Aeromicrobium sp.SZW-1, separation method thereof and application of Aeromicrobium sp.SZW-1 in plastic degradation

Abstract

The invention discloses a halophilic aeromicrobe strain Aeromicrobe sp. SZW-1, a separation method of the halophilic aeromicrobe strain Aeromicrobe sp. SZW-1 and application of the halophilic aeromicrobe strain Aeromicrobe sp. SZW-1 in plastic degradation. The invention discloses halophilic aeromicrobe Aeromicrobe sp. SZW-1, and the preservation number of the halophilic aeromicrobe Aeromicrobe sp. SZW-1 is GDMCC No: 67081. The NaCl concentration tolerance range of the Aeromicrobium sp.SZW-1 is 0 to 0.15 g / mL, the optimal NaCl concentration is 0.03 g / mL, and the Aeromicrobium sp.SZW-1 has hydrolytic activity on PET (Polyethylene Terephthalate). The strain has the advantages that the strain has natural adaptability to a high-salt environment and PET degradation activity, key technical materials and solutions are provided for PET pollution treatment, especially for high-salt environment treatment which is difficult to reach by a traditional biotechnology, and remarkable environmental protection value and application potential are also shown.

Description

Technical Field

[0001] This invention belongs to the field of biodegradation technology, specifically relating to a halophilic aeromicrobium sp. SZW-1, its isolation method, and its application in plastic degradation. Background Technology

[0002] Polyethylene terephthalate (PET) plastic is widely used due to its excellent physicochemical properties, but its waste constitutes a global "white pollution" problem. PET plastic entering the marine environment, in particular, is difficult to degrade naturally, persisting for a long time and breaking down into microplastics, posing a serious threat to ecosystems and human health. Biodegradation, especially the use of microorganisms or enzymes to hydrolyze PET, is considered an environmentally friendly green solution. Currently, although several strains, including *Ideonella sakaiensis*, have been found to have PET-degrading activity, their practical application still faces significant bottlenecks. These conventional strains and their enzymes typically require mild conditions to function, their biological processes are highly susceptible to contamination by other microorganisms, and they exhibit extremely poor adaptability to extreme environments such as high salinity and high osmotic pressure. This makes them unsuitable for direct application to high-salinity industrial wastewater rich in PET microplastics, and even more difficult for in-situ treatment of marine plastic pollution.

[0003] Microorganisms in environments such as seawater possess a natural adaptability to high-salinity conditions. On the one hand, seawater or high-salinity industrial wastewater can be directly used as a culture medium, eliminating the need for expensive pretreatment and enabling in-situ, low-cost biological removal of PET contamination from saline media. This provides a novel technological approach for the treatment of marine plastic pollution and special industrial wastewater. On the other hand, their high-salinity adaptability can create a naturally sterile operating environment, significantly reducing the risk of contamination and sterilization costs during fermentation or biological treatment, and improving the robustness and economy of the process. Therefore, identifying PET-degrading strains that can adapt to harsh environments such as high salinity has become an urgent need to solve pollution problems in these specific scenarios. Summary of the Invention

[0004] The first objective of this invention is to provide a halophilic aeromicrobium sp. SZW-1, which was deposited on October 13, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67081.

[0005] A second objective of this invention is to provide a microbial agent containing the cells of the aforementioned halophilic aeromicrobium sp. SZW-1 and / or its metabolites.

[0006] Preferably, the microbial agent is the fermentation broth of the halophilic aeromicrobium sp. SZW-1.

[0007] Further preferably, the fermentation broth is obtained by inoculating SZW-1 into LB culture medium containing 0.03 g / mL NaCl, shaking culture at 37°C, and then removing the bacterial cells.

[0008] A third objective of this invention is to provide the application of the aforementioned halophilic aeromicrobium sp. SZW-1 or the aforementioned microbial agents in the degradation and conversion of plastics.

[0009] Preferably, the plastic is polyethylene terephthalate (PET).

[0010] Further preferred, the degradation products of polyethylene terephthalate (PET) include terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET).

[0011] A fourth objective of this invention is to provide a method for the degradation and transformation of plastics, comprising the step of using the aforementioned halophilic aeromicrobium sp. SZW-1 or the aforementioned microbial agent to degrade the plastics.

[0012] Preferably, the plastic is polyethylene terephthalate (PET).

[0013] The beneficial effects of this invention are as follows:

[0014] The strain SZW-1 described in this invention is a halophilic aerobic microbacterium with an optimal growth temperature of 30-37℃, a tolerance range of NaCl concentration of 0-0.15 g / mL, and an optimal NaCl concentration of 0.03 g / mL. It also exhibits hydrolytic activity against PET. The advantages of this strain lie in its natural adaptability to high-salt environments and its PET degradation activity. It not only provides key technical materials and solutions for PET pollution remediation, especially for high-salt environments that are difficult to access using traditional biotechnology, but also demonstrates significant environmental value and application potential.

[0015] The halophilic aeromicrobium sp. SZW-1 was deposited on October 13, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67081. Attached Figure Description

[0016] Figure 1The isolation method for strain SZW-1 in Example 1 includes: (A) screening of strains with PET hydrolysis activity using transparent agar plates; and (B) streak-purified strain SZW-1.

[0017] Figure 2 This is the phylogenetic tree of strain SZW-1 in Example 1.

[0018] Figure 3 The growth curves of strain SZW-1 in Example 2 at different concentrations of NaCl are shown.

[0019] Figure 4 The degradation products of PET by the fermentation broth of strain SZW-1 in Example 3 were analyzed by HPLC. CK was the control group, and SZW-1 was the reaction group of the fermentation broth of strain SZW-1. Detailed Implementation

[0020] The following are specific implementation examples of the present invention. It should be noted that these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Modifications and substitutions to the details and form of the implementation schemes made within the scope and spirit of the present invention all fall within the protection scope of the present invention.

[0021] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0022] Example 1: Isolation and Identification of Plastic-Degrading Strains SZW-1

[0023] 1. Isolation of strain SZW-1

[0024] Preparation of PET screening plates: Weigh 1 g of PET (polyethylene terephthalate) and dissolve it in 200 mL of hexafluoroisopropanol. Add the solution dropwise to 700 mL of pre-cooled deionized water while stirring vigorously to obtain a PET suspension. Remove the organic solvent by rotary evaporation at 40 °C to obtain the final PET nanoparticle solution. Add 1 mL of PET nanoparticle solution to every 100 mL of high-salt LB medium (each high-salt LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, 30 g / L NaCl, 15 g / L agar, solvent: water), mix well, and pour into plates to obtain high-salt LB plates containing PET.

[0025] Screening of PET-degrading strains: 10 g of a mixture of sediment and seawater collected from Shenzhen Bay was added to 90 mL of sterile water and incubated with shaking at 37℃ for 2 h to prepare a bacterial suspension. The bacterial suspension was serially diluted 10-fold, and the diluted bacterial solution was spread on high-salt LB agar plates containing PET and incubated at 37℃. The presence of PET hydrolysis clear zones around the colonies was observed. Figure 1 As shown in Figure A, some colonies exhibit a transparent PET hydrolysis zone, indicating that they are strains with PET hydrolysis activity.

[0026] Purification of PET-degrading strains: Colonies with PET hydrolysis clear zones were picked and streaked onto high-salt LB agar plates containing PET, and incubated at 37°C. After three consecutive subcultures, the purified strain SZW-1 was obtained. Figure 1 As shown in Figure B, the purified strain SZW-1 grew on a PET plate and exhibited a hydrolysis clear zone, indicating that the strain possesses PET hydrolysis activity.

[0027] 2. Genome sequencing and identification of strain SZW-1

[0028] Strain SZW-1 was inoculated into LB liquid medium containing a final concentration of 0.03 g / mL NaCl and cultured at 37°C with shaking until the logarithmic growth phase. The cells were collected and sent to Shanghai Meiji Biotechnology Co., Ltd. for genome sequencing. The sequences were assembled using SOAPdenovo2 software, and the coding sequences in the genome were predicted using Prodigal software. The 16S rRNA gene sequence of strain SZW-1 (nucleotide sequence shown in SEQ ID NO.1) showed the highest homology (98.40%) with the published type species of the genus *Aeromicrobium*, *Aeromicrobium lacus* CPCC 204604(T). The phylogenetic tree constructed based on 16S rRNA alignment is shown below. Figure 2 As shown in Table 1, the simulated DNA-DNA hybridization values ​​(dDDH) of strain SZW-1 with published closely related strains were calculated using the online tool TYGS (https: / / tygs.dsmz.de / user_requests / new). The results show that the dDDH of strain SZW-1 with published closely related species in the genus *Aeromicrobium* ranged from 17.2% to 27.3%, which is lower than the internationally recognized critical value of 70% for dDDH. These results indicate that strain SZW-1 represents a new species in the genus *Aeromicrobium*.

[0029] Table 1. Simulated DNA-DNA hybridization values ​​(dDDH) of strain SZW-1 and published closely related strains.

[0030] The 16S rRNA gene sequence of SZW-1 is shown in SEQ ID NO.1, specifically:

[0031] The strain SZW-1 was named Aeromicrobium sp. SZW-1 and was deposited on October 13, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No. 67081.

[0032] Example 2: Halophilicity of strain SZW-1

[0033] Aeromicrobium sp. SZW-1 was inoculated into LB liquid medium and cultured overnight at 37°C with shaking to obtain seed culture. The seed culture was then inoculated at a volume ratio of 1% into fresh LB medium containing 0%–20% (w / v) NaCl (i.e., 0–0.2 g / mL NaCl), with three replicates for each concentration. The medium was cultured at 37°C with shaking, and samples were taken at 20-h intervals. OD values ​​were measured using a spectrophotometer. 600 .

[0034] The results are as follows Figure 3 As shown, Aeromicrobium sp. SZW-1 can grow in LB medium containing 0-0.15 g / mL NaCl, with the optimal NaCl concentration being 0.03 g / mL, indicating that this strain is a halophilic bacterium.

[0035] Example 3: Application of strain SZW-1 in PET degradation

[0036] Aeromicrobium sp. SZW-1 was inoculated into LB liquid medium containing a final concentration of 0.03 g / mL NaCl and cultured overnight at 37°C with shaking. The mixture was then centrifuged at 12000 g for 20 min at 4°C. The supernatant was collected and filtered through a sterile 0.22 mm filter to remove bacterial cells, yielding the Aeromicrobium sp. SZW-1 fermentation broth. The reaction system contained 0.01 g PET plastic and 1 mL of Aeromicrobium sp. SZW-1 fermentation broth, and was incubated at 37°C with shaking at 1000 rpm for 24 h. The control group consisted of 1 mL of LB liquid medium containing a final concentration of 0.03 g / mL NaCl, replacing the SZW-1 fermentation broth; all other steps were the same. After the reaction, the mixture was centrifuged at 12000 g for 10 min, and 150 μL of the supernatant was collected. 150 μL of methanol and 6.5 μL of HCl (6 N) were added, and the mixture was filtered through a 0.22 μm filter. The reaction products were detected by HPLC. The chromatographic column was a C18 reversed-phase column (4.6×250 mm, 5 mm). The column temperature was 25 ℃. The mobile phase was 32% methanol aqueous solution with 0.056‰ (v / v) H2SO4. The flow rate was 1 mL / min. The injection volume was 20 μL. Isocratic elution was performed for 30 min. The detection wavelength was 240 nm.

[0037] The results are as follows Figure 4 As shown, the fermentation broth of Aeromicrobium sp. SZW-1 can hydrolyze PET into monomers TPA (terephthalic acid) and MHET (mono(2-hydroxyethyl) terephthalate).

Claims

1. A halophilic aerobic microbacterium Aeromicrobium sp. SZW-1, characterized in that, The accession number is: GDMCC No: 67081.

2. A microbial inoculant, characterized in that, Contains the halophilic aerobic microorganisms as described in claim 1 Aeromicrobium The cells of sp. SZW-1 and / or its metabolites.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a halophilic aerobic microorganism. Aeromicrobium Fermentation broth of sp. SZW-1.

4. The microbial agent according to claim 3, characterized in that, The fermentation broth is made from Aeromicrobium sp. SZW-1 was inoculated into LB medium containing 0.03 g / mL NaCl, cultured at 37°C with shaking, and then the bacterial cells were removed to obtain the product.

5. The halophilic aerobic microbe as described in claim 1 Aeromicrobium The application of the microbial agent as described in sp. SZW-1 or any one of claims 2-4 in the degradation and conversion of plastics.

6. The application according to claim 5, characterized in that, The plastic is polyethylene terephthalate.

7. The application according to claim 6, characterized in that, Degradation products of polyethylene terephthalate include terephthalic acid and mono(2-hydroxyethyl) terephthalate.

8. A method for the degradation and conversion of plastics, characterized in that, Including the use of the halophilic aerobic microbes as described in claim 1 Aeromicrobium The step of degrading plastics using the microbial agent as described in sp. SZW-1 or any one of claims 2-4.

9. The method according to claim 8, characterized in that, The plastic is polyethylene terephthalate.

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