A strain of microplastic-degrading bacteria for mulch film and its application

Abstract

The present invention belongs to the field of microbial technology and relates to a microplastic-degrading bacterium for mulch film and its application. The microplastic-degrading bacterium for mulch film is characterized by being Stenotrophomonas maltophilia MD1Y, deposited with the Guangdong Provincial Microbiological Culture Collection Center of China under the deposit number GDMCC No: 62745; the deposit date is August 25, 2022. The isolated and screened Stenotrophomonas maltophilia MD1Y has a strong ability to degrade residual microplastics in mulch film, can provide an efficient degradation strain resource for the bioremediation of mulch film-contaminated environments, and has excellent application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a strain of bacteria for degrading mulch microplastics and applications thereof. Background Art

[0002] Microplastic pollution has become a global environmental concern. The terrestrial environment is a more significant sink for microplastics, with microplastic content potentially 4 to 23 times higher than in the ocean. Mulch films are widely used in agricultural production, helping to retain water and heat in the soil, improve fertilizer utilization, and reduce pests and diseases, thereby improving crop yield and quality. my country is a major producer and user of mulch films. With increasing population demand for agricultural products, the use of mulch films in agriculture has continued to increase, leading to large amounts of film entering farmland soils. Mulch film residue has become a major source of microplastic pollution in farmland soils. The accumulation of microplastics from mulch films can negatively impact farmland soil ecosystems, hinder crop growth, and even be absorbed by crops, posing a potential threat to human health through the food chain. As farmland soils are crucial for human survival and development, the issue of microplastic pollution from mulch films warrants significant attention.

[0003] Currently, the main treatment and disposal methods for waste plastics include classified recycling, landfill, incineration, and pyrolysis. These methods are simple to operate, but they come with high investment costs. The treatment and disposal process also generates secondary pollutants, posing a serious threat to the ecological environment. Furthermore, the small particle size of microplastics formed by residual mulch film makes existing recycling technology relatively backward, making it difficult to recover them from the soil and carry out further treatment and disposal. Therefore, conducting in situ soil remediation experiments is a feasible method for removing microplastics from mulch film residues. Microbial degradation is the process by which microorganisms use their metabolic functions to break down highly toxic and complex pollutants into simpler substances, thereby reducing their toxicity or rendering them harmless. Microbial remediation offers the advantages of simple treatment methods, high efficacy, low cost, and environmental friendliness, making it an effective method for removing pollutants from the environment. Microplastics can be degraded by some microorganisms. Both bacteria and fungi can participate in the degradation process of microplastics. The specific process is as follows: (1) microbial attachment to the plastic surface; (2) biofilm formation; (3) secretion of enzymes (amines, oxidases, peroxidases, laccases, ureases, proteases and esterases, etc.) and extracellular polysaccharides; (4) biorupture (formation of oligomers, dimers and monomers); (5) mineralization (production of carbon dioxide, methane and water). However, there is currently a lack of bacterial species for microbial degradation of microplastics, resulting in low efficiency in the treatment of microplastics and limiting the application of microbial remediation technology. Summary of the Invention

[0004] In response to the problems existing in the prior art, the purpose of the present invention is to provide a strain of film-degrading microplastic bacteria for isolation and screening and its application.

[0005] The purpose of the present invention is achieved by adopting the following technical solutions:

[0006] In the first aspect, the present invention discloses a film-degrading microplastic bacterium MD1Y, which is Stenotrophomonas maltophilia, deposited in the Guangdong Provincial Microbiological Culture Collection Center of China, with the deposit number GDMCC No: 62745; the deposit time is August 25, 2022.

[0007] In a second aspect, the present invention discloses the application of a strain of mulch film microplastic-degrading bacteria in the degradation of residual microplastics in mulch film.

[0008] In a third aspect, the present invention discloses a method for degrading residual microplastics in mulch by a strain of mulch microplastic-degrading bacteria, comprising the following steps:

[0009] (1) Cut the plastic mulch into small pieces, crush them with a crusher, and sieve them;

[0010] (2) The sieved plastic fragments were sequentially soaked in SDS solution, low-concentration ethanol solution, and high-concentration ethanol solution after ultrasonication, rinsed three times with sterile water on a clean bench, and then dried and sterilized to obtain mulch microplastics;

[0011] (3) The domesticated mulch microplastic-degrading bacteria MD1Y were transferred to MSM culture medium, mulch microplastics were added, and mulch microplastics were used as the sole carbon source, and the culture was shaken;

[0012] (4) After the shaking culture is completed, the supernatant is poured out, and after washing, it is dried and weighed to detect the degradation rate of microplastics in the film.

[0013] Preferably, the plastic ground film is a polyethylene ground film, and the size of the small film piece is 1×1.5 cm.

[0014] Preferably, the mass fraction of the SDS solution is 2%, the mass fraction of the low-concentration ethanol solution is 75%, and the mass fraction of the high-concentration ethanol solution is 95%.

[0015] Preferably, the ultrasonic time is 30 minutes and the soaking time is more than 2 hours.

[0016] Preferably, the sterilization is ultraviolet lamp sterilization, the time is 15 minutes; the drying temperature is 30°C.

[0017] Preferably, the shaking culture conditions are: shaking culture at 30° C. and 140 r / min for 50 days.

[0018] The beneficial effects of the present invention are:

[0019] The Stenotrophomonas maltophilia MD1Y isolated and screened by the present invention has a strong ability to degrade microplastics residual in mulch films, can provide an efficient degradation bacterial resource for the bioremediation of mulch film-polluted environments, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention is further described with reference to the accompanying drawings. However, the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative effort.

[0021] Figure 1 The morphology of the mulch microplastic-degrading bacteria MD1Y on the NB plate in Example 1 of the present invention;

[0022] Figure 2 This is the growth curve of the mulch microplastic-degrading bacteria MD1Y in MSM medium in Example 1 of the present invention;

[0023] Figure 3 This is a comparison of the removal effect of microplastics from mulch film in the control group without the addition of the degrading bacteria MD1Y and the treatment group with the addition of the degrading bacteria MD1Y in Example 1 of the present invention;

[0024] Figure 4 The surface micromorphology of the mulch microplastics before and after degradation in the control group without the addition of the degrading bacteria MD1Y and the treatment group with the addition of the degrading bacteria MD1Y in Example 1 of the present invention (a: before treatment; b. control group; c. MD1Y treatment group);

[0025] Figure 5 This is the infrared spectrum of the mulch microplastics after 50 days of cultivation in the control group without the addition of the degrading bacteria MD1Y and the treatment group with the addition of the mulch microplastic degrading bacteria MD1Y in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] In order to more clearly illustrate the present invention and have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0027] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0028] The present invention is further described with reference to the following examples.

[0029] Example 1

[0030] A method for isolating and screening Stenotrophomonas maltophilia MD1Y comprises the following steps:

[0031] (1) Weigh 10g of soil sample (soil from agricultural land contaminated by mulch film for a long time) into a triangular flask containing 10mL of micro-carbon source culture medium, add a 1×1.5cm small piece of membrane, and culture at 30℃, 140rpm for 12 days. Transfer the membrane to a new micro-carbon source culture medium and continue enrichment for 12 days. Then use phosphate buffer to shake and wash the membrane, make a bacterial suspension, transfer all of it to MSM, add mulch film microplastics at a ratio of 0.35% (w / v), and the total volume is 50mL. Culture at 30℃, 140rpm for 70 days. Then take 5mL of supernatant and transfer it to MSM culture medium with mulch film microplastics as the only carbon source, culture for 30 days, repeat the above steps, and perform acclimatization 3 times.

[0032] (2) 0.1 mL of the culture solution from the last acclimation cycle was taken and spread on NB medium. The plate was placed upside down in a 30°C biochemical incubator. After 3 to 4 days, colonies of different morphologies were picked from the plate for streak purification to obtain a pure strain, which was numbered MD1Y.

[0033] The trace carbon source medium consisted of: 0.5 g yeast extract; 2.0 g NH4SO4; 100 mL trace element solution (FeSO4·7H2O 1.0 g / L, MgSO4·7H2O 1.0 g / L, CuSO4·5H2O 0.1 g / L, MnSO4·H2O 0.1 g / L, ZnSO4·7H2O 0.1 g / L, pH 7.0, and was diluted to 1 L.

[0034] Mineral salt medium (MSM): 0.92g K2HPO4·3H2O, 0.7g KH2PO4, 0.7g MgSO4·7H2O, 1.0g NH4Cl, 0.005g NaCl, 0.002g FeSO4·7H2O, 0.002g ZnSO4·7H2O, 0.001g MnSO4·H2O, pH 7.2, fixed volume to 1L.

[0035] Nutrient broth agar solid medium (NB): 18 g nutrient broth, 18 g technical agar powder, 1 L distilled water.

[0036] Example 2

[0037] The application of a mulch film microplastic-degrading bacterium in the degradation of residual microplastics in mulch film comprises the following steps:

[0038] (1) Cut the plastic mulch into smaller pieces (1×1.5 cm), and then crush them using a crusher to form mulch microplastics;

[0039] (2) Soaking the mulch microplastics in SDS solution, low-concentration ethanol solution, and high-concentration ethanol solution in sequence after ultrasonication, with the ultrasonication time being 30 minutes and the soaking time being more than 2 hours. After rinsing with sterile water three times on a clean bench, drying at 30°C, and sterilizing with ultraviolet light for 15 minutes, the mulch microplastics are obtained; wherein the mass fraction of the SDS solution is 2%, the mass fraction of the low-concentration ethanol solution is 75%, and the mass fraction of the high-concentration ethanol solution is 95%;

[0040] (3) The mulch microplastic-degrading bacteria MD1Y obtained in Example 1 was transferred to MSM culture medium, mulch microplastics were added, and mulch microplastics were used as the sole carbon source, and the culture was shaken;

[0041] (4) The conditions for shaking culture are: shaking culture at 30°C and 140 r / min for 50 days, then pouring out the supernatant, washing, drying and weighing, and detecting the degradation rate of microplastics in the film.

[0042] control group

[0043] A control group without the addition of bacteria MD1Y was set up.

[0044] Experimental Example 1

[0045] Study on the degradation performance of mulch film microplastics by the mulch film microplastics-degrading bacteria MD1Y obtained in Example 1:

[0046] (1) Growth of strain MD1Y in MSM medium

[0047] The MD1Y bacterial suspension was transferred into MSM culture medium, 0.1 g of mulch microplastics was added, the total volume was 100 mL, the culture was shaken at 30 ° C, 140 r / min, and the OD600 of the bacteria was measured regularly to draw a growth curve.

[0048] (2) Degradation rate of microplastics in mulch film by strain MD1Y

[0049] The MD1Y bacterial suspension was transferred into MSM medium (100 mL) with mulch microplastics (0.1 g) as the sole carbon source, which served as the treatment group. A control group without the addition of bacteria MD1Y was set up, and the culture was shaken at 30°C and 140 r / min for 50 days. After centrifugation, the supernatant was discarded, and the mulch microplastics were soaked in 75% ethanol, 95% ethanol and 100% ethanol for more than 2 hours, rinsed three times with sterile water on a clean bench, and then dried naturally. The weight was used to determine the degradation rate of the mulch microplastics.

[0050] Degradation rate (%) = (mass of microplastics in the ground film before cultivation - mass of microplastics in the ground film after cultivation) ÷ mass of microplastics in the ground film before cultivation × 100%

[0051] (3) Changes in the microscopic morphology of the surface of microplastics in mulch films

[0052] After 50 days of cultivation, the supernatant was discarded after centrifugation to obtain the mulch microplastics of the treatment group and the control group. The mulch was cleaned by soaking in 75% ethanol, 95% ethanol and 100% ethanol for more than 2 hours, rinsed with sterile water three times on a clean bench and dried naturally. The microscopic characteristics of the mulch microplastics were observed by scanning electron microscopy.

[0053] (4) Changes in surface functional groups of microplastics in mulch films

[0054] After 50 days of cultivation, the supernatant was discarded after centrifugation to obtain the mulch microplastic samples of the treatment group and the control group. After washing and drying according to method (3), the functional group changes of the mulch microplastics before and after degradation were analyzed using Fourier transform infrared spectrometer.

[0055] Experimental Example 2

[0056] 1. Identification of strain MD1Y

[0057] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to complete the DNA extraction of the strain MD1Y, and the 16S rDNA was amplified and sequenced using the 27F / 1492R primers. The sequence length was 1480bp, and the 16S rDNA sequence is shown in Seq ID No: 1. The sequence of MD1Y was compared and analyzed for homology in the ribosomal database http: / / rdp.cme.msu.edu / index.jsp, and the similarity with Stenotrophomonas maltophilia was 99.93%. The strain was classified as Stenotrophomonas maltophilia. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 25, 2022, with the deposit number: GDMCC No: 62745. The morphology of strain MD1Y on the plate is as follows Figure 1 As shown, the colonies are light yellow, with a smooth and moist surface, are opaque, have neat edges, and are easy to pick.

[0058] 2. Growth curve of MD1Y in MSM medium

[0059] The growth curve of the degrading bacteria MD1Y in MSM medium with mulch microplastics as the only carbon source is shown in the figure. Figure 2 MD1Y grew slowly during the first 10 days of culture, with an OD600 increase of 0.004. It then showed a rapid growth trend, reaching an OD600 increase of 0.073 after 50 days. This suggests that MD1Y can utilize mulch microplastics as a carbon source for growth and reproduction.

[0060] 3. Degradation rate of microplastics in mulch by MD1Y

[0061] The reduction in the weight of film microplastics is one of the indicators for determining whether they have been degraded. The weight loss rate of MD1Y before and after degradation of film microplastics is as follows: Figure 3 As shown. After 50 days of incubation, in the control group without MD1Y, the weight loss rate of mulch microplastics was 0.89%. This may be due to damage caused by collisions between the microplastics and the flask during oscillation, resulting in abiotic losses. In the treatment group with MD1Y added and mulch microplastics as the sole carbon source, the degradation effect of MD1Y on microplastics gradually increased over time, especially after 20 days. At 50 days, the removal rate of mulch microplastics reached 8.67%. Compared with the control group, this indicates that Stenotrophomonas maltophilia MD1Y has excellent degradation ability.

[0062] 4. Surface microscopic characteristics of MD1Y before and after degradation of microplastics in mulch film

[0063] The results of scanning electron microscopy observations are as follows Figure 4 As shown. Untreated original film microplastics ( Figure 4 a) The surface was smooth and flat. After 50 days of culture, the control group ( Figure 4 b) showed very slight roughness on the surface of the microplastics, but the whole was intact; the treatment group ( Figure 4 The microplastics in c) showed obvious holes, grooves, wrinkles and depressions, showing large areas of damage and shedding, which shows that the bacteria have a good degradation effect on the microplastics in the mulch.

[0064] 5. Changes in functional groups before and after degradation of microplastics in mulch by strain MD1Y

[0065] After 50 days of cultivation, some wavelength peaks in the infrared spectrum of the mulch microplastics degraded by bacteria MD1Y changed compared with the control group, such as Figure 5 As shown in Figure 2, the increase in the intensity of polar functional groups significantly increases the hydrophilicity of microplastics in mulch films and increases their structural instability, thereby promoting the colonization and degradation of microorganisms on their surfaces. The addition of degrading bacteria MD1Y significantly enhanced the absorption peak intensity of polar functional groups, such as 2917 cm -1 and 2849cm -1 C-H bond (-CH2-), 1470 cm -1 C-OH bending vibration at 600-800cm -1 The absorption peaks in the range are related to crystallinity. After 50 days of degradation, the absorption peaks at 622 cm -1 and 640cm -1 The decrease in the peak intensity at indicates that the crystallinity of the microplastics in the mulch film has decreased, thereby increasing the polarity of the microplastics. All of this indicates that the bacterium MD1Y has a biodegradation effect on the microplastics in the mulch film.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A strain of microplastic-degrading bacteria for mulch film, characterized by: It is Stenotrophomonas maltophilia MD1Y, deposited in the Guangdong Provincial Microbiological Culture Collection Center of China, with the deposit number GDMCC No: 62745; the deposit date is August 25, 2022.

2. A use of a mulch film microplastic-degrading bacterium according to claim 1 in the degradation of residual microplastics in mulch films, characterized in that: The mulch film microplastic is polyethylene mulch film microplastic.

3. A method for degrading residual microplastics in mulch by a strain of mulch microplastic-degrading bacteria according to claim 1, characterized in that: The following steps are involved: (1) Cut the polyethylene mulch into small pieces, crush them with a crusher, and sieve them; (2) The sieved plastic fragments were sequentially soaked in SDS solution, low-concentration ethanol solution, and high-concentration ethanol solution after ultrasonication, rinsed three times with sterile water on a clean bench, and then dried and sterilized to obtain mulch microplastics; The mass fraction of the low-concentration ethanol solution is 75%, and the mass fraction of the high-concentration ethanol solution is 95%; (3) The domesticated mulch microplastic-degrading bacteria MD1Y were transferred to MSM culture medium, mulch microplastics were added, and mulch microplastics were used as the sole carbon source, and the culture was shaken; (4) After the shaking culture is completed, the supernatant is poured out, and after washing, it is dried and weighed to detect the degradation rate of microplastics in the film.

4. The method for degrading residual microplastics in mulch by a mulch film microplastic-degrading bacterium according to claim 3, characterized in that: The size of the small membrane piece is 1×1.5 cm.

5. The method for degrading residual microplastics in mulch by a mulch microplastic-degrading bacterium according to claim 3, characterized in that: The mass fraction of the SDS solution is 2%.

6. The method for degrading residual microplastics in mulch by a mulch microplastic-degrading bacterium according to claim 3, characterized in that: The ultrasonic time is 30 minutes and the soaking time is more than 2 hours.

7. The method for degrading residual microplastics in mulch by a strain of mulch microplastic-degrading bacteria according to claim 3, characterized in that: The sterilization is carried out using an ultraviolet lamp for 15 minutes and the drying temperature is 30°C.

8. The method for degrading residual microplastics in mulch by a strain of mulch microplastic-degrading bacteria according to claim 3, characterized in that: The shaking culture conditions are: shaking culture at 30° C. and 140 r / min for 50 days.

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