Use of a microalgae extract to inhibit nitrification, nitrification-denitrification and / or nitrate denitrification processes in soil

Abstract

Use of microalgae extracts to inhibit nitrification or denitrification in soil The present invention finds application in the agricultural field and relates to the use of microalgae extracts to control nitrification in soils and reduce losses of gaseous nitrogen in the form of N2O. The invention also relates to a product for agricultural use comprising a microalgae extract and a method for fertilizing a soil. Figure for abstract: Fig. 1.

Description

Title of the invention: Use of a microalgae extract to inhibit the processes of nitrification, nitrification-denitrification and / or denitrification of nitrates in a soil Technical field

[0001] The invention relates to the use of microalgae extracts for inhibiting the nitrification process and / or the nitrification-denitrification process in soils 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 (NO3) 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 makes it possible to develop a new additive making it possible to control both the nitrogen supply to the soil and the supply of fertilizers to the plant, but also to limit emissions of the greenhouse gas N2O. Prior art

[0003] Nitrogen fertilization plays an essential role in crop growth and yield. Nitrogen is the characteristic constituent of amino acids and proteins, so it is a very important growth and quality factor. As it is subject to 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, assimilation, 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 of all the bacteria and archaea of ​​the Nitrosomas group, AOB (Ammonia-Oxidising Bacteria) and AOA (Ammonia-Oxidising archaea) allow to oxidize ammonium into hydroxylamine (NH20H) then into nitrite (NO2 ). Finally it is the Nitrite-Oxidising Bacteria (NOB), Nitrobacter and Nitrospira, which allow to oxidize nitrites into nitrate. In order to transform ammonium into nitrites, four protons are released as well as two water molecules, this phenomenon causes an acidification of the environment, in particular around the roots. The activity of the bacteria, AOB and AOA depends strongly from the environment, so that humidity, pH, temperature and ammonium availability have a great influence on the process. Finally, nitrous oxide (N2O) emissions caused by nitrification can be observed during the oxidation of ammonium to nitrite.

[0006] Nitrification is an important biological step in the nitrogen cycle in the soil. From an agronomic point of view, its operation is considered a limiting step in the supply of nitrogen by soils. Too slow or too fast compared to the needs of plants, it can cause low nitrogen use efficiency and can contribute to groundwater pollution and greenhouse gas emissions (N2O). 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 assimilation by plants leads to a loss of nitrogen applied to the soil via nitrate leaching and nitrous oxide (N2O) emissions. These losses can affect one-fifth of the applied nitrogen. This leads to an increase in the cost of agricultural production, environmental pollution (surface water and deep water), human and animal diseases, degradation of the ozone layer 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 based on the transformation of nitrates resulting from nitrification into nitrogen. This second biological mechanism constituting denitrification corresponds to the reduction of nitrates (NO3) into gaseous nitrogen (N2) by bacteria in an anoxic situation. Denitrifying bacteria reduce the nitrate ion (NO3) successively into nitrite ion (NO2), then into nitric oxide (NO), into nitrous oxide (N2O), and finally into nitrogen (N2).

[0009] Denitrification therefore constitutes another source of emission of the greenhouse gas N2O by bacterial use of nitrates, which competes with the use of nitrates by plants.

[0010] There is also a nitrification-denitrification process carried out by nitrifying organisms 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.

[0011] For non-nitrogen-fixing plants, nitrogen is absorbed by plants mainly in nitric (NO3) or ammoniacal (NH4+) form. Depending on the plant's needs, the latter is capable of absorbing significant quantities to support its growth and development. Furthermore, a lack of nitrogen caused by a deficient supply from the soil or by losses in the environment, via ammoniacal volatilization, nitrate leaching or denitrification, can cause nitrogen deficiencies and have a negative impact on plant growth.

[0012] 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 by fertilization can be reduced, which can present a financial gain for farmers, help reduce nitrogen losses and reduce risks to the environment.

[0013] In order for the plant to use the nitrogen present in the fertilizing compositions more efficiently and to control the nitrogen transformation process in the soil, formulations comprising nitrification inhibitors are of interest to develop with the aim of controlling the nitrate supply according to the needs of the plant and optimizing its use by the crops. This allows better utilization of nitrogen by the plant, while reducing the negative consequences for the environment.

[0014] 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 present a danger to the environment and soil life.

[0015] 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 enzyme Nitrate reductase.

[0016] 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 envisage reducing nitrate losses by leaching. Statement of the invention

[0017] The use of microalgae extracts in agriculture to control one or more processes of the nitrogen cycle constitutes an innovative approach. This is the main agronomic challenge of the invention.

[0018] Thus, a first subject of the present invention relates to the use of an aqueous extract of at least one eukaryotic unicellular 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.

[0019] A second subject of the present invention relates to an agricultural fertilization method comprising a step of providing a plant with an aqueous extract of at least one microalgae, said extract being able to be applied to a soil provided by a culture medium or provided by a culture solution.

[0020] A third subject of the present invention relates to a product for agricultural use comprising an aqueous extract of a microalgae and at least one soil nutrient ingredient or a plant nutrient ingredient chosen in particular from a fertilizer, an amendment, a biostimulant and a micronutrient.

[0021] Finally, a fourth subject of the present invention relates to a method for preparing an aqueous extract of a microalgae comprising an extraction step by introducing the microalgae 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

[0022] [Fig.l] [Fig.l] represents 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 decrease from 0.4 to 0.12 mg N-NH / .g 1 Soil. Ammonium levels increase from 0.4 to 0.31 mg N-NH / .g 1 Soil in the soil that includes the microalgae extract, and from 0.4 to 0.42 mg N-NH4+.g 1 Soil in the soil that includes the functionalized microalgae extract, indicating an inhibition of the nitrification process in the soil.

[0023] [Fig.2] [Fig.2] represents the evolution of nitrate levels (NO3 ; mg N-NO3 .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 increase in nitrate levels between 0 and 12 days of incubation in the soil that does not does not include extract (Control): these contents increase from 0 to 0.22 mg N- NO3 .g 1 Soil in the soil. Ammonium contents increase from 0 to 0.15 mg N- NO3 .g 1 Soil in the soil that includes the microalgae extract, and from 0 to 0.09 mg N- NO3 .g 1 Soil in the soil that includes the functionalized microalgae extract, indicating an inhibition of the nitrification process in the soil.

[0024] [Fig.3] [Fig.3] represents the evolution of nitrous oxide (N2O) contents; 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 contents 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 the soil that includes the functionalized microalgae extract, indicating an inhibition of N2O production thanks to the functionalized microalgae extract.

[0025] [Fig.4] [Fig.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). Description of the embodiments

[0026] A first subject of the invention relates to the use of an aqueous extract of at least one eukaryotic unicellular 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 denitrification process and a nitrification-denitrification process.

[0027] Microalgae are understood to mean unicellular microorganisms which have the capacity to carry out photosynthesis.

[0028] The term "nitrification process" means all the biochemical reactions leading to the formation of nitrates from ammonium which are induced by nitrifying bacteria such as ammonia-oxidizing bacteria (AOB) and / or by nitrite-oxidizing bacteria (NOB), for example Nitrosomonas spp., Nitrosospira spp. and Nitrosococcus spp. Nitrosomonas spp., Nitrosospira spp. and Nitrosococcus spp., for example chosen from Nitrosomonas europaea, Nitrosomonas eutropha, Nitrosomonas communais, Nitrosospira multiformise and Nitrosospira lacus.

[0029] The term "nitrification process inhibitor", also called nitrification inhibitor, designates a product whose activity slows down one of the biochemical reactions of the nitrification process. A nitrification inhibitor advantageously makes it possible 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.

[0030] 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 assimilable by a plant grown on the soil.

[0031] An aqueous extract of microalgae targeted by the present invention may be a nitrification inhibitor within the meaning of the invention. In a particular embodiment of the invention, an aqueous extract of microalgae 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 an 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 an aqueous extract of microalgae are detailed in the examples.The decrease in the quantity 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 of, for example, at least 2 days after application of the extract to the soil, preferably at least 6 days or 10 days.

[0032] The term "denitrification process" refers to the reduction of nitrates (NO3) to nitrogen gas (N2) by bacteria in an anoxic situation. More precisely, denitrifying bacteria successively reduce the nitrate ion (NO3) to nitrite ion (NO2) by the action of the enzyme N2O reductase, to nitric oxide (NO), to nitrous oxide (N2O), and then to nitrogen (N2).

[0033] 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 reduction 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.

[0034] 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 nitrate reductase enzyme, and reduction of nitrous oxide emission.

[0035] The term "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 organisms and denitrifying organisms, in which the nitrates produced by the nitrifying organisms are immediately denitrified by the denitrifying organisms.

[0036] The term "nitrification-denitrification process inhibitor" designates the activity of limiting the nitrification-denitrification process which can result in an inhibition of the production of N2O. Also, the inhibition of a nitrification-denitrification process can have the effect of reducing the production of nitrous oxide. The reduction in the production of nitrous oxide can be greater than or equal to a value chosen from 5%, 10%, 20%, 30%, 40%, 50%, after a duration for example of at least 100 hours, 150 hours or 200 hours.

[0037] In one embodiment of the invention, an aqueous extract of microalgae is a nitrification-denitrification inhibitor when it reduces by at least 5% the quantity of nitrous oxide in a soil amended with microalgae extract in comparison with the same unamended soil under nitrification conditions (aerated medium).

[0038] The term "microalgae extract" refers to a product resulting from a process carried out by man to isolate a part of a microalga. The microalga is preferably a eukaryotic unicellular microalga delimited by a membrane which contains organelles, including a cytoplasm and a nucleus.

[0039] The term "aqueous microalgae extract" refers to a product resulting from the extraction of the cellular contents of a microalgae with a liquid containing water. An aqueous extract does not necessarily contain water, since 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 microalgae. The aqueous extract is therefore likely to be obtained by extraction of the molecules contained in the microalgae, with a liquid medium containing water.

[0040] The aqueous extract of at least one eukaryotic unicellular 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 chosen from carbohydrases and pectinases to the liquid medium.

[0041] The microalgae which can be used in the context of the present invention is advantageously chosen from the group consisting of Nannochloropsis sp., Amphora sp., Chaetoceros sp., Chlamydomonas sp., Chlorella sp., Dunaliella sp., Euglena sp., Fragilaria sp., Haematococcus sp., Isochrysis sp. lisochrysis sp., Nannochloris sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacylum sp., Scenedesmus sp., Schyzotrium sp., Skeletonema sp., Thalassosira sp. and Tetraselmis sp.

[0042] Agricultural fertilization process

[0043] The invention, in its second subject, relates to an agricultural fertilization method comprising a step of providing a plant with 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., Euglena sp., Fragilaria sp., Haematococcus sp., Isochrysis sp. Tisochrysis sp., Nannochloris sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacylum sp., Scenedesmus sp., Schyzotrium sp., Skeletonema sp., Thalassosira sp. and Tetraselmis sp., said extract being able to be applied to a soil provided by a culture medium or provided by a culture solution.

[0044] The term “soil” designates the substrate explored by the roots of cultivated plants. The term soil includes any type of substrate on which a plant can grow, such as, for example, artificial soil, cultivated agricultural soil, uncultivated agricultural soil, peat, potting soil, rock wool or coconut fiber. The term soil therefore also covers growing media which can in particular be used for so-called “soil-less” cultivation, for example in pots. The soil may have an acidic or basic pH. In a particular embodiment, the soil has a pH greater than or equal to a value chosen from 6, 7, or 8.

[0045] For the purposes of the present invention, the term “growing medium” means any soil or medium on which a crop can grow, such as, for example, artificial soil, cultivated agricultural soil, uncultivated agricultural soil, peat, compost, rock wool or coconut fiber.

[0046] For the purposes of the present invention, the term “culture solution” means any solution which allows the cultivation of a plant such as, for example, irrigation water, solution liquid intended for fertigation by sprinkling or dripping, hydroponic bath or aeroponic solution.

[0047] The aqueous microalgae extract which is the subject of the use of the invention, or the agricultural product of the invention, can be applied to a soil or to a plant by the root route, in the open field or above ground. They can be applied to the surface of the soil or mixed with the soil, for example with the first layers of the soil. The application can be carried out directly on the soil, over the entire surface of the soil or locally at the level of the roots of plants, by any suitable distribution means.

[0048] The application can be carried out before and / or during the growth of the cultivated plants, before and / or after the germination of the plants, or during transplanting in the case of plants requiring transplanting.

[0049] The fertilization method of the invention may comprise a step of applying the aqueous microalgae extract, and a step of applying a fertilizer, for example a solid or liquid fertilizer, an amendment, and / or a biostimulant. This may be, for example, a nitrogen fertilizer, the two steps being simultaneous or sequential. 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.

[0050] In a particular embodiment of the invention, the agricultural fertilization method further comprises a step of applying at least one nitrogen fertilizer to the soil or to the plant grown on the soil, said application step being prior to, concomitant with or subsequent to the step of supplying the aqueous extract. The nitrogen fertilizer may be a nitrogen fertilizer comprising an ammoniacal form of nitrogen, a nitrogen fertilizer releasing ammonium into the soil, or a nitrogen fertilizer comprising nitrate.

[0051] Product for agricultural use

[0052] The third subject of the invention is a product for agricultural use comprising 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., Euglena sp., Fragilaria sp., Haematococcus sp., Isochrysis sp. Tisochrysis sp., Nannochloris sp., Nitzchi sp., Odonthella sp., Porphyridium sp., Phaeodacylum sp., Scenedesmus sp., Schyzotrium sp., Skeletonema sp., Thalassosira sp. and Tetraselmis sp., and 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.

[0053] 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 the quality of soils, and particularly intended to improve the pH of soils. 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.

[0054] The fertilizer is advantageously a simple, binary or ternary solid fertilizer, organo-mineral or organic fertilizer, liquid or a water-soluble fertilizer.

[0055] 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.

[0056] In particular embodiments, the nitrogen fertilizer contains ammonium, nitrate, ammonia, and / or urea. 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).

[0057] The amendment may be an organic amendment or a mineral amendment, such as calcium carbonate.

[0058] Micronutrients such as molybdenum, zinc, boron and copper are preferably provided as salts soluble in water or organic acids.

[0059] A biostimulant may be, for example, a liquid root or foliar biostimulant

[0060] 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.

[0061] The agricultural product of the invention may be in the form of a fertilizer, a growing medium or a growing solution.

[0062] In particular embodiments, the agricultural product according to the invention is a culture solution such as irrigation water, a solution intended for fertigation by sprinkling or drip irrigation, a hydroponic bath or an aeroponic solution.

[0063] The agricultural product according to the invention may be in solid form, in particular in the form of powder, granules or microgranules, in the form of a liquid suspension or in the form of a gel or in water-soluble form.

[0064] The expression "plant" is intended to designate in the present application the plant considered as a whole, including its root system, its vegetative system, the seeds, grains and fruits.

[0065] The agricultural method or use according to the invention finds application in the treatment of soil in which a very large variety of plants can be planted. These include in particular 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 market gardening (lettuce, spinach, onion, shallot, tomato, melon), vines, arboriculture (pears, apples, nectarines), or horticulture.

[0066] In particular, the plant may belong to the order of monocotyledons, preferably to the family of Poaceae. Poaceae, commonly called grasses, include in particular most of the species commonly called "grasses" and "cereals". Among the Poaceae, mention may be made of wheat, rice, barley, oats, rye, sugarcane, meadow and corn.

[0067] Process for preparing the extract

[0068] The fourth subject of the invention relates to a process for preparing an aqueous extract of at least one microalgae selected from the group consisting of Nannochloropsis sp., Amphora sp., Chaetoceros sp., Chlamydomonas sp., Chlorella sp., Dunaliella sp., Euglena sp., Fragilaria sp., Haematococcus sp., Isochrysis sp.Tisochrysis sp., Nannochloris sp., Nitzchi sp., Odonthella sp., Porphyridium sp; Phaeodacylum sp., Scenedesmus sp., Schyzotrium sp., Skeletonema sp., Thalassosira sp. and Tetraselmis sp., said process 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.

[0069] The extraction of the aqueous extract of the microalgae can 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 can be carried out at a temperature ranging from 4°C to 300°C, preferably at a temperature ranging from 19°C to 25°C.

[0070] The liquid extraction medium comprising water preferably comprises distilled water and optionally an aliphatic alcohol, for example ethanol or a glycol.

[0071] After the extraction step, the solids may be removed by centrifugation or filtration, to recover a filtrate, which may be dried, for example by lyophilization or evaporation.

[0072] In a particular embodiment, the invention relates to a method for preparing an extract of the microalga Nannochloropsis sp., said method comprising a step of extracting Nannochloropsis sp. with distilled water, followed by a filtration step to recover the filtrate containing the aqueous extract and remove the solid residues of the microalgae. The method may comprise a step of concentrating the filtrate to dryness by freeze-drying or by evaporation at 20°C to 80°C. In this method, Nannochloropsis sp. may be Nannochloropsis salina, Nannochloropsis oculata or Nannochloropsis oceanica.

[0073] In a more particular embodiment, the method comprises a step of extracting Nannochloropsis sp. in a liquid medium consisting of distilled water at a temperature of 4°C to 300°C, for a period ranging from 30 minutes to 24 hours.

[0074] The extraction step may comprise the addition to the liquid extraction medium of at least one enzyme allowing 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.

[0075] The invention is 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.

[0076] Example 1: Preparation of microalgae extracts First excerpt#:

[0077] Distilled water and a microalgae Nannochloropsis salina, Nannochloropsis oculata or Nannochloropsis oceanica cultivated in phototrophy are introduced into a glass reactor at a mass concentration of 2.5%. After stirring (for 4 hours) and decantation, the filtrate is recovered and constitutes the “Extract”. Second extract#:

[0078] Water and the microalgae Nannochloropsis sp. 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 recovered and constitutes the “Functionalized Extract”.

[0079] Example 2: Measurement of nitrification inhibition by the determination of ammonium in a soil

[0080] Preparation of a soil which comprises a microalgae extract (Extract or Functionalized Extract) and of a soil which does not comprise a microalgae extract (Control) 5 g of dry soil (the characteristics of which are detailed in Table 1) sieved with a sieve with a mesh diameter of 2 mm, are 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) are added.

[0081] Table 1: Main soil characteristics Texture Silty clay PH 8.1 Organic matter (%) 1.9 Cation exchange capacity (meq / Kg) 187

[0082] After 24 hours of incubation, 0.4 mg of N-NH / .g 1 Sol, in the form of ammonium sulfate, are added to 1.8 ml of water. This volume allows 80% of the field capacity of the soil studied to be reached.

[0083] The bottles 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 carrying out ammonium measurements at 2; 6; 9 and 12 days.

[0084] Extraction and determination of ammonium from soil Extraction is performed by adding 15 mL of 1M KCl solution 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 qm filter to remove any particles.

[0085] The ammonium assay is carried out using the Ammonium test-Spectroquant kit (Supelco). After dilution of the filtrate, 5 ml are used to carry out 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.

[0086] For each of the incubation conditions (Control, Extract and Functionalized Extract), four batches of soil were created (1 batch = 1 biological repetition).

[0087] All treatments were carried out systematically for each of the biological repetitions, 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.

[0088] The ammonium dosage is shown in [Fig.l].

[0089] Conclusion: Soils treated with the Extract or Functionalized Extract have ammoniacal nitrogen (N-NH4+) contents, i.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.

[0090] Example 3: Measurement of nitrification inhibition by measuring nitrates in soil

[0091] Preparation of a soil which comprises a microalgae extract (Extract or Functionalized Extract) and a soil which does not comprise a microalgae extract (Control)

[0092] Flasks were prepared under the same conditions as those described in Example 2.

[0093] 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.

[0094] The nitrate assay is carried out using the Nitrate test-Spectroquant kit (Supelco). After dilution of the filtrate, 5 ml are used to carry out the assay. 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.

[0095] For each of the incubation conditions (Control, Extract and Functionalized Extract), four batches of soil were created (1 batch = 1 biological repetition).

[0096] All treatments were carried out systematically for each of the biological repetitions, 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.

[0097] The result of the nitrate ion dosage is shown in [Fig.2].

[0098] Conclusion: Soils treated with the Extract or the 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 Functionalized Extract is observed after 12 days of incubation, reflecting a decrease in nitrifying activity also called inhibition of nitrification.

[0099] Example 4: Determination of Nitrous Oxide (N 2 O) in soil

[0100] Preparation of a soil which comprises a microalgae extract (Functionalized extract) and a soil which does not comprise a microalgae 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 1 Soil, in the form of ammonium sulfate, are added. The microalgae extract prepared according to Example 1 was applied at a dose of 25 mg / bottle.

[0101] The flasks are then hermetically sealed and incubated at a temperature of 20°C for a period of up to 216 hours. During this period, the nitrification kinetics associated with the appearance of N2O are established by carrying out N2O measurements at 0; 48; 120; 168 and 216 hours.

[0102] The nitrification kinetics were also evaluated under the same conditions as previously on the same soil not including any microalgae extract used as a control.

[0103] Sampling and measurement of N 2 O 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).

[0104] 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.

[0105] The dosage of N2O is presented in [Fig.3].

[0106] 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.

[0107] Example 5: Measurement of the inhibition of the denitrification process by measuring the activity of the enzyme Nitrate Reductase

[0108] 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 or Functionalized Extract of Example 1) at a dose of 50 mg / Bottle 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). The results are shown in [Fig.4].

[0109] Conclusion: The enzyme treated with the Extract or the Functionalized Extract 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 is observed, reflecting an inhibition of the denitrifying activity.

Claims

Claims

1. Use of an aqueous extract of at least one eukaryotic unicellular microalga for inducing 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 microalga being chosen from the group consisting of Nannochloropsis sp. and Chlorella sp.

2. Use according to claim 1, characterized in that the inhibition of a nitrification process is 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 amount of ammonium available in the soil, an improvement in the growth of a plant grown on the soil, a reduction in nitrous oxide emissions, and an increase in the amount of nitrogen assimilable by a plant grown on the soil.

3. Use according to claims 1 and 2, characterized in that the inhibition of a denitrification process is 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.

4. Use according to claim 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.

5. Use according to the preceding claim, characterized in that the aqueous extract is functionalized by adding at least one enzyme chosen from carbohydrases and pectinases in the liquid medium.

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