Stenotrophomonas and application thereof in degradation of PVC (polyvinyl chloride) plastics

Abstract

The invention discloses a stenotrophomonas sp. P2112 strain and an application of the stenotrophomonas sp. P2112 strain in degradation of PVC (polyvinyl chloride) plastics. The stenotrophomonas sp. Is stenotrophomonas sp. P2112, the stenotrophomonas sp. Is preserved in the China Center for Type Culture Collection, and the preservation number of the stenotrophomonas sp. Is CCTCC (China Center for Type Culture Collection) NO: M20241725. The stenotrophomonas sp. P2112 can grow by taking the PVC plastic as a unique carbon source, and the growth of the biomass of the thallus in the fermentation process is accompanied by the weight loss of the PVC plastic. The stenotrophomonas sp. P2112 can be used for catalyzing PVC (polyvinyl chloride) plastics to generate a compound 1 and catalytically degrading a structural analogue 5-chlorovaleric acid of the PVC plastics to generate a compound 2. The microbial stenotrophomonas sp. P2112 for degrading the PVC plastic, which is provided by the invention, provides a new microbial strain resource for the biodegradation of the PVC plastic.

Description

Technical Field

[0001] The invention relates to the field of plastic degradation and environmental protection, and in particular to a strain of oligotrophic monocytogenes and application thereof in degrading PVC plastic. Background Art

[0002] Polyvinyl chloride (PVC), abbreviated as PVC in English, is the third largest synthetic polymer plastic in the world (after polyethylene and polypropylene), with an annual production of about 40 million tons of PVC. PVC was once the world's largest general-purpose plastic with a wide range of applications. However, the pollution caused by discarded PVC plastics is a serious problem. Many studies have reported the thermal degradation and photodegradation of PVC. When PVC is exposed to short-wavelength radiation, its photodegradation is accelerated. In addition to photodegradation and thermal degradation, biodegradation is a solution to PVC plastic pollution with great potential. At present, researchers have isolated PVC-degradable strains from soil with discarded PVC and tested their degradation ability. Sharma et al. isolated a PVC-degrading bacterium with high degradation ability from the soil. After 30 days of cultivation, the weight loss of the PVC film was 22.22%. The subsequent strain was identified as Bacillus cereus. Some researchers have isolated Bacillus and Micrococcus that can degrade polyvinyl chloride, and compared the degradation ability of the two bacteria. The culture time was six months. The results showed that Bacillus had a higher degradation potential than Micrococcus, and the weight loss of its PVC film was 12.7%. Danko et al. found that Pseudomonas putida and Chromobacterium versicolor isolated from hazardous waste sites had a degradation effect on vinyl chloride, and believed that the degradation of vinyl chloride by the two strains was initiated by an olefin monooxygenase. Giacomucci et al. observed through experiments that Pseudomonas citronella can form a dense biofilm on the surface of PVC film, and detected a large number of proteins. The researchers believe that the high expression of these proteins contributes to the formation of biofilms. After 45 days of co-culture of Pseudomonas citronella and PVC film, it was found that the weight loss of PVC film was about 13%; after 90 days of culture, it was found that the relative molecular mass of PVC decreased, indicating that Pseudomonas citronella can cause long-chain rupture of polyvinyl chloride. Kumari et al. used the carbon dioxide release method to estimate the remineralization degree of plastic film by Bacillus AIIW2, and used scanning electron microscopy and atomic force microscopy to observe the morphology of the surface of PVC film co-cultured with Bacillus AIIW2. The image showed that the surface of the PVC film became rough, which may be caused by bacterial attachment and biodegradation of the PVC film. Subsequent analysis of the structural changes of the PVC film revealed the formation of alcohol, carboxylic acid and ester groups, indicating that an oxidation reaction occurred during the degradation process. The destruction of the polymer chain triggered initial oxidation and chain breakage, which in turn caused the degradation products to enter β-oxidation and citric acid cycle, and finally released carbon dioxide and water. Anwar et al. co-cultured bacterial flora with PVC film, and the results showed that the average relative molecular mass of the PVC film decreased significantly with the increase of culture time. Patil et al. isolated and screened yellow micrococci with PVC degradation ability, and cultured them with pure PVC as the only carbon source. It was found that within 70 days, the yellow micrococci released 0.36% of chlorine and 8.87% of mineralization products, respectively.Peng et al. found that T. molitor larvae can rapidly degrade polymers and can degrade PVC into organic chloride intermediates, but the degree of mineralization is very limited, with only 2.9% of PVC converted into chloride. When the intestinal microorganisms are inhibited by the antibiotic gentamicin, depolymerization stops, indicating that T. molitor larvae rely on intestinal microorganisms to degrade PVC.

[0003] Zhang et al. isolated a bacterium (Klebsiella EMBL-1) from the intestines of insect larvae that can degrade and use PVC as the only carbon source for growth. With PVC film as the only carbon source, 90 days after inoculation, the EMBL-1 strain formed a dense biofilm on the surface of the PVC film, and the average weight loss of the PVC film was finally detected to be 19.57%, indicating that the strain had a depolymerization effect on the long-chain structure of PVC. Through proteomic analysis of EMBL-1 degradation of PVC, five proteins were found to be likely involved in the PVC degradation process, namely catalase-peroxidase, dehalogenase, enolase, aldehyde dehydrogenase and oxygenase, and a PVC biodegradation pathway was proposed. Catalase-peroxidase is a key enzyme for PVC degradation, but the degradation product is not a single product, which means that the degradation mechanism of the enzyme remains to be studied. Laccase is an oxidoreductase that has the ability to oxidize phenolic compounds and can degrade macromolecules such as cellulose and lignin. Sumathi et al. have proved through experiments that laccase can break long chains of PVC, but the specific mechanism is still unclear. It is speculated that the oxygen free radicals in laccase may attack carbon-carbon single bonds, and the short-chain compounds produced by the reaction will be subsequently degraded by other unknown enzymes. Although some microbial strains have been proven to have the ability to degrade PVC, and some degrading enzymes that can degrade PVC have been discovered, the degradation enzymes and degradation mechanisms of PVC are still not very clear, and the resources of PVC-degrading bacteria are not rich enough. Summary of the invention

[0004] In view of this, one of the purposes of the present invention is to provide a Stenotrophomonas sp. that can be used to degrade PVC plastics in order to address the problem of insufficient resources of existing PVC plastic-degrading microbial strains. The Stenotrophomonas sp. is Stenotrophomonas sp. P2112, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20241725 and a deposit date of August 2, 2024.

[0005] The second purpose of the present invention is to provide a method and application of Stenotrophomonas sp. P2112 for degrading PVC plastic. The method is that Stenotrophomonas sp. P2112 is grown by both solid plate culture and liquid fermentation culture using PVC plastic as the only carbon source; the application is that after culturing Stenotrophomonas sp. P2112 to obtain bacterial cells, the bacterial cells are used to catalytically degrade PVC plastic.

[0006] The specific technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a strain of Stenotrophomonas sp., which is Stenotrophomonas sp. P2112, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20241725.

[0008] Furthermore, the 16S rDNA sequence of the Stenotrophomonas sp. P2112 strain is shown in SEQ ID NO:1.

[0009] The second aspect of the present invention provides a method for degrading PVC plastic using Stenotrophomonas sp. P2112.

[0010] Specifically, the present invention provides a method for degrading PVC plastic by Stenotrophomonas sp. P2112, comprising the following steps:

[0011] The Stenotrophomonas sp. P2112 strain is inoculated into a mineral salt liquid culture medium or a mineral salt solid culture medium containing PVC plastic for fermentation culture. The strain degrades the PVC plastic to achieve growth, and the growth of the bacterial biomass during the fermentation process is accompanied by a reduction in the weight of the PVC plastic.

[0012] Furthermore, the fermentation temperature is 25-40°C; the pH value of the fermentation is 6.0-9.0; the dissolved oxygen of the fermentation is 20%-100%, and the fermentation time is 1-7 days.

[0013] Furthermore, the mineral salt liquid culture medium is composed of 10.0 g of ammonium sulfate, 1.0 g of potassium dihydrogen phosphate, 0.4 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, 2.5 g of urea, 0.01 g of ferrous sulfate heptahydrate, 0.01 g of manganese sulfate tetrahydrate, 0.05 mg of biotin and water, and the volume is adjusted to 1 liter with water; the mineral salt solid culture medium is added with 20 g of agar per liter on the basis of the liquid culture medium.

[0014] Furthermore, the Stenotrophomonas sp. P2112 cultured on PVC plastic on a mineral salt solid culture medium was observed using a scanning electron microscope, and it was found that the bacterial cells adhered to the surface of the PVC plastic and grew well.

[0015] The invention also provides the use of Stenotrophomonas sp. P2112 in degrading PVC plastics and / or 5-chlorovaleric acid.

[0016] Specifically, firstly, Stenotrophomonas sp. P2112 is propagated and cultured to obtain bacterial cells, and then the bacterial cells are applied to catalytic degradation of PVC plastic to obtain the degradation product compound 1.

[0017] Furthermore, firstly, Stenotrophomonas sp. P2112 is propagated and cultured to obtain bacterial cells, and then the bacterial cells are applied to catalytic degradation of 5-chlorovaleric acid to obtain the degradation product compound 2.

[0018] The degradation reaction is carried out in a 50 mM phosphate buffer solution (pH 7.0-9.0); the reaction temperature is 25-40° C.; the reaction is an aerobic reaction and needs to be carried out in a shaking incubator at 180-300 rpm for 2 days.

[0019] Furthermore, after the degradation reaction was completed, high performance liquid chromatography was used to detect and obtain degradation products, compound 1 and compound 2, respectively. It can be seen that Stenotrophomonas sp. P2112 degrades PVC plastic and PVC plastic structural analog 5-chlorovaleric acid through the degradation enzyme displayed on the bacterial cell surface.

[0020] The beneficial effects of the present invention are:

[0021] The invention provides a strain of Stenotrophomonas sp. P2112 for degrading PVC plastic, and provides a method and application of the strain for degrading PVC plastic, thereby providing a new method for biodegradation of PVC plastic. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the phylogenetic tree of Stenotrophomonas sp. P2112 based on 16S rDNA.

[0023] Figure 2This is a photo of the culture of Stenotrophomonas sp. P2112 on a mineral salt solid culture plate with PVC plastic film as the only carbon source;

[0024] Figure 3 This is a scanning electron microscope photo of Stenotrophomonas sp. P2112 growing on PVC plastic film;

[0025] Figure 4 This is the bacterial growth curve of Stenotrophomonas sp. P2112 cultured in a shake flask in a mineral salt liquid medium with PVC plastic film as the only carbon source. No PVC plastic was added to the control group.

[0026] Figure 5 The weight loss statistics of PVC plastics in shake flask culture of Stenotrophomonas sp. P2112 in mineral salt liquid medium with PVC plastic film as the only carbon source;

[0027] Figure 6 It is the high performance liquid chromatography detection of compound 1, a degradation product of PVC plastic catalyzed by the bacterial cells of Stenotrophomonas sp. P2112;

[0028] Figure 7 It is the high performance liquid chromatography detection of the degradation product compound 2 of 5-chlorovaleric acid catalyzed by Stenotrophomonas sp. P2112 bacterial cells. DETAILED DESCRIPTION

[0029] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0030] The experimental methods used in the following examples are all conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] The bacterial species used in the following examples are all Stenotrophomonas sp. P2112, and the culture medium formula used in the examples is as follows:

[0033] The mineral salt liquid culture medium contains 10.0 g of ammonium sulfate, 1.0 g of potassium dihydrogen phosphate, 0.4 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, 2.5 g of urea, 0.01 g of ferrous sulfate heptahydrate, 0.01 g of manganese sulfate tetrahydrate, and 0.05 mg of biotin per liter. It is made up to 1 liter with water and has a pH value of 7.0.

[0034] Mineral salt solid culture medium is mineral salt liquid culture medium with 20g agar added per liter.

[0035] Each liter of 2216E liquid culture medium contains 5.0 g of peptone, 1.0 g of yeast powder, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 5.98 g of magnesium chloride, 3.24 g of sodium sulfate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium carbonate, 0.08 g of potassium bromide, 0.034 g of strontium chloride, 0.022 g of boric acid, 0.004 g of sodium silicate, 0.0024 g of sodium fluoride, 0.0016 g of sodium nitrate, and 0.008 g of disodium hydrogen phosphate. Make up to 1 L with water, and the pH value is 7.6±0.2.

[0036] 2216E solid agar medium is based on 2216E liquid medium, with 20g of agar added per liter.

[0037] LB medium contains 10.0 g of peptone, 5.0 g of yeast powder, and 10.0 g of sodium chloride per liter, and is made up to 1 L with water, with a pH value of 7.0.

[0038] Example 1

[0039] Isolation and identification of PVC plastic-degrading strain P2112

[0040] PVC plastic powder was added to the mineral salt liquid culture medium as a carbon source, and the obtained deep-sea sediments were enriched by shake flask culture for 1 week, diluted with sterile water and coated on 2216E solid culture medium plates, and single colonies were selected for streaking purification culture. The obtained pure culture strain was inoculated into the mineral salt liquid culture medium containing PVC plastic film for test tube culture, and the ability of the pure culture to degrade PVC plastic was verified. A strain with the ability to degrade PVC plastic was obtained, numbered P2112. The genome of the strain was extracted and whole genome sequencing was performed, and the obtained 16S rDNA sequence is shown in SEQ ID NO:1. The 16S rDNA of strain P2112 was compared on the website https: / / www.ezbiocloud.net / , and it was found that it was the same as the standard strain Stenotrophomonas sepilia SM-16975 TThe similarity was 99.93%, indicating that strain P2112 was Stenotrophomonas sp., and the strain was named Stenotrophomonas sp. P2112. The phylogenetic tree based on the 16S rDNA sequence of the strain was constructed using Mega 6.0, showing that strain P2112 and strain Stenotrophomonas geniculateATCC 19374 (T) were clustered in the same branch and had a close relationship ( Figure 1 ). Stenotrophomonas sp. P2112 has been deposited in China Center for Type Culture Collection (CCTCC) in 2024, address: Wuhan University, Wuhan, China, zip code 430027, and its deposit number is: CCTCC NO: M20241725.

[0041] Example 2

[0042] Detection of PVC degradation function of Stenotrophomonas sp. P2112 cultured on solid plates

[0043] Stenotrophomonas sp. P2112 was inoculated into a test tube containing LB liquid medium for cultivation. After cultivation, the bacteria were obtained by centrifugation and washed three times with sterile water. The bacterial cells were resuspended with an equal volume of sterile water, and 10 μl of dilution was spread on a mineral salt solid medium plate. The plate was covered with a sterile PVC plastic film and cultured at 30°C for one week. It was found that bacteria grew on the edge of the PVC plastic film ( Figure 2 ). The bacteria at the edge of the plastic film were selected for culture, and the genome was extracted, amplified, and sequenced by 16S rDNA, which was confirmed to be Stenotrophomonas sp. P2112. The PVC plastic film was peeled off from the culture plate and observed by scanning electron microscopy, which showed that a large number of bacteria grew on the plastic film ( Figure 3 ).

[0044] Example 3

[0045] Growth test of Stenotrophomonas sp. P2112 in shake flask culture

[0046] Stenotrophomonas sp. P2112 was inoculated into a test tube containing LB liquid medium for cultivation. The cultured seeds were inoculated into a 500 ml shake flask containing 150 ml of mineral salt liquid medium at 1% and 2.8 g of PVC plastic film was added to the shake flask. The culture temperature was 30°C, the shaker speed was 220 rpm, and the culture time was 7 days. The biomass OD of the bacterial growth was measured. 600 The control group did not add PVC plastic film, and the other conditions were the same as the experimental group. The results showed that the OD of Stenotrophomonas sp. P2112 600 The value increased with the increase of culture time and reached 0.3 on the 7th day. The bacterial OD of the control group 600 The value does not increase ( Figure 4 ), indicating that Stenotrophomonas sp. P2112 degrades PVC plastics to grow. Through the weight loss test of PVC plastics cultured with Stenotrophomonas sp. P2112 for 3 and 6 days, it was found that the weight loss was 14.6 mg in 3 days, with a weight loss rate of 0.51%, and the weight loss was 20.6 mg in 6 days, with a weight loss rate of 0.7%. ( Figure 5 ).

[0047] Example 4

[0048] Degradation of PVC Plastics Catalyzed by Stenotrophomonas sp. P2112 Cells

[0049] Stenotrophomonas sp. P2112 was inoculated into LB liquid medium and cultured overnight. The cells were collected by centrifugation and washed three times with sterile water. The cells were added to 300 mM phosphate buffer solution (pH 7). The catalytic system was 1 L. The final cell concentration was OD 600 is 2.0, PVC plastic film 66g, temperature is 30℃, shaking speed is 220 rpm, catalytic time is 13 days, no PVC plastic is added to the control group, and the other conditions are the same as the experimental group. After the catalysis is completed, the supernatant is centrifuged and extracted with an equal volume of ethyl acetate. The organic phase is rotary evaporated and dissolved in 500μl methanol for high performance liquid chromatography detection. The high performance liquid chromatography conditions are: chromatographic column COSMOSIL 5C18-AR-II, methanol 45%, 20mM phosphate (pH 1.5) 55%, column temperature 30℃, detection wavelength 240nm. The test results show that the experimental group of Stenotrophomonas sp. P2112 degrading PVC plastic has an additional obvious chromatographic peak compared with the control group, with a retention time of 6.1min, named compound 1 ( Figure 6).

[0050] Example 5

[0051] Degradation of 5-chlorovaleric acid, a PVC plastic analogue, by Stenotrophomonas sp. P2112

[0052] Stenotrophomonas sp. P2112 was inoculated into LB liquid medium and cultured overnight. The cells were collected by centrifugation and washed three times with sterile water. The cells were added to 300 mM phosphate buffer solution (pH 7), 150 mL of the catalyst system, 5 mM 5-chlorovaleric acid, and the final cell concentration OD 600 The reaction mixture was stirred at 2.0, the temperature was 30°C, the shaking speed was 220 rpm, the catalytic time was 1 day, and no 5-chlorovaleric acid or bacteria was added to the control group. The other conditions were the same as those of the experimental group. After the catalysis was completed, the supernatant was centrifuged and filtered for HPLC detection. The HPLC conditions were: chromatographic column COSMOSIL 5C18-AR-II, 150mM ammonium sulfate (pH7) 50%, water 50%, column temperature 30°C, and detection wavelength 240nm. The test results showed that the experimental group of Stenotrophomonas sp. P2112 degrading 5-chlorovaleric acid had an additional obvious chromatographic peak compared with the control group, with a retention time of 8.1min, named compound 2 ( Figure 7 ).

[0053] Although the above embodiments have been described, once those skilled in the art know the basic creative concepts, they can make additional changes and modifications to these embodiments. Therefore, the above description is only an embodiment of the present invention and does not limit the patent protection scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the specification and drawings of the present invention, or directly or indirectly used in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A strain of Oligotrophomonas, characterized in that The Stenotrophomonas sp. is Stenotrophomonas sp. P2112, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20241725.

2. A strain of oligotrophomonas according to claim 1, characterized in that The 16S rDNA sequence of the strain is shown as SEQ ID NO:

1.

3. A method for degrading PVC plastics by oligotrophic monocytogenes as claimed in claim 1 or 2, characterized in that: The method is that Stenotrophomonas sp. P2112 uses PVC plastic as the only carbon source for solid plate culture or liquid fermentation culture, and the culture process is the process of degrading PVC plastic.

4. The method according to claim 3, characterized in that The fermentation temperature is 25-40°C; the pH value of the fermentation is 6.0-9.0; the dissolved oxygen of the fermentation is 20%-100%; and the fermentation time is 1-7 days.

5. The method according to claim 3, characterized in that: The mineral salt liquid culture medium used for liquid culture consists of 10.0 g of ammonium sulfate, 1.0 g of potassium dihydrogen phosphate, 0.4 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, 2.5 g of urea, 0.01 g of ferrous sulfate heptahydrate, 0.01 g of manganese sulfate tetrahydrate, 0.05 mg of biotin and water, and the volume is adjusted to 1 liter with water; the mineral salt solid culture medium used for solid plate culture is prepared by adding 20 g of agar per liter to the mineral salt liquid culture medium.

6. Use of Stenotrophomonas sp. P2112 as claimed in claim 1 or 2 in degrading PVC plastics and / or 5-chlorovaleric acid.

7. The use according to claim 6, characterized in that: First, Stenotrophomonas sp. P2112 was propagated and cultured to obtain bacterial cells, and then the bacterial cells were used to catalytically degrade PVC plastic to obtain the degradation product compound 1.

8. The use according to claim 6, characterized in that: First, Stenotrophomonas sp. P2112 was propagated and cultured to obtain bacterial cells, and then the bacterial cells were used to catalytically degrade 5-chlorovaleric acid to obtain the degradation product compound 2.

9. According to the use of claim 7 or 8, the degradation reaction is carried out in a 50 mM phosphate buffer solution (pH 7.0-9.0); the reaction temperature is 25-40°C; the reaction is an aerobic reaction and needs to be carried out in a shaker at 180-300 rpm.

10. According to the use of claim 7 or 8, the degradation products Compound 1 and Compound 2 are both obtained by high performance liquid chromatography.

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