Polyvinyl chloride plastic degrading bacteria and application thereof

Abstract

The application belongs to the technical field of microbial application, and specifically discloses a polyvinyl chloride plastic degrading bacterium and application thereof.The degrading bacterium is Kosakoniapseudosacchari FR-4, which was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on August 27, 2024, and the address of the GDMCC is the 5th floor of the Experimental Building, 100, Jiefang Road, Yuexiu District, Guangzhou, Guangdong, and the preservation number is GDMCC No: 65062.The Kosakoniapseudosacchari FR-4 of the application is separated from the soil of a waterfowl farm, can grow by taking PVC without additives as the only carbon source, and can degrade polyvinyl chloride plastic.The strain provided by the application provides a new resource for biodegradation of polyvinyl chloride plastic, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial application, and particularly relates to a PVC plastic degrading bacterium and application thereof. BACKGROUND

[0002] With the development of chemical industry, plastic products are widely used in various packaging materials and the like due to lightness, durability, cheapness and convenience. Polyvinyl chloride (PVC) is a high polymer material polymerized from vinyl chloride monomers, which has good flame retardance and chemical stability, poor environmental degradation capacity, is one of the plastics with more serious plastic pollution in the world, and seriously affects the environment, animals and plants and human safety, so it is urgent to find an efficient and green way to solve the problem of PVC plastic pollution.

[0003] At present, the methods for treating PVC plastic pollution that are more commonly used include photodegradation of plastic, cracking degradation and microbial degradation. Microorganisms are important promoters of material circulation in ecological environment due to the large number of species and strong decomposition and metabolism capacity. Therefore, in recent years, microbial degradation has become the mainstream due to the advantages of low cost, low energy consumption and less negative impact on the environment. The microbial remediation method can make up for the shortcomings of physical and chemical remediation technologies, utilizes the characteristics of PVC degrading bacteria that use PVC as carbon source and energy, and achieves the purpose of eliminating pollution and restoring ecological balance, and is a green and promising PVC degradation technology. However, the resources of bacteria with PVC degradation function that have been found at present are less, so screening and preparing PVC plastic degrading bacteria have great application prospect. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a PVC plastic degrading bacterium and application thereof, which provides new bacterial resources and technical means for solving the problem of PVC plastic pollution.

[0005] The first purpose of the present application is to provide a Kosakoniapseudosacchari FR-4 with PVC degradation function, which was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on August 27, 2024, and the address of the GDMCC is Experimental Building 5th Floor, 100, Jiefang Road, Yuexiu District, Guangzhou, Guangdong, China, and the postcode is 510070, and the preservation number is GDMCC No: 65062.

[0006] The degrading bacteria described in the application is isolated from the soil of waterfowl farms, named Kosakonia pseudosacchari FR-4, and can grow and degrade PVC with no additives as the sole carbon source. It is found that the strain can reduce the weight of PVC by weight measurement method; the infrared spectrum detection shows that the bacteria produce new O-H and C=C absorption peaks on the surface of the PVC film; under the electron microscope, it is observed that the bacteria can form obvious grooves and holes on the surface of the PVC film, causing “corrosion pits”, indicating that the bacterial bodies grow in large quantities on the surface of the PVC plastic; the water contact angle of the PVC film treated by the bacteria is reduced, indicating that the hydrophilicity is increased. The application proves that the bacteria Kosakonia pseudosacchari FR-4 has the ability to degrade PVC from the microbial level and the polymer material.

[0007] A second object of the application is to provide the application of the above-mentioned Kosakonia pseudosacchari FR-4 in biodegradation of PVC.

[0008] Preferably, the PVC plastic is a particle, a film or a powder of PVC plastic.

[0009] Preferably, the degradation is to grow and colonize on the surface of the plastic with PVC plastic as the sole carbon source, and gradually degrade the plastic.

[0010] A third object of the application is to provide the application of the above-mentioned Kosakonia pseudosacchari FR-4 in preparation of a preparation for degrading PVC.

[0011] In a specific embodiment of the application, a method for preparing the above-mentioned preparation for degrading PVC is provided, comprising the following steps:

[0012] (1) the above-mentioned Kosakonia pseudosacchari FR-4 is activated on an LB plate culture medium to prepare a primary seed liquid;

[0013] (2) then the primary seed liquid is cultured, and the strain is cultured in a constant temperature incubator for 24 h at a speed of 180 rpm and a temperature of 28-37℃;

[0014] (3) then the bacterial liquid is mixed with 50% glycerol water at a ratio of 1:1, and preserved at-80℃, to finally obtain a strain preparation.

[0015] The fourth object of the present application is to provide a preparation for degrading polyvinyl chloride, wherein the active ingredient of the preparation comprises the above-mentioned Kosakonia niapseudosacchari FR-4 or its microbial inoculant, and can further comprise other auxiliary materials or carriers.

[0016] The fifth object of the present application is to provide the use of the above-mentioned preparation in biodegrading polyvinyl chloride.

[0017] Preferably, the polyvinyl chloride is polyvinyl chloride plastic particles, films or powders.

[0018] The present application also provides a method for degrading polyvinyl chloride plastic, which comprises culturing the above-mentioned Kosakonia niapseudosacchari FR-4 or the above-mentioned preparation, and then contacting the polyvinyl chloride plastic to be degraded with the culture solution or culture.

[0019] Compared with the prior art, the present application has the following advantages:

[0020] (1) The Kosakonia niapseudosacchari FR-4 of the present application is isolated from the soil of a waterfowl farm, and is grown by using polyvinyl chloride plastic as the sole carbon source. After strain treatment, the weight loss rate of the PVC plastic film can reach 21.36%. Through electron microscopy, laser infrared imaging spectroscopy and contact angle testing and analysis, it is found that the surface of the degraded polyvinyl chloride plastic is oxidized, uniform erosion and gully traces appear, and the hydrophilic groups on the surface of the PVC increase, which proves that the Kosakonia niapseudosacchari FR-4 has a certain degrading effect on PVC plastic.

[0021] (2) The Kosakonia niapseudosacchari FR-4 provided by the present application is a prokaryote, which is easy to genetically manipulate, and provides a research basis for revealing the mechanism of biodegrading PVC at the molecular biology level.

[0022] (3) The Kosakonia niapseudosacchari FR-4 provided by the present application can be mass-produced by culture, and can be used for preparing polyvinyl chloride degrading preparations, which are low in production cost, environmentally friendly, easy to use, provide a new resource for biodegrading polyvinyl chloride plastic, and have good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure is the growth curve of the strain Kosakonia niapseudosacchari FR-4 of the present application grown by using PVC as the sole carbon source.

[0024] Figure 2 Growth morphology of strain Kosakonia pseudosacchari FR-4 in LB medium.

[0025] Figure 3 Gram staining microscopic observation of strain Kosakonia pseudosacchari FR-4.

[0026] Figure 4 Genetic evolution tree map of strain Kosakonia pseudosacchari FR-4.

[0027] Figure 5 PVC membrane degradation rate line graph after treatment of PVC membrane by strain Kosakonia pseudosacchari FR-4 for 120 days.

[0028] Figure 6 Infrared spectrum of PVC membrane surface after treatment of PVC membrane by strain Kosakonia pseudosacchari FR-4 for 120 days.

[0029] Figure 7 Electronic microscope graph of PVC membrane surface after treatment of PVC membrane by strain Kosakonia pseudosacchari FR-4 for 120 days: A is before treatment; B is after treatment.

[0030] Figure 8 Change schematic diagram of water contact angle of PVC membrane surface after treatment of PVC membrane by strain Kosakonia pseudosacchari FR-4 for 120 days: A is negative control group; B is treatment group. DETAILED DESCRIPTION

[0031] The technical solutions of the present application will be described in detail below with specific embodiments of the present application. It should be pointed out that the provided embodiments only represent a part of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0032] The test methods used in the embodiments of the present application are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available from commercial channels unless otherwise specified.

[0033] Isolation, purification and identification of PVC-degrading bacterium Kosakoniapseudosacchari FR-4

[0034] 1. Isolation and purification of PVC-degrading bacterium Kosakoniapseudosacchari FR-4

[0035] (1) The sediment sample was collected from the soil of a waterfowl farm in Jinli Town, Zhaoqing. 5 g of the sediment sample was placed in a culture bottle containing 90 mL of normal saline and incubated in a constant temperature shaker at 30°C and 180 rpm for 12 h. Then, 2 mL of the suspension was added to 30 mL of a normal inorganic salt medium MM30 liquid medium (MM30 carbon-free medium / L: 1 g (NH4)2SO4, 3 g KH2PO4, 6 g Na2PO4, 0.5 mL trace metal salt solution; Mixture 44 solution / 100 mL: 17.7 mg Na2B4O7·10H2O, 39.2 mg CuSO4·5H2O, 20.1 mg CaCl2·6H2O, 1095 mg ZnSO4·7H2O, 250 mg EDTA, 100-150 μL concentrated H2SO4; trace metal salt solution / 100 mL: 0.5 mg EDTA, 1 mg CaCO3, 0.5 mg FeSO4·H2O, 10 mg MgSO4·7H2O, 10 mg MnSO4·H2O, 10 mL Mixture 44) and mixed well in a shaker at 30°C and 180 rpm / min. Then, 0.2 g of PVC was added and incubated for 10 days. 2 mL of the bacterial suspension was added to MM30 liquid medium containing 0.2 g of PVC for subculture for 6 times, with PVC as the sole energy and carbon source.

[0036] (2) Isolation and culture: normal saline was used as the diluent, and 100 μL of the bacterial solution was serially diluted, with three parallel gradients for each gradient. For screening of PVC-degrading bacteria, MM30 solid medium containing 0.2 g of PVC was used, and the bacterial solution was diluted to a concentration of 10 -6 The single colony was the best. All solid media were incubated in a constant temperature incubator at 37°C for 48 h.

[0037] (3) Purification: for the single strain with PVC-degrading ability obtained by continued purification culture, MM30 solid medium was used with 0.1 g of PVC as the sole carbon source and energy source. It was placed in a constant temperature incubator at 37°C and incubated for 48 h, and then single colonies growing around the PVC were selected for the next experiment.

[0038] 2. Detection of growth characteristics of PVC-degrading bacterium Kosakoniapseudosacchari FR-4 and identification of the strain

[0039] (1) PVC powder pretreatment

[0040] PVC powder was placed in a dry sterile petri dish and irradiated under UV light for 6 h, turning every 30 min to ensure that the PVC powder was fully sterilized. Then it was inoculated on LB solid medium and incubated at 30°C for 3 d. If no colonies grew around the powder, it was considered sterile and could be used directly in the following experiments.

[0041] (2) Growth of degrading bacteria in MM30 liquid medium with PVC as the sole carbon source

[0042] The isolated degrading bacteria were inoculated in LB liquid medium and cultured until the bacteria reached the logarithmic growth phase. Then they were centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and physiological saline was added and centrifuged for 10 min. The above steps were repeated twice, and finally physiological saline was added to dilute the bacterial solution to OD600 = 1.0. 4 mL of the bacterial solution was transferred to 36 mL of MM30 liquid medium containing 0.2 g of PVC powder, and the total volume of the medium was 40 mL. The MM30 liquid medium without inoculation of degrading bacteria was used as a blank control and was incubated in a 30°C 180 rpm shaker. Each group had 3 parallel experiments, and the absorbance value at 600 nm was measured every 12 h.

[0043] As shown in Figure 1 , the degrading bacteria Kosakoniapseudosacchari FR-4 were cultured in a conical flask containing MM30 medium with PVC powder for 180 h, and the OD value of the bacterial solution was measured every 12 h. The results showed that the degrading bacteria FR-4 had three growth stages, entered the logarithmic growth phase at 12 h, reached the peak OD600 at 120 h, and entered the stationary phase at 132 h.

[0044] (3) Morphological observation of degrading bacteria

[0045] The pure culture of bacteria was streaked on LB solid medium and inverted in a constant temperature incubator for 12 h. The colony morphology was observed with the naked eye and recorded.

[0046] As shown in Figure 2 , the degrading bacteria Kosakoniapseudosacchari FR-4 showed round, neat-edged, smooth-surfaced, convex, and milky-white small colonies on LB agar medium.

[0047] (4) Gram staining and microscopic observation of degrading bacteria

[0048] The bacterial pure culture was spread on a slide, Gram staining was performed, and microscopic examination was performed to observe and record its morphology and staining characteristics, as follows:

[0049] Smear: pick a single colony onto a slide with distilled water, spread evenly, and lightly heat on a flame to fix the bacteria.

[0050] Primary staining: stained with crystal violet stain for 1 min, then washed with distilled water to remove the stain.

[0051] Mordanting: mordant with iodine solution for 1 min, and then washed with water.

[0052] Decolorization: add 95% de-coloring alcohol dropwise, act for 30 s; or cover the slide with de-coloring alcohol, immediately pour it off, add de-coloring alcohol dropwise again, act for 10 s, then wash with water and absorb the water.

[0053] Counterstaining: counterstain with saffron aqueous solution for 0.5-1 min, and then wash with water until colorless.

[0054] Microscopic examination: observe under an optical microscope after the slide is dried and free of water.

[0055] The results are shown in Figure 3 The degrading bacteria Kosakoniapseudosacchari FR-4 is a Gram-negative bacteria, observed under an oil immersion lens as pink rod-shaped or nearly spherical rod-shaped, single or paired, and some arranged in clusters.

[0056] (5) Degrading bacteria gene identification

[0057] The TIANGEN bacterial genomic DNA extraction kit (DP302) was used to extract the degrading bacteria DNA, and the specific operation steps are shown in the instruction manual. 2 μL of DNA, 6 μL of ddH2O, 10 μL of enzyme, and 1 μL of universal primer 338F / 806R were mixed by oscillation, and then PCR amplification was performed.

[0058] The PCR product was then sequenced and analyzed. The 16S rRNA gene sequence of the strain is shown in SEQ ID No. 1. The identified bacteria were classified using homology comparison with the 16S rDNA sequences already listed in the NCBI database. In addition, a phylogenetic tree was constructed using the Neighbor Joining algorithm in MEGA 7.0 software, and the results are shown in Figure 4 . Finally, the PVC degrading bacteria Kosakoniapseudosacchari FR-4 was obtained.

[0059] (6) Bacterial strain preservation and recovery

[0060] The single colony was transplanted in LB liquid medium for culture, and the culture liquid in logarithmic phase was used for strain preservation. 0.75 mL of the bacterial liquid and glycerol were added to the strain preservation tube to make the final concentration of glycerol 25%, and after shaking, it was placed in a refrigerator at -80°C for storage. When recovering, the stored strain was thawed, streaked on MM30 solid medium containing PVC powder, and then the single colony on the MM30 solid medium was transferred to MM30 liquid medium.

[0061] Example 2 Degradation of PVC plastic film by degradation bacteria Kosakoniapseudosacchari FR-4

[0062] 1. Pretreatment of PVC film

[0063] The PVC film was placed in a sterile petri dish and irradiated under a UV lamp for 6 h, turning every 30 min to ensure that the PVC film was fully sterilized. Then it was inoculated on LB solid medium and cultured at 30°C for 3 d. If no colonies grew around the film, it was considered to be sterile and could be used directly in the following experiments.

[0064] 2. Weight loss rate of PVC film

[0065] The film was cultured for 120 d using the method described in Example 1, and then weighed and observed under a microscope at 30 d, 60 d, 90 d, and 120 d. To accurately determine the weight loss rate of the PVC film, the PVC was recovered from the culture medium by the following steps: PBS soaking for 2 min; 2% SDS soaking for 2 h; discarding the liquid and washing with warm distilled water for 2-3 times; 2% pentanediol soaking for 2 h; discarding the liquid and sonicating with 50% ethanol for 2 times (30 min each time, and the ethanol was replaced each time); 75% ethanol overnight; discarding the liquid and washing with 100% anhydrous ethanol for 3 times (30 min each time, and the ethanol was replaced each time), discarding the liquid and drying; the weight of the residue was measured using a one-hundredth analytical balance, and the weight loss rate of the PVC was calculated using the following formula:

[0066] Weight loss rate% = (initial weight - weight after culture) / initial weight x 100%

[0067] The results are shown in Figure 5 After 120 d, the weight loss rate of the PVC film by the degradation bacteria Kosakoniapseudosacchari FR-4 was 21.63%, and there was no significant downward trend in the control CK group among all groups. The results showed that the degradation bacteria Kosakoniapseudosacchari FR-4 could degrade PVC to some extent.

[0068] 3. Characterization analysis of PVC plastic film after biological treatment by degradation bacteria

[0069] (1) Agilent 8700L D I R laser infrared imaging spectrometer detection

[0070] During the culture, a certain amount of MM30 liquid medium needs to be added every 10 days to maintain the reaction system at 40 mL, and 3 parallel experiments are set for each group. After being cultured for 120 days, the bacteria are removed according to the above method, and the change of the functional groups on the surface of the PVC film is detected by using the LDIR laser infrared imaging spectrometer. The attenuated total reflection (ATR) mode is used, the wavelength range is 4000-400 cm -1 , and the analysis software is OriginPro 2024.

[0071] The infrared spectrum is shown in Figure 6 Compared with the negative control Control without bacterial treatment, the treated group FR-4 generates new characteristic peaks near 3297.09 cm -1 and 1596.88 cm -1 , respectively, corresponding to the stretching vibration of O-H and the C=C double bond peak, which indicates that the surface of the PVC film is oxidized, and thus it can be seen that the strain FR-4 can play a degradation role on the surface of the PVC film.

[0072] (2) Electron microscope imaging observation

[0073] Since the plastic film is not conductive, the dried PVC film is gold-plated for 120 s, and then photographed using a Zeiss sigma300 scanning electron microscope (SEM) to observe the morphological changes on the surface of the PVC film before and after being treated by the degradation bacterium Kosakoniapseudosacchari FR-4.

[0074] The results are shown in Figure 7 After being treated by the degradation bacterium Kosakoniapseudosacchari FR-4 for 120 days, the PVC film has changed in morphology, the film surface has become rough, and uniform erosion and pit marks have appeared. It is shown that the degradation bacterium Kosakoniapseudosacchari FR-4 has a certain degradation effect on the PVC film.

[0075] (3) Water contact angle test

[0076] The water contact angle is a parameter characterizing the hydrophilicity or hydrophobicity of a material surface and can be measured at room temperature using an optical video contact angle meter. After thoroughly cleaning the PVC membrane with a 2% SDS solution and sterile water, it was dried overnight at room temperature. The membrane surface was then dried using a syringe rubber bulb, and 5 μL of deionized water was dropped onto the PVC surface. The contact angle was measured at three different locations on each PVC membrane.

[0077] like Figure 8 As shown, after treatment with Kosakonia pseudosacchari FR-4, the water contact angle on the plastic film surface decreased by about 15° compared with the negative control group, indicating that the strain enhanced the hydrophilicity of the PVC surface. This was caused by the increase in oxygen-containing groups and roughness, which indirectly verified the degradation effect of the strain.

[0078] Example 3: Preparation of Polyvinyl Chloride Plastic Degrading Bacterial Agent

[0079] A method for preparing a polyvinyl chloride (PVC) plastic degrading microbial agent includes the following steps:

[0080] (1) Pick the above Kosakonia pseudosacchari FR-4 and activate it in LB plate medium to prepare primary seed culture;

[0081] (2) The seed culture was then expanded, and the strain was placed in a constant temperature shaker for 24 hours at a speed of 180 rpm and a temperature of 28-37℃.

[0082] (3) The bacterial solution was then mixed with 50% glycerol water at a ratio of 1:1 and stored at -80℃ to obtain the bacterial preparation.

[0083] The degradation bacteria preparation prepared in Example 3 of this invention can react directly with polyvinyl chloride plastic and achieve the same degradation effect as in Example 2.

[0084] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A polyvinyl chloride degrading bacterium, characterized in that, The degrading bacteria mentioned are Cossackella ( Kosakonia pseudosacchari FR-4 was deposited on August 27, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCCNo:65062.

2. The application of the polyvinyl chloride degrading bacteria according to claim 1 in the biodegradation of polyvinyl chloride, characterized in that, The degrading bacteria mentioned are Cossackella ( Kosakonia pseudosacchari FR-4 was deposited on August 27, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCCNo:65062.

3. The application according to claim 2, characterized in that, The polyvinyl chloride mentioned is polyvinyl chloride plastic granules, films, or powders.

4. The application according to claim 3, characterized in that, The degradation process involves using polyvinyl chloride (PVC) plastic as the sole carbon source to grow and colonize the plastic surface, gradually degrading the plastic.

5. The application of the polyvinyl chloride degrading bacteria according to claim 1 in the preparation of polyvinyl chloride degrading agents, characterized in that, The degrading bacteria mentioned are Cossackella ( Kosakonia pseudosacchari FR-4 was deposited on August 27, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCCNo:65062.

6. A formulation for degrading polyvinyl chloride, characterized in that, The active ingredient of the formulation contains Cossackie FR-4 or an agent thereof as described in claim 1.

7. The application of the formulation according to claim 6 in the biodegradation of polyvinyl chloride.

8. The application according to claim 7, characterized in that, The polyvinyl chloride is polyvinyl chloride plastic granules, film, or powder.

9. A method for degrading polyvinyl chloride plastic, characterized in that, The method includes culturing Cossackie FR-4 as described in claim 1 or the preparation as described in claim 6, and then contacting the polyvinyl chloride plastic to be degraded with the culture medium or culture.

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