A black soil prokaryote separation culture medium, and a preparation method and application thereof

Abstract

The application discloses a black soil prokaryote separation culture medium and a preparation method and application thereof. The preparation raw materials of the separation culture medium include A solution, B solution and C solution, wherein components of the A solution include a nitrogen source, glucose, soluble starch and sodium pyruvate; components of the B solution include nitrilotriacetic acid and inorganic salt; and components of the C solution include vitamins, thioctic acid and p-aminobenzoic acid. The separation culture medium is designed and developed based on a complex microbial community structure in black soil. Compared with conventional culture media, the separation culture medium has lower nutrient concentration, can separate more prokaryote species and has better effect, and has important value and significance for development and utilization of black soil microbial resources.

Description

Technical Field

[0001] This invention relates to the field of microbial isolation and culture technology, and in particular to a black soil prokaryotic organism isolation culture medium, its preparation method and application. Background Technology

[0002] Black soil is a black or dark-colored humus-rich soil layer, characterized by its high organic matter and nutrient content. As one of the major grain-producing areas, black soil regions bear the heavy responsibility of maintaining stable grain production and supply. However, highly intensive farming practices and long-term unreasonable fertilization methods easily lead to a series of ecological problems in black soil, such as soil erosion, soil compaction and hardening, and soil acidification. In related research, soil microorganisms are one of the biological indicators in soil health assessment systems. Transplanting soil or soil microorganisms and restoring the soil microbiome helps to build a more diverse, stable, and functional ecosystem. Furthermore, exploring the core microbial resources native to black soil and reconstructing the structure and function of the black soil microbial community can help more effectively solve the problem of black soil degradation and promote sustainable agricultural development.

[0003] Among related technologies, culture-free techniques such as metagenomics and metatranscriptomics are important means to reveal the diversity and function of microbial communities. However, obtaining pure cultures of microorganisms is crucial for directly analyzing their metabolism and physiological functions, and is also the foundation for developing and utilizing microbial resources. However, in traditional pure culture methods, due to the difference between the nutritional conditions, temperature, and humidity in the laboratory and their original ecological environment, the types and quantities of microorganisms obtained are far lower than the actual microbial content in black soil, thus limiting the development and utilization of black soil microbial resources.

[0004] Therefore, in order to explore the diversity of culturable microorganisms in black soil and obtain more pure cultures of microorganisms, it is essential to develop an isolation culture medium specifically for black soil. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a black soil prokaryotic organism isolation culture medium, its preparation method, and its application. Using this black soil prokaryotic organism isolation culture medium, more prokaryotic groups can be isolated from black soil, providing technical support for the development of black soil microbial resources.

[0006] This invention also proposes a method for preparing a prokaryotic isolation culture medium in black soil.

[0007] This invention also proposes a method for isolating prokaryotes from black soil.

[0008] This invention also proposes a black soil prokaryotic isolation culture medium or a method for isolating prokaryotes from black soil, which is applied in the isolation of prokaryotes.

[0009] In a first aspect, the present invention provides a black soil prokaryotic organism isolation culture medium, the raw materials for which preparation includes solution A, solution B and solution C, wherein: The components of solution A include: nitrogen source, glucose, soluble starch, and sodium pyruvate; The components of solution B include: hyponitrotriacetic acid and inorganic salts; The components of solution C include: vitamins, lipoic acid, and para-aminobenzoic acid.

[0010] The black soil prokaryotic organism isolation culture medium according to embodiments of the present invention has at least the following beneficial effects: (1) In this invention, sodium pyruvate is added as a carbon source. Sodium pyruvate is a carbon source and an energy source. It can be metabolized by microorganisms into pyruvate and then participate in various biochemical reactions. When added to the prokaryotic isolation culture medium, it helps to improve the growth of prokaryotes through synergy with other components, especially those strains that can use pyruvate as the main nutrient source. In addition, it also helps to enrich the types of isolated prokaryotes.

[0011] (2) The black soil prokaryotic separation culture medium of the present invention also contains nitric acid triacetic acid, which can act as a chelating agent in the separation culture medium and combine with divalent metal ions (such as calcium, magnesium, zinc, etc.) in the culture medium. This helps to regulate the effective concentration of metal ions in the culture system, reduce the interference of high concentration metal ions on the growth of prokaryotes, and help stabilize the culture environment.

[0012] (3) The black soil prokaryotic isolation culture medium of the present invention also contains p-aminobenzoic acid. On the one hand, since it is one of the precursors of folic acid, adding a certain amount of p-aminobenzoic acid to the culture system is beneficial to promoting the healthy growth and metabolic activities of microorganisms. On the other hand, adding a certain amount of p-aminobenzoic acid helps to improve the selectivity of strains, such as improving the selectivity of strains that are dependent on p-aminobenzoic acid.

[0013] (4) The black soil prokaryotic isolation medium of the present invention is designed and developed for the complex microbial community structure in black soil. The nutrient concentration of the isolation medium is low and belongs to the oligotrophic level. Compared with the conventional medium, the isolation medium of the present invention isolates more types of prokaryotes and has better effect, which has important value and significance for the development and utilization of black soil microbial resources.

[0014] In some embodiments of the present invention, the nitrogen source in the components of solution A is selected from at least one of peptone, yeast extract, and acid-hydrolyzed casein.

[0015] In some embodiments of the present invention, the inorganic salt in solution B is selected from at least one of K2HPO4, MgSO4, MnSO4, NaCl, FeSO4, CoCl2, CaCl2, ZnSO4, CuSO4, AlK(SO4)2, H3BO3, and NaMoO4.

[0016] In some embodiments of the present invention, the vitamin in solution C is selected from biotin, folic acid, pyridoxine hydrochloride, thiamine, niacin, pantothenic acid, and vitamin B1. 12 At least one of them.

[0017] In some embodiments of the present invention, solution A comprises 0.01~0.1 g / L peptone, 0.01~0.1 g / L yeast extract, 0.01~0.1 g / L acid-hydrolyzed casein, 0.01~0.1 g / L glucose, 0.01~0.1 g / L soluble starch, and 0.01~0.05 g / L sodium pyruvate.

[0018] In some embodiments of the present invention, the B solution comprises 1.0~2.0 g / L nitrotriacetic acid, 2.0~4.0 g / L K2HPO4, 1.0~3.0 g / L MgSO4, 0.2~1 g / L MnSO4, 0.5~1.5 g / L NaCl, 0.5~0.1 g / L FeSO4, 0.05~0.15 g / L CoCl2, 0.05~0.1 g / L CaCl2, 0.05~0.12 g / L ZnSO4, 0.005~0.01 g / L CuSO4, 0.005~0.02 g / L AlK(SO4)2, 0.005~0.02 g / L H3BO3, and 0.005~0.01 g / L NaMoO4.

[0019] In some embodiments of the present invention, solution C comprises 0.001~0.01 g / L biotin, 0.001~0.01 g / L folic acid, 0.01~0.04 g / L pyridoxine hydrochloride, 0.005~0.025 g / L thiamine, 0.005~0.025 g / L niacin, 0.005~0.025 g / L pantothenic acid, and 0.001~0.01 g / L vitamin B. 12 0.005~0.025g / L lipoic acid, 0.005~0.025g / L p-aminobenzoic acid.

[0020] In some embodiments of the present invention, the components of solution A further include agar.

[0021] In some embodiments of the present invention, the concentration of the agar is 16-20 g / L.

[0022] In some embodiments of the present invention, the volume ratio of solution A, solution B and solution C is 80~120:1:0.05~0.2.

[0023] In some embodiments of the present invention, the raw materials for preparing the black soil prokaryotic isolation culture medium also include nystatin and nalidixic acid.

[0024] In some embodiments of the present invention, the final concentrations of the nystatin and the nalidixic acid are 40-60 mg / L.

[0025] Adding nystatin and nalidixic acid to the culture medium of this invention helps to inhibit the growth of non-target microbial groups (such as fungi and Gram-negative bacteria).

[0026] In some embodiments of the present invention, the solvents of solutions A, B, and C include water.

[0027] In a second aspect, the present invention provides a method for preparing a black soil prokaryotic organism separation culture medium, wherein the preparation raw materials of the black soil prokaryotic organism separation culture medium described in any one of the first aspects are mixed to obtain the culture medium.

[0028] In some embodiments of the present invention, the preparation of the black soil prokaryotic organism isolation culture medium includes sterilization and / or sterilization steps before mixing the raw materials.

[0029] In some embodiments of the present invention, the sterilization step specifically includes: sterilizing the A solution at 115~121°C for 15~30 min.

[0030] In some embodiments of the present invention, the sterilization step specifically includes: filtration sterilization of the B solution and the C solution; Preferably, the filtration sterilization includes filtration using a 0.22 μm filter membrane.

[0031] In a third aspect, the present invention provides a method for isolating prokaryotes from black soil, wherein a black soil sample is diluted and spread on the black soil prokaryotic isolation culture medium described in any one of the first aspects, cultured, and a single colony is picked.

[0032] In some embodiments of the present invention, the diluted solvent includes water, preferably distilled water.

[0033] In some embodiments of the present invention, the dilution factor can be adjusted according to actual conditions; specifically, it can be 10. -2 ~10 -7 Preferably, it can be 10 -2 10 -3 10 -4 10 -5 wait.

[0034] In some embodiments of the present invention, the culture temperature is 10~28°C.

[0035] In some embodiments of the present invention, the culture time is 7 to 14 days.

[0036] In some embodiments of the present invention, the step of picking a single colony also includes strain identification.

[0037] In a fourth aspect of the present invention, the application of the black soil prokaryotic isolation culture medium described in any one of the first aspects or the method for isolating prokaryotes from black soil described in any one of the third aspects is provided in the isolation of prokaryotes.

[0038] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 To demonstrate the separation effect of the separation culture medium in Example 1 of this invention; Figure 2 Results of detecting the number of pure cultured bacteria isolated from different black soil prokaryotic isolation media. Detailed Implementation

[0040] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0041] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0042] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0043] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0044] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0045] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0046] Example 1: Solid-state culture medium for separating prokaryotic organisms in black soil This embodiment provides a solid separation culture medium for separating prokaryotes from black soil and its preparation method. The raw materials for preparing the solid separation culture medium include solution A, solution B and solution C, and the composition of each solution is shown in Tables 1 to 3.

[0047] Table 1: Components of Solution A

[0048] Table 2: Components of Solution B

[0049] Table 3: Components of Solution C

[0050] After preparing solution A, place it in an Erlenmeyer flask and seal it. Autoclave at 121°C for 25 minutes and set aside. After preparing solutions B and C, filter them through a 0.22μm filter membrane for sterilization and set aside.

[0051] The sterilized or disinfected solutions A, B, and C were mixed in a volume ratio of 100:1:0.1, and then nystatin and nalidixic acid, each with a final concentration of 50 mg / L, were added to obtain the final solution.

[0052] Example 2: Method for Isolating Prokaryotes from Black Soil This embodiment provides a method for isolating prokaryotes from black soil, using the culture medium of this invention for microbial isolation and culture, specifically including the following steps: (1) Culture medium preparation: Pour the prepared solid isolation medium for separating prokaryotes from black soil into plates, let stand for one day to ensure the plates solidify and remove excess moisture from the surface, and obtain solid plates for later use.

[0053] (2) Serial dilution and culture: Take approximately 5g of black soil sample and add it to a sterile Erlenmeyer flask containing 50mL of sterile water and 10 sterile glass beads. Shake on a shaker at 180rpm / min for 30min to prepare a soil suspension. Record 1mL of this solution as 10. -1 and serially diluted with sterile water to 10. -5 Then take 10 in sequence. -5 10 -4 10 -3 Each dilution of 100 μL was evenly spread on a solid plate and incubated upside down in incubators at 10℃ and 28℃ for 7-14 days. Single colonies were then picked and cultured to obtain the final product.

[0054] Comparative Example 1: 1 / 10 R2A solid culture medium This comparative example provides a conventional solid isolation medium (1 / 10 R2A solid medium) for isolating prokaryotes, the raw materials of which include the following components: Tryptone 0.025 g / L, acid-hydrolyzed casein 0.05 g / L, yeast extract 0.05 g / L, soluble starch 0.05 g / L, potassium dihydrogen phosphate 0.03 g / L, magnesium sulfate 0.01 g / L, sodium pyruvate 0.03 g / L, peptone 0.025 g / L, glucose 0.05 g / L, agar 18 g / L.

[0055] After mixing the above-mentioned raw materials, dilute the volume to 1L with water and autoclave at 121℃ for 25 minutes to obtain the final product.

[0056] Comparative Example 2: 1 / 10 NA solid culture medium This comparative example provides another commonly used solid isolation medium (1 / 10 NA solid medium) for isolating prokaryotes, which is prepared from the following components: Peptone 1.0 g / L, beef extract 0.3 g / L, NaCl 0.5 g / L, agar 18 g / L.

[0057] After mixing the above-mentioned raw materials, dilute the volume to 1L with water and autoclave at 121℃ for 25 minutes to obtain the final product.

[0058] Application examples This application example uses black soil from the Heilongjiang Farmland Conservation and Agricultural Environment Science Observation and Experiment Station of the Ministry of Agriculture and Rural Affairs as the sample. The prokaryotic solid isolation culture medium prepared in Example 1 and Comparative Examples 1-2 of this invention were used as the isolation culture medium. Microbial isolation and culture were conducted in the Microbial Resources and Ecology Laboratory of the School of Life Sciences, Sun Yat-sen University. The specific isolation method is as follows: First, pour the prepared solid isolation medium for separating prokaryotes from black soil into plates and let them stand for one day to ensure solidification and remove excess moisture from the surface, obtaining solid plates for later use. Then, take about 5g of the black soil sample and add it to a sterile Erlenmeyer flask containing 50mL of sterile water and 10 sterile glass beads. Shake on a shaker at 180rpm / min for 30min to prepare a soil suspension. Record 1mL of this solution as 10. -1 and serially diluted with sterile water to 10. -5 Then take 10 in sequence. -5 10 -4 10 -3 100 μL of each dilution was evenly spread onto different solid plates and incubated upside down in incubators at 10℃ and 28℃ for 7–14 days, respectively. After incubation, single colonies were picked and placed on new solid isolation medium for further incubation. Single colonies were obtained and identified by 16S rRNA gene sequencing to count the species and number of pure cultured bacteria in the black soil samples. Each group was replicated in triplicate.

[0059] The separation effect of the black soil prokaryotic organism separation medium of Example 1 of this invention is shown in the figure below. Figure 1 As shown, the colonies on the isolation plate exhibit diverse morphologies and abundant numbers, demonstrating a good isolation effect.

[0060] The results of detecting the number of pure cultured bacteria isolated from black soil samples using different culture media of the present invention are as follows: Figure 2As shown, the black soil prokaryotic isolation medium of Example 1 of the present invention has the largest population of prokaryotes, containing more than 2,000 bacterial strains from 5 phyla and 127 genera. In addition to the common Actinobacteria and Pseudomonas phyla, it also includes Bacillus, Bacteroidetes and Heterococcus, which account for 56.26%, 22.57%, 19.95%, 0.87% and 0.34% of the total number of isolated strains, respectively. Moreover, more than 70 of the isolated strains are potential new taxonomic units, as shown in Example 1 in Table 4.

[0061] Table 4: Summary of some new strains isolated using the culture medium of Example 1

[0062] Approximately 550 strains were isolated and identified on comparative 1 / 10 R2A solid medium, distributed across 3 phyla (Actinomycetes, Pseudomonas, and Bacillus) and 74 genera, of which 15 strains were potential new species, as shown in Table 5.

[0063] Table 5: Summary of some potential new taxa isolated from 1 / 10 R2A culture medium

[0064] Approximately 400 strains were isolated using comparative 1 / 10 NA medium, distributed across 3 phyla (Actinomycetes, Pseudomonas, and Bacillus) and 70 genera, among which 9 strains were potential new species, as shown in Table 6.

[0065] Table 6: Summary of some potential new taxa isolated from 1 / 10 NA culture medium

[0066] Based on the above results of detecting the number of pure cultured bacteria isolated from black soil samples using different culture media, it can be concluded that, compared with conventional isolation media, the solid isolation media of the present invention for isolating prokaryotes in black soil can separate more types of prokaryotes and achieve better isolation results, which is of great significance for the development of black soil microbial resources.

[0067] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A culture medium for isolating prokaryotic organisms from black soil, characterized in that: The raw materials for preparation include solution A, solution B, and solution C, wherein: Solution A consists of 0.01-0.1 g / L peptone, 0.01-0.1 g / L yeast extract, 0.01-0.1 g / L acid-hydrolyzed casein, 0.01-0.1 g / L glucose, 0.01-0.1 g / L soluble starch, 0.01-0.05 g / L sodium pyruvate, 16-20 g / L agar, and water. Solution B consists of 1.0–2.0 g / L hypozonotriacetic acid, 2.0–4.0 g / L K₂HPO₄, 1.0–3.0 g / L MgSO₄·7H₂O, 0.2–1 g / L MnSO₄·2H₂O, 0.5–1.5 g / L NaCl, 0.5–0.1 g / L FeSO₄·7H₂O, 0.05–0.15 g / L CoCl₂, 0.05–0.1 g / L CaCl₂·2H₂O, 0.05–0.12 g / L ZnSO₄, 0.005–0.01 g / L CuSO₄·5H₂O, 0.005–0.02 g / L AlK(SO₄)₂, 0.005–0.02 g / L H₃BO₃, and 0.005–0.01 g / L... Composed of Na2MoO4·2H2O and water; Solution C consists of 0.001–0.01 g / L biotin, 0.001–0.01 g / L folic acid, 0.01–0.04 g / L pyridoxine hydrochloride, 0.005–0.025 g / L thiamine, 0.005–0.025 g / L niacin, 0.005–0.025 g / L pantothenic acid, and 0.001–0.01 g / L vitamin B. 12 It consists of 0.005~0.025 g / L lipoic acid, 0.005~0.025 g / L p-aminobenzoic acid, and water; The volume ratio of solutions A, B, and C is 80-120:1:0.05-0.2; the raw materials for preparing the black soil prokaryotic organism isolation culture medium also include nystatin and naphthidone acid, and the final concentrations of nystatin and naphthidone acid are both 40-60 mg / L.

2. A method for preparing a prokaryotic isolation culture medium from black soil, characterized in that: The raw materials for preparing the black soil prokaryotic organism isolation culture medium according to claim 1 are mixed together to obtain the medium.

3. A method for isolating prokaryotes from black soil, characterized in that: The black soil sample is diluted and spread on the black soil prokaryotic isolation culture medium described in claim 1, cultured, and a single colony is picked.

4. The method according to claim 3, characterized in that: The culture temperature is 10~28℃; and / or the culture time is 7~14 days.

5. The application of the black soil prokaryotic isolation culture medium as described in claim 1 or the method for isolating prokaryotes from black soil as described in any one of claims 3 to 4 in the isolation of prokaryotes.

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