A method for isolating and cultivating new species of difficult-to-cultivate microorganisms

Abstract

The present invention discloses a method for isolating and culturing new, difficult-to-cultivate microbial species. A 6-dimethylaminopurine (6-DMAP) solution, sterilized by filtration through a PTFE filter, is added to a sterilized, liquid agar medium to a final 6-DMAP concentration of 1-3 μg / mL. The solution is then solidified to obtain a solid medium, which is then inoculated with a natural sample for cultivation. The method is highly efficient in isolating new microbial species, with approximately 50% of pure cultures yielding potential new species.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for separating and culturing new species of difficult-to-cultivate microorganisms. Background Art

[0002] New microbial resources obtained from diverse environments are a reliable source for natural product discovery. These diverse and biotechnologically important natural products include antibiotics for therapeutic purposes, biopesticides and biofertilizers in agriculture, secondary metabolites for bioremediation, and biofuels. Pure cultures of novel microbial strains are crucial for the efficient discovery of these valuable natural products.

[0003] Despite this, the majority of microorganisms in the environment cannot be cultured in standard culture media. Culturable cells in natural samples only account for approximately 1% of the total. Most bacterial species are difficult to isolate using standard isolation techniques because, compared to other fast-growing bacteria (such as Pseudomonas spp. or Bacillus spp., which are easily isolated under laboratory conditions), these difficult-to-isolate bacterial species are often less adaptable to standard culture conditions.

[0004] The prior art has disclosed a variety of separation techniques to increase the diversity of cultured microorganisms, such as physically separating cells to modify growth conditions to avoid competition or reduce inhibitors of microbial growth (Connon and Giovannoni, 2002; Zengler et al., 2002), simulating natural environments (Kaeberlein et al., 2002; Nichols et al., 2010), extending culture times (Davis et al., 2005), reducing nutrient concentrations (Janssen et al., 2002), and using alternative gelling agents (Tamaki et al., 2005). The application of these methods has made it possible to cultivate some previously difficult-to-cultivate microbial species. However, the majority of microorganisms remain uncultivable. Most of these methods require specialized materials and complex procedures, making them difficult to apply on a large scale in laboratory and commercial settings. For example, the Connon and Giovannoni method requires expensive equipment and advanced techniques (e.g., microscopic observation or fluorescence-activated cell sorting (FACS), while simulating natural environments requires the use of specialized materials and complex equipment. Other methods among these are relatively simple but cannot be commercialized due to low screening efficiency. For example, Tamaki et al. used gellan gum as an alternative gelling agent. Although the application is not complicated, it has not been widely used due to its low efficiency.

[0005] In summary, existing standard culture techniques lack the necessary chemical factors for the growth of many new species of difficult-to-culture microorganisms. Therefore, it is particularly important to develop new methods for isolating and culturing difficult microorganisms. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a method for isolating and culturing a new species of difficult-to-cultivate microorganisms.

[0007] Another object of the present invention is to provide the use of 6-DMAP in preparing a culture medium for new species of difficult-to-cultivate microorganisms.

[0008] Another object of the present invention is to provide a culture medium for new species of difficult-to-culture microorganisms.

[0009] The technical solutions of the present invention are as follows:

[0010] A method for isolating and culturing new species of difficult-to-cultivate microorganisms comprises adding a 6-DMAP (6-dimethylaminopurine) solution sterilized by filtration through a PTFE filter to a sterilized liquid agar medium to a final 6-DMAP concentration of 1-3 μg / mL. The solution is then solidified to obtain a solid medium, and a natural sample is inoculated into the solid medium for cultivation.

[0011] In a preferred embodiment of the present invention, the final concentration of 6-DMAP is 2-3 μg / mL.

[0012] More preferably, the final concentration of 6-DMAP is 2.5 μg / mL.

[0013] Another technical solution of the present invention is as follows:

[0014] Application of 6-DMAP in the preparation of culture medium for new species of difficult-to-cultivate microorganisms.

[0015] In a preferred embodiment of the present invention, the final concentration of 6-DMAP is 1-3 μg / mL.

[0016] More preferably, the final concentration of 6-DMAP is 2-3 μg / mL.

[0017] More preferably, the final concentration of 6-DMAP is 2.5 μg / mL.

[0018] Yet another technical solution of the present invention is as follows:

[0019] A culture medium for new species of difficult-to-culture microorganisms comprises an agar culture medium containing 6-DMAP with a final concentration of 1-3 μg / mL.

[0020] In a preferred embodiment of the present invention, the final concentration of 6-DMAP is 2-3 μg / mL.

[0021] More preferably, the final concentration of 6-DMAP is 2.5 μg / mL.

[0022] The beneficial effects of the present invention are:

[0023] 1. Compared with the previous standard method, the present invention is simple to operate, economical and has a wide range of applications.

[0024] 2. The efficiency of isolating new microbial species resources of the present invention is very high, and about 50% of the strains obtained by culture are potential new species. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation process of Example 1 of the present invention.

[0026] Figure 2 This is a diagram showing the experimental results of Example 2 of the present invention.

[0027] Figure 3 This is a diagram of the experimental results of Example 3 of the present invention.

[0028] Figure 4 This is one of the experimental result diagrams of Example 4 of the present invention.

[0029] Figure 5 This is the second diagram of the experimental results of Example 4 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0031] Example 1

[0032] like Figure 1 As shown, the preparation method of the culture medium for new species of difficult-to-cultivate microorganisms of the present invention comprises:

[0033] (1) Filter the 6-DMAP solution through a PTFE filter membrane with a pore size of 0.2 μm;

[0034] (2) sterilizing the agar medium under high pressure;

[0035] (3) Add the filtered 6-DMAP solution to the autoclaved agar medium at 50°C and mix thoroughly; then cool naturally to room temperature and wait for the agarose to solidify.

[0036] Example 2

[0037] The Bacteroidetes and Proteobacteria involved in this embodiment are: NBU2953 (its 16S rRNA sequence has a similarity of 97% to the closest known type strain Erythromicrobium ramosum), NBU2952 (its 16S rRNA sequence has a similarity of 97% to the closest known type strain Pseudooceanicola nitratireducens), NBU2954 (its 16S rRNA sequence has a similarity of 97% to the closest known type strain Formosa maritima), NBU2956 (its 16S rRNA sequence has a similarity of 97% to the closest known type strain Marinicella sediminis), and NBU2955 (its 16S rRNA sequence has a similarity of 97% to the closest known type strain Hyphobacterium vulgare).

[0038] In this example, the above Bacteroides and Proteobacteria were used to optimize the optimal concentration of 6-DMAP in the culture medium of the new species of difficult-to-culture microorganisms prepared in Example 1.

[0039] Environmental samples were cultured using the culture medium prepared in Example 1 to screen for the aforementioned Bacteroidetes and Proteobacteria. The effects of different concentrations of 6-DMAP were examined by calculating the colony formation efficiency ratios of the culture medium containing 6-DMAP and the culture medium without 6-DMAP for each microorganism. Specifically, each strain was cultured in 5 mL of a 1:10 diluted R2A medium (10% of the recommended concentration, Hopebio, Qingdao, China) containing 2% artificial sea salt for 3-5 days (cells were harvested at the onset of the stationary phase). The harvested strains were diluted with artificial seawater (1:100) and then cultured at 5°C for 3 days to reduce microbial activity. The liquid cultures of varying dilutions were then inoculated in triplicate onto the culture medium for new species of difficult-to-cultivate microorganisms prepared in Example 1 (1:10 diluted R2A agar medium containing 6-DMAP at final concentrations of 0.25 μg / mL, 2.5 μg / mL, and 25 μg / mL) and the corresponding agar medium without 6-DMAP. After culturing at 26°C for 5 days, count the CFU on each agar plate. Select the dilution that produces 30-500 CFU (colony forming units) for colony counting. For different applied concentrations of 6-DMAP, determine the ratio of the number of colonies between the culture medium containing and without 6-DMAP. Figure 2 As shown, except for NBU2952, most strains showed the highest efficiency under condition b (final 6-DMAP concentration of 2.5 μg / mL).

[0040] Example 3

[0041] In this example, Escherichia coli and Pseudomonas aeruginosa were used to detect the effect of the culture medium for the new species of difficult-to-cultivate microorganisms prepared in Example 1, which corresponds to Example 2, on the growth of microorganisms.

[0042] The two test strains were cultured in 5 mL of 1:10 diluted R2A medium containing 2% artificial sea salt (10% of the recommended concentration, Hopebio, Qingdao, China) at 26°C for 3-5 days (cells were collected at the beginning of the stationary phase). The collected strains were diluted with PBS (phosphate buffered saline) and then cultured at 5°C for 3 days to reduce microbial activity. The liquid cultures of different dilutions were then inoculated in triplicate into the following culture media: 1:10 diluted R2A agar medium (6-DMAP final concentration was 2.5 μg / mL) and the corresponding agar medium without 6-DMAP. After culturing at 26°C for 5 days, the CFU of each agar plate was calculated. For each test strain, the ratio of the number of colonies between the culture media containing and not containing 6-DMAP was determined. The results are shown in Figure 2. Figure 3 As shown, adding a specific concentration of 6-DMAP to the culture medium can improve the growth ability of the tested strains.

[0043] Example 4

[0044] In this example, the culture medium for the new species of difficult-to-cultivate microorganisms prepared in Example 1, corresponding to Example 2, was used to culture environmental samples. The basic culture medium used in this example was diluted R2A medium (1:10). The isolation and culture method using 6-DMAP was applied to the following environmental samples: marine sediments, seawater, lake water, and soil. The proportion of new strains (whose 16S rRNA sequence had a similarity of 97% or less to the closest known model strain) obtained using this method was compared with the corresponding results using a standard culture method (without 6-DMAP) to test the efficiency of this example's method.

[0045] To prepare inoculum for marine sediment and soil samples, 1 g of sample was placed in a 15 mL conical tube containing 10 mL of sterile artificial seawater (for marine sediment samples) or PBS (for soil samples) and then homogenized using a vortex machine for 10 min. Seawater and lake water samples were used directly as inoculum. Each sample was diluted 10 mL with sterile artificial seawater or PBS. -2 to 10 -5 , and then inoculated on agar medium.

[0046] To examine the microbial colony formation in the presence and absence of 6-DMAP, the number of bacteria in each sample was counted under a microscope and compared on agar plates for CFU. For all environmental samples, the number of colonies in the culture medium containing 6-DMAP was higher and exceeded the number of colonies in the control group using standard culture methods by 1.5-2 times (e.g. Figure 4 The results showed that the addition of 6-DMAP could obtain some microorganisms that were difficult to culture using traditional standard culture methods.

[0047] Next, 100 strains were randomly selected from the agar plates for each sample and cultured on fresh agar medium (R2A medium diluted 1:10) in the presence and absence of 6-DMAP. After one week of culture, the grown colonies were used as templates for PCR, and a 750-bp fragment of the 16S rRNA gene was sequenced for taxonomic identification. The 16S rRNA gene was amplified using universal primers 27F (5′-agagtttgatcctggctcag-3′, SEQ ID NO. 01) and 1492R (5′-ggttaccttgttacgactt-3′, SEQ ID NO. 02) according to the manufacturer's recommended PCR system (Sangon Biotech, China). PCR products were sequenced using a commercial fluorescent dye terminator sequencing method (Sangon Biotech, China). The obtained sequences were compared with the database in EzBioCloud (https: / / www.ezbiocloud.net) to determine the similarity with the closest model strain sequence.

[0048] like Figure 5 As shown, among the strains isolated using the 6-DMAP-addition method of the present invention, 57%, 35%, 24%, and 16% of the strains from marine sediments, seawater, freshwater, and soil, respectively, were novel species (using a standard of ≤97% similarity to the closest 16S rRNA sequence in GenBank). In comparison, in the control group without 6-DMAP addition, only 4%, 9%, 2%, and 3% were novel species, respectively. These results demonstrate that the novel isolation and culture method of the present invention using 6-DMAP can culture significantly more novel microbial species than standard culture methods, and these novel bacterial species are difficult to culture using standard culture methods.

[0049] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention. Sequence Listing <110> Ningbo University <120> A method for isolating and cultivating new species of difficult-to-cultivate microorganisms <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 ggttaccttg ttacgactt 19

Claims

1. A method for isolating and culturing a new species of difficult-to-cultivate microorganisms, characterized by: The 6-DMAP solution sterilized by filtration through a PTEE filter is added to a sterilized liquid agar medium to a final 6-DMAP concentration of 1-3 μg / mL, and then solidified to obtain a solid culture medium. The natural sample is inoculated into the solid culture medium for culture.

2. The separation and culture method according to claim 1, wherein: The final concentration of 6-DMAP was 2-3 μg / mL.

3. The separation and culture method according to claim 2, wherein: The final concentration of 6-DMAP was 2.5 μg / mL.

4. Application of 6-DMAP in the preparation of culture medium for new species of difficult-to-cultivate microorganisms.

5. The use according to claim 4, characterized in that: The final concentration of 6-DMAP is 1-3 μg / mL.

6. The use according to claim 5, characterized in that: The final concentration of 6-DMAP was 2-3 μg / mL.

7. The use according to claim 6, characterized in that: The final concentration of 6-DMAP was 2.5 μg / mL.

8. A culture medium for new species of difficult-to-cultivate microorganisms, characterized by: Includes agar medium containing 6-DMAP at a final concentration of 1-3 μg / mL.

9. A culture medium for a new species of difficult-to-cultivate microorganisms according to claim 8, characterized in that: The final concentration of 6-DMAP was 2-3 μg / mL.

10. A culture medium for new species of difficult-to-cultivate microorganisms according to claim 9, characterized in that: The final concentration of 6-DMAP was 2.5 μg / mL.

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