Microalgae extracts for inhibiting nitrification and / or denitrification processes in soil

Abstract

The present invention is suitable for use in the agricultural field and relates to the use of eukaryotic microalgae extracts for controlling nitrification in soils and reducing nitrogen gas losses in the form of N 2 O.

Description

Description Title of the invention: Microalgae extracts for inhibiting nitrification and / or denitrification processes in soil Technical Field

[0001] The invention relates to the use of microalgae extracts in a soil fertilization process to inhibit the nitrification process and / or the nitrification-denitrification process as well as the nitrate denitrification process. These extracts have the property of controlling the functioning of nitrifying bacteria and / or nitrifying-denitrifying bacteria which are responsible for the transformation of ammoniacal nitrogen (NH4 + ) into nitric nitrogen (NO ) with the release of nitrous oxide (N2O) during this process. These extracts also have the property of inhibiting the functioning of the nitrate reductase enzyme.

[0002] The invention proposes a new method for controlling both the nitrogen supply to the soil and the supply of fertilizers to the plant, but also for limiting emissions of the greenhouse gas N2O. Prior art

[0003] Nitrogen fertilization plays a vital role in crop growth and yield. Nitrogen is the characteristic constituent of amino acids and proteins, making it a very important growth and quality factor. As it undergoes a natural cycle in the air, soil, and water, it undergoes various chemical and biological transformations—this is the nitrogen cycle.

[0004] The global nitrogen cycle describes the transformations of gaseous nitrogen, mineral nitrogen, and nitrogen-rich organic compounds present on Earth. It is a set of processes involving soil microorganisms. This includes biological nitrogen fixation, nitrogen assimilation by plants, ammonification, nitrification, and denitrification.

[0005] Among all these processes, nitrification corresponds to the oxidation of ammonium (NH4 +) into nitrate (NO3), it goes through several stages. First, bacteria and archaea of ​​the Nitrosomas group, AOB (Ammonia-Oxidising Bacteria) and AOA (Ammonia-Oxidising archaea) oxidize ammonium into hydroxylamine (NH2OH) and then into nitrite (NO2). Finally, the bacteria Nitrite-Oxidising Bacteria (NOB), Nitrobacter and Nitrospira, oxidize nitrites into nitrate. In order to transform ammonium into nitrites, four protons are released as well as two water molecules; this phenomenon causes acidification of the environment, particularly around the roots. The activity of bacteria, AOB and AOA, is highly dependent on the environment, so that humidity, pH, temperature and the availability of ammonium have a great influence on the process. Finally, emissions of nitrous oxide (N2O) caused by nitrification can be observed during the oxidation of ammonium to nitrite.

[0006] Nitrification is an important biological step in the soil nitrogen cycle. From an agronomic perspective, its operation is considered a limiting step in the nitrogen supply of soils. If it occurs too slowly or too quickly compared to plant needs, it can lead to low nitrogen use efficiency and can contribute to groundwater pollution and greenhouse gas (N2O) emissions. The nitrification rate can vary depending on the nature of the soil. Factors regulating the nitrification process include soil pH, temperature, humidity, nitrogen fertilizer applied to the soil, microorganisms, and the physical nature of the soil.

[0007] Nitrification poorly synchronized with nitrogen uptake by plants results in a loss of nitrogen applied to the soil through nitrate leaching and nitrous oxide (N2O) emissions. These losses can account for one-fifth of the applied nitrogen. This leads to increased agricultural production costs, environmental pollution (surface and deep waters), human and animal diseases, ozone depletion, and greenhouse gas emissions that contribute to climate change. Therefore, controlling the nitrification process has become a major issue in agriculture.

[0008] Another important process in the nitrogen cycle is the transformation of nitrates resulting from nitrification into nitrogen. This second biological mechanism, denitrification, involves the reduction of nitrates (NO3') to nitrogen gas (N2) by bacteria in anoxic conditions. Denitrifying bacteria successively reduce the nitrate ion (NO3) to nitrite ion (NO2'), then to nitric oxide (NO), to nitrous oxide (N2O), and finally to nitrogen (N2).

[0009] Denitrification therefore constitutes another source of emission of the greenhouse gas N2O through bacterial use of nitrates, which competes with the use of nitrates by plants. There is also a nitrification-denitrification process carried out by nitrifying organisms that are also capable of denitrification, or carried out jointly by nitrifying organisms and separate denitrifying organisms, in which the nitrates produced by the nitrifying organisms are immediately denitrified by the denitrifying organisms. N2O emissions can also be observed at the stage of the nitrification-denitrification process.

[0010] For non-nitrogen-fixing plants, nitrogen is absorbed by plants mainly in nitric (NO3') or ammoniacal (NH4) form +). Depending on the plant's needs, it is able to absorb significant quantities to support its growth and development. Furthermore, a lack of nitrogen caused by a deficiency in supply from the soil or by losses in the environment, via ammonia volatilization, nitrate leaching or denitrification, can cause nitrogen deficiencies and have a negative impact on plant growth.

[0011] Providing fertilizer solutions that are less prone to environmental losses helps avoid certain risks of deficiencies and limit the environmental impacts of fertilizers. Thus, the quantities of nitrogen added through fertilization can be reduced, which can represent a financial gain for farmers, help reduce nitrogen losses and reduce environmental risks.

[0012] In order to control the nitrogen transformation process in the soil, formulations containing nitrification inhibitors are interesting to develop with the aim of controlling the nitrate supply according to the needs of the plant and optimizing its use by crops. This allows better nitrogen utilization by the plant, while reducing the negative consequences for the environment.

[0013] Today, there are several chemical molecules capable of inhibiting the nitrification process in the soil and limiting greenhouse gas emissions. However, these are synthetic molecules that can pose a danger to the environment and soil life.

[0014] It is in this context that the applicant has demonstrated, and this constitutes the basis of the present invention, that certain natural extracts of microalgae make it possible to inhibit the nitrification and / or nitrification-denitrification process in a soil and consequently to improve the agro-environmental balance of crops. The applicant has also demonstrated that these microalgae extracts make it possible to inhibit the functioning of the nitrate reductase enzyme.

[0015] Thus, the present invention, which finds application in the agricultural field, aims to propose the use of natural extracts of microalgae as a solution to slow down nitrification and thus better regulate the supply of nitrogen to plants, reducing losses of gaseous nitrogen in the form of N2O. It is also possible to consider reducing nitrate losses by leaching. Statement of the invention

[0016] The use of microalgae extracts in agriculture to control one or more processes of the nitrogen cycle is an innovative approach. This is the main agronomic challenge of the invention.

[0017] Thus, the present invention is based on the use of an extract of at least one eukaryotic microalga to induce an inhibition of at least one step of the nitrogen cycle in a soil, said step being chosen from a nitrification process, a denitrification process and a nitrification-denitrification process. The eukaryotic microalga extract may be an aqueous extract or an oily extract.

[0018] The present invention relates to an agricultural fertilization method comprising a step of adding to a soil an extract of at least one microalgae eukaryote, said extract being able to be applied in the open field or on a culture medium, provided by a culture medium containing it, or provided by a culture solution.

[0019] The extract may be provided by a product for agricultural use comprising, in addition to ((extract of a eukaryotic microalgae, also at least one soil nutrient ingredient or a plant nutrient ingredient chosen in particular from a fertilizer, an amendment, a biostimulant and a micronutrient.

[0020] A method for preparing an aqueous extract of a microalga useful in the context of the invention may comprise an extraction step by introducing the microalga into a liquid extraction medium comprising water, a filtration step, and a step of recovering the filtrate containing the aqueous extract. Brief description of the drawings

[0021] Figure 1 shows the evolution of ammonium contents (NH4 +; mg N-NH .g -1 Soil) in soil (i) with a soil that includes a functionalized microalgae extract, (ii) with a soil that includes a microalgae extract (Extract), and (iii) with a soil that does not include an extract (Control). The graph shows a progressive decrease in ammonium levels between 0 and 12 days of incubation in the soil that does not include an extract (Control): these levels increase from 0.4 to 0.12 mg N-NH .g' 1 Soil. Ammonium levels increase from 0.4 to 0.31 mg N- NH4 + .g 1 Soil in soil which includes microalgae extract, and 0.4 to 0.42 mg N- NH4 + .g _1 Soil in soil that includes functionalized microalgae extract, indicating inhibition of the nitrification process in the soil.

[0022] Figure 2 shows the evolution of nitrate levels (NO3'; mg N-NO / .g -1Soil) in soil (i) with a soil that includes a functionalized microalgae extract, (ii) with a soil that includes a microalgae extract (Extract), and (iii) with a soil that does not include an extract (Control). The graph shows a progressive increase in nitrate levels between 0 and 12 days of incubation in the soil that does not include an extract (Control): these levels increase from 0 to 0.22 mg N- NO / .g -1 Soil in the soil. Ammonium levels increase from 0 to 0.15 mg N- NO3'.g 1 Soil in soil which includes microalgae extract, and 0 to 0.09 mg N- NO / .g -1 Soil in soil that includes functionalized microalgae extract, indicating inhibition of the nitrification process in the soil.

[0023] Figure 3 shows the evolution of nitrous oxide (N2O) levels; represented by the peak area of ​​the GC (gas chromatography) analysis in the soil (i) with a soil that includes a functionalized microalgae extract (Functionalized Extract), and (ii) with a soil that does not include an extract (Control). The graph shows a progressive increase in the peak area associated with an increase in N2O levels in the air between 0 and 216 hours of incubation in the soil that does not include an extract (Control): the peak area increases from 0 to 1200. The peak area increases from 0 to 600 in soil that includes the functionalized microalgae extract, indicating an inhibition of N2O production due to the functionalized microalgae extract.

[0024] Figure 4 represents the percentage inhibition (%) of nitrate reductase function (i) with a solution that includes a functionalized microalgae extract, (ii) with a solution that includes a microalgae extract (Extract), and (iii) with a solution that does not include an extract (Control). The graph shows an inhibition of nitrate reductase activity of 24% in the solution that includes the microalgae extract, and of 99% in the solution that includes the functionalized microalgae extract, compared to the solution that does not include an extract (Control).

[0025] Figure 5 represents the percentage inhibition (%) of nitrate reductase function (i) with a solution that includes a functionalized microalgae extract, (ii) with a solution that includes a microalgae extract (Extract), and (iii) with a solution that does not include an extract (Control). The graph shows an inhibition of nitrate reductase activity of 28.9% in the solution that includes the spirulina extract, and of 59.6% in the solution that includes the functionalized spirulina extract, compared to the solution that does not include an extract (Control). The graph also shows an inhibition of nitrate reductase activity of 13.8% in the solution that includes the chlorella extract, and of 49.9% in the solution that includes the functionalized chlorella extract, compared to the solution that does not include an extract (Control).

[0026] Figure 6 represents the percentage inhibition (%) of nitrate reductase function (i) with a rapeseed oil-based solution (HC), (ii) with a soybean oil-based solution (HS), (iii) with a solution that includes a microalgae extract in rapeseed oil, (iv) with a solution that includes a microalgae extract in soybean oil, and (v) with a solution that does not include an extract (Control). The graph shows an inhibition of nitrate reductase activity of 19.6% in the solution that includes rapeseed oil (HC bar), and of 36.3% in the solution that includes chlorella extract in rapeseed oil (Chlorella Extract-HC bar), compared to the solution that does not include an extract (Control).The graph also shows an inhibition of nitrate reductase activity of 27.2% in the solution that includes soybean oil (HS bar), and 36.8% in the solution that includes chlorella extract in soybean oil (Chlorella Extract-HS bar), and 37.9% in the solution that includes Nannochloropsis extract in soybean oil (Nannochloropsis Extract-HS bar), compared to the solution that does not include extract (Control). Description of the embodiments

[0027] The invention therefore relates to the use of an extract of at least one eukaryotic microalgae to induce an inhibition of at least one step of the nitrogen cycle in a soil, said step being chosen from a nitrification process, a process of denitrification and a nitrification-denitrification process, and relates in particular to a process for fertilizing soil harboring bacteria, by supplying said soil with an agricultural product comprising an aqueous extract of eukaryotic microalgae(s).

[0028] The microalgae extract can be an aqueous extract or an oily extract. In a particular implementation mode, the oily extract of eukaryotic microalgae makes it possible to inhibit a denitrification process in a soil.

[0029] Eukaryotic microalgae are unicellular microorganisms that have the ability to photosynthesize. Nitrification processes are defined as the set of biochemical reactions leading to the formation of nitrates from ammonium that are induced by nitrifying bacteria such as ammonia-oxidizing bacteria (AOB) and / or nitrite-oxidizing bacteria (NOB). Examples of nitrifying bacteria genera are Nitrobacter spp., Nitrosomonas spp., Nitrosospira spp. and Nitrosococcus spp. Nitrosomonas spp., Nitrosospira spp. and Nitrosococcus spp., including the species Nitrosomonas europaea, Nitrosomonas eutropha, Nitrosomonas communis, Nitrosospira multiformis and Nitrosospira iacus.

[0030] The term "nitrification process inhibitor," also known as a nitrification inhibitor, refers to a product whose activity slows down one of the biochemical reactions in the nitrification process. A nitrification inhibitor advantageously helps to better synchronize the supply of nitrogen from the soil to the needs of the plant and consequently to reduce losses of nitrogen fertilizers (leaching of nitrates from the soil, for example). In addition, since the operation of nitrification can produce N2O, a nitrification inhibitor can reduce N2O emissions from the soil.

[0031] Inhibition of a nitrification process within the scope of the invention may have at least one effect selected from inhibition of the activity of nitrifying bacteria present in the soil, reduction of fertilizer loss from the soil, reduction of nitrate leaching in the soil, increase in the amount of ammonium available in the soil, improvement of the growth of a plant grown on the soil, reduction of nitrous oxide emissions, and increase in the amount of nitrogen available for assimilation by a plant grown on the soil.

[0032] A microalgal extract covered by the present invention may be a nitrification inhibitor within the meaning of the invention. In a particular embodiment of the invention, a microalgal extract is a nitrification inhibitor when it reduces by at least 5% the quantity of nitrate nitrogen (N-NO3') in an amended soil compared to the same soil not amended with the extract, and / or when it increases by at least 5% the quantity of ammoniacal nitrogen (N-NH4 + ) in amended soil compared to the same soil not amended with the extract, after a period of at least 2 days after application of the extract to the soil, preferably at least 6 days or 10 days. Several methods for measuring the nitrification inhibition activity of a microalgae extract are detailed in the examples. The reduction in the amount of nitrate nitrogen or the increase in the quantity of ammoniacal nitrogen may be independently of one another greater than or equal to a value chosen from 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% and 250%, after a period for example of at least 2 days after application of the extract to the soil, preferably of at least 6 days or 10 days.

[0033] The term "denitrification process" refers to the reduction of nitrates (NCh ) to nitrogen gas (N2) by denitrifying bacteria in anoxic conditions. More precisely, the bacteria successively reduce the nitrate ion (NO3') to nitrite ion (NO ) by the action of the enzyme N2O reductase, to nitric oxide (NO), to nitrous oxide (N2O), and then to nitrogen (N2). The denitrifying bacteria involved include Thiobacillus denitrificans, Micrococcus denitrificans, Pseudomonas aeruginosa, Pseudomonas stutzeri, Serratia spp., and Achromobacter spp.

[0034] A “denitrification inhibitor” is a product for slowing down at least one step of the denitrification process. In a particular embodiment, a denitrification inhibitor reduces the activity of the nitrate reductase enzyme by at least 5% in a medium supplemented with a denitrification inhibitor compared to the same medium without supplementation after a period of at least 10 minutes. The decrease in the activity of the Nitrate Reductase enzyme may be greater than a value chosen from 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%, after a period of at least 10 minutes, for example.

[0035] Inhibition of a denitrification process may have at least one effect selected from inhibition of the activity of denitrifying bacteria present in the soil, inhibition of the nitrate reduction process, inhibition of the activity of the enzyme nitrate reductase, and reduction of nitrous oxide emission.

[0036] A "nitrification-denitrification process" means a process carried out by organisms capable of simultaneous nitrification and denitrification, or a process carried out jointly by separate nitrifying and denitrifying organisms, in which the nitrates produced by the nitrifying organisms are immediately denitrified by the denitrifying organisms.

[0037] The term "nitrification-denitrification process inhibitor" refers to the activity of limiting the nitrification-denitrification process which may result in an inhibition of N2O production. Also, the inhibition of a nitrification-denitrification process may have the effect of reducing the production of nitrous oxide. The reduction in nitrous oxide production may be greater than or equal to a value chosen from 5%, 10%, 20%, 30%, 40%, 50%, after a period of, for example, at least 100 hours, 150 hours or 200 hours.

[0038] In one embodiment of the invention, a microalgae extract is a nitrification-denitrification inhibitor when it reduces by at least 5% the quantity of nitrous oxide in a soil amended with microalgae extract compared to the same unamended soil under nitrification conditions (aerated environment).

[0039] When the soil harbors denitrifying bacteria, the step of adding the microalgae extract causes the inhibition of the activity of these bacteria.

[0040] When the soil harbors nitrifying bacteria, the step of adding the microalgae extract causes the inhibition of the activity of these bacteria.

[0041] The term "microalgae extract" refers to a product resulting from a process carried out by humans to isolate a part of a microalga. The microalga is preferably a eukaryotic microalga: it is delimited by a membrane and contains organelles, including a cytoplasm and a nucleus.

[0042] The term "aqueous microalgal extract" refers to a product resulting from the extraction of the cellular contents of a microalga with a liquid containing water. An aqueous extract does not necessarily contain water, as the extraction step may be followed by a step of removing the water used as an extraction solvent. The aqueous extract preferably contains the hydrophilic molecules contained within the microalga. The aqueous extract is therefore likely to be obtained by extracting the molecules contained within the microalga, with a liquid medium containing water.

[0043] The aqueous extract of at least one eukaryotic microalga can be obtained by a step of extracting the organelles contained in the cell, using a liquid extraction medium preferably consisting of water, more preferably distilled water, and the extraction step preferably being carried out at a temperature ranging from 4°C to 300°C. The aqueous extract can be functionalized by adding at least one enzyme to the liquid medium. The enzyme can be chosen from proteases, carboxypeptidases, catalases, cellulases, glucanases, hemicellulases, lipases, amylase, phospholipases. In a particular embodiment, the enzyme is chosen from carbohydrases and pectinases. Examples of carbohydrases are cellulases, hemicellulases, beta-glucanases, and xylanases.

[0044] The term "microalgal oil extract" refers to a product resulting from the extraction of the cellular contents of a microalgae with a liquid containing an oil. The oil may be a vegetable oil, such as soybean oil or rapeseed oil. The oil extract contains lipophilic molecules contained within the microalgal cells. The microalgal oil extract may be obtained by a lysis process consisting of breaking the cell membrane to release the liquid contents of the microalgae.

[0045] The oily extract of at least one eukaryotic microalgae can alternatively be obtained by a step of extracting the molecules contained in the cell, by placing the microalgae in an oil at a temperature ranging from 4°C to 300°C. The oil loaded with lipophilic molecules from the contents of the microalgae can then be separated from the treated microalgae, by filtration or by centrifugation.

[0046] The microalgae which can be used in the context of the present invention is for example a microalgae belonging to a genus chosen from the group consisting of Amphora sp., Arthrospira sp., Chaetoceros sp., Ch / amydomonas sp., Chlorella sp., Dunaliella sp., Euglena sp., Fragilaria sp., Haematococcus sp., Isochrysis sp. Tisochrysis sp., Nannoch / oris sp., Nannochioropsis sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacyium sp., Scenedesmus sp., Schyzotrium sp., Skeietonema sp., Tha / assosira sp. and Tetraseimis sp. The microalgae belonging to one of the genera mentioned above may be any species of microalgae known to those skilled in the art belonging to one of these genera.

[0047] Examples of particular microalgae species are Nannoch / oropsis satina, Nannoch / oropsis ocuiata, Nannoch / oropsis ocean ica, Arthrospira piatensis and Chioreüa sorokiniana.

[0048] The invention applies to a method of fertilizing a soil comprising a step of providing a microalgae extract which is applied to the soil, provided to the soil by a culture solution, or which is integrated into the soil, when the soil is a culture medium.

[0049] The term "soil" refers to the substrate explored by the roots of cultivated plants on which they develop. The term soil includes any type of substrate on which a plant can develop, such as, for example, artificial soil, cultivated agricultural soil, uncultivated agricultural soil, peat, potting soil, rock wool or coconut fiber. The term soil therefore covers growing media used for so-called "soil-less" cultivation, for example in pots. In a particular embodiment of the invention, a "soil" does not include a plant, and in particular does not include the root system of a plant. A soil may comprise a mixture of minerals, organisms, organic matter and air. It may comprise microorganisms, among which are nitrifying microorganisms and / or denitrifying microorganisms known to those skilled in the art, some of which have been described previously.Microorganisms may include nitrate reductase enzymes.

[0050] The inventors have surprisingly found that a eukaryotic microalgae extract can act on soil microorganisms involved in the nitrification and denitrification stages of the nitrogen cycle. The microalgae extract is preferably supplied to a soil comprising nitrifying microorganisms and / or denitrifying microorganisms. In a particular embodiment of the invention, the fertilization method does not comprise a step of supplying a eukaryotic microalgae extract to a plant.

[0051] Soil can have an acidic, neutral, or basic pH. Acidic soil is defined as a soil with a pH less than or equal to 6.2. Neutral soil is defined as a soil with a pH between 6.2 and 7.5. Finally, basic soil has a pH greater than or equal to 7.5.

[0052] In a particular embodiment, the soil has a pH greater than or equal to a value selected from 6, 6.5, 7, 7.5, or 8.

[0053] For example, soil has a basic pH, that is, a pH greater than or equal to 7.5. In a particular embodiment, the basic pH soil has a pH ranging from 7.5 to 8.5, including a pH close to 8.0 with measurement variations.

[0054] Soil can come in different textures. Soil textures can be chosen from heavy clay, silty clay, clay, silty clay loam, clay loam, very fine silt, fine silt, sandy clay, silt, sandy clay loam, sandy loam, silty sand, sand, and mixtures thereof.

[0055] In a particular implementation mode, the soil has a cation exchange capacity (CEC) ranging from 10 meq / kg to 350 meq / kg, for example ranging from 50 meq / kg to 250 meq / kg, or ranging from 180 meq / kg to 200 meq / kg.

[0056] The grain size of the soil can be between 1 mm and 5 mm, for example between 1.5 mm and 3 mm.

[0057] Finally, the soil can contain organic matter in a content ranging from 0.1% to 15% by mass relative to the mass of the soil.

[0058] The cation exchange capacity, the granulometry and the organic matter content of a soil are quantities known to those skilled in the art who know how to measure them using standard methods.

[0059] The microalgae extract can be added to the soil via a culture solution. For the purposes of the present invention, the term "culture solution" means any solution that allows the cultivation of a plant, such as, for example, irrigation water, a liquid solution intended for fertigation by sprinkling or drip irrigation, or a hydroponic bath.

[0060] The microalgae extract or the agricultural product comprising the microalgae extract is applied to soil, for example in open fields or by a soilless method. The aqueous extract may be applied to the soil surface or mixed with the soil, for example with the first layers of soil. The application may be carried out directly on the soil, over the entire soil surface or locally, by any suitable distribution means.

[0061] The addition of microalgae extract to the soil can be carried out before and / or during the growth of cultivated plants, before and / or after germination of plants, or during transplanting in the case of plants requiring transplanting.

[0062] The fertilization method of the invention may comprise one or more steps of applying the microalgae extract, as well as one or more steps of applying a fertilizer, such as for example a solid or liquid fertilizer, an amendment, and / or a biostimulant. The person skilled in the art will be able to adapt the sequence of steps and the number of repetitions of the steps according to the nature of the soil and / or the cultivated plant.

[0063] In a particular embodiment of the invention, the aqueous extract is formulated into a fertilizer before its application to the soil, so that the application of the fertilizer and the application of the microalgae extract are simultaneous. The fertilizer is advantageously chosen from nitrogen fertilizers comprising an ammoniacal form of nitrogen, nitrogen fertilizers releasing ammonium into the soil, nitrogen fertilizers comprising nitrate, and combinations thereof.

[0064] Thus, in a particular embodiment of the invention, the fertilization method involves the use of an agricultural product comprising the microalgae extract described above, and optionally at least one nutrient ingredient of the soil and / or a nutrient ingredient of a plant grown on the soil chosen from a fertilizer, an amendment, a biostimulant and a micronutrient.

[0065] The term "fertilizer" refers to fertilizing materials whose main function is to provide plants with elements directly useful for their nutrition (major fertilizing elements, secondary fertilizing elements and trace elements). The term "amendment" refers to a substance intended to improve soil quality, and in particular intended to improve soil pH. Advantageously, the amendment is chosen from basic mineral amendments of the limestone and / or limestone and magnesium type, humus amendments of the compost type or manure.

[0066] The fertilizer is advantageously a simple, binary or ternary solid fertilizer, organo-mineral or organic fertilizer, liquid or a water-soluble fertilizer.

[0067] In one embodiment, the fertilizer is a nitrogen fertilizer. It is preferably a nitrogen fertilizer comprising an ammoniacal form of nitrogen, a nitrogen fertilizer releasing ammonium into the soil, or a nitrogen fertilizer comprising nitrate.

[0068] In particular embodiments, the nitrogen fertilizer contains ammonium, nitrate, ammonia, and / or urea, wherein the urea may be formulated to slow the release of nitrogen into the soil. Examples of ammonium-containing fertilizers are calcium ammonium nitrate, calcium ammonium nitrate, ammonium sulfate nitrate, ammonium sulfate, and ammonium phosphate. An example of an ammonia-containing fertilizer is ammonium nitrate. The nitrogen fertilizer may also provide other nutrients, for example, potassium (K) and / or phosphorus (P).

[0069] The amendment can be an organic amendment or a mineral amendment, such as calcium carbonate.

[0070] Micronutrients such as molybdenum, zinc, boron and copper are preferably supplied as salts soluble in water or organic acids.

[0071] A biostimulant can be, for example, a liquid root or foliar biostimulant.

[0072] The agricultural product may also comprise at least one formulation aid chosen from a solvent, a solid support, a dispersant or an emulsifier, an organic or inorganic thickener, a pesticidal agent, an antifreeze agent, an anti-foaming agent, where appropriate a colorant, a sticking agent or a binder.

[0073] The agricultural product may be in the form of a fertilizer or a growing solution that is applied to the soil. When the soil is a growing medium, the microalgae extract may have been introduced into the growing medium during its manufacture.

[0074] In particular embodiments, the agricultural product according to the invention is a culture solution such as irrigation water, a solution intended for sprinkler or drip fertigation, a hydroponic bath or an aeroponic solution.

[0075] The agricultural product according to the invention may be in solid form, in particular in the form of powder, granules or microgranules, or in liquid form, in particular in the form of a liquid suspension, gel or solution in water.

[0076] The expression "plant" is intended to designate in this application the plant considered as a whole, including its root system, its vegetative system, the seeds, grains and fruits.

[0077] The invention finds application in the treatment of soil in which a very wide variety of plants can be planted. Among these, mention will be made in particular of large-scale crops such as cereals (wheat, corn, barley), protein crops (peas), oilseeds (soybeans, sunflowers), solanaceous crops (potatoes), amaranthaceae crops (beetroot), specialized crops such as in particular market gardening (lettuce, spinach, onion, shallot, tomato, melon), vines, arboriculture (pears, apples, nectarines), or horticulture.

[0078] The plant may belong to the order Monocotyledons, preferably to the Poaceae family. Poaceae, commonly called grasses, includes most of the species commonly called "grasses" and "cereals." Poaceae include wheat, rice, barley, oats, rye, sugarcane, meadow, and corn.

[0079] The microalgae extract can be obtained by any method known to those skilled in the art.

[0080] The microalgae used to prepare the extract are cultivated under culture conditions according to the general knowledge of the person skilled in the art using one of the following three culture modes: autotrophic, mixotrophic or heterotrophic. The culture method may comprise a step under the conditions of one of the three previous modes, combined with a passage in phototrophy.

[0081] In a particular embodiment, the microalgae extract is obtained by a process comprising the following steps: - An extraction step by introducing the microalgae into a liquid extraction medium, the extraction being carried out at a temperature ranging from 4°C to 300°C for a sufficient time to extract the molecules contained in the microalgae, - A filtration or centrifugation step to remove solids, and - A step of recovering the filtrate including the extract.

[0082] The method may comprise a step of culturing the microalgae in accordance with the preceding description. The preparation of the microalgae extract may alternatively be carried out by any extraction method well known to those skilled in the art such as hot decoction, maceration, extrusion, extraction under subcritical conditions, extraction assisted by ultrasound, with or without grinding such as ultrasonic grinding or using a mixer. The extraction may be carried out at a temperature ranging from 4°C to 300°C, preferably at a temperature ranging from 19°C to 25°C.

[0083] The liquid extraction medium may comprise water, preferably distilled water and optionally an aliphatic alcohol, for example ethanol or a glycol. In this case, the method for preparing the microalgae extract may comprise an additional step of drying or concentration, for example by freeze-drying, or by evaporation at a temperature ranging from 20°C to 80°C.

[0084] In another exemplary embodiment, the liquid extraction medium contains an oil, preferably a vegetable oil, and more preferably an oilseed oil such as rapeseed oil or soybean oil.

[0085] In a particular embodiment, the aqueous microalgae extract is prepared according to a method comprising the introduction of the microalgae into a liquid medium consisting of distilled water, followed by a filtration step to separate the filtrate containing the hydrophilic molecules and organelles contained in the cells, and the solid residues of the microalgae. The extraction step can be carried out at a temperature of 4°C to 300°C, for a period of time ranging from 30 minutes to 24 hours.

[0086] The extraction step may comprise the addition to the aqueous extraction medium of at least one enzyme enabling the degradation of the wall of the microalgae and the release of their contents into the liquid extraction medium. At least one enzyme chosen from pectinases and carbohydrases may thus be added to the liquid extraction medium during the extraction step to obtain a functionalized aqueous extract. Examples of carbohydrases are cellulases, hemicellulases, beta-glucanases, and xylanases. This description provides the following list of particular embodiments: 1. Use of an aqueous extract of at least one eukaryotic unicellular microalga to induce inhibition of at least one step of the nitrogen cycle in a soil, said step being chosen from a nitrification process, a denitrification process and a nitrification-denitrification process. 2. Use according to mode 1, characterized in that the microalgae is chosen from the group consisting of Nannochloropsis sp., Amphora sp., Chaetoceros sp., Chlamydomonas sp., Chlorella sp., Duna lie! la sp., Euglena sp., Fragi / aria sp., Haematococcus sp., Isochrysis sp. Tisochrysis sp., Na nnoch loris sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacylum sp., Scenedesmus sp., Schyzotrium sp., Skeletonema sp., Tha / assosira sp. and Tetrase / mis sp. 3. Use according to method 1 or 2, characterized in that the inhibition of a nitrification process has at least one effect chosen from an inhibition of the activity of nitrifying bacteria present in the soil, a reduction in the loss of fertilizer from the soil, a reduction in the leaching of nitrates in the soil, an increase in the quantity of ammonium available in the soil, an improvement in the growth of a plant grown on the ground, a decrease in nitrous oxide emissions, and an increase in the amount of nitrogen available to a plant grown on the ground. 4. Use according to method 1 or 2, characterized in that the inhibition of a denitrification process has at least one effect chosen from an inhibition of the activity of denitrifying bacteria present in the soil, an inhibition of the nitrate reduction process, an inhibition of the activity of the nitrate reductase enzyme, and a reduction in the emission of nitrous oxide. 5. Use according to method 1, characterized in that the aqueous extract is capable of being obtained by extraction of the molecules that the microalgae contains, with a liquid medium containing water. 6. Use according to the preceding method, characterized in that the aqueous extract is functionalized by adding at least one enzyme chosen from carbohydrases and pectinases in the liquid medium. 7. A method of agricultural fertilization comprising a step of providing a plant with an aqueous extract of at least one microalgae selected from the group consisting of Nannoch / oropsis sp., Amphora sp., Chaetoceros sp., Ch / amydomonas sp., Chlorella sp., Dunaiiella sp., Eug / ena sp., Fragilaria sp., Haematococcus sp., Isochrysis sp. Tisochrysis sp., Nannoch / oris sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacy / um sp., Scenedesmus sp., Schyzotrium sp., Ske / etonema sp., Thalassosira sp. and Tetrase / mis sp., said extract being able to be applied to a soil provided by a culture medium or provided by a culture solution. 8. Method of agricultural fertilization according to the preceding method, further comprising a step of applying at least one nitrogen fertilizer to the soil or to the plant cultivated on the soil, said application step being prior to, concomitant with or subsequent to the step of supplying the aqueous extract. 9. Agricultural fertilization method according to the preceding method, characterized in that the nitrogen fertilizer is a nitrogen fertilizer comprising an ammoniacal form of nitrogen, a nitrogen fertilizer releasing ammonium into the soil, or a nitrogen fertilizer comprising nitrate. 10. Product for agricultural use comprising an aqueous extract of at least one microalgae chosen from the group consisting of Nannoch / oropsis sp., Amphora sp., Chaetoceros sp., Ch / amydomonas sp., Chlorella sp., Dunaiiella sp., Euglena sp., Fragilaria sp., Haematococcus sp., Isochrysis sp. Tisochrysis sp., Nannochioris sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacyium sp., Scenedesmus sp., Schyzotrium sp., Skeletonema sp., Thalassosira sp. and Tetraselmis sp., and at least one soil nutrient ingredient and / or one nutrient ingredient of a plant grown on the soil chosen from a fertilizer, an amendment, a biostimulant and a micronutrient. 11. Product for agricultural use according to the preceding method, characterized in that it is in the form of a fertilizer, a culture medium or a culture solution. 12. Process for preparing an aqueous extract of at least one microalgae chosen from the group consisting of Nannochloropsis sp., Amphora sp., Chaetoceros sp., Chlamydomonas sp., Chlorella sp., Dunaliella sp., Eu g ten a sp., Fragi / aria sp., Haematococcus sp., Isochrysis sp. Tisochrysis sp., Nannochteris sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacytem sp., Scenedesmus sp., Schyzotrium sp., Sketetonema sp., Thalassosira sp. and Tetraselmis sp., said method comprising the following steps: An extraction step by introducing the microalgae into a liquid extraction medium comprising water, the extraction being carried out at a temperature ranging from 4°C to 300°C for a time sufficient to extract the molecules contained in the microalgae, A filtration step to remove solids, and A step of recovering the filtrate including the aqueous extract. 13. Method according to the preceding mode, characterized in that at least one enzyme chosen from pectinases and carbohydrases is added to the liquid extraction medium during the extraction step to obtain a functionalized aqueous extract.

[0087] The invention is also illustrated by the following examples. Unless otherwise stated, the temperature is room temperature between 20°C and 25°C and the pressure is atmospheric pressure.

[0088] Example 1: Preparation of microalaue extracts First excerpt:

[0089] Distilled water and a microalga cultivated in phototrophy are introduced into a glass reactor at a mass concentration of 2.5%. After stirring (for 4 hours) and decanting, the filtrate is recovered and constitutes the “Extract”. The microalga is, for example, a microalga of the genus Nannochloropsis sp., of the genus Arthrospira sp. or of the genus Chlorella, in particular a microalga of the species Nannochloropsis salina, Nannochloropsis ocuiata, Nannochloropsis oceanica, Arthrospira p / atensis, Chlorella sorokiniana or Chlorella vu / garis. Second excerpt:

[0090] Water and microalgae are introduced into a glass reactor at a mass concentration of 2.5%. After stirring (for 4 hours), the Viscozyme® L enzyme complex is added at a concentration of 0.1% (enzyme / raw material weight). The enzymatic functionalization of the extract lasts 5 hours with stirring. After decantation, the filtrate is collected and constitutes the “Functionalized Extract”. The microalgae is, for example, a microalga of the genus Nannochloropsis sp., of the genus Arthrospira sp. or of the genus Chlorella, in particular a microalga of the species Nannochloropsis satina, Nannochloropsis ocuiata, Nannochloropsis oceanica, Arthrospira piatensis, Chlorella sorokiniana or Chlorella vu / garis. Third excerpt:

[0091] In a glass reactor, oil and a microalgae cultivated in phototrophy are introduced, at a mass concentration of 2.5%. After stirring (for 16 hours) and decantation, the filtrate is collected and constitutes the "Extract in oil". The microalgae are, for example, Chlorella sorokiniana or Nannochloropsis oceanica.

[0092] Example 2: Measurement of nitrification inhibition by measuring ammonium in soil Preparation of a soil that includes a microalgae extract (Extract or Functionalized Extract) and a soil that does not include a microalgae extract (Control)

[0093] 5 g of dry soil from a surface soil located in France (the characteristics of which are detailed in Table 1) were sieved with a sieve with a mesh diameter of 2 mm, and placed in 60 ml glass bottles to which 0.8 ml of water containing the treatment (Extract or Functionalized Extract of Example 1) at a dose of 50 mg / Bottle or not containing it (Control) were added. Table 1: Main soil characteristics Texture Silty clay pH 8.1 Organic matter (%) 1.9 Cation exchange capacity (meq / Kg) 187

[0094] After 24 hours of incubation, 0.4 mg of N-NH .g -1 Soil, in the form of ammonium sulfate, is added to 1.8 ml of water. This volume allows to reach 80% of the field capacity of the soil studied.

[0095] The flasks are then hermetically sealed and incubated at 20°C to 22°C for up to 12 days. During this period, the nitrification kinetics in the soil are established by performing ammonium measurements at 2; 6; 9 and 12 days. Extraction and determination of ammonium from soil Extraction is carried out by adding 15 mL of a 1M KCl solution to each flask and then shaking with a rotary shaker for one hour. The flasks are left to settle for 10 min. The supernatant is collected and centrifuged at 11,000 rpm (rotations per minute) for 5 min at 4°C and then filtered through a 0.25 µm filter to remove any particles.

[0096] The ammonium dosage is carried out using the Ammonium test kit- Spectroquant (Supelco). After dilution of the filtrate, 5 ml is used to perform the assay. The Optical Density (OD) of the samples is measured using a spectrophotometer at 692 nm and the NH4 content + was estimated from a calibration curve.

[0097] For each of the incubation conditions (Control, Extract and Functionalized Extract), four batches of soil were created (1 batch = 1 biological repetition).

[0098] All treatments were carried out systematically for each of the biological replicates, i.e. in quadruplicate. The data obtained were presented as the mean and the variability of the results was given as the standard deviation of the mean for n=4.

[0099] The ammonium dosage is shown in Figure 1. Conclusion: Soils treated with the Extract or Functionalized Extract have ammoniacal nitrogen (N-NH4) contents+ ), e. the mass of the nitrogen element present in the form of ammonium, higher than that found in the control soil. An increase of +158% in the presence of the extract and +250% in the presence of the functionalized extract is observed after 12 days of incubation, reflecting a decrease in nitrifying activity also called inhibition of nitrification.

[0100] Example 3: Measurement of nitrification inhibition by measuring nitrates in soil Preparation of a soil that includes a microalgae extract [Extract or Functionalized Extract] and a soil that does not include a microalgae extract [Control]

[0101] Vials were prepared under the same conditions as described in Example 2.

[0102] The kinetics of nitrification associated with the appearance of nitrate in the soil were established by carrying out nitrate measurements at 2; 6; 9 and 12 days. Extraction and determination of nitrates from soil Extraction is performed by adding 15 mL of pure water to each flask and then shaking with a rotary shaker for one hour. The flasks are allowed to settle for 10 min. The supernatant is collected and centrifuged at 11,000 rpm (rotations per minute) for 5 min at 4°C and then filtered through a 0.25 µm filter to remove any particles.

[0103] Nitrate determination is carried out using the Nitrate test-Spectroquant kit (Supelco). After dilution of the filtrate, 5 ml are used to perform the determination. The Optical Density (OD) of the samples is measured using a spectrophotometer at 692 nm and the nitrate nitrogen content (N-NO3'), i.e. the mass of the nitrogen element present in the nitrate, was estimated from a calibration curve.

[0104] For each of the incubation conditions (Control, Extract and Functionalized Extract), four batches of soil were created (1 batch = 1 biological repetition).

[0105] All treatments were carried out systematically for each of the biological replicates, i.e. in quadruplicate. The data obtained were presented as the mean and the variability of the results was given as the standard error of the mean for n=4.

[0106] The result of the nitrate ion dosage is presented in Figure 2. Conclusion: Soils treated with the Extract or Functionalized Extract have lower nitrate nitrogen (N-NO3) contents than those found in the control soil. A decrease of -31% in the presence of the Extract and of -59% in the presence of the Extract functionalized is observed after 12 days of incubation, reflecting a decrease in nitrifying activity also called inhibition of nitrification.

[0107] Example 4: Determination of Nitrous Oxide (N2O) in soil Preparation of a soil that includes a microalka extract (Functionalized extract) and a soil that does not include a microalka extract (Control) 4.4 g of dry soil (kept at 15% mass humidity) sieved through a sieve with a mesh diameter of 1 mm, are placed in 37 ml glass bottles to which 0.6 ml of water containing 0.4 mg of N-NH .g' are added 1 Soil, in the form of ammonium sulfate. The microalgae extract prepared according to Example 1 was applied at a dose of 25 mg / bottle.

[0108] The flasks are then hermetically sealed and incubated at 20°C for up to 216 hours. During this period, the nitrification kinetics associated with the appearance of N2O are established by performing N2O measurements at 0; 48; 120; 168 and 216 hours.

[0109] Nitrification kinetics were also evaluated under the same conditions as previously on the same soil not including any microalgae extract used as a control. Sampling and dosage of NO gas The gas is taken from the gas atmosphere of the vials with a syringe, and the dosage is carried out on a microGC (Agilent 990) equipped with a TCD detector and a Porapak Q column. For each of the incubation conditions (Control and Functionalized Extract), three batches of soil were created (1 batch = 1 biological repetition).

[0110] All treatments were carried out systematically for each of the biological repetitions, i.e. in triplicate. The data obtained were presented as the mean and the variability of the results was given as the standard deviation of the mean for n=3.

[0111] The N2O dosage is shown in Figure 3. Conclusion: Soils treated with the Functionalized Extract have lower Nitrous Oxide (N2O, here represented by the peak area) contents than those found in the control soil. A decrease of -50% in the presence of the functionalized extract is observed after 216 hours of incubation.

[0112] Example 5: Measurement of the inhibition of the denitrification process by measuring the activity of the enzyme nitrate reductase

[0113] In a 96-well round-bottom plate are placed a 10 mM phosphate buffer pH 7.5, the enzyme nitrate reductase (Sigma Aldrich, N0163), and a substrate buffer pH 7.5 (containing 37.5 mM phosphate buffer pH 7.5, 15 mM potassium nitrate, 0.075 mM EDTA, 0.15 mM NADH) to which are added 0.8 ml of water containing the treatment (Extract, Functionalized Extract or Extract in Oil of Example 1) at a dose of 50 mg / Vial or not containing it (Control). The plate was then incubated at 30 °C for 40 minutes with shaking in the dark and followed by the addition of 58 mM sulfanilamide (Sigma Aldrich, S9251) with freshly prepared N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma Aldrich, 222488). The plate was again incubated for 20 minutes at room temperature in the dark for complete color development. Then, the absorbance was measured at 540 nm in a UV spectrophotometer (ClarioStar Plus, BMG Labtech).

[0114] The results are shown in Figure 4, Figure 5 and Figure 6. Conclusion: The enzyme treated with the Extract or Functionalized Extract of Nannochloropsis sa / ina, Nannochloropsis ocu / ata or Nannochloropsis oceanica shows a greater inhibition of the activity of the nitrate reductase enzyme than that found in the control solution. An inhibition of denitrification of 24% in the presence of the Extract and 99% in the presence of the Functionalized Extract of Nannochloropsis sa / ina, Nannochloropsis ocu / ata, Nannochloropsis oceanica is observed, reflecting an inhibition of the denitrifying activity (Figure 4). The enzyme treated with the Extract or Functionalized Extract of Arthrospira platensis (spirulina) or Chiore / ia sorokiniana (chlorella) shows a greater inhibition of the activity of the nitrate reductase enzyme than that found in the control solution. An inhibition of denitrification of 59.6% in the presence of the Functionalized Spirulina Extract and 49.9% in the presence of the Functionalized Chlorella Extract is observed, reflecting an inhibition of the denitrifying activity (Figure 5). The enzyme treated with Chlorella sorokiniana (chlorella) or Nannochloropsis oceanica oil or oil extract showed a greater inhibition of nitrate reductase enzyme activity than that found in the control solution. A 19.6% inhibition of denitrification in the presence of rapeseed oil and 36.3% in the presence of chlorella extract in rapeseed oil was observed, indicating an inhibition of denitrifying activity. A 27.2% inhibition of denitrification in the presence of soybean oil and 36.8% in the presence of chlorella extract in soybean oil, and 37.9% in the presence of nannochloropsis extract in soybean oil was observed, indicating an inhibition of denitrifying activity (Figure 6).

Claims

Claims

1. A method of fertilizing soil harboring microorganisms, said method comprising a step of providing said soil with at least one eukaryotic microalgae extract.

2. A method of fertilizing a soil according to claim 1, characterized in that the microalgae is selected from the group consisting of Amphora sp., Arthrospira sp., Chaetoceros sp., Ch / amydomonas sp., Chlorella sp., Dunaliella sp., Eug / ena sp., Fragiiaria sp., Haematococcus sp., Isochrysis sp. Tisochrysis sp., Nannochloris sp., Nannochloropsis sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacylum sp., Scenedesmus sp., Schyzotrium sp., Ske / etonema sp., Thalassosira sp. and Tetraselmis sp.

3. Method for fertilizing a soil according to claim 1 or 2, characterized in that the step of adding the microalgae extract causes the inhibition of the activity of the microorganisms.

4. Method for fertilizing a soil according to one of claims 1 to 3, characterized in that the supply step induces an inhibition of at least one step of the nitrogen cycle in the soil, said step being chosen from a nitrification process, a denitrification process and a nitrification-denitrification process.

5. A method of fertilizing a soil according to claim 4, characterized in that the inhibition of a nitrification process has at least one effect chosen from a reduction in the loss of fertilizer from the soil, a reduction in the leaching of nitrates in the soil, an increase in the amount of ammonium available in the soil, a reduction in nitrous oxide emissions, and an increase in the amount of nitrogen assimilable by a plant grown on the soil.

6. A method of fertilizing a soil according to claim 4, characterized in that the inhibition of a denitrification process has at least one effect chosen from an inhibition of the nitrate reduction process, an inhibition of the activity of the nitrate reductase enzyme, and a reduction in the emission of nitrous oxide.

7. Method for fertilizing a soil according to one of the preceding claims, characterized in that the extract is obtained by an extraction process in a liquid medium which may contain water or an oil, for example a vegetable oil.

8. Method for fertilizing a soil according to claim 7, characterized in that the liquid medium comprises water and at least one enzyme, which can be chosen from carbohydrases and pectinases.

9. Method for fertilizing a soil according to one of the preceding claims, characterized in that the soil has a pH greater than or equal to 6.

10. Method for agricultural fertilization of soil according to the preceding claim, characterized in that the agricultural product or the soil comprises a nitrogen fertilizer.

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