FR3147813A1 - Method of producing microorganisms and installation for implementation - Google Patents

Abstract

The invention relates to a method and an installation for producing microorganisms comprising the following steps: a first closed envelope (18) is provided containing a given strain of microorganisms in a dormant state, said given strain of microorganisms being adapted to promote the growth of at least one predefined plant variety in a given medium; a second envelope (14) is provided, independent of said first envelope; a connecting device (20) is provided to be able to connect said first envelope (18) and said second envelope (14) together, and to be able to transfer said strain of microorganisms from said first envelope to said second envelope; nutrients are introduced inside said second envelope (14), so as to be able to cause the proliferation of the microorganisms inside said second envelope; and the microorganisms having proliferated inside said second envelope (14) are recovered. Figure to be published with the abstract: Fig. 1

Description

Method of producing microorganisms and installation for implementation

The present invention relates to a method for producing microorganisms and to an installation for implementing said method.

One area of application considered is that of agricultural production and more particularly plant production.

It is known to amend soils and spread fertilizers on them to promote plant growth. Amendments, whether mineral or organic, aim to improve soil quality, while fertilizers are nutrients that are directly used by plants to promote their growth.

In addition, it is known to use phytosanitary products to protect plant production from phytopathogenic agents and pests.

Thus, the resulting continuous improvement in agricultural yields has made it possible to feed an ever-growing population across the world.

On the other hand, the large-scale use of agrochemical fertilizers and phytosanitary products leads to significant emissions of greenhouse gases including carbon dioxide and nitrous oxide; it also contributes to the pollution of soils and subsoils, water tables, watercourses, oceans and air.

As a result, it was decided to implement more natural, and above all less polluting, fertilizing and/or phytosanitary elements.

Thus, strains of microorganisms have been used to promote plant growth. For example, it has been imagined to introduce into the soil strains of Azotobacter , a bacterium promoting the fixation of atmospheric nitrogen, precisely with the aim of providing the plants then planted with the nitrogen necessary for their growth, thereby reducing the spreading of nitrogen fertilizers such as urea or ammonium nitrate for example.

However, microorganisms are living beings requiring very specific production and storage conditions before they can be associated alive with the plants implanted, in comparison with conventional mineral or synthetic fertilizers which can be stored easily under certain conditions, for long periods. In addition, these fertilizers retain their simple chemical formula and their constant activity over a long period.

Thus, a problem that arises and that the present invention aims to solve is to provide a method for producing microorganisms having a capacity for survival in the soil and increased agronomic benefits, and an installation for being able to implement it, making it possible to produce the microorganisms in order to be able to associate them easily and effectively with plant crops, and this, at advantageous costs.

In order to solve this problem, and according to a first object, a method for producing microorganisms is proposed comprising the following steps: providing a first closed envelope containing a given strain of microorganisms in a dormant state, said given strain of microorganisms being adapted to promote the growth of at least one predefined plant variety in a given medium; providing a second envelope independent of said first envelope; providing a connecting device for being able to connect said first envelope and said second envelope together, and for being able to transfer said strain of microorganisms from said first envelope to said second envelope; introducing nutrients inside said second envelope, so as to be able to cause the proliferation of the microorganisms inside said second envelope; and recovering the microorganisms which have proliferated inside said second envelope to bring them into contact with said at least one predefined plant variety and/or said given medium.

Thus, a feature of the invention lies in the implementation of the microorganisms extemporaneously. In other words, the microorganisms are produced in large numbers and implemented in situ. For example, the population of microorganisms is multiplied by more than 10, by more than 100, or even by more than 1000. To do this, production must be simple and efficient. Two envelopes are then implemented, independent of each other. The first envelope is advantageously sealed and it contains the microorganisms in a dormant state. In other words, the microorganisms can be stored alive inside a sanitized envelope.

This first envelope has a limited volume, for example less than 1000 cm³. It not only allows the storage of microorganisms in optimal conditions, but also their transport to the place of implementation. Furthermore, and as will be explained in the rest of the description, the first envelope is for single use.

For some microorganisms, storage is carried out at low temperatures, for example below 5 °C, or even below -20 °C, so as to prevent any growth and to keep them in a dormant state. For other microorganisms, storage is carried out in the solid state, for example after a freeze-drying step.

Thus, thanks to the connection device which connects in a sealed manner the first envelope containing the microorganisms and the second envelope, these microorganisms are transferred from the first envelope into the second envelope. The connection device is aseptic and the second envelope is also aseptic so that the transfer of the microorganisms can be ensured without contamination by other microorganisms.

Also, the connection device and a plurality of second envelopes can be stored on site at the user's disposal.

It will be observed that the nutrients, for example in powder form or in an aqueous medium, may be initially contained in the second envelope or may be introduced as soon as the strain of microorganisms is transferred therein. They may, for example, be introduced via the connection device so as to avoid the introduction of undesirable germs into the second envelope.

The second envelope has a larger volume than the first envelope, since the transferred microorganisms will proliferate thanks to the nutrients introduced inside the second envelope. The volume of the second envelope can be greater than three times the volume of the first envelope or ten times, or even one hundred times this volume, depending on the proliferation capacities of the microorganisms.

And when the proliferation of micro-organisms has reached the desired stage, they are collected in the second envelope to be able to put them in contact with the plant already planted, with the seed or directly in the soil prior to sowing.

Therefore, there is no need to store large quantities of microorganisms in order to be able to use them in the soil or on plant crops.

According to a particularly advantageous embodiment of the invention, the quantity of microorganisms having proliferated inside said second envelope is measured, and the microorganisms having proliferated are recovered when said quantity reaches a predetermined threshold.

The growth of the microorganisms inside the second envelope follows a substantially sigmoidal curve over time. It has two plateaus, a first plateau corresponding to low growth and a second plateau corresponding to the end of the growth of the microorganisms. And it is generally appropriate to recover the microorganisms that have proliferated preferentially between the middle of the exponential growth phase, or logarithmic phase called " log phase " in English, and the beginning of the second plateau. Consequently, measuring the quantity of microorganisms contained in the second envelope, as a function of time, makes it possible to recover the microorganisms that have proliferated at the appropriate time. As will be explained in more detail in the remainder of the description, the quantity of microorganisms produced can be measured by measuring the turbidity of the medium of the second envelope. This measurement can be carried out by spectrophotometry in the essentially visible range, for example at 600 nm. It is also possible to operate at 850 nm. In other words, we measure the optical density of the medium.

Furthermore, in a particularly advantageous manner, the recovered microorganisms are brought into contact with said at least one predefined plant variety and/or said given medium, preferably less than 24 hours, and at most 96 hours after having recovered the microorganisms. In this way, the production of microorganisms can not only be optimal, but in addition, the quantity of those which die is very low.

Preferably, the temperature of said second envelope is brought to a predetermined value. This value is generally between 10 °C and 60 °C and more precisely between 20 °C and 40 °C, for example 35 °C. As will be explained in more detail in the remainder of the description, the temperature control can be carried out using Peltier effect modules.

In addition, the contents of said second envelope are advantageously moved. In this way, the contents of the second envelope are contributed to by homogenization and aeration and, consequently, the growth of these micro-organisms is encouraged.

According to a preferred embodiment, an aqueous mixture is injected inside said second envelope. For example, buffered water containing certain salts is injected therein. In other words, the microorganisms and nutrients bathe in an aqueous medium.

Advantageously, the pH inside said second envelope is maintained between two predetermined pH values when the microorganisms proliferate. The microorganisms are in fact sensitive to the pH of the medium in which they proliferate. Therefore, the pH of the contents of the second envelope is measured and the necessary corrections are made by injecting an acidic compound or a basic compound inside the second envelope.

Also, the oxygen concentration inside said second envelope is maintained between two predetermined concentration values, when the microorganisms proliferate. Some soil microorganisms are in fact obligate anaerobic and must therefore be cultivated in the absence of oxygen. Other microorganisms are micro-aerobic, they therefore grow in the presence of a reduced oxygen concentration between optimal values, and it is appropriate to regulate the oxygen concentration inside the second envelope. The injection of a neutral gas, for example nitrogen, inside the second envelope can make it possible to expel excess oxygen. The injection of oxygen inside the second envelope makes it possible to provide a supplement if necessary. Depending on the needs, the control of gas concentrations can be extended to other gases such as carbon dioxide CO ₂ or dinitrogen N ₂ .

Furthermore, and particularly advantageously, any first envelope, depending on the type of microorganisms it contains, is associated with proliferation implementation parameters. These implementation parameters can also be adjusted, for a given type of microorganisms, depending on the use for which the microorganisms are intended after their multiplication, for example the type of plant variety. Thus, many “recipes” previously empirically validated during agronomic trials can be implemented simply in order to create an optimized product.

Furthermore, and according to a particularly advantageous embodiment, said first and second envelopes are single-use. Thus, the first and second envelopes are sterile. The second envelope is initially free of microorganisms and can thus be stored for a long period. After its implementation, the second envelope cannot be reused. The same applies to the first envelope. Therefore, it is preferable that the material of the first and second envelopes can be recycled and used as a polymer in other applications, or be biodegradable.

According to a particular embodiment of the invention, the first envelope is connected to the second envelope and its contents are poured into the latter. Another first envelope containing the same microorganisms, or different microorganisms, is in turn connected to the second envelope and its contents are also poured into it, thus adding to the microorganisms already poured.

Furthermore, according to an alternative embodiment, a third envelope containing a concentrated or non-concentrated culture medium, or an aqueous mixture, can in turn be connected to the second envelope, to be poured into it.

Also, according to a particularly advantageous embodiment variant, said first closed envelope is provided further containing a given additional strain of microorganisms in a dormant state. In certain situations, it may be appropriate to associate two strains of microorganisms with the same plant culture to promote its growth. Also, these two strains may be initially contained in the same first envelope in equal parts for example and then proliferate in a second envelope after they have been transferred inside. And moreover, for certain microorganisms, such a co-culture is synergistic and it promotes the growth of the two strains beyond what could be obtained by cultivating them independently of each other.

By extension, a plurality of strains of microorganisms can initially be formulated and inserted inside a first envelope. In this way, different types of microorganism can be chosen and combined, making it possible to provide different elements to the plant culture. For example, a strain of Azotobacter promotes the fixation of atmospheric nitrogen, while another strain produces substances making it possible to promote the solubilization of phosphorus compounds contained in the soil and their use by the roots of the plant culture. In this way, several properties necessary for the growth of the plant can be provided in a single operation.

According to yet another embodiment, the microorganisms that have proliferated are recovered and implemented in a specific formulation before being brought into contact with the plant culture and/or the environment. For example, the microorganisms are formulated in a liquid matrix that promotes the attachment of said microorganisms to the seeds or roots of the plants.

Preferably, the proliferated microorganisms are diluted before being applied, for example by spraying on the ground or more precisely in the trench when burying the seeds. They can also be applied directly to the seed before burial. The proliferated microorganisms can also be sprayed on the crops, and mainly their leaves, via manual devices, airplanes or, preferably, drones. The proliferated microorganisms can also be introduced, immediately after their production, into the supply circuit of fertigation devices, which combine a supply of fertilizer and water via precise irrigation. In a particular case, the proliferated microorganisms can be introduced into the growing medium of plants grown hydroponically. In certain cases, a formulation step can be included before the application of the microorganisms.

According to another object, there is provided a plant for producing microorganisms comprising: a first housing for receiving a first closed envelope containing a given strain of microorganisms in a dormant state, said given strain of microorganisms being adapted to promote the growth of at least one predefined plant variety in a given medium; a second housing for receiving a second envelope independent of said first envelope; a connection device extending between said first and second housings for being able to connect together said first envelope and said second envelope, and for being able to transfer said strain of microorganisms from said first envelope to said second envelope; an introduction device for introducing nutrients inside said second envelope, so as to be able to cause the proliferation of the microorganisms inside said second envelope; and, said second envelope is extracted from said second housing for being able to recover the microorganisms having proliferated inside said second envelope and to bring them into contact with said at least one predefined plant variety and/or said given medium.

Thus, the production installation in accordance with the invention makes it possible to implement the method according to the aforementioned invention.

As will be explained in more detail below, the production facility allows people who are not specialists in the cultivation of microorganisms to be able to produce in situ microorganisms that can be used as biofertilizers, biostimulants or as biological products for crop protection, soil remediation or carbon fixation, without any particular constraints and nevertheless under optimal proliferation conditions.

According to a particularly advantageous embodiment of the invention, but in no way limiting, the production installation comprises a receptacle adapted to receive said nutrients, and said receptacle is connected to said second envelope to introduce said nutrients inside. Thus, the first envelope containing a given strain of microorganisms can be provided with the receptacle containing the nutrients adapted to the growth of said given strain. The receptacle can advantageously enclose these nutrients in a sealed manner so as not to be able to be contaminated by other microorganisms or germs.

Furthermore, the receptacle is preferably adapted to be connected to the connection device to ensure the watertight transfer of nutrients inside the second envelope.

In addition, the connection device preferably comprises an airlock allowing the sealed connection of the second envelope to the first envelope to ensure the transfer of the strain of microorganisms, but also possibly to the receptacle to introduce the nutrients inside the second envelope. Thus, the contents of the second envelope cannot in any way be contaminated by other microorganisms or germs likely to affect the proliferation of the microorganisms of said strain.

Preferably, the installation comprises a spectrophotometer for measuring the quantity of microorganisms that have proliferated inside said second envelope. To implement such a spectrophotometer, the second envelope comprises, for example, a transparent conduit installed in a loop and sealed to the wall of the second envelope. The transparent conduit then passes through the cell of the spectrophotometer. Preferably, the optimum absorption wavelength of the microorganisms is 600 nm. By means of calibration, the spectrophotometer provides a measurement of the turbidity of the contents of the second envelope, a measurement that can then be correlated with the quantity of microorganisms generated. Furthermore, by carrying out such a measurement at a predetermined frequency, it is thus possible to measure the proliferation rate of the microorganisms.

Advantageously, the installation comprises a temperature control member located around said second housing to bring the temperature of said second envelope to a predetermined value. The temperature control member then comprises a probe to be able to measure the temperature of the contents of the second envelope. It further comprises elements for cooling or heating the interior of the second envelope. Such elements advantageously comprise Peltier effect modules, which make it possible to precisely adjust the required temperature, for example to 35 °C.

In addition, the device according to the invention comprises drive members for driving the contents of said second envelope into motion. These drive members comprise, for example, a peristaltic pump, making it possible to suck the contents of the second envelope into a given zone through a tapping and to discharge them into a separate zone, so as to drive the contents of the second envelope into motion by forced convection. Thanks to the peristaltic pump, there is no risk of external contamination of the contents of the second envelope.

According to another embodiment, the drive members comprise a disposable rotating part of the propeller type, permanently installed inside the second envelope and adapted to be driven in rotation by means external to the envelope. According to yet another embodiment, the second envelope is itself driven in movement alternately in order to be able to drive its contents in movement, and thus homogenize them. For example, the second envelope is driven in movement with a wave agitation device, or "wave bioreactor" in English, or with a device driving it on itself. According to yet another embodiment, the injection of air or gas bubbles into the second envelope is sufficient to set the contents of the second envelope in motion ("air lift bioreactor" in English).

Also, the installation according to the invention preferably comprises an injection member for injecting an aqueous mixture inside said second envelope. The injection member is for example coupled to the connection device which makes it possible to avoid contamination of the second envelope. The injection member preferably comprises a pump, preferably a peristaltic pump immersed in a sterile aqueous mixture.

Furthermore, in a non-limiting manner, the production installation according to the invention comprises a pH measuring device to be able to maintain the pH inside said second envelope when the microorganisms proliferate, between two predetermined pH values. The pH measuring device preferably comprises a pH meter. The latter comprises a probe placed in contact with the contents of the second envelope to be able to measure its acidity. Automatic additions of an acid or a base are provided to adjust the pH value between two predetermined values.

Also, in a non-limiting manner, the production facility advantageously comprises an oxygen probe for maintaining, inside said second envelope, the oxygen concentration between two predetermined concentration values, when the microorganisms proliferate. The oxygen probe comprises, for example, a cell receiving a cathode and an anode electrically connected by an electrolyte. The electrolyte is then separated from a fraction of the contents of the second envelope by a membrane permeable to dissolved gaseous oxygen. The oxygen can then pass through the membrane to be reduced at the cathode, producing an electric current revealing the oxygen concentration.

Preferably, the production facility comprises means for the second envelope to exchange air with the outside or with an external tank. Preferably, the connection comprises a filter to prevent contamination.

This information is then processed in a microcontroller mounted on the system. In addition, the microcontroller is connected to a display screen also mounted on the system.

Also, all other measuring and control organs are connected to the microcontroller so that they can be controlled automatically and the values they measure can be displayed.

Other features and advantages of the invention will emerge from reading the description given below of particular embodiments of the invention, given for information purposes only but not as a limitation, with reference to the appended drawings in which:

is a schematic view of a plant for producing microorganisms according to the invention; and,

is a flowchart of the method of producing microorganisms in accordance with the invention.

We will first describe the installation 10 as illustrated in the before describing more precisely with reference to the , the method of producing microorganisms that it allows to be implemented.

The microorganism production facility 10 according to the invention comprises a large housing 12, inside which a culture envelope 14 is installed. This culture envelope 14 defines an interior space 15 whose volume is adapted to the need. This interior space can be greater than one liter, ten liters or 100 liters.

The envelope 14 is made of a flexible and recyclable or biodegradable polymer material. Its thickness is less than 0.5 mm. In addition, the polymer used is impermeable to gas to a certain extent. The main suitable recyclable polymers are polypropylene, polyethylene terephthalate, high-density polyethylene or low-density polyethylene. As regards biodegradable polymers, polyvinyl alcohol or polycaprolactone can be considered.

The installation 10 also includes a small housing 16 in which a storage envelope 18 is installed. The storage envelope 18 is made of a recyclable polymer material of the type described above. Also, it must be waterproof and not very fragile to be able to ensure the storage and transport of microorganisms in a dormant state.

The small housing 16 surmounts the large housing 12 and they are connected together in a sealed material by means of a substantially cylindrical connecting device 20 with a circular directrix curve.

Also, the culture envelope 14 has a neck 22, sealed to the connection device 20. The connection device 20 comprises, inside, a tubular member 24 having a lower end 26 opening into the neck 22 of the culture envelope 14, and a pointed upper end 28 opening into the small housing 16.

As will be explained in more detail in the remainder of the description, the upper end 28 extends inside the storage envelope 18 so as to be able to put it in communication with the culture envelope 14. In addition, the tubular member 24 comprises inside, an endless screw 30 making it possible to transfer the contents of the storage envelope 18 to the inside of the culture envelope 14.

The assembly is designed so that the transfer of the contents of the storage envelope 18 to the culture envelope 14 takes place in a sterile manner.

The installation 10 comprises a receptacle 32 containing nutrients adapted to the proliferation of the microorganisms which it is intended to produce.

Also, the installation 10 comprises a first conduit 34 extending from the receptacle 32 to the connection device 10 which it seals, and it extends through the neck 22 and inside 15 of the culture envelope 14.

Furthermore, the installation 10 is equipped with a feed pump (not shown) to be able to convey nutrients 32 into the culture envelope 14.

In addition, the installation 10 comprises a first peristaltic pump 36 connected to an inlet of an aqueous mixture 38, essentially comprising water and free of germs.

Also, the peristaltic pump 36 comprises a first long conduit 40 passing in a sealed manner through the connection device 20 and extending inside 15 of the culture envelope 14 to the bottom, and a first short conduit 42 also passing in a sealed manner through the connection device 20 to extend inside 15 of the culture envelope 14 in the vicinity of the neck 22.

Thus, the first peristaltic pump 36 has two functions. On the one hand, it allows the culture envelope 14 to be supplied with an aqueous mixture, and on the other hand, it ensures the homogenization of the contents of the culture envelope 14 by sucking it up via the long conduit 40 and discharging it via the short conduit 42 as will be explained below.

The installation 10 comprises a second peristaltic pump 44, connected to a second pumping conduit 46 and to a second discharge conduit 48. The pumping conduit 46 passes through a plurality 50 of measuring cells which it supplies and which will be detailed below. The pumping conduits 46 and discharge conduits 48 pass through the connection device 20 in a sealed manner and extend inside 15 of the culture envelope 14 to the bottom.

Thus, the second pumping conduit 46 supplies a first cell 52 of a spectrophotometer. This first cell 52 makes it possible to measure the absorbance of the liquid passing through it at a wavelength of 600 nm. This measurement is correlated with a concentration of microorganisms in said passing liquid.

In addition, the second pumping conduit 46 supplies a second cell 54 for measuring the pH of the liquid passing through it. The second cell 54 is equipped with a glass electrode and a calomel reference electrode. The potential difference between the two electrodes provides access to the pH value of the liquid.

Also, the second pumping conduit 46 supplies a third cell 56 including an oxygen probe. It thus makes it possible to measure the oxygen concentration of the liquid. The cell comprises two electrodes immersed in an electrolyte, which electrolyte is separated from the liquid of the pumping conduit 46 by a membrane permeable to oxygen. A measurement of the oxygen concentration is thus obtained by means of a measurement of the potential difference between two electrodes.

The plurality 50 of measuring cells is controlled by a microcontroller 58. And the results of the measurements carried out by the cells 52, 54, 56 are then processed in the microcontroller 58 and can be displayed on a display screen 60.

In addition, the installation 10 includes a temperature probe 62 located under the culture envelope 14 to be able to measure its temperature. In addition, the large housing 12 is equipped with Peltier effect modules 64 making it possible to maintain the temperature of the culture envelope 14 between predetermined values. These temperature values are advantageously between 30 °C and 40 °C.

We will now describe with regard to the flowchart shown in the and in view of the installation 10 of the , the method of producing microorganisms in accordance with the invention.

Thus, in a first step 66, a strain of a type of microorganism is chosen which is adapted to promoting the growth of a predefined plant variety, for example, a variety of rice.

For example, bacteria of the genus Pseudomonas and in particular of the species Pseudomonas fluorescens are chosen as microorganisms. These bacteria are involved in particular in the phosphorus cycle and they promote the absorption of phosphate in ionic form by the plant.

Thus, in a second step 68 a strain of the species Pseudomonas fluorescens is provided, packaged inside an envelope of the type of the storage envelope 18 as shown in the . The storage envelope 18 delimits a volume of, for example, between 10 cm³ and 500 cm³. Here it has a volume of 300 cm³ which is entirely filled with a strain of the bacterial species Pseudomonas fluorescens in a liquid medium.

Thus, this strain can be preserved, stored and transported to its place of use, kept refrigerated at 4°C, in the vicinity of a field of the aforementioned plant variety, without being contaminated by other germs.

This particular bacterial strain is associated with an optimal proliferation protocol only in terms of the temperature of the environment in which it can develop and the nutrients necessary for its growth .

This information is then recorded in the microcontroller 58 of the installation 10, in a third step 70, a recording step.

According to a fourth step 72, the culture envelope 14, initially containing the culture medium , is installed inside the large housing 12 and it is connected in a sealed and aseptic manner to the connection device 20. The interior 15 of the culture envelope 14 is obviously sterile. It contains one liter of a culture medium adapted to the culture of Pseudomonas fluorescens . Here, LB medium, for English “Lurya broth”, buffered to pH 7, is chosen.

Then, according to a fifth step 74, the storage envelope 18 containing the bacterial strain is first loaded inside the second receptacle 16.

The second receptacle 16 delimits a sealed cavity and after the storage envelope 18 has been inserted inside, it is closed. And only then is the storage envelope 18 perforated via the upper end 28 at the tip of the tubular member 24, so as to avoid any contamination of the bacterial strain.

The perforation of the storage envelope 18 makes it possible to release the microorganisms inside the second receptacle and to be able to convey them inside the culture envelope 14, in particular by means of the worm screw 30.

In addition, the Peltier effect modules 64 are controlled to raise the temperature inside the culture envelope 14 to between 28°C and 32°C.

Furthermore, when the water loading of the culture envelope 14 has made it possible to reach the desired volume, the arrival of the aqueous mixture 38 is cut off, while the peristaltic pump 36 draws the liquid from the culture envelope 14 through the first long conduit 40 and discharges it through the first short conduit 42 to cause agitation of the medium. In this way, the contents of the culture envelope 14 are homogenized and the access of the bacteria to their nutrients is promoted.

Consequently, the bacteria of the strain will come out of their dormant state and will first grow independently of each other before multiplying. Thus, several phases appear in the growth of bacteria, a lag phase, in which the bacteria come out of their dormant state, an acceleration phase where the bacteria begin to divide, an exponential, or logarithmic, phase, a deceleration phase and then a stationary phase.

This gives a growth rate over time that follows a substantially sigmoidal curve. And it is appropriate to use the bacteria that have proliferated in this way at the right time and in an advantageous manner, between the location of their exponential growth phase and the start of the stationary phase.

Where appropriate, and not limited to, in order to optimize the proliferation of the microorganisms contained in the culture envelope 14, the following checks are carried out simultaneously, using the plurality 50 of cells.

Thus, thanks to the second peristaltic pump 44, the liquid contained inside 15 of the culture envelope 14, in which the microorganisms proliferate, is sucked up and conveyed to the right of the first cell 52 allowing the concentration of microorganisms to be measured. This measurement is carried out sequentially and at regular time intervals, according to a first sixth parallel step 76, so as to be able to establish the growth curve of the microorganisms inside 15 of the culture envelope 14.

Also, the aspirated liquid is directed to the right of the second cell 54 allowing, according to a first sixth step 78, to measure the pH. To obtain optimal proliferation of the bacteria present, the pH must be between 5.5 and 8.5.

If the second cell 54 records a value outside these limits, a device not shown makes it possible to automatically inject an acid buffer when the pH is greater than 7.5 or a basic buffer when it is less than 6.5.

And to the right of the third cell 56, is controlled, in a third sixth parallel stage 80, the oxygen concentration of the aspirated liquid. The species Pseudomonas fluorescens is an aerobic bacterium and it can develop when the oxygen concentration is for example between 5 g per liter and 10 g per liter.

The third cell 56, which provides a measurement of this concentration, makes it possible to know whether the liquid in the culture envelope 14 is at the optimum concentration.

If not, either oxygen is injected directly into the culture envelope 14, when oxygen is lacking, or a neutral gas, for example nitrogen, is injected precisely to expel the excess oxygen in the medium.

In this way, by monitoring these three parameters at regular intervals, while maintaining the temperature between 34°C and 36°C, and by agitating the medium, optimal growth of the bacteria Pseudomonas fluorescens is obtained.

By monitoring its concentration through the first cell 52, the bacterial growth rate is checked according to a seventh control step 82, and if it has reached the aforementioned stationary phase, the culture envelope 14 is then removed from the first receptacle 12, and its contents are recovered according to an eighth recovery step 84 so as to be able to implement its contents, either directly on the plants, or advantageously on the soil where they will be cultivated.

According to a particularly advantageous implementation method, the storage envelope initially contains the microorganisms but also their nutrients in concentrated form. The storage envelope is then kept at a temperature below 4 °C to prevent the proliferation of the microorganisms. In addition, the culture envelope initially contains water. Thus, the two envelopes can initially be mass-produced and sealed to avoid any contamination, so that they can be implemented in situ without additional supply of nutrients or any aqueous medium.

The example device described above makes it possible to implement the method according to the invention. Other, simpler examples of devices, making it possible to control the quantity of microorganisms and to agitate the culture envelope can be implemented.

Also, among microorganisms, plant growth promoting bacteria, or PGPB, can play a role as biofertilizers by fixing nitrogen for example or by promoting the solubilization of compounds containing phosphorus. They can also have a role as biostimulants, for example by secreting components, such as indole 3-acetic acid, playing the role of growth factors for certain plants. They can also have a role in crop protection as a pesticide for example.

In a particular embodiment, the microorganisms can be used, alone or in combination with other living organisms such as fungi and with various mineral inputs, for example rock dust as part of a forced weathering remediation operation or organic inputs, for example biochar, to promote carbon fixation within soils.

These microorganisms can be aerobic, strictly anaerobic, facultative anaerobic, facultative aerobic or micro-aerobic. They can be gram-positive or gram-negative bacteria. They can be bacteria capable of forming spores or which do not form spores.

The method according to the invention allows, without this list being considered exhaustive, the multiplication of bacteria of the genus Azotobacter , such as Azotrobacter vinelaandii, Azotobacter chroococcum ; of bacteria of the genus Bacillus , such as Bacillus circulans , Bacillus subtilis, Bacillus Megaterium ; of bacteria of the genus Pseudomonas , such as Pseudomonas putida , Pseudomonas fluorescens , Pseudomonas megaterium, Pseudomonas bilaiae ; of bacteria of the genus Azospirillum , such as Azospirillum brasilense ; of Gluconacetobacter diazotrophicus ; of Kosakonia sacchari ; of Klebsiella variicola or Klebsiella pneumoniae ; of Xanthobacter autotrophicus ; of pigmented heterotrophic bacteria, in particular facultative methanotrophic pink bacteria; bacteria of the genus Rhizobium or Bradyrhizobium , for example Bradyrhizobium japonicum .

The method according to the invention also allows the multiplication of fungi such as Aspergillus or Trichoderma, for example Trichoderma harzianum .

It also allows the multiplication of yeasts such as Saccharomyces or Penicillium .

The method according to the invention also allows the multiplication of unicellular algae, for example Chlorella , or cyanobacteria, for example Arthrospira, Nostoc , or Plectonema. In this case, a source of natural or artificial light can be provided continuously or intermittently, to the culture of these photosynthetic microorganisms.

In addition, wild strains of these microorganisms can be used. Strains modified by random mutagenesis can also be used.

Also, genetically engineered strains may be used. Genetic engineering may involve the transfer of gene(s) from microorganisms of a different genus to the modified microorganism (transgenesis) or editing the genome of the modified microorganism without intergeneric transfer of gene(s), for example via methods derived from CRISPR or using transposons or referred to in English as " prime editing ".

Typically, after multiplication of microorganisms by the method according to the invention, concentrations of microorganisms in the medium ranging from 1x10 ⁶ to 1x10 ¹⁰ CFU/mL, preferably 1x10 ⁷ to 1x10 ⁹ CFU/mL are obtained; “CFU” meaning “Colony Forming Unit”.

Thus, an amount of 1x10 ⁸ to 1x10 ¹³ CFU/ha, preferably 1x10 ¹¹ to 1x10 ¹³ CFU/ha is typically used for subsequent agronomic application.

Thanks to the method according to the invention, the culture conditions are better controlled and consequently, the quantity of living microorganisms produced is also better controlled.

In addition, the microorganisms are implemented in their maximum multiplication phase and consequently, their possibility of survival in the soil is increased. This advantage is likely to lead to significant agronomic benefits compared to the methods of implementing microorganisms used in agriculture according to the state of the art.

Also, thanks to the method according to the invention, it is possible to adjust the culture conditions to prepare the microorganisms for the stress they will encounter in the soil. In other words, the microorganisms are conditioned and their possibility of survival in the soil is increased accordingly.

Furthermore, the method according to the invention, or the production of microorganisms in situ, makes it possible to reduce the production of microorganisms upstream, typically by a factor greater than 10 or greater than 100, or even greater than 1000.

This reduces the overall cost of the process, and makes it possible to reduce the investment costs of the production unit for micro-organisms intended for the first envelope and makes it possible, for the same investment, to produce after multiplication in the second envelope, 10 times or 100 times more micro-organisms making it possible to treat an agricultural area 10 times or 100 times larger than if the micro-organisms had been produced in a comparable production unit and directly implemented conventionally without multiplication by the process according to the invention.

This results in a reduction in the logistical footprint, since much smaller quantities of microorganisms are transported.

Furthermore, since the conditions for keeping microorganisms alive, for example between -20°C and 4°C, are relatively costly and complex from a logistical point of view, it is advantageous to be able to transport quantities 10 or 100 times smaller than the quantities needed to be implemented in situ.

A very low dose of micro-organisms allowing, after multiplication by the process according to the invention, to treat a relatively large agricultural area, it is acceptable, from the point of view of the cost, to implement relatively expensive physical and/or chemical methods to protect or optimize these micro-organisms.

Also, thanks to the method according to the invention, the speed of proliferation of the microorganisms can be controlled. And consequently, for certain microorganisms, growth in the culture envelope was carried out relatively slowly.

Under these conditions, it appeared that the resulting microorganisms were much more resistant in the soil to a potentially stressful physicochemical environment and to the competition they face with the microorganisms already present in the soil.

Claims (18)

Method for producing microorganisms characterized in that it comprises the following steps:
- a first closed envelope (18) is provided containing a given strain of microorganisms in a dormant state, said given strain of microorganisms being adapted to promote the growth of at least one predefined plant variety in a given medium;
- a second envelope (14) is provided, independent of said first envelope (18);
- a connecting device (20) is provided for being able to connect together said first envelope (18) and said second envelope (14), and for being able to transfer said strain of microorganisms from said first envelope to said second envelope;
- nutrients are introduced inside said second envelope (14), so as to be able to cause the proliferation of microorganisms inside said second envelope;
and the microorganisms having proliferated inside said second envelope (14) are recovered to put them in contact with said at least one predefined plant variety and/or said given medium. Method according to claim 1, characterized in that the quantity of microorganisms having proliferated inside said second envelope (14) is measured, and in that the microorganisms having proliferated are recovered when said quantity reaches a predetermined threshold. Method according to claim 1 or 2, characterized in that the recovered microorganisms are brought into contact with said at least one predefined plant variety and/or said given medium, at most 96 hours after having recovered the microorganisms. Method according to any one of claims 1 to 3, characterized in that the temperature of said second envelope (14) is brought to a predetermined value. Method according to any one of claims 1 to 4, characterized in that the contents of said second envelope (14) are moved. Method according to any one of claims 1 to 5, characterized in that an aqueous mixture is injected inside said second envelope (14). Method according to any one of claims 1 to 6, characterized in that the pH inside said second envelope (14) is maintained between two predetermined pH values when the microorganisms proliferate. Method according to any one of claims 1 to 7, characterized in that the oxygen concentration inside said second envelope (14) is maintained between two predetermined concentration values, when the microorganisms proliferate. Method according to any one of claims 1 to 8, characterized in that said first and second envelopes (14, 18) are single use. Method according to any one of claims 1 to 9, characterized in that said first closed envelope (18) is provided, further containing an additional strain. data from dormant microorganisms. Microorganism production facility (10) characterized in that it comprises:
- a first housing (16) for receiving a first closed envelope (18) containing a given strain of microorganisms in a dormant state, said given strain of microorganisms being adapted to promote the growth of at least one predefined plant variety in a given medium ;
- a second housing (12) for receiving a second envelope (14) independent of said first envelope (18);
- a connecting device (20) extending between said first (16) and second (12) housings to be able to connect together said first envelope (18) and said second envelope (14), and to be able to transfer said strain of microorganisms from said first envelope (18) to said second envelope (14);
- an introduction device (32, 34) for introducing nutrients inside said second envelope (14), so as to be able to cause the proliferation of microorganisms inside said second envelope; and,
- said second envelope (14) is extracted from said second housing (12) in order to be able to recover the microorganisms which have proliferated inside said second envelope and to put them in contact with said at least one predefined plant variety and/or said given environment. Production plant according to claim 11, characterized in that said introduction device comprises a receptacle (32) adapted to receive said nutrients, and in that said receptacle (32) is connected to said second casing (14) to introduce said nutrients inside (15). Production installation according to claim 11 or 12, characterized in that it comprises a spectrophotometer (52) for measuring the quantity of microorganisms having proliferated inside said second envelope (14). Production installation according to any one of claims 11 to 13, characterized in that it comprises a temperature control member (62, 64) located around said second housing (14) to bring the temperature of said second envelope (14) to a predetermined value. Production installation according to any one of claims 11 to 14, characterized in that it comprises drive members (36) for driving the contents of said second envelope (14) in movement. Production installation according to any one of claims 11 to 15, characterized in that it comprises an injection member (36, 38, 42) for injecting an aqueous mixture inside said second envelope (14). Production plant according to any one of claims 11 to 16, characterized in that it comprises a pH measuring member (54) to be able to maintain the pH inside said second envelope (14) when the microorganisms proliferate, between two predetermined pH values. Production plant according to any one of claims 11 to 17, characterized in that it comprises an oxygen probe (56) for maintaining, inside said second envelope (14), the oxygen concentration between two predetermined concentration values, when the microorganisms proliferate.