Bioactivating composition, preparation method, and use

Abstract

The invention relates to a bioactivating composition for providing phosphorus that is readily available to plant crops, comprising i) a combination comprising: (a) an organic waste source as a phosphorus-rich carrier matrix composed of excreta stabilised by means of aerobic composting; (b) enzymes that mineralise phytate; (c) a bacterial consortium that solubilises phosphorus, comprising bacteria from the groups Klebsiella, Stenotrophomonas, Serratia, and (ii) a polymer for homogenising and granulating the composition. The invention also relates to the method for obtaining said bioactivating composition and the use thereof.

Description

[0001] BIOACTIVATING COMPOSITION; PREPARATION METHOD; AND USE

[0002] DESCRIPTIVE MEMORANDUM

[0003] FIELD OF INVENTION

[0004] The present invention provides a bioactivating composition, its method of obtaining and its use, comprising a combination that includes an organic source of phosphorus, phosphorus-solubilizing bacteria and phosphate-mineralizing enzymes, for use in the agricultural industry, in particular, in the industry related to the production of biofertilizers where it is useful for providing readily available phosphorus to plant crops.

[0005] STATE OF THE ART

[0006] Phosphorus is an essential nutrient involved in various biochemical processes. Plants acquire it from the soil and use it for their growth and development. Therefore, its availability in plant roots is vital, and it is primarily supplied through fertilizers.

[0007] The primary source of phosphate fertilizer production is phosphate rock, a finite resource produced naturally over millions of years through the circulation of phosphorus between the lithosphere and hydrosphere. According to studies by the United States Geological Survey (USGS), the largest phosphate rock deposits are located in Morocco (more than 70% of total reserves). Furthermore, considering its high rate of consumption, scientific studies reported by Cordell and White (2011) project that phosphate rock deposits will be depleted within the next 60 to 100 years, depending on the consumption rate and population growth projections.

[0008] On the other hand, phosphate fertilization is very inefficient, since of 100% of the phosphorus applied in the field, only about 10% to 20% of the phosphorus is able to be captured by the plants, which is why it is considered to be between 80% and 90% excess with respect to the requirements of the crops to have productive systems.

[0009] According to the Global Fertilizer Outlook (FAO, 2019 https: / / www.fao.org / 3 / ca6746en / ca6746en.pdf), global consumption of agricultural fertilizers for 2021 was estimated to increase by 1.5% compared to 2020, reaching 197.9 million tons of nutrients. Consequently, phosphate (P2O5) consumption was projected to increase by 1.8%, reaching 48.3 million tons. Looking ahead, the FAO predicts that global fertilizer demand will increase by 1.5% compared to 2021, reaching 200.9 million tons of nutrients. This is expected to result in an increase of 1.6% for P2O5, reaching 49.1 million tons of fertilizer.If production versus demand is considered to balance maximum achievable production with total demand at the regional level, FAO (2019) reports that Latin America, South Asia, Oceania, Central and Eastern Europe will have a P2O5 deficit by 2022.

[0010] Consequently, it is imperative to seek new sources of phosphorus to overcome the limited lifespan of phosphate rock and develop phosphate fertilizers, which are essential nutrients for life. Furthermore, in addition to enabling agricultural systems to provide sufficient food, these new phosphorus sources will help reduce the negative environmental impacts caused by chemical fertilization and the improper disposal or reuse of agricultural waste.

[0011] Regarding the characteristics of volcanic soils (Andisols) found in Chile, these are characterized by a low pH and low phosphorus bioavailability for plant uptake. These characteristics result in low efficiency of conventional fertilizers, leading to a high total phosphorus content in the soil. Simultaneously, soil acidification is a natural process that can accelerate over time in intensive agricultural systems. This acidification favors an increase in phytotoxic aluminum (Al). 3+ ) (Mora et al., 2005), resulting in a loss of forage yield and quality in large pasture areas in southern Chile (Mora et al., 2002, 2006). The primary toxic effect of Al 3+ It is a rapid inhibition of root growth that limits water and nutrient uptake by plants (Kochian et al., 2004). Therefore, phosphorus deficiency and aluminum toxicity3+ They coexist in the Chilean Andisols and threaten efficient crop production (Mora et al., 2002, 2007).

[0012] The use of biofertilizers is known in the prior art. For example, document CN104761299A provides an organic fertilizer using a phytate mineralizing enzyme (phytase) and a method for preparing the same, where said method includes the steps of (1) mixing weathered coal of a certain granularity and bentonite according to a mass ratio of 5-8:1 to prepare humidified bentonite; (2) adding commercially available phytase according to a certain proportion to the humidified bentonite to prepare immobilized phytase; and (3) mixing the immobilized phytase with composted dried feces of monogastric animals according to a mass ratio of 10-15:100 with uniform stirring to obtain the organic phytase fertilizer.

[0013] Studies can also be found on excrement or excrement compost, which mostly comes from agricultural waste rich in phosphorus. However, this phosphorus is not fully available to plants, so in many cases, inoculation with phosphorus-solubilizing bacteria or the direct addition of enzymes capable of mineralizing phosphate is required.

[0014] Menezes-Blackburn et al. (2014) propose combining cattle manure with (i) phosphate-solubilizing bacteria (Bacillus sp.) or (ii) phosphate-mineralizing enzymes (phytase); and finally (iii) the use of phytase applied to the soil. This work reports that the combination of manure and immobilized enzymes increases inorganic phosphorus in Andisols, ultimately resulting in a beneficial effect for plants. In the work of Menezes-Blackburn et al. (2014), using cattle manure inoculated with Bacillus MQH-19, an increase in dry matter production in wheat plants of approximately 21% by weight was obtained compared to inorganic fertilization, applying 130 mg of phosphorus / kg of soil (see Figure 2a of Menezes-Blackburn et al., 2014).In contrast, the present invention uses a combination comprising a bacterial consortium capable of solubilizing phosphate and encapsulated in an organic matrix (compost), which also includes an immobilized phytase. This combination is subsequently mixed with a polymer, such as sodium alginate, resulting in a stable, dry preparation that is easy to store and protected for activation in the crop. The present invention generates a biomass increase of over 40% compared to the control fertilized with triple superphosphate, exhibiting a 20% higher performance than that observed in Menezes-Blackburn et al. (2014).

[0015] Martínez et al. (2015) proposed the use of different phosphate-solubilizing bacteria to increase inorganic phosphorus by incubating them in Andisols. The results of this research demonstrated that, of the different strains used, only the inoculation of Serratia sp. N0-29PA in soils with 131 mg of inorganic phosphorus / kg of soil showed a 29% increase in dry matter production, a 14% increase in phosphorus content compared to the control, and a 47% increase in phosphorus uptake compared to the control, using oat plants as a model organism. In contrast, with the composition of the present invention, biomass is increased by 10% compared to the work of Martínez et al.(2015), the phosphorus content was greater than 100% compared to the control, as for phosphorus uptake the present invention achieved an increase of 159% greater in relation to the control, showing a significant improvement compared to the results reported by Martínez et al. (2015).

[0016] The state of the art includes the work of María de la Luz Mora, Rolando Demanet, Jacquelinne J. Acuña, Sharon Viscardi, Milko Jorquera, Zed Rengel, and Paola Durán, which deals with “Aluminum-tolerant bacteria improve the plant growth and phosphorus content in ryegrass grown in a volcanic soil amended with cattle dung manure,” Applied Soil Ecology 115 (2017) 19-26. In this work, aluminum-tolerant bacteria were isolated. 3+ The mechanism of tolerance to Al was studied from the endosphere and nzosphere of perennial ryegrass (Lolium perenne) grown in Andisols. 3+and its effect on ryegrass growth in the presence of cattle manure. Twenty aluminum-tolerant bacteria (10 mM) were isolated, from which five were selected: Klebsiella sp. RC3, Stenotrophomonas sp. RC5, Klebsiella sp. RCJ4, Serratia sp. RCJ6, and Serratia sp. RJAL6, due to their ability to solubilize phosphorus, phthalate mineralization activity, and siderophore production, among other properties. This work suggests that these bacteria are capable of alleviating the stress caused by aluminum. 3+ by generating a complex Al 3+-siderophore. In addition, mixing this bacterial consortium with cattle manure resulted in increased ryegrass growth, reflected in an approximate 30% increase in dry mass compared to using manure alone, without showing a beneficial effect on phosphorus content in plant shoots. However, in this patent application, using the bioactivator composition, superior ryegrass growth is achieved compared to using only cattle manure, with an increase in phosphorus content in shoots exceeding 200% compared to using manure alone (when fertilizing with 300 kg of P2O5 ha). -1 ) see technical effect in Figure 3.

[0017] Furthermore, the state of the art includes the work of Patricio J. Barra, Sharon Viscardi, Milko A. Jorquera, Paola A. Duran, Alexander J. Valentine, and María de la Luz Mora, entitled “Understanding the Strategies to Overcome Phosphorus-Deficiency and Aluminum-Toxicity by Ryegrass Endophytic and Rhizosphere Phosphobacteria,” published in Frontiers in Microbiology on June 1, 2018. This work proposes that phosphate-mineralizing bacteria use plant-like strategies to overcome aluminum toxicity. 3+ In this work, they analyzed the growth of bacteria in the presence of phosphorus and aluminum, the production of organic acids, the expression of the enzyme malate dehydrogenase (mdh), and phosphatase activity in 5 Al bacteria 3+tolerant (Klebsiella sp. RC3, Stenotrophomonas sp. RC5, Klebsiella sp. RCJ4, Serratia sp. RCJ6 and Serratia sp. RJAL6). The results of this work suggest a significant detriment to bacterial growth at low phosphorus concentrations (greater than the presence of Al 3+ In addition to the above, they show an increase in the production of organic acids (melic and citric acids) and an increase in mdh expression in some of the bacteria under low phosphorus and aluminum conditions. However, this work does not disclose a bioactivating composition like the one disclosed in the present patent application.

[0018] The present invention provides a phosphate bioactivating composition comprising a combination that includes an organic source of phosphorus, a phosphorus-solubilizing bacterial consortium, and phosphate-mineralizing enzymes, wherein said composition allows, in addition to its fertilizing effect, the mobilization of phosphorus that is found as a reservoir in the soil as a result of the previous use of mineral fertilizers.

[0019] Consequently, the bioactivating composition can complement the application of mineral fertilizers, given its ability to mobilize phosphorus fixed in the soil, thus increasing the efficiency of conventional fertilizers. The invention can be applied to annual crops, pastures, and fruit trees. Therefore, the invention aims to mobilize phosphorus that is fixed in the soil, known as "legacy phosphorus," resulting from repeated phosphate fertilizations during various planting cycles in soil that has previously undergone such fertilization.

[0020] The main advantage of the present invention over the prior art is the combination of biotechnological strategies comprising a consortium of phosphorus-solubilizing bacteria and phthalate-mineralizing enzymes, along with an organic phosphorus source, which is cattle manure compost acting as a carrier matrix. This combination provides essential nutrients for plant development and offers greater protection to the incorporated bacteria and enzymes. The invention allows for the input of phosphorus, which acts as a starter for crop nutrition, while the biotechnological strategies produce a controlled and prolonged release of the nutrient during plant growth, increasing its use efficiency.

[0021] Another advantage over the prior art is that the invention is formulated for easy integration into conventional agricultural processes, as it is prepared in granular form. Thus, the fertilizer composition can be used with existing machinery, and its use does not alter the production system, thereby avoiding any additional costs associated with applying the fertilizer composition of the invention.

[0022] An additional advantage is the increased concentration of foliar phosphorus in crops when using the bioactivating composition CBE (Compost, bacteria and enzymes), delivering a highly beneficial effect to improve the nutrition of animals that feed on the leaves of fertilized plants.

[0023] BRIEF DESCRIPTION OF THE FIGURES

[0024] Figure 1. Study of the effect of bioactivators at three phosphorus doses on biomass production at the shoot and root level. Where: TSP: Triple superphosphate, C: Compost pellets, CB: Compost pellets+ (~10 15 UFC bacterial consortium xg compost), EC: compost pellets phytase complex (QUANTUM BLUE EXP, E. coli 1,000 U g -1 ) 1:1 ratio, CBE: compost pearls + bacterial consortium + phytase complexes.

[0025] Figure 2. Study of the effect of bioactivators at three phosphorus doses on stem P concentration. Where: TSP: Triple superphosphate, C: Compost pellets, CB: Compost pellets+ (~10 15 UFC bacterial consortium xg compost), EC: compost pellets phytase complex (QUANTUM BLUE EXP, E. coli 1,000 U g -1 ) 1:1 ratio, CBE: compost pearls + bacterial consortium + phytase complexes.

[0026] Figure 3. Study of the effect of bioactivators at three phosphorus doses on P uptake by the plant. Where: TSP: Triple superphosphate, C: compost pellets, CB: compost pellets+ (~10 15 UFC bacterial consortium xg compost), EC: compost pellets phytase complex (QUANTUM BLUE EXP, E. coli 1,000 U g -1 ) 1:1 ratio, CBE: compost pearls + bacterial consortium + phytase complexes.

[0027] Figure 4. Study of the effect of bioactivators on stem biomass production over time. Where: TSP: Triple superphosphate and BioAct (CBE): compost pellets + bacterial consortium + phytase complexes.

[0028] Figure 5. Study of the effect of bioactivators on stem P concentration over time. Where: TSP: Triple Superphosphate and BioAct (CBE): compost pellets + bacterial consortium + phytase complexes.

[0029] Figure 6. Study of the effect of bioactivators on ryegrass production. Where: TSP: Triple superphosphate and BioAct (CBE): compost pellets + bacterial consortium + phytase complexes.

[0030] Figure 7. Evaluation of foliar and root biomass production as a result of the fertilization treatment in the presence and absence of N (200 kgN ha). -1 ) and K (66 kgtoO ha -1 ) at a dose of P of 300 kg P2O5 ha -1 Where TSP: Triple superphosphate, C: Compost pellets, CB: Compost pellet+ (~10 15 UFC bacterial consortium xg compost), EC: compost pellets phytase complex (QUANTUM BLUE EXP, E. coli 1,000 U g -1 ) 1:1 ratio, CBE: compost pearls + bacterial consortium + phytase complexes, and (KN): means treatment with incorporation of nitrogen and potassium.

[0031] Figure 8. Evaluation of P translocation to the plant as a result of fertilization treatment in the presence and absence of N (200 kgN ha). -1 ) and K (66 kgtoO ha -1 ) at a dose of P of 300 kg P2O5 ha -1 Where TSP: Triple superphosphate, C: compost pellets, CB: compost pellet+ (~10 15 UFC bacterial consortium xg compost), EC: compost pellets phytase complex (QUANTUM BLUE EXP, E. coli 1,000 U g -1 ) 1:1 ratio, CBE: compost pearls + bacterial consortium + phytase complexes, and (KN): means treatment with incorporation of nitrogen and potassium.

[0032] DESCRIPTION OF THE INVENTION

[0033] The present invention provides a bioactivating composition comprising:

[0034] (i) a combination comprising:

[0035] (a) a source of organic waste as a source of phosphorus;

[0036] (b) phosphate mineralizing enzymes;

[0037] (c) bacterial consortium that solubilizes phosphorus; and

[0038] (i) a polymer for homogenization and granulation of the composition.

[0039] The source of organic waste according to the present invention is a phosphorus-rich carrier matrix composed of excreta stabilized by aerobic composting, which has the function of protecting and ensuring the proper functioning of the other components of the composition, bacteria and enzymes, and which also allows a controlled delivery of phosphorus to the crops.

[0040] In a preferred embodiment of the invention, the bioactivator composition comprises an organic waste source of livestock excrement, particularly excrement from cattle, pigs, sheep, horses, and other livestock. In an even more preferred embodiment, the organic phosphorus source comprises cattle excrement. The excrement is stabilized through a composting process, primarily to eliminate pathogens and germinating seeds, and to produce more stabilized organic matter that does not generate only a fleeting effect on microbial activation.

[0041] The enzymes that mineralize phytate according to the present invention belong to the phosphatases, which catalyze the hydrolysis of organic phosphorus, of interest to the invention being phytic acid, an unassimilable organic form of phosphorus found in grains, oilseeds, soil, animal excrement, among others. This enzyme allows the release of an assimilable form of inorganic phosphorus (phosphate).

[0042] In a preferred embodiment of the invention, the bioactivating composition comprises a phthalate-mineralizing enzyme selected from the group of phytases, which are phosphatases or phosphohydrolases capable of hydrolyzing the phthaate complex or inositol polyphosphates found in plants and soil. Through the catalytic activity of this enzyme, phosphorus is released and taken up by plants.

[0043] In a more preferred embodiment, the phthalate mineralizing enzyme is a phytase isolated from Escherichia coli immobilized on a synthetic polymer with the trade name Quantum Blue 5G.

[0044] The phosphorus-solubilizing bacteria according to the invention are those bacteria capable of making inorganic phosphorus available from insoluble organic phosphorus compounds through a mineral phosphate solubilization mechanism. This is achieved by releasing low molecular weight organic acids that chelate the phosphate-bound cations, thus converting them into soluble forms. These bacteria can be selected from among the Klebsiella, Stenotrophomonas, Enterobacter, Serratia, Pantoea, Microbacterium, and Pseudomonas groups, among others.

[0045] The present invention comprises the use of a consortium of phosphate-solubilizing bacteria, which can be obtained from the roots and shoots of perennial ryegrass (Lolium perenne), as well as from the rhizosphere. In particular, the selected bacteria exhibit plant growth promotion and tolerance to aluminum (10 mM). The bacteria used in the present invention can be selected from the genera Klebsiella sp. and Stenotrophomonas sp. isolated from the endosphere of perennial ryegrass (endophytic bacteria), along with the genera Klebsiella sp., Serratia sp., and Serratia sp. isolated from the rhizosphere (rhizosphere bacteria).

[0046] The genera of bacteria in the consortium used in the bioactivating composition and their capabilities to mineralize and solubilize phosphorus are described in Table 1. Likewise, different plant growth-promoting capabilities identified in the bacteria used in the composition of the present invention are noted.

[0047] Table 1. Characteristics of the bacteria used in the combination of the fertilizer composition of the present invention (taken from Mora et al., 2017).

[0048] IAA: indoleacetic acid production; ACCD: 1-aminocyclopropane-1-carboxylate deaminase activity; PM: phytate mineralizing activity; PS: phosphorus solubilization; SID: siderophore production; +: means positive reaction, ++: means more positive reaction.

[0049] The bioactivating composition of the present invention further comprises a polymer and a solvent (aqueous solutions and / or organic solvents miscible in water), for homogenization and subsequent granulation.

[0050] The polymer for granulation is selected from the group comprising biodegradable polymers of modified polysaccharides with a high permeability coefficient (K> 4000 cm 2 s' 1 Pa -1These include, but are not limited to, water-soluble petroleum-based polymers such as starches, alginates, and agars. Other polymers that could be used include polysulfone, polyacrylonitrile, polyvinyl chloride, polyurethane, and polystyrene, which are currently used for coating agrochemical compounds. Additionally, synthetic polymers such as polysulfone and polyacrylonitrile, as well as biodegradable cellulose acetate, are used for developing slow-release fertilizers. In a preferred case, the polymer used is sodium alginate.

[0051] In a preference, the solvent of the composition of the present invention is water.

[0052] The bioactivating composition of the present invention comprises:

[0053] (i) a combination comprising:

[0054] (a) a source of organic waste as a source of phosphorus;

[0055] (b) phosphate-mineralizing enzymes; (c) a phosphorus-solubilizing bacterial consortium; and

[0056] (i) a polymer for homogenization and granulation of the composition.

[0057] The present invention also discloses a method for obtaining a bioactivator composition that mixes a combination comprising an organic waste source as a phosphorus source, a bacterial consortium, and a phthalate-mineralizing enzyme, with a polymer. The organic waste source provides an optimal environment for the development and release of the bacteria, which in turn allows for the controlled release of phosphorus through the action of the bacteria and enzymes present in the bioactivator composition. The bacterial consortium and the phthalate-mineralizing enzyme mobilize phosphorus that is fixed in the soil as a result of repeated phosphate fertilization during various planting processes, making it available for uptake by plants.Secondary benefits include the provision of other essential nutrients, as well as organic matter, which would improve plant nutrition and soil quality (see Figures 7 and 8).

[0058] The method for preparing the bioactivating composition of the present invention in granular form comprises the following steps: a) preparing a combination comprising an organic waste source, a bacterial consortium and enzymes that mineralize phosphate by the following steps:

[0059] (i) prepare compost from an organic waste source as a source of phosphorus;

[0060] (i) mix the compost from step (i) with water until a homogeneous mixture is generated;

[0061] (iii) add enzymes that mineralize f tate to the mixture of step (i);

[0062] (iv) adding a phosphorus-solubilizing bacterial consortium to the mixture from step (iii); (b) adding a polymer to the combination from step (a); (c) combining and homogenizing the mixture from step (b); (d) adding the mixture from step (c) to a solidifying solution to generate the granules; (e) separating and washing the decanted material; and (f) drying the decanted material that constitutes the granulated bioactivator composition.

[0063] In one preference, step (i) comprises the stabilization of an organic residue and the enrichment of phosphorus sources through composting, where saprophytic fungi aid in the decomposition of organic matter.

[0064] The composting process in stage (i) comprises the aerobic degradation of excreta in thermally insulated reactors. For this purpose, manure is added where the carbon-to-nitrogen ratio (C:N) is adjusted between 25:1 and 35:1 by adding glucose. The reactor is covered with a lid, and the mixture is left to decompose during the composting process. After 12 to 16 days, when the temperature in the reactors reaches approximately 25 Q C, the composting mixture is inoculated with 200 ml of a mixed fungal broth. The fungi used in the present invention are saprophytes, which aid in the degradation of organic matter and are isolated from the excrement itself. For this purpose, the environmental conditions are facilitated (25 QC with a moisture content exceeding 60%) for the development of these fungi originating from the same excrement. Finally, the composting process is carried out over a period of 80-100 days, yielding a stabilized material. This process enriches the phosphorus content and stabilizes the organic matter present in the excrement, generating a safe and suitable carrier material for the use of the bioactivator composition of the present invention.

[0065] In a preference of the invention, the ratio of compost to water in step (i) is between 1:3 and 1:4 to generate a homogeneous mixture. The water used in this step may be distilled water without limiting the invention.

[0066] The phytate mineralizing enzymes of stage (iii) are added between compost / enzyme ratios of 1:0.5 and 1:1.5, enzymes that are selected from the group of phytases, which are phosphatases or phosphohydrolases.

[0067] The phosphorus-solubilizing bacterial consortium from step (iv) is pre-hydrated in ratios between 1:6 and 1:10 with water, to obtain a bacterial load of 10 14 10 18 UFC. The water used in this stage may be sterile water without limiting the invention.

[0068] The bacterial consortium that solubilizes previously hydrated phosphorus is mixed with the preparation at compost-to-consortium ratios of 100,000:1 and 140,000:1. The bacterial consortium, composed of the five strains mentioned above, is compatible with each other, as antagonism tests were positive, confirming their suitability for the combination, and no inhibition was observed between the different bacterial strains. Furthermore, these bacteria are tolerant to aluminum, which is advantageous when working with acidic volcanic soils, which often suffer from high aluminum saturation.

[0069] The polymer for homogenization and granulation of stage b) is pre-hydrated with water to generate a solution between 2% w / v and 4% w / v.

[0070] The polymer for homogenization and previously hydrated granulation is mixed with the preparation at a final concentration that varies between 1% w / v and 3% w / v.

[0071] Stage c) is performed in a stirred reactor between 10 and 30 rpm for a time between 20 and 80 min.

[0072] In one preference, step c) is performed at 20 rpm for 60 min.

[0073] Step d) is carried out by passing the mixture through a tray with circular perforations of diameter between 4 and 6 mm, which allows the generation of the granules, which are directly mixed in the solidifying agent at an agitation between 400 and 600 rpm.

[0074] The solidifying agent can be selected from a calcium-rich solution of, for example, calcium chloride, gluconolactate, calcium acetate monohydrate, calcium lactate, among others.

[0075] In one preference, the solidifying agent in step d) is CaCh.

[0076] Once the mixture is placed in the solidifying agent, it is kept for 30 to 90 minutes with agitation between 400 and 600 rpm.

[0077] The mixture that generates the solidification of the granules is produced by cross-linking the polymer chains with calcium chloride where an ion exchange reaction occurs, in which sodium is replaced by calcium, giving the resulting gel a characteristic solid consistency.

[0078] Step e) is carried out by centrifugation, filtration, or any combination thereof.

[0079] In a preference, step e) is performed by filtering.

[0080] Once filtered, the granules are washed with water or distilled water to remove excess solidifying agent. Step f) is carried out at a temperature not exceeding 30°C and until a relative humidity of 10-16% is reached in the bioactivator composition. This is to prevent product decomposition, as higher humidity levels can degrade both the bacterial consortium and the phthalate-mineralizing enzyme. This method protects the bacteria and the enzyme through encapsulation, allowing for dry storage of the bioactivator composition.

[0081] In one preference, the drying phase is carried out on metal mesh with aeration.

[0082] In one embodiment of the invention, the bioactivating composition is in granular form (perlites of approximately 4 to 6 mm external diameter), similar to conventional nitrogen or triple superphosphate fertilizers, thus making it suitable for use by any conventional agricultural machinery.

[0083] The composition of the present invention provides a fertilizing effect, in addition to activating and increasing the efficiency of conventional mineral fertilizers.

[0084] The present invention also discloses the use of the bioactivating composition as a fertilizer, particularly in the biofertilizer production industry, which is useful for providing readily available phosphorus to plant crops.

[0085] Application Examples

[0086] The following are application examples which demonstrate preferred options for the bioactivating fertilizer composition, as well as the method of production thereof, but they do not limit the scope of the present invention.

[0087] Example 1: Obtaining the CBE bioactivating composition in granules.

[0088] The combination comprising the organic waste source, bacterial consortium, and phthalate-mineralizing enzymes is prepared by beginning with a composting process involving the aerobic degradation of cattle manure in three independent, thermally insulated cylindrical reactors, each with a capacity of 8 kg. For this purpose, 6 kg of manure were added to each reactor, where the carbon-to-nitrogen (C:N) ratio was adjusted to 35:1 by adding glucose. The reactors were covered with lids, and the mixture was allowed to decompose during the composting process. After 14 days of composting, when the temperature in the reactors reached approximately 25°C, the mixture was prepared. QIn step C, the compost mixture was inoculated with 200 mL of a mixed fungal broth. This fungal broth was obtained after providing the necessary humidity and temperature conditions to promote the growth of saprophytic fungi (fungi native to the processed cattle manure) that aided in the degradation of the organic matter. The composting process was carried out over 95 days.

[0089] Saprophytic fungi were isolated from the same bovine excrement. For this purpose, the environmental conditions were created (25 Q C with a humidity above 60%) so that these fungi from the same excrement could develop.

[0090] The composting process of organic waste with a high phosphorus content allows for the enrichment of phosphorus and its conversion into forms more readily available to plants and microorganisms. The results of the composting process of cattle manure according to the described technology are shown in Table 2. In particular, the composting process of cattle manure inoculated with saprophytic fungi increased the total phosphorus content from an initial 7.6 ± 0.3 g P / kg of manure to a final 10.2 ± 0.6 g P / kg of compost, representing an approximate 35% increase in the phosphorus content of the stabilized organic waste source (after composting).

[0091] Table 2. Chemical composition of stabilized cattle manure compost obtained after the aerobic composting process

[0092] The combination was prepared according to the composition described in Table 3. Then, in a first dry stage, 250 g of compost were homogenized with 250 g of commercial immobilized enzyme (Quantum Blue Exp, E. coll 1000 U g -1 ) in a 1:1 ratio, once homogeneous the mixture was hydrated with 950 mL of distilled water. Separately, 2.5 mg of the lyophilized bacterial consortium of load 10 was weighed 20 UFC to finally reach a load of 10 15 CFU per mg of compost, comprising microorganisms of the genera Klebsiella sp. and Stenotrophomonas sp. (endophytic bacteria), along with Klebsiella sp., Serratia sp. and Serratia sp. (rhizophene bacteria), which was hydrated in 20 mL of cold, sterile distilled water (water sterilization is by autoclave at 121 Q C for 15 min), and then incorporate the bacterial consortium suspension into the mixture.

[0093] In parallel, a sodium alginate solution was prepared using 1,830 mL of distilled water and 45 g of sodium alginate, so that the sodium alginate concentration in the total volume of water used in the process (3,000 L) would be 1.5% (w / v). The sodium alginate solution was then added to the homogeneous mixture of the previously prepared combination. The process was carried out in a mechanically stirred reactor at room temperature under constant stirring at 20 rpm for 1 hour. Subsequently, the resulting homogeneous mixture was dropped into the 0.1 M CaCl₂ solution, which was under constant stirring at 500 rpm, through plastic trays with circular perforations approximately 4 mm in diameter. Once the mixture was deposited in the 0.1 M CaCl₂, it was maintained for 1 hour under constant stirring at 500 rpm. The granules were then filtered and washed with distilled water to remove excess calcium chloride.The drying phase was carried out on metal mesh with aeration and a constant temperature not exceeding 30. Q C.

[0094] Table 3: CBE bioactivator composition

[0095] Example 2: Greenhouse trial

[0096] A greenhouse trial was conducted to evaluate the effect of the bioactivating composition of the present invention (BCI) compared to other bioactivating formulations on crop yield and phosphorus adsorption capacity, as well as its concentration in plant tissue. The trial was carried out in plastic pots containing 1.5 kg of Andisol soil, Barros Arana series, using perennial ryegrass (Lolium perenne cultivar Nui) as a control plant.

[0097] The trials were completely randomized with 50 Lolium perenne plants, where different bioactivator formulations were evaluated considering compost (acting as carrier material and phosphorus source), the bacterial consortium, and the phytate mineralizing enzymes. The composition of the invention is CBE: comprising the combination of the 3 main components compost + bacterial consortium + phytase complex in a ratio of 45.9% (w / w), 0.0005% (w / w), and 45.9% (w / w), respectively, which is described in application example 1.

[0098] Before sowing, a portion of Andisol soil (300 g) was taken from each pot to homogenize with the different bioactivator media. Granulated triple superphosphate (TSP) was used as a positive control at different doses: 100, 200, and 300 kg P₂O₅ ha⁻¹. -1The phosphorus bioactivating formulations were supplied according to their total phosphorus content. To supply the nutritional requirements for nitrogen (N) and potassium (K), 150 kg of N per hectare (ha) were applied as urea fertilizer (divided into three applications: at the start of the trial, in the first third of the trial, and in the second third of the trial) and 66 kg of K₂O per hectare (ha) were applied. -1 as muriate of potassium. Sixty seeds of L. perenne were planted in each pot, and after two weeks, the seedlings were thinned to 50. During the experiment, the plants were manually irrigated with distilled water as needed. Three harvest stages were considered: the first at 12 weeks, the second at 17 weeks after sowing, and the last at the end of the experiment at 27 weeks.

[0099] The granulated bioactivating formulations used in the invention were the following: C: compost, CB: compost + bacterial consortium, CE: compost + phytase complex, and CBE: compost + bacterial consortium + phytase complexes.

[0100] In Figure 1 when 100 kg of P2O5 were applied ha -1 No significant differences in biomass production were observed compared to the control, meaning that all formulations performed as well as conventional fertilization. In the case of 200 kg of P2O5 ha -1 It was observed that CE and CBE showed a significant increase of around 30% in the biomass production of Lolium perenne (perennial ryegrass) compared to inorganic fertilization with triple superphosphate. Increasing the phosphorus dose to 300 kg P₂O₅ ha⁻¹ further enhanced the biomass production. -1The increase in the CE composition remained at 30% compared to the control in dry matter production. However, the CBE composition increased production by over 40% compared to the control.

[0101] The application of the different bioactivator formulations showed that the various phosphorus application rates resulted in a significant increase in phosphorus concentration in plant tissue (Figure 2). The best response in terms of increased phosphorus concentration in plant tissue was obtained at the highest phosphorus rate, 300 kg P₂O₅ ha⁻¹. -1 In CBE, it triples the phosphorus concentration in plant tissue, resulting in a highly beneficial effect for improving the nutrition of animals that feed on the leaves of fertilized plants.

[0102] Figure 3 shows that the bioactivator composition CBE resulted in an approximate 160% increase in P uptake by plants compared to the control. This significantly exceeded the increases observed with the other bioactivator formulations, which reported 42% with CE, 21% with CB, and 2% with C.

[0103] The results indicate that an enhanced effect from the combination of the main components of the CBE bioactivator composition is best suited to mobilize fixed phosphorus in the soil and make it available to plants. Example 3: Field trial

[0104] The field trial began in May 2017, where 50% of the phosphorus requirement was incorporated as triple superphosphate inorganic fertilizer and the remaining 50% was applied through the CBE bioactivating composition of the present invention, CBE: compost + bacterial consortium + phthate mineralizing enzyme.

[0105] Trial location

[0106] This trial was conducted at the “Santa Elena” farm located in the town of Barros Arana, 50 km from the city of Temuco, Teodoro Schmidt Commune, Cautín Province, Chile.

[0107] Soil characteristics The Santa Elena farm has an Andisol of the Barros Arana Series, whose chemical composition evaluated prior to the start of the test is presented in Table 4.

[0108] Table 4. Chemical composition of the soil.

[0109] Add bases: exchangeable cations, such as Ca +2 , Na + , Mg +2 , K +CEC: Cation exchange capacity. Source: Soil Chemical Analysis Laboratory. Agroindustry University of La Frontera. Prior to the trial, a chemical fallow was carried out, for which 6 L of the herbicide Glyphosate + 200 cc of surfactant L1 700 were used. In addition, an amendment was made by applying 2 tons of Magnecal per 15 ha -1 .

[0110] Sowing

[0111] The trial was conducted on May 9, 2017, using the species Lolium perenne cultivar Nui with a dose of 20 kg ha -1 The planting system was manual with a row spacing of 17.5 cm, in plots of 7.9 m². 2 , with four repetitions.

[0112] Fertilization

[0113] A base fertilization of 46 kg N; 77.5 kg K2O; 22.5 kg S; and 22.5 kg MgO ha was used at the time of the trial. -1 Phosphate fertilization was applied according to treatment: as a control, 100 kg of P2O5 ha was applied -1of triple superphosphate, the application of the bioactivating composition CBE (BioAct-P) was 50% of the P / ha plus 50% of the P ha -1 as triple superphosphate. In addition, 46 kg N ha were applied after each cutting. -1

[0114] Results

[0115] The results presented in Figure 4 show that the plots fertilized with the CBE bioactivator composition exhibited the same biomass production as the plots fertilized with triple superphosphate at the different cutting periods. Similarly, Figure 5 shows that the phosphorus concentration in the plant tissue did not differ between the plots fertilized with triple superphosphate and those fertilized with the CBE bioactivator composition in a 1:1 ratio (triple superphosphate:CBE bioactivator) at the different cutting periods. Figure 6 shows a trend in November and December of increased ryegrass production resulting from the use of the CBE bioactivator composition, with a significant increase in December of over 25% compared to the control with triple superphosphate.

[0116] Finally, the results presented demonstrate the suitability of the CBE bioactivator composition for field use, reducing inorganic fertilizer (triple superphosphate) consumption by at least 50%. Considering the reported differences, the synergistic effect of combining compost, bacterial consortium, and enzyme (CBE bioactivator) is evident, as is its superiority over other bioactivator formulations. Furthermore, it is clear that the CBE bioactivator composition has a greater beneficial effect than that reported by the other products analyzed by Menezes-Blackburn et al. (2014) and Martínez et al. (2015), considering the parameters studied. Example 4: Greenhouse Trial

[0117] A trial was conducted to evaluate the effect of CBE on crop yield and phosphorus adsorption capacity, as well as phosphorus concentration in plant tissue, versus its ability to provide N and K. This study was developed in conjunction with the study described in Example 2, where trials were conducted to evaluate the capacity of different biofertilization strategies in the absence of inorganic N and K supply (urea and potassium chloride), considering that the compost used for product development contains N and K as described in Table 2. The evacuation was performed at the highest applied phosphorus dose of 300 kg P₂O₅ ha⁻¹. -1The protocol followed was the same as that described in Example 2, considering it was part of the same trial. The results show that root biomass production (Figure 7) was not affected by the treatments or by the absence of N and K supplied as conventional fertilizer. Regarding foliar biomass production, no significant differences in ryegrass production were observed compared to the ideal treatment under conventional TSP(KN) production system conditions in most treatments. However, in the case of CBE, a significant increase of 25% was observed compared to the positive control TSP(KN). As for the capacity to capture P by plant tissue (Figure 8), it was observed that in all cases the bioformulations in the absence of exogenous N and K were significantly better than conventional management, with CBE standing out with an increase of over 50%.These results show that the application of the bioactivating composition could generate a high reduction in the cost of establishing pastures due to the possibility of reducing the incorporation of conventional N, P and K fertilizers by at least 50%, reporting important benefits for both farmers and the environment.

Claims

CLAIMS 1. A bioactivating composition, for providing readily available phosphorus to plant crops, CHARACTERIZED in that it comprises: (i) a combination comprising: (a) a source of organic waste as a phosphorus-rich carrier matrix that is composed of excreta stabilized by aerobic composting; (b) enzymes that mineralize phthate; (c) phosphorus-solubilizing bacterial consortium comprising bacteria from the Klebsiella, Stenotrophomonas, Serratia-, and (i) a polymer for homogenization and granulation of the composition.

2. The bioactivating composition according to claim 1, CHARACTERIZED in that the source of organic waste is selected from livestock animal excrement such as excrement from cattle, pigs, sheep, horses, among others 3. The bioactivating composition according to claim 2, CHARACTERIZED in that the organic source of phosphorus comprises bovine excreta.

4. The bioactivating composition according to claim 1, CHARACTERIZED in that the phytate mineralizing enzymes are selected from the phytase group.

5. The bioactivating composition according to claim 4, CHARACTERIZED in that the phytate mineralizing enzyme is a phytase isolated from Escherichia coll immobilized on a synthetic polymer.

6. The bioactivating composition according to claim 1, CHARACTERIZED in that the bacterial consortium is made up of Klebsiella sp. RC3 (KU697293), Stenotrophomonas sp. RC5 (KU697294), Serratia sp. RJAL6 (KU697295), Klebsiella sp. RCJ4 (KU697296) and Serratia sp. RCJ6 (KU697297), obtained from Ryeica roots and shoots that exhibit plant growth promotion and tolerance to aluminum (10 mM).

7. The bioactivating composition according to claim 1, CHARACTERIZED in that the polymer is selected from the group comprising biodegradable polymers of modified polysaccharides with a high permeability coefficient (K> 4000 cm 2 s -1 Pa -1 ) such as: starches, alginates, agars; water-soluble petroleum-based polymers such as: polysulfone, polyacrylonitrile, polyvinyl chloride, polyurethane and polystyrene; synthetic polymers polysulfone and polyacrylonitrile and cellulose acetate 8. The bioactivating composition according to claim 10, CHARACTERIZED in that the polymer is sodium alginate.

9. A method for preparing the bioactivator composition in granular form, to provide readily available phosphorus to plant crops, CHARACTERIZED in that it comprises the following steps: a) preparing a combination comprising an organic source of phosphorus, phosphorus-solubilizing bacteria, and phosphate-mineralizing enzymes by the following steps: (i) prepare compost from an organic waste source as a source of phosphorus; (i) mix the compost from stage (i) with water until a homogeneous mixture is generated; (iii) add enzymes that mineralize f tate to the mixture of step (i); (iv) adding a phosphorus-solubilizing bacterial consortium to the mixture from step (iii); (b) adding a polymer to the combination from step (a); (c) combining and homogenizing the mixture from step (b); (d) adding the mixture from step (c) to a solidifying solution to generate granules; (e) separating and washing the precipitated material from step (vii); (f) drying the precipitated material that constitutes the granulated bioactivating composition.

10. The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that step (i) comprises the following steps: a) aerobically degrading the excreta with a fungal broth of saprophytic fungi in thermally insulated reactors where glucose is added until a carbon source to nitrogen source (C:N) ratio of between 25:1 and 35:1 is obtained, a process lasting between 12 and 16 days. b) allowing the reaction of step a) to take place for a period of 80 to 100 days to obtain the stabilized material. 1 1 . The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that the compost-to-water ratio in step (i) is between 1:3 and 1:

4.

12. The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that the enzymes selected from the group of the rates of step (iii) are added between compost / enzyme ratios of 1:0.5 and 1:1.

5.

13. The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that the phosphorus-solubilizing bacterial consortium of step (iv) is pre-hydrated in ratios between 1:6 and 1:10 with water to obtain a bacterial load of 10 14 10 18 UFC.

14. The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that the bacterial consortium is added to the mixture of step (iii) between compost / consortium ratios of 100,000:1 and 140,000:

1.

15. The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that the polymer used in step (b) is selected from the group comprising biodegradable polymers of modified polysaccharides with a high permeability coefficient (K> 4000 cm⁻¹). 2 s -1 Pa -1 ) such as: starches, alginates, agars; water-soluble petroleum-based polymers such as: polysulfone, polyacrylonitrile, polyvinyl chloride, polyurethane and polystyrene; synthetic polymers polysulfone and polyacrylonitrile and cellulose acetate.

16. The method for preparing the bioactivating composition according to claim 15, CHARACTERIZED in that the polymer previously hydrated with water between 2% 4% w / v is added to combination a) until a final concentration is obtained that varies between 1% w / v and 3% w / v.

17. The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that step c) is carried out in a stirred reactor between 10 and 30 rpm for a time between 20 and 80 min.

18. The method for preparing the bioactivating composition of claim 9, CHARACTERIZED in that step d) is carried out by passing the mixture of step (c) through a tray with circular perforations of diameter between 4 to 6 mm allowing the generation of granules which are directly mixed with the solidifying agent at an agitation between 400 and 600 rpm for 30 to 90 min.

19. The method for preparing the bioactivating composition according to claim 18, CHARACTERIZED in that the solidifying agent is selected from calcium chloride, gluconolactate, calcium acetate monohydrate, calcium lactate.

20. The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that step e) is carried out by centrifugation, filtration, or any combination thereof.

21. The method for preparing the bioactivating composition according to claim 9, CHARACTERIZED in that in step e) the washing of the granules is carried out with water or distilled water to remove the excess solidifying agent.

22. The method for preparing the bioactivating composition of claim 9, CHARACTERIZED in that step f) is carried out at a temperature not exceeding 30°C and until a relative humidity of 10-16% is reached in the bioactivating composition.

23. Use of the bioactivating composition according to claims 1 to 8, CHARACTERIZED in that it serves as a fertilizer enhancer or activator of a fertilizer or fertilizer.

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