Separation method of marine bacteria

Abstract

The invention relates to the technical field of bacterium separation and purification, in particular to a marine bacterium separation method, which comprises the following steps: soaking micro-plastic particles in seawater to obtain micro-plastic particles covered with a biofilm, collecting the biofilm, and providing new micro-plastic particles as a bacterium colonization medium. Competitive colonization of a microbial biofilm in a growth stage is realized by controlling conditions such as culture time and temperature, and bacteria with strong colonization ability can be quickly screened out by matching with a pure culture step.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial separation and purification, and in particular to a method for separating marine bacteria. Background Art

[0002] Microorganisms in the natural environment are often in a complex microbial environment, with competition, cooperation and multiple interactions between species, and in a complex dynamic balance. Through different culture media and culture environments, we can selectively screen and culture target strains from complex microbial communities, separate and purify strains, obtain pure culture strains, and conduct in-depth research.

[0003] The ocean has a rich variety of bacterial groups, among which the bacterial communities in the biofilm state have higher diversity than the bacteria in the floating state in seawater. However, in the prior art, the plate streaking separation method can only separate high-abundance bacteria in the marine microbial community, and it is difficult to effectively separate rare marine bacteria or bacteria with biofilm colonization ability or slow growth. Therefore, it is urgent to provide a method for separating marine bacteria. Summary of the invention

[0004] In order to develop a method for quickly screening bacteria with strong colonization ability, the present invention provides a method for isolating marine bacteria. The method for isolating marine bacteria provided by the present invention can effectively separate biofilm bacteria on the surface of marine microplastic particles, which is not only conducive to subsequent genome sequencing and analysis and research on the properties of bacteria on the surface of plastic particles, but also conducive to subsequent research on the acquisition and transfer of antibiotic resistance of microplastic strains.

[0005] A method for separating marine bacteria comprises the following steps: Obtain culture medium material: select a medium with a diameter of 0.3 mm to 5 mm and a density of 0.94 g / cm 3 ~0.96 g / cm 3 The microplastic particles are placed in a water permeable device and sterilized after cleaning.

[0006] The culture medium material used in the present invention is microplastic particles with a diameter of 0.3 mm to 5 mm. This is because the bacterial attachment medium, i.e., the material type and size, will affect the type of bacteria finally separated and obtained within a certain range. Bacterial colonization will be affected by the size of the medium and its own motility.

[0007] Formation of biofilm: Soak the permeable device containing microplastic particles in seawater for 8 to 12 days to obtain microplastic particles covered with biofilm.

[0008] Competitive colonization: Collect biofilms from microplastic particles covered with biofilms, allow the bacteria in the biofilms to be free in the liquid culture medium, transfer them to new liquid culture medium containing sterile microplastic particles, and culture them at 20℃~30℃ for 8 h~16 h to obtain microplastic particles colonized with bacteria.

[0009] Conduct pure culture: Collect bacterial strains from microplastic particles colonized with bacteria, disperse them in liquid culture medium to obtain seed solution, and inoculate it into new liquid culture medium for pure culture at 20℃~30℃ and a rotation speed of 160 r / min~200 r / min.

[0010] The method for separating marine bacteria provided by the present invention comprises soaking microplastic particles in seawater to obtain microplastic particles covered with biofilms, controlling the culture time, temperature and other conditions to achieve competitive colonization in the growth stage of microbial biofilms, and combining the pure culture step to quickly screen out bacteria with strong colonization ability. The present invention sequenced and analyzed the genomes of pure culture strains and found that among the isolated strains, 75 strains contained movement genes and 84 strains showed resistance to 3 or more antibiotics, which is not only conducive to subsequent genome sequencing and analysis and research on the properties of bacteria on plastic surfaces, but also conducive to subsequent research on the acquisition and transfer of antibiotic resistance of microplastic strains.

[0011] Furthermore, the microplastic particles are polyethylene particles, polypropylene particles or polyvinyl chloride particles.

[0012] Furthermore, the water-permeable device is a mesh bag with a mesh size of 72 μm to 76 μm, a mesh bag width of 18 cm to 22 cm, and a mesh bag height of 28 cm to 32 cm.

[0013] Furthermore, the mesh bag containing the microplastic particles is soaked in seawater for 10 days.

[0014] Furthermore, the liquid culture medium is 2216E liquid culture medium.

[0015] Furthermore, the method for collecting biofilm from microplastic particles covered with biofilm is: vortexing the microplastic particles covered with biofilm at 1800 r / min~2200 r / min for 8 min~12 min.

[0016] Furthermore, the competitive colonization process is repeated 9 to 12 times.

[0017] The present invention repeats the cycle colonization step multiple times. This is because the competitive attachment of bacteria is random. Even the same material may be colonized with different bacteria after multiple attachments, dissociations, and attachments, so more diverse microplastic bacteria can be obtained.

[0018] Furthermore, the culture conditions in the competitive colonization process are: static culture at 20°C to 30°C for 10 h to 14 h.

[0019] Furthermore, during the pure culture process, the volume fraction of the seed solution inoculated in the new liquid culture medium is 1% to 3%.

[0020] Furthermore, the pure culture conditions are: culturing at 20°C~30°C and 170 r / min~190 r / min for 10 h~14 h.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a method for isolating marine bacteria. After obtaining environmental microplastic particles, biofilms are collected, and new microplastic particles are provided as bacterial colonization media. By controlling the culture time, temperature and other conditions to achieve competitive colonization in the growth stage of microbial biofilms, and then combining with a pure culture step, bacteria with strong colonization ability can be quickly screened out.

[0022] 2. The present invention found that the strains isolated from microplastic particles have high diversity, and the high diversity of strains is related to bacterial colonization. More abundant bacteria can be obtained through colonization, dissociation and other techniques. Among the strains isolated by the present invention, 75 strains contain movement genes, and 84 strains show resistance to 3 or more antibiotics, which is not only conducive to subsequent genome sequencing and analysis and the study of the properties of bacteria on the surface of plastic particles, but also conducive to subsequent research on the acquisition and transfer of antibiotic resistance of microplastic strains.

[0023] 3. Existing methods based on culture medium composition and single cell separation all focus on the living and culture environment of marine microorganisms, and rarely involve separation and screening related to the motility characteristics of bacteria in the ocean. The present invention uses 2216E liquid culture medium, repeated competitive colonization and other technical means, and cooperates with conditions such as "static culture at 20℃~30℃ for 10 h~14 h", which can screen out 75 strains of bacteria containing motility genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 To obtain bacterial accumulation curve.

[0026] Figure 2 This is the phylogenetic tree of 94 non-redundant strains.

[0027] Figure 3 The drug resistance gene types of 94 isolated strains were predicted based on genome analysis. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0029] Example 1: A method for isolating marine bacteria.

[0030] S1, obtain culture medium material: select a medium with a diameter of 0.3 mm and a density of 0.95 g / cm 3 The microplastic particles were used as culture medium materials, and the microplastic particles were filled in a nylon mesh bag to one-fifth of the bag volume. The microplastic particles and nylon mesh bag were then washed three times with sterile seawater and then autoclaved at 115°C for 30 min. The microplastic particles were polyethylene particles purchased from Guangzhou Ruixiang Plastic Chemical Co., Ltd.

[0031] The nylon mesh bag has a mesh size of 74 μm, a mesh width of 20 cm, and a mesh height of 30 cm; it was purchased from Sangon Biotechnology Co., Ltd.

[0032] The liquid culture medium selected was 2216E liquid culture medium for marine bacteria. The components of each liter of 2216E liquid culture medium were as follows: 10 g agar powder, 5.0 g peptone, 1.0 g yeast powder, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.034 g strontium chloride, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.0016 g ammonium nitrate and 0.008 g disodium hydrogen phosphate, and the volume was made up to 1 L with distilled water.

[0033] S2, biofilm formation: nylon mesh bags containing microplastic particles were immersed in the seawater at a depth of 2 m in the subtidal zone of Dingjiazui, Huangdao, Qingdao, and the position of the bags was marked with buoys. The bags were placed in the in situ environment for 10 days to obtain microplastic particles covered with biofilm.

[0034] The specific location of Dingjiazui in Qingdao City is 120°8'19''E, 35°54'55''N.

[0035] S3, competitive colonization: After the microplastic particles covered with biofilm are transferred to the laboratory, sterile seawater is aspirated with a pipette to gently rinse the surface of the microplastic particles to remove loosely attached bacteria, and microplastic particles covered only with biofilm are obtained. These particles are transferred to new liquid culture medium containing sterile microplastic particles and cultured at 16℃~37℃ for 8 h~16 h to obtain microplastic particles colonized with bacteria.

[0036] The specific steps are as follows.

[0037] Step 1: Use sterile tweezers to transfer the microplastic particles covered with biofilm into 0.5 mL of sterile 2216E liquid culture medium.

[0038] Step ②: Immediately shake the centrifuge tube using a vortex mixer at 2000 r / min for 10 min until the biofilm falls off and disperses into the culture medium.

[0039] Step 3: Immediately transfer the culture medium containing only free bacteria to a new tube containing new sterile microplastic particles and 2216E liquid culture medium.

[0040] Step ④: Incubate at 25°C for 12 h to allow bacteria to colonize on the new particles and obtain bacteria-colonized microplastic particles.

[0041] Step ⑤: Transfer the bacteria-colonized microplastic particles to a new tube containing sterile 2216E liquid culture medium.

[0042] Repeat steps ① to ⑤ for 10 cycles to obtain new bacteria-colonized microplastic particles.

[0043] S4, pure culture: disperse the bacterial strains that form biofilm into new 2216E liquid culture medium by vortex oscillation, inoculate the culture medium containing bacteria into the new 2216E liquid culture medium at a 1% inoculum, and culture it on a shaking table at 25°C at a speed of 180 r / min for 12 h. Grow the bacteria to a sufficient concentration, which is judged by an absorbance value greater than 0.6 at 600 nm on a microplate reader.

[0044] S5, strain identification: DNA extraction and identification of cells were performed by 16S rRNA PCR amplification and Sanger sequencing. Bacterial DNA was extracted using the FastPure® Bacterial DNA Isolation Mini Kit according to the manufacturer's instructions. PCR amplification of the 16S rRNA gene was performed in Prime Star Max premix with the primer pair of 27F with the nucleotide sequence shown in SEQ ID NO.1: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R with the nucleotide sequence shown in SEQ ID NO.2: 5′-GGTTACCTTGTTACGACTTC-3′. The PCR products were subjected to Sanger sequencing at Sangon Biotech to obtain the full-length 16S rRNA gene. Duplicate 16S rRNA gene sequences were removed by cd-hit software to obtain non-redundant strains. Cumulative curves were plotted based on the number of isolated strains and the number of non-redundant strains.

[0045] The CD-HIT software similarity cutoff value was >99.99%.

[0046] The FastPure® Bacterial DNA Isolation Mini Kit was purchased from Nanjing Novazon Biotech Co., Ltd.

[0047] The Prime Star Max premix was purchased from Baoriyi Biological Co., Ltd.

[0048] Example 2: A method for isolating marine bacteria.

[0049] S1, obtain culture medium material: select a medium with a diameter of 0.2 mm and a density of 0.94 g / cm 3 The microplastic particles were used as culture medium materials, and the microplastic particles were filled in a nylon mesh bag to one-fifth of the bag volume. The microplastic particles and nylon mesh bag were then washed three times with sterile seawater and then autoclaved at 115°C for 30 min. The microplastic particles were polyethylene particles purchased from Guangzhou Ruixiang Plastic Chemical Co., Ltd.

[0050] The nylon mesh bag has a mesh size of 72 μm, a mesh width of 18 cm, and a mesh height of 28 cm; it was purchased from Sangon Biotechnology Co., Ltd.

[0051] The liquid bacterial culture medium selected was 2216E liquid culture medium for marine bacteria. The components of each liter of 2216E liquid culture medium were as follows: 10 g agar powder, 5.0 g peptone, 1.0 g yeast powder, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.034 g strontium chloride, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.0016 g ammonium nitrate and 0.008 g disodium hydrogen phosphate, and the volume was made up to 1 L with distilled water.

[0052] S2, biofilm formation: nylon mesh bags containing microplastic particles were immersed in the seawater at a depth of 2 m in the subtidal zone of Dingjiazui, Huangdao, Qingdao, and the position of the bags was marked with buoys. The bags were placed in the in situ environment for 10 days to obtain microplastic particles covered with biofilm.

[0053] The specific location of Dingjiazui in Qingdao City is 120°8'19''E, 35°54'55''N.

[0054] S3, competitive colonization: After the microplastic particles covered with biofilm are transferred to the laboratory, sterile seawater is aspirated with a pipette to gently rinse the surface of the microplastic particles to remove loosely attached bacteria, and microplastic particles covered only with biofilm are obtained. The particles are transferred to new liquid culture medium containing sterile microplastic particles and cultured at 16℃~37℃ for 8 h~16 h to obtain microplastic particles colonized with bacteria.

[0055] The specific steps are as follows.

[0056] Step 1: Use sterile tweezers to transfer the microplastic particles covered with biofilm into 0.5 mL of sterile 2216E culture medium.

[0057] Step ②: Immediately shake the centrifuge tube using a vortex mixer at 1800 r / min for 8 min until the biofilm falls off and disperses into the culture medium.

[0058] Step 3: Immediately transfer the culture medium containing only free bacteria to a new tube containing new sterile microplastic particles and 2216E liquid culture medium.

[0059] Step ④: Incubate at 20°C for 10 h to allow bacteria to colonize on the new particles and obtain bacteria-colonized microplastic particles.

[0060] Step ⑤: Transfer the bacteria-colonized microplastic particles to a new tube containing sterile 2216E liquid culture medium.

[0061] Repeat steps ① to ⑤ for 9 cycles to obtain new bacterial-colonized microplastic particles.

[0062] S4, pure culture: disperse the bacterial strains that form biofilm into new 2216E liquid culture medium by vortex oscillation, inoculate the culture medium containing bacteria into the new 2216E liquid culture medium at a 1% inoculum, and culture on a shaking table at 20°C at a speed of 170 r / min for 10 h. Grow the bacteria to a sufficient concentration, which is judged by an absorbance value greater than 0.6 at 600 nm on a microplate reader.

[0063] S5, strain identification: DNA extraction and identification of cells were performed by 16S rRNA PCR amplification and Sanger sequencing. Bacterial DNA was extracted using the FastPure® Bacterial DNA Isolation Mini Kit according to the manufacturer's instructions. PCR amplification of the 16S rRNA gene was performed in Prime Star Max premix with the primer pair of 27F with the nucleotide sequence shown in SEQ ID NO.1: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R with the nucleotide sequence shown in SEQ ID NO.2: 5′-GGTTACCTTGTTACGACTTC-3′. The PCR products were subjected to Sanger sequencing at Sangon Biotech to obtain the full-length 16S rRNA gene. Duplicate 16S rRNA gene sequences were removed by cd-hit software to obtain non-redundant strains. Cumulative curves were plotted based on the number of isolated strains and the number of non-redundant strains.

[0064] The CD-HIT software similarity cutoff value was >99.99%.

[0065] The FastPure® Bacterial DNA Isolation Mini Kit was purchased from Nanjing Novazon Biotech Co., Ltd.

[0066] The Prime Star Max premix was purchased from Baoriyi Biological Co., Ltd.

[0067] Example 3: A method for isolating marine bacteria.

[0068] S1, obtain culture medium material: select a medium with a diameter of 5 mm and a density of 0.96 g / cm 3 The microplastic particles were used as culture medium materials, and the microplastic particles were filled in a nylon mesh bag to one-fifth of the bag volume. The microplastic particles and nylon mesh bag were then washed three times with sterile seawater and then autoclaved at 115°C for 30 min. The microplastic particles were polyethylene particles purchased from Guangzhou Ruixiang Plastic Chemical Co., Ltd.

[0069] The nylon mesh bag has a mesh size of 76 μm, a width of 22 cm, and a height of 32 cm; it was purchased from Sangon Biotechnology Co., Ltd.

[0070] The liquid bacterial culture medium selected was 2216E liquid culture medium for marine bacteria. The components of each liter of 2216E liquid culture medium were as follows: 10 g agar powder, 5.0 g peptone, 1.0 g yeast powder, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.034 g strontium chloride, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.0016 g ammonium nitrate and 0.008 g disodium hydrogen phosphate, and the volume was made up to 1 L with distilled water.

[0071] S2, biofilm formation: nylon mesh bags containing microplastic particles were immersed in the seawater at a depth of 2 m in the subtidal zone of Dingjiazui, Huangdao, Qingdao, and the position of the bags was marked with buoys. The bags were placed in the in situ environment for 10 days to obtain microplastic particles covered with biofilm.

[0072] The specific location of Dingjiazui in Qingdao City is 120°8'19''E, 35°54'55''N.

[0073] S3, competitive colonization: After the microplastic particles covered with biofilm are transferred to the laboratory, sterile seawater is aspirated with a pipette to gently rinse the surface of the microplastic particles to remove loosely attached bacteria, and microplastic particles covered only with biofilm are obtained. The particles are transferred to new liquid culture medium containing sterile microplastic particles and cultured at 16℃~37℃ for 8 h~16 h to obtain microplastic particles colonized with bacteria.

[0074] The specific steps are as follows.

[0075] Step 1: Use sterile tweezers to transfer the microplastic particles covered with biofilm into 0.5 mL of sterile 2216E liquid culture medium.

[0076] Step ②: Immediately shake the centrifuge tube using a vortex mixer at 2200 r / min for 12 min until the biofilm falls off and disperses into the culture medium.

[0077] Step 3: Immediately transfer the culture medium containing only free bacteria to a new tube containing new sterile microplastic particles and 2216E liquid culture medium.

[0078] Step ④: Culture at 30°C for 14 hours to allow bacteria to colonize on the new particles and obtain bacteria-colonized microplastic particles.

[0079] Step ⑤: Transfer the bacteria-colonized microplastic particles to a new tube containing sterile 2216E liquid culture medium.

[0080] Repeat steps ① to ⑤ for 12 cycles to obtain new bacteria-colonized microplastic particles.

[0081] S4, pure culture: disperse the bacterial strains that form biofilm into new 2216E liquid culture medium by vortex oscillation, inoculate the culture medium containing bacteria into the new 2216E liquid culture medium at a 1% inoculum, and culture at 30°C on a shaking table at a speed of 190 r / min for 14 h. Grow the bacteria to a sufficient concentration, which is judged by an absorbance value greater than 0.6 at 600 nm on a microplate reader.

[0082] S5, strain identification: DNA extraction and identification of cells were performed by 16S rRNA PCR amplification and Sanger sequencing. Bacterial DNA was extracted using the FastPure® Bacterial DNA Isolation Mini Kit according to the manufacturer's instructions. PCR amplification of the 16S rRNA gene was performed in Prime Star Max premix with the primer pair of 27F with the nucleotide sequence shown in SEQ ID NO.1: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R with the nucleotide sequence shown in SEQ ID NO.2: 5′-GGTTACCTTGTTACGACTTC-3′. The PCR products were subjected to Sanger sequencing at Sangon Biotech to obtain the full-length 16S rRNA gene. Duplicate 16S rRNA gene sequences were removed by cd-hit software to obtain non-redundant strains. Cumulative curves were plotted based on the number of isolated strains and the number of non-redundant strains.

[0083] The CD-HIT software similarity cutoff value was >99.99%.

[0084] The FastPure® Bacterial DNA Isolation Mini Kit was purchased from Nanjing Novazon Biotech Co., Ltd.

[0085] The Prime Star Max premix was purchased from Baoriyi Biological Co., Ltd.

[0086] Test results: Examples 1 to 3 have similar effects. For the convenience of subsequent discussion and reference, the test results of Example 1 shall prevail. The following are the test results of Example 1.

[0087] In the first round of bacterial isolation using the present invention, we obtained 149 bacterial strains. Subsequent 16SrRNA gene sequencing and analysis revealed 94 non-redundant strains. To further evaluate the performance of the present invention, we plotted a cumulative curve based on the number of isolated strains, such as Figure 1 The curve still shows an increasing trend and has not reached a plateau, indicating that if more separation work is done, there is still great potential to obtain more different strains.

[0088] A total of 21 strains among the 94 non-redundant strains could not be classified to the species level, such as Figure 2 As shown, this indicates that the present invention can be used to obtain new species. Gene reading frames from 94 genomes were annotated based on a comprehensive antibiotic resistance database to identify potential antibiotic resistance genes, such as Figure 3 As shown. Each strain has at least one antibiotic resistance gene, and on average each strain is predicted to have 11 antibiotic resistance genes. The top three strains with the most resistance genes are Streptomyces taurus MPVC58, Acinetobacter beijerinii MPP60B, and Acinetobacter beijerinii MPE71, with 41, 33, and 33 resistance genes, respectively. The top three antibiotics with the most resistance genes are fluoroquinolone antibiotics, phenytoin antibiotics, and peptide antibiotics, and it is speculated that 67, 61, and 58 strains are resistant to them, respectively. The above results indicate that antibiotic resistance is common in the isolated strains.

[0089] Through the implementation of the technical solution of the present invention, 94 pure cultures were obtained in the first bacterial isolation test, which proved the high efficiency of the present invention. The results of rarefaction analysis showed that there is great potential for obtaining non-redundant strains using the technical solution of the present invention. Most of the isolated strains have antibiotic resistance, which is conducive to the subsequent study of antibiotic resistance acquisition and transfer of microplastic strains.

[0090] The strains obtained by the present invention include many bacteria that have not been isolated from the ocean before. For example, members of the genus Bacillus often live in oil field environments and rely on petroleum hydrocarbons as their main carbon source. The strong motility enables Bacillus to grow at the oil-water interface to obtain resources. Similarly, very few Acinetobacter bacteria have been isolated from the ocean before, and members of this genus are well-known for causing clinical infections and drug resistance. The close phylogenetic relationship between the Acinetobacter strains isolated by the present invention and human-related biological strains highlights the important relevance of Acinetobacter bacteria to antibiotic resistance research.

[0091] It should be noted that the culture medium material used in the present invention can be microplastic particles that simulate the marine environment, which can effectively separate bacteria attached to the microplastic particles; it can also be microplastic particles that exist in the ocean itself and have a size and density that are consistent with those described in the present invention.

[0092] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0093] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for separating marine bacteria, characterized in that: The steps include: Obtain culture medium material: select a medium with a diameter of 0.2 mm to 5 mm and a density of 0.94 g / cm 3 ~0.96 g / cm 3 The microplastic particles are placed in a water permeable device and sterilized after cleaning; Formation of biofilm: Soak the permeable device containing microplastic particles in seawater for 8 to 12 days to obtain microplastic particles covered with biofilm; Competitive colonization: Collect biofilms from microplastic particles covered with biofilms, so that the bacteria in the biofilms are free in the liquid culture medium, transfer them to new liquid culture medium containing sterile microplastic particles, and culture them at 16℃~37℃ for 8 h~16 h to obtain microplastic particles colonized with bacteria; Conduct pure culture: Collect bacterial strains from microplastic particles colonized with bacteria, disperse them in liquid culture medium to obtain seed solution, and inoculate them into new liquid culture medium for pure culture at a temperature of 16°C~37°C and a rotation speed of 160 r / min~200 r / min.

2. The method for separating marine bacteria according to claim 1, characterized in that: The microplastic particles are polyethylene particles, polypropylene particles or polyvinyl chloride particles.

3. The method for separating marine bacteria according to claim 1, characterized in that: The water permeable device is a nylon mesh bag with a mesh size of 72 μm to 76 μm, a mesh bag width of 18 cm to 22 cm, and a mesh bag height of 28 cm to 32 cm.

4. The method for separating marine bacteria according to claim 1, characterized in that: The water-permeable device containing microplastic particles was immersed in seawater for 10 days.

5. The method for separating marine bacteria according to claim 1, characterized in that: The liquid culture medium is 2216E liquid culture medium.

6. The method for separating marine bacteria according to claim 1, characterized in that: The method for collecting biofilm from microplastic particles covered with biofilm is: vortexing the microplastic particles covered with biofilm at 1800 r / min~2200 r / min for 8 min~12 min.

7. The method for separating marine bacteria according to claim 1, characterized in that: The competitive colonization process was repeated 9 to 12 times.

8. The method for separating marine bacteria according to claim 1, characterized in that: The culture conditions in the competitive colonization process are: static culture at 20°C to 30°C for 10 h to 14 h.

9. The method for separating marine bacteria according to claim 1, characterized in that: During pure culture, the volume fraction of the seed liquid inoculated in the new liquid culture medium is 1%~3%.

10. The method for separating marine bacteria according to claim 1, characterized in that: The pure culture conditions are: culturing at 20°C~30°C and 170 r / min~190 r / min for 10 h~14 h.

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