Natural organic edaphic biostimulant, preparation process and use thereof for improving the availability of nutrients confined in the soil

Abstract

The product of the present invention is a biostimulant, a natural product having the function of improving the availability of nutrients confined in the soil (for plants), in particular P, K, and Mn. Therefore, the use thereof allows increasing the efficiency and effectiveness of nutrients in the soil and / or of fertilizers, thus reducing the need for external inputs. The invention relates to both the starting enzymatic nucleus forming a complex, adaptive organic system to be used in the processes of biostabilization of animal excretions, and the process for the application thereof to soils to be cultivated for which conditioning is required in order to promote the uptake of nutrients by plants. Advantageously, in a preferred embodiment of the present invention, the enzymatic nucleus is not applied directly to the soil to be cultivated, but to a substrate of manure / animal excretions, which is activated and after a maturation period in which the availability of the existing nutrients increases comes to form a natural biostimulant product that can be packaged for storage and sale, similarly to treated universal potting soils, to then be incorporated into the top 20 cm of the surface layer of the soil by tedding.

Description

[0001] NATURAL ORGANIC EDAPHIC BIOSTIMULANT, PREPARATION PROCESS AND USE THEREOF FOR IMPROVING THE AVAILABILITY OF NUTRIENTS CONFINED IN THE SOIL DESCRIPTION SUMMARY

[0002] The present invention relates to the field of fertilizers and technical means for agricultural production, in particular a process for improving the availability of nutrients in soils, by optimally using animal manure.

[0003] Such a process falls under a circular economy practice, also applicable to the fields of sustainable agriculture in general, organic / biological and regenerative agriculture. This approach allows for savings in raw materials (including critical ones such as P), since it improves efficiency in the use of resources, thus reducing the need for additional resources.

[0004] More in particular, modes are provided herein through which a natural biostimulant can be produced and used, having the function of improving the availability of nutrients that are confined in the soil, for the plants intended to be cultivated and for which conditioning is required, in particular P, K, and Mn. Therefore, the use of such a biostimulant allows increasing the efficiency and effectiveness of nutrients in the soil and / or of fertilizers, thus reducing the need for external inputs. In practice, the consumption of resources is reduced, especially critical ones, and therefore energy consumption and greenhouse gas emissions are reduced.

[0005] The process for producing and applying the natural biostimulant to the soil according to the present invention includes 3 steps.

[0006] Step I consists in preparing, from the mixture of available and obtainable natural raw materials, at low cost, an enzymatic nucleus forming a complex, adaptive organic system to be used in processes of biostabilization of animal excretions.

[0007] Step II consists in activating said enzymatic nucleus within a substrate, substantially consisting of animal excretions, by adding said nucleus to the substrate itself, which allows for enrichment in microorganisms that degrade theorganic matter and which, in addition to having hydrolytic enzymes that enhance the activity, are also rich in C and nutrients to enhance, for example, phosphatase.

[0008] As is known, enzymes are produced in an inactive form and are activated only when they reach the site in which they must act: in the present case, the enzyme interacts with the substrate by means of weak non-covalent bonds. In enzymatic molecules, there is a special pocket or groove, referred to as the active site: it contains side chains of amino acids which create a three-dimensional surface complementary to the substrate.

[0009] Step III consists in applying, to the soil for which conditioning is required, by adding the activated substrate (nucleus + substrate) in a homogeneous manner, in the surface layer up to a 20 cm depth, the same soil so as to simultaneously provide enzymes and nutrients and stabilized organic matter.

[0010] SCOPE OF THE INVENTION AND CURRENT PROBLEMS

[0011] The population is increasing. According to United Nations estimates, the world population is expected to reach about 9.7 billion people by 2050, with respect to the current 7.9 billion. This demographic increase will contribute to an increase in the demand for food. The FAO estimates a necessary increase of about 70% in global food production by 2050. This increase in food production must take into account the limitations of natural resources, such as arable land, water, and biodiversity, which are under pressure due to climate change and other factors. In order to achieve this objective, significant improvements will be required in the efficiency of agricultural production, reduction of food waste, and sustainable management of natural resources.

[0012] Therefore, there is a strong need to maintain the quality and amount of agricultural products, while reducing the use of resources to produce them. In a sustainable agriculture perspective, it is desirable to obtain a natural product that enables quality agricultural production while reducing the environmental impact of fertilizers, allowing a more effective use thereof and thus reducing the amounts to be employed, the use of virgin resources, of raw materials (sometimes nonrenewable and critical, such as phosphorus, for example), the consumption of energy, as well as greenhouse gas emissions.Therefore, it is a first object of the present invention to suggest / identify natural products that allow, in a renewable cycle, reducing the inputs of new resources, reducing greenhouse gas emissions, so as to reduce the carbon footprint of the agricultural production.

[0013] All of the above is aimed at allowing an agricultural production which also withstands climate change, enhancing the response of living, interactive, and adaptive systems, making the best use of what is available.

[0014] BACKGROUND ART

[0015] The agricultural production of both food and plants requires plant nutrients. Nutrients such as nitrogen (N), phosphorus (P), and potassium (K) are essential elements for organisms, including plants, animals, and bacteria. And indeed N, P, K are the primary macronutrients used as fertilizers in agriculture to ensure high yields and quality in the products (Resource Efficiency in Practice -Closing Mineral Cycles | Final report EU).

[0016] Micronutrients and trace elements such as Zn, Fe, Mn, Mg, Ca, Cu, B, are also very important for agriculture. Organic soil conditioners can incorporate any one thereof so as to have a positive impact on the fertility and productivity of soils.

[0017] Historically, the following have been developed:

[0018] Fertilizers and manures which could provide plants with the nutrients required for the development and growth thereof; as well as

[0019] Products (compost, soil improvers, soil conditioners, soil correctives...) which could improve the functionalities and performance of soil.

[0020] The term "organic fertilizer" typically relates to a soil conditioner originating from natural sources which ensure at least the minimum percentage of nitrogen, phosphate, and potassium. Examples include plant and animal byproducts, rock dust, seaweed, inoculants, and conditioners.

[0021] “Biofertilizer” is another term used in this field. It refers to a substance containing live microorganisms which, when applied to seeds, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promote growth by increasing the supply or availability of primary nutrients to the host plant.Biofertilizers add nutrients through the natural processes of nitrogen fixation, phosphorus solubilization, and stimulation of plant growth through the synthesis of growth-promoting substances. The use of biofertilizers can supplement the use of chemical fertilizers, reducing the use thereof. The microorganisms found in biofertilizers restore the natural cycle of soil nutrients and build the organic matter of the soil. Using biofertilizers, healthy plants can be cultivated, while improving the health of the soil.

[0022] The GREEN DEAL “Ensuring sustainable food production Action 3” for 2030 introduces, as recommended measures for CAP STRATEGIC PLANS, the reduction of nutrient loss by at least 50%, and the corresponding reduction of fertilizer use by 20%, ensuring that there is no deterioration of soil fertility.

[0023] In a perspective of sustainability and optimal use of resources (in Europe, see the Green Deal and Farm2Fork), the development and production of traditional fertilizers (mineral and organic fertilizers) have been complemented by the development of new categories of products such as bio stimulants.

[0024] The concept of biostimulant is due to the empirical use thereof. The term biostimulant began to appear at the end of the 1990s, as a “non-fertilizer product, with beneficial effects on plant development”, particularly in low-input systems (Patrick du Jardin, 2015).

[0025] Du Jardin P (2015) suggested the following definition of biostimulant: “A plant biostimulant is any substance or microorganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and / or crop quality traits, regardless of its nutrient content. By extension, plant biostimulants also designate commercial products containing mixtures of such substances and / or microorganisms.”

[0026] In the last decade there has been a conceptual shift. Biostimulants are not defined by what they contain, but by what they do. Rather than providing nutrients, they support physiological processes of plants, root development, assimilation of nutrients, or stress response: therefore, they are not “fertilizers”, but “function enhancers”.

[0027] A fundamental contribution to this perspective came from Du Jardin(2015), who suggested an operational definition which emphasizes the effects over the composition, helping to establish a framework which distinguished biostimulants from fertilizers and pesticides. This model shifts the focus from the “formulation” to the “function”.

[0028] The concept has been introduced into technical legislation. ISO / TC 134 (ISO, 2022) describes biostimulants as “substances, microorganisms, or mixtures thereof that support a plant’s natural nutritional processes."

[0029] The new European regulation on fertilizers (Regulation EC / 1009 / 2019) in force since 2022 has introduced a new definition for biostimulants, which reads: “Plant biostimulant is any product that stimulates the plant nutrition processes independently of the product’s nutrient content, with the sole aim of improving one or more of the following characteristics of the plant or rhizosphere of the plant:

[0030] a) nutrient use efficiency;

[0031] b) tolerance to abiotic stress;

[0032] c) quality traits; and

[0033] d) availability of confined nutrients in the soil or rhizosphere. (Art.47)”.

[0034] In Regulation EC 2019 / 1009, biostimulants are thus defined based on the function thereof and not on the composition thereof nor by modes of operation, and can be either microbial or non-microbial.

[0035] Microbial plant biostimulants PBs include beneficial fungi and bacteria, such as arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR).

[0036] Non-microbial biostimulants are distinguished into various subcategories, based on the main active component: chitosan oligosaccharides / chitosan (Chi), humic and fulvic acids (HFA), animal and plant protein hydrolysates (PH), phosphorus and silicon compounds (phosphites, Phi), seaweed extracts (SWE), silicon (Si) compounds, inorganic salts (salts of K and Mg with organic matter), and plant extract-based biostimulants (PE) (excluding SWE) (Li et al., 2022). Seaweed extracts have been used in agriculture since Roman times. Biostimulants based on algae / humic acids excel at carbon sequestration.Emerging techniques focus on nano-biostimulants (e.g., SiO2 nanoparticles) for targeted release, as well as on the application of PGPR / AMF and hybrids (e.g., hydrolysates + Bacillus) to obtain a synergy. The use of modeling by means of artificial intelligence is under development.

[0037] Li et al. (2022) report increases in yield in open-field cultivation following the application of bio stimulants, comparing various parameters: biostimulant category, method of application, plant species, weather conditions, and soil properties. The overall results showed that the additional benefit in terms of yield across all categories of biostimulants is on average 17.9% and has reached the maximum potential by means of soil treatment. PE proved to be the most effective biostimulant. The authors have connected the efficacy of PE to the complex composition of plant metabolites thereof, containing several macros and minerals, osmoprotectants, and antioxidants. PE probably also contains plant hormones which, in small amounts, are known for their ability to stimulate agricultural production. The responses depend on several parameters, type of application, types of soil, weather conditions, etc. The "environment" can influence the response.

[0038] The new definition better aligns with the currently available data. At present indeed, in practice, the distribution of a fertilizer in the soil for providing nutrient elements to plants does not necessarily result in the direct use thereof. The nutrients are added to the complex soil system and interact therewith and with microorganisms contained therein. Hence, in general, the function of biostimulants is to make such nutrients available to plants and to improve use efficiency (NUE).

[0039] By improving yields, biostimulants could reduce the requirement for (synthetic) fertilizers by 15 / 25% through an increase in NUE, aligning with the requirements of the EU Green Deal.

[0040] Since there is a general consensus on the emergence of synthetic fertilizers which represent an environmental threat to local and global ecosystems, plant biostimulants (PB) are potentially a tool to mitigate climate change-induced stress and reduce dependence on chemical fertilizers.As for nutrient availability, particular attention has been devoted to phosphorus (P), which is an essential and non-renewable resource, fundamental for agricultural production, as shown below.

[0041] PHOSPHORUS AND FEATURES THEREOF

[0042] • P is one of the 6 building blocks of life (Oxygen, Hydrogen, Potassium, Nitrogen, and Carbon) and is vital for life on the planet, in both plants and animals, including humans, as well as for bio-based economic processes occurring in the global economy. P is vital for plant growth and is involved in all stages of development. Several key processes for plants require an adequate uptake of P by the crop, such as the biosynthesis of cellular constituents (e.g., nucleic acids, phospholipids, ATP, etc.), photosynthesis, and fixation of N2 by legumes (Bargaz et al., 2021)

[0043] • P is a limited and non-renewable resource. Phosphate rock and phosphorus are included within the list of critical raw materials (Critical Raw Materials, Strategic Raw Materials) by the EC.

[0044] Globally, 91% of phosphate rock is used for producing fertilizers; in Europe, 85%. There is no substitute for phosphate rock for producing fertilizers. For the application thereof in agriculture, it can be partially replaced by secondary sources of P such as manures, sludges, and food residues (biogenic waste flows).

[0045] Similarly, Magnesium, Manganese, Nickel and Copper are also recognized as critical materials (other micronutrients for plants).

[0046] • P is often strongly fixed in soils, thus becoming unavailable to plants. Only 1 to 5% of the total P present in the soil is in a form available to plants (Arcand & Schneider, 2006). The excessive application of phosphorus, in combination with the poor utilization efficiency thereof by plants, leads to a significant accumulation thereof in the soil.

[0047] Due to the limited availability of phosphorus in many soils, the excessive application of fertilizers is often used to ensure optimal plant growth and maximize the annual crop yields (Liu et al., 2022), which is then sometimes a cause of eutrophication of water.

[0048] The soil cycle of P is a dynamic process which involves the transformationof P by geochemical and biological processes.

[0049] Phosphorus is present in three main groups: organic P (Po), inorganic P bound to Ca, and inorganic P bound to Fe and Al.

[0050] The fraction of P available to plants is found in the soil solution as orthophosphate anions, mainly FhPCU-and HPCL2.

[0051] The fraction of P available to the plant, or P solution, is in equilibrium with a relatively labile fraction of P which is adsorbed by hydrated aluminum or ferric oxides, clays, calcium carbonates, and organic matter, and is thus associated with the solid phase of the soil. (Arcand & Schneider, 2006).

[0052] Organic phosphorus Po is the dominant form in many soils, typically about 50% of Ptot in agricultural soils, but in some cases it can reach 95%. (Liu et al., 2022). In fact, inorganic P (Pi) in fertilized soils is often converted / transformed by microorganisms and plants (see Fig. 1).

[0053] When soluble P sources are added to soils, for example from fertilizers and manure, they are fixed and highly insoluble compounds are formed over time, which are unavailable to plants. Fixation reactions in soils can allow only small fractions (10% to 15%) of the P content in fertilizers and manures to be absorbed by plants in the year of application. Therefore, it is common practice to apply greater amounts to ensure the uptake by plants.

[0054] HOW TO PROMOTE THE AVAILABILITY FOR PLANTS

[0055] Precisely because mobilization from plant-unavailable P to plant-available P is a prerequisite to support agricultural productivity (Bat et al., 2017), the processes, conditions, and factors allowing such a transformation have been studied over time, all linked to enzymatic activities, in particular (acid and alkaline) phosphatase and phytase.

[0056] The biological processes are made possible by enzymes. It is for good reason that the distribution of enzymes in the soil is also studied as an indicator of the quality thereof. In the following Table 1, different types of enzymes are grouped for merely indicative purposes, as well as the type of reaction catalyzed and the reference cycle.

[0057] TABLE 1Soil enzymes (adapted from Adetunjil et al., 2017; Bautista-Cruz & Ortiz- Hernandez, 2015)

[0058]

[0059] where:

[0060] a single * indicates an enzyme that catalyzes hydrolysis and biodegradation of various P-glucosidases found in the organic matter;

[0061] two ** indicate the phosphatase which catalyzes the hydrolysis of esters and anhydrides of H3PO4 for releasing inorganic P, assimilable by plants, and three *** indicate enzymes involved in the progressive decomposition of the N content in the proteins.

[0062] In order to evaluate the soil quality, subjected to different types of management, among hydrolases, the following were especially used:

[0063] Glucosidase, relative to the enzymes involved in the carbon cycle;Phosphatase: the activity was the most frequently used parameter to estimate changes in soil activity, also in the presence of contaminants; Urease, relative to the nitrogen cycle (Bautista-Cruz & Ortiz-Hernandez, 2015).

[0064] Acid and alkaline phosphatases are a group of enzymes that catalyze the hydrolysis of esters and anhydrides of phosphoric acid, releasing inorganic P and phosphate ions assimilable for plants. Plants and microorganisms are the main source of phosphatase enzymes in soil (Adetunji et al., 2017). Especially for P, the conditions that allow promoting the action of phosphatase to make it available for the plants have been investigated. Some results from the literature are given below.

[0065] Adetunji et al. (2017) report variations in the phosphatase activity with the pH of the soil, and a reduction in the presence of drought conditions and in the presence of heavy metals and Pb. They report greater excretion of phosphatase enzymes by legumes as compared to other plants. They also report the increase in the activity of the phosphatase enzymes in soils enriched with organic soil conditioners / fertilizers (plant residues, sewage sludge, animal excretions, compost, and vermicompost). It can be connected to the stimulation of microbial growth and the enrichment of the organic matter of soils.

[0066] Bargaz et al. (2021) suggest the use of bacteria which improve the use of P, in particular PSBs (P Solubilizing Bacteria) as inoculum in the soil to improve the efficiency of use of P and the agronomic performance, promoting the cooptimization of PSBs- Fertilizers - processes involving the roots. In fact, PSBs are involved in the mechanism of P uptake by plants through the steps of solubilization, mineralization, and mobilization of P, with the production of organic acids and P-hydrolytic enzymes. In fact, PSBs release different types of organic acids (oxalic, citric, gluconic, succinic, a-ketoglutaric and fumaric acids), responsible for the transformation of the insoluble forms of P into soluble forms. In fact, these compounds chelate the cations (Al, Fe, and Ca) bound to P through the hydroxyl and carboxyl groups thereof, thus offering a form of P which is readily assimilable by plants. They also mediate the mineralization processthrough the production of P-hydrolyzing enzymes, such as phytase and (acid and alkaline) phosphatase that catalyze the hydrolysis of P-ester bonds.

[0067] Bargaz et al. (2021) also report the solubilization of certain forms such as tricalcium phosphate by PSBs through secretion of various organic acids (malic acid, lactic acid, acetic acid, citric acid, oxalic acid, and succinic acid) and of the enzyme acid phosphatase.

[0068] Therefore, inoculation with PSBs is suggested to simultaneously mobilize the unavailable forms of P in the soil and significantly improve the use efficiency of P-containing fertilizers, providing unique opportunities for a better availability of P in the soil-root- microorganism continuum.

[0069] They further highlight how the efficiency in the agronomic use of P by PSBs is enhanced in combination with mineral and especially organic (manures in general - poultry, goat and bovine) fertilizers, and generally with co-application of bio-organic P (mixture of compost, biogas residue, and P rock). The partial replacement of mineral fertilizers (NPK) with manures can also enhance the availability of P in the soil and reshape the PSB community in the soil with improvement in P mineralization and uptake by plants.

[0070] Bargaz et al. (2021) conclude that the inoculation of crops with PSBs in combination with mineral or organic fertilization can be considered a promising route for an integrated strategy to improve the availability of P by improving the agronomic use / efficiency of P-containing fertilizers, in order to increase the fertility of soils and support for agriculture.

[0071] Arcand & Schneider (2006) studied the solubilization of phosphate rocks by plants and microorganisms.

[0072] Some plant species exhibit mechanisms localized in the rhizosphere which enable an efficient use of P through the dissolution of PR. Examples of non-mycorrhizal plants are those of the Cruciferae family, or plants such as white lupin (Lupinus albus L.) that form cluster roots, which solubilize P Rock (PR) in case of P lack in the soil.

[0073] Some microorganisms are capable of solubilizing PR and are collectively referred to as phosphate- solubilizing microorganisms (PSBs).Rhizobacteria, of the genera Pseudomonas, Bacillus and Rhizobium, are among the most potent phosphate-solubilizing bacteria.

[0074] Plants operate through excretion of H+ by the roots in the rhizosphere, modifying the pH. Similarly, microorganisms also release H+ (e.g., Bacillus). Both plants and bacteria release organic acids.

[0075] The solubilization of Prock occurs through acidification mechanism, chelation, and exchange reactions. Citric (from Penicillum or Aspergillus niger). oxalic, and tartaric acid are particularly effective.

[0076] Arcand & Schneider (2006) already recognize the role of PSM inoculation in the soil to promote the uptake of P from Prock by plants, but they also recognize the survival difficulties of such microorganisms in the rhizosphere, in competition with native soil microorganisms. They suggest the use of fermentation substrates for PSMs prior to field application, recognizing that “the pre-incub ation of waste materials is a key factor for the PSM-Proc system”. It is assumed that Aspergillus niger can release organic acids (citric and oxalic acids) through an aerobic fermentation process.

[0077] Both plants and microorganisms are capable of solubilizing Prock through the production of H+ ions and organic acids.

[0078] By optimizing the culture conditions of microorganisms, in a controlled environment for producing organic acids, the dissolution of PRock can be enhanced prior to field application.

[0079] The use of PSMs which produce organic acids in a pre-treatment step, under controlled conditions prior to field application, is a low-input and appropriate technology to increase the agronomic effectiveness of Prock.

[0080] Liu et al. (2022) report that even the utilization of P in the form of phytate by plants often proceeds through solubilization of phytate by means of organic acids, to then proceed through phytase (EC 3.1.3.8, EC 3.1.3.72, EC 3.1.3.26). Organic acids can be released by plants (e.g., malonic, malic, citric acids) or more often by microorganisms (e.g., citric, succinic, lactic, oxalic, gluconic, tartaric acids) and fungi (e.g., citric, succinic, lactic, oxalic, gluconic, tartaric, acetic, fumaric acids).Bautista-Cruz & Ortiz-Hernandez (2015) report that enzymatic activity decreases with the depth of the soil, due to the decline of microbial activity and macronutrients such as organic C and organic N. They further report that mineral N fertilization stimulates phosphatase activity, most likely because phosphatase requires a substantial investment in N; NP fertilization also stimulates in the same manner. N can control the enzymatic activity of phosphatase, because the phosphatase cannot be produced if there is not sufficient N.

[0081] The activity of hydrolytic enzymes in soil is responsive to different forms of fertilizers. Organic inputs stimulate the activity of hydrolytic enzymes in soil.

[0082] The increase in the enzymatic activity of soils treated with organic residues can be attributed to the combined effect of (i) a higher degree of enzyme stabilization with the formation of organo-mineral complexes as a result of the increase in humus content, and (ii) an increase in microbial biomass due to the increase in organic C content (Bautista-Cruz & Ortiz-Hernandez, 2015).

[0083] In fact, the organic matter of soil plays a key role in soil structure and contributes to the reactions that occur, such as the penetration of water, the development of roots, etc. (IPCC 2001). It is the storage sphere of macronutrients, such as N, P, S, and micronutrients. From an agronomic viewpoint, enhancing the organic pool to an optimal level improves crop and pasture yields through various processes (Lal 2010): i) increasing water retention, ii) improving nutrient supply to plants, iii) restoring soil structure, iv) minimizing erosion risks.

[0084] Common elements to promote the transformation activity of P into plant-assimilable forms are represented by: the presence of organic acids to promote solubilization and the increase in the presence of fertilization, especially organic fertilization.

[0085] As highlighted by multiple sources, the issue of the survival of microorganisms / enzymes inoculated in soil is crucial. The problem of the survival of enzymes and microorganisms in hostile environments had already been addressed by the use of “additives” for compost as early as the ‘90s. Products have been developed, which allow optimizing consumptions, reducing odor emissions, targeting the quality of the finished product, for example bymaximizing the nutrients. Precisely to improve survival and thus efficacy, extracellular enzymes (especially hydrolases to accelerate the composting step I, the slowest one) and / or microorganisms have gradually been incorporated within complex products, consisting of at least one of the following categories: microorganisms, enzymes, carriers, supplements, and nutrients (co-formulation). In order to compete with the natural micro flora / fauna in a highly competitive and microbiologically active environment, co-formulations have been introduced.

[0086] Mineral supplements / nutrients are intended to prevent the deficiency of trace elements which could limit or halt microorganism activity.

[0087] In this specific case, reference will be made to the techniques described in European patent EP1314710 entitled “A process of maturing and stabilizing biomasses under reduction of smelling emissions”, and in Italian patent No.

[0088] 1393404 entitled “Procedimento per ridurre la produzione e le emissioni di metano da discariche e da siti di recupero rifiuti”.

[0089] As clearly indicated in the title and widely illustrated, patent EP1314710 relates to the treatment of fermentable matrices, biomass in general, for the stabilization and maturation thereof in landfills, using enzymatic preparations, and introduces and describes the procedure for preparing an enzymatic composition, based on the use of natural products such as plants and milk, and possibly acetic substances. Such an enzymatic composition is used in doses of 0.1-2 kg / m3 of matrix to be stabilized, evenly distributed at the beginning of the treatment on heaps of biomass located in landfills.

[0090] The plant components belong to 5 families. Unpasteurized milk is obviously used because it is rich in ferments, including bifidobacteria which produce lactic and acetic acids, reducing CO2 emissions.

[0091] It is a stated task of the described treatment to accelerate the biostabilization processes, reducing the tedding operations on biomass heaps, and therefore the number of steps in which the temperature of the heap is reduced and odor emissions are greater. The thermophilic conditions are thus maintained within the heap, avoiding slowdowns during the first moments after the tedding operation. The maintenance of thermophilic conditions in the heap (T> 55 °C forat least 3 consecutive days) is further claimed to ensure sanitation. Furthermore, the other goal related to the reduction of the olfactometric impact is promoted by the use of vegetal components / plants which can mitigate the bad odors of the animal excretion collecting and treating system.

[0092] A recent application of such a patent was in the European project LIFE17 ENV / IT / 333 POREM (Poultry manure-based bioactivator for better soil management through bioremediation (see Dall’ar Alice “New strategies for better soil management via remediation with poultry manure based bioactivator”, 8 December 2021 (2021-12-09) pages 1-35. XP093319526).

[0093] As highlighted in the abstract, “POREM BIOACTIVATOR is a sustainable fertilizer for degraded soils, based on natural resources: it is a product obtained from poultry manure modified with a natural enzymatic preparation derived from plants.” It was produced on a pilot scale during the Project: the POREM bioactivator proved to be effective for the remediation of degraded soils, with retention of C in the soils, biodiversity growth and water retention of the same treated soils.

[0094] Similarly, patent IT RM20080500 Al discloses a procedure for the preparation of an enzymatic composition obtained from plants and milk. They are different from those of EP1314710, since they are rich in Vitamins A, C, and E, belonging to the families asteraceae, gramineae, malvaceae, rosaceae and Rutaceae; specifically, 8 species are indicated: Jerusalem artichoke, dandelion, sunflower, wheat, sorghum, rosehip, mallow, and rue. The enzymatic compositions are used together with a tailored compost for a specific purpose: for the reduction of methane production and emissions in landfills and waste disposal sites.

[0095] Also claimed is an accelerated process of a tailored compost, with maintenance of thermophilic conditions for at least 1 week between 55 and 75 °C.

[0096] Other techniques based on the use of common raw materials have been introduced in CN 105 254 365 A (20 January 2016) and CN 107 721 483 A (23 February 2018).

[0097] Both have a similar approach: they use natural products (milk and plants)and manure to produce special fertilizers. In the case of the latter two documents, the fertilizers are enriched agricultural products mulberry leaves with improved medicinal properties and selenium-enriched chestnuts, a functional food or superfood, respectively. It is the objective of our work to enrich soils instead.

[0098] Both relate to fertilizers in general, from natural products (including milk and plants) and manure. In both cases, the manure is added at a percentage of the final fertilizer between 10 and 25%, while in our process it is used to multiply the amount thereof, forming almost the totality of the fertilizer (> 99%).

[0099] In particular, CN105254365A relates to a special fertilizer (preparation method and fertilizer) intended for mulberry leaves to improve the medicinal value thereof. This fertilizer is applied to the soil and can be used long term to promote the absorption of medicinal components by mulberry leaves. The raw materials are: milk and plants, some of which originating from traditional Chinese medicine, and a small amount of manure. Milk is the main component of the fertilizer; manure is about half of the milk. The plants are completely different. The composition and the preparation procedure are summarized in Figure 1.

[0100] With respect to our results, it is immediately verified that:

[0101] - the composition is different,

[0102] - the preparation method is different, and

[0103] - the final product (fertilizer) and the function thereof (improvement of the medicinal value of mulberry leaves) are different.

[0104] Patent CN107721483A relates to a method for planting selenium-enriched chestnuts. It is organized in 3 steps:

[0105] - soil improvement,

[0106] - preparation and use of an acidic base fertilizer,

[0107] - management of diseases.

[0108] The patent describes a selenium-rich liquid root fertilizer and an acidic base fertilizer.

[0109] The preparation of the acidic fertilizer is based on the use of rice straw (43%), wine distillation residues, milk, yeast, and decomposed sheep manure. The final fertilizer has a pH equal to 5. Milk is used to increase protein and traceelement content. The function of the acidic fertilizer is the provision of a liquid fertilizer rich in nitrogen, phosphorus, potassium, trace elements, and humus, readily absorbable by the chestnut tree.

[0110] The basic acidic fertilizer serves the function of improving the alkaline soil, improving the fertility thereof, reducing the pH value, and making the soil suitable for the growth of the chestnut tree, while providing a rich humus. The use in combination with a selenium-rich root fertilizer allows the chestnut tree to effectively absorb abundant nutrients and selenium.

[0111] As highlighted by the diagrams given, the composition of the acidic base fertilizer is completely different from that of the biostimulant according to the present application (Figs. 2 and 3), and the preparation procedure is also different, since it is the objective here to improve the absorption of selenium by the roots with liquid fertilizers in a soil acidified by the use of acidic fertilizers in large amounts (15-20 cm layer in the drainage channels). The function is also different from that of the biostimulant.

[0112] For the biostimulant, an average of 99% of the fertilizer consists of manure, the basis for the environmental and economic sustainability of the suggested fertilizer. The use thereof is provided in a range of 1-5 tons / ha, therefore from 0.2 to 3 liters of milk / ha are required, where the lower values refer to the treatment of large amounts of manure.

[0113] PURPOSE AND OBJECT OF THE INVENTION

[0114] In a sustainable agriculture perspective, it is the task of the present invention to provide a product which allows quality agricultural production while reducing the environmental impact of fertilizers, allowing a more effective use thereof and therefore capable of reducing the amounts to be employed, the use of virgin resources, of raw materials (sometimes non-renewable and critical, such as phosphorus, for example), the consumption of energy, as well as greenhouse gas emissions.

[0115] To do so, the techniques of patent EP1314710 were used to handle the plant and milk components but, according to two peculiar features of the present invention, for the purpose of enhancing the production of (lactic) acid, in the stepof preparing an enzymatic nucleus that, unlike what is known, must serve the function of a biostimulant capable of supporting the natural nutritional processes of a plant, the % of milk was significantly modified, to be considerably increased, and a fundamental 10-day fermentation step after the milk-fertilizer mixing step was also introduced.

[0116] This is because, with a view to going beyond the POREM program, which was limited to providing a medicine for the recovery of degraded soils, it is the task of the present finding to use enzymatic activities not only for the nutrients found in the fertilizer / substrate, regulating the release thereof to the environment and plants, but also to transfer the same enzymatic activities found in the nucleus, into the «soil environment» also directly affecting the nutrients found in the soil, thus providing a medium with long shelf life which introduces vitality (measured as “basal microbic respiration”) capable of enhancing the soil activity, entirely benefiting productivity and biodiversity.

[0117] In other words, it is the object of the present invention to produce and utilize the vitality of an edaphic biostimulant to interact with the soil and improve the availability of nutrients for the plants, in accordance with the definition of biostimulant, and to extend the application thereof to all types of agricultural activities in the perspective of a circular agriculture.

[0118] While POREM is substantially a specialized medicine for degraded soils, specifically tested in soils poor in Organic carbon, the edaphic biostimulant according to the present invention can be applied to all agricultural practices (conventional, organic, and regenerative agriculture), as well as to soilless techniques, horticulture, gardening, and nursery.

[0119] Accordingly, there is a need for a system which adapts to the various soil conditions and cultivation practices. The biostimulant production process described allows operating under high thermophilic conditions (T> 65 °C for weeks), useful for both the sanitization of the final fertilizer and the selection of microfauna, disadvantaging mineralizers (disfavored above 60°C, in the hot compost range).

[0120] It is another object of the present invention to also ensure an agriculturalproduction which withstands climate change, enhancing the response of living, interactive, and adaptive systems, making the best use of what is available.

[0121] As previously stated, the product of the present invention is a biostimulant, a natural product having the function of improving the availability of nutrients confined in the soil (for plants), in particular P, K, and Mn. Therefore, the use thereof allows increasing the efficiency and effectiveness of nutrients in the soil and / or of fertilizers, thus reducing the need for external inputs.

[0122] The invention relates to both the starting enzymatic nucleus to be used as a complex, adaptive system in the processes of bio stabilization of animal excretions and the process for the application thereof to soils to be cultivated, for which conditioning is required, in order to promote the uptake of nutrients by the plants.

[0123] Advantageously, according to a peculiar feature of the invention, in a preferred embodiment of the present invention, the enzymatic nucleus is not applied directly to the soil to be cultivated, but to a substrate of manure / animal excretions, which is activated and after a maturation period in which the availability of the existing nutrients increases comes to form, per se, a natural biostimulant product that can be packaged for storage and sale, similarly to treated universal potting soils, to then be transported easily to the place of use and incorporated into the top 20 cm of the surface layer of the soil by tedding.

[0124] Therefore, the invention also relates to such a natural biostimulant product adapted to be applied to the soil and capable of simultaneously providing both enzymes and nutrients, and stabilized organic matter, to the soil for which conditioning is required.

[0125] The results of the main chemical analyses have shown that, after the application of such a product to the soil, total P doubled while the available form increased 25 times, total K increased by 12% while the available form tripled. As for Mg concentration, it remained substantially constant (+3%), while the availability thereof increased by 20%.

[0126] Therefore, the natural organic product thus obtained is consistent with the definition of biostimulant contained in Regulation (EC) 2019 / 1009.

[0127] DETAILED DESCRIPTION OF THE INVENTIONThe description of the invention will be better understood with reference to the accompanying drawings, which show, by way of non-limiting example, some process diagrams and physicochemical features of the natural biostimulant according to a preferred embodiment.

[0128] In the drawings:

[0129] Fig. 1 shows the phosphorus cycle in the soil: from the sources of phosphorus in the soil to the pathways through which phosphorus becomes available / unavailable for absorption by plants;

[0130] Figure 2 is a diagram of the various steps of the process for producing the natural biostimulant in hand, according to the finding, with the formation of an enzymatic nucleus from enzymes and organic acids, which is added to a substrate of manure / animal excretions to give rise to an activated substrate that is left to mature before the application thereof to the soil to be fertilized, so that the substrate and the nucleus are two activators working in tandem to increase the availability of nutrients in the soil for plants;

[0131] Figs. 3A and 3B show the steps of preparing and activating the substrate; Figs. 4A, 4B and 4C show the trend of the electrical conductivity EC and nutrients over the maturation / activation time (N, P2O5 and K2O);

[0132] Figs. 5A and 5B show the XRD profiles (X-ray diffractometry analysis) for the initial poultry manure sample at T=0, where a small presence of struvite is evident, and for the internal sample at t= 40, 80 and 120 days, where peaks of calcite and struvite are observed for SO and Sinl20, respectively;

[0133] Figs. 6A and 6B show insertion of the enzymatic nucleus into a heap of animal excretions for producing the biostimulant, and the final heap of the biostimulant, respectively;

[0134] Fig. 6C shows the organic biostimulant product marketed in the form of pellets and the dimensional relation thereof with the hand of an operator;

[0135] Fig. 7 shows the temperature profile over time in a heap of animal excretion substrate;

[0136] Fig. 8 shows the complex, adaptive enzymatic system as packaged for marketing.From the above, the inventive concept underlying the invention is the provision of a starting enzymatic nucleus to be combined with a substrate of animal excretions for the time required to allow the enzymes to remain active and be protected from inactivation in the hostile environment to which they are be introduced, prior to the application thereof to the soil, forming a natural product that has the functions of improving the availability of nutrients in the soil for the plants, and thus classifiable as a biostimulant, capable of promoting the uptake of nutrients by the plants, and susceptible to being stored and sold as an innovative product.

[0137] The objective is to increase the available fraction of the nutrients with respect to the conditions without / before the use of the biostimulant, allowing greater efficiency of the use of nutrients. The amounts of nutrients to be supplied for cultivation from outside, in particular chemical fertilizers, are thus reduced. The finding is in accordance with the definition of biostimulant contained in Regulation (EC) 2019 / 1009 (art. 47 pt d).

[0138] In order to overcome the drawbacks and limitations of the techniques employed so far, a natural organic biostimulant, the process for the preparation thereof, and the process for the application thereof to the soil for which conditioning is desired are provided herein, which allow simultaneously achieving the following results:

[0139] • improvement of the availability of nutrients in the soil for plants, in particular P, K, but also micronutrients such as Mn, Zn;

[0140] • reduction in the amount of necessary fertilizers for cultivation, in particular chemical fertilizers, with the simultaneous reduction of greenhouse gas emissions and resources, and

[0141] • recovery of organic matter to be supplied to soils (Carbon sink) using stabilized manure.

[0142] According to the present invention, the process of producing and applying a natural biostimulant is conceptually structured into three steps as follows:

[0143] I. an enzymatic nucleus is structured:

[0144] the enzymes needed are taken where they are in the plants, andthe substances (organic acids) which allow the enzymes to be effective and work better are added, and

[0145] they are structured into a nucleus which survives and adapts to the environment in which it is introduced, to have a complex adaptive system. A Complex adaptive system, or CAS, is defined as a dynamic complex system with capacity for self-organization, consisting of multiple parts, mixed together, which adapt to the environment in which they are introduced (organic matrix, soil) and develop differently depending on what they have available, giving rise to global behaviors that cannot be explained by a single physical law.; such a complex adaptive system is marketable per se and autonomously usable;

[0146] II. a carrier is used for said enzymatic nucleus:

[0147] a carrier is used, which increases the available amount while also enhancing the activity of the enzymes / nucleus without losing the biostimulant capacity of the nucleus, better if obtained from by-products with recycling, which provides amounts at low cost. Such a carrier is selected so as to supply the C and N needed (C and organic matter to maximize the phosphatase activity, the amounts of N which are needed) and the micronutrients (Cu, Mn, Zn, ...) necessary for the soil to avoid production blockages, according to the Leibig’s law of the minimum.

[0148] Liebig's law of the minimum is a principle developed in the agricultural science by Carl Sprengel (1840) and then made popular by Justus von Liebig. It states that growth is not dictated by the total resources available, but by the poorest resource (limiting factor).

[0149] III. the obtained product is brought to the soil:

[0150] the biostimulant product (nucleus + substrate) is then applied to the soil, incorporating it in the surface layer (e.g., 0-20 cm), which is richer in microorganisms and with the presence of air (microaerophilic zone);

[0151] the function thereof is to improve the availability of nutrients in the soil for the plants, thus classifiable as a biostimulant, increasing the available fraction of nutrients with respect to the conditions without / before the use of thebiostimulant, allowing for a greater efficiency of the use of nutrients.

[0152] The process of producing and applying the natural biostimulant is diagrammed in Figure 2.

[0153] As can be seen, already from step I such a process is characterized in that it operates through complex adaptive systems (CAS).

[0154] With reference to the same Figure 2, the 3 steps of the process are described in sequence.

[0155] Step I - Nucleus preparation

[0156] The first step consists in preparing the enzymatic nucleus to be used, by means of a substrate, in the formulation of the biostimulant capable of making soil nutrients more available to plants from natural raw materials.

[0157] The nucleus consists of two essential parts, the enzymes (a) involved in the activity of making nutrients available and the organic acids (b) which facilitate the action of the enzymes themselves and make them effective, and is formulated so as to be active even in hostile environments, as described below:

[0158] a) enzymes

[0159] Based on the previous statement, the enzymes that have been selected are those which make nutrients available, i.e., essentially hydrolytic enzymes: phosphatases and phytases for P, hydrolases in general for K and micronutrients, including P-glucosidases related to the C cycle, because they degrade the organic matter, making nutrients available. For example, phosphatase plays a key role in the availability of P from manure or agricultural residues.

[0160] Selection of enzymes to be taken

[0161] The inventors have sourced the useful enzymes from plant-based raw materials, using plants and / or parts thereof containing the enzymes to be used (e.g., hydrolytic enzymes, ...) and the vitamins required to activate them.

[0162] Plants were further used, which by virtue of the fragrance thereof can be used to counteract the unpleasant odors produced by the biostabilization processes of step II, involving application of the nucleus to an organic substrate before application to soils (step II).

[0163] The selected plant families are: gramineae, cucurbitaceae, labiatae,umbelliferae, and rutaceae.

[0164] In particular:

[0165] Gramineae are rich in enzymes and widely studied. The abundance of enzymes, e.g., in wheat (Triticum), has been studied for both the beneficial effects of food and to prevent undesired alterations of flours during the storage thereof, by humidity, for example (Leitgeb et al., 2022). Enzymatic activity is a quality parameter of wheat (Khalid et al., 2023).

[0166] The chemical composition of wheat grains, obviously a function of the species, variety, and growth conditions, is fairly stable and distributed among carbohydrates (> 70%, 58% of which is starch), proteins (~ 11%), lipids (~ 2%), and minerals (~ 2%), with a moisture content of about 13% (Weiser et al. 2020).

[0167] Furthermore, the wheat grains are important sources of vitamin E (a-tocopherol) and B vitamins, especially thiamin (Bl), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), and folates (B9). They are rich in minerals, such as K, P, Mg, Ca, Zn, Mn, and Fe; Cu and Se are present in trace amounts. (Weiser et al. 2020).

[0168] Metabolic proteins include hydrolytic enzymes: hydrolases, which cleave starch (amylases), proteins (peptidases), and lipids (lipases), together with oxidoreductases, transferases. Also included are protective proteins which inhibit external amylase and peptidase activities (Weiser et al. 2020).

[0169] The green part (wheat grass) also contains a large amount of biologically active constituents, such as chlorophyll, amino acids, minerals, vitamins, and enzymes. In particular, it is rich in vitamins (A, Bl, 2, 3, 5, 6, 8, and 12; C, E and K, ascorbic acid, carotene) and minerals (S, Na, Al, Cu, Ca, I, P, Mg, alkaline earth metals such as K, Se, Zn, B, and Mo) (Singh et al., 2012).

[0170] Characteristic enzymes are: Proteases, amylases, lipases, cytochrome oxidases, transhydrogenases, superoxide dismutase (SOD); SOD is an antioxidant enzyme, which converts ROS (dangerous free radicals of reactive oxygen species) into H2O2 and one molecule of O (Singh et al., 2012).

[0171] Wheat is rich in hydrolytic enzymes, in particular: enzymes that hydrolyze carbohydrates (a-amylases, cellulases); enzymes that hydrolyze proteins(proteases); enzymes that act on fats and oils, such as lipases and lipoxygenases, polyphenol oxidases, and peroxidases. The enzymatic activities have also been quantified (Rani et al., 2001).

[0172] They are the enzymes that operate on the components of the seed, and that further allow it to germinate and utilize the reserves contained therein. Therefore, they are also available for the hydrolysis of carbohydrates, proteins, and lipids found in the matrices of the substrates to be biostabilized.

[0173] The use of the grain in its entirety allows also incorporating within the nucleus, in addition to enzymes, nutrients, minerals, vitamins, and carbohydrates which are useful for the metabolism of lactic acid bacteria, an integral part of the nucleus. Some of them are inactivated depending on the pH.

[0174] The family Rutaceae, in addition to hydrolytic enzymes and vitamins A and C, also contains vitamin P which enhances vitamin C.

[0175] A summary diagram of the selected plants is given in the following Table 2, together with the % of use (indicative).

[0176] TABLE 2

[0177] Plants selected for the nucleus

[0178]

[0179] b) organic acids

[0180] The presence of organic acids is essential to make the enzymes effective, since the processes of transformation of P from unavailable forms to forms available for plants proceed through the steps of acidification, chelation, andexchange reactions.

[0181] For example, PSBs (P Solubilizing Bacteria), in addition to secreting enzymes which hydrolyze, produce weak organic acids to ensure the first step of acidification (malic, lactic, acetic and oxalic, citric, and succinic acids) (Bargaz et al., 2021).

[0182] Liu et al. (2022) report that organic acids can be released by plants (e.g., malonic, malic, citric acids) or more often by microorganisms (e.g., citric, succinic, lactic, oxalic, gluconic, tartaric acids) and fungi (e.g., citric, succinic, lactic, oxalic, gluconic, tartaric, acetic, fumaric acids) during the processes of solubilization and availability of P.

[0183] In the present invention, the organic acids are introduced by adding natural raw materials such as milk. In the reported examples, non-pasteurized cow milk was used, kept at T < 60°C, containing organic acids, such as lactic, propionic (propanoic) acids, as well as fatty acids such as butanoic, stearic, palmitic, and oleic acids. Furthermore, the milk includes / contains bifidobacteria, lactic acid and acetic acid in a 2:3 ratio, without producing gas (lactic fermentation ...), and the presence of lactoperoxidase and others capable of promoting the production of humus and detoxification.

[0184] The range of acids can be expanded if the milk is used in combination with acetic substances, in a proportion from 1 to 14 by weight.

[0185] c) the protection system which allows the enzymes to be active and protects them from inactivation in the hostile environment in which they are introduced (substrate, soil). As also introduced by Bautista-Cruz & Ortiz-Hernandez (2015), only a low percentage of the enzymes exiting the cells is stabilized, since they are released into a hostile environment where they can be subject to non-biological denaturation, adsorption, or degradation.

[0186] In order to ensure the survival of the nucleus, the technique described in patent EP1314710 “A process of maturing and stabilizing biomasses under reduction of smelling emissions” is adopted. This protection is obtained by using the plants or a part thereof in their entirety, without opting for separation of the enzymes or active substances which are to be used. This involves both a realprotective step and the presence of nutrients.

[0187] Avoiding a separation / extraction step allows saving a processing step, with the related labor and energy costs as well as the creation of by-products.

[0188] From a practical viewpoint, the preparation of the nucleus, in the form of a complex and protected, marketable product, is carried out as follows:

[0189] A. Plant components containing enzymes, rich in vitamins, and plant components which develop fragrances and aromas are selected; both components are harvested in the balsamic period (full vegetative development and in the phase of mature seeds or fruits), to have an enzymatic Pool which makes P and other nutrients and micronutrients available for plants;

[0190] B. The damaged or broken parts are removed,

[0191] C. They are chopped to a size smaller than a grain of rice, between 3 and 10 mm;

[0192] D. The chopped parts are mixed all together, in the expected proportions, as shown in Table 2,

[0193] E. To the mixed plants, unpasteurized milk, kept at T < 40°C (mesophilia phase), is added, in doses 1:1.5 and 1: 2 by weight and the whole is mixed until a pasty texture is obtained. The milk can be supplemented with vinegar / acetic substances

[0194] in proportion from 14 to ’A by weight.

[0195] F a step of fermenting the mixture is carried out at ambient temperature for about ten days, typically 8-12 days, preferably 10.

[0196] The enzymatic nucleus in the form of a fermented pasty product can now be packaged into blocks enclosed in containers such as nylon bags, cartons, evacuated of air and kept in the dark, even for months, prior to use.

[0197] Step II. substrate activation

[0198] Step II consists in activating a substrate (animal excretions) by adding the nucleus to the substrate itself.

[0199] As already reported in the literature (Garg & Bahl, 2008); Arcand & Schneider, 2006), the phosphatase activity improves through the application ofanimal excretions (various organic manures), which occurs as enhanced availability of P in the soil. The activity of PSBs inoculated in the soil is also improved by the simultaneous addition of organic and inorganic phosphate fertilizers in the soil (Bargaz et al., 2021).

[0200] The innovation suggested in the present patent application lies in that, unlike the direct use of enzymes / microorganisms that solubilize P and organic / mineral fertilizers containing P / manure in the soil, the nucleus is incorporated into a substrate based on animal excretions / manure, and left under preparation, before the application thereof to the soil for which conditioning is required.

[0201] Unlike the direct use of the nucleus in the soil, step II consists in preparing a substrate with the nucleus before use in the soil. In practice, they are two activators working together in tandem. By combining them, complexity clearly increases. The substrate used consists of animal excretions, a by-product of agrifood production, thus allowing an optimization of a circular economy practice.

[0202] The choice to operate with a substrate allows:

[0203] • Obtaining a product which, in turn, contains both the hydrolytic enzymes and the organic acids required to improve the availability of nutrients in the soil, particularly in the root zone;

[0204] • Increasing the amounts to be used (with dosages of 1 kg nucleus per m3, i.e., about 2 kg / t means bringing amounts increased at least 500 times), if with similar enzymatic activities,

[0205] • Providing sufficient C - organic matter and N, required to maintain the phosphatase activity (2017);

[0206] • Also containing all those nutrients (N) and microelements which, in the absence or lack thereof, could block or reduce the productivity.

[0207] The selection of animal excretions as substrate was driven by the following factors:

[0208] • Utilizing a matrix with a complex system rich in microorganisms, with enzymatic activities, both hydrolytic and with development of organic acids; e.g., poultry excretions are rich in microorganisms,Proteobacteria, Actinobacteria, Bacteroideties, Firmicutes, and Planctomycetes which provide active enzymes for the cycle of C, N and S (Minkina T et al., 2023).

[0209] • Supplying organic matter (after stabilization) to the soil, giving fertility to the soil and enabling those “ecosystem services” that a fertile soil can carry out, with an improvement of the soil structure, porosity and aeration thereof (better root development). Supplying biostabilized organic matter.

[0210] Non-exhaustive examples of substrate are: bovine manure, buffalo manure, poultry litter and manure from any poultry farm, swine excretions after solid / liquid separation.

[0211] As reported in the examples, applications have been developed for poultry excretions, richer in nutrients and enzyme phytase which is found in avian diets and is thus also present in the excretions. It is also the one in which it is easier to observe the biogenic formation of Struvite, a slow-release P and N fertilizer.

[0212] As already identified in EP1314710 and in IT Patent No. 1393404, in the step of preparing the biostimulant to be applied to the soil, the nucleus is inserted, for example, at the base of the substrate heap during the formation thereof and left in a sort of “no disturbance” fermentation. According to a further feature, unlike EP1314710 and IT Patent No. 1393404, the process can also be conducted in static form, in a thermophilic phase, which can also maintain temperatures > 65°C for weeks. Indeed, temperature ranges of 65-75°C are observed for weeks. Processes conducted in a fluid phase can also be provided.

[0213] Given the type of process, i.e., static, we have the advantage of reducing odorous emissions and greenhouse gases typical of these steps.

[0214] Step III: application to the soil

[0215] Step III consists in applying the biostimulant (substrate activated by the nucleus) to the soil , homogeneously in the surface layer, up to a depth of 20 cm, so as to simultaneously supply both enzymes and nutrients and stabilized organic matter into the surface layer of soil where both nutrients and organic C are more present.Usage doses

[0216] Usage doses of the nucleus within the substrate in the proportion

[0217] 0.2-2 kg per m3of substrate, density between 0.5-0.7 ton / m3.

[0218] The usage doses of biostimulant depend on soil conditions, requirements and type of crops, between 1-5 t / ha.

[0219] Timing

[0220] Nucleus preparation / fermentation time: about 8-12 days, easily 10 days. The preparation time for substrate activation varies depending on the substrate, the weather conditions (and the usage doses). As reported by the examples for poultry excretions, it ranges from 60 days to 120 days.

[0221] Effect time on soils (from) 40 days.

[0222] Use modes

[0223]

[0224] The product is simple to use: the biostimulant can be used by anyone, there is no need for specific knowledge or particular expertise.

[0225] Examples are the farmer who uses the biostimulant with a view to both a regenerative intervention on soil and a programmed fertilization plan, the horticulturist (professional, hobbyist) for use in the vegetable garden or in lawn fertilization, or the private individual who purchases the biofertilizer for the fertilization of plants and flowers on balconies and in the garden.

[0226] It can be used for any type of soil, potting soils, and substrates.

[0227] It can be used either as such, in bulk, or pelletized.

[0228] No specific soil requirements are necessary, it is adaptable and simple to use. It can be used by everyone because it is harmless.

[0229] It has no contraindications with respect to overdoses.

[0230] The simplicity and production mode of the biostimulant from the nucleus also allows for the self-production. In fact:

[0231] the nucleus can be employed as such by agricultural and / or livestock farms for a maturation process of the manure, obtaining the on-farm biofertilizer / biostimulant,

[0232] the product can be employed by a company which produces fertilizers for the creation of an innovative product with all the features indicated above.Another objective is the reduction of the emissions of methane and CO2. The use of biostimulants can enhance yield and thus reduce the necessary fertilizer units, thus also reducing the emissions connected to the production thereof.

[0233] EXPERIMENTAL TESTS

[0234] The results obtained are reported in the following Examples:

[0235] • EXAMPLE 1. Preparation of the nucleus: physicochemical characterization and biochemical parameters (enzymatic activity relative to the C, N and P cycles),

[0236] • EXAMPLE 2. Preparation and activation of the substrate

[0237] • EXAMPLE 3. Application of the biostimulant to the soil

[0238] • The specific methods are reported in the specific chapter at the end of the Materials and Methods section.

[0239] EXAMPLE 1: - NUCLEUS PREPARATION

[0240] Physicochemical characterization and biochemical parameters The first step consists in preparing the enzymatic nucleus to be used - by means of substrate - in soils to improve the availability of nutrients (biostimulant function for plants) from natural raw materials available at low cost.

[0241] Such a nucleus was prepared from plant-based raw materials and milk. The plant-based raw materials used are those plants or parts thereof containing the enzymes to be used (e.g., hydrolytic enzymes such as phosphatases, beta-glucosidases) and the vitamins required to activate them. Furthermore, there are used plants that, for the fragrance thereof, can be used to counteract the unpleasant odors produced by the fermentations, also including the use of the activated substrate in greenhouses. The plants were harvested in the balsamic period thereof.

[0242] In the experimental test which is described, cow milk, unpasteurized and kept at T < 40°C was used.

[0243] The plants used for the preparation of the nucleus belong to the following families, together with the % of use:

[0244] Cucurbitaceae at 5%,Graminaceae Wheat at 30%,

[0245] Labiatae at 0%,

[0246] Uumbrelliferae at 0%,

[0247] Rutaceae e.g., rue at 5%.

[0248] The preparation of the enzymatic nucleus was carried out as follows:

[0249] A. The individual plant components, as above, were selected,

[0250] B. The damaged or broken parts were removed,

[0251] C. They were chopped to a smaller size than a grain of rice,

[0252] D. The chopped parts were mixed all together.

[0253] The nucleus was obtained by adding a 1:2 by weight dose of milk as described above, obtaining a preparation with a pasty texture, and after fermentation at room temperature, preferably for 10 days. The nucleus can then be packaged so as to preserve the functions thereof even for months and at room temperature.

[0254] At the time of use, the enzymes become activated upon contact with the biomass to be treated, as explained in example 2. The usage doses for addition to the substrate were in the range of 0.2 - 2 kg per m3of substrate.

[0255] The final nucleus is a complex organic product, which can survive in a hostile environment, such as different soils or animal excretions and manures, which adapts to the environment (contains extracellular enzymes, ferments, nutrients, carriers) linked to the components and preparation thereof. The characterization thereof is reported in Table 3, where the results of the physicochemical and biochemical characterization are shown (enzymatic activity with respect to the C, N and P cycles). The data show the following features:

[0256] E. It is an acidic product, which can promote acid catalysis;

[0257] F. It is an organic, moist - water at about 50% - product with a high concentration of Carbon, almost entirely organic, with a soluble fraction, therefore available, very high, greater than 50%;

[0258] G. Presence of N (N:C is about 1:15), N more than half available, and K (K:C is about 1:15) ,

[0259] H. Low content of other nutrients (Ca, Mg, Na, P, S) and lack of heavymetals;

[0260] I. Presence of enzymatic activity relative to the cycles for C, P, and N (P- Glucosidase, Phosphatase, and Urease, respectively).

[0261] The analysis of two samples tested at different times is reported.

[0262] TABLE 3

[0263]

[0264]

[0265] EXAMPLE 2: SUBSTRATE PREPARATION AND ACTIVATION on a pilot scale.

[0266] The Nucleus of example 1 was used on a substrate consisting of poultry excretions, in particular on poultry manure from intensively reared laying hens after drying.

[0267] With reference to Figure 3A, the poultry manure was piled in a heap and positioned inside an authorized shelter, with the possibility of air exchange but sheltered from the weather. The nucleus was inserted at the base of the heap during the formation thereof.

[0268] The whole was subjected to maturation through a discontinuous process, under static conditions and without air insufflation, with a supply of air / oxygen by means of natural convection for 120 days (simplified passive composting, with aeration by natural convection) (Fig. 3B).

[0269] Usage doses: Nucleus: 30 kg in a heap of 30 m3(base 20 m2) MATERIALS AND METHODS

[0270] The substrate was sampled at t= 0, initial conditions, then at time t=40, 80 and 120 days.

[0271] At each time point, samples were collected from 4 separate points (see Figure, 3 samples from the surface layer of the heap - top, center, and bottom, and 1 internal sample - SIN). Each sample consisted of 4 sub-samples.The concentrations of the main nutrients were measured, both total and plant-available, and water-soluble.

[0272] X-ray diffractometry (XRD) analyses were also conducted on substrate samples dried at 60°C.

[0273] RESULTS

[0274] The results of the characterization of the initial and final samples are reported in Table 4; the trends of electrical conductivity and total and ammoniacal N over the maturation / activation time are in Figs. 4A, 4B and 4C.

[0275] Starting from wet material (45%, in any case below 50%, recognized as a threshold value to avoid anoxic phenomena and substrate compaction) to then obtain a highly dried material in the surface layer which retains moisture in the internal zone. In fact, the “microenvironmental” conditions induced by the activation process lead to the creation, within the heap, of at least 2 zones: a surface layer, in contact with the air, under completely aerobic conditions, and an inner zone, under microaerophilic conditions, i.e., at reduced O2 concentration, where the high mineralization processes requiring a lot of oxygen are thus slowed down.

[0276] TABLE 4

[0277] Characterization of substrate nutrients at the beginning and end of treatment

[0278]

[0279]

[0280] The pH evolves from neutral values (about 7) to basic values (> 9), probably connected to ammoniacal compounds. As for electrical conductivity, the curve shows a maximum at 80 days and a sharp drop with a final value lower than the initial one (Figure 4A). The electrical conductivity value is correlated with the salinity value. The salinity of poultry manure is one of the critical aspects for the application thereof in the field: indeed, the use of immature poultry excretions can "burn" the plant due to the onset of reverse osmosis phenomena and the competition for nitrogen between plants and soil microorganisms. The reduction of the salinity is correlated with the maturity of the product.

[0281] The process has highlighted:

[0282] The biostabilization of the substrate, verified by i) physicochemical characterization, with a reduction of Volatile Organic Compound (VOC) in parallel with the reduction of TOC (from 70 to 50% and from 36 to 21%, respectively).

[0283] The increase in the availability of nutrients in the mature substrate:

[0284] - P2O5 soluble in H2O goes from 40 to 5% in the final substrate, subject to leaching; available P2O5 is over 15% of the total in the final substrate. Such a variation is also consistent with the biogenic presence / formation of struvite which is detected especially in the final samples. Struvite is a slow-release N and P fertilizer. It is a form of phosphate which is sparingly soluble in water, yet available to plants.

[0285] - K2O soluble in H2O increases in the final product from 45 to > 60 % while > 15 % is available.

[0286] The phase analysis was carried out by examining the Powder DiffractionFile (PDF) limited to phases containing the following elements: C, N, H, P, O, K, Ca, Mg, S, Na, Cl, Fe, Mn, B, Zn, Cu. The difficulties encountered in the identification of the phases are common for biogenic minerals, since they are poorly crystallized and have imperfect stoichiometries. Generally, the spectra shown in the Figure exhibit two different types of profile; there is a hump-shaped curve in the region between 15° and 35°, which indicates the presence of amorphous material (probably an organic phase) and an inorganic phase above the previous ones, which indicates the mineral part of the sample.

[0287] The phase identification was generally found to be almost certain for the presence of Calcite CaCO3 and for a partial presence of struvite H16MgNO10P (JCPDS Card 00-0015-0762), a complex compound containing N and P.

[0288] In the poultry manure samples at T=0 (SO in Figure 5A), a small presence of struvite is observed, probably covered by an amorphous area. Struvite is characterized by multiple peaks about 15° and by an intense peak (1=100%) about 21°. In this sample the most abundant phase is (JCPDS Card 00-005-0586) with a very intense peak about 30° (1=100%).. These results are in good agreement with our expectations, as already previously reported, because the absence of struvite could be a particular feature of fresh poultry manure. In the case of external sampling, struvite and calcite are present in all the samples. After 80 and 120 days, the amorphous phase decreases and the samples show a peak of maximum intensity of struvite about 21°. In general, all struvite peaks have a lower intensity. As for internal sampling (Figure 5 B), we can observe calcite peaks for SO and Sinl20. Furthermore, the figure shows for Snuo and Sinso a strong presence of amorphous area, unlike the external samples with the same aging time. This feature is not detectable in Sinl20, where the XRD pattern for the external samples is comparable to the XRD for the internal samples. Therefore, in the latter sample, the presence of struvite is probably predominant as compared to Sin4o and Sinso.

[0289] EXAMPLE 2 BIS: SUBSTRATE PREPARATION AND ACTIVATION TEST on a laboratory scale (enzymatic activities)

[0290] The nucleus of Example 1 was used in a laboratory- scale test on asubstrate consisting of poultry excretions, in particular on poultry manure from intensively reared laying hens.

[0291] The objective was to verify the effect of the activation of the Nucleus on a specific substrate to obtain a biostimulant, a product which improves the availability of the nutrients in the soil. In this case, the effect on enzymatic activities particularly correlated with the C, N and P cycles was studied.

[0292] Containers with a volume of 25 1 were prepared: they were filled with about 12 kg of poultry manure with 50 g of nucleus added, corresponding to a dose of 2 kg / m3.

[0293] For the purposes of the study, the monitoring was carried out by substrate sampling and subsequent analyses of the sample. The sampling times were set at t= 0, 15, 30, 60 and 90 days. At each monitoring time, 3 containers were used for sampling; for each sample, 3 measurement replicates were performed, specifically to account for the intrinsic variability of the matrix. Therefore, at each time, 9 values were available. For the 5 monitoring times, the average value and the standard deviations were calculated.

[0294] The analytical methods are described in the Materials and Methods Section after Example 3.

[0295] RESULTS

[0296] The main results are summarized in Table 5, for the initial and final values.

[0297] The data show a low-mineralization process, demonstrated by small variations in the Volatile organic compound (VOC). The pH showed the maintenance of neutral conditions over time. The salinity also returned to the initial values (EC).

[0298] TABLE 5.

[0299] Physical and biochemical characterization of the substrate under initial conditions and after 90 days; the data are expressed as Mean value ± standard deviation

[0300]

[0301] As for enzymatic activities in the substrate, linked to the C, N and P cycles, the final values are significantly higher than the initial values for P- Glucosidase and Urease, and of the same order of magnitude as the nucleus itself. As for phosphatase, there was a reduction of about 20% from the initial conditions to the final conditions, while maintaining the same order of magnitude as the starting Nucleus.

[0302] The activated substrate is characterized by enzymatic activities similar to the Nucleus (Example 1), but the amount thereof is 250 times!

[0303] The product is active, as demonstrated by basal respiration measurements.

[0304] EXAMPLE 2 TER: SUBSTRATE PREPARATION AND ACTIVATION on a pilot scale of litter

[0305] The Nucleus of example 1 was used on a substrate consisting of poultry litter.

[0306] The poultry manure was piled into a heap and positioned inside an authorized shelter, with air exchange and sheltered from the weather. The nucleus was inserted at the base of the heap during the formation thereof (6A and 6B). The whole was subjected to maturation by a discontinuous process, under static conditions and without air insufflation, with a supply of air / oxygen by means of natural convection for 100 days (simplified passive composting, with aeration by natural convection). Given the low initial moisture of the substrate, wetting withwater was carried out during the heap formation step. After 100 days, the substrate was subjected to pelletization with a low-temperature process.

[0307] Nucleus usage doses: 11 kg in a heap of 15 tons.

[0308] MATERIALS AND METHODS

[0309] The substrate was sampled at t= 0, initial conditions, then in bulk state, after 75 gg of maturation, and pelletized after 100 gg of maturation (Fig. 6C).

[0310] At each time, 3 samples were collected from different positions. Each sample consisted of 3 sub- samples.

[0311] A physicochemical characterization was performed, the concentrations of the main nutrients were measured, as well as the enzymatic activities. The analytical methods are reported in the specific chapter at the end of the Materials and Methods section. X-ray diffractometry (XRD) analyses were also conducted on the pelletized samples.

[0312] RESULTS

[0313] The results of the characterization of the initial samples are reported in Table 6.

[0314] It starts from low-humidity material rich in organic matter.

[0315] The final fertilizer is a mature product, pelletized at low temperature, with neutral pH and low salinity, such as that of bovine manure, for example. It is a “living” product and adapts to the soils to which it is added. It is a hygienically safe product, because obtained through a thermophilic process (T> 65°C) for weeks.

[0316] The final fertilizer - the edaphic biostimulant - is characterized by:

[0317] • A high Content of stable organic matter (organic carbon > 35% and Organic matter over 70%), with 8% of humic matter and only 8% immediately available,

[0318] • The high Nitrogen content (N > 3.5% as such), much greater than a compost, of which about 30% water-soluble, immediately available (nitrates and ammonium), and the large part being organic, which can be mineralized and used by plants and microorganisms over time, compatible with slow-release N,• High values for P and K (about 2% for P2O5 and 2.68% for K2O); this is indicative of this product not only having the conditions to be a good organic soil conditioner, with a part of the carbon thereof stable and another active, but also maintaining appreciable values of macronutrients such as N, P and especially K,

[0319] • Presence of micronutrients (B, Cu, Fe, Al, Mn, Mo, Zn) together with Ca, S are retained by the organic matrix and the solubility and bioavailability thereof are mediated by the organic matter, as with many organic fertilizers / soil conditioners,

[0320] • Enzymatic activity, with respect to the C, N and P cycles, showing a decrease in the final values, with higher values for phosphatase (of the order of ten) with respect to P-glucosidase and urease; the very low urease value is a further indication of a process of nitrogen release which proceeds through urea decomposition, which is slowed;

[0321] • Respiration is a parameter widely used to estimate microbial activity. The final product is a viable “living” product, with microbial activity that has increased after maturation and is also maintained with low-temperature pelletization.

[0322] • The C / N ratio about 8-10, with 10 being typical of the surface layer of soils,

[0323] • Presence of mineralogical phases attributable to struvite.TABLE 6

[0324] Characterization of the substrate at the beginning and end of treatment

[0325]

[0326] EXAMPLE 3: APPLICATION OF THE BIOSTIMULANT TO THE SOIL The enzymatic Nucleus was first prepared as described in Example 1. The nucleus was then added to the substrate (poultry manure from laying hens) for the activation thereof, as described in example 2.

[0327] The activated substrate was applied to an agricultural soil characterized by a deficiency of organic matter, at dosages of 5 t / ha. It was distributed over an area of 2000 m2and incorporated into the top 20 cm of the surface layer by tedding with appropriate equipment.

[0328] It was the object of the work / study to verify the biostimulant effect of the finding, i.e., the improvement of the availability of nutrients in the soil, in the area of the roots. The soil was then used for the cultivation of chili pepper.

[0329] The chemical analyses of the soil were carried out before and then after the application of the biostimulant. A comparison with virgin soil, rich in organic Carbon, was also introduced.

[0330] MATERIALS AND METHODS

[0331] Sampling

[0332] The soils were sampled at 0-20 cm depth (composite samples from 3 subsamples). Carbon and nutrients were measured, both as total concentration and as amount available for plants. The analytical Methods are reported in the appropriate experimental section.

[0333] RESULTS

[0334] The results of the main chemical analyses of the soil are shown in Table 7. They showed an excellent initial performance of the application of the biostimulant, doubling total organic carbon and total nitrogen in a soil very poor in organic matter, maintaining a C / N ratio of about 4. The values are still lower as compared to a rich and fertile soil (reference).

[0335] After the application, increases in the concentration of nutrients but especially in the availability thereof were measured: the total P doubled after the application of the biostimulant, while the available form increased 25 times; the total K increased by 12% while the available form tripled. The concentration of Mg remained virtually constant (+3%) while the availability thereof gained +20%.With respect to the virgin soil, the rich reference soil, the concentration of P of the soil treated with the finding was lower, but the availability of P was greater.

[0336] The effect of the improvement in availability has also been observed for some micronutrients. The concentration of Cu and Zn increased (by 23% and 43%, respectively), while the concentration of Fe and Mn remained substantially the same. The effect on the availability of Mn and Zn was significant (20 and 15 times after the application of the biostimulant, respectively). Based on these data, the effect on availability was selective.

[0337] The addition of the biostimulant / finding had an overall impact on EC, which increased by nearly 4 times the initial soil conditions and nearly double the value of the reference soil. It had a positive effect on the exchange capacity, however lower than that of the reference soil, very rich in organic matter.

[0338] Particularly interesting is the behavior of the available fraction with respect to the total concentration of the nutrients, reported in Table 7, expressed as the ratio between the concentration of available nutrient and the total concentration found:

[0339] NUTRIENT available

[0340] Ratio = - NUTRIENT tot.

[0341] In particular, in Table 8 the characterization of the nutrients in the soil before and after the application of the biostimulant is shown, compared with a soil rich in organic matter (reference). Also reported are the variations with respect to the initial value (%).TABLE 7.

[0342] Characterization of the soil before and after application of the biostimulant

[0343]

[0344] TABLE 8 .

[0345] Ratio of available fraction / total concentration of nutrients in the soil before and after

[0346]

[0347] In summary,, the application of the biostimulant induced in the surface layer of the soil (range 0-20 cm):• Ratio Passimiiabie / Ptot increased by 13 times,

[0348] • Ratio Kassimiiabie / Ktot increased by 2.8 times,

[0349] • Ratio Mgassimiiabie / Mgtot increased by 12%,

[0350] • Ratio Mnassimiiabie / Mntot increased by 20 times, (it is still half with respect to the reference soil)

[0351] • Ratio Znassimiiabie / Zn tot increased by 10 times, (and it is double with respect to the reference soil).

[0352] Therefore, the availability of nutrients in the soil has been improved. Chili pepper was cultivated in the area, whereby both the distribution of the fertilizer and the samplings were performed in the layer of interest of the roots.

[0353] To complete the Experimental tests section, the following are reported below:

[0354] ANALYTICAL METHODS USED IN EXAMPLES NO. 1 - 3 Physicochemical parameters

[0355] Moisture: Loss on drying at 105 °C for 12 h and OM concentration by loss-on-ignition.

[0356] Dry Substance (DS): residue after drying at 105 °C for 12 h.

[0357] pH and EC (electrical conductivity): were extracted by stirring a mixture of soil / substrate and distilled water, with a solid / liquid ratio of 1:5 for 2 hours, and then measured with a pH-meter and an EC-meter, respectively, after centrifugation and filtration through ashless filter paper (Albet 145 110).

[0358] TOC (Total organic C) and Ntot were measured with an elemental analyzer LECO C, N, S macro samples Ash: residue after combustion at 550°C for 6 h.

[0359] TOCsol and Nsol: from the same extraction, they were quantified with a TOC analyzer (TOC- 5050A, Shimadzu).

[0360] Volatile Organic Compound (VOC): loss on ignition at 550°C for 6 h. Humic Substance: The quantification of the various humic fractions in the samples was obtained in the organic fraction extractable with 0.1 M Na4P2O? (EXC) and in the fulvic acid fraction (FA), after precipitation of humic acids atpH 2.0. The humic acid fraction (HA) was obtained by subtracting FA from HS. Organic C was measured in the alkaline extracts after oxidation with KiCnO? and spectrophotometric determination of Cr3+at 590 nm.

[0361] Macro and micro-elements: Ptot, Ktot, metals and microelements were quantified, obtained by inductively coupled plasma emission spectroscopy (ICP-OES), 6000 Series ICP Emission Spectrometer (Thermo Scientific), after microwave acid digestion according to the soil analysis methods suggested by the Soil Science Society of America.

[0362] P2O5 available: based on the method developed by Olsen et al., (1954) by extraction using an alkaline sodium bicarbonate solution (pH 8.5) and determining the concentration of P colorimetrically (at 630 nm in Technicon AutoAnalyzer II).

[0363] Ca, Cu, Zn, K, Fe, Mn, Mg available according to the methods provided by “Soil analysis”

[0364] Enzymatic activities

[0365] Urease activity: was determined with the buffered method of Kandeler and Gerber (1988). In this procedure, 0.5 ml of 0.48% urea and 4 ml of borate buffer at pH 10 were added to 1 g of soil in a hermetically sealed flask, and then incubated for 2 hours at 37°C. The ammonium content of the centrifuged extract was determined using a colorimetric method.

[0366] P-glucosidase activity: was determined using the method of Tabatabai (1982) and Eivazi & Tabatabai (1988), based on the spectrophotometric determination of p-nitrophenol released when soil samples were incubated for 1 hour at 37°C with a buffered solution (pH=6.5) of the artificial substrate p-nitrophenyl-P-D-glucopyranoside (PNG).

[0367] Phosphatase activity: was determined using the method of Tabatabai and Bremer (1969), based on the spectrophotometric determination of p-nitrophenol released when the soil samples were incubated for 1 hour at 37°C with a buffered solution (pH 11) of the artificial substrate p-nitrophenyl phosphate (PNF).

[0368] Basal respiration was analyzed by placing 15 g of each sample, moistened to 50-60% of the water-holding capacity (WHC), in a hermetically sealed flask provided with a rubber septum for taking glass samples. The samples were thenincubated at 28°C for 20 days, and the CO2 emitted was measured at predetermined time intervals, with an infrared gas analyzer (PBI-Dansensor, CheckMate II) (Hernandez and Garcia, 2003).

[0369] X-ray diffractometry (XRDf.

[0370] X-ray diffraction (XRD) profiles were recorded between 10° and 70° (26) using a Philips powder diffractometer with Bragg-Brentano geometry, provided with a copper anode operated at 40 kV and 30 mA (0.02° step, 6 sec step time). The phase analysis was carried out using the X'pertHigh Snucleus PC software version 2.2a (Panalytical B.V., Almelo, the Netherlands).

[0371] REFERENCES

[0372] Adetunji et al. 2017. Adewole T. Adetunji, Francis B. Lewu, Reckson Mulidzi, Bongani Ncube. The biological activities of P-glucosidase, phosphatase and urease as soil quality indicators: a review. Journal of Soil Science and Plant Nutrition, 2017, 17 (3), 794-807

[0373] Arcand & Schneider, 2006. Plant- and microbial-based mechanisms to improve the agronomic effectiveness of phosphate rock: a review. Anais da Academia Brasileira de Ciencias (2006) 78 (4): 791-807 (Annals of the Brazilian Academy of Sciences) https: / / doi.or / 10.1590 / S0001-37652006000400Q13

[0374] Bargaz A, Wissal Elhaissoufi, Said Khourchi, Bouchra Benmrid, Kira A. Borden, Zineb Rchiad. 2021. Benefits of phosphate solubilizing bacteria on belowground crop performance for improved crop acquisition of phosphorus. Microbiological Research 252 (2021) 126842

[0375] Bautista-Cruz & Ortiz-Hernandez, 2015. Hydrolytic soil enzymes and their response to fertilization: a short review. Com. Sci., Bom Jesus, v.6, n.3, p.255-262, Jul. / Sep. 2015.

[0376] du Jardin P, (2015) Review Plant biostimulants: Definition, concept, main categories and Regulation. Scientia Horticulturae 196 (2015) 3-14

[0377] Garg S. and Bahl G.S., 2008. Phosphorus availability to maize as influenced by organic manures and fertilizer P associated phosphatase activity in soils.

[0378] Bioresource Technology, Vol 99, Issue 13, Pages 5773-5777

[0379] Khalid A, Hameed A and Tahir MF (2023) Wheat quality: A review on chemicalcomposition, nutritional attributes, grain anatomy, types, classification, and function of seed storage proteins in bread making quality. Front. Nutr.

[0380] 10:1053196. doi: 10.3389 / fnut.2023.1053196.

[0381] Lal R., 2010. Managing Soils and Ecosystems for Mitigating Anthropogenic Carbon Emissions and Advancing Global Food Security. BioScience, October 2010

[0382] Liu Xue, Ran Han, Yue Cao, Benjamin L. Turner, and Lena Q. Ma, 2022. Enhancing Phytate Availability in Soils and Phytate-P Acquisition by Plants: A Review. Environ. Sci. Technol. 2022, 56, 9196-9219

[0383] Minkina, T., Sushkova, S., Delegan, Y. et al. 2023. Effect of chicken manure on soil microbial community diversity in poultry keeping areas. Environ Geochem Health 45, 9303-9319 (2023). https: / / doi.org / 10.1007 / sl0653-022-01447-x Rani K. U., U. J. S. Prasada Rao, K. Leelavathi and P. Haridas Rao. Distribution of Enzymes in Wheat Flour Mill Streams. Journal of Cereal Science 34 (2001) 233-242

[0384] Singh N., P. Verma, B. R. Pandey. 2012. Therapeutic Potential of Organic Triticum aestivum Linn. (Wheat Grass) in Prevention and Treatment of Chronic Diseases: An Overview. International Journal of Pharmaceutical Sciences and Drug Research 2012; 4(1): 10-14

[0385] Wieser, H, Koehler, P, and Scherf, KA. The two faces of Wheat. Front Nutr. (2020) 7:517313. doi: 10.3389 / fnut.2020.517313

[0386] Consulted websites of EC

[0387] European Commission: Directorate- General for Environment, Frelih-Larsen, A., Koeijer, T., Ding, H., Michels, R. et al., Resource Efficiency in Practice - Closing Mineral Cycles - Final Report, Publications Office, 2016, https: / / data.europa.eu / doi / 10.2779 / 710012

[0388] European Commission: Directorate- General for Internal Market, Industry, Entrepreneurship and SMEs, Grohol, M. and Veeh, C., Study on the Critical Raw Materials for the EU 2023 - Final Report, Publications Office of European Union, 2023, https: / / data.europa.eu / doi / 10.2873 / 725585European Commission: Joint Research Centre, Georgitzikis, K., Giner Santonja, G., Roudier, S., Scalet, B. et al., Best available techniques (BAT) reference document for the intensive rearing of poultry or pigs - Industrial Emissions Directive 2010 / 75 / EU (Integrated Pollution Prevention and Control), Publications Office, 2017, https: / / data.europa.eu / doi / 10.2760 / 020485

[0389] Best Available Techniques (BAT) Reference Document for the Intensive Rearing of Poultry or Pigs https: / / eippcb.irc.ec.europa.eu / reference / intensive-rearing-poultry-or-pigs-0

Claims

CLAIMS:1) A process for producing a complex, adaptive enzymatic system, also referred to as enzymatic nucleus, to be used in processes of biostabilization of animal excretions, of the type comprising the following steps:A) mixing natural raw materials, for the preparation of an enzymatic nucleus comprising enzymes involved in the activity of making the soil nutrients for which conditioning is required available;said natural raw materials used being:those plants or parts thereof containing the enzymes to be used and the vitamins required to activate them; andthose plants which, due to the fragrance thereof, can be used to counteract unpleasant odors produced by fermentations,B) adding organic acids to said plants or parts thereof, which facilitate the action of the enzymes themselves, making them effective;characterized in thatfor making an edaphic biostimulant, having the function of improving the availability of nutrients that are confined in the soil for which conditioning is required,the addition of such organic acids occurs by adding a dose between 1:1.5 and 1:2: by weight of unpasteurized milk and kept at T< 40 °C, andafter mixing, there is included a step of fermenting the pasty material thus obtained at ambient temperature for a period of 8-12 days, preferably 10, useful to enhance the production of lactic acid and other organic acids such as succinic, maleic, and oxalic acid; thus obtaining an enzymatic preparation with a pasty texture with a high concentration of Carbon, almost all organic, of an acidic nature to promote acid catalysis, with a very high and thus available, soluble fraction greater than 30%. by weight, ready for packaging.2) The process for producing a complex, adaptive enzymatic system according to claim 1, characterized in that the plants used for the preparation of said enzymatic nucleus are selected from at least three of the following families and with the following % of use by weight:- Cucurbitaceae at 1 -5%;Graminaceae Wheat at 20-35%;Labiatae at 20-35%,Umbrelliferae at 20 -35%,Rutaceae e.g., rue at 1-5%.3) The process for producing a complex, adaptive enzymatic system according to claim 1, characterized in that in order to ensure the survival of the enzymatic nucleus allowing the enzymes to be active and protect them from inactivation in the hostile environment to which they are introduced, a protective step is carried out, which avoids the separation of the enzymes themselves and of the active ingredients to be used, and which includes the following steps:- the plants or part thereof are all harvested in the balsamic period thereof; - are chopped down to particles having a smaller particle size than that of a grain of wheat, i.e., of the order of 1 mm,- the chopped parts are all mixed together, in the proportions by weight of use provided in claim 2.4) The process for producing a complex, adaptive enzymatic system according to claim 1, characterized in that the plants used for the preparation of said enzymatic nucleus are selected from at least three of the following families and with the following % of use by weight:glucosidase, related to the carbon cycle;phosphatase, the activity of which allows the transformation of P in organic form into forms available for plants such as orthophosphate anions, and urease, relative to the nitrogen cycle.5) The complex, adaptive enzymatic system obtained according to one of claims 1 to 4, characterized in that it is marketed in the form of blocks of pasty material enclosed in containers such as nylon bags, or cartons, evacuated of air and kept in the dark, so as to preserve the functions thereof and also marketable for months.6) The complex, adaptive enzymatic system obtained according to one of claims 1 to 4, characterized in that the presence of the organic acids introducedby adding natural raw materials such as milk is increased if the milk is used in combination with acetic substances, in a proportion from 1 to V2 by weight, so as to introduce acetic, tartaric, oxalic acid, before the fermentation step.7) The complex, adaptive enzymatic system obtained according to claim 1, characterized in that:it is an acidic product, with pH between 4 and 6.5, which can promote acid catalysis;it is a wet organic product - water greater than 50% - with high Carbon concentration, almost all organic, with a very high and thus available soluble fraction between 25 and 60%;N is present with an N:C ratio equal to about 1:15 - 1: 20, where N over half available, andK is present with a K:C ratio equal to about 1: 15 - 1: 20;it has a low content of other nutrients (Ca, Mg, Na, P, S) and lacks heavy metals; andenzymatic activity relative to the cycles for C and P (P-Glucosidase, urease, and phosphatase, respectively) is present:P-Glucosidase 1 - 20 pmol INTF / g*hPhosphatase 1 - 50 pmol N-NH4 / g*hUrease 0.1 - 10 pg INTF / g*h.8) Use of the complex, adaptive enzymatic system according to one of claims 5 to 7 for producing an organic edaphic biostimulant product characterized in that it includes:the activation of a substrate consisting of animal excretions, by adding said complex system to the substrate itself, in an amount between 0.2-2 kg per m3of substrate, of density equal to 0.5 - 0.7 ton / m3so as to simultaneously provide both enzymes and nutrients and stabilized organic matter for the enrichment thereof with microorganisms that degrade the organic matter and have hydrolytic enzymes which enhance the activity, but are also rich in C and nutrients to enhance the function.9) Use of the complex, adaptive enzymatic system, or enzymaticnucleus, according to claim 8 for producing an organic edaphic biostimulant product having the function of improving the availability of nutrients that are confined in the soil for which conditioning is required, characterized in that:- said substrate consists of poultry excretions, in particular poultry manure from laying hens intensively reared in cages, after drying the manure;- the poultry manure is piled up in a heap and has a humidity below 50%;- said enzymatic nucleus is inserted at the base of the heap during the formation thereof;- the whole is subjected to maturation through a discontinuous process, under static conditions and without air insufflation, with a supply of air / oxygen by means of natural convection, i.e., through passive simplified composting, with aeration by natural convection for 60-130 days, preferably 120 days; - the maturation / activation proceeds through a high thermophilic phase which allows maintaining temperatures >65°C for 3 consecutive weeks.10) Use of the complex, adaptive enzymatic system for producing an organic edaphic biostimulant product having the function of improving the availability of nutrients that are confined in the soil for which conditioning is required, according to claim 8, wherein said substrate is represented by cattle manure, buffalo manure, litter and poultry manure from any poultry farm, and pig excretions after solid / liquid separation.11) A complex, organic edaphic biostimulant product, having the function of improving the availability of nutrients that are confined in the soil for which conditioning is required, characterized in that it consists of:A) a complex, adaptive enzymatic system or enzymatic nucleus prepared from natural raw materials available at low costs, such as vegetables and milk wherethe vegetable raw materials used are:those plants or parts thereof containing the enzymes to be used and the vitamins required to activate them; andthose plants which, due to the fragrance thereof, can be used to counteract unpleasant odors produced by fermentations,wherethe plants or parts thereof are all harvested in the balsamic period thereof; andwherea dose of 1-1.5 and 1-2 by weight of unpasteurized milk kept at T< 40 °C is added to said plants or parts thereof,thus obtaining an organic preparation with a pasty texture that can be packaged so as to preserve the functions thereof even for months and at room temperature ready for use after a period of between eight and twelve days, preferably ten, with a high concentration of Carbon, of an acidic nature to promote acid catalysis, with a pH between 4 and 6.5, and with a very high and thus available soluble fraction greater than 30%;andB) a substrate consisting of animal excretions to which said enzymatic nucleus is added in an amount between 0.2-2 kg per m3of substrate of density equal to 0.5-0.7 ton / m3, so as to simultaneously provide both enzymes and nutrients and stabilized organic matter for the enrichment thereof with microorganisms that degrade the organic matter and have hydrolytic enzymes which enhance the activity, but are also rich in C and nutrients to enhance the function,wherethe enzymatic nucleus is inserted at the base of the heap of said animal excretions during the formation thereof, and the whole is subjected to maturation through a discontinuous process, under static conditions, without tedding, with a supply of air / oxygen by means of natural convection, i.e., for simplified passive composting, with aeration by natural convection for a period between 60 and 130 days, preferably of 120 days, proceeding through thermophilic conditions with temperatures greater than or equal to 65 °C for at least three weeks.12) The complex, organic edaphic biostimulant product according to claim 11, having the function of improving the availability of nutrients that are confined in the soil for which conditioning is required, characterized in that:it is an organic product, with high carbon concentration, almost all organic, with a very high and thus available soluble fraction between 25 and 60%;N is present with an N:C ratio equal to about 1:15 - 1: 20, where N over half available, andK is present with a K:C ratio equal to about 1 :(15- 20);it contains magnesium and other micronutrients (B, Ca, Mg, Na, P, S) and lacks heavy metals; andenzymatic activity relative to the cycles for C and P (P-Glucosidase, Urease, and Phosphatase, respectively) is present:P-Glucosidase 1 - 20 pmol INTF / g*hPhosphatase 1 - 50 pmol N-NH4 / g*hUrease 0.1 - 10 pg INTF / g*hliving, with basal respiration > 2000 mg C-CO2 / kg*g.13) The complex, organic edaphic biostimulant product obtained according to claim 11, characterized in that it is marketed as such or pelletized by means of a low-temperature process, less than 70°C, with a neutral pH and with low salinity, lower than 5mS / cm.14) A process for applying the organic edaphic biostimulant product according to claim 13 to the soil, characterized in that it includes the activated / edaphic biostimulant substrate, comprising the enzymatic nucleus + the substrate, being added homogeneously in the surface layer of the soil, up to a depth of 20 cm, so as to provide, simultaneously, both enzymes and nutrients, and stabilized organic matter, where the usage doses of the organic edaphic biostimulant product, which depend on the conditions of the soil and on the requirements and type of crops, are between 1 and 5 t / ha.15) The process for applying the organic biostimulant product obtained according to the process of claim 11 to the soil, characterized in that it is possible to increase the use amounts, improving the availability of nutrients in the soil, particularly in the root zone.16) The complex, organic edaphic biostimulant product, having the function of improving the availability of nutrients that are confined in the soil forwhich conditioning is required, according to claim 11, characterized in that said substrate consists of poultry excretions, preferably poultry manure from laying hens intensively reared in cages, after drying the manure, and where the poultry excretions (poultry manure) are piled up in a heap and have a moisture below 50% to avoid anoxic phenomena and compaction of the substrate, and then obtain a very dry material in the surface layer which maintains moisture in the inner zone.17) The organic edaphic biostimulant product - the final product -obtained from poultry excretions according to claim 16, having the function of improving the availability of nutrients that are confined in the soil for which conditioning is required, characterized in that:it is an organic product, with a high concentration of Carbon, almost all organic,it is at about neutral pH, within a range of 6-8, low or medium salinity (electrical conductivity < 5 mS / cm),presence of nutrients by weight (total Nitrogen (N) 2.5 - 4.0 %, total Phosphorus (P2O5) 2.0 - 3.5 %, total Potassium (k) > 2 and total Magnesium (Mg) > 0.3%, essential for the formation of struvite,presence of micronutrients such as B, Ca, Mg, Na, P, S, Fe, Al, Mn) so as to prevent production blocks and a lack of heavy metals in the soil;presence of enzymatic activities useful for making nutrients available relative to the cycles for C, and P , P-Glucosidase, Urease, and Phosphatase, respectively:P-Glucosidase 0.1 - 10 pmol PNF / g*hPhosphatase 5 - 60 pmol INTF / g*hUrease 0.01 - 10 pg N-NH4 / g*h, andit also contains P in the form of struvite;it is an active product, as demonstrated by n basal respiration measurements (> 2000 mg C-CO2 / kg*g.

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