BIOFERTILIZERS BASED ON NATURAL POLYMERS

Abstract

The present disclosure relates to a composition, in particular a biofertilizer or a probiotic, comprising a polymer matrix based on chitosan and anionic polysaccharides, in particular carrageenan, and microorganisms, as well as a method for its preparation. Abstract figure: Figure 2

Description

Title of the invention: BIOFERTILIZERS BASED ON NATURAL POLYMERS FIELD OF THE INVENTION

[0001] The present invention relates to a composition, in particular a biofertilizer or a probiotic, comprising a polymer matrix based on chitosan and anionic polysaccharides, in particular carrageenan, and microorganisms immobilized by the polymer matrix, as well as its preparation process. STATE OF THE ART

[0002] In recent years, biofertilizers have been identified as viable alternatives to synthetic fertilizers, capable of promoting plant growth without harmful effects on the environment and humans. Biofertilizers are agricultural inputs containing microorganisms that promote plant growth. They are intended to be applied to seeds, plants or soils.

[0003] Biofertilizers can be in solid or liquid form. In solid forms, microorganisms are delivered in association with materials. The choice of the appropriate material to transport these microorganisms and the way in which the bacteria are associated with the carrier are essential to ensure the survival and release of these microorganisms.

[0004] Alginate is known to be the most commonly tested biodegradable polymer as a carrier. However, calcium alginate is not stable in soils and requires additional processing steps, such as coating, to ensure product stability1.

[0005] Mixtures comprising chitosan and starch have also been proposed2. However, to manufacture a hydrogel-type support based on chitosan and starch, the use of an ionic crosslinker is necessary, sodium tripolyphosphate. Although it is known to be generally non-toxic, the formation of complex insoluble forms can occur in soils in which this crosslinker can be trapped3. In addition, crosslinkers induce complex and slow degradation processes in soils and pose difficulties for large-scale use. Furthermore, the preparation of this type of support requires an autoclave sterilization step before immobilizing the bacteria on the support2. This additional step makes it possible to double the swelling capacity of the hydrogel and therefore to increase the number of immobilized bacteria but proves restrictive for large-scale production.

[0006] Thus, a need remains for the provision of a stable, high-performance and environmentally friendly biofertilizer, which can be prepared using a simple process that can be easily transposed to an industrial scale. Summary of the invention

[0007] The present invention relates to a composition comprising:

[0008] - a polymer matrix based on chitosan and anionic polysaccharides; and

[0009] - microorganisms.

[0010] The present invention also relates to two methods for preparing such a composition.

[0011] Thus, the composition can be prepared by a process comprising the following steps:

[0012] (a) preparation of an aqueous solution of chitosan at acidic pH, the aqueous solution chitosan which may include potassium ions and microorganisms;

[0013] (b) preparation of an aqueous solution of anionic polysaccharides, in particular of carrageenan, at pH ranging from 5 to 8, the aqueous solution of anionic polysaccharides which may include potassium ions and microorganisms;

[0014] at least one of the two solutions comprising potassium ions and at least one of the two solutions comprising microorganisms;

[0015] (c) dropwise introduction of the solution prepared in (a) into the solution prepared in (b);

[0016] (d) maturation of the gels obtained at the end of step (c) for a period of at least 15 hours at a temperature of 25 to 30°C;

[0017] (e) drying the gel obtained at the end of the maturation step.

[0018] The composition may also be prepared by a process comprising the following steps:

[0019] (a') preparation of an aqueous solution of chitosan at acidic pH, the aqueous solution chitosan which may include potassium ions and microorganisms;

[0020] (b') preparation of an aqueous solution of anionic polysaccharides, in particular of carrageenan, at pH ranging from 5 to 8, the aqueous solution of anionic polysaccharides which may include potassium ions and microorganisms;

[0021] at least one of the two solutions comprising potassium ions and at least one of the two solutions comprising microorganisms;

[0022] (c') introduction and mixing of the solutions prepared in steps (a') and (b') in a extruder;

[0023] (d') extrusion of the formed gel;

[0024] (e') cutting the extruded gel;

[0025] (f') optionally maturation of the extruded gel;

[0026] (g') drying of the gel fragments.

[0027] Finally, the present invention relates to a method for fertilizing a soil comprising spreading on and / or in the soil a composition according to the invention or the use of the composition for the delayed release of microorganisms immobilized by the polymer matrix.

[0028] Other aspects of the invention are as described below. DESCRIPTION OF FIGURES

[0029] [Fig.l] Confirmation of immobilization of bacteria by confocal microscopy (arrows indicating immobilized bacteria).

[0030] [Fig.2] Average number of bacteria released from air-dried biofertilizer granules loaded with Azospirillum baldaniorum Sp245 pOTAe(ppdC-egfp) compared to free bacteria.

[0031] [Fig.3] Confocal microscopy image of a wheat apex inoculated with granules loaded with Azospirillum baldanorium Sp245 pOTAe(ppdC-egfp) (left image; arrows indicating areas colonized by the bacteria) compared to control experimental conditions: wheat apex with uninoculated matrix (center image) and wheat apex without any particles (right image).

[0032] [Fig.4] Storage module for chitosan-carrageenan samples as extruded at different chitosan / carrageenan isovolumic charge ratios. DETAILED DESCRIPTION OF THE INVENTION

[0033] The inventors have developed a composition based on natural polymers that meets the expressed needs. The composition has good stability. Thus, it can be stored for several months, for example up to 3 months or more, in a dry place.

[0034] Furthermore, advantageously, the composition allows the delayed release of microorganisms over several weeks or even several months, for example from 8 weeks to 3 months.

[0035] The composition is particularly useful as a biofertilizer or probiotic.

[0036] The composition can be prepared by methods that are respectful of microorganisms and do not require the use of a crosslinking agent.

[0037] Thus, the present invention relates to a composition, in particular a biofertilizer or a probiotic, comprising:

[0038] - a polymer matrix based on chitosan and anionic polysaccharides, in par particular carrageenan; and

[0039] - microorganisms.

[0040] The composition is preferably in the form of granules. When the composition is a biofertilizer, the granules preferably have an average diameter ranging from 0.1 to 5 mm, preferably from 100 microns to 300 microns, typically around 200 microns, when the granules are in dry form (around 400 microns in hydrated form).

[0041] When the composition is a probiotic, the granules preferably have an average diameter ranging from 100 microns to 1 mm (dry form). The average diameter of the granules can be measured according to various methods known to those skilled in the art. In the context of the invention, it corresponds to the average diameter obtained by observation with a binocular magnifying glass and image processing.

[0042] Polymer matrix based on chitosan and carrageenan

[0043] The polymer matrix based on chitosan and anionic polysaccharides, in particular carrageenan, makes it possible to immobilize microorganisms. The combination of chitosan and anionic polysaccharides, in particular carrageenan, forms a polyelectrolyte-type complex.

[0044] A polyelectrolyte complex is an assembly of at least two oppositely charged polymers associated by attractive electrostatic interactions. The complex forms spontaneously by mixing solutions of oppositely charged polyelectrolytes in the absence of chemical or physical crosslinking agents.

[0045] Chitosan is a biodegradable biopolymer. It is a polysaccharide produced by chemical (in an alkaline medium) or enzymatic deacetylation, total or partial, of chitin, a component of the exoskeleton of arthropods (crustaceans), the cuticle of insects, the endoskeleton of cephalopods or even the wall of fungi. Chitosan is composed of D-glucosamine units (deacetylated units) and N-acetyl-D-glucosamine units (acetylated units), linked together by B-(l-4) glycosidic bonds. It can in particular be obtained by laboratory extraction from chitin from the shells of blue crabs (Portunus pelagicus) from the Philippines.

[0046] In an acidic medium, chitosan is in a polycationic form, due to the protonation of the amine groups of the glucosamine units.

[0047] The chitosan useful in the context of the present invention typically has a degree of acetylation ranging from 1 to 35%, preferably from 15 to 25%.

[0048] The chitosan useful in the context of the present invention typically has a weight-average molar mass ranging from 50 to 600 kg / mol, preferably from 100 to 400 kg / mol or from 300 to 400 kg / mol.

[0049] The anionic polysaccharides may be alginates, carrageenans, dextrans sulfates, polygalacturonic acids, ulvans, hyaluronic acid, xanthan, heparin or chondroitin sulfate, preferably carrageenans.

[0050] Carrageenans are linear polysulfated anionic polysaccharides, gener- commonly extracted from red algae such as Kappaphycus alvarezii, Eucheuma denticulatum and Kappaphycus cotonii. The main chain of carrageenans is composed of galactose and anhydrogalactose units linked by glycosidic units. Depending on the extraction methods and sources, there are three types of carrageenans which differ mainly in the degree of substitution of the sulfate group: K-carrageenan (kappa-carrageenan); i-carrageenan (iota-carrageenan); and X-carrageenan (lambda-carrageenan). All three types of carrageenans can be used in the present invention.

[0051] The K-carrageenan useful in the context of the present invention typically has a weight-average molar mass ranging from 100 to 1,000 kg / mol, preferably from 500 to 700 kg / mol.

[0052] The K-carrageenan useful in the context of the present invention typically has a degree of sulfation ranging from 50 to 80%, preferably from 60 to 75%. Microorganisms

[0053] Microorganisms are generally rhizospheric or endophytic microorganisms that promote plant growth. Such microorganisms are particularly useful for the design of biofertilizers.

[0054] The microorganisms can be bacteria or yeasts.

[0055] Preferably, the microorganisms are bacteria.

[0056] The microorganisms are preferably bacteria selected from the group consisting of phytobeneficial, biostimulatory or biocontrol bacteria, also referred to as PGPR or PGPB (for Plant Growth Promoting (Rhizo)Bacterid).

[0057] Preferably, the microorganisms are rhizobacteria, in particular from the family Rhizobiaceae, Azospirillacaceae, Pseudomonadaceae or Bacillaceae, preferably chosen from the genera Rhizobium, Azospirillum, Pseudomonas, and Bacillus, and even more preferably from Rhizobium leguminosarum, Azospirillum baldanorium, Azospirillum brasilense, species from the group of fluorescent Pseudomonas or Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus velezensis, Bacillus subtilis.

[0058] The microorganisms can also be bacteria of several genera: Lactobacillus spp. (Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus aci-dophilus, Lactobacillus plantarum, Lactobacillus reuterï) and Bifidobacterium spp (Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium bifidum), or the genera Streptococcus, Lactococcus. Such microorganisms are particularly useful for the design of probiotics.

[0059] Yeasts can be selected from the genus Saccharomyces (e.g. Saccharomyces cerevisiae, Saccharomyces boulardii). Such yeasts are particularly useful for the design of probiotics.

[0060] The composition, in particular the biofertilizer, generally comprises from 2.5 x 10A 8 to 3.1 x 10^8 CFU / g, preferably from 2 x 10A8 to 5 x 10A8 CFU / g of bacteria relative to the weight of the biofertilizer. Method of preparing the composition

[0061] The composition of the present invention can be prepared according to two alternative methods. Advantageously, the methods according to the invention do not require the use of a crosslinking agent. Method 1

[0062] A first method for preparing the composition, in particular the biofertilizer or the probiotic, according to the invention comprises the following steps:

[0063] (a) preparation of an aqueous solution of chitosan at acidic pH, the aqueous solution chitosan which may include potassium ions and microorganisms;

[0064] (b) preparation of an aqueous solution of anionic polysaccharides, in particular of carrageenan, at pH ranging from 5 to 8, preferably from 6.5 to 7.5, the aqueous solution of anionic polysaccharides possibly comprising potassium ions and microorganisms;

[0065] at least one of the two solutions comprising potassium ions and at least one of the two solutions comprising microorganisms;

[0066] (c) dropwise introduction of the solution prepared in (a) into the solution prepared in (b);

[0067] (d) maturation of the gels obtained at the end of step (c) for a period of at least 15 hours at a temperature of 25 to 30°C;

[0068] (e) drying the gel obtained at the end of the maturation step. Steps (a) and (b)

[0069] The aqueous chitosan solution has an acidic pH, i.e. less than 6.5. Typically, the pH of the aqueous chitosan solution varies from 2 to 6.5, preferably from 5.20 to 6.

[0070] The aqueous solution of anionic polysaccharides, in particular carrageenan, has a pH ranging from 5 to 8, preferably ranging from 6.5 to 7.5, even more preferably ranging from 6.9 to 7.3.

[0071] At least one of the two aqueous solutions comprises potassium ions. In some preferred embodiments, both solutions comprise potassium ions.

[0072] Potassium ions partially screen the intra-chain electrostatic interactions of chitosan and anionic polysaccharide (carrageenan), which makes them more flexible and thus promotes complexation. Potassium ions also contribute to strengthening the mechanical properties of formed products4.

[0073] The potassium ion concentration in either solution generally ranges from 0 to 15 mM.

[0074] In some embodiments, the aqueous chitosan solution comprises from 3 to 10 mM potassium ions, preferably from 4 to 6 mM potassium ions.

[0075] In some embodiments, the aqueous carrageenan solution comprises from 3 to 10 mM potassium ions, preferably from 4 to 6 mM potassium ions.

[0076] The potassium ions are typically provided in the form of potassium acetate.

[0077] The aqueous chitosan solution typically comprises from 3 to 20 g / L of chitosan, preferably from 5 to 15 g / L of chitosan, for example around 10 g / L.

[0078] The aqueous solution of anionic polysaccharides, in particular carrageenan, typically comprises from 3 to 10 g / L of anionic polysaccharides, in particular carrageenan, preferably from 3 to 8 g / L of anionic polysaccharides, in particular carrageenan, for example around 5 g / L.

[0079] In certain preferred embodiments, the aqueous solution of chitosan comprises from 3 to 20 g / L, preferably from 5 to 15 g / L of chitosan, and from 3 to 10 mM, preferably from 4 to 6 mM, of potassium ions and the aqueous solution of anionic polysaccharides, in particular carrageenan, comprises from 3 to 10 g / L, preferably from 3 to 8 g / L, of anionic polysaccharides, in particular carrageenan, and from 3 to 10 mM, preferably from 4 to 6 mM of potassium ions.

[0080] At least one of the two aqueous solutions comprises microorganisms. In certain preferred embodiments, the aqueous solution of anionic polysaccharides, in particular carrageenan, comprises the microorganisms (the aqueous solution of chitosan does not). Depending on the sensitivity of the microorganisms to pH, a person skilled in the art will be able to determine in which solution to add the microorganisms (or even both).

[0081] The concentration of microorganisms in either solution generally varies from 1 x 10A7 to 15 x 10A7 CFU / mL.

[0082] The solutions may be sterilized before proceeding to subsequent steps, for example for 20 minutes at 121°C, 20 psi. Steps (c) and (d)

[0083] The aqueous solution of chitosan is introduced drop by drop into the aqueous solution comprising the anionic polysaccharide, in particular carrageenan.

[0084] In preferred embodiments, an aqueous solution comprising chitosan and potassium ions (no microorganism) is introduced dropwise into an aqueous solution comprising the anionic polysaccharide, in particular carrageenan, potassium ions and microorganisms.

[0085] Dropwise addition can be done using the prilling technique. Prilling allows the production of calibrated size drops.

[0086] The whole is matured for a period of at least 15 hours, preferably ranging from 19 to 24 hours, at a temperature of 20 to 40°C, preferably from 25 to 30°C. Moderate stirring, of the order of 50 rpm, is typically carried out during maturation.

[0087] The introduction of the aqueous chitosan solution dropwise into the aqueous solution of anionic polysaccharides, in the presence of potassium ions, forms a gel in the form of a capsule / bead. The maturation step makes it possible to obtain a stable gel. A reorganization of the gel takes place during this step leading to its strengthening. The term "gel" designates a semi-solid material consisting of a polymer matrix swollen by a quantity of solvent. When the solvent is water, the gel is a hydrogel. Step (e)

[0088] The drying of the gel obtained at the end of the maturation step is typically carried out at moderate temperatures in order to preserve the survival of the microorganisms. Those skilled in the art will know how to adapt the drying temperature to the nature of the microorganisms.

[0089] In certain embodiments, the drying is carried out at a temperature ranging from 15 to 30°C, preferably from 25 to 30°C.

[0090] Those skilled in the art will also be able to adapt the drying time. In certain embodiments, the drying is carried out for a period ranging from 48 hours to 4 days.

[0091] In certain particular embodiments, the gel is air-dried at a temperature ranging from 25 to 30°C for a period ranging from 48 hours to 4 days.

[0092] The composition obtained after drying is typically in the form of beads / granules. The beads / granules generally have a core-shell structure comprising a core rich in chitosan, an outer shell rich in anionic polysaccharide and an intermediate layer comprising chitosan and the anionic polysaccharide. Method 2

[0093] A second method for preparing the composition according to the invention, in particular the biofertilizer or the probiotic, comprises the following steps:

[0094] (a') preparation of an aqueous solution of chitosan at acidic pH, the aqueous solution chitosan which may include potassium ions and microorganisms;

[0095] (b') preparation of an aqueous solution of anionic polysaccharides, in particular of carrageenan, at pH ranging from 5 to 8, preferably from 6.5 to 7.5, the aqueous solution of anionic polysaccharide possibly comprising potassium ions and microorganisms;

[0096] at least one of the two solutions comprising potassium ions and at least one of the two solutions comprising microorganisms;

[0097] (c') introduction and mixing of the solutions prepared in steps (a') and (b') in a extruder, preferably twin-screw;

[0098] (d') extrusion of the formed gel;

[0099] (e') cutting the extruded gel;

[0100] (f') optionally maturation of the extruded gel;

[0101] (g') drying of the gel fragments.

[0102] This second process has the advantage of being easily applicable on an industrial scale.

[0103] It allows preparation of the composition on a large scale.

[0104] Steps (a') and (b') are as described previously in relation to the first method (steps (a) and (b)).

[0105] Mixing and extrusion can be carried out at room temperature (15-20°C) or by heating. Those skilled in the art will know how to adapt the mixing and extrusion temperature to the nature and sensitivity of the microorganisms in order to preserve them while ensuring good processability conditions. In certain embodiments, mixing and extrusion are carried out at temperatures ranging from 20 to 40°C.

[0106] The residence time in the extruder typically varies from 20 to 30 minutes.

[0107] The solutions are typically introduced into the extruder at a rate ranging from 0.5 to 1.5 kg / hour for the anionic polysaccharide solution and from 0.7 to 1.5 kg / hour for the chitosan solution.

[0108] The cutting of the gel can be carried out by any suitable means known to those skilled in the art. The size of the fragments formed will be adapted to the uses of the gel.

[0109] The drying (step (g')) of the gel fragments obtained at the end of step (e') or (f') is carried out as described previously in connection with the first method (step (e)).

[0110] The gel fragments can then be transformed into granules or powder form.

[0111] The granules (obtained by process 1 or 2) generally have an average weight ranging from 20 to 500 mg per granule.

[0112] The compositions of the present invention are stable. Indeed, it has been observed that after 3 months of storage at room temperature (20-25°C; relative humidity 35%), a similar number of bacteria is released (see examples). Uses

[0113] The composition of the present invention can be used as a biofertilizer.

[0114] The biofertilizer of the present invention can be applied to seeds, soil or the surface of plants.

[0115] It allows the delayed release of microorganisms immobilized by the polymer matrix. Advantageously, the microorganisms are released in a prolonged manner over time.

[0116] The biofertilizer may be particularly useful in a soil fertilization method. The present invention thus also relates to a method of fertilizing a soil comprising spreading the biofertilizer of the present invention on and / or in the soil.

[0117] The present invention also relates to the use of a biofertilizer according to the invention for the delayed administration of microorganisms in the field of agriculture, in particular the promotion of plant growth.

[0118] The composition of the present invention can be used as a probiotic for the formulation of probiotics containing bacteria (bifidobacteria, lactobacilli, lactococci, etc.), but also yeasts (saccharomycetes). The composition can be administered orally. It thus allows the delivery of microorganisms into the intestine. The origin of the microorganisms can be fecal matter (fecal transplantation). EXAMPLES

[0119] Example 1: preparation of a biofertilizer according to the invention

[0120] In this example, the rhizobacterium Azospirillum baldanorium Sp245 pOTle(ppdC-egfp), a non-sporulating strain that promotes plant growth, was successfully immobilized in a chitosan-carrageenan-based matrix.

[0121] Chitosan was prepared from chitin extracted in the laboratory from the shells of Philippine blue crabs (Portunus pelagicus)5. Carrageenan, extracted from the seaweed Kappaphycus cotonii, was purchased from Marine Resources Development Corporation.

[0122] The preparation method was carried out as follows.

[0123] A 1% by mass chitosan solution comprising 5 mM potassium (acetate potassium) was added dropwise to a 0.5% by mass carrageenan solution comprising 5 mM potassium (potassium acetate) and the rhizobacterium Azospirillum baldanorium Sp245 pOTAe(ppdC-egfp), 1.10 x 10A7 to 1.43 x 10A8 cells / mL of solution.

[0124] Ratio of aqueous chitosan solution / aqueous carrageenan solution (mL / mL): 2.5 to 100.

[0125] An opaque, gel-like structure is collected after an overnight maturation period (approximately 12 hours). Granules in the form of substantially spherical beads are then obtained after drying at 28°C for 2 to 4 days, with an average weight of 0.63 ± 0.02 mg / bead. Confirmation of immobilization

[0126] Imaging of biofertilizer granules was performed by confocal microscopy to confirm the immobilization of bacteria. The green fluorescent protein, or Enhanced Green Fluorescent Protein (EGFP), is produced by the bacterium Azos-pirillum baldanorium Sp245 pOTAe(ppdC-EGFP). It allows the inclusion in the granules and subsequent release of bacteria from the granules to be monitored by confocal microscopy.

[0127] Thin sections of the different samples, namely wet and air-dried, were prepared. The wet sample is a biofertilizer granule that has not undergone drying and was transferred into an aqueous solution of magnesium sulfate, the air-dried and freeze-dried biofertilizer samples were rehydrated in an aqueous solution of magnesium sulfate. These granules were carefully sliced to reveal their respective cross-sections and were then placed on glass slides covered with a drop of Aqua-Polymount mounting fluid and a coverslip. The confocal microscope parameters used include excitation and emission wavelengths of 488 nm and 504-555 nm, respectively, for Egfp and 650 nm emission and 646-700 nm excitation for the matrix.The chosen excitation wavelength depended on the autofluorescence emission of the polymer carrier matrix, which gives a visible red color in the micrograph images (appears as light gray in the black and white images). All samples were projected onto a plane and constructed in 3D (see [Fig.l]).

[0128] Measurements of the concentration of bacteria released over time

[0129] Three granules of the biofertilizer prototype were immersed in three milliliters of sterilized ultrapure water contained in sterile tubes. Six repetitions of these experimental devices were prepared. One hundred and fifty microliters of sample were taken at each sampling step. From this sample volume, the quantification of the bacteria was carried out using a method of counting by dilution and depositing drops of the suspensions on nutrient agar medium. To do this, seven serial dilutions were prepared, with serial dilutions of 10 by 10. In each dilution, 100 qL was added to 900 qL of 10 mM MgSO4. For each dilution, five replicates of 10 qL were deposited as droplets on each Petri dish containing nutrient agar broth (NAB) with gent-tamycin (at 15 qg / mL) as antibiotic (plasmid tagging for the ppdC::egfp' construct).The plates were incubated at 28°C for at least two days, after which the colonies in each drop were counted and the CFU / mL was calculated.

[0130] The results are shown in [Fig.2].

[0131] After rehydration of the granules loaded with bacteria in water, the maximum number of bacteria released is close to 2.8 x 10A8 ± 0.3 x 10A8 CFU / g of biofertilizer; a quantity comparable to previous tests showing the positive impact of free bacteria on plant growth (from wheat and rice grains)67. After three months of storage at room temperature, a similar number of released bacteria was still observed, 1.38 x 10A8 ± 0.62 x 10A8 CFU / g, which shows that the biofertilizer retains its properties in the long term. It is interesting to note that the release of bacteria extends over a long period, and allows for a delayed bacterial supply to the plants. For example; on the 17th day, the release of bacteria is still 5.8 x 10A7 ± 1.8 x 10A7 CFU / g of biofertilizer; a corresponding decrease of 19% in the logi0 value compared to that after the first 48 hours. The presence of unreleased bacteria still trapped in the biofertilizer was also observed by confocal microscopy.However, according to an experiment conducted on the biofertilizer prototype stored in water for six months, bacteria were still released after the prototype was washed several times with water and transferred to a new volume of water. This finding was confirmed by epi-fluorescence microscopy, which revealed the presence of numerous bacteria in the biofertilizer prototype. This is a good indication of an effective carrier because it can allow longer survival of bacteria until the polymer carrier degrades in the soil.

[0132] Example 2: In vitro tests (wheat seeds) and greenhouses

[0133] The in vitro plant growth study was conducted to evaluate the effect of the biofertilizer prototype on wheat plant growth. The five main experimental steps are (1) production of the biofertilizer prototype, (2) disinfection of the seed surface and pregermination of wheat seeds, (3) inoculation of the biofertilizer prototype, (4) growth of wheat plants for one week and (5) harvesting of plants and analysis of root and aerial biomass and morphological characteristics of roots.

[0134] In vitro tests: the tests were carried out in Petri dishes on an agar medium containing only plant agar at 8 g / L.

[0135] Greenhouse trials: The trials were carried out in a semi-controlled environment using an unsterilized soil mixture and a determined temperature and relative humidity.

[0136] Wheat seeds were disinfected by several alternating washes of sodium hypochlorite and sodium thiosulfate solutions and water. The disinfected seeds were then pre-germinated in agar plates. The following day, the seeds that started to produce sprouts were selected and used for the study of plant growth in vitro. Three experimental conditions were prepared: (1) control - only pre-germinated wheat seeds were transferred to plant agar plates, (2) polymer-carrying matrix only - wheat seeds treated with the matrix only, and (3) biofertilizer prototype - treated wheat seeds with the biofertilizer prototype. A total of 20, 14, and 19 seeds were used for each of these experimental conditions, respectively, and these experiments were carried out in two replicates in total. The water-hydrated biofertilizer prototypes, in the form of granules approximately 0.2 cm in diameter, were placed near the radicle of the pre-germinated seeds. Ten granules were applied to each seed, which contained an average of 2.18 x 10A6 ± 0.99 x 10A6 total cell number / granule. The dishes were maintained in a Phytotron (Sanyo), with a temperature of 20°C and a relative humidity of 60%. The plants were then harvested on the seventh day by clipping the aerial parts and scanning the agar-free roots. The fresh weight of the aerial parts and roots (without the seed) was measured, then these samples were placed in an oven at 105°C for three days for the determination of the dry weight.For measurements of root morphological characteristics, WinRhizo software was used.

[0137] Various plant parameters, including root and aerial biomasses and root morphological characteristics, were measured and evaluated. Box plots of all collected data were constructed. Statistical analyses were also performed. The Shapiro-Wilk test was used to verify the normal distribution of the collected data, after which the significance test was assessed using ANOVA and post-hoc non-parametric tests that include Mann Whitney (comparison between 2 treatments) or Kruskal-Wallis (comparison between 3 treatments), as applicable. The results are presented in Table 1.

[0138] [Tables 1] Morphological characteristics of roots in in vitro plant growth experiments Biofertilizer vs. control Biofertilizer vs. matrix Number of roots +33.2 +51.9 Leaf dry weight 0 +9.76 Root dry weight + 11.2 +19.3 Total biomass +4.3 +13.5 Root to leaf dry weight ratio + 12.7 +19.3 Total root length + 15.7 +61 Total root surface area + 17.9 +19.9 Total root volume + 14.8 +8.3

[0139] Table 1: Rate of increase (%) showing improvement with the use of the biofertilizer prototype in in vitro plant growth experiments.

[0140] The in vivo greenhouse plant growth study was conducted to evaluate the effect of the biofertilizer prototype on the growth of wheat plants grown in non-sterile soil, but still under controlled environmental conditions. Three experimental conditions were prepared: (1) control - only wheat seeds were planted, (2) polymer-bearing matrix only - wheat seeds planted with the matrix only, and (2) biofertilizer prototype - wheat seeds planted with the biofertilizer prototype. Ten seeds were planted for each of these experimental conditions.Seven major experimental steps were carried out: (1) preparation of the soil mixture; (2) preparation of the planting pots and addition of water; (3) swelling of the seeds and biofertilizer; (4) planting and inoculation; (5) growth of the plants for three weeks; (6) harvesting period: cleaning of the roots and collection of the biomass; and (7) analyses of the biomass of the roots and shoots and the morphological characteristics of the roots.

[0141] A soil mixture consisting of soil, compost, and vermiculite (a soil conditioner) was initially prepared. At least thirty pots containing 1.4 L (average weight of 826 g) of soil mixture were arranged, and the required water was added according to the water-holding capacity of the soil. Similar to the in vitro experiments, wheat seeds were also used for the greenhouse experiment. However, the seeds were no longer pre-germinated. The seeds were soaked in water for a few hours before planting. Similarly, the polymer carrier matrix and the biofertilizer prototype were rehydrated with water before being applied to the wheat seeds. One seed was placed in a small, shallow cavity located in the top center of each planting pot.The rehydrated polymer matrix and the biofertilizer prototype were applied directly to the seed, separately according to the experimental conditions of each pot. When using the biofertilizer prototype, 11 granules were applied to each seed, which contained 7.32 x 10A5 ± 3.74 x 10A5 total number of cells / granule, on average. An equivalent amount of the bacteria-free polymer carrier matrix was also used for this type of treatment, i.e., 11 matrix units. The wheat seeds were grown for three weeks and watered every other day except on weekends. The volume of water added is in reference to the water-holding capacity of the soil. The plants were then harvested, the shoots collected and cut, and the roots carefully cleaned of soil mixture particles in a saline solution.The roots were then scanned for morphological analysis using WinRhizo software. The fresh weight of shoots and roots (without seeds) was measured, and these samples were then placed in an oven at 105 °C. for four to five days for dry weight determination.

[0142] The data analyses performed for the greenhouse experiment are the same as those performed for the in vitro experiment.

[0143] The results are presented in Table 2:

[0144] [Tables2] Morphological characteristics of roots in greenhouse plant growth experiments Biofertilizer vs. control Biofertilizer vs. matrix Number of leaves +5.9 +5.9 Leaf dry weight + 12.2 -4.5 Root dry weight +42.1 +20.7 Total biomass +20.9 +2.8 Root to leaf dry weight ratio +26.7 +28.9 Total root length +24.9 +9.7 Total root surface area +19.3 +2.3

[0145] Table 2: Rate of increase (%) showing improvement with the use of the biofertilizer prototype in greenhouse plant growth experiments. Conclusions

[0146] In vitro and greenhouse experiments carried out with the bacteria-laden granules applied directly to wheat seeds showed successful colonization of the roots by the bacteria and a promising positive effect on plant growth by analyzing plant biomasses and root characteristics. The biofertilizer prototype therefore proves capable of providing plant roots with phytobeneficial bacterial strains, known to be sensitive, such as strains of the genus Azos-pirillum, and of promisingly improving plant growth.

[0147] Example 3: preparation of a composition according to the invention by extrusion

[0148] Solutions at concentrations of 2% w / w (i.e. mass fraction in the solution) of chitosan (commercially available from Mahtani Pvt. Ltd.) and 2% w / w carrageenan both containing 5 mM potassium acetate were prepared.

[0149] The complexation of these polyelectrolyte solutions was carried out in a co-rotating twin-screw extruder (model Leistriz ZSE18HPe-60D, diameter 18 mm, L / D=60) at 40°C and a screw speed of 500 rpm. The carrageenan solution is fed into the extruder by a peristaltic pump whose injector has a diameter of 0.5 mm. On the other hand, a Roth Cyclo II cyclic pump is used for feeding the chitosan solution with a similar injector diameter. A counter-current gear was also incorporated into the screw profile (i.e., GFA-15-15-L). Different flow rates were used to obtain different types of chitosan / carrageenan loading ratio, r, such that r = 1 which corresponds to the stoichiometric complexation of chitosan and carrageenan and r > 1 which feeds excess chitosan.

[0150] The results showed that at r = 1, the measured storage modulus, G' = 17,298.40 Pa, exceeded that of carrageenan, G' = 14,796.7 Pa, indicating that a gel was extruded ([Fig.4]). The complexation of the chitosan and carrageenan solutions is therefore successful.

[0151] The ARES 2000ex was used to measure the storage modules through the frequency sweep test using the 25 mm aluminum Peltier plate, with a 4° cone. The procedure included conditioning the sample at 25°C with a soak time of 60 seconds, and an oscillation frequency at 25°C, a strain of 0.10%, from 0.01 to 600 rad / s, and continuous oscillation. BIBLIOGRAPHICAL REFERENCES

[0152] 1 Saberi-Riseh R, Moradi-Pour M, Mohammadinejad R, Thakur VK. Biopolymers for Biological Control of Plant Pathogens: Advances in Microencapsulation of Beneficial Microorganisms. Polymers 2021; 13:1938.

[0153] 2 Perez JJ, François NJ, Maroniche GA, Borrajo MP, Pereyra MA, Creus CM. A novel, green, low-cost chitosan-starch hydrogel as potential delivery System for plant growth-promoting bacteria. Carbohydrate Polymers 2018; 202: 409-417.

[0154] 3 HERA. Human & Environmental Risk Assessment on ingrédients of European household cleaning products: Sodium tripolyphosphate (STPP) CAS: 7758-29-4. 2003.https: / / www.heraproject.com / files / 13-f-04-%20hera%20stpp%20full%20web%2 0wd.pdf.

[0155] 4 Hermansson A-M, Eriksson E, Jordansson E. Effects of potassium, sodium and calcium on the microstructure and rheological behavior of kappa-carrageenan gels. Carbohydrate Polymers 1991; 16: 297-230.

[0156] 5 Ongkiko AGM, Fernando LAT, Diaz LJL. Continuous Extraction Process of Chitin from D. iscarded Shells of Philippine Blue Swimming Crab (Portunus pelagicus). g J 2016; : 15.

[0157] 6 Bashan Y, de-Bashan LE, Prabhu SR, Hernandez J-P. Advances in plant growth- promoting bacterial inoculant technology: formulations and practical perspectives (1998-2013). Plant Soil 2014; 378: 1-33.

[0158] 7 Dlaz-Zorita M, Canigia MVF, Bravo OÂ, Berger A, Satorre EH. Field Evaluation of Extensive Crops Inoculated with Azospirillum sp. In: Cassân FD, Okon Y, Creus CM (eds). Handbook for Azospirillum. Springer International Publishing: Cham, 2015, pp 435-445.

Claims

Claims

1. Composition comprising: - a polymer matrix based on chitosan and anionic polysaccharides; and - microorganisms.

2. A composition according to claim 1 wherein the anionic polysaccharide is a carrageenan.

3. A composition according to claim 1 or 2, wherein the microorganisms are rhizobacteria, preferably from the family Rhi-zobiaceae, Azospirillacaceae, Pseudomonadaceae or Bacillaceae.

4. Composition according to claim 1 or 2, wherein the microorganisms are yeasts of the genus Saccharomyces, preferably Sac-charomyces cerevisiae or Saccharomyces boulardii.

5. Composition according to one of the preceding claims in which the chitosan has a weight-average molar mass ranging from 50 to 600 kg / mol, preferably from 100 to 400 kg / mol.

6. Composition according to one of the preceding claims in the form of granules.

7. A method for preparing a composition according to any one of the preceding claims comprising the following steps: (a) preparing an aqueous solution of chitosan at acidic pH, the aqueous solution of chitosan possibly comprising potassium ions and microorganisms; (b) preparing an aqueous solution of anionic polysaccharides, in particular carrageenan, at pH ranging from 5 to 8, the aqueous solution of anionic polysaccharides possibly comprising potassium ions and microorganisms; at least one of the two solutions comprising potassium ions and at least one of the two solutions comprising microorganisms; (c) introducing dropwise the solution prepared in (a) into the solution prepared in (b); (d) maturing the gels obtained at the end of step (c) for a period of at least 15 h at a temperature of 25 to 30°C; (e) drying the gel obtained at the end of the maturing step.

8. A process for preparing a composition according to any one of claims 1 to 6 comprising the following steps: (a') preparing an aqueous solution of chitosan at acidic pH, the

9.

10.

11. aqueous chitosan solution which may include potassium ions and microorganisms; (b') preparation of an aqueous solution of anionic polysaccharides, in particular carrageenan, at pH ranging from 5 to 8, the aqueous solution of anionic polysaccharides possibly comprising potassium ions and microorganisms; at least one of the two solutions comprising potassium ions and at least one of the two solutions comprising microorganisms; (c') introducing and mixing the solutions prepared in steps (a') and (b') in an extruder; (d') extrusion of the formed gel; (e') cutting the extruded gel; (f') optionally maturation of the extruded gel; (g') drying of gel fragments. The method of claim 7 or 8 wherein the aqueous solution comprising chitosan further comprises potassium ions and the aqueous solution comprising the anionic polysaccharides, in particular carrageenan, further comprises potassium ions and microorganisms. Method for fertilizing a soil comprising spreading on and / or in the soil a composition according to one of claims 1 to 6. Use of a composition according to one of claims 1 to 6 for the delayed release of microorganisms immobilized by the polymer matrix.

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