WO2023222956A1

WO2023222956A1 - Method for producing a microbial carrier, associated product and use thereof

Info

Publication number: WO2023222956A1
Application number: PCT/FR2023/000102
Authority: WO
Inventors: Magali GARCIA; Paul Thomas; Yannick MANON
Original assignee: Ab7 Industries
Priority date: 2022-05-18
Filing date: 2023-05-16
Publication date: 2023-11-23
Also published as: FR3135730A1

Abstract

The present invention relates to a method for manufacturing a microbial carrier. The method according to the invention aims to produce solid carriers on which microorganisms are immobilized, said supports being intended to be used in various biological applications and making it possible to maintain cell viability over time.

Description

DESCRIPTION

TITLE: Process for manufacturing a microbial support, associated product and its use

The present invention relates to a method of manufacturing a microbial support. The invention also relates to a product resulting from this process, namely the support on which microorganisms are immobilized, as well as its use.

The method according to the invention aims to produce supports on which microorganisms are immobilized; said supports are intended to be used in various biological applications.

Such applications are based on the use of microorganisms, particularly in the fields of agriculture, oenology, water treatment, soil remediation, surface treatment, and even veterinary medicine.

More generally, it is appropriate to distinguish the use of free microorganisms or in planktonic form as opposed to the use of microorganisms in non-free form, namely fixed or immobilized.

Indeed, it is known that non-free forms of microorganisms can be more resistant and more persistent over time for the same species. Sometimes, this aggregation is likely to pose a problem when it involves pathogenic microorganisms, but it can nevertheless prove to be beneficial with non-pathogenic microorganisms that are described as beneficial or effectors.

This benefit then takes on its full meaning in the context of agriculture as well as in the development of new phytosanitary techniques or even in that of biocontrol in plants or animals in a leading context that is that of ecology and respect for the environment.

There is a need to rethink sustainable agriculture and treatments using fewer pesticides and chemical fertilizers, by limiting soil inputs, but we also need to reposition ourselves on biocontrol to limit the use of chemical parasiticide in plants or animals. In this respect, we know of microorganisms having proven effects on parasites or which act as a real growth or protection agent in plants.

In the phytosanitary field, we find bioinoculant or biofertilizer type products in the form of powder or liquids intended for agricultural spreading. These However, products require repeated applications over time, which leads to an increase in costs and the more frequent mobilization of resources. Furthermore, these uses are not entirely satisfactory because the viability of the microorganisms poses a problem and constitutes a major obstacle before and after use.

Indeed, the population of microorganisms tends to drop drastically after use; several hypotheses can explain this.

Concerning liquid forms, there is a certain competition between the indigenous soil microbiota and exogenous microorganisms, introduced during treatment. We can add to this the stress suffered by the latter, who must adapt to a new environment to survive under different constraints such as hygrometry, ultraviolet (UV) rays, pH or pollution, we then speak of need for acclimatization.

Concerning the powder forms of bioinoculants, the microorganisms are mixed with mineral or inorganic powders, for example based on talc, clays or alumino-silicate. After being cultivated in a humid or liquid environment, the latter are often subjected to desiccation or freeze-drying before mixing with mineral powders and then spreading. Here again, cell viability is contested, with current techniques being too aggressive to be able to claim an economical and long-term stable biological product.

The development of better formulations to ensure survival and activity in the field on the one hand, and compatibility with chemical and biological treatments on the other hand is attracting increasing attention. Approaches include optimization of growth conditions prior to formulation and development of improved carrier and application technology. As such, maintaining the stability, effectiveness and viability of effector strains is a real challenge which must be done in compliance with the standards associated with bioinoculants.

Immobilization techniques for microorganisms on supports can be a response in various applications. Thus, it is understood for the purposes of this application that the process according to the invention has no limiting nature concerning its field of application.

The development of formulations or products must take into account the change in scale, the shelf life as well as compatibility with current practices.

Among the known means using microorganisms on support, there are known methods of encapsulation or inclusion of microorganisms with mineral powders containing nutrient medium for the development of the microorganisms after spreading. But the stability over time, the survival of the microorganisms remains less than 5 months, which is not satisfactory.

We also find, in agriculture, means aimed at preserving the microorganisms of the rhizosphere, notably with the use of alginate in the form of micro-beads. The alginate matrix covering the seed must ensure protection and release of microbes in the field, near the seed or the plant rhizosphere.

Encapsulated formulations provide physical protection against harsh environments as well as a three-dimensional structure to which cells can adhere but these do not last over time. All these methods and processes are intended for seed coating. Unfortunately, there are still many downsides to this.

Indeed, when applying directly to the seed or seeds, the phytosanitary constraint requires strict compliance with certain health standards regarding microorganisms. The handling of microorganisms on seeds relating to foodstuffs must be able to be done in the presence of an antifungal, the latter does not make the development of all microorganisms conducive, which can constitute a barrier in certain applications.

We can also add that working on seeds is not without risk for the development of the plant itself; germination problems are frequently encountered among coated or treated grains. There is thus a need for a means of preparing a product based on a microorganism, capable of responding to the disadvantages cited, respectful of the environment, which can respond to a problem of viability and stability over time of said microorganisms.

An aim of the invention is thus to provide a process for manufacturing a solid microbial support allowing better stability and viability of microorganisms over time.

The invention also relates to the product obtained by this process and its use in improving the survival of microorganisms over time. The microbial support of the invention is defined by a solid support, which after the different stages of the microorganism immobilization process, will serve as a microbial vector.

By microbial vector is meant the microbial support corresponding to the solid support, after immobilization of the microorganisms, obtained at the end of the manufacturing process according to the invention. Said microbial vector will allow the supply of a quantity of microorganisms in various biological applications, in particular agriculture. In the following the text of the present invention, the notion of microbial vector, microbial support or solid microbial support will be used interchangeably.

The process for manufacturing the microbial support proposed by the applicant makes it possible to obtain a product which overcomes the preceding obstacles, and which has multiple applications. It is understood that the use of the product resulting from this process is not limited to the agricultural sector.

The process for manufacturing a solid microbial support according to the invention comprises the following steps: a) Preparation of the solid support b) Immobilization of the microorganisms on the support c) Drying

By immobilization we mean an adhesion of microorganisms on the support, comparable to a biofilm, and acting as a stable microbial coating. For the purposes of this application, it is appropriate to define immobilization as an adsorption or absorption of microorganisms on the support which allows stable and effective fixation of the latter over time. For the purposes of this application, stable means the capacity of a microorganism to be preserved or renewed over time.

The solid supports according to the invention can be porous materials, capable of increasing in volume when they are brought into contact with a liquid and which have good tolerance to drying. The latter must be easily accessible, physically and chemically stable, non-toxic in use, non-toxic to microorganisms, free of pollutants, easy to process, and possibly have a high moisture retention capacity. In certain applications according to the invention, the support is chosen for its ability to retain water. Advantageously, the solid supports of the invention have a water retention capacity of 0.001 to 5 times their weight.

Solid supports can be chosen from organic materials, fibrous materials, biodegradable polymers as well as materials of plant origin, and any other biosourced materials having a good specific surface area.

By good specific surface area, we mean a ratio of the surface area of the real surface of the material and the apparent volume of the object, sufficient to allow surface phenomena such as adsorption or absorption of microorganisms. Ideally, the support has good surface roughness which can be defined as the ability of the material to be a support for microorganisms while being porous. In one embodiment, the solid supports used in the process according to the invention are therefore preferably in the form of granules, chips or powder whose particle size is between 200 pm and 15000 pm, preferably between 500 pm and 10000 pm, more preferably between 1500 pm and 5000 pm, more preferably between 2000 pm and 4500 pm and even more preferably between 2500 pm and 3800 pm.

In a variant, the supports can also be membrane type materials, particularly in the context of supports immersed in a liquid, or even materials resulting from three-dimensional printing, said materials being able to be biosourced.

In another embodiment, the solid supports have a density of between 100 kg/m ³ and 5000 kg/m ³ or between 200 kg/m ³ and 1200 kg/m ³ , or between 300 kg/m ³ and 500 kg/m ³ , preferably between 360 kg/m ³ and 460 kg/m ³ .

In a preferred embodiment, the solid support is a biosourced and biodegradable material.

In a preferred embodiment of all, the solid support is corn cob.

By microorganisms we mean organisms belonging to the category of bacteria, fungi, microalgae or even yeast. The supports according to the invention can thus be used for the immobilization of these categories of microorganisms taken individually or in the form of a consortium. Thus the process according to the invention can allow the immobilization of several species, or categories, on the same support.

The bacteria capable of being immobilized according to the invention are chosen from the genera Bacillus, Pseudomonas, Rhizobium, Azospirillum, Nitrosomonas, Nitrobacter, Oenococcus, Lactobacillus, Lactococcus, Leuconostoc, Arthrospira, Limnospira, Aphanizomenon, Azotobacter or their mixture. The preferred bacteria according to the invention are chosen from Bacillus Thuringiensis, Oenococcus oeni, Pseudomonas fluorescens, Rhizobium leguminorosarum or their mixture.

The fungi capable of being immobilized according to the invention are chosen from the genera Aspergillus, Trichoderma, Phanerochaete, Phlebiopsis, Coniothyrium, Rhizophagus, preferably Aspergillus brasiliensis and Trichoderma harzianum or their mixture.

The microalgae capable of being immobilized according to the invention are chosen from the genera Chlorella, Duneliella, Odontella, Haematococcus, Scenedesmus, Porphyridium, Ascophyllum, preferably Chlorella vulgaris and Haematococcus pluvialis or their mixture.

The yeasts capable of being immobilized according to the process of the invention are chosen from yeasts, such as the genus Saccharomyces, Kluyveromyces, Pichia, Rhodotorula, Yarrowia, Candida preferably Saccharomyces cerevisiae and Pichia pastoris or their mixture.

In addition to the microorganisms mentioned above, the process according to the invention does not exclude the use of strains of probiotics known to those skilled in the art.

One embodiment of the invention relates to a process for manufacturing a microbial support corresponding to the solid support on which bacteria, fungi, yeasts, microalgae or a mixture of these can be immobilized. The support being a solid support chosen from a biodegradable polymer, a compostable polymer, a biodegradable polyester, a biodegradable copolyester, cellulose acetate, polylactic acid, cellulose powder, grape marc, grape stalk, preferably corn stalk.

In a preferred variant, the invention relates to such a process involving bacteria or fungi on corncobs.

The first step of the process according to the invention is that of the preparation of the solid support. Indeed, it is possible that spores of undesirable microorganisms may still be present on solid supports. However, this sporulating form is more resistant and therefore more difficult to eliminate. In order to guarantee the elimination of these spores, the applicant included in this first stage of preparation of solid supports, a germination stage. Germination is defined as the fact that a microorganism potentially present in the form of a spore on the starting support exits its sporulation state.

Said germination step consists, after rinsing the solid supports with water and a first sanitization, of placing the wet supports for 1 hour to 24 hours, preferably for 10 hours to 20 hours at a temperature between 20 °C and 40 °C in a suitable closed container, which a person skilled in the art will be able to choose and adapt. The purpose of this germination is to germinate any spores of unwanted microorganisms which will then be able to be eliminated during sanitization.

It is understood within the meaning of the invention that the means for sanitizing solid supports are conventional means commonly used by those skilled in the art such as the use of dry or wet thermal sterilization means, such as an autoclave. or any other equivalent means known to those skilled in the art. The preparation step of the process according to the invention comprises at least one sanitizing step after germination. In a variant, there is a pre-germination sanitization step and a post-germination sanitization step, said germination being an intermediate step between two sanitization phases.

The second step of the process according to the invention is a step of immobilizing the microorganisms on a solid support. This immobilization step is divided into two successive and complementary phases: inoculation and incubation of the supports in a seeding solution. Incubation also allows the growth and attachment of cells to the support.

Inoculation within the meaning of the invention means the action which consists of bringing a previously prepared seeding solution into contact with the supports obtained during the first stage of the process, namely that of the preparation of the support.

By contacting we mean the immersion of the support in an adequate volume of a seeding solution.

In order to ensure optimal immobilization of the microorganisms, the applicant has found that a ratio of between 0.1:10 and 9.5:10, i.e. between 0.1 gram of support for 10ml of seeding solution and 9 .5 grams of support for 10ml of seeding solution, preferably between 2.1:10 and 7:10, i.e. between 2.1 grams of support for 10ml of seeding solution and 7 grams of support for 10ml of seeding solution, even more preferably 2.5:10, or 2.5 grams of supports for 10 ml of seeding solution, allowed lasting immobilization of the microorganisms on said supports.

Said seeding solution consists of a mixture of a suspension of microorganism and a minimally depleted medium. This seeding solution can be defined and understood as serving for immobilization according to the process of the invention.

By suspension of microorganism is meant a mixture of microorganisms with an isotonic aqueous diluent known to those skilled in the art such as a tryptone-salt type diluent, or even a mixture of microorganisms with a culture medium or any other equivalent diluent. known to those skilled in the art.

By depleted minimum medium is meant a minimum medium known to those skilled in the art, in which the applicant has reduced the total quantity of carbon and possibly the quantity of trace elements. This is a minimal nutrient-depleted environment. Advantageously, the use of this medium makes it possible to cause beneficial stress among the microorganisms used in the present process, the latter will in fact have a tendency to modify their behavior and their metabolism in order to survive.

These non-limiting variants allow the process according to the invention to be applied to a wide diversity of microorganisms.

Microorganisms being sensitive to environmental conditions, the applicant was able to observe that the pH tended to decrease with the addition of a biosourced support, in particular corn cob. To counteract the acidity relating to the use of such solid supports, the applicant therefore proposes an inoculation of said supports with a seeding solution containing carbonate or carbonate species in a concentration preferably between 2 and 100 mmol/L, preferably between 3 and 45 mmol/L, even more preferably between 4 and 15 mmol/L.

The carbonates and carbonate species usable in the invention can be chosen from carbonate ions (CO ₃ ² ), hydrogen carbonate ions (HCO ₃ ), carbonic acid (H2CO3) or their mixture.

Sodium hydrogen carbonate (NaHCOs) or calcium carbonate (CaCOs) are the preferred alkaline carbonates within the meaning of the invention.

In a preferred embodiment, the immobilization of the microorganisms on the supports is carried out with a seeding solution consisting of a mixture containing a suspension of microorganisms, a minimum depleted medium in an equivalent ratio in respective volume of 5:95 (v/v) as well as the addition of 0.067% by weight of this mixture of sodium carbonate (NaHCOs). It is understood within the meaning of the invention that this ratio is adjustable depending on the quantity and nature of the support.

In order to finalize the immobilization step, it is necessary to incubate the mixture consisting of the supports and the seeding solution. This step will allow the adhesion of the microorganisms to the support by adsorption and/or absorption as well as their growth.

By incubation we mean the maintenance of microorganisms in favorable growth conditions. The incubation conditions within the meaning of the invention include an incubation temperature of between 20°C and 37°C, an incubation time of between 2 hours and 10 days with agitation or recirculation, if necessary. .

Incubation times can be between 2 hours and 240 hours, between 12 hours and 216 hours, between 24 hours and 192 hours, preferably between 48 hours and 168 hours.

It is understood that these conditions apply to living microorganisms, and that it will be necessary to modulate them according to the microorganism concerned. For the purposes of the invention, it is defined in an undifferentiated manner that the immobilized supports are considered to be supports obtained at the end of the incubation.

The third step of the process according to the invention concerns drying. The immobilized supports must be dried to be conditioned. This is a critical step which avoids freeze-drying and other deleterious processes for the survival of microorganisms. Indeed, freeze-drying and known processes pose problems regarding the survival of microorganisms. In the context of freeze-drying, we observe a significant drop in the survival percentage of microorganisms during drying and storage in freeze-dried form, which is a means conventionally used to preserve them before their use.

Advantageously, the process according to the invention makes it possible to maintain an average survival percentage at least twice greater than that obtained with freeze-dried microorganisms after one year.

Drying can be done using a vacuum oven or any other equivalent and suitable drying means that a person skilled in the art will know how to use.

Thus, the applicant was able to develop drying conditions allowing the obtaining of a microbial support having a residual water content of between 7% and 25% by total weight of support, preferably between 15 and 23%. Surprisingly, the applicant found that residual humidity was necessary to ensure cell viability.

This residual water content ensures good maintenance of the adhesion of microorganisms to the support and their survival during storage periods.

It is understood within the meaning of the invention that the drying parameters are dependent on the equipment and strains used.

Good results were obtained when the drying conditions included a temperature between 0°C and 50°C, a drying time between 1 hour and 24 hours, preferably between 2 hours and 12 hours, as well as a pressure ranging from 1 mbar to 500 mbar, preferably from 10 mbar to 250 mbar, even more preferably from 15 mbar to 100 mbar.

The applicant was able to demonstrate that the best results were obtained when the drying conditions cumulatively combined, on a laboratory scale, the following parameters: a temperature of 35°C, a drying time of 4 hours and a pressure of 30 mbar. The average residual water content obtained after drying under these conditions is 21%. It is understood within the meaning of the invention that those skilled in the art will be able to adapt these parameters for any other scale. The measurement of the residual water content is carried out after obtaining the supports according to the process, by drying said supports at 105°C until a stable mass is obtained.

Thanks to this process, the applicant was able to develop an effective means of preserving the cellular viability of microorganisms, particularly with regard to bacteria inoculated on corncobs.

The process according to the invention allows homogeneous and lasting immobilization of the microorganisms on the support, which results in maintaining at least 25%, preferably 30% and more preferably 50%, of the survival of the microorganisms after storage for 12 months and at least 15%, preferably 20% and more preferably 40% of the survival of the microorganisms after storage for 18 months.

The present invention therefore also relates to the microbial vector obtained according to the method according to the invention and having significantly improved bacterial survival compared to the freeze-dried bacteria obtained in the state of the art.

The invention also relates to the use of the microbial vector according to the invention to improve the survival of microorganisms over time, characterized in that said improvement in the survival of microorganisms after storage is at least 25%, preferably 30% and more. preferably 50% for 12 months and at least 15%, preferably 20% and more preferably 40% for 18 months.

The microbial support manufacturing process as well as said microbial support will be better understood through the examples and figures.

Example 1: Progress of the process according to the invention with the P.fluorescens strain on corncobs

1.Preparation of media

The solid support used for this test is corn cob marketed under the name Eu-Grits 6/8 by Eurocob®.

A 500 ml glass bottle is filled to 1/3 of its volume with the stalk rinsed with water before autoclaving for 25 min at 121°C. This bottle is then closed and incubated for 12 hours at 30°C, to germinate the unwanted spores potentially contained on the solid support. 250 ml Erlenmeyer flasks are filled with 25 g of corncobs and autoclaved again. The Erlenmeyer flasks with supports are then left open under a microbiological safety station for 15 min.

Before proceeding with immobilization, a P.fluorescens seeding solution is then prepared by subculturing the strain onto a Petri dish containing TSA agar medium and incubated for 24 h at 30°C. The colonies are then removed and suspended in 20 ml of Tryptone-Salt broth.

2. Inoculation and incubation of media

The solid supports are placed in contact with a mixture containing 95 ml of depleted minimum medium, 0.067% sodium carbonate (NaHCOs) and 5 ml of the bacterial suspension of P.fluorescens previously prepared, this mixture constituting the seeding solution.

Everything is then incubated for 48 h at 30°C with shaking at 150 rpm and an eccentricity of 5 cm. After incubation, the immobilized supports are obtained.

3. Drying

1 g of inoculated immobilized support is taken from the Erlenmeyer flasks, then the supports are placed in a petri dish with a diameter of 55 mm. The latter are dried in a vacuum oven for 4 hours at 35°C under a pressure of 30 mbar. The supports obtained correspond to the microbial vectors.

Example 2: Comparative study of the survival of microorganisms resulting from freeze-drying and those resulting from the process according to the invention

In order to compare the effectiveness of the process of the invention in maintaining the survival of microorganisms over time, the microbial vector corresponding to the solid support on which a strain of Pseudomonas fluorescens bacteria is immobilized according to the process of the invention has was compared with this same strain marketed in freeze-dried form.

After drying, each dried 1 g sample is stored in a sterile polypropylene (PP) bottle placed in the dark. At each stability time, two samples of 1 g are analyzed. These samples are then placed in 9 ml of tryptone-salt then subjected to a vortex for 1 min before being placed in an ultrasound bath for 10 min. The suspension obtained is diluted in cascade and the dilutions are counted on the surface on TSA agar medium, then incubated at a temperature between 30 and 32°C for 24 h.

After incubation, the colonies are counted in order to deduce the survival percentage. Finally, the average of the bacterial concentrations of the two samples analyzed is taken.

These results show that, when bacteria are freeze-dried, the survival rate drops drastically during the first 10 weeks, with a loss of more than 50% of bacterial survival. On the other hand, with the microbial vector obtained according to the process of the invention, the loss of 50% of bacterial survival only appears after 30 weeks.

Over a full year, or approximately 52 weeks, these results are even more convincing on the effectiveness of the applicant's process in maintaining the survival of bacteria.

Indeed, the average survival remains around 50% after one year for the microbial vector while it continues to decrease for freeze-dried bacteria to reach 10% after one year. The same observation can be demonstrated after 78 weeks, or after 18 months, where the average bacterial survival is around 40% for the microbial vector according to the process of the invention unlike freeze-dried bacteria for which the survival rate only reaches 5% after 78 weeks, or after 18 months.

The applicant has also demonstrated that the survival rate of the microbial vector according to the process of the invention maintains a survival rate of around 40% after 104 weeks, or after 24 months.

These results thus show that the applicant's process allows the survival of microorganisms to be maintained over time. The microbial vectors obtained according to the present invention therefore have significantly improved bacterial survival compared to the freeze-dried bacteria obtained in the state of the art.

[FIG1] shows a comparison of the percentage of survival of the Pseudomonas fluorescens bacteria immobilized on the corn cob according to the manufacturing process of the microbial support of the invention and of this same strain in freeze-dried form.

The invention therefore relates to a process for manufacturing a solid microbial support comprising the following steps: - Preparation of a solid support, in which a germination step of the spores of undesirable microorganisms is carried out and which comprises at least one aseptization phase;

- Immobilization of the microorganisms by inoculation then incubation of the support using a seeding solution comprising a depleted minimum medium and a suspension of microorganisms, the inoculation consisting of the immersion of said support with said seeding solution;

- Drying, in which the temperature is between 0 °C and 50 °C, the drying time is between 1 h and 24 h and in which the pressure is between 1 mbar and 500 mbar.

In a variant, during the support preparation step, germination is carried out at a temperature of 30°C for 1 h to 24 h, said germination being an intermediate step between two aseptization phases.

In one embodiment of the method, the supports are inoculated with the seeding solution in a volume proportional to the weight of supports equivalent to a ratio between 0.1: 10 (w/v) and 9.5: 10 (p /v).

In a variant of this embodiment, the seeding solution of the inoculation step further comprises carbonates or carbonate species.

In another embodiment, the seeding solution is a mixture containing a suspension of microorganisms, a minimum depleted medium in a respective volume equivalent ratio of 5:95 (v/v) as well as 0.067% by total weight of said mixture (w/v) in alkaline carbonate.

In a variant embodiment, the incubation of the immobilization step takes place after inoculation and is carried out at a temperature between 20°C and 37°C, over a period of between 2 h and 240 h.

In another variant of the process, the drying step is carried out, on a laboratory scale, at a temperature of 35°C, for a drying time of 4 hours and at a pressure of 30 mbar.

According to one embodiment, the solid support is a biosourced and biodegradable material with an average particle size of between 200 pm and 15,000 pm.

In a variant, the solid support is chosen from a biodegradable polymer, a compostable polymer, a biodegradable polyester, a biodegradable copolyester, cellulose acetate, polylactic acid, cellulose powder, grape marc, grape stalks, preferably corn stalks.

The microorganisms capable of being used in the process of the invention are chosen from bacteria, fungi, yeasts, microalgae, probiotics or their mixture.

The invention also relates to the microbial vectors obtained by the method, and their use to improve the survival of microorganisms over time, characterized in that said improvement in the survival of microorganisms after storage is in particular at least 50% for 12 months. and at least 40% for 18 months.

Claims

1. Process for manufacturing a solid microbial support comprising the following steps:

- Preparation of a solid support, in which a germination step of the spores of undesirable microorganisms is carried out, comprising at least one sanitization phase;

- Immobilization of the microorganisms by inoculation then incubation of the support using a seeding solution consisting of a mixture of a depleted minimum medium and a suspension of microorganism, the inoculation consisting of the immersion of said support with said seeding solution;

2. Method according to claim 1, in which during the step of preparing the support, the germination is carried out at a temperature of 30 ° C for 1 h to 24 h, said germination being an intermediate step between two phases d sanitization.

3. Method according to claim 1 or 2, in which the supports are inoculated with the seeding solution with a volume proportional to the weight of supports equivalent to a ratio between 0.1: 10 (w/v) and 9.5 : 10 (w/v).

4. Method according to claim 3, wherein the seeding solution of the inoculation step further comprises carbonates or carbonate species.

5. Method according to claims 1 to 4, in which the seeding solution is a mixture containing a suspension of microorganisms and a minimum depleted medium in a respective volume equivalent ratio of 5: 95 (v/v) as well as 0.067%. in total weight of said mixture (w/v) in alkaline carbonate.

6. Method according to claims 1 to 5, in which the incubation of the immobilization step is carried out at a temperature between 20 °C and 37 °C, over a period of between 2 h and 240 h.

7. Method according to claims 1 to 6, in which during the drying step on a laboratory scale, the temperature is 35 ° C, the drying time is 4 hours and the pressure is 30 mbar.

8. Method according to one of the preceding claims, in which the solid support is a biosourced and biodegradable material with an average particle size of between 200 pm and 15,000 pm.

9. Method according to claim 8, in which the solid support is chosen from a biodegradable polymer, a compostable polymer, a biodegradable polyester, a biodegradable copolyester, cellulose acetate, polylactic acid, cellulose powder , grape marc, grape stalks, corn stalks.

10. Method according to claims 1, 8 or 9, in which the microorganisms are chosen from bacteria, fungi, yeasts, microalgae, probiotics or their mixture.

11. Microbial vector obtained by the method according to one of claims 1 to 10.

12. Use of a microbial vector according to claim 1 1 to improve the survival of microorganisms over time characterized in that said improvement in the survival of microorganisms after storage is at least 50% for 12 months and at least 40% for 18 months.