FR2647213A1 - AUTOMATIC SAMPLING SYSTEM OF A STERILE BIOLOGICAL LIQUID - Google Patents

Abstract

The invention relates to a system of automatic sterile sampling of a biological fluid. A filter module (1) is placed at the end of the sampling module (2) in the reactor (R). The sampling vessel (3) is directly connected to the outlet of the sampling module (2). A fluid injection circuit (40, 41, 42) is provided for controlling the sampling circuits which consist of the filter module (1), the sampling module (2) and the sampling vessel (3), said injection circuit being sequentially controlled by a control and calculation computer (5). Applications in laboratory or industrial sampling systems.

Description

 The present invention relates to an automatic sampler system for a sterile sampling biological fluid.

 The development of new "in situ" sensors that can be used online in biological reactors, such as fermenters, is currently severely limited by the need to sterilize these materials.

 More conventionally, only physicochemical sensors for measuring temperature, pressure, ORP potential, oxygen partial pressure and dissolved CO 2 or for measuring gaseous and volatile molecules in gases or using membranes are available. Sterile sampling of liquid samples therefore appears as a solution to the lack of "in situ" sensors, because in this case, it is possible to couple many conventional analyzers for the online detection of the molecules to be quantified.

 At the present time, many authors have proposed different systems for sampling filtered liquid biological medium, such as fermentation media, in order to monitor or control the evolution of biotechnological processes.

 It is noted that most of these systems include continuous recycling of medium to be filtered. The principle of these systems consists of a continuous pumping of the biological medium circulating in a pipe external to the reactor, with subsequent reinjection into the reactor. The sampling can be sequential or continuous.

 Such systems have been the subject of a particular description in US Patent 4,561,161 issued February 26, 1985. The system described in the aforementioned patent comprises two membranes of average diameter comprising pores ranging between I and 10 μm and forming an external recirculation loop with sequential sampling.

 Another system of this type has been described in the article "An automatic and sterilizable sampler for laboratory Fermentors: Application to the on-line control of glucose concentration" published by Mohamed Ghoul, Evelyne Ronat and 3ean- Marc Engasser in the journal Biotechnology and Bioengineering Vol. XXVII p. 119-121 (1986) John Wiley & Sons, Inc.

 The system described in the aforementioned article also uses a recirculation loop and steam sterilization of the sampling circuit to the filtration module must be provided.

More recently, a number of systems have been proposed by different manufacturers. Among these, a filtration system using an asymmetric multilayer ceramic module has been described in 86 1100459 "On Line Aseptic Autosampler" published by Komatsugawa Chemical Engineering Co., Ltd., 10-5,
Iwamoto-cho 1- Chome, Chiyoda-ku, Tokyo Japan. This type of apparatus is, however, subject to steam sterilization.

 Thus, the various systems previously described are, for the most part, usable under limited working conditions, fermentation of yeasts or living cells of large size. The problems most frequently encountered are related to the polarization and clogging more or less fast depending on the type of filtration membrane used, especially in the case of frontal filtrations, the latency period to obtain a representative sample in the case of filtrations tangential, the size of pores which, too important, does not allow to work with bacteria, the complexity of systems that include either a large number of pumps, or complex steam sterilization systems, too large volume of the recycling loop with respect to the volume of a small reactor or laboratory fermenter, of the order of two liters, and the ease of sterile sampling and therefore the respect of sterility.

 The automatic sampler system of a sterile sampling biological fluid, object of the invention, aims to remedy all of the aforementioned drawbacks.

 Another object of the present invention is the implementation of a system that does not include a loop for recirculating the biological fluid subjected to sampling.

 Another object of the present invention is the implementation of a system in which the steam sterilization members are removed.

 Another object of the present invention is also the implementation of a system for ensuring the taking of samples under conditions of reliability and sterility satisfactory in the absence of effective sterilization of the corresponding circuits during successive samples.

 The automatic sampler system of a biological fluid for sterile sampling, which is the subject of the invention, comprises a sampling module in a biological fluid reactor, a filtration module connected to a sampling pot, a distribution circuit of the one or more samples to analyzer means and control and calculation means interconnected respectively to the sampling module, the filtration module, the sampling pot and the distribution circuit of the sample or samples to ensure sequential control of the taking of samples .

 It is remarkable, on the one hand, in that the filtration module is placed at the end of the sampling module in the reactor, the sampling pot being directly connected at the outlet of the sampling module and, secondly, in it comprises a circuit for injecting sampling circuit control fluids constituted by the sampling module, the sampling pot, the control fluid injection circuit being sequentially controlled by the control means and calculation.

The system, object of the invention, will be described in more detail in connection with the drawings below in which
FIG. 1 represents a general view of the automatic sampler system of a sterile sampling liquid which is the subject of the invention;
FIG. 2 represents a sectional view along a longitudinal plane of symmetry of a sampling pot allowing the realization of the system according to the invention, as represented in FIG.
FIGS. 3 and 4 show in section along a longitudinal plane of symmetry respectively of a filtration module and of a cartridge or filter sleeve, in accordance with the subject of the present invention,
FIG. 5 represents a simplified flowchart making it possible to carry out sequential sampling (s) in accordance with the system according to the invention.

 The complex metabolic reactions involved in microorganisms (bacteria, yeasts, fungi, plant and animal cells) during "fermentation" processes are sensitive to many external parameters, such as pH, temperature, pressure, as well as the age or physiological state of the cells.

 From an industrial and practical point of view, it is essential to control the evolution of the processes by acting on the physico-chemical parameters or by controlling the composition of the culture medium in some of its essential constituents. Quantitative control of the processes is an essential element in obtaining high productivities or products of constant quality. At present, process monitoring is, in the vast majority of cases, carried out from an offline sampling. This sample is composed of a liquid phase and a solid phase comprising microorganisms and other materials such as cellular debris or from the raw material.

Quantitative analysis of biochemical compounds, substrates, and metabolism products, for example by conventional gas or liquid chromatography methods, requires the removal of solid particles.

 However, offline sampling has a number of problems and disadvantages such as the risk of contamination or non-representativity of the sample due to sampling and solid-liquid separation techniques, particularly in the case of volatile metabolites.

 On the other hand, online sampling in principle allows better control of a process if it meets a certain number of criteria. The volume sampled must be low, constant and representative of the state of the fermentation medium thanks to a simple and reliable system which, by design, minimizes losses so that it can be used in limited capacity reactors (from 2 liters). While ensuring efficient filtration, fast and repetitive at regular but variable time intervals, the device must ensure absolute sterility vis-à-vis the culture medium.

 The automatic sampler system of a sterile sampling biological fluid, object of the present invention, will first be described in connection with FIG.

 According to the aforementioned figure, the sampler system, which is the subject of the invention, comprises a sampling module 2 for samples in a reactor R or fermenter of the biological fluid in question. It also comprises a filtration module 1, the filtration module I and the sampling module 2 being connected to a sampling pot 3. A distribution circuit CD of the sample or samples taken to the analyzer means is also provided. The sampler system, object of the invention.

further comprises control and calculation means 5 interconnected respectively to the sampling module 2 and the filtration module I to the sampling pot 3 and to the distribution circuit CD of the sample or samples to ensure a sequential control of the intake of aforementioned sample.

 According to a particularly advantageous characteristic of the system which is the subject of the invention, the filtration module 1 is placed at the end of the sampling module 2 in the reactor R or fermenter.

The sampling pot, in accordance with another particularly advantageous aspect of the system which is the subject of the invention, is then directly connected at the outlet of the sampling module 2, in the absence of a loop for recycling the biological fluid sampled.

 As has also been shown in FIG. 1, the sampler system also comprises a circuit 40, 41, 42 for injecting sampling circuit control fluids constituted by the filtration module I, the sampling module 2. , the sampling pot 3.

 According to another particularly advantageous aspect of the sampler system, object of the invention, as will be described in more detail below in the description, the control fluid injection circuit is sequentially controlled by the control means and calculation 5.

In order to perform all the various functions of the automatic sampler system for a sterile sampling liquid which is the subject of the present invention, the circuit for injecting sampling circuit control fluids advantageously comprises, as can be seen in FIG. FIG. 1 shows a first circuit 40 for injecting an inert gas into the biological fluid to be sampled. This circuit advantageously ensures pre-cleaning and / or unclogging of the filtration module
By injection of the inert gas to the reactor or fermenter R.

 As will be understood, the inert gas with respect to the biological fluid to perform the predecolmatage or declogging functions may be constituted, depending on the nature of the biological fluid to be sampled, by air, nitrogen , carbon dioxide for example.

 As has also been shown in FIG. 1, the control fluid injection circuit for sampling circuits comprises a second circuit 41 for injecting a rinsing water into the circuits connecting the reactor or fermenter R and sampling pot 3.

 Similarly, in FIG. 1 above, there is shown a third dry air injection circuit 42 in the circuits connecting the reactor or fermentor R and the sampling pot 3. The previously mentioned circuit 42 is therefore also one of the constituent elements of the control fluid injection circuit, in accordance with the object of the present invention, this circuit 42 for ensuring a drying of the circuits providing the connection between the reactor R and the pot of sampling 3.

 An advantageous embodiment of the first 40, second 41 and third 42 injection circuits of a rinse water gas and inert dry air will be described in connection with Figure 1, this embodiment to ensure the injection of the aforementioned inert gas from the rinsing water and the dry air.

 As has been shown in FIG. 1 above, the first 40, second 41 and third 42 injection circuits may advantageously consist of a first three-way solenoid valve 421 interconnected in connection with the end of the sampling module. This first three-way solenoid valve 421 receives on an auxiliary input the rinsing water or drying air, as will be described below.

 In order to supply the aforementioned auxiliary input of the first three-way solenoid valve 421 with rinsing water or drying air; the second injection circuit 41 may, as shown in FIG. 1, advantageously be constituted by a second three-way solenoid valve 422 connected in connection between the rinse water supply circuit and the auxiliary inlet of the first solenoid valve 421 previously mentioned. The second three-way solenoid valve 422 receives on an auxiliary input drying air intended to ensure the aforementioned drying.

 Finally, as shown in FIG. 1, a third three-way solenoid valve 423 is provided, this third solenoid valve being connected in connection between the first solenoid valve 421, at the outlet thereof, and the sampling pot 3. An auxiliary input the third solenoid valve 423 receives the inert gas to the biological fluid to perform the predecolmatage or declogging function mentioned above. The third solenoid valve 423 thus constitutes the inlet of the first injection circuit 40 mentioned above. It will be noted that the supply of inert gas with respect to the biological liquid can be carried out from a tank of inert gas under pressure. It is the same, with regard to the supply of drying air, of the second three-way solenoid valve 422. Of course, this previously mentioned solenoid valve 422 is fed from a distilled water reservoir, which distributes the rinse water previously mentioned.

In order to ensure the collection of samples of biological fluid, on the one hand, and the various predecolmatage, declogging, rinsing and drying functions, as shown in FIG. 1, the sampling means 2 furthermore comprise a peristaltic pump type pump 20 ensuring the interconnection between the third solenoid valve 423 and the sampling pot 3. It will be noted that, advantageously, the peristaltic pump 20 is associated with a valve or solenoid valve 22 mounted in bypass on the aforementioned pump. The sudden opening of the solenoid valve 22 makes it possible to unclog the filtration module 1 during sampling. Indeed, this opening causes the suction to stop upstream of the pump 20, and thus a take-off of the particles that have agglomerated on the outer layer or membrane 113 of the filtration module
Finally, it will be noted that the junction between the sampling element 2 and the filtration module 1 can advantageously be achieved by means of a manual valve 23. This manual valve makes it possible to provide a safety function in order to isolate totally where appropriate, the reactor R, and the biological medium contained therein, of the entire installation.

 According to another advantageous characteristic of the system, object of the invention, the sample pot 3 is a refrigerated pot. Thus, the sampling pot 3 comprises a double wall, in which is formed a stream of coolant to maintain the sample of biological fluid taken at a predetermined temperature depending on the nature of the biological fluid considered. The refrigeration system of the sampling pot 3 will not be described because it can be achieved by any means usually used in the laboratory allowing refrigeration conditions compatible with obtaining and maintaining the sterility of the sampled samples.

 A more detailed description of an advantageous embodiment of a sampling pot 3 will be given in connection with FIG.

 Figure 2 above shows a sectional view along a longitudinal plane of symmetry of a sampling pot 3 according to an advantageous embodiment of the system object of the invention.

 According to the above-mentioned figure, the sampling pot comprises a sample inlet orifice 30, this orifice 30 being directly connected at the outlet of the peristaltic pump 20. The sample inlet orifice 30 is itself connected to a sample inlet chamber 31, this inlet chamber being provided with a distributor 310. A vent 311 is provided, this vent being in communication with the aforementioned admission chamber 31. The distributor 310 may advantageously be constituted by a conical element of revolution provided with through-holes parallel to the longitudinal axis of the sampling pot 3. The aforementioned through-holes are in communication with a sample receiving chamber 32. Sample receiving 32 is itself in communication with a sample outlet 33, which is itself connected to the distribution circuit CD, as will be described later in the description.

 As further shown in FIG. 2, the sample receiving chamber 32 is provided with a plurality of electrodes 32G, 321, 322. The aforementioned electrodes are sampled biological fluid level probes. non-limitatively, the electrode 329 may correspond to a minimum sample volume taken, the electrode 321 may correspond to a volume of samples taken intermediate, and the electrode 322 may correspond to a volume of samples taken maximum .

 The body of the sampling pot can then be made of a material inert to the biological fluid, such as for example food plastics or the like.

As furthermore shown in FIG. 1, the outlet orifice 35 of the sampling pot is connected to the distribution circuit
The same two-way solenoid valve 35 is itself interconnected with a three-way solenoid valve 36, one input of which is connected to the two-way solenoid valve 35, a first two-way solenoid valve 35. direct outlet is connected to an evacuation or sewer line and a second controlled outlet is connected to a dispensing tube or to a sample fraction collector.

 In addition, as shown in FIG. 1, the vent 311 in FIG. 2 is itself connected via a three-way solenoid valve 37, on the one hand, to a duct drainage or sewer in direct connection and, secondly, in controlled connection to a pressure source of an inert gas or biological fluid sampled.

 In an advantageous embodiment of the system, object of the invention, as shown in FIG. 1, the connection between the first three-way solenoid valve 421 and the end of the sampling module connected to the filtration module 1 is effected by the The multiple inputs of the selection valve 21 can be connected sequentially to a different reactor determined, each reactor may contain different biological solutions or different batches of the same biological solution. The output of the multichannel selection valve 21 is directly connected to the first three-way solenoid valve 421.

In FIG. 1, the two-way solenoid valves and the three-way solenoid valves have been represented symbolically, the two-way solenoid valves comprising an inlet inlet A and a discharge outlet r, the energizing of the solenoid valve causing the admission by the inlet A and the discharge through the outlet r of the fluid considered. Similarly, the three-way solenoid valves have been shown as having an inlet inlet A, a discharge outlet or inlet r and an inlet auxiliary P, the absence of control in voltage of
the three-way solenoid valve considered to allow the transmission of a
fluid between the inlet inlet A and the discharge outlet r or
reciprocally, while the voltage control of the three-way solenoid valve
considered routes results in the transmission of a different fluid between
the auxiliary input P and the discharge outlet r. The operation of
the entire device or system object of the invention, as shown in Figure 1, will be described later in the description.

 A more detailed description of the filtration module 1 will now be given in connection with FIG.

 According to the aforementioned figure, the filtration module 1 advantageously comprises a central filter body 10 forming a mounting axis of a filter element and a removable filter element 11 formed by a sleeve of ceramic material. The removable filtration element It is engaged on the mounting axis 10. The connection between the straight-section edges of the filtration element 11 and the bearing surfaces of the mounting shaft 10 is effected by means of seals 100, 110 in Figure 3. The seals can advantageously be constituted by silicone seals.

 According to a particularly advantageous aspect of the system which is the subject of the invention, the filter element 11 is constituted by a cylindrical sleeve 112, as shown in FIG. 4, in section along a longitudinal plane of symmetry of the sleeve. This cylindrical sleeve 112 may advantageously be made of ceramic with an average pore diameter of the order of 10 μm. In addition, as shown in this same figure 4, an inner layer 114 and an outer layer 113 grafted ceramic are provided on the inner and outer side surfaces of the aforementioned cylindrical sleeve. The inner and outer grafted layers 114 and 114 have an average pore diameter of less than or equal to 0.2 μm and act as a filter membrane.

In addition, in order to guarantee absolute tightness of the assembly, an enameling or deposition of an epoxy resin layer 115, 116 is provided at both ends of the filter element 11. The above-mentioned enameling or deposit may cover the aforementioned ends over a length of
About 1 cm. The outer 113 and inner 114 layers are made of alumina, and have an average pore diameter of between 0.1 and 0.4 μm, preferably 0.2 μm, and the cylindrical sleeve 112 of alumina has a diameter of pores from 10 pm to 15 pm approximately. The cylindrical sleeve 112 and the inner 114 and outer 113 layers are connected monolithically by sintering.

As furthermore shown in FIG. 3, the mounting axle 10 advantageously comprises a central internal channel 105 extending to the opposite portion of the distal portion of the mounting axis.
10 above. The central internal pipe 105 thus abuts inside the filter element 11 or the aforementioned cylindrical sleeve 112 and constitutes the end of the sampling means 2. In FIG. 3, the orifices through which the central internal channel 105 passes through in the sleeve 112 are marked 155G and 1051.

 A more detailed description of the control and calculation means 5 will be given in connection with FIGS. 1 and 5.

 As shown in FIG. 1, the control and calculation means 5 may advantageously be constituted by a CPU microprocessor interconnected to interface circuits 51 and 52 and further comprising a program memory 50.

 The aforementioned interface circuits advantageously comprise a first series 51 of sampling control interface circuits.

These interface circuits of the first series 51 may each consist of an analog digital conversion circuit delivering each of the three-way solenoid valves previously described and the peristaltic pump 20 a control voltage on or off for example.

 Similarly, the second series 52 of sample level detection interface circuits may consist, for each of the interfaces, in a digital analog conversion circuit delivering to the microprocessor CPU corresponding messages of sample level in the sample pot. sampling 3.

 The program memory 50 may advantageously be constituted by a RONI ROM in which a sequential sample collection program is implemented.

 As shown in FIG. 5, the sequential sampling control program may advantageously comprise a first sub-program 1000 making it possible to ensure a pre-decanting phase of the filtration element I by admission of an inert gas. to the reactor or fermenter R. In this case, the third three-way solenoid valve is controlled voltage, solenoid valve 423, and the first and second three-way solenoid valves are at rest, solenoid valves 421, 422.

 The first sub-program 1000 is then followed by a second sub-program 1001 making it possible to place the system, object of the invention, in a waiting phase for a duration of a few seconds for example. In this case, none of the sampling elements are excited.

 The second subroutine 1000 previously mentioned is then followed by a third subroutine 1002 to ensure a proper sampling phase. In this case, the peristaltic pump 20 is then periodically controlled and the stopping of the sampling phase is carried out on detection by the microprocessor CPU of the corresponding sample level messages in the sampling pot 3.

 The third sub-program 1002 is then followed by a fourth subprogram 1003 making it possible to ensure a transfer phase of the sample contained in the sampling pot 2 to the analyzer circuits or, at least, to the distribution circuits CD via the solenoid valves 35 and 36. The fourth sub-program 1003 allows for example, following the opening of the solenoid valves 35 and 36, to control the voltage solenoid valve 37 so as to send in the sampling pot 3 at the upper part thereof relative to the sample contained in the aforementioned sampling pot an overpressure of inert gas to the biological fluid constituting the sample. This overpressure makes it possible to ensure that the entire sample is transferred to the fractionation collector in a predetermined sequence.

 The fourth subroutine 1003 previously described is then followed by a fifth subroutine 1004 to ensure a water rinse by voltage control of the first solenoid valve 421, the second solenoid valve 422 in the unexcited state delivering the rinsing water to the sampling pot 3 via the peristaltic pump 20 itself controlled voltage. Rinsing, of course, can be performed continuously for a fixed period.

 In addition, following the fifth subroutine 1004 mentioned above, succeeds a sixth subroutine 1005 of successive washes lasting a few seconds, for example sampling pot 3 and pipes via the first three-way solenoid valve 421 and of the peristaltic pump 20.

 Finally, a seventh sub-program 1006 for drying the sampling pot is provided in order to ensure the drying of the aforementioned sampling pot and the pipes for a few minutes by the internal diaire of the voltage control of the first 421 and second solenoid valve. three-way 422 and the peristaltic pump 20.

 Of course, the succession of the aforementioned programs is not limiting, a subroutine may be called arbitrarily so as to constitute a sequence appropriate to the application in question. Thus, it is possible between the sampling phase subroutine 1002 and the sample transfer phase sub-program to the analyzer circuits 10G3 to insert a call of the unclogging phase subroutine 1000 to constitute a new declogging of the element. Filtration I.

A table summarizing a preferential automatic sampling cycle is given below in the description, this table indicating by way of nonlimiting example, on the one hand, the called subprograms, the corresponding sequence numbers, the unit operations corresponding to each sequence, the duration of these operations and the solenoid valves and system elements shown in Figure I controlled voltage.

<Tb>

SEQUENCES <SEP> OPERATIONS <SEP> DURATIONS <SEP> ORGANS <SEP> UNDER
<tb><SEP> UNITS <SEP> VOLTAGE
<tb><SEP> 2 <SEP> Predecolmate <SEP> e <SEP> 0 <SEP> 5 <SEP> s <SEP> 423
<tb><SEP> 3 <SEP> Waiting <SEP> 10 <SEP> s
<tb><SEP> 4 <SEP> Sampling <SEP> to <SEP> Pump <SEP> 20, <SEP>
<tb><SEP> contact <SEP> 22 <SEP> to <SEP> reason
<tb><SEP> probe <SEP> ni- <SEP> from <SEP> 0.2 <SEP> s <SEP> all
<tb><SEP> calf <SEP> selected <SEP><SEP> 30 <SEP> s
<tb><SEP> 5 <SEP> Decolmate <SEP> e <SEP> 0 <SEP> 5 <SEP> s <SEP> 423
<tb><SEP> 6 <SEP> Transfer <SEP> to <SEP> on <SEP> 35, <SEP> 37,
<tb><SEP> collector <SEP> 36 <SEP>
<tb><SEP> 7 <SEP> Rinse <SEP> to <SEP> water <SEP> 1 <SEP> min <SEP> 20 <SEP> 35, <SEP> 421
<tb><SEP> 7 '<SEP> 3 <SEP> successive washes <SEP>
<tb><SEP> of <SEP> pot <SEP> and <SEP> of <SEP> 3s <SEP> 25, <SEP> 421
<tb><SEP> pipes <SEP> lOs <SEP> 20 <SEP> 35 <SEP> 421
<tb><SEP> 8 <SEP> Drying <SEP> pot <SEP> + <SEP><SEP> 2 <SEP> mn <SEP> 2G, <SEP> 35, <SEP> 422
<tb><SEP> pipes <SEP> 421, <SEP> 22
<tb><SEP> 9 <SEP> Feed <SEP> of a <SEP> tube
<tb><SEP> 10 <SEP> Waiting <SEP> preprogram
<tb><SEP> m <SEP> of a new <SEP>
<tb><SEP> cycle
<Tb>
Of course, the aforementioned times are given for information only and may be modified for adaptation to different types of culture or biological media. For example, the duration of the unclogging sequence can be increased up to 3 to 4 seconds if necessary and the transfer sequence can be of the order of 10 seconds, that of the sequence 9 is very fast and that of the sequence 10 is any.

 It will be noted that in a particularly advantageous embodiment of the system that is the subject of the invention, the microprocessor CPU may advantageously be constituted by an 8-bit microprocessor that is normally available commercially. In this case, the microprocessor CPU can be interconnected by a bus-type connection to a microcomputer, which makes it possible to ensure interactive operation of the system that is the subject of the invention with a user, the system, object of the invention mentioned above, being able to then be programmed.

 In the case where the microprocessor CPU is constituted by an 8-bit microprocessor, it is advantageous to assign each bit of the 8-bit word processed by the microprocessor in question to one of the elements such as a two-way or three-way solenoid valve. peristaltic pump of the device shown in Figure 1 through the corresponding control interface 51. Thus, each sequence simply corresponds to an 8-bit word constituting the subroutine 1000 to 1006 shown in Figure 5. Each bit of value 0 or 1 may then allow, via the aforementioned control interfaces 51, to control voltage or, on the contrary, to remove this voltage command for the electrovaes or the peristaltic pump considered.

 In a particular non-limiting embodiment, the microprocessor CPL 'and the interface circuits are replaced by a programmable controller. The programmable logic controller used was a KLÖCKNER-MOELLER programmable controller type PS3-DC.

Such an embodiment allows a more flexible use of the system.

 An automatic sampling system for a particularly advantageous sterile sampling liquid has thus been described.

Indeed, the system, object of the invention, allows an implementation "in situ" in the absence of recycling loop. Next, the system, which is the subject of the invention, makes it possible to ensure sampling in
the absence of stress on the animal, plant or microorganism cells of the biological medium in which the sample is to be taken, the system according to the invention being non-destructive in this respect.

 In addition, the system according to the invention makes it possible to obtain sterile behavior in the absence of the need for steam sterilization in accordance with the devices of the prior art.

 Similarly, the use of a filtration element having a porosity of 0.2 μm or less in an outer layer, single or double, makes it possible to carry out a sampling of biological medium comprising bacteria.

 In addition, the simplicity of design of the system shown in FIG. 1 makes it possible, on the one hand, to obtain a high reliability of operation of the assembly with a very low dead volume, which allows a small volume filtration with a short time. latency.

 Of course, another advantage of the system which is the subject of the invention is the permanent control of the clogging of the filter element by a first predecolmatage sequence and then a declogging sequence, following the sampling sequence.

 In addition, the system, object of the invention, allows the choice of a constant sampling volume regardless of the amount of dissolved gas and desorbent, the cell concentration of the biological medium in question, the size of these cells or the internal pressure in the reactor R.

 In addition, the cartridges of the removable filter element I are easily reusable and interchangeable.

 The system, object of the invention, also allows modular use from the size of small reactors or laboratory fermenters of two liters capacity up to the industrial size.

 The use of the system, as shown in FIG. 1, is facilitated by the pressurization of the reactor or fermentor R, which makes it possible to envisage use with relatively viscous media or biological liquids.

 Finally, a precise analysis of volatile products can be carried out thanks to the refrigeration of the sample pot or of molecules that are not or only slightly volatile.

Tests of sterility and good functioning of the device or system according to the invention, as represented in FIG. 1, were carried out according to two different processes: by repeated sampling simulation at intervals of 2 hours during
a duration of 2 weeks on a conventional synthetic medium of
fermentation, sterile and without seeding of microorganisms,. then by injection of medium heavily loaded with small bacteria
from the outside of the filtration module to the inside of the fermenter,
that is, in a sterile environment.

 The results obtained were very satisfactory since no contamination of the medium contained in the fermenter was observed.

The good functioning of the system was also tested on the quality of the samples from fermentations using different microorganisms with yeasts such as Saccharomyces uvarum and
Saccharomyces cerevisiae, bacteria like Escherichia coli and
Clostridium acetonobutylicum and finally a mixture of bacteria, yeasts and filamentous fungi. Whatever the operating conditions,
The sample taken has always been free of microorganisms. In addition, on-line monitoring, thanks to the gas chromatograph / sampler coupling, of anaerobic ferrentrations such as the production of ethanol by S. uvarum or of acetone-butanol with C acetonobutylicum, showed an excellent agreement between the analyzes of line and those performed by manual off-line sampling.

Claims (16)

 An automatic sampler system for a sterile sampling liquid, said sampler system comprising a sampling module in a biological fluid reactor, a filtration module connected to a sampling pot, a distribution circuit of the sample or samples to analyzer means and control and calculation means interconnected respectively to the sampling module, to the filtration module, to the sampling pot and to the distribution circuit of the sample or samples to ensure sequential control of the taking of samples, characterized in that said filtration module (I) is placed at the end of said sampling module (2) in the reactor (R), said sampling vessel (3) being directly connected at the outlet of the sampling module (2) and in said sampler system further comprises a pre-circuit control fluid injection circuit (40, 41, 42) the filter module (1), the sampling module (2), the sampling pot (3), said control fluid injection circuit being sequentially controlled by said control and calculation means (5). ).  2. System according to claim 1, characterized in that said sampling circuit control fluid injection circuit comprises a first circuit (40) for injecting an inert gas into the biological fluid to ensure pre-cleaning and / or a declogging of said filtration module (I) by injection of said gas to the reactor (R).  3. System according to one of claims 1 or 2, characterized in that said sampling circuit control fluid injection circuit comprises a second circuit (41) for injecting a rinsing water circuits providing the connection between said reactor (R) and the sampling pot (3).  4. System according to one of claims 1 to 3, characterized in that said sampling circuit control fluid injection circuit further comprises a third circuit (42) for injecting dry air into the circuits providing the connection between said reactor (R) and the sampling pot (3).  5. System according to one of claims 2 to 4 above, characterized in that said first (40), second (41), third (42) injection circuits of an inert gas, a rinsing water, of a dry air are constituted by:. a first three-way solenoid valve (421) interconnected in connection  between the end of said sampling module connected to the module of  filtration (1) and said sampling pot (3), and receiving, on a  auxiliary input, rinse water or drying air,. a second three-way solenoid valve (422) connected in connection between  the rinse water supply circuit and said auxiliary inlet of the  first solenoid valve, and receiving, on an auxiliary input, said air  drying,. a third three-way solenoid valve (423) connected in connection between  the first solenoid valve and said sampling pot (3) and one of which  auxiliary input receives said inert gas to the biological fluid.  6. System according to claim 5, characterized in that said third solenoid valve is interconnected to said sampling pot via a peristaltic pump (20).  7. System according to one of the preceding claims, characterized in that said sampling pot (3) is a refrigerated pot.  8. System according to one of claims 1 to 7, characterized in that said sampling pot (3) comprises:. a sample inlet (30), a sample inlet chamber (31) provided with a distributor (310), a vent (311) in communication with said inlet chamber (31), a sample receiving chamber (32) in communication with  said intake chamber (31), said sample receiving chamber  spindle (32) being provided with a plurality of electrodes (320, 321, 322)  constituting sampled biological fluid level probes.  9. System according to one of the preceding claims, characterized in that the connection between said first (421) three-way solenoid valve and the end of said sampling module connected to the filtration module (1) is effected via a multichannel selection valve (21) whose multiple inputs can be connected sequentially to a specific reactor and whose output is directly connected to said first three-way solenoid valve (421).  10. System according to one of the preceding claims, characterized in that said filter module (1) comprises a central filter body (10) forming a mounting axis of a filter element.  filtration, a removable filter element (11) formed by a sleeve of material  ceramic and engaged on said mounting axis (10), the connection between the  straight section edges of said filter element (II) and surfaces  supporting said mounting pin (10) being effected by means of joints  sealing (100, 110).  11. System according to claim 10, characterized in that said filter element (11) is constituted by. a cylindrical sleeve (112) of ceramic having an average pore diameter of  the order of 10 μm, an inner layer (114) and an outer ceramic layer (113)  grafted on the inner and outer lateral surfaces of said sleeve  cylindrical, said inner (114) and outer (113) graft layers  having a pore diameter of less than or equal to 0.2 μm.  12. The system of claim 10, characterized in that said mounting axis comprises a central inner pipe (105) abutting the opposite portion to the distal portion of said mounting axis and constituting the end of said sampling means (2).  13. System according to one of claims 1 to 4, characterized in that said means (5) for control and calculation are constituted by a microprocessor interconnected to interface circuits (51, 52) and having a program memory (50).  The system of claim 13, characterized in that said interface circuits comprise: a first series (51) of control interface circuits of  sampling, said interface circuits of the first series consisting of  each in a digital-to-analog conversion circuit delivering  each of the three-way solenoid valves and said peristaltic pump a  control voltage on or off ,.  the sampling pot.  microprocessor the corresponding sample level messages in  each in a digital analog conversion circuit delivering audit  sample, said interface circuits of the second series consisting of  . a second series (52) of level detection interface circuits  15. System according to one of claims 13 or 14, characterized in that said microprocessor comprises, implanted in the read-only memory (5G) associated with said microprocessor, a sequential sample collection program program, said program comprising at least. a first sub-program (1000) to ensure a phase of  pre-cleaning of said filter element by admission of an inert gas  to the reactor, said third three-way solenoid valve being  controlled voltage and said first and second solenoid valves to  three tracks being idle, a second subroutine (l0 (j1) allowing the system to be placed in  waiting for a period of a few seconds, none of the  sampling elements not being excited, a third sub-program (1002) to ensure a phase of  actual sampling, said peristaltic pump being controlled  in tension periodically, stopping the sampling phase being  performed on detection by said microprocessor corresponding messages  sample level in the sampling pot, a fourth sub-program (1003) to ensure a phase of  transfer of the sample contained in the sampling pot to the  analyzer circuits,. a fifth sub-program (1004) to ensure a rinse to  water by voltage control of said first solenoid valve, said  second solenoid valve, in the unexcited state, delivering said water of  rinsing to the sample cup, via the pump  peristaltic controlled voltage, said rinsing being performed during  a predetermined duration, a sixth sub-program (1005) of successive washes of durations of  a few seconds from the sampling pot and pipes by  via the first three-way solenoid valve and the pump  peristaltic,. a seventh subroutine (1006) for drying the sample pot  and pipes for a few minutes, via the  voltage control of the first and second solenoid valves  tracks, of the peristaltic pump.  16. System according to one of claims 1 to 4, characterized in that said means (5) for controlling are constituted by a programmable controller.