Screening method for functional microbiomes in local soil and its application

Abstract

This invention provides a bioremediation method (a method of decomposing or absorbing harmful substances using microorganisms) that is widely applicable to various soil conditions and can significantly reduce the time required for soil recovery, making it effective for soil regeneration. [Solution] The present invention provides a method for screening functional microbiomes and its applications. The method includes providing a local soil sample, pre-treating and filtering the local soil sample to obtain a first bacterial suspension, culturing the first bacterial suspension according to a culture formula to obtain a second bacterial suspension, and identifying the second bacterial suspension by the spread plate method. Subsequently, the functional microbiome is domesticated, and the domesticated functional microbiome is introduced into the local soil to achieve soil restoration.

Description

Technical Field

[0001] The present invention relates to a screening system and method for microbiota for soil restoration, and particularly to screening and domesticating a group of microorganisms having a soil restoration function from in-situ soil and applying them to the restoration of the in-situ soil.

Background Art

[0002] Soil restoration refers to taking improvement and restoration measures for a soil environment that has lost its balance and solving various situations faced by the soil. Such situations include problems caused by naturally existing environmental factors or anthropogenic factors. Examples of environmental factors include that crops are difficult to grow in permafrost in cold regions and that vegetation cannot survive due to too high salt concentration in the soil. Examples of anthropogenic factors include the discharge of industrial waste, the excessive use of chemical fertilizers and pesticides, and the release of nuclear waste.

[0003] Methods of soil restoration include bioremediation and physicochemical treatment, which are selected according to the characteristics of the region and the type of pollution. <, Among them, bioremediation is mainly a method of using microorganisms to decompose or absorb harmful substances. For example, hydrocarbons can be decomposed using specific bacteria after an oil spill. It is also possible to improve pollutants in the soil using microorganisms. As a common method, microorganisms having the ability to decompose pollutants are introduced into the contaminated soil, and the pollutants are converted into harmless substances by metabolic action, or microorganisms are introduced to release beneficial substances into the soil, increasing the organic matter content of the soil and enhancing its fertility. Compared with conventional chemical treatment methods, bioremediation has the advantages of low cost and environmental friendliness, and has attracted increasing attention and wider application in recent years. ]

Summary of the Invention

[0004] However, the bioremediation methods currently used for soil regeneration always involve introducing a single beneficial bacterial strain into the environment. In actual applications, however, these beneficial strains often fail to adapt to the local environment, resulting in insufficient bioremediation capabilities or extremely long adaptation times. Therefore, developing a bioremediation method that can be widely applied to various soil conditions and significantly reduce the time required for soil recovery is a crucial challenge in this field. [Means for solving the problem]

[0005] In view of these, the present invention is To provide local soil samples, The aforementioned local soil sample is pretreated and filtered to obtain the first bacterial suspension. The first bacterial solution is cultured according to the culture formula to obtain the second bacterial solution, and The second bacterial solution is applied to a plate to measure the content of Bacillus subtilis, and if the content of Bacillus subtilis accounts for 5% or more of the total content of all bacterial species, the second bacterial solution is the screened functional microbiome, including the following: This provides a screening method for functional microbiomes.

[0006] In some specific embodiments, the local soil sample contains earthworms and / or substances metabolized by earthworms.

[0007] In some specific embodiments, the pretreatment includes heat treatment and / or treatment with an extractant.

[0008] In some specific embodiments, the heat treatment involves adding the local soil sample to the extract and heating it, with a heating temperature of 60-90°C and a treatment time of 30-60 minutes.

[0009] In some specific embodiments, the treatment with the extract involves adding the extract to the local soil sample and performing the extraction.

[0010] In some specific embodiments, the extract comprises at least one selected from the group consisting of water, physiological saline, general culture medium, bacitracin-containing culture medium, single colony culture supernatant, salt solution, and metal ion solution.

[0011] In some specific examples, the culture formulation includes at least one selected from the group consisting of glucose, glycerol, molasses, starch, arabinose, fructose, galactose, lactose, maltose, mannose, sucrose, peanut flour, soybean flour, corn gluten flour, beef extract, peptone, yeast flour, fish meal, urea, spent mycelium, distilled meal ammonium salt, nitrate, ammonia water, yeast extract, sodium chloride, magnesium chloride, sodium bicarbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, ammonium chloride, sodium citrate, sodium malate, sodium carbonate, and potassium carbonate.

[0012] In another respect, the present invention is To provide a functional microbiome screened by the method described above, This involves domesticating microorganisms in the functional microbiome, culturing the functional microbiome in a bioreactor, and controlling the microenvironmental parameters within the bioreactor to simulate the environment of the local soil sample, and This includes introducing the domesticated functional microbiome into the local soil. We provide a method for soil regeneration.

[0013] In some specific embodiments, the microenvironmental parameters include the components of the nutrient source, the nutrient source content, the pH value, temperature, humidity, external force, and the growing medium.

[0014] In some specific embodiments, the growth medium includes wood chips, rice husks, and soil.

[0015] The conventional bioremediation method for soil was to select a single strain beneficial to the soil and introduce the single strain into the soil for restoration. However, in actual applications, there are differences in soil environments in different regions. For example, due to differences in humidity, temperature, pH value, and natural microbial flora, a single strain cannot well adapt to the growth conditions of different environments, so it was difficult to achieve the restoration effect.

Advantages of the Invention

[0016] The present invention directly obtains a complete microbial flora of local strains from the soil environment to be restored, removes harmful bacteria by pretreatment, then cultures them with a special culture formula, and further confirms the culture results by methods such as the spread plate method, thereby screening for a functional microbiome. After that, by introducing the functional microbiome into the soil environment originally intended to be restored, specific effects such as environmental restoration and improvement of environmental health are achieved. The functional microbiome according to the present invention can be domesticated before being introduced into the soil environment originally intended to be restored. That is, the functional microbiome is cultured in a bioreactor, and the parameters of the bioreactor are adjusted to simulate the state of the natural environment, so that the functional microbiome can be domesticated to adapt to the microbial flora of the original environment. After that, by introducing the domesticated functional microbiome into the area actually requiring restoration, the time required for environmental regeneration can be significantly shortened.

Brief Description of the Drawings

[0017] [Figure 1] Figure 1 is a flowchart of a method for screening a functional microbiome according to the present invention. [Figure 2]Figure 2 is a flowchart of the soil restoration method according to the present invention. [Figure 3] Figure 3 is a diagram showing the results of coating on a plate after pretreatment at different heating temperatures and times. [Figure 4] Figure 4 is a diagram showing the results of identifying different microbial group samples by the spread plate method in a selective medium. [Figure 5] Figure 5 is a comparison diagram of seed germination results after introducing microbiome, functional microbiome, and single strains into in-situ soil (bitter melon cultivation land) and culturing for 3 days. [Figure 6A] Figure 6A is a photographic view of a bitter melon cultivation land before applying the functional microbiome according to the present invention. [Figure 6B] Figure 6B is a photographic view of a bitter melon cultivation land after applying the functional microbiome according to the present invention. [Figure 7] Figure 7 is a comparison diagram of seed germination results after introducing microbiome, functional microbiome, and single strains into in-situ soil (castor aralia cultivation land) and culturing for 3 days. [Figure 8A] Figure 8A is a photographic view of a castor aralia cultivation land before applying the functional microbiome according to the present invention. [Figure 8B] Figure 8B is a photographic view of a castor aralia cultivation land after applying the functional microbiome according to the present invention. [Figure 9A] Figure 9A is a photographic view of a komatsuna cultivation land before applying the functional microbiome according to the present invention. [Figure 9B] Figure 9B is a photographic view of a komatsuna cultivation land after applying the functional microbiome according to the present invention.

Embodiments for Carrying Out the Invention

[0018] Regarding other technical contents, features, and effects of the present invention, they are clearly shown in the following detailed description of preferred embodiments in accordance with the attached drawings.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0020] In one respect, the present invention provides a method for screening functional microbiomes.

[0021] Referring to Figure 1, the method includes steps 11 to 14.

[0022] Step 11: Provide a local soil sample.

[0023] In some specific embodiments, the local soil sample contains earthworms and / or substances metabolized by earthworms. In some specific examples, the local samples contain microbial communities specific to the region of origin.

[0024] Step 12: The first bacterial suspension is obtained by pre-treating and filtering the aforementioned local soil sample.

[0025] In some specific embodiments, the pretreatment includes heat treatment and / or treatment with an extractant. The aforementioned pretreatment involves one or more physical or chemical processes. In the present invention, the order of pretreatment is not restricted. For example, heat treatment can be performed first, followed by treatment with the extract, or treatment with the extract can be performed first, followed by heat treatment.

[0026] In some specific embodiments, the heat treatment involves adding the local soil sample to the extract and heating it. Of these, the heating temperature is 60 to 90°C, for example, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, or 90°C. Of these, the processing time is 30 to 60 minutes, for example, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes.

[0027] In some specific embodiments, the treatment with the extract involves adding the extract to the local soil sample and performing the extraction.

[0028] In some specific embodiments, the extract is a liquid capable of eluting microorganisms and includes at least one selected from the group consisting of water, physiological saline, general culture medium, bacitracin-containing culture medium, culture supernatant of a single colony strain, salt solution, and metal ion solution.

[0029] The extract used in this invention is not necessarily bacitracin. The effect of bacitracin is to suppress harmful bacteria and thereby increase the proportion of beneficial bacteria, and any antimicrobial agent familiar in this field can be substituted for bacitracin. Of these, the culture supernatant of a single colony strain exerts an effect of suppressing other bacterial species and facilitates the growth of the single colony strain. In some specific examples, the single colony strain is Bacillus subtilis. Furthermore, substances in the supernatant (postbiotics) also have the effect of promoting the growth of probiotics that coexist with other organisms.

[0030] In some specific embodiments, the filtration is centrifugal filtration or gravity filtration using filter paper. Its main purpose is to remove interference from solid components.

[0031] Step 13: The first bacterial suspension is cultured according to the culture formula to obtain the second bacterial suspension.

[0032] In some specific examples, the culture formulation includes at least one selected from the group consisting of glucose, glycerol, molasses, starch, arabinose, fructose, galactose, lactose, maltose, mannose, sucrose, peanut flour, soybean flour, corn gluten flour, beef extract, peptone, yeast flour, fish meal, urea, spent mycelium, distilled meal ammonium salt, nitrate, ammonia water, yeast extract, sodium chloride, magnesium chloride, sodium bicarbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, ammonium chloride, sodium citrate, sodium malate, sodium carbonate, and potassium carbonate.

[0033] In some specific examples, the culture formulation includes elements such as carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, potassium, calcium, magnesium, boron, manganese, zinc, molybdenum, cobalt, iodine, and copper.

[0034] In some specific examples, the culture formulation is a culture material that is favorable for the growth of Bacillus subtilis.

[0035] In some specific examples, different local soil samples are subjected to different culture formulations. For example, if the local soil is slightly acidic or slightly alkaline, it may be necessary to fine-tune the pH value in the culture formula. Furthermore, since different local soils contain different trace elements (e.g., metal ions), the cultivation formula in this invention needs to be finely adjusted according to the local soil environment.

[0036] Step 14: The second bacterial solution is spread onto a plate to measure the amount of Bacillus subtilis. If the amount of Bacillus subtilis accounts for 5% or more of the total amount of all bacterial species, the second bacterial solution is the screened functional microbiome.

[0037] In some specific examples, the content of Bacillus subtilis accounts for 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% or more of the total content of all bacterial species.

[0038] More specifically, in this invention, identification involves determining the microbial community by spreading the cultured microbiome onto a plate and observing the growth status of Bacillus subtilis to confirm the proportion of Bacillus subtilis in the microbiome. If Bacillus subtilis accounts for at least 5% or more of the total colony composition, the microbiome is a functional microbiome according to the present invention and can be used for subsequent soil restoration.

[0039] In other respects, the present invention provides a method for soil regeneration. Referring to Figure 2, the method includes steps 21 to 26.

[0040] In some specific examples, the functional microbiome screened by the screening method according to the present invention is subjected to the domestication of microorganisms. The domestication of the microorganisms involves culturing the functional microbiome in a bioreactor and controlling the microenvironmental parameters within the bioreactor to simulate the environment of the local soil sample. This facilitates further adaptation of the functional microbiome to the local soil environment during repeated cultivation and expansion. The aforementioned microenvironmental parameters include the components of the nutrient source, the nutrient source content, pH value, temperature, humidity, external force, growth medium, and the content of other bacterial species. The growing medium includes solid and liquid growing media, such as wood chips, rice husks, soil, and other porous materials.

[0041] In some specific embodiments, the domesticated functional microbiome can be further immobilized. The immobilization carrier includes, but is not limited to, wood chips or calcium carbonate. The aforementioned immobilization process involves first placing the solid carrier in a stirring device, then starting the mixing and stirring device, and finally adding the bacterial solution. The mixing ratio is in the range of 1:2 to 1:4. After complete mixing, the carrier is dried and stored.

[0042] In some specific embodiments, the immobilized functional microbiome can exist in various forms, such as solid powder, granules, or solid particles.

[0043] In some specific embodiments, the soil regeneration method according to the present invention involves introducing the domesticated functional microbiome into the local soil, and includes, but is not limited to, scattering, irrigating, or mixing it into the environment.

[0044] In some specific examples, the domesticated functional microbiome is introduced into an unbalanced environment along with its nutrients. The aforementioned nutrients are sources of nutrients for the growth of the microbiome, and examples include food processing and organic waste from agriculture and livestock for composting. In some specific embodiments, the region to which the functional microbiome screened and domesticated according to the present invention is most applicable is its place of origin. However, the present invention is not limited to introducing the functional microbiome to its place of origin, but can also be introduced to regions with similar environmental problems in order to achieve environmental restoration effects.

[0045] It should be understood that the general descriptions above and the detailed descriptions below are all illustrative and explanatory, and are not intended to limit the rights of the present invention. Some details of one or more embodiments of the present invention are described below. Other features or advantages of the present invention are evident from the following representative examples, which are not exhaustive, as well as from the attached claims.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. The purpose of the references to the technologies used in this text is to illustrate technologies commonly understood in the art, including variations, equivalents, or alternative technologies developed in the future. This will be obvious to those skilled in the art.

[0047] It should be noted that the singular forms "one," "one kind," and "the aforementioned" used in this text include plural references unless otherwise explicitly indicated. Unless otherwise explicitly stated in the surrounding text, the terms "or" and "and / or" are used alternately. The terms "approximately," "roughly," or "close to" used in this text substantially indicate that the numerical value or range is within 3%, preferably within 1%, and more preferably within 0.5%. The digitized quantities shown in this text are approximations and can be inferred even without the use of the terms "approximately," "roughly," or "close to."

[0048] As used in this text, the term "contains" is open, indicating that such examples may contain additional elements. Conversely, the term "consisting of" is closed, indicating that such examples do not contain additional elements (except for trace impurities). The phrase "essentially consisting of" is somewhat closed, indicating that such embodiments may also contain elements that do not substantially alter the fundamental characteristics of such embodiments.

[0049] Unless otherwise defined, technical and scientific terms used in conjunction with this text have the same meaning as those commonly understood by those skilled in the art. Furthermore, unless otherwise required by the surrounding text, singular terms must encompass plural forms, and plural terms must encompass singular forms. Generally speaking, the nomenclature used in relation to the following technologies described in this text, as well as the techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, immunology, medicine, pharmacy, cell culture, and bacterial culture, are all well-known and commonly used in this field. Unless otherwise described, the methods and techniques of the present invention are carried out by conventional methods commonly known in the art, and are further described in various general and more specific references cited and discussed herein.

[0050] The term "functional microbiome" as used in this text generally refers to a group of microorganisms that have the function of soil restoration, cultured and screened by the method according to the present invention. It should be understood that this is not a single specific strain, but rather a group of different bacterial species, and that the group of bacterial species has been confirmed by evaluation to satisfy specific conditions.

[0051] In this invention, the specific condition is defined as satisfying the definition of a "functional microbiome" when the content of Bacillus subtilis accounts for 5% or more of the total content of all bacterial species.

[0052] The term "microbiome" as used in this text generally refers to a group of microorganisms cultured and screened by the method according to the present invention. More specifically, it refers to a group of microorganisms obtained by culture using the pretreatment and culture formulation of the present invention, and if it is determined that the content of Bacillus subtilis accounts for 5% or more of the total content of all microbial species, it is named a "functional microbiome."

[0053] In this text, the term "local soil sample" generally refers to a sample obtained from local soil, and its main purpose is to obtain a microbial community specific to a particular region. Since the sample originates from the aforementioned region, the microbial community in the sample has the ability to adapt to the environment of that region. For example, field soil samples are obtained directly from the soil of the region or from the rhizosphere of plants in the region. From the above samples, a microbial community specific to the aforementioned region can be obtained. Furthermore, by taking samples from inside earthworms, it is possible to obtain microbial communities unique to that particular location.

[0054] As used in this text, the term "domestication of microorganisms" refers to cultivating microorganisms in a non-natural environment and growing the microbial community to a stable state of artificial expectation. A non-natural environment includes, but is not limited to, a biological reactor.

[0055] As used in this text, the term "microenvironmental parameters" generally refers to the numerical values ​​of various indicators in the natural environment, including, but not limited to, organic matter components, nutrient components, nutrient content, pH value, temperature, humidity, growing medium, initial microbial species and / or initial microbial content, as well as solid / liquid growing media such as wood chips, rice husks, soil, and other porous materials.

[0056] As used in this text, the term "soil fertility" generally refers to the soil's ability to support plant growth and development, particularly by providing necessary nutrients, water, gases, and physical structure, and promoting the healthy development of plant root systems. The higher the soil fertility, the more suitable the soil is for the growth of crops and / or plants.

[0057] As used in this text, the term "soil restoration" generally refers to restoring soil ecosystems damaged by human activities, natural disasters, and / or climatic factors, returning them to a natural or near-original state, and further improving the environment. This includes, for example, promoting biodiversity and increasing organic matter content.

[0058] The present invention will be further illustrated by the following examples and should not be interpreted as any further limitation in any form. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, apparatus and equipment used in the following examples are all commercially available.

[0059] Example 1: Soil restoration in a bitter melon cultivation area

[0060] In this specific example, a sample of local soil was obtained from the natural environment, and beneficial bacteria in the local soil were screened and cultured using the functional microbiome screening method according to the present invention. These bacteria were then introduced into the local soil, and changes in soil fertility were observed.

[0061] In this specific embodiment, after obtaining a soil sample from a barren bitter melon cultivation area (location: Xinshe District, Taichung City, Taiwan), the soil sample was subjected to heat treatment and filtration, cultured using the culture formulation of the present invention, and then spread onto a plate to observe the microbial community. Samples with a Bacillus subtilis content higher than 5% were screened and designated as the functional microbiome in this embodiment. Subsequently, the functional microbiome was cultured on a larger scale and applied to the barren cultivation area.

[0062] [Screening of functional microbiomes]

[0063] 1. Pretreatment of soil samples

[0064] 10 g of soil sample was added to 100 mL of physiological saline (0.85% NaCl + ddH2O) in an Erlenmeyer flask. The flask opening was sealed with aluminum foil, and each sample was heat-treated in a water bath apparatus at 60°C, 70°C, and 90°C for 30 minutes or 60 minutes, respectively. The heat-treated sample was subjected to gravity filtration. A funnel was placed over an Erlenmeyer flask, a folded filter paper was placed in the funnel, and the heat-treated sample was gradually poured along the edge of the filter paper until 50 mL of the sample filtrate was collected. Subsequently, the filtration device was removed to obtain the first bacterial suspension according to the present invention. The first bacterial suspension samples obtained under each heat treatment condition (treatment at 60°C for 60 minutes, treatment at 70°C for 60 minutes, treatment at 90°C for 60 minutes, treatment at 60°C for 30 minutes, treatment at 70°C for 30 minutes, and treatment at 90°C for 30 minutes) were identified using the spread plate method. The control group consisted of samples that had not undergone heat treatment. As shown in Figure 3, different heat treatment temperatures and times resulted in clear changes in the microbial community. Furthermore, different temperature and heating time conditions are applied to different local soil environments. In this example, when heat treatment was performed at 90°C, many contaminants in the sample were killed, resulting in a relatively simpler microbial community sample used for subsequent bacterial species culture.

[0065] 2. Bacterial species culture

[0066] The first bacterial suspension sample (1 mL) was taken using a pipette, added to 100 mL of the culture formulation, and incubated in an incubator at 30°C and 150 rpm for 24 hours to obtain the second bacterial suspension according to the present invention. The culture formulation includes at least one selected from the group consisting of glucose, glycerol, molasses, starch, arabinose, fructose, galactose, lactose, maltose, mannose, sucrose, peanut flour, soybean flour, corn gluten flour, beef extract, peptone, yeast flour, fish meal, urea, spent mycelium, distilled meal ammonium salt, nitrate, ammonia water, yeast extract, sodium chloride, magnesium chloride, sodium bicarbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, ammonium chloride, sodium citrate, sodium malate, sodium carbonate, and potassium carbonate.

[0067] In this specific example, the culture medium is an acidic medium containing 1-4 g / L of an inorganic nitrogen source selected from ammonium sulfate, ammonium nitrate, ammonium chloride, or a combination thereof; 10-25 g / L of a phosphate buffer system containing K2HPO4 and KH2PO4; 0.5-2 g / L of an organic acid salt selected from sodium citrate, sodium malate, or a combination thereof; 0.05-0.5 g / L of magnesium ions; and 3-10 g / L of a carbon source selected from glucose, sucrose, fructose, molasses, or a combination thereof.

[0068] 3. Spreadplate method identification

[0069] Second bacterial suspensions, obtained by culturing under several different conditions, were identified using the spread plate method. For the spread plate method, HIMEDIA Bacillus medium (HiCrome Bacillus Agar Base, purchased from HIMEDIA) was used, and the above bacterial suspensions were cultured at room temperature for 1 day. The control group consists of local microorganisms that have not undergone the bacterial species culture described in this invention. See Figure 4 for the results of coating the plate. In this embodiment, the color distribution and proportion of each petri dish were further calculated using an image analysis system. Different bacterial species form colonies of different colors, with the green colonies representing Bacillus subtilis. The calculation results are shown in Table 1 below. In the control group, Bacillus subtilis accounted for only 1.02% of the colony composition. However, in the cultured microbial group sample A, Bacillus subtilis accounted for 9.18% of the colony composition, satisfying the condition in this invention that "the content accounts for 5% or more." Therefore, microbial group sample A is a screened functional microbiome and will be used in subsequent experiments and in the soil restoration process. In contrast, Bacillus subtilis in cultured microbial sample B accounts for only 3.82% of the colony composition, and does not satisfy the condition defined in the screening method according to the present invention, which states that "the proportion of the content is 5% or more." Therefore, microbial sample B is not a functional microbiome according to the present invention, but is simply called a microbiome.

[0070] Table 1. Analysis of Bacillus subtilis in each microbial group sample. TIFF2026106438000002.tif85145

[0071] [Seed Germination Test] In this example, 38g of local soil was placed in each petri dish, and 2g each of the functional microbiome according to the present invention, a microbiome (sample with a Bacillus subtilis content of less than 5%), and a single bacterial strain (Bacillus subtilis) were added to each, followed by 15 seeds of bok choy in each dish. After 3 days of incubation, the seed germination rate of each group after application of the microbial community was observed. The control group consists of in-situ soil without added microbial communities.

[0072] See Table 2 and Figure 5 for experimental results. After three days of cultivation, only three seeds germinated in the control group, resulting in a germination rate of approximately 20%, indicating that the initial local soil was quite barren. On the other hand, in the group treated with the functional microbiome according to the present invention, 11 seeds germinated, and the germination rate reached approximately 73%, which was superior to the control group, the group treated with the microbiome, or the group treated with a single bacterial strain. This demonstrates that applying the functional microbiome obtained by the screening method according to the present invention to local soil improves seed germination rates and has a soil restoration effect.

[0073] Table 2. Germination rate test in soil from bitter melon cultivation area. TIFF2026106438000003.tif41145

[0074] [Restoration of local soil]

[0075] In this specific embodiment, the screened functional microbiome (microbial group sample A) was cultured in a bioreactor, and the environment of the local soil sample was simulated by controlling the microenvironmental parameters within the bioreactor. The aforementioned microenvironmental parameters include the components of the nutrient source, the nutrient source content, pH value, temperature, humidity, external force, and growth medium. This allowed for the domestication and large-scale cultivation of microorganisms within the aforementioned functional microbiome. In this specific example, the conditions for domesticating the microorganisms were as follows: pH value adjusted to 5.5-6.5, temperature adjusted to 28-35°C, shaking frequency set to 80-120 rpm, moisture content of culture medium controlled to 40-70% (w / w), relative humidity controlled to 40-80%, and cultured for approximately 48-72 hours.

[0076] In this specific embodiment, the domesticated functional microbiome was applied to the barren cultivation area. Referring to Figure 6A, before applying the functional microbiome obtained by the screening method according to the present invention, the soil in the cultivation area was quite barren, and plant growth was poor. However, referring to Figure 6B, three months after applying the functional microbiome obtained by the screening method according to the present invention, the soil fertility in the area clearly improved, and the plant growth also improved significantly. This indicates that the functional microbiome obtained by the screening method according to the present invention has a soil regeneration effect.

[0077] Example 2: Soil restoration in a Camellia japonica cultivation area

[0078] In this specific example, after obtaining local soil samples containing earthworms and substances metabolized by earthworms from a barren camellia cultivation area (location: Qiatonli, Hsinchu City, Taiwan), the soil samples were pre-treated and filtered, cultured using the culture formulation of the present invention, and then spread onto plates to observe the microbial community. Samples with a Bacillus subtilis content higher than 5% were screened and designated as the functional microbiome in this example. Subsequently, the functional microbiome was domesticated and cultured to expand its range, and the functional microbiome was then applied to the barren cultivation area. For the sample preparation, culture, screening, and domestication methods in this example, please refer to Example 1.

[0079] In this specific example, the culture formulation is a medium that is close to neutral and contains 3-10 g / L of a protein hydrolysate selected from tryptone, hydrolyzed soy protein, or a combination thereof; 1-5 g / L of yeast extract; 0-5 g / L of meat extract; 3-10 g / L of sodium chloride; and 0-5 g / L of a carbon source selected from glucose, molasses, glucose syrup, or a combination thereof.

[0080] [Seed Germination Test]

[0081] In this example, 38g of local soil was placed in each petri dish, and 2g each of the functional microbiome according to the present invention, a microbiome (sample with a Bacillus subtilis content of less than 5%), and a single bacterial strain (Bacillus subtilis) were added to each, and then 15 bok choy seeds were added to each. After 3 days of incubation, the seed germination rate of each group after application of the microbial community was observed. The control group consists of in-situ soil without added microbial communities.

[0082] The experimental results will be explained with reference to Table 3 and Figure 7. After three days of cultivation, only four seeds germinated in the control group, resulting in a germination rate of approximately 27%, indicating that the initial local soil was quite barren. On the other hand, in the group treated with the functional microbiome according to the present invention, 11 seeds germinated, and the germination rate reached approximately 73%, which was superior to the control group, the group treated with the microbiome, or the group treated with a single bacterial strain. This demonstrates that applying the functional microbiome obtained by the screening method according to the present invention to local soil improves seed germination rates and has a soil restoration effect.

[0083] Table 3. Germination rate tests in soil from Camellia japonica cultivation sites. TIFF2026106438000004.tif41145

[0084] [Restoration of local soil]

[0085] In this example, the screened functional microbiome was domesticated and cultured on a large scale, and then applied to the local soil. In this specific example, the conditions for domesticating the microorganisms were as follows: pH value adjusted to 6.8-7.6, temperature adjusted to 30-37°C, shaking frequency set to 80-120 rpm, aeration rate of 0.5-2 vvm supplied to the fermentation tank, moisture content of the culture medium controlled to 40-65% (w / w), relative humidity controlled to 40-70%, and cultured for approximately 24-48 hours.

[0086] Referring to Figure 8A, before applying the functional microbiome obtained by the screening method according to the present invention, the soil of the cultivation site was quite barren, lacked cover plants, and produced only a small amount of flowers and fruits. However, referring to Figure 8B, four months after applying the functional microbiome obtained by the screening method according to the present invention, the soil fertility of the area clearly improved, the vitality of cover plants recovered, plant growth was vigorous, and the production of flowers and fruits also increased significantly. This indicates that the functional microbiome obtained by the screening method according to the present invention has a soil regeneration effect.

[0087] Example 3: Soil restoration in a bok choy cultivation area

[0088] In this specific embodiment, after obtaining soil samples from a bok choy cultivation site affected by high salinity (location: Erlun Township, Yunlin County, Taiwan), the soil samples were pre-treated and filtered, cultured using the culture formulation of the present invention, and then spread onto plates to identify the microbial community. Samples with a Bacillus subtilis content higher than 5% were screened and designated as the functional microbiome in this embodiment. Subsequently, the functional microbiome was domesticated in a bioreactor, and the soil conditions in a high-salinity environment were simulated by adjusting the microenvironmental parameters of the bioreactor. The functional microbiome was domesticated for 3 to 5 days, then fixed and stored. When ready for use, it was cultured on a larger scale, and the domesticated functional microbiome was applied to the cultivation area affected by the high salinity, and the improvement in soil EC value and soil compaction was observed. For the sample preparation, culture, screening, and domestication methods in this example, please refer to Example 1.

[0089] In this specific example, the culture formulation is an alkaline medium containing 3-10 g / L of protein hydrolysate; 1-10 g / L of yeast extract; 0.5-3 g / L of phosphate; 0.05-0.5 g / L of magnesium ions; 5-15 g / L of a carbon source selected from glucose, starch, maltose, molasses, or a combination thereof; and 2-10 g / L of alkali salts selected from sodium carbonate, sodium bicarbonate, potassium carbonate, or a combination thereof.

[0090] In this specific example, the conditions for domesticating the microorganisms were as follows: pH value adjusted to 8.5-10.5, temperature adjusted to 30-42°C, shaking frequency set to 100-150 rpm, moisture content of culture medium controlled to 35-65% (w / w), relative humidity controlled to 40-70%, and cultured for approximately 4-48 hours.

[0091] In this example, the solid sample was preserved by sterilizing the wood shavings in an autoclave at 121°C for 30 minutes, followed by drying at 60°C. The aforementioned wood chips are used as a solid carrier. The fungal solution and wood shavings were uniformly mixed in a 1:2 (w / w) ratio, and then stored in a drying oven at 45°C.

[0092] Referring to Figure 9A, before applying the functional microbiome obtained by the screening method according to the present invention, the soil in the cultivation area had a considerably high EC value, a low seed germination rate, and severe soil compaction. However, as explained with reference to Figure 9B, three months after applying the functional microbiome obtained by the screening method according to the present invention, the EC value of the soil in the area clearly decreased, the seed germination rate increased, the soil compaction improved significantly, and plant yield increased by approximately 60%. This indicates that the functional microbiome obtained by the screening method according to the present invention has a soil regeneration effect.

[0093] In summary, the screening method according to the present invention performed screening using field samples. The microbial communities in the aforementioned field samples possess considerable adaptability to the region, and the screened and domesticated functional microbiomes do not cause problems by disrupting the balance of the local microbial community through the use of exotic bacteria.

[0094] Furthermore, this invention enhances the ability to adapt to different environments by replacing traditionally used single bacterial strains within the microbiome, thereby improving the survival rate of the bacterial species introduced into the area being restored and significantly increasing the success rate of environmental restoration. Furthermore, experiments have demonstrated that the functional microbiome screened by the screening method according to the present invention has effects such as improving seed germination rates, efficiently achieving soil restoration, and possessing broad application value in fields such as environmental restoration and agricultural development.

[0095] The present invention has been described with reference to the above-described examples. The above embodiments are merely selections of preferred embodiments of the present invention and are not intended to limit it. Equivalent variations or modifications made without departing from the spirit and scope of the present invention, given that a person skilled in the art has understood the aforementioned technical features and embodiments of the present invention, still fall within the scope encompassed by the present invention. Furthermore, the claims of the present invention must be limited to the scope set forth in the claims appended herein. [Explanation of symbols]

[0096] 11-14, 21-26: Step

Claims

1. To provide local soil samples, The first bacterial suspension is obtained by pre-treating and filtering the local soil sample. The first bacterial solution is cultured according to the culture formula to obtain the second bacterial solution, and The second bacterial solution is applied to a plate to measure the amount of Bacillus subtilis, and if the amount of Bacillus subtilis accounts for 5% or more of the total amount of all bacterial species, the second bacterial solution is the screened functional microbiome, including the following: A screening method for functional microbiomes.

2. The screening method according to claim 1, wherein the local soil sample contains earthworms and / or substances metabolized by earthworms.

3. The screening method according to claim 1, wherein the pretreatment includes heat treatment and / or treatment with an extractant.

4. The screening method according to claim 3, wherein the heat treatment involves adding the local soil sample to an extract and heating it, the heating temperature being 60 to 90°C, and the treatment time being 30 to 60 minutes.

5. The screening method according to claim 3, wherein the treatment with the extract is performed by adding the extract to the local soil sample and performing the extraction.

6. The screening method according to claim 4 or 5, wherein the extract comprises at least one selected from the group consisting of water, physiological saline, general culture medium, bacitracin-containing culture medium, culture supernatant of a single colony strain, salt solution, and metal ion solution.

7. The screening method according to claim 1, wherein the culture formulation comprises at least one selected from the group consisting of glucose, glycerol, molasses, starch, arabinose, fructose, galactose, lactose, maltose, mannose, sucrose, peanut flour, soybean flour, corn gluten flour, beef extract, peptone, yeast flour, fish meal, urea, spent mycelium, distilled meal ammonium salt, nitrate, ammonia water, yeast extract, sodium chloride, magnesium chloride, sodium bicarbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, ammonium chloride, sodium citrate, sodium malate, sodium carbonate, and potassium carbonate.

8. To provide a functional microbiome screened by the method described in claim 1, This involves domesticating microorganisms in the functional microbiome, culturing the functional microbiome in a bioreactor, and controlling the microenvironmental parameters within the bioreactor to simulate the environment of the local soil sample, and This includes introducing the domesticated functional microbiome into the local soil. Soil restoration methods.

9. The soil restoration method according to claim 8, wherein the microenvironmental parameters include the components of the nutrient source, the content of the nutrient source, the pH value, temperature, humidity, external force, and growing medium.

10. The soil restoration method according to claim 8, wherein the growing medium includes wood chips, rice husks, and soil.

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