Polystyrene plastic-decomposing bacteria, suspension and fermentation culture solution using the same, and method for decomposing plastics

Abstract

To provide a polystyrene plastic-decomposing bacterium, and to provide a method for decomposing plastics using the same.SOLUTION: Bacillus aryabhattai (Priestia aryabhattai) Red22 was deposited at the Guangdong Provincial Microbial Strain Depositary Center on May 8, 2023, with the deposit number GDMCC No: 63428. The polystyrene plastic-decomposing bacterium-Bacillus aryabhattai Red22 provided by the present invention has obvious decomposition ability in actual water bodies of different environments, so that the Red22 bacterium has good environmental adaptability, provides a new resource for the biodegradation of polystyrene plastics, and is expected to have relatively good applications.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to the technical field of microbial application, and more particularly to a polystyrene plastic decomposition bacterium and a plastic decomposition method using the same. [Background technology]

[0002] With the rapid development of global industrial and agricultural production and the needs of human life, the production of plastic products is increasing year by year, but only about 10% can be recycled each year, and the rest is incinerated or buried, decomposed by weathering, and disperses and moves into tiny fragments, widely distributed in soil, rivers and oceans. When plastics are 5mm or smaller in size, they become microplastics, which inhibit the immune response of living organisms and accumulate through the food chain as persistent pollutants that increase the risk of diseases such as infertility, obesity and cancer.

[0003] Polystyrene (PS), which is widely used in the food and remodeling industries, is one of the most polluting plastics in the world because of its chemical stability and the difficulty of removing it from the natural environment. Compared with treatment methods such as incineration and pyrolysis, in situ decomposition by microorganisms is an important means to solve the pollution problem of polystyrene-based plastics due to its mild conditions, simplicity, and environmental friendliness. PS can be decomposed by microorganisms, but few resources of functional bacterial strains that decompose PS have been identified, and it is urgent to increase the discovery of related functional strains and establish novel microbial treatment methods for PS. Summary of the Invention [Problem to be solved by the invention]

[0004] In view of the current situation in which resources of plastic-degrading functional bacterial strains are scarce and governance methods are lacking, the object of the present invention is to provide a polystyrene-based plastic-degrading bacterium and a plastic decomposition method using the same, in order to provide bacterial strain resources and technical means for solving the polystyrene-based plastic pollution problem. [Means for solving the problem]

[0005] The polystyrene plastic decomposition bacterium of the present invention is Bacillus aryabhattai Red22, deposited on May 8, 2023 at the Guangdong Microbial Strain Deposit Center (GDMCC, address: Building 59, Dayuan, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, 510070, postal code: GDMCC No: 63428).

[0006] A second object of the present invention is to provide a bacteriophage capable of degrading polystyrene-based plastics, which contains the above-mentioned Bacillus aryabataii Red 22 as an active ingredient.

[0007] A third object of the present invention is to provide a use of the above-mentioned Bacillus aryabataii Red 22 in decomposing polystyrene-based plastics.

[0008] Preferably, the decomposition is carried out by growing on polystyrene-based plastic as the sole carbon source, settling on the surface of the plastic, and gradually decomposing the plastic.

[0009] A fourth object of the present invention is to provide a method for decomposing polystyrene-based plastics, which comprises co-cultivating the above-mentioned Bacillus aryabataii Red 22 with polystyrene-based plastics.

[0010] Preferably, the plastic to be degraded is polystyrene particles, or a film, or a powder.

[0011] Preferably, the polystyrene plastic is degraded by Bacillus aryabataii Red 22 in different water bodies.

[0012] Preferably, the Bacillus alyabatai Red 22 is a Bacillus alyabatai Red 22 fermentation broth, and its OD 600is 1.0-1.8, and when inoculated into a liquid containing polystyrene plastic, the inoculation amount is 1%-10% v / v.

[0013] Preferably, the co-culture is performed under the following culture conditions: temperature: 25° C. to 37° C., and shaking culture rotation speed: 150 to 220 rpm. Effect of the Invention

[0014] The present invention is to sieve and isolate polystyrene plastic decomposition bacteria from plastic waste buried in environmental soil, and use them to decompose plastics. Microorganisms are widely present in soil, and have the characteristics of being easy to culture, highly active, and environmentally friendly. When decomposing polystyrene plastics, they can settle on the surface of the plastic and gradually decompose it. After a certain period of decomposition culture, the weight loss rate of polystyrene plastics caused by Red22 reaches 7.3%. Furthermore, as can be seen from the results of analysis such as scanning electron microscope, Fourier transform infrared spectroscopy, and contact angle measurement, obvious grooves and pits were observed on the surface of the decomposed polystyrene plastics, the chemical structure of the decomposed polystyrene plastics changed, the hydrophilic group increased, and the molecular weight decreased, proving that the decomposition effect of this bacterium is obvious. The polystyrene plastic decomposition bacterium Red22 provided by the present invention has obvious decomposition ability in real water areas of different environments, so that the Red22 bacterium has good environmental adaptability, provides a new resource for the biodegradation of polystyrene plastics, and is expected to have relatively good application.

[0015] Priestia aryabhattai Red22 was deposited at the Guangdong Provincial Microbial Strain Deposit Center (GDMCC, address: Building 59, Dayuan, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, 510070, Japan) on May 8, 2023, with the deposit number GDMCC No: 63428. [Brief description of the drawings]

[0016] [Figure 1]A comparative image of the growth state on the surface of a polystyrene plastic sheet after decomposing the polystyrene plastic sheet for 45 days by the Red22 bacteria described in Example 3 of the present invention, using a laser confocal microscope. The left image is the control group, and the right image is the Red22 bacteria treatment group. [Diagram 2] FIG. 11 is a graph showing the mass reduction of the polystyrene plastic sheet described in Example 3 of the present invention after it was decomposed by Red 22 bacteria for 45 days. [Diagram 3] 1 is a comparative SEM image of the polystyrene plastic sheet described in Example 3 of the present invention after it was decomposed by Red22 bacteria for 45 days. The left image is the control group, and the right image is the Red22 bacteria treated group. [Figure 4] FIG. 1 is an FTIR change diagram of the polystyrene-based plastic sheet described in Example 3 of the present invention after it was decomposed by Red 22 bacteria for 45 days. [Diagram 5] FIG. 11 is a graph showing changes in contact angle after the polystyrene plastic sheet described in Example 3 of the present invention was decomposed by Red 22 bacteria for 45 days. [Figure 6] FIG. 11 is a graph showing changes in molecular weight of the polystyrene plastic sheet described in Example 3 of the present invention after it has been decomposed by Red 22 bacteria for 45 days. [Figure 7] FIG. 11 is a graph showing the mass reduction of the polystyrene plastic described in Example 4 of the present invention after it is decomposed by Red22 bacteria in different real water areas for 50 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will now be further described with reference to the following examples, which are used to illustratively explain the present invention and are not intended to limit the present invention in any way.

[0018] Example 1 1. Isolation, purification and identification of polystyrene plastic degrading strains: (1) Isolation and purification of polystyrene plastic decomposition strains: Plastic waste buried in soil was lightly washed with sterilized water, cut into small pieces, placed in a sterilized Erlenmeyer flask, saline and glass beads were added, and the mixture was shaken and cultivated at 200 rpm in a shaker at 30°C for 6 hours to prepare a bacterial suspension. The bacterial suspension was transferred to a carbon-free mineral salt medium containing UV-sterilized polystyrene powder (2 g / L) at an inoculum volume ratio of 10%, 18% glycerol was added, and the mixture was shaken and cultivated at 180 rpm in a shaker at 30°C for 15 days, and samples were taken periodically to measure the OD. 600 After the values ​​were measured and the cultures became turbid, they were transferred to mineral salt medium containing carbon-free polystyrene powder (2 g / L) and cultured three times. The cultures with the fastest growth were selected and diluted with a gradient (10 -3 ~10 -6 ) and isolated the strain by the dilution-coated plate method (LB plate), scribing and purifying it several times to isolate a pure yellow-white strain, which we named red22. The isolated strain was stored in a slant medium and a glycerol tube.

[0019] The carbon-free mineral salt medium screened in the above isolation was obtained by dissolving 0.52g of Na2HPO4, 0.50g of KH2PO4·3H2O, 1.5g of NH4Cl, 0.03g of CaCl2, 0.8g of MgSO4·7H2O, 0.01g of FeSO4·7H2O, 0.02g of ZnSO4·7H2O, 0.02g of CuSO4·5H2O, and 0.006g of MnSO4·H2O in 1L of water, mixing uniformly, and sterilizing.

[0020] (2) Identification of polystyrene plastic degrading strain: DNA of the isolated Red22 strain was extracted, and DNA sequencing was entrusted to Shanghai Bio-Engineering Co., Ltd. Broad spectrum primers 27F: AGAGTTGATCCTGGCTCAG and 1492R: TACGGYTACCTTGTTAYGACTT were used for 16S rRNA amplification. As can be seen from the identification results, the Red22 strain is Bacillus aryabhattai and was named Bacillus aryabhattai Red22. The polystyrene plastic degrading strain used in the present invention is not limited to the field isolated strain used in the present invention.

[0021] Bacillus aryabhattai (Priestia aryabhattai) Red22 was deposited at the Guangdong Provincial Microbial Strain Deposit Center (GDMCC, address: 5th Floor, No. 59 Building, Dayuan, No. 100, Xianlie Middle Road, Guangzhou, China, Postal Code: 510070), with deposit number: GDMCC No: 63428.

[0022] The 16S rRNA gene sequence of this strain is shown below:

[0023] Example 2 1. The decomposition of polystyrene plastic sheets by the polystyrene plastic-degrading bacterium Red22 involves the following steps: (1) Preparation of polystyrene-based plastic sheet: In order to facilitate evaluation of the characteristics of the plastic and to improve the accuracy of weighing after recovery, a polystyrene-based plastic sheet was used in this experiment for the decomposition experiment. Pure polystyrene powder purchased was added to xylene (3 g / mL), mechanically stirred, and left overnight to dissolve. After stirring uniformly, 5 mL of the transparent liquid was spread evenly on a 90 mm glass petri dish and allowed to harden for 36 hours. The formed polystyrene thin film was then cut into small pieces of 0.5 x 0.5 mm, washed with 75% ethanol and sterilized water, and air-dried.

[0024] (2) Activation of the strain: Polystyrene plastic decomposition bacteria, Bacillus aryabataii Red 22, was inoculated onto an LB agar plate and cultured at 30°C for 24 hours to allow the colonies to mature.

[0025] (3) Seed culture: A single colony of Bacillus aryabataii Red22 after activation was inoculated into 10 mL of LB liquid medium and cultured with constant temperature shaking at 30°C and 180 rpm for 16 hours to obtain a seed solution.

[0026] (4) Fermentation culture: The seed culture was inoculated into LB liquid medium at an inoculum amount of 2% by volume and cultured at constant temperature with shaking at 30°C and 180 rpm for 16 hours. The bacterial suspension obtained after fermentation culture was centrifuged at 6000 x g for 5 minutes, and the bacterial precipitate was collected and washed twice with PBS buffer. The precipitate was resuspended in carbon-free mineral salt medium, and the OD of the resuspended bacterial liquid was measured. 600 The value has been adjusted to 1.5.

[0027] (5) Degradation of polystyrene by Bacillus aryabata Red 22: A 0.5 x 0.5 mm polystyrene plastic sheet was weighed, immersed in 75% ethanol for 1 hour in an ultraclean bench for sterilization, and then removed and the surface liquid was evaporated in a sterile air stream in an ultraclean bench. 15 mL of carbon-free mineral salt medium and the surface-sterilized polystyrene plastic sheet (approximately 0.01 g / mL) were added to a large test tube, and the collected Bacillus aryabata Red 22 resuspension was inoculated at an inoculum amount of 10% (v / v). The non-inoculated treatment was the control group, and six parallel samples were set up for each treatment. The test tubes were placed in a constant temperature shaking incubator at 30 °C and 180 rpm for incubation, and samples were taken periodically to analyze the mass loss, surface morphological changes, functional group changes, and surface hydrophobic changes of the polystyrene plastic sheet.

[0028] Example 3 Degradation of polystyrene plastic sheets by Bacillus Aryabata Red 22: (1) Growth of Bacillus alyabatai Red22 on polystyrene plastic surface: After decomposing polystyrene plastic sheets with Bacillus alyabatai Red22 for 45 days, three polystyrene plastic sheets were taken out and immediately washed once with PBS buffer. Then, the bacteria were stained with a viability probe dye, and the growth of the Red22 strain on the polystyrene plastic sheet surface was photographed with a laser confocal microscope. As can be seen from Figure 1, Bacillus alyabatai Red22 was uniformly attached to the polystyrene plastic sheet surface and formed a biofilm. Even after 45 days of culture and decomposition, the activity of Bacillus alyabatai Red22 was relatively good, and most of the cells were alive. This indicates that the strain Red22 can adapt to the living environment created by polystyrene plastic and grow normally. Bacillus alyabatai Red22 can attach to the polystyrene plastic surface and grow using it as a carbon source while decomposing polystyrene.

[0029] (2) Mass reduction test of polystyrene-based plastic sheets: After decomposing polystyrene with Bacillus aryabataii Red22 for 45 days, all polystyrene-based plastic sheets in the test tube were collected by filtration, and first ultrasonically cleaned with 2% SDS cleaning solution for 1 hour, then ultrasonically cleaned with 75% absolute ethanol for 20 minutes to remove the biofilm on the surface of the polystyrene-based plastic sheets, and then washed twice with sterilized water. Finally, the washed polystyrene-based plastic sheets were placed in a vacuum oven at 50°C and dried for 12 hours. The polystyrene-based plastic sheets after drying were weighed and their weight reduction rate was calculated. Weight reduction rate of polystyrene-based plastic sheets (%) = (initial mass of polystyrene - mass after decomposition) ÷ initial mass × 100%. As can be seen from the weight reduction rate data in Figure 2, after decomposing polystyrene-based plastic sheets with strain Red22 for 45 days, the weight reduction rate of the plastic sheets reached 7.3%. The weight loss rate of the non-inoculated control group was only 2.2%, indicating that Bacillus aryabataii Red 22 decomposes polystyrene plastic sheets using them as a carbon source.

[0030] (3) Surface morphology changes after polystyrene degradation by Bacillus aryabataii Red22: After washing and drying the degraded polystyrene plastic sheet, its surface morphology was observed by scanning electron microscope (SEM), as shown in Figure 3. There were obvious irregular grooves and bits on the surface of the polystyrene plastic sheet after 45 days of degradation by Red22 bacteria, while the surface of the non-inoculated control polystyrene plastic sheet was relatively smooth. During growth on the surface, the strain Red22 can use polystyrene as the only carbon source to erode the plastic sheet and destroy its surface structure, resulting in obvious decomposition effect on the polystyrene plastic sheet.

[0031] (4) Changes in surface functional groups after polystyrene decomposition: Fourier transform infrared spectroscopy (FTIR) was used to measure the changes in surface functional groups before and after the decomposition of polystyrene-based plastic sheets by the strain Red22. The spectrum measurement range was 400-4000 cm -1The results are shown in Figure 4. Compared to the polystyrene plastic sheet before decomposition, the polystyrene plastic sheet decomposed by Bacillus Aryabata Red 22 had a peak of 3400-3700 cm -1 A new characteristic peak appears at 1750 cm, which is the stretching vibration peak of the OH bond. -1 A new characteristic peak appeared in the red region, which is the stretching vibration peak of the C=O bond. It is believed that the polystyrene plastic sheet was decomposed and oxidized by the Red22 bacteria, generating active functional groups such as hydroxyl groups and carbonyl groups. After the polystyrene plastic sheet was treated with Red22, the surface was obviously decomposed, and some polymers underwent partial depolymerization or chain scission, generating new oxidation products.

[0032] (5) Change in surface hydrophobicity after polystyrene decomposition: After washing and drying the polystyrene-based plastic sheet decomposed by Bacillus Aryabata Red 22, the contact angle of a water droplet on the surface of the polystyrene-based plastic sheet was measured using an optical contact angle meter. When measuring, the polystyrene-based plastic sheet was attached flat to a slide with double-sided tape, 10 μL of water was dropped with a microsyringe, and three points were tested for each sample, and the average value was taken. The contact angle test results are shown in Figure 5, where the contact angle of the polystyrene-based plastic sheet before decomposition was 89.62°±1.51°, and the water contact angle of the polystyrene-based plastic sheet decomposed by Bacillus Aryabata Red 22 decreased to 77.84°±1.13°. This indicates that the surface hydrophobicity of the polystyrene-based plastic sheet after decomposition was significantly reduced, and together with the FTIR results, it was shown that hydrophilic functional groups were generated on the surface of the decomposed polystyrene-based plastic sheet, and further, Bacillus Aryabata Red 22 has a high decomposition ability for polystyrene.

[0033] (6) Change in molecular weight before and after polystyrene decomposition: The results of measuring the change in molecular weight before and after the decomposition of polystyrene-based plastics by Bacillus Aryabata Red 22 using gel permeation chromatography (GPC) are shown in Figure 6. The mass average molecular weight (Mw) and number average molecular weight (Mn) of polystyrene-based plastics decomposed by Red 22 bacteria were lower than before decomposition, and the molecular weight distribution index (PDI = Mw / Mn) of the decomposed plastics was higher than before decomposition. The higher the PDI value, the wider the molecular weight distribution of the polymer and the higher the degree of depolymerization, indicating that Bacillus Aryabata Red 22 has a high decomposition ability for polystyrene and can decompose long chains of polystyrene into products with shorter carbon chains.

[0034] Example 4 Effect of Bacillus Aryabatai Red 22 on degrading polystyrene plastic in different real water bodies: (1) Steps for Bacillus aryabata Red22 to degrade polystyrene plastic in different real water bodies: 0.5×0.5 mm polystyrene plastic sheets were weighed, immersed in 75% ethanol for 1 hour in an ultraclean bench for sterilization, and then removed and the surface liquid was evaporated in a sterile air stream in an ultraclean bench. 15 mL of different types of real water bodies (upstream water from a water purification plant and spring water from a polluted river) and the polystyrene plastic sheets after surface sterilization (approximately 0.01 g / mL) were added to large test tubes, and the Bacillus aryabata Red22 fermentation culture liquid of Example 2 was inoculated at an inoculum amount of 10% (v / v). The treatment without inoculation of Red22 bacteria was used as the control group, and six parallel samples were set for each treatment. The above test tubes were placed in a constant temperature shaking incubator at 30°C and 180 rpm for incubation, and samples were taken periodically to analyze the mass loss, surface morphological changes, and changes in the hydrophobicity of the polystyrene plastic sheets.

[0035] (2) Mass reduction test after polystyrene plastic sheets are decomposed by Bacillus albicans Red 22 in different water bodies: After Bacillus albicans Red 22 decomposed polystyrene in different real water bodies for 50 days, all polystyrene plastic sheets in the test tubes were collected by filtration, first ultrasonically cleaned in 2% SDS cleaning solution for 1 hour, then ultrasonically cleaned in 75% absolute ethanol for 20 minutes to remove the biofilm on the surface of the polystyrene plastic sheets, then washed twice with sterilized water, and finally the washed polystyrene plastic sheets were placed in a vacuum oven at 50°C to dry for 12 hours. The polystyrene plastic sheets after drying were weighed and their weight reduction rate was calculated. Weight reduction rate of polystyrene plastic sheets (%) = (initial mass of polystyrene - mass after decomposition) ÷ initial mass × 100%. As can be seen from the weight loss data in Figure 6, after Bacillus Aryabata Red22 decomposed polystyrene plastic sheets in clean upstream water of a water purification plant for 50 days, the weight loss rate of the plastic sheet reached 4.1%, while the weight loss rate of the non-inoculated control group was only 1.7%. After Bacillus Aryabata Red22 decomposed polystyrene plastic sheets in polluted spring water of a river for 50 days, the weight loss rate of the plastic sheet reached 4.7%, while the weight loss rate of the non-inoculated control group was only 1.9%. This proves that the strain Red22 can decompose polystyrene plastics in both relatively clean and polluted water, and further shows that the strain Red22 has good environmental adaptability and can decompose polystyrene plastics in different real environments.

[0036] (Additional Note) (Appendix 1) Bacillus aryabhattai (Priestia aryabhattai) Red 22, accession number GDMCC No: 63428.

[0037] (Appendix 2) A bacteriophage capable of degrading polystyrene-based plastics, comprising the Bacillus aryabataii Red 22 described in Appendix 1 as an active ingredient.

[0038] (Appendix 3) Use of Bacillus aryabataii Red 22 as described in appendix 1 or the bacteriophage as described in appendix 2 in decomposing polystyrene-based plastics.

[0039] (Appendix 4) The use described in Appendix 3, characterized in that the decomposition is carried out by growing on polystyrene-based plastic as the only carbon source, settling on the plastic surface, and gradually decomposing the plastic.

[0040] (Appendix 5) A method for decomposing polystyrene-based plastic, comprising co-culturing Bacillus aryabataii Red 22 according to claim 1 with polystyrene-based plastic.

[0041] (Appendix 6) The method of claim 5, wherein the polystyrene-based plastic is a polystyrene particle, thin film, or powder.

[0042] (Appendix 7) 6. The method of claim 5, wherein the polystyrene plastic is degraded by Bacillus Aryabati Red 22 in different water bodies.

[0043] (Appendix 8) The Bacillus alyabatai Red 22 is a Bacillus alyabatai Red 22 fermentation broth, and its OD 600 The method according to claim 5, wherein the inoculation amount is 1% to 10% v / v when inoculated into a liquid containing a polystyrene-based plastic.

[0044] (Appendix 9) The method according to claim 5, wherein the co-culture is performed under culture conditions of a temperature of 25° C. to 37° C. and a shaking culture rotation speed of 150 to 220 rpm.

Claims

1. Bacillus aryabhattai Red 22, deposit number GDMCC No: 63428.

2. A bacteriophage capable of degrading polystyrene plastics, comprising the Bacillus aryabataii Red 22 according to claim 1 as an active ingredient.

3. Use of the Bacillus aryabataii Red 22 according to claim 1 or the bacteriophage according to claim 2 in decomposing polystyrene-based plastics.

4. The use according to claim 3, characterized in that the decomposition is carried out by growing on polystyrene-based plastic as the only carbon source, settling on the plastic surface, and gradually decomposing the plastic.

5. A method for decomposing polystyrene-based plastics, comprising co-culturing the Bacillus aryabataii Red 22 according to claim 1 with polystyrene-based plastics.

6. 6. The method of claim 5, wherein the polystyrene-based plastic is a polystyrene particle, film, or powder.

7. 6. The method of claim 5, wherein the polystyrene-based plastics are degraded by Bacillus aryabataii Red 22 in different water bodies.

8. The Bacillus alyabatai Red 22 is a fermentation liquid of Bacillus alyabatai Red 22, and its OD ６００ 6. The method according to claim 5, wherein the inoculation amount is 1% to 10% v / v when inoculating a liquid containing polystyrene-based plastic.

9. The method according to claim 5, wherein the co-culture is performed under the following culture conditions: temperature: 25°C to 37°C, and shaking culture rotation speed: 150 to 220 rpm.

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