. 2 and 3 represent evolution curves obtained for experiments having consisted of initially filling the two reservoirs 1, 2 with nutritive media (water plus nutrient comprising glucose), and inoculating only one of said reservoirs by means of Kluyveromices marxianus (Figure 2) and

  Saccharomyces cerevisiae (Figure 3).

  It emerges from the examination of these figures, on each of which are represented, on the one hand the evolution curve of the biomass content, and on the other hand the evolution curves of the glucose content in each of it 2781391 reservoirs 1, 2, that a homogenization of the concentrations in

  glucose in the liquid phases of said reservoirs.

  Glucose being, for these experiments, the limiting substrate, it therefore emerges from the analysis of the curves of FIGS. 2 and 3, that whatever the consumption kinetics, the homogenization of the solute concentration in the liquid phases of

two tanks.

  FIG. 5 represents, for its part, the evolution curves obtained on a pair of competition microorganisms on a substrate. In the example, the strains consist of Saccharomyces cerevisiae and

  Kluyveromices marxianus, and the substrate consists of glucose.

  The choice of such strains also aims to validate the device, because the two species are morphologically different and therefore identifiable under a microscope, and the distinct 1S growth kinetics can thus be obtained, even if the microorganisms are mixed . We observe, by comparison of the growth and disappearance curves of the substrate represented in FIG. 5, obtained according to the method of the invention, and those represented in FIG. 4 obtained from a mixture of the two species in a fermenter. classic of the same capacity a perfect

similarity of said curves.

  It is therefore clear from this comparison that the reaction takes place as in a conventional medium with two species mixed due to the homogenization of the liquid phases of the two reservoirs 1, 2, resulting from the

  alternative transfer through the filtration module.

  FIG. 7 represents a second application of the method of the invention aimed at producing an industrial bioreactor

  including a sterile sampling loop.

  To this end, the bioreactor 17 constitutes one of the reservoirs and is connected to a sampling chamber 18 constituting the second reservoir,

  via a communication line 19.

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  In addition, as previously, a filtration module 20 is disposed in one of the tanks, in the example the bioreactor 17, in the

  extension of the communication line 19.

  In addition, there are also provided as above, respectively two solenoid valves 21, 22 for pressurization and two solenoid valves 23, 24 for venting, reservoirs 17, 18 controlled by an electronic unit 25 capable of controlling the inversions of transfer, and to which are connected for this purpose two level detectors, respectively maximum and

  minimal, arranged in the sampling chamber 18.

  The sampling chamber 18 is finally equipped with probes such as 26, 27 and a nozzle 28 for sampling to ensure monitoring and surveillance of the reaction in the bioreactor 17. The principle of this device allows , in particular thanks to the transfers of the liquid phase between the bioreactor 17 and the sampling chamber 18, to carry out all of the measurements and to reintroduce the excess of sample into said bioreactor without having to worry about sterility problems because the hollow fiber membranes of the filtration module are sterilizing. In addition, the various sensors 26, 27 can be isolated from the bioreactor 17 and therefore be checked periodically and even

  replaced without the need to stop production.

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Claims (12)

  1 / - Process for the homogenization of two liquid media containing solid and / or dissolved components and / or micro-organisms, characterized in that: 5. two hermetically sealed communicating tanks (1, 2; 17, 18) are used by means of a pipe (5; 19) opening into the upper volume of said tanks, and extended in at least one of these tanks by a filtration module (6; 20) consisting of hollow fiber membranes sealed at one of their ends in a nozzle (7) mounted 10 on said pipe, and closed at their opposite end, said hollow fiber membranes having a cutoff threshold lower than the minimum size of the components and / or microorganisms than the we wish to confine in each tank, we fill each tank (1, 2; 17, 18) with one of the liquid media so that the communication line (5; 19) plunges into each of the liquid media and that each filter module one (6; 20) is immersed, cyclically and alternately controls the transfer of a volume of the liquid contained in one of the reservoirs (1, 2; 17, 18) to the other reservoir, through the communication line (5; 19) and each filtration module (6; 20), and measurements are taken periodically and / or samples taken from '' samples in at least one of the tanks (1, 2 17, 18).   2 / - Homogenization process according to claim 1, characterized in that each reservoir (1, 2; 17, 18) is kept under pressure by means of a pneumatic source, and the alternating transfer of the liquid phases is controlled increasing the pneumatic pressure in one of the tanks (1, 2; 17, 18), and simultaneously putting the other tank under pressure atmospheric.   3 / - Homogenization process according to one of   claims 1 or 2, characterized in that the direction is reversed 14 2781391   liquid phase flow after transfer of a predetermined volume of each of said liquid phases.   4 / - Homogenization process according to claim 3, characterized in that the liquid level in at least one of the reservoirs (1, 2; 17, 18) is detected, and in that the reversing the direction of flow when a predetermined liquid level is obtained in said reservoir. / - Apparatus for the homogenization of two media   liquids containing solid and / or dissolved components and / or micro-   o0 organisms, characterized in that it comprises in combination: - two hermetically sealed tanks (1, 2; 17, 18) each intended to contain a liquid medium, - a line (5; 19) for connecting the two tanks (1, 2; 17, 18) provided with end sections each extending in one of said reservoirs so as to immerse in the liquid medium contained in the latter, - at least one filtration module (6; 20) mounted in the extension of one of the end sections of the pipe (5; 19) for connecting the tanks (1, 2; 17, 18) so as to be immersed in the liquid medium contained in said tank, said module filtration consisting of a bundle of hollow fiber membranes having a cut-off threshold lower than the minimum size of the solids and / or microorganisms to be confined in the tanks, said hollow fiber membranes being sealed at one of their ends, so-called superior , in a nozzle (7) mounted on the end section of the pipe (6; 19), and being closed at their   opposite end, called the lower end.   - means (8-1 1; 21-24) of circulation adapted to allow transfers of liquid in both directions between the tanks (1, 2; 17, 18), through the communication line (5 ; 19) and of each filtration module (6; 20), - means (12; 25) for controlling the circulation means (8-11; 21-24) suitable for cyclically controlling and 2781391   alternatively the transfer of a volume of the liquid contained in a reservoir (1, 2; 17, 18) to the other reservoir 6 / - Apparatus according to claim 5, characterized in that: - the means for circulating comprise pneumatic means (8, 9; 21, 22) for pressurizing each reservoir (1, 2; 17, 18), and means (10, 11 1; 23, 24) for venting each of said reservoirs.   - the servo means (12; 25) are adapted to alternatively control the pressurization of one of the tanks (1, 2   17, 18) and simultaneously venting the other tank.   7 / - Apparatus according to claim 6, characterized in that: - the pneumatic means comprise a pneumatic source connected to each of the tanks (1, 2; 17, 18) by channeling means comprising, for each of said tanks, a member shutter (8, 9; 21, 22),   - the venting members consist, for each tank (1, 2; 17, 18), of a shutter member (10, 11; 23, 24) installed on the upper wall of said tank, - the means servo (12; 25) are connected to the shutter pressurization (8, 9; 21, 22) and venting (10, 11; 23, 24) of the tanks (1, 2 ; 17, 18), and adapted to sequentially control the opening and closing cycles of said organs.   8 / - Apparatus according to one of claims 5 to 7,   characterized in that the filtration module (6; 20) is composed of membranes   hollow fibers folded into a U and sealed at their free end.   9 / - Apparatus according to one of claims 5 to 8,   characterized in that: - each reservoir (1, 2) is equipped with a detector (13, 14) of the level of liquid contained in said reservoir, 16 2781391   - the servo means (12) are connected to the level detectors (13, 14) of the reservoirs (1, 2) and programmed to control cycle reversals for predetermined levels of liquid in said tanks.   10 / - Apparatus according to one of claims 5 to 9,   characterized in that the two tanks (1, 2) have identical volumes, the communication line (5) being extended by a single filtration module   (6) disposed in one of said tanks.   11 / - Apparatus according to claim 10, characterized in that 1o that each tank (1, 2) is arranged in a container containing a thermostatically controlled liquid.   12 / - Apparatus according to one of claims 5 to 9,   characterized in that one of the reservoirs (17) consists of a bioreactor, the other reservoir (18) consisting of a sampling chamber equipped with means (26-28) for reading measurements of given parameters of the liquid enclosed in said sampling chamber.   13 / - Apparatus according to claim 12, characterized in that   that it comprises a filtration module (20) disposed in the bioreactor (17).   14 / - Apparatus according to claim 12, characterized in that it comprises a filtration module disposed in the sampling chamber (18).