WO2023175548A1

WO2023175548A1 - Nitrogen-containing fertilizer and method for the production thereof

Info

Publication number: WO2023175548A1
Application number: PCT/IB2023/052561
Authority: WO
Inventors: Alberto Bertucco; Eleonora SFORZA
Original assignee: Materias S.R.L.
Priority date: 2022-03-17
Filing date: 2023-03-16
Publication date: 2023-09-21
Also published as: IT202200005198A1

Abstract

A method for producing a nitrogen-containing fertilizer is described, comprising (i) a step of cultivating one or more nitrogen-fixing cyanobacteria of the genus Anabaena, (ii) a step of separating the Anabaena cell biomass obtained in the cultivation step from the liquid culture medium, and (iii) a step of subjecting the cells of said biomass to mechanical disruption, preferably by sonication. The cultivation step is carried out in continuous mode in a closed photobioreactor, preferably in a flat-plate photobioreactor. The fertilizer that can be obtained by means of the method of the invention is also described, which is particularly effective in nourishing and promoting the growth of plants as well as improving the fertility of the cultivation soil.

Description

Nitrogen-containing fertilizer and method for the production thereof

The present invention relates to a method for producing a natural nitrogen-containing fertilizer from nitrogen-fixing cyanobacteria of the genus Anabaena, and the biofertilizer that can be obtained by this method.

Recent statistics on the future development of the world’s population agree that by 2050 the inhabitants of the Earth will be about 30% more. As a result, the demand for food is expected to increase by approximately 70%. It will therefore be necessary to ensure a substantial increase in agricultural production, while respecting the constraints on water consumption and greenhouse gas emissions. Fertilizers are the main means available to achieve this goal. The plant’s most requested nutrient is nitrogen, which is very abundant in the earth’s atmosphere as N2, but not usable in its molecular form by most plants. Fertilizers containing nitrogen (mainly urea and ammonium nitrate) are currently obtained starting from ammonia which, in turn, is produced through Haber-Bosch-type processes starting from N2 and H2. These processes, which are the conventional method for chemical nitrogen fixation, have some disadvantages, including high energy requirements, the emission of large quantities of carbon dioxide, and the exploitation of non-renewable sources for the production of syngas from which hydrogen is obtained.

In recent years, the need to develop more sustainable agriculture has prompted the scientific world to look for new alternatives for the fixation of atmospheric nitrogen. Among the various possibilities, biological nitrogen fixation by means of different microorganisms, such as bacteria and unicellular algae, appeared to be of particular interest, since it takes place at room temperature and atmospheric pressure. However, the use of algae as fertilizers has significant limitations due to the reduced productivity of these microorganisms with conventional cultivation methods and the consequent need to supplement fertilizing formulations containing these microorganisms with additional nitrogen sources, such as wastewater or ammonia. CN105859445 discloses a fertilizer formulation comprising various ingredients including lignosulfonates, humic acid, ammonium phosphate, urea, algal amino acid trace elements, plant ash, composite bacillus, and gibberellins.

CN104119111 discloses a method for manufacturing an organic fertilizer using blue algae, reeds, and rice straw. More specifically, the method disclosed therein comprises a first step of grinding the aforementioned materials, followed by a step of wet fermentation, mixing, dry anaerobic fermentation, granulation of biogas residues and drying.

CN106699465 concerns a fertilizing formulation including brown algae, mature coal or lignite, nitrogen, phosphorus and potassium.

In the field of fertilizers, the potential of algae when applied as consortia with other organisms/biomass was also investigated. In this regard, Kholssi R., et al, “A consortium of cyanobacteria and plant growth promoting rhizobacteria for wheat growth improvement in a hydroponic system”; (2021) South African Journal of Botany, Vol. 142, p. 247-258 describe the combined use of a microbial consortium consisting of plant growth promoting rhizobacteria (PGPR) and cyanobacteria, as a biofertilizer.

The research described in Bhuvaneshwari, K, and Pawan Kumar Singh. “Response of nitrogen-fixing water fern Azolla biofertilization to rice crop.” (2015) 3 Biotech vol.5, 4: 523-529. doi:10.1007/sl3205-014-0251-8, demonstrates the beneficial effects of the combined administration of fresh-water fem Azolla, living in symbiosis with the cyanobacterium Anabaena azollae, and chemical nitrogenous fertilizers on rice crops.

Other lines of research have instead focused on the use of algae together with other matrices as unconventional fertilizers on various types of crops. For example, the publication of Renuka, N., et al. “Exploring the efficacy of wastewater-grown microalgal biomass as a biofertilizer for wheat” (2016) Environ Sci Pollut Res 23, 6608-6620, describes fertilizer formulations containing consortia of unicellular and/or filamentous microalgae from wastewater, in combination with the mineral vermiculite, while the Article by Atzori, G., et al. “Algae and Bioguano as promising source of organic fertilizers” (2020) Journal of Applied Phycology, 32 (6), pp. 3971-3981, shows the fertilizing action of a mixture of macroalgae, spirulin and guano, known as bioguano, on barley seedlings.

CN 111560319 describes the use of a mutant strain of the cyanobacterium Anabaena as a soil amender for the removal of metals, in particular cadmium. In the experimental examples set out in this patent application, the algal biomass is applied to the soil after treatment with a fertilizer containing chemically synthesized compounds, including urea.

US2021179507 relates to a method for producing ammonia which comprises cultivating in a bioreactor, in the presence of an inhibitor, a cyanobacterium genetically modified to secrete ammonium, which is then extracted and processed into nitrate for use as a nitrogenous fertilizer.

US Patent 5,797,976 describes fertilizing compositions that may also include, i.a., a mixture of cultures of different microorganisms, including cyanobacteria of the genera Gloeocapsa and Anabaena.

One object of the present invention is to provide a method that allows large-scale, high-yield production of a nitrogen-containing fertilizer, with a limited number of steps starting from naturally occurring microorganisms.

Another object of the invention is to provide a method for obtaining a nitrogenous fertilizer which is effective in supplying nourishment to plants and promoting the growth thereof, as well as enhancing the fertility of the cultivation soil, without the need for further use of other substances, in particular chemically synthesized fertilizers, thereby allowing proper management of the soil and the environment.

In consideration of these objects, one aspect of the invention is a method for producing a nitrogen-containing fertilizer, comprising the steps of:

(i) cultivating in continuous mode one or more nitrogen-fixing cyanobacteria of the genus Anabaena in a liquid culture medium in a closed photobioreactor, thereby obtaining a cell biomass of nitrogen-fixing cyanobacteria of the genus Anabaena in suspension in said liquid culture medium, wherein said liquid culture medium comprises potassium dihydrogen phosphate (KH2PO4) and one or more trace elements selected from the group consisting of iron, magnesium, manganese, zinc, copper, cobalt, and any combination thereof, and does not comprise nitrogen; wherein in said cultivating step, the one or more nitrogen-fixing cyanobacteria of the genus Anabaena are kept at a temperature comprised between 23 °C and 25 °C for a residence time of at least 15 hours and are exposed to a light radiation of a wavelength of from 350 nm to 750 nm and an intensity of from 450 to 600 micromoles of photons per square metre per second (pmol m'² s'¹);

(ii) separating the cell biomass of nitrogen-fixing cyanobacteria of the genus Anabaena obtained in step (i) from the liquid culture medium; and

(iii) subjecting the cells of said biomass of nitrogen-fixing cyanobacteria of the genus Anabaena to mechanical disruption, thereby obtaining a nitrogen-containing fertilizer.

The appended independent and dependent claims form an integral part of the present specification.

The method according to the invention is based on a step of intensive cultivation of nitrogenfixing cyanobacteria belonging to the genus Anabaena. In the context of the present invention, Anabaena cylindrica, Anabaena flos aquae and Anabaena sp. Nostoc are the most preferred species.

Microorganisms belonging to the phylum Cyanobacteria are photoautotrophic prokaryotes capable of colonizing extreme terrestrial and marine environments, which, in order to grow, require a light source (artificial, or preferably natural), inorganic carbon (CO2), water, and a minimum supply of nutrients. Among these, cyanobacteria of the genus Anabaena are able to fix atmospheric molecular nitrogen and reduce it to ammonia using the nitrogenase enzyme complex.

In the method of the invention, the step of cultivating the cyanobacteria Anabaena is carried out in a photobioreactor. The term “continuous mode” refers to a cultivation method in which the reactor is continuously fed with fresh culture medium, while the spent medium and biomass are removed from the reactor at the same flow rate as the input feed.

Within the scope of the present description, the term “closed photobioreactor” refers to a bioreactor in which the microbial culture does not come into contact with the atmosphere or other types of contaminants such as, for example, dust or other microorganisms.

In a preferred embodiment, the photobioreactor used in the method according to the invention is a flat-plate photobioreactor. As is known in the art, in this type of photobioreactor, the union of two panels, suitably spaced from each other, allows the formation of a thin plate with a thickness generally between 4 and 10 cm, inside which the microbial culture flows.

In this embodiment, the photobioreactor panels are preferably made of polycarbonate material in order to prevent the Anabaena biomass from adhering to the walls of said panels.

An example of a photobioreactor suitable for use in the method of the invention is shown in Figure 1. The profile view (on the left) shows the panels of single- or double- sided light emitters (“LEDs”) alternating with the photosynthetic panels, where the Anabaena biomass (“PBR”) is produced. The elevation view (on the right) shows the “LED” panel. Dimensions shown are in cm.

Preferably, in the method according to the invention, the photobioreactor is equipped with a system that ensures the mixing of the Anabaena biomass therein, in order to prevent the sedimentation of these cyanobacteria and the formation of shaded areas. According to the invention, a system using the bubbling of an insufflated air/CCL mixture is particularly preferred, thereby reducing any mechanical stress and additional energy costs resulting from mechanical stirring.

The liquid culture medium used in the method according to the invention is suitable to meet the nutritional requirements of Anabaena cyanobacteria, as it comprises a phosphorus source and trace elements selected from the group consisting of iron, magnesium, manganese, zinc, copper, cobalt, and any combination thereof, and is at the same time nitrogen-free since these cyanobacteria are advantageously capable of fixing atmospheric molecular nitrogen into organic compounds. According to the invention, phosphorus, a fundamental macronutrient for growth, necessary for protein synthesis and DNA replication, is supplied in the aforementioned culture medium in an inorganic form, as potassium dihydrogen phosphate (KH2PO4).

Optionally, the liquid culture medium of the method according to the invention may include additional components such as, for example, bicarbonates or other molecules suitable for buffering pH changes.

Preferably, the liquid culture medium of the method of the invention has a pH value in the range of 7.5 to 8.5, more preferably a pH value of 8.0.

Methods suitable to adjust the pH value of a culture medium are known and described in the prior art, therefore the selection and use thereof are within the skills of those of ordinary skill in the art.

In one embodiment of the method according to the invention, the pH of the liquid culture medium is adjusted by using a bicarbonate buffer system, which is based on the use of a probe in contact with said culture medium and a system for regulating the flow rate of CO2 supplied.

According to the present invention, the step of cultivating one or more nitrogen-fixing cyanobacteria of the genus Anabaena is characterized by particular operating conditions, relating to temperature, residence time, lighting and light radiation regime, which advantageously enable a high yield of nitrogen-rich biomass.

In the method according to the invention, the cultivation step is carried out at a temperature comprised between 23°C and 25°C, preferably between 23.5°C and 24.5°C, more preferably at 24°C. The time of residence of the one or more Anabaena cyanobacteria in the photobioreactor during the cultivation step is at least 15 hours, preferably between 19 and 40 hours, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 hours.

Within the scope of the present description, the term “time of residence/residence time” refers to the time spent by the microorganisms cultured inside a reactor in continuous mode. This parameter is indicated as the ratio between the volume of the photobioreactor, for example expressed in litres, and the volumetric flow rate of the culture medium entering the reactor, expressed as volume per time unit (for example, litres/hour).

As is known in the art, the emission spectrum and light intensity variables can significantly affect the growth of a photoautotropic microorganism in culture.

In the method according to the invention, the one or more nitrogen-fixing cyanobacteria of the genus Anabaena are exposed to light radiation with a wavelength in the range from 350 nm to 750 nm, said range corresponding to the photosynthetically active radiation (PAR) of the light spectrum.

The term “photosynthetically active radiation (PAR)”, as used herein, refers to light spectrum radiation capable of exciting photosystems and induce carbon fixation reactions and microalgal growth.

In the present invention, the light radiation has an intensity of from 450 to 600 micromoles of photons per square metre per second (pmol m'² s'¹), preferably an intensity of from 500 to 580 pmol m'² s'¹, more preferably from 530 to 560 pmol m'² s'¹. A light radiation intensity of 550 pmol m'² s'¹ is most preferred.

Within the scope of the present invention, the light intensity measurement can be performed using radiometers with probes specific for the PAR wavelength range. In one embodiment, the light radiation is generated by a natural light source, for example by solar radiation.

In another embodiment, the light radiation is generated by an artificial light source, for example a light-emitting diode (LED) source or a neon lamp.

The selection of the most appropriate light source to be used within the scope of the present invention falls well within the skills of those of ordinary skill in the art.

In the method of the invention, during the cycle of cultivation of the one or more cyanobacteria of the genus Anabaena, the light radiation regime may be continuous with constant lighting.

In this embodiment, the Anabaena biomass is preferably allowed to acclimatize for about 3- 5 days, then the photobioreactor is operated in continuous mode until a steady state is reached in which the concentration, composition and quality of the biomass remain constant over time.

Alternatively, in the method of the invention, the light radiation regime may be cyclical, either natural or artificial, each cycle comprising a period of lighting and a period of absence or reduction of light.

Preferably, during the cycle of cultivation of the one or more cyanobacteria of the genus Anabaena, the cyclical light radiation regime comprises a daily exposure to light radiation for a period of at least 10 hours, more preferably for a time interval between 12 hours and 16 hours.

In this embodiment, the use of a natural light source is particularly preferred.

The preferred operating conditions of the cultivation step are as follows:

- temperature: 24°C; - light radiation wavelength: light composed of the following three wavelengths: 450 nm, 630 nm and 680 nm;

- light radiation intensity: 550 pmol m’² s’¹;

- residence time: 22 hours;

- light source: LEDs;

- radiation regime: continuous with artificial radiation.

The method of the invention also comprises a step of separating the Anabaena cell biomass obtained in the cultivation step from the liquid culture medium, and a further step of subjecting the cells of said biomass to mechanical disruption.

Methods suitable for use in the above separation step include, but are not limited to, sedimentation, filtration, centrifugation and flocculation procedures.

Filtration through a membrane, preferably through a polypropylene membrane, is particularly preferred.

Techniques for operating the mechanical disruption of cell biomass are known and described in the state of the art, therefore the selection and use thereof are within the skills of those of ordinary skill in the art. The use of ultrasound (sonication), grinding, and crushing by centrifugation is mentioned by way of non-limiting example.

The preferred embodiments of the method according to the invention described above can be combined with each other as required, and the implementation of these combinations falls within the skills of the person skilled in the art.

As mentioned above, advantageously, the present invention provides a method that allows large-scale production of a purely natural fertilizer suitable for industrial applications, with simpler operating procedures and lower costs than traditional processes.

As will be explained in more detail in the following experimental section, surprisingly, the method of the present invention makes it possible to obtain a fertilizer that is capable as such of fully satisfying the nutritional requirements of crops in agronomic practices, without the need for further use of other substances, in particular chemical nitrogen fertilizers which are often harmful to the environment.

Therefore, the present invention also relates to a nitrogen-containing fertilizer that can be obtained by means of the aforementioned method of the present invention, which comprises nitrogen in a concentration by weight of from 0.06% to 0.09% w/w based on the total weight of the fertilizer.

A method for fertilizing a plant, comprising the step of applying a fertilizer as previously defined to the plant, parts of the plant, plant propagation material, and/or plant growth site, is also within the scope of the invention.

The term “fertilizing”, as used herein, refers to the agronomic procedure of supplying nourishment to a plant in order, for example, to accelerate the growth of the plant, increase leaf density, increase the number of flowers per plant, increase root development, increase the number of stems per plant and/or improve the colour of a plant's leaves after seeding.

According to the method of the invention, the fertilizer can be applied to a variety of plants in various forms or parts of a plant, such as for example leaves, gems, branches, stems, bark, flowers, flower buds, fruits, roots, seeds, bulbs, tubers and/or sprouts.

The term “propagation material”, as used herein, refers to any plant material from which a plant or part of a plant can be derived. Seeds, seedlings, cuttings, scions, rootstocks, explants, bulbs, tubers, and combinations thereof, are mentioned by way of non-limiting example.

Additionally or alternatively, the fertilizer according to the invention can be applied to the plant growth site.

In one embodiment, the plant is grown in the soil and the application of the fertilizer according to the invention can take place, for example, over the entire cultivation surface, in one or more furrows and/or around them, in the sowing holes, in the area below the stem or trunk, and/or in the area between the roots.

In another embodiment, the plant is grown out of the soil or soilless. The hydroponic cultivation technique is mentioned by way of non-limiting example among the out-of-the- soil or soilless cultivation methods, in which soil is replaced by an inert substrate, such as for example expanded clay, coconut fibre, rock wool or zeolite, and the plant assimilates nutrients thanks to a solution composed of water and inorganic elements, which have the purpose of providing all the substances required for the normal mineral nutrition of the plant organism.

According to a preferred embodiment, the fertilizer of the present invention is applied by irrigation, i.e., directly into the soil, for example in the form of an irrigation liquid. The distribution of a fertilizer together with irrigation water is commonly referred to as ‘fertigation’.

In this embodiment of the invention, the method comprises applying the fertilizer of the invention at least twice, preferably 4 times, more preferably 5 times.

Preferably, the time interval between one application of the fertilizer by fertigation, for example a first, second, third, fourth or fifth application, and the subsequent application may be approximately 4 days to approximately 4 weeks, more preferably approximately 5 days to approximately 3 weeks, even more preferably approximately 6 days to approximately 2 weeks, e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

In the case of a hydroponic cultivation, the method according to the invention provides that the fertilizer is administered to the plants in the nutrient solution.

In the methods of use as previously defined, the fertilizer according to the invention can optionally be diluted before its application depending on the type of cultivar. A further aspect of the present invention is the use of the fertilizer as previously defined to fertilize a plant.

The preferred embodiments described above can be combined with each other as required, and the implementation of these combinations falls within the skills of the person skilled in the art.

The examples that follow are provided for illustration purposes and do not limit the scope of the invention as defined in the appended claims.

EXAMPLES

1. Method for producing the fertilizer according to the invention

For their experiments, the present inventors employed a flat plate photobioreactor, 4-10 cm thick, sized to ensure effective light absorption by Anabaena cyanobacteria (Figure 1).

A liquid culture medium, after sterilization, was introduced into the reactor, said medium having the following composition: Ippm Na₂Mg EDTA; 6.2 ppm FeC13 6 H2O; 6 ppm Citric acid; 36 ppm CaCl₂ -2H₂O; 75 ppm MgSO₄ • 7H₂O; 75 ppm K₂HPO₄; 3 ppm H3BO3; 1.81 ppm MnCl₂ • 4H₂O; 0.2 ppm ZnSO₄ • 7H₂O; 0.08 ppm CuSO₄ • 5H₂O; 0.05 ppm COC1₂ • 6H₂O; 0.4 ppm Na₂MoO₄ • 2H₂O; 20 ppm Na₂CO3; 1500 ppm NaHCCh.

Subsequently, the biomass of nitrogen-fixing cyanobacteria of the genus Anabaena was inoculated, in ratios between 1-5% of the total volume of the reactor. A gas mixture consisting of air and CO₂, with a percentage between 1-5% v:v (CO₂:air), in atomized form, was then fed through an appropriate bubbling system, with a flow rate of 0.5- 1.0 L/h per litre of culture.

The biomass was allowed to acclimatize for about 3-5 days. Next, the fresh medium feed pump and the liquid suspension extraction pump were started, both at the same flow rate to keep the volume of liquid suspension constant inside the photobioreactor. The photobioreactor was operated in continuous mode until a steady state was reached in which the concentration and composition of the biomass remained constant over time.

The step of cultivating the cyanobacterium Anabaena in continuous mode was performed at a temperature of 24°C under the condition of continuous light emitted by LED lamps, with a wavelength between 350 and 750 nm, the typical spectrum of warm white light, and an incident light intensity of 550 pmol m’² s’¹, as measured by a radiometer equipped with a PAR probe. The cultivation of the cyanobacteria Anabaena was carried out for a residence time of about 19 to 31 hours (corresponding to a residence time interval between 0.8 and 1.3 days). The reactor was operated continuously for 90-100 days.

The present inventors, taking into account the different residence times, as calculated from the ratio between the reactor volume and the input flow rate, thus obtained different biomass concentrations in the steady state. Moreover, depending on the light intensity used, the present inventors established the optimum value for the residence time which guarantees maximum productivity, i.e., for the cyanobacterium Anabaena.

Under the above operating conditions, a biomass productivity of 1.2- 1.7 g/L/day was obtained, with an intracellular nitrogen content of 7-8%.

The flow leaving the reactor, containing spent medium and biomass, was subjected to separation and post-treatment processes. The biomass produced, consisting of filamentous Anabaena organisms, was separated, for example, by using a 30 g/m² filter made of polypropylene, having a water porosity of 950 L/m²/s.

The separation step resulted in an Anabaena biomass paste with a concentration of approximately 9-11 g/L, which was sonicated to disrupt cell structures. Sonication parameters used were as follows: 10 minutes at 3.7 W s’¹, 50% amplitude and -10 offset, and the equipment was calibrated to prevent overheating of the biomass. The separated water was recirculated at the top of the process for the formulation of the culture medium, in order to minimize water consumption. The recirculated water was not mixed with fresh medium to prevent contamination, but was directly reintroduced into the reactor.

2. Assessment of the effectiveness of the fertilizer according to the invention

In order to verify the effectiveness of the fertilizing action of the product obtained with the method of the invention, the present inventors carried out dedicated studies on spinach plants grown in pots in greenhouses. The germination tests were carried out during the two-month period of November-December 2020, at the plant phenotyping platform (“Piattaforma di Fenotipizzazione delle Piante”) of ALSIA, “Centro Ricerche Metapontum Agrobios”, in Metaponto (Southern Italy).

In the studies carried out by the inventors, visible light image analysis (RGB) was used to assess the effectiveness of the fertilizer obtained by means of the method of the invention on phenotypic parameters of spinach. The experimental activity was based on growth tests on spinach seedlings subjected to different control treatments, without fertilizers, and treatments with commercial fertilizers, compared with the fertilizer of the invention. The product, used in a single dosage, was compared with the commercial fertilizers Entec® (46%) and Slowenne® (14.7.14) and with a non-fertilized control. These products were distributed by root system on spinach variety Marten RZ Fl (Rijk Zwaan) grown in pots.

The compost that was used for cultivation (Vigorplant) is a sandy loam with a sub-alkaline reaction and very low salinity. It has a low content of nitrates, phosphorus, potassium, and exchangeable cations, as well as low availability of trace elements. As a result, in all samples subjected to fertilization a basal dressing was carried out with 0.2 grams per pot, equivalent to 60 kg (8.2 units of N) per hectare, of slow-release fertilizer (by administering the product Slowenne® of Valagro Spa). This treatment was not carried out in the negative control (“non-fertilized”). Treatments of spinach plants were carried out according to the scheme set out in Table 1 below.

Table 1

The fertilizers of the invention were stored in plastic tanks and kept at 4°C until use. Prior to the preparation of the nutrient solution, the fertilizing products of the invention were stirred and mixed and, once weighed, diluted in water and administered to the soil in the immediate vicinity of the spinach plants by fertigation.

Assessment of effects

The objective of the study was to verify, for the parameters analysed and the cultivation conditions applied, the effectiveness of the action of the fertilizer according to the invention, compared with some slow-release fertilizers and with a non-fertilized control. The phenotype of the plants was examined by image analysis, in particular by analysing RGB images (visible spectrum), to obtain information about the growth of the plants based on some digital traits (leaf area, height, compactness, colour).

After administration of the products, non-destructive image analyses were carried out during the growth of the plants for the size index (in pixels, three images per plant), leaf area, height, leaf coverage, plant compactness, and colour index.

The plant was observed by acquiring 3 images through RGB sensors, two of which laterally (RGB_side) from two different angles (0 and 90°), and one from above (RGB_TV).

The set of images obtained from the different perspectives allowed more information to be obtained about the morphology, size or biomass of the analysed plant. In particular, the formula as proposed by J. Guerra (KeyGene, R.V. Berloo, J. Guerra, 2009) was used to estimate the plant size index.

In their study on effectiveness, the inventors examined the following parameters:

- "Projected Shoot Area", which corresponds to the sum of the projected area, silhouette, of the plant from three orthogonal projections (views), two lateral and one from above, as a function of time;

- “Projected Shoot Area Growth”, which corresponds to the change rate over time of the "Projected Shoot Area" parameter;

- "Height", which corresponds to the lateral view of the height of the plant, as a function of time;

- "Top Area View DIFF”, which corresponds to the increase in the projection of the foliage on the ground from the initial value, the one measured 11 days after seeding. It is a parameter that can be correlated with the Leaf Area Index (LAI).

2a. Projected Shoot Area In this study, the measured values of the “Projected Shoot Area” as defined above were converted from units of pixel area to SI, measured as cm² or mm². The parameter was calculated from colour images of a single plant.

As illustrated by the graph in Figure 2, which shows the means of 10 independent measurements, spinach plants treated with the fertilizing products of the invention exhibited a higher increase in volume during the treatment period than the other samples under examination.

2b. Projected Shoot Area Growth

The values of the "Projected Shoot Area Growth" parameter as defined above were obtained as an increase in area over time, per survey date. From the graph illustrated in Figure 3, which shows the means of 10 independent measurements, a higher growth is observed in the samples treated with the test products compared to the SlowenneO-treated samples, with the EntecO-treated samples in an intermediate position.

2c. Height

The values relating to the "Height" parameter as defined above are illustrated in the graph in Figure 4, which shows the means of 10 independent measurements. Statistical analysis with a 90% confidence interval showed significant differences between the compared samples. In particular, the plants treated with the test products were found to have a greater height, starting from the 2^nd survey and maintaining this advantage until the end of the test.

2d. Top Area View

The results obtained by the present inventors for the parameter “Top Area View” as defined above, illustrated by the graph in Figure 5, show that, for each of the days considered, the leaf coverage was significantly increased in the plants treated with the fertilizer of the invention compared to the other samples under examination. The graph shows the means of 10 independent measurements. 3. Conclusion

In conclusion, the results from the study on effectiveness carried out by the inventors and explained above indicate that all fertilizer theses showed higher production compared to the negative control without added fertilizer. Moreover, although statistical analysis performed with a 90% confidence interval (alpha = 0.1) showed no significant differences between the compared theses, the Anabaena biomass -containing fertilizer exhibited higher effectiveness than commercial control products over time. These data were also confirmed by the final production (fresh weight) of the plants. Therefore, the results shown herein demonstrate the possibility of completely replacing commercial chemical fertilizers with the Anabaena- containing fertilizer.

Claims

1. A method for producing a nitrogen-containing fertilizer, comprising the steps of:

(i) cultivating in continuous mode one or more nitrogen-fixing cyanobacteria of the genus Anabaena in a liquid culture medium in a closed photobioreactor, thereby obtaining a cell biomass of nitrogen-fixing cyanobacteria of the genus Anabaena in suspension in said liquid culture medium, wherein said liquid culture medium comprises potassium dihydrogen phosphate (KH2PO4) and one or more trace elements selected from the group consisting of iron, magnesium, manganese, zinc, copper, cobalt, and any combination thereof, and said liquid culture medium does not comprise nitrogen; wherein in said cultivating step, the one or more nitrogen-fixing cyanobacteria of the genus Anabaena are kept at a temperature comprised between 23°C and 25°C for a residence time of at least 15 hours and are exposed to a light radiation of a wavelength of from 350 nm to 750 nm and an intensity of from 450 to 600 micromoles of photons per square metre per second (pmol m'² s'¹);

2. The method according to claim 2, wherein the one or more nitrogen-fixing cyanobacteria of the genus Anabaena belong to a species selected from the group consisting of Anabaena cylindrica, Anabaena flos aquae and Anabaena sp. Nostoc, and any combination thereof.

3. The method according to claim 1 or 2, wherein the closed photobioreactor is a flatplate photobioreactor.

4. The method according to any of claims 1 to 3, wherein the light radiation is generated by a natural or an artificial light source.

5. The method according to any of claims 1 to 4, wherein the duration of the residence time is comprised between 19 and 40 hours.

6. The method according to any of claims 1 to 5, wherein in step (iii) the biomass of nitrogen-fixing cyanobacteria of the genus Anabaena is separated from the liquid culture medium by means of filtration.

7. A nitrogen-containing fertilizer obtainable by the method according to any of claims 1 to 6, the fertilizer comprising nitrogen at a concentration by weight comprised within the range of from 0.06% to 0.09% w/w based on the total weight of the fertilizer.

8. A method for fertilizing a plant, comprising the step of applying a nitro gen-containing fertilizer according to claim 7 to the plant, parts of the plant, plant propagation material, and/or plant growth site.

9. The method according to claim 8, wherein the nitrogen-containing fertilizer is applied by irrigation or in a hydroponic solution.

10. The use of the nitrogen-containing fertilizer according to claim 7, for fertilizing a plant.