Stenotrophomonas maltophilia, fungicide, application and method

Abstract

The invention discloses stenotrophomonas maltophilia, a microbial inoculum, application and a method. The stenotrophomonas maltophilia QY1 strain provided by the invention can rapidly grow by taking nano polystyrene as a unique carbon source, the concentration and molecular weight of plastic can be remarkably reduced, and polystyrene is degraded into styrene monomers. Plastic pollution commonly existing in the environment still has wide requirements for degrading bacteria, and the technology enriches a plastic degrading strain resource library. The plastic degrading strain enrichment method provided by the invention can create an enrichment scene beneficial to growth stimulation and gradual domestication aiming at the difficulty of plastic degrading strain enrichment, is simple to operate and low in cost, and can enrich the plastic degrading strains with high throughput.

Description

Technical Field

[0001] The present invention generally relates to microbial technology, and in particular to a kind of Stenotrophomonas maltophilia, a bacterial agent containing the same, a use thereof, a method for degrading polystyrene, and a method for enriching plastic-degrading bacteria. Background Art

[0002] Plastic pollution is one of the most pressing ecological and health issues in the world today. Micro-nano plastics affect surface water, soil, air quality and even the geobiochemical cycle, and even threaten the health of plants, animals and humans. Plastics are everywhere, and production and emissions continue to rise. Micro-nano plastics have been widely detected in surface waters such as oceans, rivers and lakes, in the atmosphere, and in soil. According to the Global Plastics Outlook: Economic Drivers, Environmental Impacts and Policy Options released by the OECD, the global annual production of plastics soared from 234 million tons in 2000 to 460 million tons in 2019, and plastic waste increased from 156 million tons in 2000 to 353 million tons in 2019. Among them, polystyrene is the fourth largest general-purpose plastic in my country, and is used in large quantities to produce foam lunch boxes, water cups, etc. Its use process easily produces microplastics and enters the human body through diet, threatening health.

[0003] Microorganisms are widely distributed, have strong vitality, are low-cost, separable, and environmentally friendly. Using microorganisms to degrade microplastics is an important method to remove and control microplastics in the environment. Enriching plastic-degrading bacteria is a key cornerstone of biodegradable plastics. The current screening and domestication of plastic-degrading bacteria may only be the "tip of the iceberg" of environmental microbial dark matter. The large-scale and widespread plastic pollution in the environment still has an important demand for degradation bacteria. Summary of the invention

[0004] Based on this, the present application provides a Stenotrophomonas maltophilia, the Stenotrophomonas maltophilia is Stenotrophomonas maltophilia QY1 (Stenotrophomonas maltophilia QY1), and the preservation number is: CCTCC No: M 20241862.

[0005] In another aspect, the present application also provides a bacterial agent comprising the Stenotrophomonas maltophilia QY1 described herein.

[0006] On the other hand, the present application also provides the use of the Stenotrophomonas maltophilia QY1 described herein or the bacterial agent described herein in degrading polystyrene. Through plate culture and laser confocal high-content imaging microscopy, it was observed that the strain can rapidly proliferate and grow well when cultured with polystyrene as the only carbon source, and can tolerate 1-5000 mg / L of polystyrene plastic. It was found that after 7 days of treatment with the Stenotrophomonas maltophilia QY1 strain, the concentration of plastic decreased as observed by the fluorescent microplate reader, the molecular weight of plastic decreased as observed by gel permeation chromatography, and the plastic degradation monomer was identified by pyrolysis gas chromatography-mass spectrometry, indicating that the strain has the ability to degrade polystyrene.

[0007] In another aspect, the present application also provides a method for degrading polystyrene, comprising co-culturing the Stenotrophomonas maltophilia or the bacterial agent described herein with polystyrene.

[0008] On the other hand, the present application also provides a method for enriching plastic degrading bacteria, comprising the following steps:

[0009] 1) Cultivating the mixed strains in the sample in mineral water to form a bacterial solution;

[0010] 2) Cultivating the strains in the bacterial solution using a liquid plastic-degrading bacteria enrichment medium (PEM medium);

[0011] 3) culturing the strains that survived in step 2) using a solid plastic degrading bacteria purification medium (PPM medium) with plastic added as the sole carbon source to obtain strains that can survive and grow well;

[0012] 4) The strain obtained in step 3) is diluted and divided into a microplate containing a solid plastic degrading bacteria purification medium (PPM medium) with plastic added as the only carbon source for further cultivation to screen out strains that can degrade plastic.

[0013] The present application provides a strain of Stenotrophomonas maltophilia, which is named as Stenotrophomonas maltophilia QY1. It is deposited in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit date is August 28, 2024. The deposit number is CCTCC No: M 20241862.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The plastic-degrading strain enrichment method provided in the present application can address the difficulties in enriching plastic-degrading bacteria and create an enrichment scenario that is conducive to stimulating growth and gradual domestication. It is simple to operate, low in cost, and can enrich plastic-degrading strains in a high-throughput manner.

[0016] (2) The strain QY1 of Oligotrophomonas maltophilia provided in the present application can grow rapidly with nano-polystyrene as the sole carbon source, can significantly reduce the concentration and molecular weight of plastics, and degrade polystyrene into styrene monomers. The widespread plastic pollution in the environment still has a wide demand for degrading bacteria, and this technology enriches the resource library of plastic degrading bacteria.

[0017] (3) The strain QY1 of Stenotrophomonas maltophilia provided in this application can provide basic support for in-depth research on the microbial degradation mechanism of polystyrene and improving the degradation function through genetic engineering.

[0018] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0020] Figure 1 This is the growth condition of Stenotrophomonas maltophilia QY1 using polystyrene as the sole carbon source in one embodiment of the present application.

[0021] Figure 2 This is a laser confocal high-content imaging microscope image showing the change in the number of Stenotrophomonas maltophilia QY1 cells in a degradation system according to an embodiment of the present application, wherein the left image is 0h, and the right image is 7d.

[0022] Figure 3 The figure shows the tolerance growth of Stenotrophomonas maltophilia QY1 to different concentrations of polystyrene plastics in a degradation system according to an embodiment of the present application, wherein the upper figure is 3d and the lower figure is 7d.

[0023] Figure 4 This is a diagram of polystyrene degradation monomers detected by pyrolysis mass spectrometry in a degradation system according to one embodiment of the present application.

[0024] Figure 5 This is a graph showing changes in polystyrene concentration in a degradation system according to an embodiment of the present application.

[0025] Figure 6 This is a graph showing the change in molecular weight of polystyrene in a degradation system according to one embodiment of the present application. DETAILED DESCRIPTION

[0026] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the application belongs. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0027] The terms "about" and "approximately" when used with a numerical variable generally refer to the value of that variable and all values ​​of that variable are within experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0028] The expression "comprising" or its synonymous similar expressions "including", "containing" and "having" etc. are open-ended and do not exclude additional unrecited elements, steps or ingredients. The expression "consisting of excludes any element, step or ingredient not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or ingredients, plus the optional elements, steps or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0029] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0030] The present application belongs to the exploration and development of microbial resources, and provides a method for high-throughput screening and enrichment of plastic-degrading strains. The application of the enrichment method is illustrated by taking polystyrene as an example, and the degradation of polystyrene by the enriched strain is proved. The strain can grow and metabolize polystyrene with polystyrene as the sole carbon source, and has been identified as Oligotrophomonas maltophilia by 16S rRNA sequencing. Using a laser confocal high-content imaging microscope, it was observed that the strain proliferated in the polystyrene carbon source and the plastic decreased; using a multifunctional microplate reader, it was detected that the concentration of polystyrene decreased under the action of the strain; using a pyrolysis mass spectrometer, it was detected that the monomer styrene degraded by polystyrene; using gel permeation chromatography, it was observed that the number average, weight average and Z average molecular weight of the plastic all decreased. In short, the strain can grow using plastic and has the ability to degrade polystyrene. The present application provides a scientific basis for the exploration of new species of microbial resources and their application in the degradation of polystyrene.

[0031] In one aspect, the present application provides a Stenotrophomonas maltophilia, wherein the Stenotrophomonas maltophilia is Stenotrophomonas maltophilia QY1 (Stenotrophomonas maltophilia QY1), and the deposit number is: CCTCC No: M 20241862.

[0032] In another aspect, the present application also provides a bacterial agent comprising the Stenotrophomonas maltophilia QY1 described herein.

[0033] In another aspect, the present application also provides use of the Stenotrophomonas maltophilia QY1 described herein or the bacterial agent described herein in degrading polystyrene.

[0034] In some embodiments, the degradation is that Stenotrophomonas maltophilia QY1 grows on polystyrene as the sole carbon source and colonizes on the surface of polystyrene, gradually degrading the plastic.

[0035] In another aspect, the present application also provides a method for degrading polystyrene, comprising co-culturing the Stenotrophomonas maltophilia QY1 described herein or the bacterial agent described herein with polystyrene.

[0036] In some embodiments, the polystyrene is a granule, a film or a powder of polystyrene. In some embodiments, the polystyrene includes at least one of various particle sizes and specifications.

[0037] In some embodiments, the present application also provides a method for degrading polystyrene plastics, comprising co-culturing the Stenotrophomonas maltophilia QY1 described herein or the bacterial agent described herein with polystyrene plastics. In some embodiments, the plastics include plastics of various types and sources. In some embodiments, the polystyrene plastics include at least one of various particle sizes and specifications.

[0038] In some embodiments, polystyrene is degraded by Stenotrophomonas maltophilia QY1 in different water bodies (eg, river water, lake water, ocean, or sewage).

[0039] On the other hand, the present application also provides a method for enriching plastic degrading bacteria, comprising the following steps:

[0040] 1) Cultivating the mixed strains in the sample in mineral water to form a bacterial solution;

[0041] 2) Cultivating the strains in the bacterial solution using a liquid plastic degrading bacteria enrichment medium (PEM medium);

[0042] 3) culturing the strains that survived in step 2) with a solid plastic degrading bacteria purification medium (Plastic Degrading Bacteria Purification Medium, PPM medium) using plastic as the only carbon source to obtain strains that can survive and grow well;

[0043] 4) The strain obtained in step 3) is diluted and divided into a microplate containing a purified medium for solid plastic degrading bacteria (PPM medium) with plastic as the only carbon source for further cultivation to screen out strains that can degrade plastic.

[0044] In some embodiments, the formula of the liquid plastic degrading bacteria enrichment medium is: 3g (NH4)2SO4, 0.05g KNO3, 0.0085g NaCl, 0.0001g C5H 14 ClNO, 0.0001 g C 18 H 32 CaN2O 10 , 0.0001g C 19 H 19 N7O6, 0.0001gC6H6N2O, 0.0001g C8H9NO3, 0.00001g C 17 H 20 N4O6, 0.0001g C 12 H 17 ClN4OS·HCl and 0.0002gC6H 12 O6, 1g KH2PO4, 3g K2HPO4·3H2O, 0.2g MgSO4, 1g NaCl, 0.01g CaCl2, 0.006gNa2SeO35H2O, 0.008g Na2WO42H2O, 0.0005g EDTA, 0.0002g 7H2O·Fe2SO4, 0.00001g7H2O·ZnSO4, 0.000003g 4H2O·MnCl2, 0.00003g H3BO3, 0.00002g 6H2O·CoCl2, 0.000001g2H2O·CuCl2, 0.000002g 6H2O·NiCl2, 0.000003g 2H2O·NaMoO4 and 1L ultrapure water.

[0045] In some embodiments, the formula of the purified culture medium of solid plastic degrading bacteria without carbon source is 1g KH2PO4, 3g K2HPO4·3H2O, 0.2g MgSO4, 1g NaCl, 0.01g CaCl2, 0.006g Na2SeO35H2O, 0.008g Na2WO42H2O, 0.5g EDTA, 0.2g 7H2O·Fe2SO4, 0.01g 7H2O·ZnSO4, 0.003g 4H2O·MnCl2, 0.03g H3BO3, 0.02g 6H2O·CoCl2, 0.001g 2H2O·CuCl2, 0.002g 6H2O·NiCl2, 0.003g2H2O·NaMoO4, 15g agar and 1L ultrapure water. Plastic can be added to a solid plastic-degrading bacteria purification culture medium that does not contain a carbon source to obtain a solid plastic-degrading bacteria purification culture medium with plastic as the sole carbon source.

[0046] In some embodiments, the sample can have multiple sources. In some embodiments, the sample can be a soil sample from a contaminated site. In some embodiments, the sample can be a sewage sample. In some embodiments, the sample can be plastic waste buried in environmental soil. In some embodiments, the sample can be a sediment sample in various water bodies (such as the ocean).

[0047] In some embodiments, the plastic is aged plastic.

[0048] In some embodiments, the aged plastic is in the form of nanoparticles. In some embodiments, the aged plastic is prepared into a plastic mother solution of a certain concentration and added to the PPM medium described herein for screening and purification of plastic degrading bacteria. The concentration of the plastic mother solution can be adjusted according to the type of strain to be isolated or the type of plastic.

[0049] In some embodiments, step 4) is repeated one or more times to isolate and purify a single strain.

[0050] In some embodiments, the plastic is polystyrene plastic.

[0051] The present application provides a high-throughput plastic-degrading bacteria enrichment method, which first adds mineral water to activate the soil of the contaminated site for 24 hours to prepare a bacterial suspension; then adds the activated bacterial solution to a liquid plastic-degrading bacteria enrichment medium (PEM medium), adds a pretreated polystyrene solution and oscillates for 48 hours; finally, in a solid plastic-degrading bacteria purification medium (PPM medium) with polystyrene as the only carbon source, further separates and purifies the polystyrene-degrading bacteria. The relevant specific processes are as follows.

[0052] During the pretreatment of polystyrene, polystyrene nanoplastics were added to 100 mL of water to prepare a 10 g / L plastic mother liquor; under an LED lamp (395 nm wavelength) simulating natural ultraviolet radiation, the beaker was placed in a magnetic stirrer and stirred continuously to simulate environmental aging, and incubated at room temperature of 25°C for 7 days.

[0053] The formula of liquid plastic degrading bacteria enrichment medium (PEM medium) is 3g (NH4)2SO4, 0.05g KNO3, 0.0085g NaCl, 0.0001g C5H 14 ClNO, 0.0001 g C 18 H 32 CaN2O 10 , 0.0001g C 19 H 19 N7O6, 0.0001gC6H6N2O, 0.0001g C8H9NO3, 0.00001g C 17 H 20 N4O6, 0.0001g C 12 H 17 ClN4OS·HCl and 0.0002gC6H 12 O6, 1g KH2PO4, 3g K2HPO4·3H2O, 0.2g MgSO4, 1g NaCl, 0.01g CaCl2, 0.006gNa2SeO35H2O, 0.008g Na2WO42H2O, 0.0005g EDTA, 0.0002g 7H2O·Fe2SO4, 0.00001g 7H2O·ZnSO4, 0.000003g 4H2O·MnCl2, 0.00003g H3BO3, 0.00002g 6H2O·CoCl2, 0.000001g 2H2O·CuCl2, 0.000002g 6H2O·NiCl2, 0.000003g 2H2O·NaMoO4 and 1L ultrapure water.

[0054] Preparation of purified medium for solid plastic-degrading bacteria (PPM medium) with plastic as the sole carbon source: weigh 1g KH2PO4, 3g K2HPO4·3H2O, 0.2g MgSO4, 1g NaCl, 0.01g CaCl2, 0.006g Na2SeO35H2O, 0.008g Na2WO42H2O, 0.5g EDTA, 0.2g 7H2O·Fe2SO4, 0.01g 7H2O·ZnSO4, 0.003g4H2O·MnCl2, 0.03g H3BO3, 0.02g 6H2O·CoCl2, 0.001g 2H2O·CuCl2, 0.002g 6H2O·NiCl2, 0.003g2H2O·NaMoO4, and 15g agar, then mix thoroughly and make up to 1L of ultrapure water. After high temperature and high pressure sterilization, add plastic mother liquor and mix well, pour into the wells of the microplate and cool and solidify for use.

[0055] The present application describes a plurality of embodiments, but the description is exemplary rather than restrictive, and it is apparent to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application. Although many possible feature combinations are shown in the drawings and discussed in the specific embodiments, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

[0056] The present application includes and contemplates combinations of features known to those of ordinary skill in the art. The embodiments and features disclosed in the present application may also be combined with any conventional features to form a unique invention scheme defined by the claims. Any feature of any embodiment may also be combined with features from other invention schemes to form another unique invention scheme defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in the present application may be implemented individually or in any appropriate combination. Therefore, except for the limitations made according to the attached claims and their equivalents, the embodiments are not subject to other restrictions. In addition, various modifications and changes may be made within the scope of protection of the attached claims.

[0057] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps described herein, the method or process should not be limited to the steps of the specific order described. As will be understood by those of ordinary skill in the art, other sequences of steps are also possible. Therefore, the specific sequence of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for the method and / or process should not be limited to the steps of performing them in the order written, and those skilled in the art can easily understand that these sequences can be changed and still remain within the spirit and scope of the embodiments of the present application.

[0058] The experimental methods in the following examples without specifying specific conditions are usually measured according to national standards. The experimental materials in the following examples without specifying the source are all commercially available raw materials. The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise defined or specified, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of this application.

[0059] Example

[0060] The relevant materials used in the examples are as follows:

[0061] 1. Reagents and Materials

[0062] (NH4)2SO4, KNO3, NaCl, C5H 14 ClNO, C 18 H 32 CaN2O 10 , C 19 H 19 N7O6, C6H6N2O, C8H9NO3, C 17 H 20 N4O6、C 12 H 17 ClN4OS·HCl and C6H 12O6, KH2PO4, K2HPO4·3H2O, MgSO4, NaCl, CaCl2, Na2SeO35H2O, Na2WO42H2O, EDTA, 7H2O·Fe2SO4, 7H2O·ZnSO4, 4H2O·MnCl2, H3BO3, 6H2O·CoCl2, 2H2O·CuCl2, 6H2O·NiCl2, 2H2O·NaMoO4 and other culture medium formula reagents are all analytically pure and purchased from Sinopharm Group, Maclean, Aladdin, Sigma, etc. Agar is purified agar. Microplastics are 20nm red fluorescent microplastics (Jiangsu Zhichuan Technology).

[0063] 2. Instruments

[0064] Routine operation and culture instruments include high-temperature and high-pressure sterilizers, clean benches, shakers, constant temperature incubators, etc. LED ultraviolet lamps with a wavelength of 395nm are used to irradiate aged microplastics. Detection instruments include multifunctional microplate reader (TECAN, Spark), laser confocal high-content imaging microscope (PerkinElmer, Opera Phenix), gel permeation chromatograph (Waters, Breeze2), pyrolysis chromatography mass spectrometer (Shimadzu, GCMS-QP2010SE), etc.

[0065] Example 1. High-throughput plastic-degrading bacteria screening method to isolate polystyrene-degrading bacteria

[0066] This example uses polystyrene as an example to illustrate the process of separating plastic-degrading bacteria by the well plate plastic enrichment method of the present application. First, add 10g of soil from a plastic-contaminated site (isolated from the garbage dump in Xiawa Town, Aohan, Inner Mongolia) and 100mL of Nongfu Spring Changbai Mountain mineral water to a conical flask, and culture at 30°C, 180rpm / min on a shaker for 24h to prepare a soil bacterial suspension; then add 100μL of soil bacterial suspension and 800μL of low-nutrient plastic-degrading bacteria enrichment medium PEM medium to each well of a 48-well plate and activate by shaking for 24h; continue to add 100μL of aged 20nm polystyrene mother solution (1000mg / L), and culture at 30°C, 80rpm / min on a shaker for 4h. 8h, further acclimate the degradation bacteria; finally, add polystyrene mother liquor to the plastic degradation bacteria purification medium PPM medium, use no polystyrene as a carbon-free nitrogen control, inoculate and streak the degradation bacteria acclimatized in the previous step to the solid PPM medium, culture at 30°C for 7-20d, observe and photograph the growth of bacterial colonies, and bacteria that can form colonies on the PPM plate with polystyrene as the only carbon source but cannot grow in the solid PPM medium without plastic are judged to have plastic degradation potential; select single colonies with plastic degradation potential for multiple separation and purification, and finally obtain pure polystyrene degradation bacteria, which are identified as Stenotrophomonas maltophilia strains after 16S rRNA sequencing (its 16S rRNA sequence is shown in SEQ ID NO: 1). It has a high similarity with the Stenotrophomonas maltophilia strain ZT1, and is named Stenotrophomonas maltophilia QY1. It was deposited in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on August 28, 2024. The deposit number is CCTCC No: M 20241862.

[0067] Example 2. Growth of degrading bacteria in polystyrene culture medium

[0068] Growth of degrading bacteria in solid culture medium. In the PPM plastic degrading bacteria purified microplate solid culture medium with polystyrene plastic as the only carbon source, the growth of degrading bacteria without adding plastic and adding 1mg / L plastic was investigated. A single colony of the QY1 strain of Oligotrophomonas maltophilia was streaked on the plate on the PPM solid culture medium, and then cultured in a constant temperature incubator at 30℃ for 2-7 days, and the growth of the colony was recorded by taking photos. The results showed that ( Figure 1 ), no colony proliferation was observed in the control without added plastic, while the colony number of degrading bacteria in the microwells with added plastic increased significantly, and obvious colonies were formed on the second day.

[0069] Growth of degrading bacteria in liquid culture medium. Inoculate the strain of Stenotrophomonas maltophilia QY1 in LB culture medium at a ratio of 1%, and culture overnight at 30°C and 180rpm / min to activate and rejuvenate the strain. Then, remove the LB culture medium by centrifugation at 8000rpm / min, resuspend with sterile ultrapure water, vortex and centrifuge for 3 times to remove nutrients in the system. Add the washed bacteria to the PPM liquid culture medium with 1000mg / L fluorescent nano-polystyrene plastic as the only carbon to adjust the OD 600 =0.2, and then cultured in a shaking incubator at 30°C and 180 rpm / min. The culture solution was taken to observe the changes of degradation bacteria and plastics under a laser confocal high-content imaging microscope. The results showed that ( Figure 2 ), after 7 days of culture, the number of bacteria increased and the amount of plastic decreased, indicating that the strain was able to grow and reproduce in the polystyrene culture medium.

[0070] Example 3. Growth of degrading bacteria in polystyrene culture medium and tolerance thresholds to different concentrations of polystyrene

[0071] The tolerance threshold of degrading bacteria to different concentrations of polystyrene was verified in a 9 cm culture dish. Different plastic concentrations (0, 1 mg / L, 20 mg / L, 100 mg / L, 1000 mg / L, 5000 mg / L, 20000 mg / L) were set, and single colonies of the QY1 strain of Oligotrophomonas maltophilia were streaked on the plate, and cultured in a 30°C constant temperature shaker for 2-10 days. The growth of the colonies was observed and photographed to determine the range of plastic concentrations that can grow colonies. The results showed that ( Figure 3 ), the QY1 strain of Stenotrophomonas maltophilia could not grow in a carbon-free and nitrogen-free culture medium without plastic, and there was no difference in growth on 1 mg / L, 20 mg / L, and 100 mg / L plastics. The number of colonies that could be formed under the three concentrations of plastic of 1000 mg / L, 5000 mg / L, and 20000 mg / L decreased with the increase of plastic concentration; this indicated that the QY1 strain of Stenotrophomonas maltophilia could grow with polystyrene as the only carbon source, and could tolerate polystyrene plastic concentration of 1-5000 mg / L.

[0072] Example 4. Changes in plastics in the degradation system

[0073] 1. Detection of plastic concentration

[0074] The liquid of QY1, a strain of Stenotrophomonas maltophilia grown with polystyrene as the sole carbon source, was collected at different culture times, and the fluorescence (excitation 310nm, emission 680nm) was detected using a multifunctional microplate reader. The fluorescence intensity reflects the concentration change of polystyrene plastic in the bacterial liquid. The results show that ( Figure 4), when the strain QY1 of Stenotrophomonas maltophilia was grown in a culture medium with polystyrene as the sole carbon source, the fluorescence intensity of the detectable polystyrene plastic decreased with time, indicating that the concentration of plastic in the culture system decreased.

[0075] 2. Detection of plastic monomers

[0076] When detecting the degradation monomers of plastic, the bacterial solution treated with the strain for 7 days was collected, freeze-dried, and about 3 mg of powder was weighed. The degradation monomers of polystyrene plastic were detected by pyrolysis chromatography-mass spectrometry ( Figure 5 ). By comparing the characteristic peaks identified in the spectral library, it was found that the strain could degrade polystyrene plastic into monomer styrene.

[0077] 3. Gel permeation chromatography

[0078] In the PPM medium with polystyrene plastic added, the bacterial liquid of the treatment group of Stenotrophomonas maltophilia QY1 and the control group without Stenotrophomonas maltophilia QY1 after 7 days of reaction was taken, and then the polystyrene plastic was dissolved and dried using N,N-dimethylformamide, and the changes in weight average, Z average and peak molecular weight were detected by gel permeation chromatography ( Figure 6 The results showed that when the strain QY1 of Stenotrophomonas maltophilia was grown in a culture medium with polystyrene as the sole carbon source, the weight average, Z average and peak molecular weight of the polystyrene plastic that could be detected decreased with time, indicating that the plastic in the culture system was degraded.

Claims

1. A strain of Stenotrophomonas maltophilia, characterized in that: The Stenotrophomonas maltophilia is Stenotrophomonas maltophilia QY1 (Stenotrophomonas maltophilia QY1), and the preservation number is: CCTCC No: M 20241862.

2. A bacterial agent comprising the Stenotrophomonas maltophilia QY1 according to claim 1.

3. Use of the Stenotrophomonas maltophilia QY1 according to claim 1 or the bacterial agent according to claim 2 in degrading polystyrene.

4. The use according to claim 3, characterized in that The degradation is that the bacterium Stenotrophomonas maltophilia QY1 grows on polystyrene as the only carbon source and colonizes on the surface of polystyrene, thereby gradually degrading the plastic.

5. A method for degrading polystyrene, characterized in that: The Stenotrophomonas maltophilia QY1 according to claim 1 or the bacterial agent according to claim 2 is co-cultured with polystyrene.

6. The method according to claim 5, characterized in that The polystyrene is polystyrene particles, films or powders.

7. The method according to claim 5, characterized in that The polystyrene was degraded by the Stenotrophomonas maltophilia QY1 in different water bodies.

8. A method for enriching plastic-degrading bacteria, characterized in that: The following steps are involved: 1) Cultivating the mixed strains in the sample in mineral water to form a bacterial solution; 2) culturing the strains in the bacterial solution using a liquid plastic-degrading bacteria enrichment medium; 3) culturing the strain that survived in step 2) using a solid plastic-degrading bacteria purification medium with plastic added as the sole carbon source to obtain a strain that can survive and grow well; 4) The strain obtained in step 3) is diluted and divided into a microplate containing a solid plastic degrading bacteria purification culture medium with plastic added as the sole carbon source for further cultivation to screen out strains that can degrade plastic.

9. The method according to claim 8, characterized in that The formula of the liquid plastic degrading bacteria enrichment medium is: 3g (NH4)2SO4, 0.05g KNO3, 0.0085g NaCl, 0.0001g C5H 14 ClNO, 0.0001 g C 18 H 32 CaN2O 10 , 0.0001g C 19 H 19 N7O6, 0.0001g C6H6N2O, 0.0001g C8H9NO3, 0.00001g C 17 H 20 N4O6, 0.0001gC 12 H 17 ClN4OS·HCl and 0.0002 g C6H 12 O6, 1g KH2PO4, 3g K2HPO4·3H2O, 0.2g MgSO4, 1gNaCl, 0.01g CaCl2, 0.006g Na2SeO35H2O, 0.008g Na2WO42H2O, 0.0005g EDTA, 0.0002g7H2O·Fe2SO4, 0.00001g 7H2O·ZnSO4, 0.000003g 4H2O·MnCl2, 0.00003g H3BO3, 0.00002g6H2O·CoCl2, 0.000001g 2H2O·CuCl2, 0.000002g 6H2O·NiCl2, 0.000003g 2H2O·NaMoO4 and 1L ultrapure water.

10. The method according to claim 8, characterized in that The formula of the solid plastic degrading bacteria purification culture medium is 1g KH2PO4, 3g K2HPO4·3H2O, 0.2g MgSO4, 1g NaCl, 0.01g CaCl2, 0.006g Na2SeO35H2O, 0.008g Na2WO42H2O, 0.5g EDTA, 0.2g 7H2O·Fe2SO4, 0.01g 7H2O·ZnSO4, 0.003g4H2O·MnCl2, 0.03g H3BO3, 0.02g 6H2O·CoCl2, 0.001g 2H2O·CuCl2, 0.002g 6H2O·NiCl2, 0.003g2H2O·NaMoO4, 15g agar and 1L ultrapure water.

11. The method according to any one of claims 8 to 10, characterized in that: Repeat step 4) once or multiple times to isolate and purify a single strain.

12. The method according to any one of claims 8 to 11, characterized in that The plastic is polystyrene plastic.

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