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    • 专利摘要:
    Liquid biofertilizer that comprises strains of Azospirillum brasilense and Pantoea dispersa and method of obtaining it. Liquid biofertilizer comprising the strains Azospirillum brasilense CECT 5802 and Pantoea dispersa CECT 5801. Method for stimulating the growth of a plant, which comprises applying said liquid biofertilizer to said plant. Method of obtaining a liquid biofertilizer, which comprises cultivating said strains in first liquid culture media and adding the culture broths obtained to a second liquid culture medium. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2789973A1
    申请号:ES201930369
    申请日:2019-04-26
    公开日:2020-10-26
    发明作者:López-Román Jorge Malo；Soto-Navarro Carmen María Mengual；Carpena Juan José García；Cayuelas Alberto Botella；Gómez Arturo Jesús Carrillo；Fernández Guillermo Vázquez；Picó Isidro Blanca；Belmonte José Manuel Casanova
    申请人:Probelte S A U；
    IPC主号:
    专利说明:

    [0004] TECHNICAL SECTOR
    [0006] The present invention refers to a product for biological fertilization consisting of a liquid formulation that contains two strains of bacteria of the genera Azospinllum and Pantoea, with the ability to fix atmospheric nitrogen, solubilize phosphates as well as other mineral nutrients in the soil and produce high amounts of substances that stimulate plant growth. Said microorganisms have been formulated in liquid form, which guarantees easy application and high stability in cell viability and provides sufficient organic and mineral nutrients to facilitate the colonization of plant roots.
    [0008] BACKGROUND OF THE INVENTION
    [0010] The use of fertilizers is essential for maintaining high yields in crops. Through chemical fertilization, significant amounts of nitrogen, phosphorus and potassium are added to the soil, as well as other mineral elements, however, their availability is very low, since a fraction remains immobilized in the soil, forming insoluble compounds that cannot be assimilated by the plants and another is washed through a leaching process, which in addition to economic losses, generates a significant environmental pollution problem.
    [0012] In the case of phosphorus, in particular, a significant part of the added soluble phosphates is insolubilized by iron and aluminum in acidic soils and by calcium in calcareous soils, progressively converting into less assimilable forms. As a result of the various retention mechanisms, most of the phosphorus applied through fertilization cannot be used by crops and is retained in the soil in an insoluble form. Given this phenomenon and the cyclical application of fertilizers, the concentration of phosphorus in the soil has increased remarkably, so that in many soils long-term crops could be established if these reserves could be exploited economically. Adding excess inorganic fertilizers can also lead to environmental problems such as groundwater pollution and eutrophication of waterways. Therefore, it is of great interest to investigate management strategies that are capable of improving the efficiency of phosphorus fertilization, increasing crop yields and reducing environmental pollution caused by the loss of phosphorus from the soil (Alori et al., 2017).
    [0014] The importance of microorganisms in the cycle of nutrients in the soil and their role in plant nutrition is well known. Its active participation in the decomposition and mineralization of organic matter, as well as in the fixation and release of nutrients from the soil, is crucial for the maintenance of plant productivity. The interactions established between soil microorganisms and plant roots satisfy important nutritional requirements for both. The roots are directly influenced by the composition and density of the microbial community that develops in them, this being known as the "Rhizosphere Effect", which depends particularly on the plant and its physiological maturity. The practice of inoculating plants with microorganisms is well known in the state of the art (Vessey 2003).
    [0016] In nature, there are numerous microorganisms in the rhizosphere capable of releasing phosphorus from the soil through solubilization and mineralization (Bhattacharyya and Jha, 2012). This group of microorganisms is known as phosphorous solubilizing microorganisms (PSM). Many species of soil fungi and bacteria can solubilize phosphorus in vitro and some of them increase the bioavailability of insoluble phosphorus in the soil for use by plants (Zhu et al., 2011). They solubilize insoluble inorganic (mineral) phosphorus and mineralize insoluble organic phosphorus (Sharma et al., 2013).
    [0018] A group of microorganisms that is of notable importance in this phenomenon is that which participates in the solubilization of phosphorus from sources that would otherwise be inaccessible to plants.
    [0020] Phosphate solubilizing microorganisms have been isolated in practically all the soils tested, although the number and proportion of these varies according to the type of soil, the climate and other factors such as the historical evolution of the soil. Many microorganisms are capable of assimilating insoluble phosphorus from the soil, releasing a part of it in the form of soluble phosphates that in turn can be used by plants, thus contributing to plant nutrition. In general it is accepted that the solubilization of phosphates in the soil is due to the production of chelating organic acids and oxo acids, from sugars (Seshachala and Tallapragada, 2012).
    [0021] Procedures have been described in which phosphate solubilizing microorganisms have been used in fertilization (Yu et al., 2011).
    [0023] The methods used in the isolation of phosphate solubilizing microorganisms are based fundamentally on the use of insoluble inorganic phosphorus salts, such as hydroxyapatite and tricalcium phosphate, in agarized culture media. For the use of these salts, the microorganism produces organic acids that, when diffused in the medium, cause a decrease in pH and consequently the formation of a halo of transparency in the vicinity of the colony. However, it has been pointed out that agarized culture media do not always detect the most suitable microorganisms for the solubilization of phosphates, a scheme and selection of this type of microorganisms has been proposed as an alternative, using shaken liquid cultures as an alternative, highlighting the importance of certain components of the medium and their concentration in the results achieved and proposing a culture medium to carry out the selection (Nautiyal 1999). Other authors, working with agarized media, concluded that the pH buffering capacity of the culture medium used in the isolation has a decisive importance in the selection of strains that produce sufficient quantities of organic acids so that they present a real interest in biofertilization, they described that gluconic acid is the main component of organic acids produced by Enterobacter asburiae and that the enzyme glucose dehydrogenase, responsible for the production of gluconic acid, is regulated by phosphate starvation, that is, low concentrations of soluble phosphates stimulate the production of acids in this type of microorganism (Gyaneshwar et al., 1999).
    [0025] The Enterobacter and Pantoea genera have been used in agriculture as phosphate solubilizers and for protection against plant diseases (Beltrán-Pineda, 2014). Pantoea dispersa is a species with genes that encode the enzyme for the detoxification of albicidin. Said genes and enzyme have been used to obtain transgenic plants resistant to fungal diseases, which demonstrates their ability to protect against fungal plant diseases. There are different documents of the state of the art that point to Pantoea dispersa as a solubilizing species of phosphates (Fernández et al., 2008).
    [0027] Another aspect that occupies a very important place in practice is the use of atmospheric nitrogen-fixing microorganisms in the rhizosphere. Numerous microorganisms have been used for this function, among which are bacteria of genera such as Rhizobium, Azotobacter and Azospirillum and fungi of the genera Saccharomyces, Hansenula and Aspergillus, among others.
    [0029] Nitrogen is an abundant element, which makes up almost 80% of the earth's atmosphere and a very scarce nutritional part. The paradox is easily solved: atmospheric nitrogen is inert and cannot be used by most organisms, and can only be incorporated into biological synthesis when it has been "fixed" or combined with certain elements such as hydrogen or oxygen. Bacteria are capable of fixing 1.5 x 108 metric tons per year, a significant part of which is synthesized using the Haber-Bosch process.
    [0031] Experiments carried out in Brazil determined the significant contribution of N2 fixed to plants by different microorganisms, with Azospirillum being among the main genera. Subsequent studies to quantify the biological fixation of nitrogen (BNF) in sugar cane in this same country showed that practically 65% of the total accumulated N2 was derived from BNF, which represents around 150 kg N2 x ha- 1 x year-1, which led to the recommendation to minimize the use of nitrogen fertilizers.
    [0033] It is known that different plant species produce different effects on the rhizosphere, and it is also known that the strains isolated in a type of plant species have a totally different effect on the rhizosphere from which it was isolated compared to other crops. However, Azospirillum brasilense is able to colonize plants of different species by attaching itself to the roots. There are works that have demonstrated the ubiquity of the genus Azospirillum, carrying out its isolation in varied crops, as well as in regions with very dissimilar climates (Fukami et al., 2016).
    [0035] It has been described that when inoculating different plant cultures with species of the genus Azospirillum, changes in the morphology of the roots appeared and it has been determined that, in the first three weeks after germination, the number of root hairs and roots increases. with inoculation, which produces an increase in the absorption surface of the roots.
    [0037] In studies of inoculation of wheat plants with Azospirillum brasilense, it has been observed that small cell aggregates formed on the surface of the inoculated roots, and when cutting, it has been found that some bacteria penetrated the young cells of roots, but there were many more in the intercellular spaces, as well as in the epidemic layers of the cells. It has been found, in turn, that the adsorption of this bacterium to the roots is weak and is carried out by metabolism processes dependent on the bacteria, mediated by recognition factors. The ability of this species to move in the soil to colonize plant roots and the ability to colonize, in addition to cereals, different crops has also been described, producing morphological changes similar to those that occurred in the Graminaceae family on their roots. .
    [0039] The main element involved in the Azospirillum-plant relationship is not only nitrogen, also phosphorus and potassium play a fundamental role in this relationship. Depending on the strain used, there is a quantitative change in the uptake of minerals by the plant, significantly increasing some crops. According to the "theory of multiple mechanisms", the bacterium acts with a pattern of cumulative or sequential effects, as a result of mechanisms that occur simultaneously or consecutively (Bashan and De-Bashan 2010).
    [0041] At present, the use of this genus is very common in the production of biological fertilizers.
    [0043] It should be noted that many authors coincide in pointing out that the beneficial effects produced by inoculation with microorganisms on plant growth are not only due to the solubilization of phosphates or the biological fixation of nitrogen. There are mechanisms such as the production of phytohormones and siderophores or the activity of the enzyme 1-aminocyclopropane 1-carboxylate deaminase, among others, that notably contribute to this effect (Fukami et al., 2018).
    [0045] Among the microbial relationships that take place in the rhizosphere are the cooperation calls that are established between those microbial groups that carry out complementary metabolisms, this may be the case of some phosphate-solubilizing bacteria that excrete organic acids into the environment. which, in turn, constitute the main source of carbon in some genera of nitro-fixing bacteria, thus allowing both groups to coexist, at the same time having a double effect on improving crops and protecting against diseases.
    [0047] The associative development of the genera Azospirillum and Pantoea has been carried out successfully in the biological fixation of nitrogen, showing that there are no incompatibilities between both genders and that by showing differences in metabolic regulation mechanisms they can perform different functions in a given ecosystem (Flores et al., 2010; Del Amor and Cuadra, 2011; Schoebitz et al., 2014; Mengual et al., 2014). Mixed cultures of soil microorganisms with different metabolic capacities can develop cooperative relationships among themselves and with plants that make the adsorption of nutrients by them more efficient and can also produce substances that stimulate plant growth, increasing the productivity of crops and plants. protecting them against pathogenic microorganisms.
    [0049] The practice of inoculating plants with microorganisms using mixed cultures (also called consortia) that enhance phenomena such as the increase in the efficiency of phosphorus uptake by the roots, the biological fixation of nitrogen, the stimulation of plant growth by the production of substances Regulators of plant growth, as well as siderophores and protection against diseases produced by pathogenic microorganisms, among others, has been revealed as the most effective in biofertilization (Nuti and Giovannetti, 2015).
    [0051] The physical form of a biofertilizer is a determining factor in the practical result that said biofertilizer provides. The viability of the organisms present in a biofertilizer during production, formulation, storage, transport / distribution and application in the field is directly related to the growth potential of the plants. This limits its use due to compatibility, stability and survival problems in different soil conditions. Therefore, an improved shelf life could be the key to a greater generalization of the application of biofertilizers (Brar et al., 2012).
    [0053] In a biological fertilizer, the microorganisms must remain viable, dormant or metabolically active. This factor has two determining aspects, the durability of the product and its ability to colonize the roots of the crops to be stimulated or protected, once applied in the field. The use of cell-free preparations is a common practice in agricultural biotechnology and is very effective when working with microorganisms capable of forming resistance structures, as in the case of bacteria of the Bacillus genus . This problem has another character when it comes to cells that do not have the capacity to form resistance structures, since the crops lose viability over time and also their survival capacity in the soil is very low. Many of the failures that have occurred in the field of Biofertilization and bioprotection are related to this phenomenon. The inoculation of soils with microorganisms capable of promoting the productivity of plants is a very complex process in which a number of factors can have a determining effect. For this reason, it is essential that the biological fertilizer is capable of preserving cell viability under adverse conditions for long periods of time and guaranteeing as far as possible the colonization capacity of the roots once applied in the field. One of the most important criteria to take into account for this is to achieve biological fertilizer preparations that consistently release an appreciable number of viable cells (Pindi and Satyanarayana, 2012).
    [0055] In common practice, for a better shelf life of the biofertilizer formulation, a carrier or a mixture of such carrier materials is selected based on the viability of the microorganisms mixed with them. Similarly, its enrichment with nutrients is the other strategy to improve shelf life by allowing the microorganism to maintain and grow in a non-competitive microenvironment (Yardin et al., 2000).
    [0057] Document ES2234417A1 describes a biological fertilizer comprising bacterial strains of Azospirillum brasilense and Pantoea dispersa immobilized on a solid support. Specifically, said document describes the Azospirillum brasilense CECT 5802 strain and the Pantoea dispersa strain CECT 5801.
    [0059] Document WO2009027544A1 describes a biological fertilizer that comprises a solid support on which the bacterial strains Azospirillum brasilense CECT 5802 and Pantoea dispersa CECT 5801 and, additionally, indole-3-acetic acid or an indole-3-acetic acid promoter have been immobilized. .
    [0061] The change from a biofertilizer that comprises microorganisms immobilized on a solid support to a biofertilizer in liquid form is associated with a significant reduction in the viability of said microorganisms and a decrease in the period in which the product remains viable while maintaining efficacy. The shelf life of a liquid biofertilizer is a major concern (Brar et al., 2012).
    [0063] However, liquid biofertilizers have advantages over biofertilizers in which the microorganisms contained in them are immobilized on a solid support, such as improvements in the mode of application, being able to apply by irrigation by dripping, spraying or by aerial means. It is not possible to apply biofertilizers immobilized on a solid support by drip irrigation or by liquid spraying. Furthermore, liquid biofertilizers allow their application to other parts of the plants other than the roots, as they can be sprayed on the aerial parts of the plants (stems, leaves, fruits, flowers, etc.). Unlike biofertilizers based on solid carriers, liquid formulations allow to include a sufficient amount of nutrients, cell protectants and inducers of cell formation to guarantee a long shelf life.
    [0065] In order for obtaining a biofertilizer to be attractive from an economic point of view, it is necessary to make an appropriate selection of the medium in which its production is to be carried out. For this, it is essential to carefully select the raw materials and materials to be used, so that costs are minimized. For this purpose, it is necessary that the raw materials and their concentration in the fermentation medium make it possible to obtain a high cellular concentration or of the desired product at a minimum cost. Products of natural origin are very appropriate for this purpose, given their low cost and their harmlessness for the environment. These can have the additional advantage of stimulating desired effects if they are included in the final preparation. Tomato concentrates have been used in the production of gibberellins, as a nitrogen source and as a carbon source for growth and the production of metabolites of microbial origin due to their high content of organic carbon. On the other hand, collagen hydrolyzate for use in agriculture is a very suitable and cheap source of organic nitrogen, even for microorganisms incapable of producing proteolytic enzymes. The combination of these two sources offers a universal culture medium, very low cost and therefore very attractive for carrying out large-scale fermentation processes. In addition, its natural origin allows a more ecological agriculture that today's society demands.
    [0067] From all the above, the interest that arouses the development of a liquid biofertilizer that includes bacterial strains is derived, in which said bacterial strains maintain their viability for a long time while maintaining their effectiveness, thus obtaining the advantages, previously mentioned, that they provide liquid biofertilizers versus biofertilizers immobilized on solid supports.
    [0069] OBJECT OF THE INVENTION
    [0071] The object of the invention is a product for biological fertilization consisting of a liquid formulation containing two strains of bacteria of the genera Azospinllum and Pantoea, with the ability to fix atmospheric nitrogen, solubilize phosphates as well as other mineral nutrients from the soil and produce high amounts of substances that stimulate plant growth. These microorganisms have been formulated in liquid form, which guarantees easy application and high stability in cell viability and provides sufficient organic and mineral nutrients to facilitate the colonization of plant roots. Another object of the invention is a method of stimulating the growth of a plant, which comprises applying the liquid biofertilizer of the invention to said plant, a method of obtaining the liquid biofertilizer of the invention and a liquid biofertilizer obtainable according to said method of obtaining.
    [0073] DESCRIPTION OF THE INVENTION
    [0075] The present invention refers to a liquid biofertilizer that comprises a strain of Azospirillum brasilense deposited in the Spanish Collection of Type Cultures (CECT) with deposit number CECT 5802, a strain of Pantoea dispersed deposited in the CECT with deposit number CECT 5801, at least one inorganic salt and a soluble molasses concentrate.
    [0077] The present invention differs from the state of the art in that it is a biofertilizer formulated in liquid form, preferably in an aqueous medium suitable to maintain the viability of the biofertilizer strains, maintaining between 60% and 70% cell viability of both strains after one year of conservation, at temperatures not exceeding 30 ° C.
    [0079] Said suitable aqueous medium is responsible for maintaining the viability of the strains in the liquid formulation. Without such a suitable aqueous medium, the strains would lose their viability in a very short time.
    [0081] The present invention provides such an aqueous medium suitable for the maintenance of the strains in the liquid biofertilizer. Said liquid medium must comprise at least one inorganic salt and a soluble molasses concentrate to maintain the viability of the biofertilizer strains for a long time. Said liquid medium may comprise pH regulating agents and one or more antifoaming agents.
    [0083] The application dose is significantly reduced in the liquid biofertilizer of the invention, which comprises the bacterial strains Azospirillum brasilense CECT 5802 and Pantoea dispersa CECT 5801, with respect to a biofertilizer comprising the same strains, immobilized on a solid support, because said liquid biofertilizer can make bacteria available to plants faster than in the form of immobilized on a solid support. This reduction in the dose is approximately 20 times, going from an application of biofertilizer immobilized on a solid support of 300 kg / ha to an application of said liquid biofertilizer of 15 L / ha. Additionally, the bacterial strains present in the liquid biofertilizer of the invention remain viable, in said liquid biofertilizer, for at least one year maintaining their efficacy.
    [0085] The complex technical problem to be solved would therefore consist in providing a biofertilizer comprising bacterial strains, formulated in liquid form, which allows its application in plants with a reduced dose and in which said bacterial strains remain viable for at least one year. maintaining its effectiveness.
    [0087] The present invention, defined by the object of the claims, provides a solution to said technical problem.
    [0089] The advantages of said liquid biofertilizer over biofertilizers immobilized on a solid support are based on the liquid formulation form itself. Said liquid biofertilizer, as it is a liquid product where the bacteria remain viable for at least one year, favors the mode of application thereof, and can be applied by drip irrigation, spraying or by aerial means. The application by drip irrigation and liquid spray is not possible with biofertilizers immobilized on a solid support.
    [0091] In a preferred embodiment, said biofertilizer is an aqueous suspension.
    [0093] In another preferred embodiment, said biofertilizer has a pH between 6 and 8. For this, suitable pH regulating agents are used.
    [0095] In another preferred embodiment, said biofertilizer further comprises at least one pH regulating agent and at least one antifoam agent. Even more preferably, said biofertilizer comprises the pH regulating agents malic acid and KOH. Even more preferably, said antifoam agent is polypropylene glycol.
    [0097] In a more preferred embodiment, said inorganic salt is selected from the group consisting of (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4 and CaCl2. In one embodiment still more preferred, said biofertilizer comprises the inorganic salts (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4 and CaCl2.
    [0098] Inorganic salts and soluble molasses concentrate are ingredients used as sources of nutrients in the liquid culture medium in which the strains of the invention grow, necessary to maintain the viability of the microorganisms present in the biofertilizer.
    [0100] In another preferred embodiment, said pH regulating agents are malic acid and KOH.
    [0102] In another preferred embodiment, said biofertilizer of the invention comprises (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4, CaCl2, soluble molasses concentrate, malic acid, KOH and polypropylene glycol. More preferably, said biofertilizer of the invention comprises (NH4) 2SO40.5-3.5% (w / w), K2HPO40.1-1% (w / w), ZnSO4-7H2O 0.005-0.1% (w / p), MnSO4-H2O 0.005-0.1% (p / p), CuSO4-5H2O 0.001-0.01% (p / p), MgSO4-7H2O 0.001-0.01% (p / p), CaCl2- 2H2O 0.001-0.01% (w / w), soluble molasses concentrate 0.05-2% (w / w), malic acid 0.01-0.1% (w / w), KOH 0.005-1% (w / w) and polypropylene glycol 0.001-0.1% (w / w). Even more preferably, said biofertilizer of the invention comprises (NH4) 2SO41.5-2.5% (w / w), K2HPO40.18-0.20% (w / w), ZnSO4-7H2O 0.01-0, 02% (p / p), MnSO4-H2O 0.01-0.02% (p / p), CuSO4-5H2O 0.001-0.002% (p / p), MgSO4-7H2O 0.003-0.004% (p / p) , CaCl2-2H2O 0.001-0.002% (w / w), soluble molasses concentrate 0.25-0.75% (w / w), malic acid 0.035-0.045% (w / w), KOH 0.02-0 , 1% (w / w) and polypropylene glycol 0.005-0.01% (w / w).
    [0104] In another preferred embodiment, said biofertilizer further comprises KH2PO4 and / or NH4Cl.
    [0106] In another preferred embodiment, said biofertilizer further comprises one or more ingredients selected from the group consisting of tomato paste, collagen hydrolyzate and yeast extract. In another even more preferred embodiment, said biofertilizer comprises tomato paste, collagen hydrolyzate and yeast extract.
    [0108] Tomato paste is a natural product used as a carbon source in the liquid culture medium in which the strains of the invention grow.
    [0110] Collagen hydrolyzate is a natural product used as a nitrogen source in the liquid culture medium in which the strains of the invention grow.
    [0111] Natural yeast extract is a natural product rich in vitamins, especially B complex, amino acids and other growth factors. It is used as a source of nutrients in the liquid culture medium in which the strains of the invention grow.
    [0113] In another preferred embodiment, said biofertilizer further comprises KH2PO4, NH4Cl, tomato paste, collagen hydrolyzate and yeast extract. More preferably, said biofertilizer comprises KH2PO40.05-0.3% (w / w), NH4Cl 0.01-2% (w / w), tomato paste 0.5-3% (w / w), hydrolyzed of collagen 0.01-2% (w / w) and yeast extract 0.01-2% (w / w).
    [0115] The present invention also relates to a method of stimulating the growth of a plant, which comprises applying said liquid biofertilizer to said plant.
    [0117] In a preferred embodiment of said method of stimulating the growth of a plant, the application of said liquid biofertilizer is selected from the group consisting of fertigation, spraying and drip irrigation.
    [0119] In another preferred embodiment of said method of stimulating the growth of a plant, the application dose of said liquid biofertilizer is between 5 and 30 L / ha. In a more preferred embodiment of said method of stimulating the growth of a plant, said application dose is between 10 and 20 L / ha.
    [0121] In another preferred embodiment of said method of stimulating the growth of a plant, said plant is selected from the group consisting of lettuce, broccoli and tomato.
    [0123] The present invention also relates to a method for obtaining a liquid biofertilizer, comprising:
    [0124] (a) cultivate a strain of Azospirillum brasilense deposited in the Spanish Collection of Type Cultures (CECT) with deposit number CECT 5802 and a strain of Pantoea dispersed deposited in the CECT with deposit number CECT 5801 in separate first liquid fermentation media and appropriate for each strain, at a temperature between 25 ° C and 35 ° C, under stirring and aeration and
    [0125] (b) cold add the fermentation broths obtained in the previous stage to a second fresh liquid medium, previously sterilized.
    [0127] In a preferred embodiment of said method for obtaining the biofertilizer of the invention, said biofertilizer is aqueous, more specifically an aqueous suspension.
    [0128] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, the first liquid fermentation media comprise at least one of the ingredients selected from the group consisting of tomato paste, collagen hydrolyzate and yeast extract. In another more preferred embodiment of said production method, the first liquid fermentation media comprise tomato paste, collagen hydrolyzate and yeast extract. The addition of these products of natural origin is especially appropriate to use these biofertilizers in organic farming.
    [0130] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, the first liquid fermentation media also comprise at least one inorganic salt selected from the group consisting of K2HPO4, KH2PO4 and NH4Cl. In another more preferred embodiment of said method for obtaining the biofertilizer of the invention, the first liquid fermentation media comprise the inorganic salts K2HPO4, KH2PO4 and NH4Cl.
    [0132] In another embodiment of said method for obtaining the biofertilizer of the invention, the first liquid fermentation media comprise K2HPO4, KH2PO4, NH4Cl, tomato paste, collagen hydrolyzate and yeast extract. More preferably, the first liquid fermentation media comprise K2HPO41-6% (w / w), KH2PO40.01-0.6% (w / w), NH4Cl 0.02-4% (w / w), tomato paste 1-6% (w / w), collagen hydrolyzate 0.02-4% (w / w) and yeast extract 0.02-4% (w / w).
    [0134] In another embodiment of said method for obtaining the biofertilizer of the invention, the second liquid fresh medium comprises at least one inorganic salt, a soluble molasses concentrate, at least one pH regulating agent and at least one antifoam agent.
    [0136] In a more preferred embodiment of said method for obtaining the biofertilizer of the invention, said inorganic salt is selected from the group consisting of (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4 and CaCl2. In a more preferred embodiment of the method for obtaining the biofertilizer of the invention, the second liquid fresh medium comprises the inorganic salts (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4 and CaCl2.
    [0137] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, the second fresh liquid medium comprises the pH regulating agents malic acid and KOH.
    [0139] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, said antifoam agent is polypropylene glycol.
    [0141] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, the second fresh liquid medium comprises (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4, CaCl2, a soluble concentrate of molasses, malic acid, KOH and polypropylene glycol . More preferably, the second fresh liquid medium comprises (NH4) 2SO41-7% (w / w), K2HPO40.001-0.2% (w / w), ZnSO4-7H2O 0.001-0.2% (w / w) , MnSO4-H2O 0.001-0.2% (p / p), CuSO4-5H2O 0.001-0.02% (p / p), MgSO4-7H2O 0.001-0.02% (p / p), CaCl2-2H2O 0.001 -0.02% (w / w), soluble molasses concentrate 0.1-2% (w / w), malic acid 0.02-0.2% (w / w), KOH 0.01-2% (w / w) and polypropylene glycol 0.001-0.2% (w / w). More preferably, the second fresh liquid medium comprises (NH4) 2SO43-5% (w / w), K2HPO40.04-0.06% (w / w), ZnSO4-7H2O 0.02-0.04% (w / p), MnSO4-H2O 0.02-0.04% (p / p), CuSO4-5H2O 0.002-0.004% (p / p), MgSO4-7H2O 0.006-0.008% (p / p), CaCl2-2H2O 0.002 -0.004% (w / w), soluble molasses concentrate 0.5-1.5% (w / w), malic acid 0.07-0.09% (w / w), KOH 0.04-0, 2% (w / w) and polypropylene glycol 0.01-0.02% (w / w).
    [0143] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, between 20% and 30% (w / w) of the fermentation broths obtained in step (a) are added to said second fresh liquid medium.
    [0145] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, the cultures of step (a) are carried out for a time between 8 and 20 hours.
    [0147] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, the stirring speed in step (a) is between 400 and 800 r.p.m.
    [0149] In another preferred embodiment of said method for obtaining the biofertilizer of the invention, the aeration in step (a) is between 0.5 and 2 L / min.
    [0151] The present invention also relates to a liquid biofertilizer obtainable according to said method of obtaining the invention.
    [0152] Field trials have been carried out under production conditions in the open air and in the greenhouse with said liquid biofertilizer, with very satisfactory results. Such field tests are described in Examples 3-5.
    [0154] Unless otherwise defined, all technical and scientific terms have the same meaning as those commonly understood by a person skilled in the field of the invention. Methods and materials similar and equivalent to those described herein can be used in the practice of the present invention.
    [0156] Throughout the description and claims, the term "comprises", "comprising" and their variants are not limiting in nature and therefore are not intended to exclude other technical characteristics. The term "comprises", "comprising" and their variants, throughout the description and claims, specifically includes the term "consists of", "consisting of" and their variants.
    [0158] Definitions
    [0160] In the present specification, the term "viability" refers to the fact that the microorganisms in the liquid biofertilizer maintain their ability to reproduce, to colonize the roots and, therefore, maintain their ability to exert their biofertilizer function. This viability means that the biofertilizer is stable and functional during storage under suitable conditions. In particular, both strains of the liquid biofertilizer maintain between 60% and 70% cell viability after one year of conservation, at temperatures no higher than 30 ° C. The terms "viability", "viable" and the like have, for the purposes of this specification, the same meaning as the terms "stability", "stable" and the like.
    [0162] As used herein, the term "aqueous liquid biofertilizer" refers to a liquid biofertilizer in which the majority solvent is water. Said aqueous liquid biofertilizer comprises at least one inorganic salt and a soluble molasses concentrate and can comprise acids, hydroxides, polypropylene glycol and other ingredients. Examples of other ingredients are: tomato paste, collagen hydrolyzate, and yeast extract. Said liquid biofertilizer may consist of a buffered formulation.
    [0163] In the present specification, the term "soluble molasses concentrate" refers to a product obtained from industrial fermentation processes for the production of yeast, alcohol and organic acids on molasses substrate. Molasses is obtained from sugar crystallization procedures and is a residue of this process from which no more sugar can be obtained by physical methods. Molasses can be obtained from sugar cane.
    [0165] As used herein, the term "pH regulating agents" refers to chemicals that adjust the pH of a solution to a desired value or are capable of maintaining the pH of a solution within a narrow range. Examples of regulating agents are carboxylic acids, dicarboxylic acids, malic acid, metal hydroxides, such as KOH, NaOH, etc.
    [0167] As used herein, the term "antifoam agent" refers to a chemical that prevents foaming in a solution.
    [0169] In the present specification, the term "collagen hydrolyzate" refers to a product obtained from biological animal tissues, such as skin, bones, bones, scales, of porcine, beef, chicken, fish, etc. origin. The collagen present in these tissues undergoes a hydrolysis process that separates the polypeptide chains of collagen. These chains are then fragmented into smaller segments, using chemicals (chemical hydrolysis) or proteolytic enzymes (enzymatic hydrolysis). It is a product used in the preparation of culture media for microorganisms as a source of nitrogen.
    [0171] As used herein, the term "yeast extract" refers to a water soluble extract formed by the autolysate of yeast cells. It is a product rich in vitamins, especially the B complex, amino acids and other growth factors. It is used in the preparation of culture media for microorganisms as a source of nutrients.
    [0173] As used herein, the term "fertigation" refers to a fertilizer application technique that allows the simultaneous application of water and fertilizers through the irrigation system.
    [0174] As used herein, the term "spraying" refers to a fertilizer application technique that distributes a particulate liquid.
    [0175] DESCRIPTION OF REALIZATION MODES
    [0177] Example 1. Obtaining the liquid biofertilizer
    [0179] Obtaining fermentation broths from the strains
    [0181] A tomato culture medium was prepared, a culture medium that comprises tomato paste as a carbon source, hydrolyzed collagen from animal skin of fertilizer use as a source of organic nitrogen and whose composition is that described in Table 1 .
    [0183] The tomato paste was a sterile product obtained from whole tomatoes ( Solanum Lycopersicum L./Lycopersicum Sculentum "Mili") , crushed, sieved, homogenized, concentrated, pasteurized and aseptically packed (commercial product "Tomato concentrate 28/30 ° Brix" of Mensajero Alimentación, SL). This product had 28-30 ° Brix, contained 30-32% (w / w) dry extract, pH 4.0-4.4, acidity (citric acid) 1.6-2.2, Bostwick viscosity (20 ° C ) 6-11 cm / 30 seconds, mesh size 0.6 mm and Howard count less than 60%. The average values per 100 g of this product were: energy value 92 Kcal / 387 Kj, total fat 0.2 g, saturated fatty acids less than 0.1 g, carbohydrates 22 g, sugars 12 g, dietary fiber 7 g , proteins 5 g and salt less than 0.1 g.
    [0185] The collagen hydrolyzate was a powdered product consisting of a powdered mixture of amino acids and peptides obtained by chemical hydrolysis (commercial product "Protifert P N 142 from Sicit 2000 S.p.A.). The product was an ivory-white powder, it contained dry matter 95% (w / w), total nitrogen 14% (w / w), volumetric mass 300 g / L, pH 6.0-7.5 in 10% solution (w / w), thermal stability greater than 200 ° C oxidation in static air and greater than 300 ° C in an inert atmosphere.
    [0187] The yeast extract used is a powdered product (commercial product "Yeast Extract Powder" from Oxoid Ltd). The product was a pale beige powder.
    [0189] Table 1. Composition of the tomato culture medium
    [0192] The pH of the tomato culture medium was 7.0. The tomato culture medium was sterilized at 121 ° C for 30 minutes.
    [0194] With the tomato culture medium, high cell counts were reached in 12-14 hours of fermentation for both strains. The culture medium is very inexpensive and what is more important, high cell counts of the order of 109-1010 cells / mL are reached in both cases.
    [0196] The purity of an isolate of the Azospirillum brasilense CECT 5802 strain was verified by taking an ampoule of said isolate, seeding a sample of said ampoule in plates of Congo Red medium and incubating at 30oC for 72 hours. A portion of the culture was taken from this plate with a loop, it was inoculated in a 1000 mL Erlenmeyer flask with 100 mL of tomato culture medium and incubated with shaking at 30oC for 16 hours. At the end of this time, the contents of the flask, which were in the exponential phase, were inoculated in a 3 L Braun Biotech BIOSTAT® B fermenter with 1.9 L of tomato culture medium. Fermentation was carried out for 16 hours at a stirring speed of 600 rpm, an aeration of 2 L / min (1 vvm (volume of air per volume of medium per minute)) and a temperature of 30 ° C. The pH was allowed to vary freely and reached a value of 6.8. A concentration of 8.9x109 cells / mL was reached. The specific growth rate in exponential phase (p) was 0.25 h "1.
    [0198] For the Pantoea dispersed strain CECT 5801, the same fermentation scheme was followed, with minor modifications, which are described below. The purity of the isolate of the strain was checked in MacConkey culture medium, incubating an inoculum of said strain on an orbital shaker for 12 hours. The culture in the BIOSTAT® B fermenter was carried out for 12 hours at a stirring speed of 600 rpm, an aeration of 3 L / min (1.5 vvm) and a temperature of 30oC. The pH was allowed to vary freely and reached a value of 6.9. A concentration of 9.5x109 cells / mL was reached. The specific growth rate in exponential phase for this strain was p = 0.57 h "1.
    [0200] Preparation of fresh medium
    [0202] A fresh medium was prepared. The ingredients that make up the fresh medium are described in Table 2. The concentration of the ingredients in this example was the mean value of the range indicated in Table 2.
    [0203] The soluble molasses concentrate was a concentrated liquid product obtained from the industrial fermentation processes for the production of yeast, alcohol and organic acids on molasses substrate and other draft liquors, source of nitrogen and mineral salts (commercial product "Nitrogen fertilizer fluid organic ”from ED&F Man Liquid Products Italia Srl). The product was a thick dark brown or black liquid, had a viscosity of 200-2000 cp at 20 ° C, specific gravity 1.15-1.35, flash point above 200 ° C, miscible in water and decomposition temperature higher than 45 ° C and pH between 5.5-8.5.
    [0205] The polypropylene glycol was a polymer, a liquid product (commercial product "VORANOL * 2000 L Polyol" from The Dow Chemical Company). The product was a colorless liquid, with a flash point of 230 ° C COC, vapor density (air = 1 ) greater than 1, specific gravity (water = 1) 1.01, density 1.0 g / cm3, dynamic viscosity 305-335 mPa s at 25 ° C.
    [0207] Table 2. Components of the fresh medium
    [0212] A fermentation reactor with stirring and temperature control was charged with the amount of water necessary to obtain the final volume of product desired. Said amount of water required was approximately 44% w / w. The stirrer was started and the salts were added, following the order in which said salts appear in Table 2, until their complete dissolution.
    [0214] Next, the molasses concentrate, the malic acid and, finally, the polypropylene glycol were added, maintaining stirring.
    [0215] Once a completely homogeneous mixture was obtained, the pH of the medium was adjusted to 7.00, by adding potash (KOH).
    [0217] Finally, the fresh medium was sterilized at 121 ° C for 20 minutes.
    [0219] Obtaining the liquid biofertilizer
    [0221] The fresh medium was allowed to cool. Next, the fermentation broths of the Azospirillum brasilense CECT 5802 (25% w / w) and Pantoea dispersa CECT 5801 (25% w / w) strains were added, obtained according to the procedure described in the section "Obtaining fermentation broths from the strains "of this example. These fermentation broths were added with slow stirring until a homogeneous mixture was obtained.
    [0223] Example 2. Evaluation of the cell viability of the liquid biofertilizer
    [0225] The cell viability of the liquid biofertilizer was periodically checked. The cell viability of the Azospirillum brasilense CECT 5802 and Pantoea dispersa CECT 5801 strains was tested in Congo Red medium and MacConkey medium, respectively. The liquid biofertilizer conserved more than 60% of cell viability after one year of conservation, at temperatures no higher than 30oC, of both strains.
    [0227] Example 3. Test with liquid biofertilizer on lettuce
    [0229] This trial was conducted outdoors between November 2018 and February 2018 in Santomera, Murcia.
    [0231] In some treatments of this example and of the following examples, the liquid biofertilizer obtained in Example 1 was used.
    [0233] Tables 3 and 4 show the production results obtained in the test.
    [0234] Table 3. Lettuce production by Thesis (treatment)
    [0237] Table 4. Average weight of lettuce by Thesis
    [0242] As can be seen in Tables 3 and 4, the liquid biofertilizer of the invention, at 2.5 and 5 L / ha, obtains superior yields both with respect to the untreated control and the thesis with the recommended fertilizer according to the Integrated production standards of the Region of Murcia (BORM, Order 8024).
    [0244] The liquid biofertilizer of the invention is effective in reducing the need for chemical fertilization since it obtains results similar to or superior to 100% chemical fertilization.
    [0246] Example 4. Test with liquid biofertilizer in broccoli
    [0248] This trial was conducted outdoors between November 2018 and February 2019 in Santomera, Murcia. Tables 5 and 6 show the production results obtained in the test.
    [0250] Table 5. Broccoli Production by Thesis
    [0255] Table 6. Average weight of broccoli by Thesis
    [0258] Liquid biofertilizer 5 L / ha
    [0259] As can be seen in Tables 5 and 6, the liquid biofertilizer of the invention, at 2.5 and 5 L / ha, obtains higher yields with respect to the untreated control.
    [0261] The liquid biofertilizer of the invention is effective in reducing the need for chemical fertilization since it obtains results superior to 100% chemical fertilization.
    [0263] Example 5. Test with liquid biofertilizer on tomato
    [0265] This trial was carried out in a greenhouse between September 2017 and May 2018 in Mazarrón, Murcia. Table 7 shows the production results obtained in the test.
    [0267] Table 7. Tomato production by Thesis
    [0272] Table 8. Chemical fertilizers used by treatment
    [0277] The liquid biofertilizer of the invention, at 5 L / ha, obtains an annual production increase in kg / ha of 12% in the thesis applied with the liquid biofertilizer of the invention only without any type of chemical contribution. In the thesis that 50% of chemical fertilization is maintained, the increase reaches 20%.
    [0279] For the treatment with conventional chemical fertilizer, the consumption of fertilizer products has risen to 730 kg / ha (Table 8), which in the thesis of the product alone, without chemical inputs, is totally replaced by only 20 L / ha of liquid biofertilizer .
    [0280] In the thesis in which half of the usual fertilization has been maintained, the saving has been 365 kg / ha of fertilizers, achieving a notable productive increase of 20%, providing no more than 10 L / ha of liquid biofertilizer.
    [0282] REFERENCE TO THE DEPOSITED BIOLOGICAL MATERIAL
    [0284] The Azospirillum brasilense and Pantoea dispersa strains were deposited on June 9, 2003, according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with the International Deposit Authority (IDA) Spanish Collection of Type Cultures (CECT), by the depositor Probelte, SA The depositor identified the strains as “Azospirillum brasilense M3” and “Pantoea dispersa C3”. After successful completion of the strain viability trials, the IDA accepted the strains, confirmed the above-stated date on which the IDA received the deposit, and assigned the deposit number CECT 5802 and CECT 5801, respectively, to those strains. .
    [0286] The Azospirillum brasilense CECT 5802 strain was isolated from the roots of pea plants ( Pisum sativum) collected in the Murcia region. The ability of the Azospirillum brasilense CECT 5802 strain to stimulate plant growth was verified by laboratory and greenhouse bioassays. The Azospirillum brasilense CECT 5802 strain produced the greatest growth stimulating effect of the more than 50 isolates of nitrogen-fixing bacteria tested. The production of 3-indole acetic acid and other plant growth promoting substances was verified. The presence of other phytohormones of the cytokinin type was detected. In the production of 3-indole acetic acid, values of more than 95% transformation of tryptophan were achieved in tomato medium with 150 mg x L-1 of this amino acid. The activity of the enzyme 1-aminocyclopropane 1-carboxylate deaminase present in this strain was also verified, through growth in media with 1-aminocyclopropane 1-carboxylic acid (ACC) as the sole nitrogen source.
    [0288] The Pantoea dispersed strain CECT 5801 was isolated from the rhizosphere of Shorgum halepense plants collected in the Murcia region. The characterization of the organic acids produced by the dispersed Pantoea strain CECT 5801 was carried out and it was found that it produces mostly gluconic acid, as well as other organic acids in small quantities. The selection was made through its ability to stimulate plant growth and to produce auxins. Their ability to produce siderophores was determined. Through laboratory and greenhouse bioassays, the ability to stimulate the plant growth. The activity of the enzyme 1-aminocyclopropane 1-
    [0289] carboxylate deaminase present in this strain, through growth in media with
    [0290] 1-aminocyclopropane 1-carboxylic acid (ACC) as the sole nitrogen source.
    [0292] LIST OF BIBLIOGRAPHIC REFERENCES
    [0294] Alori, ET, Glick, BR & Babalola, OO (2017). Microbial Phosphorus Solubilization and Its Potential for Use in Sustainable Agriculture. Front. Microbe!. 8: 971.
    [0296] Bashan, Y. & de-Bashan, LE (2010). How the plant growth-promoting bacterium Azospiri !! um promotes plant growth — a critical assessment. Adv Agron 108: 77-136.
    [0298] Beltrán-Pineda, ME (2014). The solubilization of phosphates as a microbial strategy to promote plant growth. Corpoica Ciencia y Tecnología Agropecuaria, Vol. 15, No.
    [0299] 1, 101-113.
    [0301] Bhattacharyya, PN, & Jha, DK (2012). Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J. Microbiol. Biotechnol. 28, 1327-1350.
    [0303] Brar, SK, Sarma, SJ & Chaabouni, E. (2012). Shelf-life of Biofertilizers: An Accord between Formulations and Genetics. J Biofertil Biopestici, 3: 5.
    [0305] Del Amor, FM & Cuadra, P. (2011). Plant growth-promoting bacteria as a tool to improve salinity tolerance in sweet pepper. Functional Plant Biology 39 (1): 82-90.
    [0307] Fernández, AI, Villaverde, M., Nicolás, JA, García-Gómez, A. & Malo, J. (2008). Pantoea dispersa ; Plant Growth Promoting Rhizobacteria (PGPR). VII SEAE Congress.
    [0308] Flores, P., Fenoll, J., Hellín, P. & Aparicio-Tejo, P. 2010. Isotopic evidence of significant assimilation of atmospheric-derived nitrogen fixed by Azospirillum brasilense co-inoculated with phosphate-solubilizing Pantoea dispersa in pepper seedling. Applied Soil Ecology, 46: 335-340.
    [0310] Fukami, J., Nogueira, MA, Araujo, RS & Hungria, M. (2016). Accessing inoculation methods of maize and wheat with Azospirillum brasilense. AMB Express, 6: 1.
    [0312] Fukami, J., Cerezini, P. & Hungria, M. (2018). Azospirillum: benefits that go far beyond biological nitrogen fixation AMB Expr, 8:73.
    [0314] Gyaneshwar, P., Parekh, LJ, Archana, G., Poole, PS, Collins, MD, Huston, RA & Kumar, GN (1999). Involvement of a phosphate starvation inducible glucose dehydrogenase in soil phosphate solubilization by Enterobacter asburiae . FEMS Microbiology Letters 171: 223-229.
    [0316] Mengual, C., Roldán, A., Caravaca, F. & Schoebitz. M. 2014. Advantages of inoculation with immobilized rhizobacteria versus amendment with olive-mill waste in the afforestation of a semiarid area with Pinus halepensis Mill. Ecological Engineering, 73: 1-8.
    [0318] Nautiyal, CS (1999). An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters Vol 170, 1: 265-270.
    [0319] Nuti, M. & Giovannetti, G. (2015). Borderline Products between Bio-fertilizers / Bio-effectors and Plant Protectants: The Role of Microbial Consortia. Journal of Agriculture! Science and Technology, A 5: 305-315.
    [0321] Pindi, PK & Satyanarayana, SDV (2012) Liquid Microbial Consortium-A Potential Tool for Sustainable Soil Health. J Biofertil Biopestici, 3: 4
    [0323] Schoebitz, M., Mengual, C. & Roldán, A. (2014). Combined effects of clay immobilized Azospirillum brasilense and Pantoea dispersa and organic olive residue on plant performance and soil properties in the revegetation semiarid area. Science of the Total Environment . 466-467 : 67-73.
    [0325] Seshachala, U. & Tallapragada, P. (2012). Phosphate solubilizers from the rhizosphere of Piper nigrum L. in Karnataka, India. Chil. J. Agric. Res. 72, 397-403.
    [0327] Sharma, SB, Sayyed, RZ, Trivedi, MH & Gobi, TA (2013). Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. Springerplus 2, 587-600.
    [0329] Vessey, JK (2003). Plant growth promoting rhizobacteria as biofertilizers. Plant and Soil, 255: 571-586.
    [0331] Yardin, MR, Kennedy, IR & Thies, JE (2000). Development of high quality carrier materials for field delivery of key microorganisms used as bio-fertilizers and bio-pesticides. Radiation Physics and Chemistry 57: 565-568.
    [0333] Yu, X., Liu, X., Hui, TZ, Liu, GH & Mao, C. (2011). Isolation and characterization of phosphate solubilizing bacteria from walnut and their effect on growth and phosphorus mobilization. Biol Fertil Soils 47: 437-446.
    [0335] Zhu, F., Qu, L., Hong, X., & Sun, X. (2011). Isolation and characterization of a phosphate solubilizing halophilic bacterium Kushneria sp. YCWA18 from Daqiao Saltern on the coast of yellow sea of China. Evid. Based Complement. Alternat. Med. 615032.
    权利要求:
    Claims (42)
    [1]
    1. A liquid biofertilizer comprising a strain of Azospirillum brasilense deposited in the Spanish Collection of Type Cultures (CECT) with deposit number CECT 5802, a strain of Pantoea dispersed deposited in the CECT with deposit number CECT 5801, at least one salt inorganic and a soluble molasses concentrate.
    [2]
    2. The biofertilizer according to claim 1, wherein said biofertilizer is an aqueous suspension.
    [3]
    The biofertilizer according to claim 1 or 2, wherein said biofertilizer has a pH between 6 and 8.
    [4]
    4. The biofertilizer according to any of claims 1-3, further comprising at least one pH regulating agent and at least one antifoam agent.
    [5]
    5. The biofertilizer according to any of claims 1-4, wherein said inorganic salt is selected from the group consisting of (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4 and CaCl2.
    [6]
    6. The biofertilizer according to claim 5, which comprises inorganic salts (NH4 ^ SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4 and CaCl2.
    [7]
    The biofertilizer according to any of claims 4-6, comprising the pH regulating agents malic acid and KOH.
    [8]
    8. The biofertilizer according to any of claims 4-7, wherein said antifoam agent is polypropylene glycol.
    [9]
    9. The biofertilizer according to any of claims 1-8, comprising (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4, CaCl2, soluble molasses concentrate, malic acid, KOH and polypropylene glycol.
    [10]
    10. The biofertilizer according to claim 9, comprising (NH4) 2SO40.5-3.5% (w / w), K2HPO40.1-1% (w / w), ZnSO4-7H2O 0.005-0.1% ( p / p), MnSO4-H2O 0.005-0.1% (p / p), CuSO4-5H2O 0.001-0.01% (p / p), MgSO4-7 ^ O 0.001-0.01% (p / p ), C aC h ^^ O 0.001-0.01% (p / p), soluble molasses concentrate 0.05-2% (p / p), malic acid 0.01-0.1% (p / p ), KOH 0.005-1% (w / w) and polypropylene glycol 0.001-0.1% (w / w).
    [11]
    The biofertilizer according to claim 10, comprising (NH4) 2SO41.5-2.5% (w / w), K2HPO40.18-0.20% (w / w), ZnSO4-7H2O 0.01-0 , 02% (p / p), MnSO4-H2O 0.01-0.02% (p / p), CuSO4-5H2O 0.001-0.002% (p / p), MgSO4-7H2O 0.003-0.004% (p / p ), CaCl2-2H2O 0.001-0.002% (p / p), soluble molasses concentrate 0.25-0.75% (p / p), malic acid 0.035-0.045% (p / p), KOH 0.02- 0.1% (w / w) and 0.005-0.01% polypropylene glycol (w / w).
    [12]
    12. The biofertilizer according to any of claims 1-11, which further comprises KH2PO4 and / or NH4CL
    [13]
    13. The biofertilizer according to any of claims 1-12, further comprising one or more ingredients selected from the group consisting of tomato paste, collagen hydrolyzate and yeast extract.
    [14]
    14. The biofertilizer according to any of claims 1-13, which further comprises tomato paste, collagen hydrolyzate and yeast extract.
    [15]
    15. The biofertilizer according to any of claims 1-14, which further comprises KH2PO4, NH4Cl, tomato paste, collagen hydrolyzate and yeast extract.
    [16]
    16. The biofertilizer according to claim 15, comprising KH2PO40.05-0.3% (w / w), NH4Cl 0.01-2% (w / w), tomato paste 0.5-3% (w / p), collagen hydrolyzate 0.01-2% (w / w) and yeast extract 0.01-2% (w / w).
    [17]
    17. A method of stimulating the growth of a plant, comprising applying to said plant a liquid biofertilizer according to any of claims 1-16.
    [18]
    18. The method according to claim 17, wherein the application of said liquid biofertilizer is selected from the group consisting of fertigation, spraying, and drip irrigation.
    [19]
    19. The method according to claim 17 or 18, wherein the application rate of said liquid biofertilizer is between 5 and 30 L / ha.
    [20]
    20. The method according to claim 19, wherein said application rate is between 10 and 20 L / ha.
    [21]
    21. The method according to any of claims 17-20, wherein said plant is selected from the group consisting of horticultural plants, ornamental plants, fruit plants and cereals.
    [22]
    22. A method of obtaining a liquid biofertilizer, comprising:
    (a) cultivate a strain of Azospirillum brasilense deposited in the Spanish Collection of Type Cultures (CECT) with deposit number CECT 5802 and a strain of Pantoea dispersed deposited in the CECT with deposit number CECT 5801 in separate first liquid fermentation media and appropriate for each strain, at a temperature between 25 ° C and 35 ° C, under stirring and aeration and
    (b) cold add the fermentation broths obtained in the previous stage to a second fresh liquid medium, previously sterilized.
    [23]
    23. The production method according to claim 22, wherein said biofertilizer is an aqueous suspension.
    [24]
    24. The production method according to claim 22 or 23, wherein the first liquid fermentation media comprise at least one of the ingredients selected from the group consisting of tomato paste, collagen hydrolyzate and yeast extract.
    [25]
    25. The production method according to any of claims 22-24, wherein the first liquid fermentation media comprise tomato paste, collagen hydrolyzate and yeast extract.
    [26]
    26. The production method according to any of claims 22-25, wherein the first liquid fermentation media further comprises at least one inorganic salt selected from the group consisting of K2HPO4, KH2PO4 and NH4Cl.
    [27]
    27. The production method according to any of claims 22-26, wherein the first liquid fermentation media comprise the inorganic salts K2HPO4, KH2PO4 and NH4Cl.
    [28]
    28. The production method according to any of claims 22-27, wherein the first liquid fermentation media comprise K2HPO4, KH2PO4, NH4Cl, tomato paste, collagen hydrolyzate and yeast extract.
    [29]
    29. The production method according to claim 28, wherein the first liquid fermentation media comprise K2HPO4 1-6% (w / w), KH2PO40.01-0.6% (w / w), NH4Cl 0.02 -4% (p / p), tomato paste 1-6% (p / p), collagen hydrolyzate 0.02-4% (p / p) and yeast extract 0.02-4% (p / p ).
    [30]
    30. The production method according to any of claims 22-29, wherein the second liquid fresh medium comprises at least one inorganic salt, a soluble molasses concentrate, at least one pH regulating agent and at least one antifoam agent.
    [31]
    31. The production method according to claim 30, wherein said inorganic salt is selected from the group consisting of (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4 and CaCl2.
    [32]
    32. The production method according to any of claims 22-31, wherein the second liquid fresh medium comprises the inorganic salts (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4 and CaCl2.
    [33]
    33. The production method according to any of claims 30-32, wherein the second liquid fresh medium comprises the pH regulating agents malic acid and KOH.
    [34]
    34. The production method according to any of claims 30-33, wherein said antifoam agent is polypropylene glycol.
    [35]
    35. The production method according to any of claims 22-34, wherein the second liquid fresh medium comprises (NH4) 2SO4, K2HPO4, ZnSO4, MnSO4, CuSO4, MgSO4, CaCl2, a soluble concentrate of molasses, malic acid, KOH and polypropylene glycol.
    [36]
    36. The production method according to claim 35, wherein the second liquid fresh medium comprises (NH4) 2SO41-7% (w / w), K2HPO40.001-0.2% (w / w), ZnSO4-7H2O 0.001-0.2% (p / p), MnSO4-H2O 0.001-0.2% (p / p), CuSO4-5H2O 0.001-0.02% (p / p), MgSO4-7H2O 0.001-0.02 % (w / w), CaCl2-2H2O 0.001-0.02% (w / w), soluble molasses concentrate 0.1-2% (w / w), malic acid 0.02-0.2% (p / w), KOH 0.01-2% (w / w) and polypropylene glycol 0.001-0.2% (w / w).
    [37]
    37. The production method according to claim 36, wherein the second liquid fresh medium comprises (NH4) 2SO43-5% (w / w), K2HPO40.04-0.06% (w / w), ZnSO4-7H2O 0.02-0.04% (p / p), MnSO4-H2O 0.02-0.04% (p / p), CuSO4-5H2O 0.002-0.004% (p / p), MgSO4-7H2O 0.006-0.008 % (w / w), CaCl2-2H2O 0.002-0.004% (w / w), soluble molasses concentrate 0.5-1.5% (w / w), malic acid 0.07-0.09% (p / w), KOH 0.04-0.2% (w / w) and polypropylene glycol 0.01-0.02% (w / w).
    [38]
    38. The obtaining method according to any of claims 22-37, wherein between 20% and 30% (w / w) of the fermentation broths obtained in step (a) is added to said second liquid fresh medium .
    [39]
    39. The production method according to any of claims 22-38, wherein the cultures of step (a) are carried out for a time between 8 and 20 hours.
    [40]
    40. The production method according to any of claims 22-39, wherein the stirring speed in step (a) is between 400 and 800 rpm.
    [41]
    41. The production method according to any of claims 22-40, wherein the aeration in step (a) is between 0.5 and 2 L / min.
    [42]
    42. A liquid biofertilizer obtainable according to the obtaining method of claims 22-41.
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    ES2789973B2|2022-02-07|LIQUID BIOFERTILIZER COMPRISING STRAINS OF AZOSPIRILLUM BRASILENSE AND PANTOEA DISPERSA AND METHOD OF OBTAINING THE SAME
    同族专利:
    公开号 | 公开日
    WO2020216978A1|2020-10-29|
    EP3960723A1|2022-03-02|
    ES2789973B2|2022-02-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2003016241A1|2001-08-13|2003-02-27|Agro.Bio Hungary Kft.|Micro-organisms for the treatment of soil and process for obtaining them|
    ES2234417A1|2003-10-24|2005-06-16|Probelte S.A.|Biological fertiliser consists of e.g. a Pantoea dispersa pure culture yielding high organic acid concentrations|
    WO2009091557A1|2008-01-15|2009-07-23|Michigan State University|Polymicrobial formulations for enhancing plant productivity|
    BRPI0722102A2|2007-08-27|2014-04-08|Probelte S A|NEW BIOLOGICAL FERTILIZER, METHOD FOR OBTAINING IT AND USING THIS AS A VEGETABLE GROWTH STIMULATOR|
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    EP20796214.3A| EP3960723A1|2019-04-26|2020-04-24|Liquid biofertiliser which comprises azospirillum brasilense and pantoea dispersa strains and method for obtaining same|
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