Black soil prokaryote separation culture medium as well as preparation method and application thereof

Abstract

The invention discloses a black soil prokaryote separation culture medium as well as a preparation method and application thereof. The isolation medium is prepared from a solution A, a solution B and a solution C, wherein the solution A comprises a nitrogen source, glucose, soluble starch and sodium pyruvate; the solution B comprises the following components: nitrilotriacetic acid and inorganic salt; the solution C comprises the following components: vitamins, lipoic acid and para aminobenzoic acid. The separation culture medium is designed and developed based on a complex microbial community structure in black soil, the nutrient concentration of the separation culture medium is low, compared with a conventional culture medium, prokaryotes separated by the separation culture medium are more in variety and better in effect, and the separation culture medium has important value and significance in development and utilization of black soil microbial resources.

Description

Technical Field

[0001] The invention relates to the technical field of microbial separation and cultivation, and in particular to a black soil prokaryotic separation culture medium and a preparation method and application thereof. Background Art

[0002] Black soil is a black or dark humus soil layer with high organic matter content and rich nutrient content. As one of the main grain producing areas, the black soil area shoulders the important task of maintaining stable grain production and supply. However, highly intensive farming patterns and long-term unreasonable fertilization methods can easily lead to a series of ecological problems in black soil, such as soil erosion, soil hardening, and soil acidification. In related studies, soil microorganisms are one of the biological indicators of the soil health evaluation system. Transplanting soil or soil microorganisms and restoring the microbiome in the soil can help build a more diverse, stable and fully functional ecosystem. Among them, exploring the core microbial resources of black soil and rebuilding 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 the related technologies, the use of culture-free technologies such as metagenomics and metatranscriptomics is an important means to reveal the diversity and function of microbial communities, but obtaining pure culture of microorganisms is crucial for directly analyzing the metabolism and physiological functions of microorganisms, and is also the basis for the development and utilization of microbial resources. However, in traditional pure culture methods, since the nutritional conditions, temperature, humidity, etc. under laboratory conditions are different from the original ecological environment in which they grow, the types and quantities of microorganisms obtained by isolated culture are far lower than the actual amount of microorganisms contained in black soil, which limits the development and utilization of black soil microbial resources.

[0004] Therefore, in order to deeply explore the diversity of culturable microorganisms in black soil and obtain more pure cultures of microorganisms, it is crucial to develop an isolation culture medium specific to black soil. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a black soil prokaryotic separation medium and a preparation method and application thereof. The black soil prokaryotic separation medium of the present invention can separate more prokaryotic groups from the black soil, which can provide technical support for the development of black soil microbial resources.

[0006] The invention also provides a method for preparing a black soil prokaryotic separation culture medium.

[0007] The invention also provides a method for separating prokaryotes from black soil.

[0008] The invention also provides a black soil prokaryotic separation medium or a method for separating prokaryotes in black soil for application in separating prokaryotes.

[0009] In a first aspect of the present invention, a black soil prokaryotic separation medium is provided, wherein the raw materials for preparing the medium include solution A, solution B and solution C, wherein: The components of the solution A include: a nitrogen source, glucose, soluble starch, and sodium pyruvate; The components of the B solution include: nitrilotriacetic acid and an inorganic salt; The components of the C solution include vitamins, lipoic acid and p-aminobenzoic acid.

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

[0011] (2) The black soil prokaryotic organism separation medium of the present invention is also added with nitrilotriacetic acid, which can act as a chelating agent in the separation medium and combine with divalent metal ions (such as calcium, magnesium, zinc, etc.) in the culture medium, thereby helping to regulate the effective concentration of metal ions in the culture system, reduce the interference of high concentrations of 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 is also supplemented with p-aminobenzoic acid. On the one hand, since p-aminobenzoic acid 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 conventional culture media, the isolation medium of the present invention can separate more prokaryotes and has better effects, which is of great value and significance to the development and utilization of black soil microbial resources.

[0014] In some embodiments of the present invention, the nitrogen source in the components of the A solution 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 the B solution 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 vitamins in the C solution are selected from biotin, folic acid, pyridoxine hydrochloride, thiamine, niacin, pantothenic acid, vitamin B 12 At least one of .

[0017] In some embodiments of the present invention, the A solution includes 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 includes 1.0~2.0g / L nitrilotriacetic acid, 2.0~4.0g / L K2HPO4, 1.0~3.0g / L MgSO4, 0.2~1g / L MnSO4, 0.5~1.5g / L NaCl, 0.5~0.1g / L FeSO4, 0.05~0.15g / L CoCl2, 0.05~0.1g / L CaCl2, 0.05~0.12g / L ZnSO4, 0.005~0.01g / L CuSO4, 0.005~0.02g / L AlK(SO4)2, 0.005~0.02g / L H3BO3, and 0.005~0.01g / L NaMoO4.

[0019] In some embodiments of the present invention, the C solution includes 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, 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 the A solution also 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 organism isolation culture medium further include nystatin and nalidixic acid.

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

[0025] Adding nystatin and nalidixic acid to the culture medium of the present 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 solvent of the A solution, the B solution and the C solution comprises water.

[0027] The second aspect of the present invention provides a method for preparing a black soil prokaryotic organism separation medium, which is prepared by mixing the raw materials for preparing the black soil prokaryotic organism separation medium described in any one of the first aspects.

[0028] In some embodiments of the present invention, the preparation of the black soil prokaryotic organism isolation medium further includes a sterilization and / or disinfection step before the raw materials are mixed.

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

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

[0031] The third aspect of the present invention provides a method for isolating prokaryotes from black soil, comprising diluting a black soil sample and applying it to any one of the black soil prokaryotic isolation mediums described in the first aspect, culturing, and picking a single colony.

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

[0033] In some embodiments of the present invention, the dilution multiple 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 culturing time is 7 to 14 days.

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

[0037] The fourth aspect of the present invention provides the use of the black soil prokaryotic isolation medium described in any one of the first aspect or the method for isolating prokaryotes in black soil described in any one of the third aspect in isolating prokaryotes.

[0038] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The separation effect of the separation medium of Example 1 of the present invention is shown; Figure 2 These are the results of the test on the number of pure cultured bacteria isolated from different black soil prokaryotic isolation media. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

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

[0042] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

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

[0044] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0045] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0046] Example 1: Black soil prokaryotic solid separation medium The present embodiment provides a solid separation medium for separating prokaryotes from black soil and a preparation method thereof. The raw materials for preparing the solid separation medium include solution A, solution B and solution C, and the components of each solution are shown in Tables 1 to 3.

[0047] Table 1: Solution A components

[0048] Table 2: Solution B composition

[0049] Table 3: Solution C composition

[0050] After the preparation of solution A is completed, it is placed in a conical flask and sealed, and sterilized by high pressure at 121°C for 25 minutes for use; after the preparation of solution B and solution C, they are filtered and sterilized with a 0.22μm filter membrane respectively and set aside.

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

[0052] Example 2: Black soil prokaryotic separation method This embodiment provides a method for isolating prokaryotes from black soil, using the culture medium of the embodiment of the present invention to isolate and culture microorganisms, specifically comprising the following steps: (1) Culture medium preparation: Pour the solid separation medium prepared above for separating prokaryotes from black soil onto a plate, let it stand for one day to ensure that the plate solidifies and remove excess water on the surface to obtain a solid plate for later use.

[0053] (2) Gradient dilution and culture: Take about 5g of black soil sample, add it to a sterile conical flask containing 50mL of sterile water and 10 sterile glass beads, and shake it on a shaker at 180rpm / min for 30min to make a soil suspension. 1mL of this solution is recorded as 10 -1 and diluted to 10 with sterile water -5 Then take 10 -5 , 10 -4 , 10 -3 100 μL of each dilution was evenly spread on a solid plate and placed in an incubator at 10°C and 28°C for inversion culture for 7 to 14 days, and a single colony was picked for culture.

[0054] Comparative Example 1: 1 / 10 R2A solid culture medium This comparative example provides a solid separation medium (1 / 10 R2A solid medium) conventionally used for separating prokaryotes, and its preparation raw materials include the following components: Tryptone 0.025g / L, acid hydrolyzed casein 0.05g / L, yeast extract powder 0.05g / L, soluble starch 0.05g / L, potassium dihydrogen phosphate 0.03g / L, magnesium sulfate 0.01g / L, sodium pyruvate 0.03g / L, peptone 0.025g / L, glucose 0.05g / L, agar 18g / L.

[0055] The above-prepared raw materials were mixed and the volume was made up to 1 L with water, and then sterilized by high pressure at 121° C. for 25 minutes.

[0056] Comparative Example 2: 1 / 10 NA solid culture medium This comparative example provides another solid separation medium (1 / 10 NA solid medium) conventionally used for separating prokaryotes, and its preparation raw materials include the following components: Peptone 1.0g / L, beef extract 0.3g / L, NaCl 0.5g / L, agar 18g / L.

[0057] The above-prepared raw materials were mixed and the volume was made up to 1 L with water, and then sterilized by high pressure at 121° C. for 25 minutes.

[0058] Application Examples This application example uses the black soil of the Heilongjiang Farmland Conservation and Agricultural Environment Science Observation Experimental Station of the Ministry of Agriculture and Rural Affairs as a sample, and uses the prokaryotic solid separation medium prepared in Example 1 of the present invention and Comparative Examples 1-2 as a separation medium, and separates and cultivates microorganisms in the Microbial Resources and Ecology Laboratory of the School of Life Sciences of Sun Yat-sen University. The specific separation method is as follows: First, pour the solid separation medium prepared above for separating prokaryotes from black soil into plates, let it stand for one day to ensure that the plates solidify and remove excess water on the surface to obtain solid plates for use. Then take about 5g of the above black soil sample, add it to a sterile conical flask containing 50mL of sterile water and 10 sterile glass beads, and shake it on a shaker at 180rpm / min for 30min to prepare a soil suspension. 1mL of this solution is recorded as 10 -1 and diluted to 10 with sterile water -5 Then take 10 -5 , 10 -4 , 10 -3 100 μL of each dilution was evenly spread on different solid plates and placed in an incubator at 10°C and 28°C for 7 to 14 days. After the culture was completed, a single colony was picked and placed on a new solid separation medium for culture. A single colony was obtained and 16S rRNA gene sequencing was performed to count the types and numbers of pure cultured bacteria in the black soil sample, with three replicates per group.

[0059] The separation effect diagram of the black soil prokaryotic separation medium using Example 1 of the present invention is as follows Figure 1 As shown, the colonies on the separation plate are diverse in morphology and abundant in number, with good separation effect.

[0060] The results of the detection of 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, it is shown that the prokaryotic organism population isolated by the black soil prokaryotic organism isolation medium of Example 1 of the present invention is the largest, including more than 2000 bacterial strains from 5 phyla and 127 genera. In addition to the common Actinobacteria and Pseudomonas, they also include Bacillus, Bacteroidetes and Deinococcus, which account for 56.26%, 22.57%, 19.95%, 0.87% and 0.34% of the total number of isolated strains, respectively, and more than 70 of the isolated strains are potential new taxa, 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] About 550 strains were isolated and identified on the comparative example 1 / 10 R2A solid culture medium, distributed in 3 phyla (Actinobacteria, 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] About 400 strains were isolated on the comparative example 1 / 10 NA medium, distributed in 3 phyla (Actinobacteria, Pseudomonas and Bacillus) and 70 genera, of 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 medium

[0066] Based on the above-mentioned detection results of the number of pure cultured bacteria separated from black soil samples using different culture media, it can be analyzed that compared with conventional separation culture media, the solid separation culture medium for separating prokaryotes in black soil of the present invention can separate more prokaryotic species and has a better separation effect, which is of great significance for the development of black soil microbial resources.

[0067] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A black soil prokaryotic separation medium, characterized in that: The preparation raw materials include solution A, solution B and solution C, wherein: The components of the solution A include: a nitrogen source, glucose, soluble starch, and sodium pyruvate; The components of the B solution include: nitrilotriacetic acid and an inorganic salt; The components of the C solution include vitamins, lipoic acid and p-aminobenzoic acid.

2. The black soil prokaryotic separation medium according to claim 1, characterized in that: The nitrogen source in the components of the A solution is selected from at least one of peptone, yeast extract, and acid-hydrolyzed casein; And / or, the inorganic salt in the B solution is selected from at least one of K2HPO4, MgSO4, MnSO4, NaCl, FeSO4, CoCl2, CaCl2, ZnSO4, CuSO4, AlK(SO4)2, H3BO3, and NaMoO4; And / or, the vitamins in the C solution are selected from biotin, folic acid, pyridoxine hydrochloride, thiamine, niacin, pantothenic acid, vitamin B 12 At least one of .

3. The black soil prokaryotic separation medium according to claim 2, characterized in that: The A solution includes 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; And / or, the B solution includes 1.0-2.0 g / L nitrilotriacetic 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; And / or, the C solution includes 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, 0.001-0.01 g / L vitamin B 12 , 0.005~0.025g / L lipoic acid, 0.005~0.025g / L p-aminobenzoic acid.

4. The black soil prokaryotic separation medium according to any one of claims 1 to 3, characterized in that: The components of the A solution also include agar; Preferably, the concentration of the agar is 16-20 g / L.

5. The black soil prokaryotic separation medium according to claim 4, characterized in that: The volume ratio of the A solution, the B solution and the C solution is 80-120:1:0.05-0.

2.

6. The black soil prokaryotic separation medium according to claim 5, characterized in that: The raw materials for preparing the black soil prokaryotic organism separation culture medium also include nystatin and nalidixic acid; Preferably, the final concentration of the nystatin and the nalidixic acid is 40-60 mg / L.

7. A method for preparing a black soil prokaryotic separation medium, characterized in that: The preparation method is obtained by mixing the raw materials for preparing the black soil prokaryotic organism separation medium according to any one of claims 1 to 6.

8. A method for separating prokaryotes from black soil, characterized in that: The black soil sample is diluted and applied to the black soil prokaryotic isolation culture medium according to any one of claims 1 to 6, cultured, and a single colony is picked.

9. The method according to claim 8, characterized in that: The culture temperature is 10-28° C.; and / or the culture time is 7-14 days.

10. Use of the black soil prokaryotic separation medium according to any one of claims 1 to 6 or the method for separating prokaryotes from black soil according to any one of claims 8 to 9 in separating prokaryotes.

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