WO2022117901A1 - Use of microalgae resistance forms in agriculture - Google Patents

Use of microalgae resistance forms in agriculture Download PDF

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Publication number
WO2022117901A1
WO2022117901A1 PCT/ES2021/070858 ES2021070858W WO2022117901A1 WO 2022117901 A1 WO2022117901 A1 WO 2022117901A1 ES 2021070858 W ES2021070858 W ES 2021070858W WO 2022117901 A1 WO2022117901 A1 WO 2022117901A1
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Prior art keywords
microalgae
resistance
forms
species
cyanobacteria
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PCT/ES2021/070858
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Spanish (es)
French (fr)
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Miguel GARCÍA GUERRERO
José Antonio DEL CAMPO CASTILLO
María SEGURA FORNIELES
Lucía MORENO GARRIDO
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Algaenergy, S.A.
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Publication of WO2022117901A1 publication Critical patent/WO2022117901A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C21/00—Methods of fertilising, sowing or planting
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C21/00—Methods of fertilising, sowing or planting
    • A01C21/005—Following a specific plan, e.g. pattern
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/03—Algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like

Definitions

  • the present invention relates to the use of resistant forms of microalgae in agriculture. Furthermore, it refers to a method that improves soil quality, plant growth and/or crop yield.
  • microalgae have immense potential to contribute to responding to the challenges facing the sector.
  • Microalgae are microorganisms that possess chlorophyll a and other pigments, which have the ability to carry out oxygenic photosynthesis.
  • This heterogeneous group includes tens of thousands of eukaryotic species with different morphology, size and habitat, as well as the prochaotic cyanobacteria, cyanophyceae or blue-green algae.
  • Microalgae inhabit fresh and brackish waters, marine systems and soils.
  • microalgae in sustainable agriculture goes beyond biostimulation. Soil quality is a determining factor in agricultural production, with some microalgae being natural inhabitants of these ecosystems, where they play an essential role in determining the health, quality and fertility of agricultural soils. In fact, microalgae are basic in all stages of soil formation, including the initial phase of the establishment of a biological crust on an abiotic mineral substrate.
  • These cells are resistant and usually have a thicker and harder envelope or wall than that of vegetative cells, from which they differentiate, as well as because they contain a reserve of nutrients. In nature, they can act as transport vectors between different geographical areas, both locally and intercontinentally.
  • Some microalgae that are part of the soil microflora are capable of developing forms of resistance. Under favorable environmental conditions, the forms of resistance germinate, developing and multiplying the resulting vegetative cells, thus giving rise to the establishment of important colonies of microalgae.
  • the set of effects translates into an increase in soil fertility and the productivity of the crops that are grown in them, as a result of a biotechnological action, which preserves the environment and avoids the undesirable addition of chemical agents. . Consequently, all of them are positive effects and results that can contribute decisively to food security and thus to one of the main UN Sustainable Development Goals, specifically number 2, ZERO HUNGER.
  • microalgae In the cultivation of microalgae, suitable media are used for the growth of each species in question. To stimulate the production of resistant forms, it is necessary to modify the culture conditions in order to induce their formation, which entails the controlled alteration of the concentration of certain nutrients (eg nitrogen, phosphorus, sulfur, iron, silicon ) or other critical culture parameters, such as agitation, air supply, light irradiance or temperature.
  • the application of forms of microalgae resistance to soils is an efficient way to improve their quality and fertility. On the one hand, to promote improvements in the production of the crops that grow on the soils treated in this way. On the other, for the transformation of mineral edaphic substrate, of an arid or desert nature, into soils for agricultural use. Additionally, the forms of resistance can be used in bioremediation actions focused on the recovery and restoration of impoverished, degraded or contaminated soils.
  • one aspect of the invention is the method for improving the productivity of agricultural crops that comprises the application of an efficient amount of forms of microalgae resistance, in solid form or in liquid solution, to the land in which the plants grow. cultivate.
  • a second aspect relates to a method for improving the health and fertility of a soil comprising applying an efficient amount of resistant forms of microalgae, in solid form or in liquid solution, to the soil.
  • a third aspect refers to a method of recovery and restoration of impoverished, degraded or contaminated soils.
  • the land where the forms of resistance are applied is not fertile land, but rather degraded land not suitable for cultivation that is recovered for agriculture.
  • efficient amount refers to the amount of the product, resistant forms of microalgae, that, when applied, is sufficient to improve crop productivity or improve soil fertility.
  • resistance forms refers to cells that are dormant, latent, quiescent, or metabolically quiescent.
  • Another aspect of the invention is a bioactive composition comprising resistant forms of microalgae in a range between 30% and 99% of the total number of cells in the composition.
  • bioactive refers to a composition that is added to the soil in order to provide basic elements for the growth of the plant or for the health of the plant or for the yield of the crops. plant fruits.
  • Another aspect of the invention refers, therefore, to the use of the resistant forms of microalgae as a bioactive composition to improve the productivity of agricultural crops and the fertility of the soil, or to recover or restore impoverished, degraded or contaminated soils.
  • Figure 1 shows a microscope image of the resistance forms of the cyanobacteria obtained according to the described process.
  • Figure 2 shows a micrograph of resistant forms of cyanobacteria in the process of germination.
  • the first aspect of the invention is the method to improve plant productivity, which comprises the application to the land or agricultural environment of an efficient amount of forms of microalgae resistance
  • a second aspect refers to a method to improve the fertility of a soil or to recover or restore impoverished, degraded or contaminated soils, which includes the application of an efficient quantity of resistant forms of microalgae to the land.
  • a third aspect of the invention refers to a bioactive composition, which comprises the resistant forms of microalgae in a range comprised between 30% and 90% of the total number of cells in the composition.
  • the bioactive composition comprises the resistance forms of microalgae and an agriculturally acceptable carrier of the resistance forms.
  • agriculturally acceptable carrier refers to a carrier that is known and accepted in the formation of formulations for use in agriculture or horticulture.
  • the forms of resistance preferably come from soil microalgae or terrestrial microalgae.
  • Soil microalgae are preferably selected species belonging to the Divisions: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heteromonyphyta and f ⁇ hodophyta.
  • Heterochyphyta More preferred among the Heterochyphyta are species of Bacillariophyceae (diatoms) and among the Cyanophyta (cyanobacteria) are strains of genera such as Anabaena and Nostoc.
  • microalgae in poor conditions for vegetative growth (suboptimal), have the ability to develop forms of resistance, which allow them to resist adverse conditions until the environmental and/or nutritional variables are again conducive to the development of vegetative cells.
  • This process can be reproduced artificially, forcing said conditions of nutritional and/or environmental stress.
  • the development of the resistance form is favored by controlling the level of phosphorus in the microalgae culture medium.
  • the development of the resistance form is favored under conditions of high carbon input to the microalgae culture medium.
  • the methods described above further comprise the steps of: a) cultivating the microalgae; b) induce the form of resistance; c) preserve the form of resistance; d) germinate the form of resistance.
  • Storage is preferably carried out in the dark. More preferably at a temperature between -20°C and 25°C.
  • the examples of the present invention demonstrate the formation of dormant forms, their possible conservation over time and their ability to germinate once they are in the right environment.
  • Example 1 Process of induction, isolation and stabilization of forms of resistance of bacillariophyceae (diatoms)
  • Cells from a diatom were cultured in standard liquid medium. After 10 days of growth, part of the vegetative cells were transferred to a medium with a low concentration (10 micromolar) of sodium nitrate and 150 micromolar of silicic acid, conditions that favor the massive production of forms of resistance. The temperature was maintained at 25°C in a thermostatted and illuminated chamber, using cycles of 12 hours of light: 12 hours of darkness.
  • the resistance forms obtained were dried with hot air at a moderate temperature, being ready for later storage. Periodic controls were carried out to verify that the resistant forms maintain their ability to germinate when placed in favorable conditions (humidity, temperature, nutrients, etc.)
  • Example 2 Process of induction, isolation and stabilization of resistant forms of cyanobacteria
  • the culture began with the transfer of the strains from solid medium (agar) to 250 mL flasks with standard culture medium maintained at 25°C and with orbital agitation.
  • the cultures of the cyanobacterial strains were subjected to nutritional stress due to phosphorus limitation.
  • the resuspended sediment was transferred in a volume of 600 mL to 2-5 L bottles with the low-phosphorus medium and cultured for 21 days. sampling every 3 days to follow its progression with measurements of absorbance at 750 nm and the formation of resistance forms by counting with a Neubauer Chamber.
  • the supernatant was removed from the centrifugation by decantation and the pellet was recovered in 600 mL of medium, repeating the process three times.
  • the resuspended sediment was transferred in a volume of 600 mL to 2-5 L bottles with the medium supplemented with carbon and cultured for 21 days, sampling every 3 days to follow the progression of the culture with absorbance measurements at 750 nm and the formation of resistance forms by counting with the Neubauer Chamber.
  • Figure 1 shows a photomicrograph of the concentrate of resistant forms of cyanobacteria.
  • the count of the resistance forms was carried out with a Neubauer chamber and the concentrate of resistance forms was centrifuged, eliminating the supernatant, leaving the sediment ready to proceed with drying and preservation tests and subsequent germination of the resistance forms.
  • the product obtained in the previous step was dried in an oven at 37°C for 24 hours. Once dried, preparations rich in resistance forms can be stored for several months in closed containers in the dark, either at room temperature (25°C) or at 4°C or even at temperatures below zero (-17°C).
  • Germination tests of the resistance forms were carried out 3 months after their production and storage. For this process to take place, it was necessary to resuspend and rehydrate the biomass of the resistant forms with complete culture medium.
  • This germination process also takes place in the soil, under conditions (moisture, temperature and nutrient availability) that allow the propagation and growth of cyanobacteria.
  • Dried resistance forms were resuspended in a small volume of complete medium.
  • the necessary amount of medium was added so that the absorbance at 750 nm was between 0.2 and 0.4.
  • the resulting resistance forms suspension was kept in a 100 mL flask with agitation in an orbital shaker and natural photoperiod.
  • Figure 2 shows the germination of the resistance form.

Abstract

The present invention relates to the use of microalgae resistance forms in agriculture. Furthermore, it relates to a process that improves plant productivity and/or crop yield and/or soil fertility.

Description

USO DE FORMAS DE RESISTENCIA DE MICROALGAS EN AGRICULTURA USE OF RESISTANCE FORMS OF MICROALGAE IN AGRICULTURE
DESCRIPCION DESCRIPTION
La presente invención se refiere al uso de formas de resistencia de microalgas en agricultura. Además, se refiere a un procedimiento que mejora la calidad del suelo, el crecimiento vegetal y/o el rendimiento de los cultivos. The present invention relates to the use of resistant forms of microalgae in agriculture. Furthermore, it refers to a method that improves soil quality, plant growth and/or crop yield.
Estado de la técnica State of the art
La sociedad se enfrenta a grandes retos en el siglo XXI, muchos de ellos estrechamente vinculados al sector agroalimentario y a su capacidad para dar respuesta a una demanda creciente de alimentos que permita abastecer a una población mundial que aumenta a un ritmo muy elevado. Se requiere, por tanto, aumentar considerablemente la productividad agrícola para poder cubrir esa acuciante necesidad. Sin embargo, y considerando otras dificultades adyacentes, como son la limitación de recursos cada vez más escasos -tierra cultivable y agua-, la seguridad alimentaria, el cambio climático y la creciente contaminación medioambiental, la solución pasa por incrementar esa productividad de una manera sostenible y respetuosa con el medio ambiente. Society faces great challenges in the 21st century, many of them closely linked to the agri-food sector and its ability to respond to a growing demand for food that allows it to supply a world population that is increasing at a very high rate. Therefore, it is necessary to considerably increase agricultural productivity in order to cover this pressing need. However, and considering other adjacent difficulties, such as the limitation of increasingly scarce resources -arable land and water-, food security, climate change and growing environmental pollution, the solution lies in increasing that productivity in a sustainable manner. and respectful with the environment.
El desafío al que se enfrenta la sociedad en algo tan esencial y básico como es garantizarse su propia alimentación, obliga al sector agroalimentario a desempeñar un papel protagonista en la búsqueda de soluciones, sobre tres pilares fundamentales: innovación, sostenibilidad y eficiencia. En este contexto, las microalgas tienen un inmenso potencial para contribuir a dar respuesta a los retos a los que se enfrenta el sector. Las microalgas son microorganismos poseedores de clorofila a y de otros pigmentos, que tienen la capacidad de llevar a cabo la fotosíntesis oxigénica. Este heterogéneo grupo comprende decenas de miles de especies eucarióticas con vahada morfología, tamaño y hábitat, así como las cianobactehas procahóticas, cianofíceas o algas verdeazuladas. Las microalgas habitan aguas dulces y salobres, sistemas marinos y suelos. The challenge that society faces in something as essential and basic as guaranteeing its own food, forces the agri-food sector to play a leading role in the search for solutions, based on three fundamental pillars: innovation, sustainability and efficiency. In this context, microalgae have immense potential to contribute to responding to the challenges facing the sector. Microalgae are microorganisms that possess chlorophyll a and other pigments, which have the ability to carry out oxygenic photosynthesis. This heterogeneous group includes tens of thousands of eukaryotic species with different morphology, size and habitat, as well as the prochaotic cyanobacteria, cyanophyceae or blue-green algae. Microalgae inhabit fresh and brackish waters, marine systems and soils.
La idoneidad de las microalgas y sus derivados, para una variedad de aplicaciones de carácter práctico, se ha puesto de manifiesto en diversidad de instancias, incluyendo algunas particularmente eficientes que están relacionadas con la agricultura. Por ejemplo, los estudios existentes de bioestimulantes agrícolas basados en microalgas dejan evidencia de que no solo favorecen la absorción de nutrientes, sino que también mejoran la eficiencia en la utilización de éstos, así como la tolerancia al estrés abiótico y finalmente, incrementan la calidad de los cultivos. En la patente con número de publicación US10701941 se describe un método basado en la microalga Chlorella para mejorar los cultivos. Igualmente se describe el uso de las microalgas vivas en la solicitud ALI2019240617, si bien en este documento nunca se hace referencia al uso de las formas latentes de microalgas en fertilizantes, ni se describe la manera de inducirlas o de preservarlas, ni se demuestra que dichas formas latentes puedan ser de nuevo activas en un medio agrícola. The suitability of microalgae and their derivatives, for a variety of practical applications, has been shown in a variety of instances, including some particularly efficient ones that are related to agriculture. For example, existing studies of agricultural biostimulants based on microalgae leave evidence that they not only favor the absorption of nutrients, but also improve the efficiency in their use, as well as tolerance to abiotic stress and finally, increase the quality of crops. Patent with publication number US10701941 describes a method based on the Chlorella microalgae to improve crops. Likewise, the use of live microalgae is described in application ALI2019240617, although this document never refers to the use of latent forms of microalgae in fertilizers, nor does it describe how to induce or preserve them, nor does it demonstrate that said dormant forms can become active again in an agricultural environment.
Sin embargo, el papel de las microalgas en la agricultura sostenible va más allá de la bioestimulación. La calidad del suelo es un factor determinante en la producción agrícola, siendo algunas microalgas pobladoras naturales de estos ecosistemas, donde desempeñan una función esencial en la determinación de la salud, calidad y fertilidad de los suelos agrícolas. De hecho, las microalgas son básicas en todas las etapas de la formación del suelo, incluyendo la fase inicial del establecimiento de una costra biológica sobre un sustrato mineral abiótico. However, the role of microalgae in sustainable agriculture goes beyond biostimulation. Soil quality is a determining factor in agricultural production, with some microalgae being natural inhabitants of these ecosystems, where they play an essential role in determining the health, quality and fertility of agricultural soils. In fact, microalgae are basic in all stages of soil formation, including the initial phase of the establishment of a biological crust on an abiotic mineral substrate.
Por ejemplo, en la solicitud de patente con número de publicación US10457610, se describen métodos y composiciones que comprenden microalgas para tratar el suelo, ya que consigue enriquecerlo en sodio. For example, in the patent application with publication number US10457610, methods and compositions are described that comprise microalgae to treat the soil, since it manages to enrich it in sodium.
Es, por tanto, de gran interés ampliar el repertorio de productos para la agricultura basados en microalgas, como el que se va a describir a continuación. It is, therefore, of great interest to expand the repertoire of products for agriculture based on microalgae, such as the one that will be described below.
Descripción Description
En los ciclos vitales de numerosas microalgas se dan diferentes tipos de células durmientes, latentes, quiescentes o en reposo metabólico, que constituyen formas de resistencia, las cuales toleran condiciones ambientales y nutricionales adversas, posibilitando que la microalga se perpetúe en el tiempo, hasta encontrar condiciones favorables para su germinación y desarrollo. In the life cycles of numerous microalgae, there are different types of dormant cells, latent, quiescent or in metabolic rest, which constitute forms of resistance, which tolerate adverse environmental and nutritional conditions, allowing the microalgae to perpetuate itself in time, until finding favorable conditions for its germination and development.
Estas células son resistentes y suelen tener una envuelta o pared más gruesa y dura que la de las células vegetativas, a partir de las que se diferencian, así como también por contener una reserva de nutrientes. En la naturaleza, pueden actuar como vectores de transporte entre diferentes zonas geográficas, tanto a escala local como intercontinental. Algunas microalgas que forman parte de la microflora de los suelos son capaces de desarrollar formas de resistencia. En condiciones ambientales favorables, las formas de resistencia germinan, desarrollándose y multiplicándose las células vegetativas resultantes, dando lugar así al establecimiento de importantes colonias de microalgas. La aportación de estas formas de resistencia a un suelo agrícola se traduce en su enriquecimiento en materia orgánica rica en carbono, nitrógeno, fósforo y otros nutrientes, así como fitohormonas y diversos compuestos bioactivos con capacidad estimulante del crecimiento vegetal, posibilitando mejorar sustancialmente la eficiencia de uso de nutrientes por los cultivos y de conferirles resistencia frente a estreses bióticos y abióticos. Productos adicionales de interés para el suelo resultantes de la actividad de las microalgas que se desarrollen son, entre otros, el oxígeno, que facilita la aireación, así como compuestos que contribuyen a la compactación y estructuración del suelo con reducción de la erosión, a la capacidad de retención de agua, así como al desarrollo de otros organismos, que asimismo contribuyen eficazmente a la calidad, salud y fertilidad de los suelos. These cells are resistant and usually have a thicker and harder envelope or wall than that of vegetative cells, from which they differentiate, as well as because they contain a reserve of nutrients. In nature, they can act as transport vectors between different geographical areas, both locally and intercontinentally. Some microalgae that are part of the soil microflora are capable of developing forms of resistance. Under favorable environmental conditions, the forms of resistance germinate, developing and multiplying the resulting vegetative cells, thus giving rise to the establishment of important colonies of microalgae. The contribution of these forms of resistance to agricultural soil translates into its enrichment in organic matter rich in carbon, nitrogen, phosphorus and other nutrients, as well as phytohormones and various bioactive compounds with the ability to stimulate plant growth, making it possible to substantially improve the efficiency of use of nutrients by crops and confer resistance to biotic and abiotic stresses. Additional products of interest for the soil resulting from the activity of the microalgae that develop are, among others, oxygen, which facilitates aeration, as well as compounds that contribute to the compaction and structuring of the soil with reduction of erosion, to the water retention capacity, as well as the development of other organisms, which also contribute effectively to the quality, health and fertility of the soil.
El conjunto de los efectos se traduce en un aumento de la fertilidad del suelo y de la productividad de las cosechas que en ellos se desarrollen, como resultado de una actuación de carácter biotecnológico, que preserva el medio ambiente y evita la indeseable adición de agentes químicos. En consecuencia, todos ellos son efectos y resultados positivos que pueden contribuir decisivamente a la seguridad alimentaria y con ello, a uno de los principales Objetivos de Desarrollo Sostenible de la ONU, concretamente el n° 2, HAMBRE CERO. The set of effects translates into an increase in soil fertility and the productivity of the crops that are grown in them, as a result of a biotechnological action, which preserves the environment and avoids the undesirable addition of chemical agents. . Consequently, all of them are positive effects and results that can contribute decisively to food security and thus to one of the main UN Sustainable Development Goals, specifically number 2, ZERO HUNGER.
En el cultivo de las microalgas se emplean medios ¡dóneos para el crecimiento de cada especie en cuestión. Para estimular la producción de formas de resistencia, se hace necesario modificar las condiciones de cultivo al objeto de inducir su formación, lo que conlleva la alteración controlada de la concentración de determinados nutrientes (p. ej. nitrógeno, fósforo, azufre, hierro, silicio) o de otros parámetros críticos de cultivo, como son la agitación, el suministro de aire, la irradiancia luminosa o la temperatura. La aplicación de formas de resistencia de microalgas a los suelos supone una forma eficiente de mejorar su calidad y fertilidad. De un lado, para propiciar mejoras de producción de los cultivos que se desarrollan en los suelos así tratados. De otro, para la transformación de sustrato edáfico mineral, de carácter árido o desértico, en suelos para uso agrícola. Adicionalmente, las formas de resistencia pueden emplearse en actuaciones de biorremediacion enfocadas a la recuperación y restauración de suelos empobrecidos, degradados o contaminados. In the cultivation of microalgae, suitable media are used for the growth of each species in question. To stimulate the production of resistant forms, it is necessary to modify the culture conditions in order to induce their formation, which entails the controlled alteration of the concentration of certain nutrients (eg nitrogen, phosphorus, sulfur, iron, silicon ) or other critical culture parameters, such as agitation, air supply, light irradiance or temperature. The application of forms of microalgae resistance to soils is an efficient way to improve their quality and fertility. On the one hand, to promote improvements in the production of the crops that grow on the soils treated in this way. On the other, for the transformation of mineral edaphic substrate, of an arid or desert nature, into soils for agricultural use. Additionally, the forms of resistance can be used in bioremediation actions focused on the recovery and restoration of impoverished, degraded or contaminated soils.
La principal ventaja del proceso que se describe aquí sobre otras alternativas, incluyendo opciones biológicas que emplean biomasa fresca, radica en el agente empleado para conseguir los fines pretendidos: formas de resistencia de microalgas. El conocido empleo de biomasa fresca de microalgas, con predominio de células vegetativas, se encuentra limitado frente al de las formas de resistencia por la escasa durabilidad del material y la dificultad para su transporte. La disponibilidad para la aplicación del agente aquí considerado, formas de resistencia, implica sin embargo un proceso mucho más complejo y en su conjunto disruptive, consistente en su generación, aislamiento, secado, almacenado y, eventualmente, estabilizado, así como su inclusión en soportes ¡dóneos para su utilización como enmiendas agrícolas, aunque no ha de descartarse a priori su aplicación directa al suelo a tratar. The main advantage of the process described here over other alternatives, including biological options that use fresh biomass, lies in the agent used to achieve the intended ends: resistant forms of microalgae. The known use of fresh biomass of microalgae, with a predominance of vegetative cells, is limited compared to the forms of resistance due to the low durability of the material and the difficulty of its transport. The availability for the application of the agent considered here, forms of resistance, however, implies a much more complex process and disruptive as a whole, consisting of its generation, isolation, drying, storage and, eventually, stabilization, as well as its inclusion in supports. They are suitable for use as agricultural amendments, although their direct application to the soil to be treated should not be ruled out a priori.
Por todo ello, un aspecto de la invención es el método para mejorar la productividad de cultivos agrícolas que comprende la aplicación de una cantidad eficiente de formas de resistencia de microalgas, en forma sólida o en solución líquida, al terreno en el cual las plantas se cultivan. For all these reasons, one aspect of the invention is the method for improving the productivity of agricultural crops that comprises the application of an efficient amount of forms of microalgae resistance, in solid form or in liquid solution, to the land in which the plants grow. cultivate.
Un segundo aspecto se refiere a un método para mejorar la salud y la fertilidad de un terreno que comprende la aplicación de una cantidad eficiente de formas de resistencia de microalgas, en forma sólida o en solución líquida, al terreno. A second aspect relates to a method for improving the health and fertility of a soil comprising applying an efficient amount of resistant forms of microalgae, in solid form or in liquid solution, to the soil.
Un tercer aspecto se refiere a un método de recuperación y restauración de suelos empobrecidos, degradados o contaminados. A third aspect refers to a method of recovery and restoration of impoverished, degraded or contaminated soils.
En este tercer aspecto el terreno donde se aplica las formas de resistencia no son terrenos fértiles, sino que son terrenos degradados no aptos para el cultivo que se recuperan para la agricultura. In this third aspect, the land where the forms of resistance are applied is not fertile land, but rather degraded land not suitable for cultivation that is recovered for agriculture.
El término “cantidad eficiente” se refiere a la cantidad del producto, formas de resistencia de microalgas que, al ser aplicado, es suficiente para mejorar la productividad de los cultivos o mejorar la fertilidad del terreno. The term “efficient amount” refers to the amount of the product, resistant forms of microalgae, that, when applied, is sufficient to improve crop productivity or improve soil fertility.
Como se ha dicho, el término “formas de resistencia” se refiere a células durmientes, latentes, quiescentes o en reposo metabólico. Otro aspecto de la invención es una composición bioactiva que comprende formas de resistencia de microalgas en un rango entre el 30% y el 99% del total de células en la composición. As stated, the term "resistance forms" refers to cells that are dormant, latent, quiescent, or metabolically quiescent. Another aspect of the invention is a bioactive composition comprising resistant forms of microalgae in a range between 30% and 99% of the total number of cells in the composition.
La forma en la que se utiliza en la presente invención el término “bioactiva” se refiere a una composición que se añade al terreno para conseguir aportar elementos básicos para el crecimiento de la planta o para la salud de la planta o para el rendimiento de los frutos de la planta. The way in which the term "bioactive" is used in the present invention refers to a composition that is added to the soil in order to provide basic elements for the growth of the plant or for the health of the plant or for the yield of the crops. plant fruits.
Otro aspecto de la invención se refiere, por tanto, al uso de las formas de resistencia de microalgas como composición bioactiva para mejorar la productividad de cultivos agrícolas y la fertilidad del suelo, o recuperar o restaurar suelos empobrecidos, degradados o contaminados. Another aspect of the invention refers, therefore, to the use of the resistant forms of microalgae as a bioactive composition to improve the productivity of agricultural crops and the fertility of the soil, or to recover or restore impoverished, degraded or contaminated soils.
FIGURAS FIGURES
La Figura 1 muestra una imagen al microscopio de las formas de resistencia de las cianobacteñas obtenidas según el proceso descrito. Figure 1 shows a microscope image of the resistance forms of the cyanobacteria obtained according to the described process.
La Figura 2 muestra una micrografía de formas de resistencia de las cianobacteñas en proceso de germinación. Figure 2 shows a micrograph of resistant forms of cyanobacteria in the process of germination.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
Como se ha indicado, el primer aspecto de la invención es el método para mejorar la productividad vegetal, que comprende la aplicación al terreno o medio agrícola de una cantidad eficiente de formas de resistencia de microalgas, un segundo aspecto se refiere a un método para mejorar la fertilidad de un suelo o recuperar o restaurar suelos empobrecidos, degradados o contaminados, que comprende la aplicación de una cantidad eficiente de formas de resistencia de microalgas al terreno. As indicated, the first aspect of the invention is the method to improve plant productivity, which comprises the application to the land or agricultural environment of an efficient amount of forms of microalgae resistance, a second aspect refers to a method to improve the fertility of a soil or to recover or restore impoverished, degraded or contaminated soils, which includes the application of an efficient quantity of resistant forms of microalgae to the land.
Un tercer aspecto de la invención se refiere a una composición bioactiva, que comprende las formas de resistencia de microalgas en un rango comprendido entre el 30% y el 90% del total de células en la composición. De manera preferente, la composición bioactiva comprende las formas de resistencia de microalgas y un portador aceptable en la agricultura de las formas de resistencia. El término “portador aceptable en la agricultura” se refiere a un portador que es conocido y aceptado en la formación de formulaciones para uso en la agricultura u horticultura. A third aspect of the invention refers to a bioactive composition, which comprises the resistant forms of microalgae in a range comprised between 30% and 90% of the total number of cells in the composition. Preferably, the bioactive composition comprises the resistance forms of microalgae and an agriculturally acceptable carrier of the resistance forms. The term "agriculturally acceptable carrier" refers to a carrier that is known and accepted in the formation of formulations for use in agriculture or horticulture.
Las formas de resistencia proceden, preferentemente, de microalgas del suelo o microalgas terrícolas. Las microalgas del suelo son, preferentemente, de especies seleccionadas pertenecientes a las Divisiones: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heterokontophyta y fíhodophyta. The forms of resistance preferably come from soil microalgae or terrestrial microalgae. Soil microalgae are preferably selected species belonging to the Divisions: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heterokontophyta and fíhodophyta.
Más preferentes entre las Heterokontophyta son especies de bacilariofíceas (diatomeas) y entre las Cyanophyta (cianobacterias) son estirpes de géneros como Anabaena y Nostoc. More preferred among the Heterokontophyta are species of Bacillariophyceae (diatoms) and among the Cyanophyta (cyanobacteria) are strains of genera such as Anabaena and Nostoc.
Estas divisiones y especies son las preferentes en los composiciones y usos de la invención. These divisions and species are preferred in the compositions and uses of the invention.
Estas microalgas, en condiciones deficientes para el crecimiento vegetativo (subóptimas), tienen la capacidad de desarrollar las formas de resistencia, que les permiten resistir condiciones adversas hasta que las variables ambientales y/o nutricionales sean de nuevo propicias para el desarrollo de células vegetativas. These microalgae, in poor conditions for vegetative growth (suboptimal), have the ability to develop forms of resistance, which allow them to resist adverse conditions until the environmental and/or nutritional variables are again conducive to the development of vegetative cells.
Este proceso puede reproducirse artificialmente, forzando dichas condiciones de estrés nutricional y/o ambiental. This process can be reproduced artificially, forcing said conditions of nutritional and/or environmental stress.
De manera preferente para la cianobacteria del género Anabaena se favorece el desarrollo de la forma de resistencia controlando el nivel de fósforo en el medio de cultivo de la microalga. Preferably, for the cyanobacteria of the genus Anabaena, the development of the resistance form is favored by controlling the level of phosphorus in the microalgae culture medium.
En otra forma preferente para la cianobacteria del género Nostoc se favorece el desarrollo de la forma de resistencia en condiciones de elevado aporte de carbono al medio de cultivo de la microalga. In another preferred way for the cyanobacteria of the Nostoc genus, the development of the resistance form is favored under conditions of high carbon input to the microalgae culture medium.
Preferentemente los métodos descritos más arriba además comprenden las etapas de: a) cultivar la microalga; b) inducir la forma de resistencia; c) conservar la forma de resistencia; d) germinar la forma de resistencia. Preferably the methods described above further comprise the steps of: a) cultivating the microalgae; b) induce the form of resistance; c) preserve the form of resistance; d) germinate the form of resistance.
Preferentemente la conservación se realiza en oscuridad. Más preferente a una temperatura comprendida entre -20°C y 25°C. Storage is preferably carried out in the dark. More preferably at a temperature between -20°C and 25°C.
En los ejemplos de la presente invención se demuestra la formación de las formas latentes, la posible conservación de las mismas durante el tiempo y la capacidad de germinación de las mismas una vez se encuentran en el entorno adecuado. The examples of the present invention demonstrate the formation of dormant forms, their possible conservation over time and their ability to germinate once they are in the right environment.
Ejemplos examples
Los siguientes ejemplos son a nivel ilustrativo y no tienen como objeto limitar la presente invención. The following examples are by way of illustration and are not intended to limit the present invention.
Ejemplo 1. Proceso de inducción, aislamiento y estabilización de formas de resistencia de bacilariofíceas (diatomeas) Example 1. Process of induction, isolation and stabilization of forms of resistance of bacillariophyceae (diatoms)
Se cultivaron células de una diatomea en medio líquido estándar. Tras 10 días de crecimiento, parte de las células vegetativas se transfirieron a un medio con baja concentración (10 micromolar) de nitrato sódico y 150 micromolar de ácido silícico, condiciones que propician la producción masiva de formas de resistencia. La temperatura se mantuvo a 25°C en cámara termostatada e iluminada, empleándose ciclos de 12 horas de luz: 12 horas de oscuridad. Cells from a diatom were cultured in standard liquid medium. After 10 days of growth, part of the vegetative cells were transferred to a medium with a low concentration (10 micromolar) of sodium nitrate and 150 micromolar of silicic acid, conditions that favor the massive production of forms of resistance. The temperature was maintained at 25°C in a thermostatted and illuminated chamber, using cycles of 12 hours of light: 12 hours of darkness.
Aislamiento y recolección de formas de resistencia (FR). Tras seis días de cultivo en el medio con bajo nitrato, se centrifugó la suspensión celular a 5000 x g durante 10 minutos, a temperatura ambiente. Se resuspendió la biomasa celular sedimentada en tampón Tris-EDTA-Mg. Tras nueva centrifugación, se resuspendió en un pequeño volumen de tampón Tris-Mg. Isolation and collection of forms of resistance (FR). After six days of culture in the low nitrate medium, the cell suspension was centrifuged at 5000 x g for 10 minutes at room temperature. The pelleted cell biomass was resuspended in Tris-EDTA-Mg buffer. After a new centrifugation, it was resuspended in a small volume of Tris-Mg buffer.
Se trató con ultrasonidos (20 % de intensidad máxima a intervalos (3-5) de 20 s), para destruir células vegetativas residuales. It was treated with ultrasound (20% maximum intensity at intervals (3-5) of 20 s), to destroy residual vegetative cells.
La preparación concentrada de formas de resistencia así obtenida puede mantenerse a 5°C en oscuridad hasta que se proceda a su desecación. Secado y almacenamiento: The concentrated preparation of forms of resistance thus obtained can be kept at 5°C in the dark until it is dried. Drying and storage:
Las formas de resistencia obtenidas se secaron con aire caliente a temperatura moderada, quedando listas para su posterior almacenamiento. Se realizaron controles periódicos para comprobar que las formas de resistencia mantienen su capacidad para germinar cuando se colocan en condiciones favorables (humedad, temperatura, nutrientes, etc.) The resistance forms obtained were dried with hot air at a moderate temperature, being ready for later storage. Periodic controls were carried out to verify that the resistant forms maintain their ability to germinate when placed in favorable conditions (humidity, temperature, nutrients, etc.)
Ejemplo 2. Proceso de inducción, aislamiento y estabilización de formas de resistencia de cianobacterias Example 2. Process of induction, isolation and stabilization of resistant forms of cyanobacteria
Cultivo de las estirpes Cultivation of the strains
Se han seleccionado dos estirpes de cianobacterias filamentosas fijadoras de nitrógeno atmosférico, pertenecientes a los géneros Anabaena y Nostoc, con el fin de evaluar la producción de formas de resistencia para su posterior uso en aplicaciones agrícolas. Two strains of filamentous atmospheric nitrogen-fixing cyanobacteria, belonging to the Anabaena and Nostoc genera, have been selected in order to evaluate the production of resistant forms for later use in agricultural applications.
El cultivo comenzó con la transferencia de las estirpes desde medio sólido (agar) a matraces de 250 mL con medio de cultivo estándar mantenidos a 25°C y con agitación orbital. The culture began with the transfer of the strains from solid medium (agar) to 250 mL flasks with standard culture medium maintained at 25°C and with orbital agitation.
Posteriormente, se trasvasaron a botellas de cultivo de 1 L con agitación por burbujeo de aire enriquecido con CO2, expuestas a una irradiancia luminosa de 80- 100 pmol fotones rm2 s’1, hasta obtener una concentración significativa de 0,5 g biomasa seca/L. Subsequently, they were transferred to 1 L culture bottles with CO2-enriched air bubbling agitation, exposed to a light irradiance of 80-100 pmol photons rm 2 s' 1 , until a significant concentration of 0.5 g dry biomass was obtained. /L.
Inducción de la diferenciación celular a formas de resistencia Induction of cell differentiation to forms of resistance
Los cultivos de las estirpes de cianobacterias se sometieron a estrés nutricional por limitación de fósforo. The cultures of the cyanobacterial strains were subjected to nutritional stress due to phosphorus limitation.
Después de 7-10 días de incubación en esas condiciones, cuando el cultivo hubo alcanzado valores de absorbancia a 750 nm cercanos a 1, se procedió a su centrifugación para, a continuación, refrescar con medio y volver a centrifugar. After 7-10 days of incubation under these conditions, when the culture had reached absorbance values at 750 nm close to 1, it was centrifuged, then refreshed with medium and centrifuged again.
Se transfirió el sedimento resuspendido en un volumen de 600 mL a botellas de 2-5 L con el medio pobre en fósforo y se procedió a su cultivo durante 21 días, maestreándose cada 3 días para seguir su progresión con mediciones de absorbancia a 750 nm y de la formación de formas de resistencia mediante conteo con Cámara de Neubauer. The resuspended sediment was transferred in a volume of 600 mL to 2-5 L bottles with the low-phosphorus medium and cultured for 21 days. sampling every 3 days to follow its progression with measurements of absorbance at 750 nm and the formation of resistance forms by counting with a Neubauer Chamber.
Para las estirpes de Nostoc, la presencia de una fuente orgánica de carbono en el medio de cultivo fue una condición inductora del desarrollo de formas de resistencia. For the Nostoc strains, the presence of an organic carbon source in the culture medium was an inducing condition for the development of forms of resistance.
Se centrifugaron 600 mL de suspensión celular a 5.000 r.p.m. a temperatura ambiente durante 10 min. 600 mL of cell suspension were centrifuged at 5,000 r.p.m. at room temperature for 10 min.
Se retiró el sobrenadante de la centrifugación por decantación y se recuperó el sedimento en 600 mL de medio, repitiéndose el proceso tres veces. Se transfirió el sedimento resuspendido en un volumen de 600 mL a botellas de 2-5 L con el medio suplementado con carbono y se procedió a su cultivo durante 21 días, maestreándose cada 3 días para seguir la progresión del cultivo con mediciones de absorbancia a 750 nm y de la formación de formas de resistencia mediante conteo con Cámara de Neubauer. The supernatant was removed from the centrifugation by decantation and the pellet was recovered in 600 mL of medium, repeating the process three times. The resuspended sediment was transferred in a volume of 600 mL to 2-5 L bottles with the medium supplemented with carbon and cultured for 21 days, sampling every 3 days to follow the progression of the culture with absorbance measurements at 750 nm and the formation of resistance forms by counting with the Neubauer Chamber.
Aislamiento de formas de resistencia de cianobacterias Isolation of resistant forms of cyanobacteria
Después de 17-21 días de cultivo en el medio para la inducción de formas de resistencia, se centrifugó a 3.000 r.p.m. a temperatura ambiente durante 10 minutos. After 17-21 days of culture in the medium for the induction of resistance forms, it was centrifuged at 3,000 r.p.m. at room temperature for 10 minutes.
Se resuspendió el sedimento en 600 mL de medio. La figura 1 muestra una microfotografía del concentrado de formas de resistencia de cianobacterias. The pellet was resuspended in 600 mL of medium. Figure 1 shows a photomicrograph of the concentrate of resistant forms of cyanobacteria.
Se realizó el conteo de las formas de resistencia con cámara de Neubauer y se centrifugó el concentrado de formas de resistencia, eliminándose el sobrenadante, quedando el sedimento listo para proceder al secado y ensayos de preservación y posterior germinación de las formas de resistencia. The count of the resistance forms was carried out with a Neubauer chamber and the concentrate of resistance forms was centrifuged, eliminating the supernatant, leaving the sediment ready to proceed with drying and preservation tests and subsequent germination of the resistance forms.
Secado y preservación de las formas de resistencia de cianobacterias Drying and preservation of resistant forms of cyanobacteria
Para estabilizar la preparación enriquecida en formas de resistencia de cianobacterias, el producto obtenido en el paso anterior se secó en estufa a 37°C durante 24 horas. Una vez secas, las preparaciones ricas en formas de resistencia pueden conservarse durante varios meses en recipientes cerrados y en oscuridad, tanto a temperatura ambiente (25°C) como a 4°C o incluso a temperaturas bajo cero (-17°C). To stabilize the preparation enriched in resistant forms of cyanobacteria, the product obtained in the previous step was dried in an oven at 37°C for 24 hours. Once dried, preparations rich in resistance forms can be stored for several months in closed containers in the dark, either at room temperature (25°C) or at 4°C or even at temperatures below zero (-17°C).
Germinación en suelos soil germination
Se realizaron ensayos de germinación de las formas de resistencia transcurridos 3 meses desde su producción y almacenamiento. Para que este proceso tuviera lugar, fue necesario resuspender y rehidratar la biomasa de las formas de resistencia con medio de cultivo completo. Germination tests of the resistance forms were carried out 3 months after their production and storage. For this process to take place, it was necessary to resuspend and rehydrate the biomass of the resistant forms with complete culture medium.
Este proceso de germinación (ver Figura 2) también tiene lugar en el suelo, en condiciones (humedad, temperatura y disponibilidad de nutrientes) que permiten la propagación y crecimiento de las cianobacterias. This germination process (see Figure 2) also takes place in the soil, under conditions (moisture, temperature and nutrient availability) that allow the propagation and growth of cyanobacteria.
Se siguió el siguiente protocolo para los ensayos de germinación de las formas de resistencia: The following protocol was followed for the germination tests of the resistance forms:
Se resuspendieron las formas de resistencia desecadas en un pequeño volumen de medio completo. Dried resistance forms were resuspended in a small volume of complete medium.
Se añadió la cantidad de medio necesaria para que la absorbancia a 750 nm quede entre 0,2 y 0,4. The necessary amount of medium was added so that the absorbance at 750 nm was between 0.2 and 0.4.
La suspensión de formas de resistencia resultante se mantuvo en un matraz de 100 mL con agitación en un agitador orbital y fotopehodo natural. The resulting resistance forms suspension was kept in a 100 mL flask with agitation in an orbital shaker and natural photoperiod.
Se siguió el progreso de la absorbancia y se observaron muestras al microscopio cada 5 días, verificándose la germinación de las formas latentes a células vegetativas y filamentos. The progress of the absorbance was followed and samples were observed under a microscope every 5 days, verifying the germination of the latent forms to vegetative cells and filaments.
Como se ha dicho, la Figura 2 muestra la germinación de la forma de resistencia. As said, Figure 2 shows the germination of the resistance form.
En este ejemplo se ha demostrado la posibilidad de generar la forma de resistencia, de almacenarla en el tiempo y de germinarla tras un largo periodo de latencia. In this example, the possibility of generating the form of resistance, storing it over time and germinating it after a long period of latency has been demonstrated.

Claims

REIVINDICACIONES
1. Método para mejorar la productividad de cultivos agrícolas caracterizado porque comprende la aplicación al terreno o medio agrícola en el cual se cultivan las plantas, de una cantidad eficiente de formas de resistencia de microalgas. 1. Method for improving the productivity of agricultural crops characterized in that it comprises the application to the land or agricultural medium in which the plants are grown, of an efficient amount of forms of microalgae resistance.
2. Método para mejorar la fertilidad de un terreno, caracterizado porque comprende la aplicación al terreno o medio de una cantidad eficiente de formas de resistencia de microalgas. 2. Method for improving the fertility of a land, characterized in that it comprises the application to the land or medium of an efficient amount of forms of microalgae resistance.
3. Método de recuperación y restauración de suelos empobrecidos, degradados o contaminados, caracterizado porque comprende la aplicación al terreno o medio de una cantidad eficiente de formas de resistencia de microalgas. 3. Method of recovery and restoration of impoverished, degraded or contaminated soils, characterized in that it comprises the application to the land or medium of an efficient quantity of forms of microalgae resistance.
4. Método según las reivindicaciones 1 a 3 caracterizado porque las formas de resistencia proceden de microalgas del suelo. 4. Method according to claims 1 to 3, characterized in that the forms of resistance come from soil microalgae.
5. Método según las reivindicaciones 1 a 4 caracterizado porque las microalgas son seleccionadas entre especies de las Divisiones: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heterokontophyta y fíhodophyta. 5. Method according to claims 1 to 4, characterized in that the microalgae are selected from species of the Divisions: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heterokontophyta and fíhodophyta.
6. Método según una cualquiera de las reivindicaciones 1 a 5 caracterizado porque las microalgas se seleccionan entre especies de bacilariofíceas (diatomeas) y entre estirpes de cianobacterias de géneros como Anabaena y Nostoc. 6. Method according to any one of claims 1 to 5, characterized in that the microalgae are selected from species of bacillariophyceae (diatoms) and from strains of cyanobacteria of genera such as Anabaena and Nostoc.
7. Método según alguna de las reivindicaciones 1 a 6 caracterizado porque además comprende las etapas de: a) cultivar la microalga; b) inducir la forma de resistencia; c) conservar la forma de resistencia; d) germinar la forma de resistencia. 7. Method according to any of claims 1 to 6, characterized in that it further comprises the steps of: a) cultivating the microalgae; b) induce the form of resistance; c) preserve the form of resistance; d) germinate the form of resistance.
8. Composición bioactiva caracterizada porque comprende formas de resistencia de microalgas en un rango comprendido entre el 30%y el 99% respecto del total de células de microalgas presentes en la composición. 8. Bioactive composition characterized in that it comprises forms of microalgae resistance in a range between 30% and 99% with respect to the total number of microalgae cells present in the composition.
9. Composición bioactiva según la reivindicación 8 caracterizada porque comprende un portador aceptable en la agricultura. 9. Bioactive composition according to claim 8, characterized in that it comprises an agriculturally acceptable carrier.
10. Composición bioactiva según las reivindicaciones 8 a 9 caracterizada porque las microalgas son seleccionadas entre especies de las Divisiones: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heterokontophyta y fíhodophyta. 10. Bioactive composition according to claims 8 to 9, characterized in that the microalgae are selected from species of the Divisions: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heterokontophyta and fíhodophyta.
11. Composición bioactiva según la reivindicación 9 caracterizada porque las algas se seleccionan entre especies de bacilariofíceas (diatomeas) y entre estirpes de cianobacterias de géneros como Anabaena y Nostoc. 11. Bioactive composition according to claim 9, characterized in that the algae are selected from species of bacillariophyceae (diatoms) and from strains of cyanobacteria of genera such as Anabaena and Nostoc.
12. Uso de las formas de resistencia de microalgas para mejorar la productividad de cultivos agrícolas y la fertilidad del suelo, o recuperar o restaurar suelos empobrecidos, degradados o contaminados. 12. Use of resistant forms of microalgae to improve the productivity of agricultural crops and soil fertility, or recover or restore impoverished, degraded or contaminated soils.
13. Uso según la reivindicación 12 caracterizado porque las microalgas son seleccionadas entre especies de las Divisiones: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heterokontophyta y Flhodophyta. 13. Use according to claim 12, characterized in that the microalgae are selected from species of the Divisions: Chlorophyta, Cryptophyta, Cyanophyta, Euglenophyta, Heterokontophyta and Flhodophyta.
14. Uso según la reivindicación 13 caracterizado porque las algas se seleccionan entre especies de bacilariofíceas (diatomeas) y entre estirpes de cianobacterias de géneros como Anabaena y Nostoc. 14. Use according to claim 13, characterized in that the algae are selected from species of bacillariophyceae (diatoms) and from strains of cyanobacteria of genera such as Anabaena and Nostoc.
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