Pseudomonas halludinii G1B capable of degrading PET (polyethylene terephthalate) plastic and application of pseudomonas halludinii G1B

Abstract

The invention relates to a pseudomonas halludinii G1B capable of degrading PET (Polyethylene Terephthalate) plastic and application of the pseudomonas halludinii G1B, and belongs to the technical field of microorganisms. The classification name of the strain is Pseudomonas hardinii, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 9, 2024, the preservation number is CGMCC NO.31904, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postcode of the preservation address is 100101. According to the present invention, the Pseudomonas hardinii G1B is prepared into the bacterial liquid, the bacterial liquid is inoculated to the inorganic salt liquid culture medium containing the PET plastic film to culture for 3 d, such that the PET plastic film can be degraded to produce the new product, the surface has wrinkles and fragments, and the peak intensities of the key functional groups such as C-H, C-O and C = O on the surface of the PET plastic film are weakened; according to the pseudomonas halludinii G1B provided by the invention, the resources of PET (Polyethylene Terephthalate) plastic degradation strains are enriched.

Description

Technical Field

[0001] The invention relates to a strain of Pseudomonas harutini G1B capable of degrading PET plastic and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Polyethylene terephthalate (PET) is one of the most commonly used plastics. It has a complex structure and can exist in nature for a long time, seriously affecting the environment and human health. How to effectively remove PET waste has become a hot topic of global concern. PET biodegradation is considered to be the most promising degradation method due to its simple operation, environmental friendliness, and low energy consumption. PET biodegradation mainly degrades PET macromolecular polymers through microbial action. However, since PET belongs to high polyester compounds and contains a large number of aromatic groups in the molecular chain, PET is more difficult to degrade under natural conditions than other types of plastics. The number of strains with the ability to degrade PET that have been reported so far is limited, and the degradation efficiency is low. Therefore, it is still necessary to further isolate and screen strains with high-efficiency PET degradation activity from the environment in order to provide rich bacterial resources for the biodegradation of PET. Summary of the invention

[0003] To solve the above technical problems, the present invention provides a strain of Pseudomonas haludini G1B that can degrade PET plastic. The strain is classified and named Pseudomonas haludini (Pseudomonas haludinis), which was deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms on September 9, 2024, with a deposit number of CGMCCNO.31904, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101; the Pseudomonas haludini G1B was isolated from soil enrichment liquid from a landfill.

[0004] The present invention also provides a bacterial agent capable of degrading PET plastic, wherein the bacterial agent contains the above-mentioned Pseudomonas harutini G1B.

[0005] The present invention also provides the use of the above-mentioned Pseudomonas harutini G1B and bacterial agent in degrading PET plastic.

[0006] The present invention also provides a method for biodegrading PET plastics, comprising preparing the above-mentioned Pseudomonas harutini G1B into a bacterial liquid, inoculating it into a liquid culture medium containing PET plastics for culturing; In one embodiment of the present invention, the OD of the bacterial solution is 600 The value is 1.0; In one embodiment of the present invention, the inoculation ratio is 4% v / v; In one embodiment of the present invention, the content of the PET plastic in the liquid culture medium is 1 g / L; In one embodiment of the present invention, the liquid culture medium is an inorganic salt liquid culture medium, and its formula is: K2PO4 1.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, (NH4)2SO4 1.5 g / L, NaCl 0.5 g / L, FeCl·6H2O 0.001 g / L, CaCl·2H2O 0.02 g / L; In one embodiment of the present invention, the culture conditions are: 30° C., 180 rpm, and dark culture.

[0007] The significant advantages of the present invention are: The present invention provides a strain of Pseudomonas halodini G1B capable of degrading PET plastic, which is prepared into OD 600 The bacterial liquid with a value of 1.0 is inoculated into an inorganic salt liquid culture medium containing 1g / L PET plastic film at an inoculum amount of 4% (v / v), and after culturing at 30°C and 180rpm in the dark for 3 days, the PET plastic film can be degraded to produce new products, wrinkles and fragments appear on the surface, and in addition, the peak intensity of several key functional groups such as CH, CO and C=O on the surface of the PET plastic film is weakened. The Pseudomonas halodini G1B provided by the present invention enriches the resources of PET plastic degradation strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 : Colony morphology and bacterial morphology of Pseudomonas harutini G1B.

[0009] Figure 2 : Phylogenetic tree of Pseudomonas harutini G1B.

[0010] Figure 3 : HPLC graph of degradation of PET plastic film by Pseudomonas harutini G1B.

[0011] Figure 4 : LC-MS graph of degradation of PET plastic film by Pseudomonas harutini G1B.

[0012] Figure 5 : SEM images of PET plastic film treated with Pseudomonas harutini G1B.

[0013] Figure 6 : FTIR images of PET plastic film treated with Pseudomonas harutini G1B. DETAILED DESCRIPTION

[0014] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0015] Embodiment 1: 1 Experimental Materials and Methods 1.1 The substrates involved in the examples are as follows: Polyethylene terephthalate (PET) film was purchased from Guangyuan Plastic Chemical Company.

[0016] 1.2 The culture medium involved in the embodiment is as follows: LB liquid culture medium: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, sterilize with high-temperature steam at 121℃ for 15min before use.

[0017] LB solid culture medium: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, and 1.5wt% agar powder. Sterilize with high-temperature steam at 121℃ for 15min before use.

[0018] Inorganic salt liquid culture medium: K2PO4 1.5g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.2g / L, (NH4)2SO4 1.5g / L, NaCl 0.5g / L, FeCl·6H2O 0.001g / L, CaCl·2H2O 0.02g / L, sterilize with high-temperature steam at 121℃ for 15min before use.

[0019] Inorganic salt solid culture medium: K2PO4 1.5g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.2g / L, (NH4)2SO4 1.5g / L, NaCl 0.5g / L, FeCl·6H2O 0.001g / L, CaCl·2H2O 0.02g / L, and add 1.5wt% agar powder. Sterilize with high-temperature steam at 121℃ for 15min and set aside.

[0020] 1.3 The method for identifying the PET-degrading bacteria involved in the examples is as follows: (1) Observation of colony morphology of PET-degrading bacteria Using PET plastic as the sole carbon source, an efficient PE plastic degradation strain was screened from the soil enrichment liquid of a landfill in Fujian by enrichment culture method, named G1B. A loop of strain G1B was scraped and inoculated into LB solid medium for streak culture. After culturing at 30℃ for 12h, its colonies were observed.

[0021] (2) Molecular biological identification of PET-degrading bacteria The total DNA of strain G1B was extracted by referring to the bacterial genome extraction kit TIANamp Bacteria DNA Kit 50. The bacterial universal primers 16S(F) (5'-AGAGTTTGSTCCTGGCTCAG-3') and 16S(R) (5'-GGTTACCTTGTTACGACTT-3') were used for 16Sr DNA amplification. The PCR used a 25μL reaction system, including 2×TaqMaster Mix 12.5μL, 16S(F) (10μmol / L) 1μL, 16S(R) (10μmol / L) 1μL, DNA template 1μL, ddH2O 9.5μL. The PCR amplification program was as follows: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 50℃ annealing for 45s, 72℃ extension for 1min40s, for a total of 35 cycles; 72℃ extension for 7min. The amplified product was detected by 1% agarose gel electrophoresis and recovered using the PCR product recovery kit SanPrep ColumnDNA Gel Extraction Kit. The recovered PCR product was connected to pMD19-TVector at 16°C and the ligated product was transformed into E. coli In DH5α competent cells, invert and culture overnight, pick single colonies for PCR verification, and detect the amplified products by 1% agarose gel electrophoresis. The successful connection is preliminarily judged based on the electrophoresis bands, and the bacterial solution containing the target bands is sent to Shanghai Biotech Co., Ltd. for sequencing.

[0022] 1.4 The method for biodegrading PET plastic involved in the embodiment is as follows: Strain G1B was cultured in LB liquid medium until OD 600 The value was 1.0, and the inoculum size was 4% (v / v) and inoculated into 40 mL of inorganic salt liquid culture medium containing 1 g / L PET plastic film. Three parallel groups were set up (the control group was taken without bacteria). After culturing in the dark at 30 ℃ and 180 rpm for 3 days, samples were taken for HPLC analysis to analyze the degradation of PET plastic film by strain G1B, and liquid chromatography-mass spectrometry (LC-MS) analysis was used to further determine the degradation products of PET plastic film.

[0023] 1.5 The sample processing and detection methods involved in the embodiments are as follows: (1) Sample treatment: After degradation, take 10 mL of culture medium and centrifuge it at 4°C and 5000 g for 10 min to obtain the supernatant. Add 10 mL of ethyl acetate at a volume ratio of 1:1. After rotary evaporation, add 2 mL of chromatographic grade methanol to dissolve, filter with a 0.22 μm organic membrane, use HPLC to detect whether there is any intermediate product, and then use LC-MS to verify the degradation product.

[0024] (2) HPLC detection conditions: Chromatographic column: Shimadzu C18 column (4.6*150mm; 5μm); column temperature: 35℃; flow rate: 0.5mL / min; detection wavelength: 240nm; injection volume: 5μL; mobile phase: 80% chromatographic grade methanol, added with 0.5% glacial acetic acid; running time: 25min.

[0025] (3) LC-MS detection conditions: Liquid chromatography: Agilent C18 column (3.0*250mm; 5μm); column temperature: 35℃; flow rate: 0.5mL / min; injection volume 5μL; mobile phase A is pure water (0.5% glacial acetic acid), B is chromatographic grade methanol (0.5% glacial acetic acid); gradient elution program is shown in Table 1. Mass spectrometry parameters: dual ESI source, negative ion mode; drying gas temperature: 350℃; drying gas flow rate: 10L / min; nebulizer gas pressure: 40psi; fragmentor: 130V; capillary voltage: 3500V; acquisition frequency: 1spectra / s; acquisition mode: 2 GHz dynamic expansion mode; scanning range: 100~350m / z. The gas used in the experiment was nitrogen (purity>99.9%), and the collision gas was high-purity nitrogen (purity>99.999%).

[0026] Table 1 Gradient elution program 1.6 Detection method of surface damage of PET plastic film: The PET plastic film treated with strain G1B was washed with deionized water for 3 to 5 times, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, oscillated at 37 ° C and 230 rpm for 2 hours, placed in an ultrasonic cleaner for 30 minutes, then washed several times with 70% anhydrous ethanol, and finally placed in a 100 ° C oven for drying, with the untreated PET plastic film as a control. A conductive tape was applied to the sample stage, and the sample was picked up with tweezers and attached to the conductive tape. The sample stage with the sample was placed in an ion sputtering instrument, and after gold was sprayed for 60 seconds under vacuum, it was placed under a scanning electron microscope (SEM) to adjust the magnification and focus to observe its microscopic morphology. After confirmation, the image was scanned, photographed and saved.

[0027] 1.7 Detection method for changes in functional groups on the surface of PET plastic film: The PET plastic film treated with strain G1B was washed with deionized water for 3 to 5 times, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, shaken at 37°C and 230 rpm for 2 hours, placed in an ultrasonic cleaner for 30 minutes, then washed several times with 70% anhydrous ethanol, and finally dried in an oven at 100°C. The untreated PET film was used as a control. Fourier transform infrared spectroscopy (FTIR) was used to characterize the changes in the surface functional groups of the PET plastic film, and the ATR mode was used to obtain the 400-4000 cm -1 The spectrum was scanned 32 times on average.

[0028] 2 Results and analysis 2.1 Colony morphology of PET-degrading bacteria G1B Scrape a loop of strain G1B and inoculate it onto LB solid medium for streak culture. After culturing at 30℃ for 12h, observe the colonies (such as Figure 1 As shown in the figure, its colonies were found to be rod-shaped, yellow-green, opaque, with neat and irregular edges.

[0029] 2.2 Molecular biological identification of PET-degrading bacteria G1B The 16S rRNA gene sequencing results of strain G1B were accessioned as OR939651 in the GenBank database. The sequencing results were compared by Nucleotide BLAST homology comparison in NCBI. The strain G1B was highly related to Pseudomonas harudinis, and the homology of 16S rDNA sequence with Pseudomonas harudinis was as high as 99.73%. Phylogenetic analysis based on 16S rDNA sequences showed that strain G1B and Pseudomonas harudinis were clustered into a branch with a bootstrap value of 99 (Fig. Figure 2 Therefore, strain G1B was identified as Pseudomonas harudinis.

[0030] Pseudomonas harutini Pseudomonas harudinis ) G1B was deposited in the China General Microbiological Culture Collection Center on September 9, 2024, with the deposit number CGMCC NO.31904. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the postal code of the deposit address is 100101.

[0031] 2.3 HPLC analysis of PET plastic film degradation by strain G1B Figure 3This is the HPLC graph of strain G1B degrading PET plastic film. After 3 days of treatment with strain G1B, a new product absorption peak was detected in the culture medium, while no product absorption peak appeared in the culture medium (Control) not treated with strain G1B, which also indicates that strain G1B has degradation activity on PET plastic film.

[0032] 2.4 LC-MS analysis of PET plastic film degradation by strain G1B In order to further identify the degradation products of PET plastic film, the culture solution of strain G1B degrading PET plastic film was analyzed by LC-MS. Figure 4 As shown, ion fragments of bis(2-hydroxyethyl)terephthalate (BHET), mono(2-hydroxyethyl)terephthalate (MHET) and terephthalic acid (TPA) were detected in the co-culture products of strain G1B and PET plastic film, thus proving that BHET, MHET and TPA were present in the degradation products.

[0033] 2.5 SEM analysis of PET plastic film degradation by strain G1B Depend on Figure 5 It can be seen that when the PET plastic film treated with strain G1B and the PET plastic film not treated with strain G1B (Control) were magnified 600 times, 1kv times and 5kv times, it can be seen that the surface of the PET plastic film not treated with strain G1B is flat and smooth (AC), but the surface of the PET plastic film after being treated with strain G1B is rough, wrinkled, and fragmented (DF).

[0034] 2.6 FTIR analysis of PET plastic film degradation by strain G1B PET is a polyester compound, and the ester bond is the most critical functional group. FTIR results are as follows Figure 6 As shown in the figure, compared with the PET plastic film not treated with strain G1B (Control), the PET plastic film treated with strain G1B showed a -1 CH at 1050cm -1 、1094cm -1 and 1240cm -1 CO at 1700-1750cm -1 The absorption peak intensity of C=O at the position was significantly reduced. The strain G1B mainly achieved the degradation of PET by breaking the ester bond of PET and converting the original large molecular insoluble matter into smaller molecular substances.

[0035] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Pseudomonas harutini G1B that can degrade PET plastic, characterized by: The strain was named Pseudomonas harutini ( Pseudomonas harudinis ), which was deposited in the China General Microbiological Culture Collection Center on September 9, 2024, with the deposit number CGMCC NO.31904, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code of the deposit address is 100101.

2. A bacterial agent for degrading PET plastic, characterized in that: Contains the Pseudomonas harutini G1B according to claim 1.

3. Use of the Pseudomonas harutini G1B described in claim 1 and the bacterial agent described in claim 2 in degrading PET plastic.

4. A method for biodegrading PET plastics, characterized in that: The Pseudomonas harutini G1B described in claim 1 is prepared into a bacterial liquid, and inoculated into a liquid culture medium containing PET plastic for cultivation.

5. The method according to claim 4, characterized in that: The OD of the bacterial solution 600 The value is 1.

0.

6. The method according to claim 4, characterized in that: The inoculation ratio was 4% v / v.

7. The method according to claim 4, characterized in that: The content of the PET plastic in the liquid culture medium is 1 g / L.

8. The method according to claim 4, characterized in that: The liquid culture medium is an inorganic salt liquid culture medium, and its formula is: K2PO4 1.5g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.2g / L, (NH4)2SO4 1.5g / L, NaCl 0.5g / L, FeCl·6H2O 0.001g / L, CaCl·2H2O 0.02g / L.

9. The method according to claim 4, characterized in that: The culture conditions are: 30° C., 180 rpm, and dark culture.

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