Soil inoculant for remediation thereof

Abstract

The present invention relates to a microencapsulation having different active substrates and vectors that meet the different needs of soil bioremediation and are made up of multilayers containing microorganisms in different substrates. In this way, their placement makes it possible to break the layers of the microencapsulation so that it releases the microorganisms until they are positioned in different soil horizons, thereby allowing bioremediation to be carried out at different levels of depth with a single object. They are used for agricultural crops, preferably vegetable crops, such as: Chenopodiaceae, Amaryllidaceae, Lamiaceae, Asteraceae, Apiaceae, Apiaceae, Solanaceae, Convolvulaceae, Boraginaceae, Brassicaceae, Cucurbitaceae, Cucurbitaceae, Asteraceae, Amaryllidaceae, Amaryllidaceae, Apiaceae, Brassicaceae, Brassicaceae, Brassicaceae, Brassicaceae, Brassicaceae, Brassicaceae, Brassicaceae, Asteraceae, Amaryllidaceae, Asparagaceae, Chenopodiaceae, Fabaceae, Fabaceae, Valerianaceae, Fabaceae, Asteraceae, Euphorbiaceae, Poaceae, Cucurbitaceae, Brassicaceae, Solanaceae, Cucurbitaceae, Apiaceae, Solanaceae, Amaryllidaceae, Brassicaceae, Asteraceae, Chenopodiaceae, Brassicaceae, Cucurbitaceae, Solanaceae, Apiaceae, Cucurbitaceae or Cucurbitaceae.

Description

[0001] SOIL INOCULANT FOR REMEDIATION

[0002] FIELD OF INVENTION

[0003] The present invention relates to a microencapsulation of different active substrates and vectors that allow to meet different needs in soil bioremediation, which are composed of multilayers that contain microorganisms in different substrates in such a way that allows their placement to break the layers of the microencapsulation in such a way that it releases the microorganisms until they are positioned in different soil horizons, which allows bioremediation to be carried out at different depth levels with a single object.

[0004] BACKGROUND

[0005] The state of the art includes patents that perform soil bioremediation in different ways, including some that contain a microorganism that replicates under climatic and natural conditions, such as the following patents.

[0006] Patent PA / A / 2004 / 001383 entitled Microorganisms for soil treatment and methods for obtaining them, The present invention relates to product(s) containing live microorganism(s) suitable for soil treatment, microorganisms that multiply under different climatic and natural conditions, as well as a method for producing the products, also a method for treating soil and plants with the products. In more detail, the invention relates to a method for preparing the products from any of the microorganisms specified below, or from mixtures thereof. Likewise, the invention relates to a method for creating cultures of the microorganisms that will be used. The subject of the invention are the microorganisms themselves as well.In more detail, the invention relates to a process for treating soil and plants with a product containing at least one of the microorganisms Azospirillum brasilense spp. SW 51 (NCAIM / P / B 001293), Azotobacter vinelandii spp. M657 (NCAIM / P / B 001291 ), Pseudomonas fluorescens var. SW11 (NCAIM / P / B 001296), Bacillus polymyxa var. SW17 / NCAIM / P / B 001295), Bacillus megaterium var. M326 (NCAIM / P / B 001301 ), also the microorganisms multiply and exist in the environment of the plant in question, which contains the listed microorganisms and their production.

[0007] Patent MX / 2012 / 014725 is called agro-inputs obtained from the transformation of agro-industrial by-products and waste and consists of a set of agro-inputs obtained from the transformation of agro-industrial by-products and waste for the care and cultivation of ornamental plants and vegetables in urban areas. Said agro-inputs may be a pulverized mineralized organic biofertilizer; a mineralized organic fertilizer in concentrated solution; an enriched, mineralized and structured germination substrate; a nutritive and structured substrate for naturalization systems and a precursor substrate for composting for domestic use.

[0008] Patent CN112592866 on a carbon-based microbial agent for soil remediation, as well as a method for preparing and applying a carbon-based microbial agent, where the invention discloses a carbon-based microbial agent for soil remediation, as well as a method for preparing and applying the carbon-based microbial agent, and relates to the technical field of biological agents for soil remediation. The carbon-based microbial agent comprises the following components in parts by weight: 100-150 parts of composite microbial bacteria, 200-300 parts of a microbial agent culture medium, 400-500 parts of woody peat / plant ash, 40-60 parts of biomass ash, 100-150 parts of sodium humate, and 30-40 parts of an amino acid nutrient solution, wherein the composite microbial bacteria comprise photosynthetic bacteria, bacilli, enzyme microorganisms, and lactic acid bacteria.The carbon-based microbial agent has the beneficial effects of degrading residual pesticides, hormones, and inorganic nutrients in the soil, preventing pollutants such as pesticides and toxic heavy metals in the air and water sources from accumulating in the soil, and blocking the uptake of heavy metal substances by plant root systems. Furthermore, it has significant effects on problems such as soil acidification, hardening, salinization, heavy metal contamination, and similar issues.

[0009] Patent CN111875444 Method of preparing a special slow-release mixed fertilizer for corn in a semi-humid area of ​​Heilongjiang where the invention discloses a method of preparing a special slow-release mixed fertilizer for corn in a semi-humid area of ​​Heilongjiang, it relates to the field of agriculture and aims to solve the problems of unreasonable mixing fertilizer ratio and low fertilizer utilization rate in a semi-humid area of ​​Heilongjiang, spring corn planting process in the semi-humid area of ​​Heilongjiang. The slow-release combined fertilizer contains large particles of urea, sulfur-coated urea, resin-coated urea, monoammonium phosphate, potassium chloride, zinc sulfate monohydrate, straw charcoal, livestock and poultry manure compost, Azotobacter chroococcum, Bacillus megatherium, Bacillus mucilaginosus and the like.The fertilizer has a reasonable formula, has a biological activity effect, and can significantly improve the utilization efficiency of chemical fertilizers.

[0010] Patent CN110373207 "Saline-alkaline soil improved microbial agent and preparation process thereof" describes a saline-alkaline soil improved microbial agent and a preparation process thereof, and belongs to the field of saline-alkaline soil improvement. The saline-alkaline soil improved microbial agent is prepared from the following raw materials in parts by weight: 140-180 parts of a composite microbial agent, 100-130 parts of biochar, 30-50 parts of humic acid, 5-10 parts of a trace element, 10-15 parts of a filler, and 30-50 parts of superphosphate. Synergistic growth and reproduction in a saline-alkaline soil environment is achieved by combining Bacillus subtilis, Bacillus licheniformis, Bacillus laterosporus,Jingyang Streptomyce and Bacillus mucilaginosus in a specific proportion. The combination of these types of microbial agents can synergistically improve alkali resistance. Patent CN109627099 "Spherical Particle Saline-Alkali Conditioner and Preparation Thereof" provides a spherical particle saline-alkali conditioner and its preparation. The conditioner is unique and can quickly improve hardened saline-alkali soil to improve soil air permeability and form soil aggregates. The main components of the conditioner are crop straw. Obtaining crop straw is simple, convenient, and easy, the cost is low, the sources are abundant, and environmental pollution caused by excess straw can be reduced; the types of crop straw used are diversified, and more complete nutrients can be provided; the conditioner comprises a large amount of crop husks,Clam shells, eggshells, calcium, other nutrients, and starch-grafted acrylate salt can achieve a better water conservation effect and a better effect on saline and alkaline environments. The conditioner is provided with a large amount of micronutrient fertilizers to provide more complete nutrients for saline-alkaline plants. The resistance of saline and alkaline soil crops is effectively improved to promote growth. The conditioner comprises a large amount of carbon substances, and thus the soil properties of saline-alkaline soil can be effectively improved to enhance soil organic matter. Some of the added flora are cultivated in the saline-alkaline environment shared by different salinities, and thus the adaptability can be greatly improved.The soil properties of saline-alkaline soil can be effectively improved to enhance soil organic matter; some of the added flora are cultivated in the saline-alkaline environment shared by different salinities, and therefore, the adaptability can be greatly improved, and therefore, the soil properties of saline-alkaline soil can be effectively improved to enhance soil organic matter; some of the added flora are cultivated in the saline-alkaline environment shared by different salinities, and therefore, the adaptability can be greatly improved.

[0011] As noted and which could affect the inventive activity of this document, the combination of all of them to achieve a microencapsulation of the different microorganisms used could be obvious to a person skilled in the art, however it is not obvious since the microorganisms have different characteristics, as survival when placed on certain substrates is not obvious and in the same way the handling of sizes to achieve a multi-layer microparticle as will be explained below.

[0012] DESCRIPTION

[0013] The characteristic details of these novel multilayered microcapsules for soil bioremediation in different horizons are clearly shown in the following description and in the accompanying figures, where the same reference signs are used to indicate the parts and figures shown.

[0014] Brief description of the figures:

[0015] Figure 1 is a representative diagram of one of the multilayer microcapsules for soil bioremediation in different horizons.

[0016] Figure 2 is a diagram representing the technical effect of multilayer microcapsules for soil bioremediation in different horizons

[0017] Based on the figures above, multilayered microcapsules for soil bioremediation in different soil horizons are the result of exhaustive research to determine the vectors that allow microorganisms (substrates) to reach different soil horizons.

[0018] The horizons of the soil.

[0019] Each of the layers contained in the soil is called a horizon, with different characteristics due to its mineral composition, amount of organic matter, degree of rock decomposition, granular texture, etc.

[0020] Horizon 0: It is the surface layer of Horizon A. It is composed of leaves, branches and plant remains (detritus). Also called the organic layer.

[0021] Horizon A: Herbaceous species take root in this layer. It is rich in decomposing matter and humus. Its color is darker than that of the lower layers. Many of its materials (organic and mineral) are susceptible to being carried downward by water.

[0022] Horizon B: This layer contains virtually no humus, which is why its color is lighter than the upper layer. Materials carried down from above, especially clayey materials, oxides, and hydroxides, are deposited here.

[0023] C Horizon: Also known as the subsoil, it is composed of more or less fragmented rock material.

[0024] D or R Horizon: Called Parent Rock or Rock Material. In it we find the rock material that makes up the unaltered soil.

[0025] Based on these horizons, the specific weight of different substrates was analyzed to be able to support a set of microorganisms with the aim of being able to penetrate by simple gravity and by the action of water, their solubility and mechanisms of action, preferring the inert ones and not necessarily the organic ones because it is a matter of encapsulating in a microcapsule and this is not viable from organic substrates, choosing only some for their particular characteristics.

[0026] Substrates have different characteristics, the most important of which are aeration and drainage, water retention, and low wet weight per volume (synonymous with bulk density). It must have a total porosity of at least 70% by volume. The porosity, or air space occupied by the substrate, is probably the most important physical property of substrates used in horticulture.

[0027] Silicates: moisture content of 10-20%, the pore can be impregnated by vigorous mechanical means. The structure features channels and cavities of variable molecular size, in which, in addition to water molecules, absorbates, and salts, the compensating cations are found. This type of microporous structure gives zeolites an extremely large internal surface area relative to their external surface area.

[0028] Rock wool: 0% moisture content, pores cause insulating condensation, are non-hydrophilic, have zero capillarity, and perform well against the action of water, ensuring that all mechanical, thermal, and acoustic properties are maintained.

[0029] Clay: 10%-15% moisture content, it is very porous and has plastic properties, meaning that when moistened, it can be easily molded. When it dries, it firmes, and when subjected to high temperatures, chemical reactions occur that, among other changes, cause the clay to become a permanently rigid material, called ceramic.

[0030] Perlite: 0%, is hydrophilic and has a great consistency, is very resistant to erosion, can be recycled and is very light approximately 100 kg per m3, that is, 15 times lighter than sand and 8 times lighter than expanded clay.

[0031] Zeolite* (M2 / nO.AI2O3.xS¡O2.yH2O), humidity of 10-20% is impregnated in an accessible way by vigorous mechanical means, the structure presents channels and cavities of variable molecular dimensions, in which, in addition to water molecules, absorbates and salts, the compensation cations are found. This type of microporous structure causes zeolites to have an extremely large internal surface area in relation to their external surface. Where - M: is an n valence cation; - "x": is the number of aluminum atoms; - ”y”: is the number of silicon atoms; - “x”: takes values ​​between 2 and 10; - “y”: takes values ​​between 2 and 8.

[0032] Activated carbon: humidity 10-20%, it is difficult to impregnate in another substrate and shows a large surface area, its characteristic porosity, pore volume, pore diameter, functional groups and the electrostatic properties that these have due to their surface chemistry. Biocarbon: humidity 40-53.73%, impregnating the pore is accessible by vigorous mechanical means that affect the size of the piece, Composed of large fragments of biomass, it is porous and its outer layer requires complex mechanisms to impregnate microorganisms but carbon saturation can threaten the life of the microorganism, it does not penetrate different soil horizons until its total decomposition.

[0033] The tendency toward microporosity and the structural characteristics of these materials allow for the transfer of matter between the intracrystalline space and the surrounding medium; this transfer is determined by the pore diameter, so only those molecules whose dimensions are smaller than a certain critical value, which varies from one zeolite to another, can enter or leave the intracrystalline space.

[0034] Zeolites can be classified as:

[0035] - Extra-large pore zeolite (0>9).

[0036] - Large pore zeolite (6<0<9).

[0037] - Medium pore zeolite (5<0<6).

[0038] - Small pore zeolite (3<0<5).

[0039] For the microcapsule, medium-pore or small-pore zeolite has been chosen.

[0040] Now, the particle size of an organic or inorganic material determines the size and distribution of pores and the water-air ratio, and consequently, the irrigation regime and plant development. Therefore, particle size analysis is considered an important practice in substrate characterization.

[0041] Particle sizes smaller than 0.5 mm have the greatest influence on air porosity and water retention, decreasing and increasing them, respectively. Particles larger than 0.5 mm increase total porosity and decrease water retention. Therefore, particle size must be modified or selected appropriately to achieve optimal physical properties.

[0042] Therefore, the microcapsule is chosen, because the influence of the particle size on the following characteristics of the size of the substrates is noted

[0043] Moreover, like plants, most microorganisms prefer an acidity level between pH 5.5 and 5.8. High or low pH levels, as well as pH fluctuations, can disrupt the development of microflora. However, some microorganisms are capable of growing, and even proliferating, under extreme conditions. The salinity or electroconductivity (EC) level of the substrate also affects the proliferation of microorganisms, as does the type and composition of the salts contained in the substrate. This is especially true for salts such as potassium chloride, which can alter the plant's atmosphere and, consequently, the types of microlife that populate the root zone.

[0044] In inorganic substrates such as rock wool, clay pellets, or perlite, most microlife is compatible with water. These microorganisms are introduced through irrigation systems, the air, or plants, and survive as long as the humidity level is high enough, even when there are only small pockets of moisture. Like soil-borne microorganisms, these can also be beneficial or pathogenic.

[0045] Beneficial microbes compete with pathogens for nutrients or glucose, and some antagonists have their own unique methods for gaining the upper hand. The fluorescent bacterium Pseudomonas, for example, can produce proteins that transform slightly soluble iron (Fe) into more easily absorbed iron chelates. This, in turn, deprives Fusarium fungi of the iron they need to grow, inhibiting their development. Competition for glucose can also cause microbiostasis, which means that the spores of the pathogenic fungus in question germinate much more slowly due to the lack of energy they obtain from glucose.

[0046] Therefore, microorganisms are sought that produce exudates that are particularly attractive to the microorganisms with which they coexist and, on the other hand, that if these microorganisms are pathogenic, then they are not viable for the microcapsule.

[0047] In this sense, the following microorganisms have been selected from an approximate list of 36 different microorganisms analyzed because they are the ones that present the following advantages: a. Different colonization speeds b. Exudates that are attractive to microorganisms c. Extremophilic microorganisms due to the type of horizon that is intended to impact d. Fixation to the different non-organic substrates, porosity of the substrate and size of the molecules to impregnate themselves in the substrate.

[0048] The following microorganisms have been chosen for this microcapsule:

[0049] • Pseudomonas spp. CM-CNRG 186

[0050] • Serratia spp. CM-CNRG 199

[0051] • Pseudomonas spp. CM-CNRG 181

[0052] • Pseudomonas spp. CM-CNRG 200

[0053] • Pseudomonas palleroniana CM-CNRG 172

[0054] • Pseudomonas extremerientalis CM-CNRG 144

[0055] • Serratia ghmesii CM-CNRG 197

[0056] • Azobacter

[0057] • Bacillus

[0058] The microorganisms CM-CNRG 186, CM-CNRG 199, CM-CNRG 181, CM-CNRG 200, CM-CNRG 172, CM-CNRG 144 and CM-CNRG 197 are deposited in the National Center for Genetic Resources of Mexico, a Center Certified before the WIPO for the deposit of genetic material since 2017 when the patent MX / A / 2017 / 004753 was filed and published in November 2018 and is available to the registration authorities, in the same way a simple copy of these certificates is attached.

[0059] Once the above characteristics have been analyzed, the following method is then carried out:

[0060] STAGE A. PREPARATION OF THE ACETAMIDE / ZEOLITE / PSEUDOMONAS MEDIUM. The internal medium (1) for the conservation of microorganisms: Pseudomonas spp. CM-CNRG 186, Serratia spp. CM-CNRG 199 and Pseudomonas spp. CM-CNRG 181

[0061] When comparing the effectiveness of a synthetic medium (m-Acetamide Medium) and an undefined medium (m-Cethmide Medium) in membrane filtration, microbiological evaluation was performed with high dilutions (10 -6 ) of control strains and simulated water samples (inoculated with Pseudomonas at a low concentration). Statistical analysis (p < 0.05) of the results obtained showed that m-Acetamide Medium is superior in terms of recovery of P. aeruginosa and is more selective since it does not allow the development of the accompanying microbiota. The sensitivity of m-Acetamide Medium is 100%, while the specificity is 100%. The average relative recovery frequency is 95.76%. Although other media such as m-Cethmide Medium surpass it in the speed of development of this microorganism, for this development it is necessary to slow its growth without having competitors in its activation.

[0062] The m-Cethmide medium has the following composition: a. peptone gelatin (in a range of 20-50 g / l); b. magnesium chloride (in a range of 1.0-4.0 g / l); c. potassium sulfate (in a range of 15.0-30.0 g / l); d. cetrimide (in a range of 0.1-0.9 g / l); e. glycerol (in a range of 0.5-15 ml / l).

[0063] m-Acetamide medium contains the following ingredients: a. acetamide (in a range of 0.5 to 20.0 g / l); b. sodium chloride (in a range of 5.0 to 10.0 g / l); c. basic potassium phosphate (in a range of 1.50 to 2.78 g / l); d. potassium hydrogen phosphate (in a range of 1.01 to 1.46 g / l); e. magnesium sulfate heptahydrate (in a range of 0.3 to 1.0 g / l); f. phenol red (in a range of 0.01 to 0.024 g / l).

[0064] Sterile cellulose absorbent pads were placed in sterile polystyrene Petri dishes as a solid support for the membrane. Four ml of the prepared membrane filtration culture medium was aseptically dispensed into the Petri dishes and used immediately.

[0065] And from this comparison, acetamide is selected, which will then be the medium that preserves a first group (2) of microorganisms, made up of Pseudomonas spp. CM-CNRG 186, Serratia spp. CM-CNRG 199 and Pseudomonas spp. CM-CNRG 181

[0066] The culture medium necessary to maintain minimum activity, acetamide houses the selected Pseudomonas (the medium represents a first substrate). Unlike growing Pseudomonas in boxes, what is required is generally a culture space but that is delayed in its replicative activity until the time when the activity of the same is required, then the selected group of Pseudomonas is impregnated in zeolite (2) at the time of preparing the culture medium by adding zeolite in a size smaller than 0.5 mm which decreases its porosity and at the same time retains the culture medium with the first group of Pseudomonas, this preparation is cooled until reaching a temperature range between 10 ° and 15 ° C to avoid its activity and is reserved for the next stage;

[0067] STAGE B. FIRST ENCAPSULATION IN A BUFFER SYSTEM. The culture medium prepared in the previous stage is presented as a block, which is crushed by mechanical action and at the same time heated to approximately 25° to facilitate the release of the zeolite but not allow the entire medium in which it has been impregnated to separate from it and then it is passed through a second substrate (4) which is a buffer system consisting of at least one salt of the bisodium or bipotassium phosphate type and at least one peptone substances to prevent pH deviation, in which a microorganism:salt preparation is previously carried out that keeps a 1:5 proportion;where the microorganism is a second group (5) of Pseudomonas, consisting of Pseudomonas palleroniana CM-CNRG 172, Pseudomonas extremoríentalis CM-CNRG 144 and Serratia grimesii CM-CNRG 197, mixing vigorously and when the zeolite with adherent characteristics passes through the first substrate (1) that it contains, it then solidifies the outer layer of it, leaving it encapsulated and retaining the substrate inside; this microcapsule is reserved for the next stage; and consists of at least one salt of the bisodium or bipotassium phosphate type and at least one peptone substance to prevent pH deviation; and;

[0068] STAGE C: SECOND ENCAPSULATION IN A CULTURE MEDIUM. A third substrate (6) is prepared consisting of a gelled medium consisting of saturated agar, by being impregnated with a third group (7) of microorganisms such as Azobacter and Bacillus where the microcapsule obtained in the previous stage is placed by vigorous mechanical action it is impregnated in this third substrate (8) and finally it is passed through activated carbon (9); and potentially the third substrate (8) can be added with microorganisms or additional agents such as crystal violet, bile salts, sodium azide, potassium teluhte, antibiotics if it is desired to convert it into an antipathogen.

[0069] This process can be iterated at least once (A, B) in such a way that a multi-layer microcapsule is made which brings together different technical effects for different soil horizons as shown below and which turns out to be selective of the type of bioremediation necessary for different types of soil, therefore it represents a selective and technically efficient agent as shown by the examples that follow below, which are intended to be illustrative, but not limiting.

[0070] Example 1 .

[0071] To achieve nutrient balance in the soil, it is necessary to adopt a strategy that allows us to maximize nutrient inputs and minimize their outputs, such as multi-layer microcapsules for bioremediation in distinct soil horizons with 20% to 30% moisture.

[0072] Nitrogen (N), potassium (P) and phosphorus (K) balance will be influenced by the mobility of the nutrient; the type of plant or crop established and its requirements; soil characteristics; management and agronomic practices applied; amount of organic matter present; and the activity of microorganisms which indicates that at the soil level, microorganisms are required that solubilize NPK in a simple, efficient and rapid manner; To correct nutrient deficiencies through bio fertilization should be considered at least: the richness (proportion) of the nutrient in the fertilizer to be used; the dose to be applied; the time of application; the form and depth of application; and the edaphic and climatic conditions. All of the above in order to reduce nutrient losses and achieve an adequate balance in the soil.At least one plot of 10x14 meters was selected and divided into 5 different zones of 2x5 meters, leaving an area of ​​one meter between each of them to avoid contamination, and they are even isolated with plastic film, additionally making a gutter that goes deep to horizon 1 between the first two, to horizon 2 between the following ones and so on until leaving the last one as a witness.

[0073] Crops such as: Chard (Chenopodiaceae), Garlic (Amarilidaceae), Basil (Lamiaceae), Artichoke (Asteraceae), Celery (Apiaceae), Celeriac (Apiaceae), Eggplant (Solanaceae), Sweet Potato (Convolvulaceae), Borage (Boraginaceae), Broccoli (Brassicaceas), Courgette (Cucurbitaceae), Pumpkin (Cucurbitaceae), Thistle (Asteraceae), Onion (Amarilidaceae), Chives (Amarilidaceae), Parsnip (Apiaceae), White Cabbage (Brassicaceas), Chinese Cabbage (Brassicaceas), Brussels Sprouts (Brassicaceas), Savoy Cabbage (Brassicaceas), Red Cabbage (Brassicaceas), Cauliflower (Brassicaceas), Swede (Brassicaceas), Endive (Asteraceae), Shallot (Amarilidaceae), Asparagus (Asparagaceae), Spinach (Chenopodiaceae), Pea (Fabaceae), Broad bean (Fabaceae), Lamb's lettuce (Valerianaceae), Green bean (Fabaceae), Lettuce (Asteraceae), Cassava (Euphorbiaceae), Corn (Poaceae), Melon (Cucurbitaceae), Turnip (Brassicaceas), Potato (Solanaceae), Cucumber (Cucurbitaceae), Parsley (Apiaceae), Pepper (Solanaceae),Leek (Amarilidaceae), Radish (Brassicaceas), Radichetta (Asteraceae), Beetroot (Chenopodiaceae), Romanesco (Brassicaceas), Watermelon (Cucurbitaceae), Tomato (Solanaceae), Carrot (Apiaceae), Zucchini (Cucurbitaceae) or Squash (Cucurbitaceae);

[0074] Once sowing is done, before the first irrigation, the multilayer microcapsules are spread in a proportion of at least 10 per square decimeter and then the first irrigation is carried out with conventional water; the carbon of the outer layer of it, is dispersed with the water, leaving exposed microorganisms such as Azobacter and Bacillus impregnated in it and if it was added with other microorganisms or agents such as crystal violet, bile salts, sodium azide, potassium tellurite, antibiotics then they begin their conventional activity with the water existing in the 0 horizon of the irrigated soil;by gravity, the weight of the microcapsule causes the water and gravity to direct it towards soil horizon 1 and at this site the peptones prevent pH deviation, and releases Pseudomonas palleroniana CM-CNRG 172, Pseudomonas extremorientalis CM-CNRG 144 and Serratia grimesii CM-CNRG 197 allowing the weight of the zeolite and the substrate it contains to enter soil horizon 2 where Pseudomonas spp. CM-CNRG 186, Serratia spp. CM-CNRG 199 and Pseudomonas spp. CM-CNRG 181 are released thus achieving solubilities of different types of nutrients by different soil horizons.;

[0075] Thus, the observed results are shown in the following table by soil zone and by humidity, climate, and microbial activity conditions. Table 1: Activity of each microcapsule layer and initial culture conditions

[0076] By carrying out a May-July cultivation period in conditions of constant humidity of rain at temperatures ranging between 25° and 35°C in the environment, and harvesting each of the crops carried out asynchronously, the following results were observed according to the type of vegetables planted which were cilantro, chard, potato, carrot, peanut, jicama, lupine, sunflower, sweet clover and alfalfa.

[0077] Table 2. Results of microcapsule application in a plot of the selected type.

[0078] Conclusion. The experiment revealed that plants show a rapid recovery compared to biofertilizers composed of a single type of nutrient, because the microcapsule, in addition to providing a bioremediation microorganism, in its composition shows nutrients that favor the microorganism's biome and which remain inert until the point where humidity releases the corresponding layer and this represents, in addition to a vector, an enrichment medium for the crop that is also composed of nutrients that favor the plant; Under general conditions similar and almost identical to those of the plot described, the percentage of recovery of it with the deficiency that occurs and being irrigated with other commercial biofertilizers available in the market are presented in Table 3. Table 3. Comparison of results of three commercial brands of biofertilizers and the microcapsules in percentage of recovery.

Claims

CLAIMS Having sufficiently described my invention, which I consider to be a novelty and therefore claim as my exclusive property the contents of the following claims.

1. A multi-layer microcapsule for soil bioremediation in different horizons characterized in that it is composed of at least one iteration to form the following layers: a. An internal medium impregnated with a first group of microorganisms contained in said internal medium as a first substrate; b. A zeolite that is impregnated with the first substrate, which is cooled until reaching a temperature range between 10° and 15°C; c. Once the zeolite cools, it is crushed and heated until reaching approximately 25° and is passed through a second substrate that acts as a buffer system; d. Which is bathed in a gelling agent and coated with activated carbon; Where the first group of microorganisms is made up of Pseudomonas spp. CM-CNRG 186, Serratia spp. CM-CNRG 199 and Pseudomonas spp. CM-CNRG 181; Where the second substrate is a buffer system consisting of at least one salt of the bisodium or bipotassium phosphate type and at least one peptone substance to prevent pH deviation; which is previously prepared microorganism:salt that keeps a 1:5 ratio; where the microorganism is a second group of Pseudomonas, consisting of Pseudomonas palleroniana CM-CNRG 172, Pseudomonas extremoríentalis CM-CNRG 144 and Serratia grimesii CM-CNRG 197 that solidifies the outer layer of it, leaving it encapsulated and retaining the substrate inside; and the salt is conventional; 2. A process for the preparation of the multi-layer microcapsule for bioremediation of soils in different horizons of claim one, characterized in that it comprises the following stages: STAGE A. PREPARATION OF THE ACETAMIDE / ZEOLITE / PSEUDOMONAS MEDIUM. The following microorganisms are preserved in a selected internal acetamide medium: Pseudomonas spp. CM-CNRG 186, Serratia spp. CM-CNRG 199 and Pseudomonas spp. CM-CNRG 181; which houses the selected Pseudomonas and the medium represents a first substrate; which is impregnated in zeolite the selected group of Pseudomonas at the time of preparing the culture medium by adding zeolite in a size less than 0.5 mm which reduces its porosity and at the same time retains the culture medium with the first group of Pseudomonas, this preparation is cooled until reaching a temperature range between 10 ° and 15 ° C to prevent its activity and is reserved for the next stage; STAGE B. FIRST ENCAPSULATION IN A BUFFER SYSTEM. The culture medium prepared in the previous stage is presented as a block, which is crushed by mechanical action and at the same time heated to approximately 25° to facilitate the release of the zeolite but not allow the entire medium in which it has been impregnated to separate from it and then it is passed through a second substrate which is a buffer system consisting of at least one salt of the bisodium or bipotassium phosphate type and at least one peptone substance to prevent pH deviation, in which a microorganism:salt preparation is previously carried out that keeps a 1:5 ratio; where the microorganism is a second group of Pseudomonas, consisting of Pseudomonas palleroniana CM-CNRG 172, Pseudomonas extremorientalis CM-CNRG 144 and Serratia grimesii CM-CNRG 197, mixed vigorously and when the zeolite with adherent characteristics passes through the first substrate it contains, its outer layer solidifies, leaving it encapsulated and retaining the substrate inside; this microcapsule is reserved for the next stage; and consists of at least one salt of the bisodium or bipotassium phosphate type and at least one peptone substance to prevent pH deviation; and STAGE C: SECOND ENCAPSULATION IN A CULTURE MEDIUM. A third substrate is prepared consisting of a gelled medium consisting of saturated agar, by being impregnated with a third group of microorganisms such as Azobacter and Bacillus where the microcapsule obtained in the previous stage is placed by vigorous mechanical action it is impregnated in this third substrate and finally it is passed through activated carbon; and potentially the third substrate can be added with microorganisms or additional agents such as crystal violet, bile salts, sodium azide, potassium tellurite, antibiotics if it is desired to convert it into an antipathogen; This process can be iterated at least once in such a way that a multi-layer microcapsule is made; Wherein the m-Acetamide medium contains the following ingredients: a. acetamide (in a range of 0.5 to 20.0 g / l); b. sodium chloride (in a range of 5.0 to 10.0 g / l); c. basic potassium phosphate (in a range of 1.50 to 2.78 g / l); d. potassium hydrogen phosphate - (in a range of 1.01 to 1.46 g / l); e. magnesium sulfate heptahydrate (in a range of 0.3 to 1.0 g / l); and f. phenol red (in a range of 0.01 to 0.024 g / l); 3. The multi-layer microcapsule for bio-remediation of soils in different horizons according to claim 1, characterized in that it is vigorously shaken by mechanical means to form a granule with a size less than 0.5 mm to improve absorption in the soil and which is achieved by iterating the process of claim 2; 4. The use of the multilayer microcapsule for soil bioremediation in different horizons according to claim 1 and 3, characterized in that it is used for soil bioremediation in crops of Chard (Chenopodiaceae), Garlic (Amarilidaceae), Basil (Lamiaceae), Artichoke (Asteraceae), Celery (Apiaceae), Celeriac (Apiaceae), Eggplant (Solanaceae), Sweet Potato (Convolvulaceae), Borage (Boraginaceae), Broccoli (Brassicaceas), Zucchini (Cucurbitaceae), Pumpkin (Cucurbitaceae), Thistle (Asteraceae), Onion (Amarilidaceae), Chives (Amarilidaceae), Chives (Apiaceae), White Cabbage (Brassicaceas), Chinese Cabbage (Brassicaceas), Brussels sprouts (Brassicaceas), Savoy cabbage (Brassicaceas), Red cabbage (Brassicaceas), Cauliflower (Brassicaceas), Swede (Brassicaceas), Endive (Asteraceae), Shallot (Amarilidaceae), Asparagus (Asparagaceae), Spinach (Chenopodiaceae), Pea (Fabaceae), Broad bean (Fabaceae), Lamb's lettuce (Valehanaceae), Green bean (Fabaceae), Lettuce (Asteraceae),Cassava (Euphorbiaceae), Corn (Poaceae), Melon (Cucurbitaceae), Turnip (Brassicaceas), Potato (Solanaceae), Cucumber (Cucurbitaceae), Parsley (Apiaceae), Pepper (Solanaceae), Leek (Amarilidaceae), Radish (Brassicaceas), Radichetta (Asteraceae), Beetroot (Chenopodiaceae), Romanesco (Brassicaceas), Watermelon (Cucurbitaceae), Tomato (Solanaceae), Carrot (Apiaceae), Zucchini, (Cucurbitaceae) or Squash (Cucurbitaceae); and which works as follows Once sowing is done, prior to the first irrigation, the multilayer microcapsules are spread in a proportion of at least 10 per square decimeter and then the first irrigation is carried out with conventional water; the carbon in the outer layer of it is dispersed with the water, leaving exposed microorganisms such as Azobacter and Bacillus impregnated in it and if it was added with other microorganisms or agents such as crystal violet, bile salts, sodium azide, potassium tellurite, antibiotics then they begin their conventional activity with the water existing in the 0 horizon of the irrigated soil;by gravity, the weight of the microcapsule causes the water and gravity to direct it towards soil horizon 1 and at this site the peptones prevent pH deviation, and releases Pseudomonas palleroniana CM-CNRG 172, Pseudomonas extremoríentalis CM-CNRG 144 and Serratia grimesii CM-CNRG 197 allowing the weight of the zeolite and the substrate it contains to enter soil horizon 2 where Pseudomonas spp. CM-CNRG 186, Serratia spp. CM-CNRG 199 and Pseudomonas spp. CM-CNRG 181 are released thus achieving solubilities of different types of nutrients by different soil horizons.;

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