Process for producing a fertilizer and related fertilizer

Abstract

The invention relates to a process for producing a fertilizer in the form of a manure from poultry manure, wherein the manure is arranged in a pile with a pile height of 4 to 15 meters, and subjected to spontaneous fermentation under aerobic conditions for at least 5 to 7 months with at least two turnings without applying an induced heat source and keeping the pile stationary before, between and after the turnings. The interruption of the fermentation occurs when the following microorganisms are determinable in the manure: (a) Bacillus licheniformis and Bacillus subtilis, and (b) Bacillus haynesii and / or Bacillus glycinifermentans. A related fertilizer is also described.

Description

[0001]85.2023 APPLICATION BODY 1 TITLE: PROCESS FOR PRODUCING A FERTILIZER AND RELATED FERTILIZER Applicant: F.O.M.E.T. S.P.A., Via Vialarga 25, 37050 San Pietro di Morubio (VR), Tax Code / VAT number IT00236940235, ITALY Inventors: Rossin, Michele; Modena, Alberto; Franceschi, Clizia; Casella, Matteo; Pagliarini, Elia * * * * * TECHNICAL FIELD The present invention relates to a process for producing a fertilizer from poultry manure by means of a fermentation under aerobic conditions, arranging the poultry manure in a pile with a certain height and for a duration of at least 5-7 months with the production of specific microorganisms during the degradation of the organic material. The invention also relates to a related fertilizer which is characterized by the presence of determined microorganisms. BACKGROUND OF THE INVENTION Several studies show that the best contribution of organic matter to the soil is given by animal manure, consisting of solid or liquid dejecta on cellulosic substrate or bedding. The organic substance thus determines a series of advantages which are manifested in the state of health of the soil and the surrounding ecosystem, keeping the contributions of carbon, water and nutrients of the plants balanced. The activity of microorganisms is a major factor in the diversification of organic matter. The microbial population present in the various stages of maturation of the manure can vary. The aerobic fermentation of manure is a process by which a base matrix (mature organic substance) is obtained for the production of organic fertilizers where high temperatures are reached inside the pile. The rise in temperature is due to the metabolic process of the microbial load present which develops and degrades the organic matrix, releasing heat. There are three steps during fermentation: the first step is called mesophilic and has a duration of 2-3 days, in which the bacteria perform a first transformation, the temperature inside the pile begins to increase to about 70 °C. In this step, the elimination of some pathogens and weeds begins. The second step follows, the thermophilic step, in which the thermophilic bacteria present inside the manure begin to develop and replace the previous ones. In this step the temperature increases, and ammonia, water and carbon dioxide are emitted by volatilization. In 85.2023 APPLICATION BODY 2 the final step, i.e., the maturation step, microbial stability is achieved with the beginning of the formation of humic substances. A progressive lowering of the temperature is obtained in this step, due in part to the end of the fermentation process. CN107857689A and CN112608196A describe processes for preparing a microbial fertilizer using poultry manure wherein the fertilizer comprises Bacillus licheniformis and Bacillus subtilis. These two bacteria are also described in CN111676174A, while CN111423271A mentions a combination of Bacillus subtilis and Bacillus glycinifermentans without the co- presence of Bacillus licheniformis. DISCLOSURE OF THE INVENTION The invention aims to identify a process for producing a fertilizer and a related fertilizer which is of manure origin and microbiologically enhances and optimizes the organic matrix so as to obtain a combination of microorganisms in the organic matrix which is beneficial for the development of plant roots. In a first aspect of the invention, the object is achieved by a process for producing a fertilizer in the form of a manure from poultry manure, preferably only manure from hens and / or turkeys and / or chickens, comprising the following steps: (I) providing exclusively poultry manure; (II) arranging the manure in a pile with a pile height of 4 to 15 meters, preferably 4 to 12 meters and even more preferably > 5 meters and ≤ 12 meters; (III) spontaneous fermentation of the manure under aerobic conditions for about 5 – 7 months with at least two turnings of the pile at intervals of 1 – 2 months, in particular a first turning about one and a half months after the arrangement of the manure in the pile and a second turning about a month after the first turning, without applying an induced heat source, and keeping the pile stationary before, between and after the turnings; (IV) interrupting fermentation when the following microorganisms are determinable in the manure: (a) Bacillus licheniformis and Bacillus subtilis, and (b) Bacillus haynesii and / or Bacillus glycinifermentans. These bacteria, in addition to enhancing the organic matrix, are also indicators for having achieved a certain progress of fermentation or maturation. While Bacillus haynesii is more 85.2023 APPLICATION BODY 3 indicative after about five months of fermentation, Bacillus glycinifermentans is more typical for fermentations with a longer duration. Bacillus licheniformis and Bacillus subtilis always occur from the fifth to the seventh month of fermentation, thus throughout the indicated period of time. The co-presence of Bacillus haynesii and Bacillus glycinifermentans in the organic matrix occurs, for example, in an intermediate interval between 5 and 7 months of fermentation, or by mixing organic matrices obtained from fermentations interrupted after five and seven months, respectively. The fact that the fermentation occurs without applying an induced heat source, means in other words that the heat formed in the pile is practically the result of the spontaneous biochemical processes occurring in the pile. Preferably, during the storage and fermentation of the manure, only two turnings of the pile take place, keeping the pile stationary for the rest of the time, thus avoiding further external interventions. A month is intended as lasting 30 days, the indication "about" provides a tolerance of + / - five days in relation to the indicated period and in the case of the period of a month a tolerance of + / - 2-3 days. The fact of leaving the pile stationary for the majority of the time and of achieving a certain height thereof allows to reach temperatures between 70 and 90 °C during the fermentation and temperatures above 80 °C for at least a period of time. Temperatures partly above 80 °C are decisive for the sporification of strongly thermophilic strains which then by adaptation stabilize as mesophilic bacteria. By virtue of these high temperatures, strains also develop which withstand high temperatures, such as those also known from volcanic valleys, for example Bacillus haynesii and Bacillus methylotrophicus. Height has a significant role in the selection of the microbiological load. The pressure exerted by the volume of manure and the temperature gradient which is created in the rising pile seem to be crucial for the development of the selected bacteria which tend to develop by stratification from the surface inside the pile. The absence of continuous turnings or movements of the pile, which basically remains stationary for most of the time, serves to create the optimal microclimatic conditions for the sporification of useful strains. In a preferred embodiment of the invention, the interruption of the fermentation of step (IV) occurs when at least one of the following microorganisms is also determinable in the manure: Bacillus methylotrophicus, Pantoea, sp. Bacillus paralicheniformis, Bacillus aryabhattai, Bacillus stratosphericus, Pseudomonas psychrotolerans, Microbacterium oleivorans, 85.2023 APPLICATION BODY 4 Curtobacterium flaccumfaciens. Not all of these microorganisms are found at all times after 5 – 7 months of fermentation, some form at the beginning of the period which goes from the fifth to the seventh month, and disappear after a while, others form towards the end of the indicated period. Depending on needs, the fermentation may be interrupted at a more or less advanced stage of fermentation. Table 6 links the presence of certain types of strains to certain moments of fermentation. In a very advantageous embodiment of the invention, the interruption of the fermentation of step (IV) occurs when the following microorganisms are also determinable in the manure: Bacillus methylotrophicus, Pantoea, Bacillus paralicheniformis and Bacillus glycinifermentans is present. These are the microorganisms present after about seven months of fermentation, while Bacillus aryabhattai, Bacillus stratosphericus, Pseudomonas psychrotolerans, Microbacterium oleivorans, Bacillus haynesii and Curtobacterium flaccumfaciens are more typical after about five months of fermentation. Preferably, the microorganisms correspond to the strains identified with a genetic analysis and therefore to microorganisms whose genetic sequence (DNA) has one of the sequences from SEQ ID NO. 11 – SEQ ID NO. 24 as related to the type of microorganism with an identity of at least 97 %, preferably at least 98 %, even more preferably at least 99 %. For Bacillus licheniformis or others, for example, more than one strain has been identified and advantageously the microorganism corresponds to at least one of these specific strains as characterized by its genetic sequence. Advantageously, the microorganisms correspond to those indicated in table 6 for the second and / or third sampling, with a sequence identity as defined herein. The term "identity" refers to the sequence identity between two nucleic acid molecules. The identity is determined by comparing a position in each sequence which can be aligned for comparison purposes. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. The degree of similarity or identity between nucleic acid sequences is a function of the number of identical or corresponding nucleotides in the positions shared by the nucleic acid sequences. Alignment algorithms and programs are used to calculate the identity between two sequences. FASTA and BLAST are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, WI) and are used with the default settings. Sequences having the degrees of identity or similarity indicated above 85.2023 APPLICATION BODY 5 or any intermediate degree of identity or similarity with the sequences described herein are contemplated and understood in the present disclosure. Advantageously, the entire process occurs at atmospheric pressure, or the pile is externally exposed to pressures corresponding to atmospheric pressure. The atmospheric pressure can vary depending on the geographical location, but advantageously no suction flows are applied to create vacuum situations or further external pressures, in order to maintain the variable pressure controllable given the size of the pile in question. The process according to the invention can include, after the fermentation step, the formation of pellets, optionally with the addition of other fertilizing substances customary in the field known to the person skilled in the art. Preferably, the duration of the fermentation is less than 9 months, preferably less than 8 months to avoid the deterioration of the formed microbial flora. Preferably, the fermentation is interrupted at a time when the manure is free of Staphylococcus, such as Staphylococcus warneri or hominis, and Proteus terrae. Further dimensions useful in order to obtain pressures and temperature trends inside the pile which then lead to the desired microbial flora, are a length of 30 – 150 m, a width of 5 – 20 meters of the pile with an overall volume preferably of 1,000 – 15,000 m3. Hen and / or turkey and / or chicken manure is preferred with respect to guinea fowl manure, in particular chicken manure on bedding. With respect to guinea fowl manure, the one chosen from hen manure, chicken manure and turkey manure and mixtures thereof is less humid and retains a higher nutrient content. A second aspect of the invention relates to a fertilizer in the form of a poultry manure comprising the following microorganisms: (a) Bacillus licheniformis and Bacillus subtilis, and (b) Bacillus haynesii and / or Bacillus glycinifermentans. The presence of these bacteria has proven particularly favorable in the application of fertilizer on different crops, in particular on horticultural plants with a long or short cycle. In another preferred embodiment of the invention, the fertilizer further comprises at least one of the following microorganisms: Bacillus methylotrophicus, Pantoea, sp. Bacillus paralicheniformis, Bacillus aryabhattai, Bacillus stratosphericus, Pseudomonas psychrotolerans, Microbacterium oleivorans, Curtobacterium flaccumfaciens. The 85.2023 APPLICATION BODY 6 simultaneous presence of Bacillus glycinifermentans and Bacillus haynesii is particularly preferred. None of the publications cited on page 2 mentions Bacillus haynesii. Another preferred combination of microorganisms includes the presence of Bacillus licheniformis, Bacillus subtilis, Bacillus haynesii, Bacillus glycinifermentans, Bacillus methylotrophicus, Pantoea, and Bacillus paralicheniformis. Preferably, the microorganisms comprise one of the sequences SEQ ID NO.11 – SEQ ID NO.24 as related to the relative type of microorganism. The fertilizer obtainable by the process according to the invention advantageously has the following chemical features: a total nitrogen content of 2.5 – 4 % by weight corresponding to the organic nitrogen content, a total phosphorus pentoxide content (P2O5determined on the dry material) of 2.0 – 3.5 % by weight, a water-soluble potassium oxide content (K2O determined on the dry material) of 2.0 – 3.0 % by weight, a moisture content of 20.0 – 30.0 % by weight, an organic carbon content of 20.0 – 35.0 % by weight, a pH of 7.0 – 8.0 and a salinity of 1.5 – 3.0 ppt. The fertilizer according to the invention may be particularly suitable for fertilizing gardens and orchards. The fertilizer according to the invention, in addition to being obtainable by the method according to the invention, can be prepared by adding the strains of interest to the manure. The fertilizer producible with the process according to the invention, combines a high contribution of nutrients, a remarkable action for the general improvement of the structure and fertility of the soil. By virtue of the humidification process during the degradation of the organic material, it has a reduced percentage of cellulose and lignin, already transformed directly into humus. The fact that it has not undergone any external heating process or artificial drying (with the use of an oven or hot air), but is only subject to the natural heating caused by fermentation, makes it rich in microbial loads useful for soil life. Always by virtue of the exclusive fermentation process, it does not contain antibiotic residues which may be harmful to the soil and the crop. On the outer walls and on the upper part of the pile, the temperatures reached are lower than inside the pile due to contact with the external temperature. To make the fermentation uniform, also in thermal terms, as well as the bacterial load, at least two turnings of the pile are thus performed. With each turning, the core temperature decreases and then rises cyclically. Advantageously, the number of turnings is limited to two to avoid excessively low temperatures and ensure periods in which the temperatures reach values greater than 80 °C. 85.2023 APPLICATION BODY 7 During the maturation step a progressive lowering of the temperature is then obtained, due in part to the end of the fermentation process, and in part to the turnings which are performed. Furthermore, the populations of active microorganisms also vary, with the transition from thermophilic to mesophilic, to psychrophilic. In this step the temperatures drop to values of 40- 45 °C and then gradually drop to a few degrees above the temperature of the surrounding environment. It is important to stop the fermentation or the maturation as the final step of the fermentation in the point where the microorganisms of interest are present. The great value of the fertilizer is its bacterial flora; humic substances are regardless a factor present. The identification of the right point for the interruption is possible by virtue of the characterization of the bacterial load present in the manure during and after the fermentation process. Preferably, a relative manure sampling is followed by microbiological analysis and colony isolation, DNA extraction and PCR amplification, colony sequencing, with sequencing of the 16s gene with real-time PCR and comparison of the sequences obtained with respective databases. For example, 10 g of the sample is mixed in 90 ml of saline for the sampling. The sample is then homogenized with a Stomacher® type homogenizer. Serial dilutions follow and the pour plate method is applied to TSA plates from which microbial counts and the isolation of bacterial colonies are carried out with the identification of the bacteria, and the DNA is extracted to perform a PCR examination and verification with agarose gel electrophoresis, PCR purification, sequencing and blast data analysis. The bacterial plate count on average gives lower values than PCR determination, and the errors are also on average higher, and have an error around about 1 log CFU / g. The features and advantages described for one aspect of the invention may be transferred mutatis mutandis to the other aspects of the invention. The industrial applicability is obvious from the moment in which a fertilizer and a process for its production have been made available which are adapted to satisfy the need to be able to bring a combination of bacteria to the roots of the plant or to the soil which are suitable for increasing the growth of the roots. The objects and advantages will be further highlighted in the description of preferred examples of embodiments of the invention given by way of non-limiting example only. Variants and further features of the invention are the subject matter of the dependent claims. The description of the preferred exemplary embodiments of the process for producing a 85.2023 APPLICATION BODY 8 fertilizer and the fertilizer according to the invention is given by way of example and not of limitation, with reference to the attached drawings. In particular, unless otherwise specified, the number, shape, size, configurations and materials of the systems described and of the individual components may vary, and equivalent elements may be applied without deviating from the inventive concept. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a pile of poultry manure with an indication of the sampling points. Fig. 2 shows the bacterial count for the different sampling points of figure 1 and for different sampling times. Fig. 3 shows for several moments the total bacteria of the manure with a sample in point 1 (external) of figure 1, determined by PCR and with a plate bacterial count. Fig. 4 shows for several moments the total bacteria of the manure with a sample in point 2 (high internal) of figure 1, determined by PCR and with a plate bacterial count. Fig. 5 shows for several moments the total bacteria of the manure with a sample in point 3 (low internal) of figure 1, determined by PCR and with a plate bacterial count. Fig. 6 shows for several moments the total fungi of the manure with a sample in point 1 (external) of figure 1. Fig. 7 shows for several moments the total fungi of the manure with a sample in point 2 (high internal) of figure 1. Fig. 8 shows for several moments the total fungi of the manure with a sample in point 3 (low internal) of figure 1. DESCRIPTION OF PREFERRED EXAMPLE EMBODIMENTS The data described below refer to a manure pile of organic matrix deriving from poultry manure on bedding (poultry farms, laying hens). The storage place was a warehouse 5-15 meters in 85.2023 APPLICATION BODY 9 height with a pile volume of 1000 – 15,000 The pile was turned twice with intervals of at least 1-2 months. The temperatures measured during the fermentation ranged from 70°C to 90°C. The fermentation was aerobic, natural and spontaneous for 5-7 months. No external heat source was included. To identify the particular bacterial flora which developed by virtue of the particular trend of manure production, samples (manure and fresh manure) were taken at various steps of the fermentation and then subjected to microbiological analysis of the matrices taken. The sampling occurred in three different points of the manure pile (low internal, high internal, external surface, indicated in figure 1 which shows the pile respectively with reference numbers 3, 2 and 1), so as to obtain a broader monitoring of the microbial population formed. Similarly, fresh manure was analysed as a starting reference: three samples were taken in the aforementioned different points of the pile to ensure the survey of the population of the matrix in its complexity. Three samplings were carried out for the manure over a period of about three months, the first when the manure was already at the end of the third month of fermentation / maturation (1st sampling), the second after about five months of fermentation / maturation (2nd sampling) and the third after about seven months of fermentation / maturation (3rd sampling). The sample of fresh manure was taken in order to have feedback on the presence of any pathogenic microorganisms and on the initial microbial population. The points sampled in the pile remained fixed so that the only variable of this analysis is the maturation phase in which the manure is found. The microbiological analyses included: (a) preparation of the culture media for the inoculation of the samples, (b) seeding the plates, (c) the count on the plate, (d) the withdrawal of at least nine colonies for each sampling, chosen based on the different morphology (obtained from the different sampling points), and (e) the purification of the pure culture, (f) the extraction of the DNA with the amplification of the 16S rDNA and (g) the identification of the isolated microorganism. In addition, total bacteria and fungi were counted on all manure samples by real-time PCR (quantitative Polymerase Chain Reaction). This is necessary for an absolute quantification of the microbial forms present (bacteria and fungi), since it is known that only 10% of soil microorganisms can be cultivated on a plate. This analysis gives value to the quantitative data which is proposed when characterizing a matrix. In this regard, the following procedure was 85.2023 APPLICATION BODY 10 performed: (1) total DNA extraction from the matrix, (2) quantification of DNA and assessment of the degree of purity, (3) amplification of 16S rDNA for bacteria and 18S rDNA for fungi. MATERIALS AND METHODS FOR CULTURE MEDIA PREPARATION The media used for this type of test are Tryptic Soy Agar (TSA) and Potato Dextrose Agar (PDA). TSA is a medium that promotes the growth of most bacteria, while PDA is a selective medium for moulds and fungi. During this type of test, a physiological solution (NaCl 0.9%) is also prepared which is used to serially dilute the samples. Subsequently, all the sample tubes are closed with cellulose caps and autoclaved. PLATE SEEDING 10 g of matrix to be analysed is weighed in a bag for a Stomacher® type homogenizer and 90 g of saline is added. After mixing with the Stomacher® for 1 minute, the solid part is allowed to settle for 30 minutes so that the microorganisms present rise in suspension. From this point, it is good to proceed by performing serial dilutions in order to reduce the bacterial load, making it countable on the plate. For this operation, 1 ml of the main solution is taken (already diluted at 10-1) and inserted into a first physiological tube, obtaining a 10-2dilution. Proceeding, 1 ml is again taken from the previous tube and inserted into another saline tube to obtain the dilution to 10-3. All the subsequent dilutions are obtained in this manner. During this procedure, it is important to always sterilize the tubes just opened and before closing, passing the opening of the tube over the flame of a Bunsen burner. Between each dilution, it is good practice to mix the contents of the tube using a vortex. Once these operations are performed, the seeding on a plate is carried out. The colonies to be sequenced were chosen based on colour, size and shape. DNA EXTRACTION FROM COLONIES This operation starts with the collection of a microbial colony (previously purified on TSA medium), which was inoculated in 9 ml in TSB culture medium (Tryptic Soy Broth). After 24 hours of incubation at 37°C the extraction of DNA can be carried out, as experience teaches that DNA extraction is more efficient if obtained from a fresh culture. The extraction procedure follows the following protocol: (1) withdraw 1 ml of the culture, (2) centrifuge at 13,000 rpm for 2 minutes. Remove the supernatant and resuspend the obtained pellet in 480 µl of 50 mM 85.2023 APPLICATION BODY 11 EDTA (ethylenediaminetetraacetic acid), (3) add the lysozyme to break the cell wall (140 µl of a 40 µl / ml solution in TE* (*10 Tris-HCl, 1 mM disodium EDTA, pH 8.0), (4) incubate at 37 °C for 60 minutes, (5) centrifuge for 2 minutes at 13,000 rpm and remove the supernatant, (6) add 600 µl of Nuclei Lysis Solution, (7) incubate at 80 °C for 5 minutes, (8) add 3 µl of RNase, (9) incubate at 37 °C for 60 minutes, (10) add 200 µl of Protein Precipitation Solution (solution for precipitating proteins to clean the DNA from the proteins), (11) incubate on ice for 5 minutes, (12) centrifuge at 13,000 rpm for 3 minutes, (13) transfer the supernatant into new Eppendorf type pipettes containing 600 µl of isopropanol (for precipitating the DNA), (14) centrifuge at 13,000 rpm for 2 minutes, (15) remove the supernatant and add 600 µl of 70% ethanol, (16) centrifuge at 13,000 rpm for 2 minutes, (17) aspirate the ethanol and add 100 µl of DNA Rehydration Solution and (18) incubate at 65 °C for 60 minutes and (19) store the DNA at -20 °C. DNA QUANTIFICTION The obtained DNA was then quantified by measuring the absorbance at 260 nm using the Infinite 200 PRO NANOQUANT instrument (Tecan Group Ltd., Switzerland). DNA purity was assessed by absorption ratio at 260 nm and 280 nm (260 / 280 ratio). This value must be between 1.9 – 2.1 for the DNA to be judged “clean”, i.e., free of proteins. DNA AMPLIFICATION Each PCR reaction was set up in MicroAmpTM Fast Reaction Tube with cap in a final volume of 30 µl, of which 28.5 µl of PCR reaction mix and 1.5 µl of DNA at the concentration of about 100 ng / µL: 85.2023 APPLICATION BODY 12 *Taq polymerase (thermostable DNA polymerase) is a polymerase from the thermophilic organism Thermus aquaticus. At the end of the amplification each sample was separated on a 1.5% agarose gel added with a cyanine dye, used as a nucleic acid dye, for example Sybr SafeTM, according to the indications reported.5 µl of PCR product is loaded into the wells. One well is loaded with 5µl of 1kb ladder (molecular weight marker). The presence of a single amplification band was verified at the GelDoc transilluminator (BioRad) and its purification occurred by means of the NucleoSpin kit (Macherey-Nagel GmbH & Co. KG, Germany) so as to eliminate any impurities due to PCR products (primers). The purified amplicon was sent to Eurofins MWG Operon for sequencing. TOTAL DNA EXTRACTION The procedure was performed with the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Sussex, USA) which envisages cell lysis using beads as described in the following protocol: 1) Add 0.250 g of soil into a PowerBead Tube. 2) Add 750µl of PowerBead solution. 3) Add 60µl of lysis solution (C1 solution of the kit) and invert the tubes several times. 4) Vortex for 8-10 minutes. 5) Centrifuge the tubes at 1,000 rpm for 30 seconds. 6) Transfer the supernatant (400-500 µl) into the 2 ml Eppendorf type pipettes provided with the kit. 7) Add 250µl of C2 solution and vortex for 5 seconds. 8) Incubate at 2-8 °C for 5 minutes. 9) Centrifuge for 1 minute at 10,000 rpm. Transfer 600 µl of the supernatant into the Eppendorf type pipettes provided with the kit. 10) Add 200 µl of the C3 solution, vortex and incubate at 2-8°C for 5 minutes. 11) Centrifuge the tubes at 10,000 rpm for 1 minute, transfer 750µl of the supernatant into the 2 ml Eppendorf pipettes provided with the kit. 12) Add 1200 µl of the C4 solution and vortex for 5 seconds. 85.2023 APPLICATION BODY 13 13) Take 675 µl of the supernatant and transfer it into the MB Column to be centrifuged at 10,000 rpm for 1 minute. Repeat the operation by taking another 675 µl of supernatant. 14) Remove the supernatant and perform a vacuum centrifuge at 10,000 rpm for 1 min. 15) Add 500 µl of the C5 solution and centrifuge at 10,000 rpm for 30 seconds. 16) Remove the supernatant and perform a vacuum centrifuge for 1 min at 10,000 rpm. 17) Insert the filter column inside new 2 ml Eppendorf type pipettes provided with the kit and add 100 µl of the C6 solution. 18) Centrifuge at 10,000 rpm for 30 seconds. 19) Store the DNA at -20°C. REAL TIME PCR PROTOCOL In order to quantify the total bacteria and fungi present in the various stages of manure maturation, DNA samples were prepared with a known quantity of target gene copies used as standard. The analyses were performed with 20 µl of an amplification mixture containing 10 µl of Fast SYBR® Green Master Mix (Applied Biosystems), an optimal concentration of primers, H2O RNAse free and 2 µl of DNA extracted from fecal samples at the concentration of 5 ng / µl for all samples. The final concentration of the primers was obtained after carrying out a series of preliminary tests. The sequences of the primers used and the conditions of the procedure are shown in table 3 and 4, respectively. The data obtained from the analysis of each species or microbial group in the fecal samples were then transformed to obtain the number of 16S rDNA copies for each microbial group tested. Table 4. Cycles and concentration of primers for real time PCR using Sybr Green 85.2023 APPLICATION BODY 14 TOTAL MANURE AEROBIC BACTERIAL COUNT RESULTS Table 5 shows the results of the bacterial count of the samples taken from various samplings, which are also represented in the graph in figure 2. DNA EXTRACTION FROM COLONIES Table 6 illustrates which strains of bacteria were detected during the various samplings in the 85.2023 APPLICATION BODY 15 The types of bacteria strains could be identified through a comparison with the NCBI database (National Center for Biotechnology Information, accessible at https: / / www.ncbi.nlm.nih.gov) in which the percentage of identity with the closest relative (microorganism of which the total or partial sequence that most closely resembles the sequenced one is available) corresponded to ≥ 99%. The sequences indicated in the sequence list correspond to the sequences identified in the samples and are located in the Genebank database within the NCBI database with the following accession numbers indicated in Table 7: PGPR are among the bacteria identified, i.e., plant growth stimulators, useful for root development and soil microflora. All three samples confirm the presence of rhizosphere bacteria. PGPR (Plant Growth Promoting Rhizobacteria) are bacteria that live in the rhizosphere, i.e., in that portion of the soil that suffers the effects of the root of the plant and promote the growth of the plant. 85.2023 APPLICATION BODY 16 Table 8 summarizes the amounts of bacteria determined by PCR at the various sampling points and at certain times of the fermentation. Figures 3 to 5 show the trend of the growth of bacteria for each sampling point as a function of the fermentation time. In the first three months there is a decrease in the bacterial load, followed by a stabilization or a slight decrease in the bacterial load in the following four months. For the reasons set out above, the values determined with PCR generally exceed those determined with the bacterial count. Table 9 summarizes the quantities of fungi determined at the various sampling points and at certain fermentation times. 85.2023 APPLICATION BODY 17 Figures 6 to 8 show the trend of the growth of fungi for each sampling point as a function of the fermentation time. In the first three months, the quantity of fungi decreased, followed by a stabilization of the quantities. In summary, a decrease in the biomass of the bacterial count over time is observed in all three sample collection locations (external, high internal, low internal). The reduction is particularly evident between fresh manure and the first sampling (3 months). The count performed with molecular methods provides generally higher values in line with the limitations already highlighted for the plate count (not all soil bacteria can be cultivated). The fungi have a similar trend to the bacteria, as they decrease in the first three months, reaching a stable concentration during maturation. The results of the molecular characterization of the bacteria isolated in the plate show a preponderance of microorganisms referable to the genus Bacillus, and this observation is consolidated in the 3rd sampling in which 12 out of 13 sequenced microorganisms belong to this genus. Some species reappear in all the samplings at different times. As stated in the literature, many of these bacteria have beneficial functions for plant growth. For example, microorganisms of the species B. licheniformis are known for their properties (Lim et al., 2013) and B. methylotrophicus shows antagonistic activity against fungal pathogens (Ge et al., 2016). No pathogenic microorganisms emerged from the data obtained, while an enrichment of beneficial microorganisms is observed with the advancement of the maturation phase of the manure. The finished manure (after seven months of maturation) is chemically characterized as follows: 85.2023 APPLICATION BODY 18 The use of the fertilizer according to the invention is indicated for all crops; in particular it is recommended on all loose soils (sandy) with accentuated macroporosity and heavy (clayey) with risk of compaction. The best results are obtained by distributing the product immediately before the last processing, mixing it with the soil during the pre-seeding or pre-transplanting step; in particular, the full-field spreading must preferably occur for a minimum depth of 2 cm and a maximum of 15 cm; the use on tree crops advantageously envisages a distance from the trunk of 18 / 20 cm and a depth of 10 / 15 cm. If the fertilization is carried out on grassy orchards (spontaneous or re-seeded turf between the rows) the product should ideally be spread evenly on the surface; the subsequent mowing of the grass may be useful in order to accelerate the dissolution of the product. It can also be distributed on crop residues (straw, corn stalks) before their burial to facilitate their disintegration and to favour the microbiological activity involved in the soil. The fertilizer can be optimally distributed in coverage, in the tillering step of autumn-winter grains. Suitable dosages include 1,000-1,500 kg / ha for wheat, barley and rice, 1,200 – 1,500 kg / ha for maize, beet and tobacco, 1,800 – 2,000 kg / ha for horticultural crops in the open field, 100 – 300 kg / 1,000 m2for horticultural crops in greenhouses, 0.5 – 4 kg / plant for orchards, citrus and olive trees, 0.5 – 3 kg / plant for vineyards, 0.250 kg / m2for turf and parks, and 1,500 – 2,000 kg / ha for floral crops on the ground. The preparation of the fertilized soil advantageously involves 4 - 6 kg of fertilizer per m3of dissolved soil, peat or soil already pre-constituted but not fertilized (all this can also contain inert material that confers porosity). A basic fertilization to enrich soil includes 0.200 kg per m2, equal to 2000 kg per hectare. Fertilization on stubble, straw, crop residues (for the recovery of nutrients and transformation into humus) instead includes, for example, 900 - 1200 kg per hectare. 85.2023 APPLICATION BODY 19 At the end of the fermentation, the manure is usually sieved, then optionally the addition of additives depending on the needs of the soil and the plants to be fertilized, then a pelleting (homogeneous pellet of cylindrical shape with an average diameter of 3 - 4 mm and an average length of 5 - 7 mm) and finally the packaging. The fermentation according to the invention ensures the absence of Salmonella and an E. coli concentration of less than 10 CFU / g, as prescribed by European regulations. The fertilizer or manure according to the invention can be mixed with other types of fertilizers, such as vegetal, feathers, protein flours of plant or animal origin, horn-hoof mixture, dried blood, oilcakes, meat meal, bone meal, hides, horsehair, ground horns and hooves. Chemical additives may be chosen from iron sulphate, calcium sulphate, magnesium salt (Kieserite), sulphur (micronized), boron ethanolamine, calcium carbonate, natural phosphate, potassium sulphate, zinc sulphate, ammonium sulphate, biammonium phosphate, urea, ammonium phosphate, other types of fertilizer. The manure preferably comprises ammonifying bacteria, nitrate reducers, cellulosolytics and schizomycetes. During implementation, to the process for producing a fertilizer and to the fertilizer, object of the invention, further non-described modifications or executive variants may be applied. If such modifications or such variants should fall within the scope of the following claims, they should all be considered protected under the present patent. Bibliography Ge, B., Liu, B., Nwet, T. T., Zhao, W., Shi, L., & Zhang, K. (2016). Bacillus methylotrophicus strain NKG-1, isolated from Changbai Mountain, China, has potential applications as a biofertilizer or biocontrol agent. PloS one, 11(11), e0166079. Lim, J. H., & Kim, S. D. (2013). Induction of drought stress resistance by multi-functional PGPR Bacillus licheniformis K11 in pepper. The plant pathology journal, 29(2), 20 Free text in the sequence list 85.2023 APPLICATION BODY 20 SEQ ID NO. 1 – 4 concern primers with their respective names or destinations.

Claims

85.2023 APPLICATION BODY 21 CLAIMS (1) Process for producing a fertilizer in the form of a manure from poultry manure, preferably only manure from hens and / or turkeys and / or chickens, comprising the following steps: (I) providing exclusively poultry manure; (II) arranging the manure in a pile with a pile height of 4 to 15 meters, preferably 4 to 12 meters and even more preferably > 5 meters and ≤ 12 meters; (III) spontaneous fermentation of the manure under aerobic conditions for at least 5 to 7 months with at least two turnings of the pile at intervals of 1 to 2 months, in particular a first turning about 1 and a half months after the arrangement of the manure in the pile and a second turnings about 1 month after the first turning without applying an induced heat source and keeping the pile stationary before, between, and after the turnings; (IV) interrupting fermentation when the following microorganisms are determinable in the manure: (a) Bacillus licheniformis and Bacillus subtilis, and (b) Bacillus haynesii and / or Bacillus glycinifermentans. 2) Process for the production of a fertilizer according to claim 1, characterized in that the interruption of the fermenation of phase (IV) occurs when at least one of the following microorganisms is also determinable in the manure: Bacillus methylotrophicus, Pantoea sp., Bacillus paralicheniformis, Bacillus aryabhattai, Bacillus stratosphericus, Pseudomonas psychrotolerans, Microbacterium oleivorans, Curtobacterium flaccumfaciens. 3) Process for the production of a fertilizer according to claim 1, characterized in that the interruption of the fermentation of phase (IV) occurs when the following microorganisms are also determinable in the manure: Bacillus methylotrophicus, Pantoea, Bacillus paralicheniformis and when Bacillus glycinifermentans is present. 4) Process for producing a fertilizer according to any of the preceding claims, characterized in that the microorganisms have in their genetic sequence one of the sequences from SEQ ID NO. 11 - SEQ ID NO. 24 as related to the type of microorganism and having an identity of at least 97 %, preferably at least 98 %, even more preferably at least 99 %.85.2023 APPLICATION BODY 22 5) Process for the production of a fertilizer according to any of the preceding claims, characterized in that the entire process takes place at atmospheric pressure or that the pile is exposed externally to pressures that correspond to atmospheric pressure. 6) Process for the production of a fertilizer according to any of the preceding claims, characterized in that the duration of fermentation is less than 9 months, preferably less than 8 months. 7) Process for the production of a fertilizer according to any of the previous claims, characterized in that said pile has a length of 30 to 150 m, a width of 5 to 20 meters with an overall volume of preferably 1,000 to 15,000 m3. 8) A fertilizer in the form of a poultry manure including the following microorganisms: (a) Bacillus licheniformis and Bacillus subtilis, and (b) Bacillus haynesii and / or Bacillus glycinifermentans. 9) A fertilizer in the form of a poultry manure according to claim 8 including the following microorganisms: Bacillus licheniformis and Bacillus subtilis and Bacillus haynesii, 10) A fertilizer in the form of a poultry manure according to claim 9 further including the microorganism Bacillus glycinifermentans. 11) The fertilizer according to anyone of the claims 8 – 10 further comprising at least one of the following microorganisms: Bacillus methylotrophicus, Pantoea sp., Bacillus paralicheniformis, Bacillus aryabhattai, Bacillus stratosphericus, Pseudomonas psychrotolerans, Microbacterium oleivorans, Curtobacterium flaccumfaciens, in particular further including the group of microorganisms formed by Bacillus methylotrophicus, Pantoea, and Bacillus paralicheniformis and comprising Bacillus glycinifermentans, wherein the microorganisms preferably comprise one of the sequences SEQ ID No.11 - SEQ ID NO.24 as related to the type of microorganism.85.2023 APPLICATION BODY 23 12) The fertilizer according to anyone of the claims 8 - 11 characterized by having a total nitrogen content of 2.5 - 4 weight % corresponding to the organic nitrogen content, a total phosphorus pentoxide content (P2O5 determined on the dry material) of 2.0 - 3, 5 weight %, a water-soluble potassium oxide content (K2O determined on the dry material) of 2.0 - 3.0 weight %, a moisture content of 20.0 - 30.0 weight. %, an organic carbon content of 20.0 - 35.0 weight %, a pH of 7.0 - 8.0 and a salinity of 1.5 - 3.0 ppt.

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