Extracts of microalgae for inhibiting nitrification and / or denitrification processes in soil

Abstract

The present invention is suitable for use in the field of agriculture and relates to the use of an eukaryotic microalgae extract for controlling nitrification in soil and reducing nitrogen loss in the form of N2O.

Description

Technical Field

[0001] This invention relates to the use of microalgae extracts in soil fertilization methods for inhibiting nitrification and / or nitrification-denitrification processes, as well as nitrate denitrification. These extracts possess the property of controlling the function of nitrifying bacteria and / or nitrification-denitrification bacteria responsible for converting ammoniacal nitrogen (NH4+) into nitrogen. + ) is converted into nitrate nitrogen (NO3) - In the process, nitrous oxide (N2O) is released. These extracts also have the property of inhibiting the function of nitrate reductase.

[0002] This invention proposes a novel method that can control nitrogen supply to soil and fertilizer supply to plants while limiting the emission of greenhouse gas N2O. Background Technology

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

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

[0005] In all these processes, nitration corresponds to ammonium (NH4) + ) is oxidized to nitrate (NO3) - This process involves several steps. First, nitrite-oxidizing bacteria and archaea (AOB (ammonia-oxidizing bacteria) and AOA (ammonia-oxidizing archaea)) oxidize ammonium to hydroxylamine (NH2OH), and then to nitrite (NO2). - Finally, nitrite-oxidizing bacteria (NOB) (Nitrobacterium spp.) Nitrobacter ) and Nitrifying Spirulina ( Nitrospira Nitrite can be oxidized to nitrate. The conversion of ammonium to nitrite releases four protons and two water molecules, leading to environmental acidification, particularly around the roots. The activities of bacteria AOB and AOA are highly dependent on the environment; therefore, humidity, pH, temperature, and ammonium availability significantly influence this process. Finally, during the oxidation of ammonium to nitrite, nitrous oxide (N₂O) emissions due to nitrification can be observed.

[0006] Nitrification is a crucial biological step in the soil nitrogen cycle. From an agronomic perspective, its activity is considered the limiting step in soil nitrogen supply. If it occurs too slowly or too quickly relative to plant needs, it can lead to low nitrogen use efficiency and may contribute to groundwater pollution and greenhouse gas emissions (N2O). Nitrification rates can vary depending on soil properties. Factors modulating the nitrification process include soil pH, temperature, humidity, nitrogen fertilizer applied to the soil, microorganisms, and soil physical properties.

[0007] Poor synchronization between nitrification and nitrogen assimilation by plants leads to the loss of nitrogen applied to the soil through nitrate leaching and nitrous oxide (N2O) emissions. These losses can account for up to one-fifth of the applied nitrogen. This results in increased agricultural production costs, environmental pollution (both surface and deep water), human and animal diseases, ozone layer depletion, and greenhouse gas emissions that contribute to climate change. Therefore, controlling nitrification has become a major problem in agriculture.

[0008] Another important process in the nitrogen cycle is the conversion of nitrates produced by nitrification into nitrogen. This second biological mechanism, denitrification, involves bacteria converting nitrates (NO3-) into nitrogen under anaerobic conditions. - ) is reduced to nitrogen gas (N2). Denitrifying bacteria then sequentially convert nitrate ions (NO3) into nitrogen gas (N2). - ) is reduced to nitrite ions (NO2) - Then it is reduced to nitric oxide (NO), then to nitrous oxide (N2O), and finally to nitrogen gas (N2).

[0009] Therefore, denitrification constitutes another source of greenhouse gas N2O emissions through bacteria using nitrates (competing with plants for nitrates).

[0010] There is also a nitrification-denitrification process, which is carried out by nitrifying organisms that are also capable of denitrification, or by a combination of nitrifying organisms and denitrifying organisms alone, in which the nitrates produced by the nitrifying organisms are immediately denitrified by the denitrifying organisms. N2O emissions can also be observed during the nitrification-denitrification process.

[0011] For non-nitrogen-fixing plants, nitrogen is mainly produced in the form of nitrate (NO3). - ) or ammonia (NH4) + Nitrogen is absorbed by plants in the form of nitrogen (N). Depending on the plant's needs, it can absorb a large amount to support its growth and development. However, nitrogen deficiency can occur due to insufficient soil supply or environmental losses caused by ammonia volatilization, nitrate leaching, or denitrification, negatively impacting plant growth.

[0012] Providing fertilizer solutions that are less likely to cause environmental damage helps avoid certain deficiencies and limit the environmental impact of fertilizers. Therefore, the amount of nitrogen added through fertilization can be reduced, which can bring economic benefits to farmers, help reduce nitrogen loss, and lower environmental risks.

[0013] To control nitrogen transformation processes in soil, it is beneficial to develop formulations containing nitrification inhibitors. The aim is to control nitrate supply according to plant needs and optimize crop nitrate utilization. This allows plants to utilize nitrogen more effectively while reducing negative environmental impacts.

[0014] Currently, several chemical molecules can inhibit nitrification in soil and limit greenhouse gas emissions. However, these are synthetic molecules, which may pose harm to the environment and soil organisms.

[0015] It is against this backdrop that the applicant demonstrated that certain natural extracts from microalgae can inhibit nitrification and / or nitrification-denitrification processes in soil, thereby improving the agricultural environmental balance of crops, which forms the basis of this invention. The applicant also demonstrated that these microalgae extracts can inhibit the function of nitrate reductase.

[0016] Therefore, this invention is applicable to the agricultural field, aiming to propose the use of natural extracts of microalgae as a solution for: slowing nitrification, thereby better regulating nitrogen supply to plants and reducing nitrogen loss in the form of N2O. It can also be considered as reducing nitrate loss due to leaching. Summary of the Invention

[0017] Using microalgae extracts to control one or more processes of the nitrogen cycle in agriculture is a novel approach. This is the main agronomic challenge of the present invention.

[0018] Therefore, this invention is based on using an extract of at least one eukaryotic microalga to induce inhibition of at least one step of the nitrogen cycle in soil, said step being selected from nitrification, denitrification, and nitrification-denitrification processes. The eukaryotic microalgae extract can be an aqueous extract or an oil extract.

[0019] This invention relates to an agricultural fertilization method comprising the step of supplying soil with an extract of at least one eukaryotic microalga, the extract being applicable to field fields or culture media, supplied via a culture media containing the extract, or supplied via a culture solution.

[0020] The extract may be provided by a product for agricultural use, which, in addition to an extract of eukaryotic microalgae, contains at least one soil nutrient or plant nutrient selected from, in particular, fertilizers, amendments, biostimulants and micronutrients.

[0021] A method for preparing an aqueous extract of microalgae usable under the present invention may include an extraction step by introducing the microalgae into a liquid extraction medium containing water, a filtration step, and a step of recovering the filtrate containing the aqueous extract. Attached Figure Description

[0022] Figure 1 The ammonium content (NH4) in the following soils is described. + mg N-NH4 +. g -1 Changes in soil composition: (i) soil containing functionalized microalgae extract, (ii) soil containing microalgae extract (extract), and (iii) soil without extract (control). The figure shows a gradual decrease in ammonium content during incubation from 0 to 12 days in soil without extract (control): these contents decreased from 0.4 mg N-NH4. + .g -1 The soil concentration increased to 0.12 mg N-NH4. + .g -1 Soil. Ammonium levels in soils containing microalgae extracts ranged from 0.4 mg N-NH4. + .g -1 The soil concentration increased to 0.31 mg N-NH4. + .g -1 Soil, ammonium levels in soil containing functionalized microalgae extracts ranged from 0.4 mg N-NH4. + .g -1 The soil concentration increased to 0.42 mg N-NH4. + .g -1 The soil indicates that nitrification is inhibited.

[0023] Figure 2 The following soil nitrate levels (NO3) are shown. - mg N-NO3 - .g -1 Changes in soil composition: (i) soil containing functionalized microalgae extract, (ii) soil containing microalgae extract (extract), and (iii) soil without extract (control). The figure shows a gradual increase in nitrate levels during incubation from 0 to 12 days in soil without extract (control): these levels increased from 0 to 0.22 mg N-NO3. - .g -1 Soil. Ammonium levels in soil containing microalgae extracts increased from 0 to 0.15 mg N-NO3. - .g -1 In soil containing functionalized microalgae extracts, ammonium levels increased from 0 to 0.09 mg N-NO3. -.g -1 The soil indicates that nitrification is inhibited.

[0024] Figure 3 The figure shows the changes in nitrous oxide (N2O) content (expressed as peak area by GC (gas chromatography) analysis) in the following soils: (i) soil containing a functionalized microalgae extract (functionalized extract) and (ii) soil without the extract (control). The figure shows a gradual increase in peak area associated with increased atmospheric N2O content during incubation from 0 to 216 hours in the soil without the extract (control): the peak area increased from 0 to 1200. In the soil containing the functionalized microalgae extract, the peak area increased from 0 to 600, indicating that N2O production was inhibited due to the functionalized microalgae extract.

[0025] Figure 4 The percentage (%) of inhibition of nitrate reductase function is shown in the following solutions: (i) solution containing functionalized microalgae extract, (ii) solution containing microalgae extract (extract), and (iii) solution without extract (control). The figure shows that the inhibition of nitrate reductase activity was 24% in the solution containing microalgae extract and 99% in the solution containing functionalized microalgae extract compared to the solution without extract (control).

[0026] Figure 5 The percentage (%) of inhibition of nitrate reductase function is shown in the following solutions: (i) solutions containing functionalized microalgae extracts, (ii) solutions containing microalgae extracts (extracts), and (iii) solutions without extracts (controls). The figure shows that, compared to the solution without extracts (control), the inhibition of nitrate reductase activity was 28.9% in the solution containing Spirulina extract and 59.6% in the solution containing functionalized Spirulina extract. The figure also shows that, compared to the solution without extracts (control), the inhibition of nitrate reductase activity was 13.8% in the solution containing Chlorella extract and 49.9% in the solution containing functionalized Chlorella extract.

[0027] Figure 6The percentage (%) of inhibition of nitrate reductase function is shown in the following solutions: (i) rapeseed oil-based solution (HC), (ii) soybean oil-based solution (HS), (iii) rapeseed oil containing microalgae extract, (iv) soybean oil containing microalgae extract, and (v) solution without extract (control). The figure shows that, compared to the solution without extract (control), the inhibition of nitrate reductase activity was 19.6% in the rapeseed oil-containing solution (HC column) and 36.3% in the rapeseed oil-containing Chlorella extract solution (Chlorella extract-HC column). The figure also shows that, compared with the solution without extract (control), the inhibition of nitrate reductase activity was 27.2% in the solution containing soybean oil (HS column), 36.8% in the solution containing Chlorella extract in soybean oil (Chlorella extract-HS column), and 37.9% in the solution containing Nannochloropsis extract in soybean oil (Nannochloropsis extract-HS column). Detailed Implementation

[0028] Therefore, the present invention relates to the use of an extract of at least one eukaryotic microalgae for inducing inhibition of at least one step of the nitrogen cycle in soil, said step being selected from nitrification, denitrification and nitrification-denitrification processes, and particularly relates to a method for fertilizing soil by supplying agricultural products containing an aqueous extract of eukaryotic microalgae to soil containing bacteria.

[0029] Microalgae extracts can be aqueous or oil-based. In certain embodiments, oil extracts of eukaryotic microalgae can inhibit denitrification processes in soil.

[0030] "Eukaryotic microalgae" are single-celled microorganisms capable of photosynthesis. Nitrification is defined as a group of biochemical reactions induced by nitrifying bacteria (e.g., ammonia-oxidizing bacteria (AOB) and / or nitrite-oxidizing bacteria (NOB)) to form nitrates from ammonium. Examples of nitrifying bacteria genera include *Nitrobacter* (…). Nitrobacter spp. ), Nitrosomonas spp. Nitrosomonas spp. ), Nitrostrophus ( Nitrosospira spp. ) and Nitrifying Cocci ( Nitrosococcus spp. ), Nitrosomonas spp. Nitrosomonas spp. ), Nitrostrophus ( Nitrosospira spp. ) and Nitrifying Cocci ( Nitrosococcus spp. ), including the following species: European nitrosomonas ( Nitrosomonas europaea ), Nitrosomonas ( Nitrosomonas eutropha ), Nitrosomonas spp. (Nitrosomonas communis ), Polymorphic Nitrospisil ( Nitrosospira multiformis ) and Lake Nitrifying Spirulina ( Nitrosospira lacus ).

[0031] The term "nitrification inhibitor," also known as a nitrification depressant, refers to a product whose activity slows down one of the biochemical reactions involved in the nitrification process. Nitrification inhibitors advantageously allow for better synchronization of soil nitrogen supply with plant needs, thereby reducing nitrogen fertilizer losses (e.g., leaching of nitrates from the soil). Furthermore, since nitrification produces N₂O, nitrification inhibitors can reduce N₂O emissions into the soil.

[0032] Within the scope of this invention, inhibition of the nitrification process can have at least one effect, selected from inhibiting the activity of nitrifying bacteria present in the soil, reducing fertilizer loss in the soil, reducing nitrate leaching in the soil, increasing the amount of ammonium available in the soil, improving the growth of plants grown in the soil, reducing nitrous oxide emissions, and increasing the amount of nitrogen that plants grown in the soil can absorb.

[0033] The microalgae extracts covered by this invention can be nitrification inhibitors within the scope of this invention. In a specific embodiment of the invention, after at least 2 days, preferably at least 6 or 10 days, following the application of the extract to the soil, when the microalgae extract improves nitrate nitrogen (N-NO3) levels in the soil... - When the amount of ammonia nitrogen (N-NH4) in the modified soil is reduced by at least 5% compared to the same soil not modified with the extract, and / or when the microalgae extract reduces the amount of ammonia nitrogen (N-NH4) in the modified soil. + When the amount of nitrate nitrogen increases by at least 5% compared to the same soil without the extract, the microalgae extract is considered a nitrification inhibitor. Several methods for measuring the nitrification inhibitory activity of the microalgae extract are detailed in the examples. After a duration of at least 2 days, preferably at least 6 days or 10 days, following application of the extract to the soil, the decrease in the amount of nitrate nitrogen or the increase in the amount of ammonia nitrogen can be independently greater than or equal to a value selected from 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, and 250%.

[0034] The term "denitrification" refers to the process by which denitrifying bacteria convert nitrates (NO3) into nitrogenous substances under anaerobic conditions. - The bacteria reduce nitrate ions (NO3) to nitrogen gas (N2). More precisely, bacteria use N2O reductase to reduce nitrate ions (NO3) to nitrogen gas (N2). - ) are successively reduced to nitrite ions (NO2) - The nitrogen dioxide is reduced to nitric oxide (NO), then to nitrous oxide (N₂O), and finally to nitrogen gas (N₂). The denitrifying bacteria involved include *Thiobacillus denitrifyingus* (…). Thiobacillus denitrificans ), denitrifying micrococci (Micrococcus denitrificans ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Pseudomonas schrenckii ( Pseudomonas stutzeri ), Serratia ( Serratia spp. ) and Achromobacterium spp. ( Achromobacter spp. ).

[0035] A "denitrification inhibitor" is a product used to slow down at least one step in the denitrification process. In a particular embodiment, the denitrification inhibitor causes, after at least 10 minutes, the activity of nitrate reductase in a medium supplemented with the denitrification inhibitor to decrease by at least 5% compared to the same medium without supplementation. For example, after a duration of at least 10 minutes, the reduction in nitrate reductase activity may be greater than a value selected from 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95%.

[0036] Inhibition of the denitrification process can have at least one effect, selected from inhibiting the activity of denitrifying bacteria present in the soil, inhibiting the nitrate reduction process, inhibiting the activity of nitrate reductase, and reducing nitrous oxide emissions.

[0037] "Nitrification-denitrification process" refers to a process carried out by organisms capable of both nitrification and denitrification, or a process carried out by different nitrifying and denitrifying organisms in combination, in which nitrates produced by nitrifying organisms are immediately denitrified by denitrifying organisms.

[0038] The term "nitrification-denitrification process inhibitor" refers to a process that limits the activity of nitrification-denitrification, thereby inhibiting N2O production. Furthermore, inhibition of the nitrification-denitrification process can reduce nitrous oxide production. After a duration of, for example, at least 100 hours, 150 hours, or 200 hours, the reduction in nitrous oxide production can be greater than or equal to a value selected from 5%, 10%, 20%, 30%, 40%, and 50%.

[0039] In one embodiment of the invention, the microalgae extract is a nitrification-denitrification inhibitor when it reduces the amount of nitrous oxide in soil improved with the microalgae extract by at least 5% under nitrification conditions (aerated environment) compared to the same unimproved soil.

[0040] When soil contains denitrifying bacteria, the addition of microalgae extracts inhibits the activity of these bacteria.

[0041] When soil contains nitrifying bacteria, the addition of microalgae extracts inhibits the activity of these bacteria.

[0042] The term "microalgae extract" refers to a product produced by a human-operated method for isolating a portion of microalgae. The microalgae are preferably eukaryotic microalgae: membrane-bound and containing organelles, including the cytoplasm and nucleus.

[0043] The term "microalgae aqueous extract" refers to a product obtained by extracting the cellular contents of microalgae using a liquid containing water. The aqueous extract does not necessarily contain water, as a step can be taken after the extraction to remove the water used as the extraction solvent. Preferably, the aqueous extract contains the hydrophilic molecules contained within the microalgae. Therefore, the aqueous extract can be obtained by extracting the molecules contained within the microalgae using a liquid medium containing water.

[0044] An aqueous extract of at least one eukaryotic microalga can be obtained by extracting organelles contained in the cells using a liquid extraction medium preferably composed of water (more preferably distilled water), the extraction 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 selected from proteases, carboxypeptidases, catalases, cellulases, glucans, hemicellulases, lipases, amylases, and phospholipases. In a particular embodiment, the enzyme is selected from carbohydrate enzymes and pectinases. Examples of carbohydrate enzymes are cellulases, hemicellulases, β-glucans, and xylanases.

[0045] The term "microalgal oil extract" refers to a product obtained by extracting the cellular contents of microalgae from a liquid containing oil. The oil can be a vegetable oil, such as soybean oil or rapeseed oil. The oil extract contains lipophilic molecules found within the microalgal cells. Microalgal oil extracts can be obtained through a lysis method that involves rupturing the cell membrane to release the liquid contents of the microalgae.

[0046] Alternatively, an oil extract of at least one eukaryotic microalga can be obtained by extracting the molecules contained in the cells by placing the microalgae in oil at a temperature ranging from 4°C to 300°C. The oil containing lipophilic molecules from the contents of the microalgae can then be separated from the treated microalgae by filtration or centrifugation.

[0047] In the context of this invention, the microalgae that can be used are, for example, those belonging to the genus Dipterocarpus (… Amphora sp. ), genus Arthrospira ( Arthrospira sp. ), Chaetoceros genus ( Chaetoceros sp. Chlamydomonas ( ) Chlamydomonas sp. Chlorella ( ) Chlorella sp. ), Dunaliella genus ( Dunaliella sp. Euglena ( ) Euglena sp. ), genus *Briarella* ( Fragilaria sp. ), genus Haematococcus ( Haematococcus sp. Isochrysis ( ) Isochrysis sp. ), Tisochrysis sp. Micrococcus (Nannochloris sp. ), Micrococcus ( Nannochloropsis sp. ), , Nitzchi sp. genus *Porphyra* ( Odonthella sp. ), genus *Phaeodactylum* ( Porphyridium sp. Phaeodacylum ), *Scenedesmus* ( sp. ), Scenedesmus sp. genus *Skeletonema* Schyzotrium sp. ), genus *Williams* ( ) and the genus *Chlorophyta* ( Skeletonema sp. Microalgae belonging to one of the genera listed above. Microalgae belonging to one of the genera can be any microalgae species known to those skilled in the art that belong to one of these genera.

[0048] An example of a specific microalgae species is Haloxylon ammodendron (Haloxylon ammodendron). Thalassosira sp. ), Eyedrop Micrococcus ( Tetraselmis sp. ), marine microbiococcus ( Nannochloropsis salina ), Spirulina obtuseum ( Nannochloropsis oculata ) and Sorokin Chlorella ( Nannochloropsis oceanica ).

[0049] The present invention relates to a method for fertilizing soil, the method comprising the step of providing a microalgae extract, wherein the microalgae extract is applied to the soil, provided to the soil by means of a culture solution, or integrated into the soil when the soil is a culture medium.

[0050] The term "soil" refers to a substrate for the root development of cultivated plants. The term soil includes any type of substrate suitable for plant development, such as artificial soil, cultivated agricultural soil, uncultivated agricultural soil, peat, potting soil, rock wool, or coconut fiber. Therefore, the term soil encompasses culture media used for so-called "soilless" cultivation (e.g., potted plants). In a particular embodiment of the invention, "soil" does not include plants, and in particular, does not include plant roots. Soil may comprise a mixture of minerals, organisms, organic matter, and air. It may contain microorganisms, including nitrifying and / or denitrifying microorganisms known to those skilled in the art (some of which have already been described above). Microorganisms may contain nitrate reductases.

[0051] The inventors unexpectedly discovered that eukaryotic microalgae extracts can act on soil microorganisms involved in the nitrification and denitrification steps of the nitrogen cycle. The microalgae extracts are preferably supplied to soils containing nitrifying and / or denitrifying microorganisms. In a particular embodiment of the invention, the fertilization method does not include the step of supplying eukaryotic microalgae extracts to plants.

[0052] Soils can have acidic, neutral, or alkaline pH values. Acidic soils are defined as soils with a pH value less than or equal to 6.2. Neutral soils are defined as soils with a pH value between 6.2 and 7.5. Finally, alkaline soils have a pH value greater than or equal to 7.5.

[0053] In a specific implementation, the soil pH value is greater than or equal to a value selected from 6, 6.5, 7, 7.5 or 8.

[0054] For example, soils with an alkaline pH value, that is, a pH value greater than or equal to 7.5. In certain embodiments, apart from measurement variations, the pH value of soils with an alkaline pH value ranges from 7.5 to 8.5, and particularly close to 8.0.

[0055] Soil can have different textures. Soil texture can be selected from heavy clay, silty clay, clay, sticky silty loam, clay loam, very fine silt, fine silt, sandy clay, silt, sandy clay loam, sandy silt, silty sand, sandy soil and mixtures thereof.

[0056] In a particular implementation, the soil has a cation exchange capacity (CEC) in the range of 10 meq / kg to 350 meq / kg, for example, in the range of 50 meq / kg to 250 meq / kg, or in the range of 180 meq / kg to 200 meq / kg.

[0057] The particle size of the soil can be between 1 mm and 5 mm, for example, between 1.5 mm and 3 mm.

[0058] Finally, the soil may contain organic matter in a range of 0.1% to 15% by mass relative to the soil mass.

[0059] The cation exchange capacity, particle size, and organic matter content of soil are quantities known to those skilled in the art, who know how to measure them using standard methods.

[0060] Microalgae extracts can be added to the soil via a culture solution. For the purposes of this invention, the term "culture solution" means any solution that can be used to cultivate plants, such as irrigation water, liquid solutions intended for irrigation and fertilization by spraying or dripping, or hydroponic baths.

[0061] Microalgae extracts, or agricultural products containing microalgae extracts, are applied to the soil, for example, in field or via soilless methods. Aqueous extracts can be applied to the soil surface or mixed with the soil, such as with the first layer of soil. Application can be made directly on the soil, across the entire soil surface, or locally by any suitable dispensing method.

[0062] Adding microalgae extracts to the soil can be done before and / or during the growth of the cultivated plants, before and / or after the plants germinate, or during transplanting if the plants need to be transplanted.

[0063] The fertilization method of the present invention may include one or more steps of applying microalgae extract, and one or more steps of applying fertilizer (e.g., solid or liquid fertilizer, amendment, and / or biostimulant). Those skilled in the art can adjust the order and number of repetitions of the steps according to the properties of the soil and / or the plants being cultivated.

[0064] In a particular embodiment of the invention, the aqueous extract is formulated into the fertilizer before application to the soil, allowing for the simultaneous application of the fertilizer and the microalgae extract. The fertilizer is advantageously selected from nitrogen fertilizers containing ammoniacal nitrogen, nitrogen fertilizers that release ammonium into the soil, nitrogen fertilizers containing nitrates, and combinations thereof.

[0065] Therefore, in a particular embodiment of the invention, the fertilization method involves using agricultural products that comprise the aforementioned microalgae extract and optionally at least one nutrient from the soil and / or at least one nutrient from plants grown in the soil, the nutrient being selected from fertilizers, amendments, biostimulants, and micronutrients.

[0066] The term "fertilizer" refers to fertilization materials whose primary function is to provide plants with elements (major fertilizing elements, minor fertilizing elements, and micronutrients) that are directly useful for their nutrition. The term "amendment" refers to substances designed to improve soil quality, particularly soil pH. Advantageously, amendments are selected from basic mineral amendments of the limestone and / or limestone and magnesium type, compost-type humus amendments, or manure.

[0067] Fertilizers are preferably simple binary or ternary solid fertilizers, organic mineral or organic fertilizers, or liquid or water-soluble fertilizers.

[0068] In one embodiment, the fertilizer is a nitrogen fertilizer. It is preferably a nitrogen fertilizer containing ammoniacal nitrogen, a nitrogen fertilizer that releases ammonium into the soil, or a nitrogen fertilizer containing nitrates.

[0069] In certain implementations, nitrogen fertilizers contain ammonium, nitrates, ammonia, and / or urea, wherein the urea can be formulated to slow the release of nitrogen into the soil. Examples of ammonium-containing fertilizers are calcium ammonium nitrate, calcium nitrate and ammonium nitrate, ammonium nitrate-ammonium sulfate, ammonium sulfate, and ammonium phosphate. Examples of ammonia-containing fertilizers are ammonium nitrate. Nitrogen fertilizers may also provide other nutrients, such as potassium (K) and / or phosphorus (P).

[0070] The modifier can be an organic modifier or a mineral modifier, such as calcium carbonate.

[0071] Micronutrients such as molybdenum, zinc, boron and copper are preferably supplied in the form of water-soluble salts or organic acids.

[0072] Biostimulants can be, for example, liquid root or leaf biostimulants.

[0073] Agricultural products may also contain at least one formulation adjuvant selected from solvents, solid carriers, dispersants or emulsifiers, organic or inorganic thickeners, pesticides, antifreeze agents, antifoaming agents, colorants where appropriate, adhesives or binders.

[0074] Agricultural products can be in the form of fertilizers applied to the soil or culture solutions. When soil is used as a culture medium, microalgae extracts can be introduced into the culture medium during its manufacturing process.

[0075] In a particular embodiment, the agricultural product according to the invention is a culture solution, such as irrigation water, a solution intended for irrigation and fertilization by spraying or drip irrigation, a hydroponic bath, or an aeroponic solution.

[0076] The agricultural products according to the invention can be in solid form, particularly powder, granules or microparticles, or in liquid form, particularly liquid suspension, gel or aqueous solution.

[0077] In this application, the term "plant" is intended to refer to a plant as a whole, including its root system, phytophysial system, seeds, grains, and fruits.

[0078] This invention is applicable to soils suitable for growing a wide variety of plants. Among these plants, large-scale crops such as cereals (wheat, corn, barley), protein crops (peas), oilseeds (soybeans, sunflowers), nightshade crops (potatoes), amaranth crops (beets), specialty crops such as, in particular, commercial vegetable cultivation (lettuce, spinach, onions, scallions, tomatoes, melons), grapevines, arboretum (pears, apples, nectarines), or horticulture are particularly noteworthy.

[0079] The plants can belong to the order Monocotyledons, but are preferably in the family Poaceae. Poaceae plants are generally called grasses, and specifically include most species commonly referred to as "grass" and "cereals." Poaceae plants include wheat, rice, barley, oats, rye, sugarcane, forage grasses, and maize.

[0080] Microalgae extracts can be obtained by any method known to those skilled in the art.

[0081] Based on the general knowledge of those skilled in the art, microalgae used for extract preparation are cultured under cultivation conditions using one of three cultivation modes: autotrophic, multitrophic, or heterotrophic. The cultivation method may include steps performed under one of the aforementioned three modes, combined with phototrophic subculturing.

[0082] In a particular implementation, the microalgae extract is obtained by a method comprising the following steps: - The extraction process involves introducing microalgae into a liquid extraction medium, with the extraction time sufficient to extract the molecules contained in the microalgae at a temperature ranging from 4°C to 300°C. - A filtration or centrifugation step to remove solids, and - The step of recovering the filtrate containing the extract.

[0083] The method may include the steps of culturing microalgae as described above. Alternatively, the preparation of the microalgae extract may 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, ultrasonic-assisted extraction, with or without grinding (e.g., ultrasonic grinding), or using a mixer. Extraction may be carried out at temperatures ranging from 4°C to 300°C, preferably from 19°C to 25°C.

[0084] The liquid extraction medium may include water (preferably distilled water) and optional aliphatic alcohols (e.g., ethanol or glycol). In this case, the method for preparing the microalgae extract may include additional drying or concentration steps, such as drying or concentration by freeze drying or evaporation at a temperature in the range of 20°C to 80°C.

[0085] In another exemplary embodiment, the liquid extraction medium contains oil, preferably vegetable oil, more preferably oilseed oil, such as rapeseed oil or soybean oil.

[0086] In a specific embodiment, a microalgae aqueous extract is prepared according to a method comprising: introducing microalgae into a liquid medium consisting of distilled water, followed by a filtration step to separate the filtrate containing hydrophilic molecules and organelles contained in the cells from the solid residue of the microalgae. The extraction step can be carried out at a temperature from 4°C to 300°C for a duration from 30 minutes to 24 hours.

[0087] The extraction step may include adding at least one enzyme to an aqueous extraction medium, said enzyme being capable of degrading the wall of the microalgae and releasing its contents into the liquid extraction medium. Therefore, during the extraction step, at least one enzyme selected from pectinases and carbohydrate enzymes may be added to the liquid extraction medium to obtain a functionalized aqueous extract. Examples of carbohydrate enzymes include cellulase, hemicellulase, β-glucanase, and xylanase.

[0088] This specification provides the following specific implementation schemes: 1. Use of an aqueous extract of at least one eukaryotic single-celled microalga for inducing inhibition of at least one step of the nitrogen cycle in soil, said step being selected from nitrification, denitrification, and nitrification-denitrification processes.

[0089] 2. The use according to method 1, characterized in that the microalgae are selected from the genus *Microcystis* (…). Arthrospira platensis ), genus Dipterocarpa ( Chlorella sorokiniana ), Chaetoceros genus ( Nannochloropsis sp. Chlamydomonas ( ) Amphora sp. Chlorella ( ) Chaetoceros sp. ), Dunaliella genus ( Chlamydomonas sp. Euglena ( ) Chlorella sp. ), genus *Briarella* ( Dunaliella sp. ), genus Haematococcus ( Euglena sp. Isochrysis ( ) Fragilaria sp. ), Haematococcus sp. Micrococcus ( Isochrysis sp. ), Tisochrysis sp. Nannochloris sp. Nitzchi sp. , Odonthella sp. genus *Porphyra* ( Porphyridium sp. ), genus *Phaeodactylum* ( Phaeodacylum sp. ), *Scenedesmus* ( Scenedesmus sp. ), Schyzotrium sp. genus *Skeletonema* Skeletonema sp. ), genus *Williams* ( Thalassosira sp. ) and the genus *Chlorophyta* ( Tetraselmis sp. ).

[0090] 3. The use according to method 1 or 2, characterized in that the inhibition of the nitrification process has at least one effect selected from the following: inhibiting the activity of nitrifying bacteria present in the soil, reducing fertilizer loss in the soil, reducing nitrate leaching in the soil, increasing the amount of ammonium available in the soil, improving the growth of plants cultivated in the soil, reducing nitrous oxide emissions, and increasing the amount of nitrogen that can be absorbed by plants cultivated in the soil.

[0091] 4. The use according to method 1 or 2, characterized in that the inhibition of the denitrification process has at least one effect selected from the following: inhibiting the activity of denitrifying bacteria present in the soil, inhibiting the nitrate reduction process, inhibiting the activity of nitrate reductase, and reducing nitrous oxide emissions.

[0092] 5. The use according to method 1, characterized in that the water extract can be obtained by extracting molecules contained in microalgae using a liquid medium containing water.

[0093] 6. The use according to the preceding method, characterized in that the water extract is functionalized by adding at least one enzyme selected from carbohydrate enzymes and pectinases to a liquid medium.

[0094] 7. An agricultural fertilization method comprising the step of supplying a plant with an aqueous extract of at least one microalgae, said microalgae being selected from the genus *Micrococcus* (…). Nannochloropsis sp. ), genus Dipterocarpa ( Amphora sp. ), Chaetoceros genus ( Chaetoceros sp. Chlamydomonas ( ) Chlamydomonas sp. Chlorella ( ) Chlorella sp. ), Dunaliella genus ( Dunaliella sp. Euglena ( ) Euglena sp. ), genus *Briarella* ( Fragilaria sp. ), genus Haematococcus ( Haematococcus sp. Isochrysis ( ) Isochrysis sp. ), Tisochrysis sp. Micrococcus ( Nannochloris sp. ), Nitzchi sp. , Odonthella sp. genus *Porphyra* ( Porphyridium sp. ), genus *Phaeodactylum* ( Phaeodacylum sp. ), *Scenedesmus* ( Scenedesmus sp. ), Schyzotrium sp. genus *Skeletonema* Skeletonema sp. ), genus *Williams* ( Thalassosira sp. ) and the genus *Chlorophyta* ( Tetraselmis sp. The extract can be applied to soil, provided by a culture medium or by a culture solution.

[0095] 8. The agricultural fertilization method according to the preceding method, further comprising the step of applying at least one nitrogen fertilizer to the soil or to plants grown in the soil, said application step being performed before, simultaneously with or after the step of adding the water extract.

[0096] 9. The agricultural fertilization method according to the preceding method, characterized in that the nitrogen fertilizer is a nitrogen fertilizer containing ammonia nitrogen, a nitrogen fertilizer that releases ammonium into the soil, or a nitrogen fertilizer containing nitrates.

[0097] 10. A product for agricultural use, comprising at least one selected from the genus *Micrococcus* (…). Nannochloropsis sp. ), genus Dipterocarpa ( Amphora sp. ), Chaetoceros genus ( Chaetoceros sp. Chlamydomonas ( ) Chlamydomonas sp. Chlorella ( ) Chlorella sp. ), Dunaliella genus ( Dunaliella sp. Euglena ( ) Euglena sp. ), genus *Briarella* ( Fragilaria sp. ), genus Haematococcus ( Haematococcus sp. Isochrysis ( ) Isochrysis sp. ), Tisochrysis sp. Micrococcus ( Nannochloris sp. ), Nitzchi sp. , Odonthella sp. genus *Porphyra* ( Porphyridium sp. ), genus *Phaeodactylum* ( Phaeodacylum sp. ), *Scenedesmus* ( Scenedesmus sp. ), Schyzotrium sp. genus *Skeletonema* Skeletonema sp. ), genus *Williams* ( Thalassosira sp. ) and the genus *Chlorophyta* ( Tetraselmis sp. The water extract of microalgae in the soil, and at least one soil nutrient or / or one plant nutrient selected from fertilizers, amendments, biostimulants and micronutrients, wherein the plant is a plant cultivated in the soil.

[0098] 11. The product for agricultural use according to the preceding method, characterized in that the product is in the form of fertilizer, culture medium or culture solution.

[0099] 12. A method for preparing an aqueous extract of at least one microalgae, said microalgae being selected from the genus *Microcystis* (…). Nannochloropsis sp. ), genus Dipterocarpa ( Amphora sp. ), Chaetoceros genus ( Chaetoceros sp. Chlamydomonas ( ) Chlamydomonas sp. Chlorella ( ) Chlorella sp. ), Dunaliella genus ( Dunaliella sp. Euglena ( ) Euglena sp. ), genus *Briarella* ( Fragilaria sp. ), genus Haematococcus ( Haematococcus sp. Isochrysis ( ) Isochrysis sp. ), Tisochrysis sp. Micrococcus ( Nannochloris sp. ), Nitzchi sp. , Odonthella sp. genus *Porphyra* ( Porphyridium sp. ), genus *Phaeodactylum* ( Phaeodacylum sp. ), *Scenedesmus* ( Scenedesmus sp. ), Schyzotrium sp. genus *Skeletonema* Skeletonema sp. ), genus *Williams* ( Thalassosira sp. ) and the genus *Chlorophyta* ( Tetraselmis sp. The method includes the following steps: - The extraction process involves introducing microalgae into a liquid extraction medium containing water, with the extraction time sufficient to extract the molecules contained in the microalgae at a temperature ranging from 4°C to 300°C. - Filtration step to remove solids, and - A step for recovering the filtrate containing the water extract.

[0100] 13. The method according to the preceding method, characterized in that, during the extraction step, at least one enzyme selected from pectinase and carbohydratease is added to the liquid extraction medium to obtain a functionalized aqueous extract.

[0101] The invention is further illustrated by the following embodiments. Unless otherwise stated, the temperature is room temperature between 20°C and 25°C, and the pressure is atmospheric pressure.

[0102] Example 1: Preparation of microalgae extract First extract: Distilled water and photocultured microalgae were introduced into a glass reactor at a concentration of 2.5% by mass. After stirring (4 hours) and decanting, the filtrate was collected, which constituted the "extract". Microalgae included, for example, those from the genera *Microchondria*, *Arthrospira*, or *Chlorella*, particularly species such as *Haloxylon ammodendron*. Nannochloropsis salina ), Eyedrop Micrococcus ( Nannochloropsis oculata ), marine microbiococcus ( Nannochloropsis oceanica ), Spirulina obtuseum ( Arthrospira platensis ), Sorokin Chlorella ( Chlorella sorokiniana ) or common Chlorella ( Chlorella vulgaris ).

[0103] The second extract: Water and microalgae were introduced into a glass reactor at a mass concentration of 2.5%. After stirring (4 hours), the Viscozyme® L-enzyme complex was added at a concentration of 0.1% (enzyme / raw material weight). Enzyme functionalization of the extract was continued for 5 hours with stirring. After decantation, the filtrate was collected, which constituted the "functionalized extract". Microalgae included, for example, those of the genera *Microchondria*, *Arthrospira*, or *Chlorella*, particularly species such as *Haloxylon ammodendron* (*Microchondria*). Nannochloropsis salina ), Eyedrop Micrococcus ( Nannochloropsis oculata ), marine microbiococcus ( Nannochloropsis oceanica ), Spirulina obtuseum ( Arthrospira platensis ), Sorokin Chlorella ( Chlorella sorokiniana ) or common Chlorella ( Chlorella vulgaris ).

[0104] The third type of extract: Oil and photocultured microalgae were introduced into a glass reactor at a concentration of 2.5% by mass. After stirring (16 hours) and decantation, the filtrate was collected, constituting the "oil extract". Microalgae, for example, were *Chlorella sorokinense* (…). Chlorella sorokiniana ) or marine micrococcus ( Nannochloropsis oceanica ).

[0105] Example 2: Measurement of nitrification inhibition by measuring ammonium in soil Preparation of soil containing microalgae extract (extract or functionalized extract) and soil not containing microalgae extract (control). Five g of dried soil from topsoil in France (characteristics detailed in Table 1) was sieved through a sieve with a mesh diameter of 2 mm and placed in a 60 ml glass bottle. 0.8 ml of water was added to the glass bottle, the water containing a treatment agent (the extract or functionalized extract of Example 1) at a dose of 50 mg / bottle or without the treatment agent (control).

[0106] Table 1: Main Soil Characteristics

[0107] After 24 hours of incubation, 0.4 mg of N-NH4 in the form of ammonium sulfate was added. + .g -1 Sol was added to 1.8 ml of water. This volume resulted in 80% of the field volume of the soil studied.

[0108] The bottles were then sealed and incubated at 20°C to 22°C for up to 12 days. During this period, nitrification kinetics in the soil were established by measuring ammonium on days 2, 6, 9, and 12.

[0109] Extraction and determination of ammonium in soil Extraction was performed as follows: 15 mL of 1M KCl solution was added to each flask, and the flask was shaken with a rotary vibrator for one hour. The flasks were allowed to stand for 10 minutes. The supernatant was collected, centrifuged at 11,000 rpm for 5 minutes at 4°C, and then filtered through a 0.25 μm filter to remove any particles.

[0110] Ammonium determination was performed using the Spectroquant Ammonium Assay Kit (Supelco). After diluting the filtrate, 5 ml was used for the determination. The optical density (OD) of the sample was measured at 692 nm using a spectrophotometer, and the NH4+ was estimated based on the calibration curve. + content.

[0111] For each incubation condition (control, extract, and functionalized extract), four batches of soil were prepared (1 batch = 1 biological replicate).

[0112] For each biological replicate, i.e., each of the four replicates, all treatments were performed systematically. The data obtained are presented as mean, and the variability of the results is given as the standard deviation of the mean with n=4.

[0113] The quantitative amount of ammonium is shown in Figure 1 middle.

[0114] Conclusion: Soil ammonia nitrogen (N-NH4) treated with extracts or functionalized extracts + The content of ammonium nitrogen (i.e., the mass of nitrogen in the form of ammonium) was higher than that found in control soils. After 12 days of incubation, an increase of +158% was observed in the presence of the extract and an increase of +250% in the presence of the functionalized extract, reflecting a decrease in nitrification activity, also known as nitrification inhibition.

[0115] Example 3: Measurement of nitrification inhibition by measuring nitrate levels in soil Preparation of soil containing microalgae extract (extract or functionalized extract) and soil not containing microalgae extract (control). The bottle was prepared under the same conditions as described in Example 2.

[0116] Nitrification kinetics associated with the presence of nitrate in the soil were established by measuring nitrate on days 2, 6, 9, and 12.

[0117] Extraction and determination of nitrates in soil Extraction was performed as follows: 15 mL of purified water was added to each vial, and the vials were shaken for one hour. The vials were then allowed to stand for 10 minutes. The supernatant was collected, centrifuged at 11,000 rpm for 5 minutes at 4°C, and then filtered through a 0.25 μm filter to remove any particles.

[0118] Nitrate levels were determined using the Spectroquant Nitrate Assay Kit (Supelco). After dilution of the filtrate, 5 ml was used for measurement. The optical density (OD) of the sample was measured at 692 nm using a spectrophotometer, and the nitrate nitrogen content (N-NO3) was estimated based on the calibration curve. - ), which is the mass of nitrogen present in nitrate.

[0119] For each incubation condition (control, extract, and functionalized extract), four batches of soil were prepared (1 batch = 1 biological replicate).

[0120] For each biological replicate, i.e., each of the four replicates, all treatments were performed systematically. The data obtained are presented as mean values, and the variability of the results is given as the standard error of the mean with n=4.

[0121] The results of nitrate ion quantification are shown in Figure 2 middle.

[0122] Conclusion: Nitrate nitrogen (N-NO3) in soil treated with extracts or functionalized extracts - The nitrate nitrogen content was lower than that found in the control soil. After 12 days of incubation, a reduction of -31% was observed in the presence of the extract and a reduction of -59% in the presence of the functionalized extract, reflecting a decrease in nitrification activity, also known as nitrification inhibition.

[0123] Example 4: Determination of nitrous oxide (N2O) in soil Preparation of soil containing microalgae extract (functionalized extract) and soil without microalgae extract (control). 4.4 g of dry soil (maintained at 15% moisture content) sieved through a 1 mm sieve was placed in a 37 ml glass bottle, and 0.6 ml of 0.4 mg N-NH4 containing ammonium sulfate was added to the bottle. + .g -1 Sol in water. The microalgae extract prepared according to Example 1 was administered at a dose of 25 mg / bottle.

[0124] The bottles were then sealed and incubated at 20°C for 216 hours. During this period, nitrification kinetics associated with the appearance of N2O were established by measuring N2O at 0, 48, 120, 168, and 216 hours.

[0125] Nitrification kinetics were evaluated under the same conditions as before, on the same soil (used as a control) without any microalgae extracts.

[0126] Sampling and measurement of N2O gas Gas was extracted from the gas environment of the bottle using a syringe and measured on a miniature GC (Agilent 990) equipped with a TCD detector and a Porapak Q column.

[0127] For each incubation condition (control and functionalized extract), three batches of soil were prepared (1 batch = 1 biological replicate).

[0128] For each biological replicate, i.e., each of three replicates, all treatments were performed systematically. The data obtained are presented as mean, and the variability of the results is given as the standard deviation of the mean over n=3.

[0129] The quantitative representation of N2O is shown in Figure 3 middle.

[0130] Conclusion: The nitrous oxide (N2O, expressed here as peak area) content in soil treated with the functionalized extract was lower than that found in the control soil. After 216 hours of incubation, a reduction of -50% was observed in the presence of the functionalized extract.

[0131] Example 5: Measurement of the inhibition of denitrification process by measuring the activity of nitrate reductase Place 10 mM phosphate buffer (pH 7.5), nitrate reductase (Sigma-Aldrich, N0163), and substrate buffer (pH 7.5 containing 37.5 mM phosphate buffer, 15 mM potassium nitrate, 0.075 mM EDTA, and 0.15 mM NADH) in a 96-well round-bottom plate. Add 0.8 ml of water containing 50 mg / vial of treatment agent (extract, functionalized extract, or oil extract from Example 1) or no treatment agent (control). Incubate the plate at 30°C in the dark with shaking for 40 minutes. Then add 58 mM sulfanilamide (Sigma-Aldrich, S9251) and freshly prepared N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma-Aldrich, 222488). Incubate the plate again at room temperature in the dark for 20 minutes for complete color development. The absorbance was then measured at 540 nm using a UV spectrophotometer (ClarioStar Plus, BMG Labtech).

[0132] The results are shown in Figure 4 , Figure 5 and Figure 6 middle.

[0133] Conclusion: Enzymes treated with extracts or functionalized extracts of *Haloxylon ammodendron*, *Micrococcus serrata*, or *Micrococcus marineus* showed greater inhibition of nitrate reductase activity than those observed in the control solution. A 24% inhibition of denitrification was observed in the presence of the extracts, while a 99% inhibition was observed in the presence of functionalized extracts of *Micrococcus ammodendron*, *Micrococcus serrata*, and *Micrococcus marineus*, reflecting the inhibition of denitrification activity. Figure 4 ).

[0134] The enzymes treated with extracts or functionalized extracts of *Spirulina platensis* or *Chlorella sorokinense* showed greater inhibition of nitrate reductase activity than those observed in the control solution. An inhibition of denitrification was observed at 59.6% in the presence of functionalized *Spirulina* extract and at 49.9% in the presence of functionalized *Chlorella* extract, reflecting the inhibition of denitrification activity. Figure 5 ).

[0135] The enzymes treated with oil or oil extracts of *Chlorella sorokinense* (Chlorella vulgaris) or *Microcystis thunbergii* showed greater inhibition of nitrate reductase activity than those observed in the control solution. An inhibition of denitrification was observed at 19.6% in the presence of rapeseed oil and at 36.3% in the presence of *Chlorella vulgaris* extract from rapeseed oil, reflecting inhibition of denitrification activity. An inhibition of denitrification was observed at 27.2% in the presence of soybean oil, at 36.8% in the presence of *Chlorella vulgaris* extract from soybean oil, and at 37.9% in the presence of *Microcystis thunbergii* extract from soybean oil, reflecting inhibition of denitrification activity. Figure 6 ).

Claims

1. A method for fertilizing soil containing microorganisms, the method comprising the step of adding at least one eukaryotic microalgae extract to the soil.

2. The method for fertilizing soil according to claim 1, characterized in that, Microalgae were selected from the genera *Amphora* and *Arthrospira*. Arthrospira sp. ), Chaetoceros genus ( Chaetoceros sp. Chlamydomonas ( ) Chlamydomonas sp. Chlorella ( ) Chlorella sp. ), Dunaliella genus ( Dunaliella sp. Euglena ( ) Euglena sp. ), genus *Briarella* ( Fragilaria sp. ), genus Haematococcus ( Haematococcus sp. Isochrysis ( ) Isochrysis sp. ), Tisochrysis sp. Micrococcus ( Nannochloris sp. ), Micrococcus ( Nannochloropsis sp. ), Nitzchi sp. , Odonthella sp. genus *Porphyra* ( Porphyridium sp. ), genus *Phaeodactylum* ( Phaeodacylum sp. ), *Scenedesmus* ( Scenedesmus sp. ), Schyzotrium sp. genus *Skeletonema* Skeletonema sp. ), genus *Williams* ( Thalassosira sp. ) and the genus *Chlorophyta* ( Tetraselmis sp. ).

3. The method for fertilizing soil according to claim 1 or 2, characterized in that, The addition of microalgae extracts inhibits the activity of microorganisms.

4. The method for fertilizing soil according to any one of claims 1 to 3, characterized in that, The added step induces inhibition of at least one step of the nitrogen cycle in the soil as described below, the step being selected from nitrification, denitrification, and nitrification-denitrification processes.

5. The method for fertilizing soil according to claim 4, characterized in that, Inhibition of nitrification has at least one of the following effects: reducing fertilizer loss in the soil, reducing nitrate leaching in the soil, increasing the amount of ammonium available in the soil, reducing nitrous oxide emissions, and increasing the amount of nitrogen that can be absorbed by plants grown in the soil.

6. The method for fertilizing soil according to claim 4, characterized in that, Inhibition of the denitrification process has at least one effect selected from the following: inhibiting nitrate reduction, inhibiting nitrate reductase activity, and reducing nitrous oxide emissions.

7. The method for fertilizing soil according to any one of the preceding claims, characterized in that, The extract is obtained by an extraction process carried out in a liquid medium that may contain water or oil, such as a vegetable oil.

8. The method for fertilizing soil according to claim 7, characterized in that, The liquid medium contains water and at least one enzyme, which can be selected from carbohydrate enzymes and pectinases.

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

10. The method for agricultural fertilization of soil according to the preceding claim, characterized in that, Agricultural products or soil contain nitrogen fertilizer.

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