Cadmium-resistant P.megaterium and application thereof in treatment of combined polluted environment

Abstract

The invention discloses cadmium-resistant P.megaterium and an application of the cadmium-resistant P.megaterium in treatment of a combined polluted environment. The invention provides a PBAT (poly (butyleneadipate-co-terephthalate)) efficient degradation bacterium, namely, a Pristia megaterium S261-3 strain, and the strain can grow at the temperature of 10-50 DEG C, has certain salt and cadmium resistance, and can normally grow in a relatively high salt or cadmium concentration range. Meanwhile, the S261-3 strain has a good degradation effect on PBAT, can also degrade PBAT under cadmium stress, and can be used for degrading plastic pollutants in cadmium-polluted soil. The microbial strain provided by the invention has stronger adaptability to polluted environment, meets the repair requirement of a combined pollution site, can be used for comprehensive treatment of micro-plastic and heavy metal combined pollution, is beneficial to improvement of soil quality and improvement of land utilization efficiency, and has important practical significance for soil combined pollution repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms and environmental governance, in particular, to a cadmium-tolerant Pseudomonas putida and its application in the governance of compound pollution environment. BACKGROUND

[0002] Polybutylene adipate-co-terephthalate (PBAT) is a widely used biodegradable plastic, mainly used to replace traditional petroleum-based plastics (such as PE, PP). It is widely used in packaging materials, agricultural mulch, disposable tableware, industrial products and other fields. PBAT is a thermoplastic biodegradable plastic copolymerized from adipic acid butanediol and terephthalic acid butanediol, which has good heat resistance, impact resistance, ductility and elongation at break. PBAT needs to be effectively degraded under industrial composting conditions (high temperature, high humidity, microbial action), and the degradation rate is very slow in the natural environment (such as soil, ocean). If it is discarded in the natural environment, it may break into microplastics, pollute the soil and water, and threaten the ecological system. As a carrier of heavy metals, microplastics seriously threaten soil quality and crop growth and development.

[0003] Cadmium (Cd) is one of the most toxic heavy metal pollutants, which can easily accumulate through the food chain and harm human health. Plastic mulch (such as PBAT) and heavy metal compound pollution has become a global environmental governance problem. Traditional physical and chemical remediation methods are high in cost and easy to cause secondary pollution, while microbial remediation has become a research hotspot due to its high efficiency and green characteristics.

[0004] Current research shows that using microorganisms to degrade PBAT mulch can effectively shorten the residual time of mulch in the field, but conventional PBAT degrading strains have low efficiency, poor environmental adaptability, and few strains that can tolerate heavy metal stress. However, the existing research on cadmium-tolerant strains is mostly aimed at degrading single heavy metal pollution environment, and the plastic degradation capacity is limited, which is difficult to meet the remediation needs of compound pollution sites. Therefore, it is of great significance to develop more microbial strains with cadmium tolerance and plastic degradation functions, which can be used for comprehensive governance of microplastic and heavy metal compound pollution, and help to improve soil quality and improve land use efficiency. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the shortage of existing microbial strains with cadmium tolerance and plastic degradation functions. The present application provides a cadmium-tolerant Pseudomonas putida and its application in the governance of compound pollution environment.

[0006] The first object of the present application is to provide a Pseudomonas putida S261-3 strain.

[0007] A second objective of this invention is to provide the application of the Priestella megaterium S261-3 strain.

[0008] A third objective of this invention is to provide a degradation agent or a heavy metal tolerance agent.

[0009] A fourth object of the present invention is to provide a method for degrading plastic articles containing polybutylene terephthalate.

[0010] The fifth objective of this invention is to provide a method for the environmental remediation of plastic pollution and / or cadmium pollution.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a strain of Priestella megaterium ( Priestia megaterium The strain S261-3 was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 14, 2025, with accession number GDMCC No. 66327.

[0012] This invention isolated and identified a strain of *Priestella giantiflora* S261-3 from the intestinal contents of *Eisenia fetida*. The colonies of this strain are white, translucent, glossy, with a raised center, a smooth, convex surface, and regular edges. Gram staining was positive, and reactions with catalase and catalase were also positive; reactions with oxidase were negative. Studies show that strain S261-3 can grow at temperatures ranging from 10 to 50°C and exhibits certain salt and cadmium tolerance. It can still grow normally at high salt or cadmium concentrations, tolerating salt concentrations of 2–8% and cadmium concentrations of 50–300 mg / L. Furthermore, strain S261-3 shows good degradation effects on polybutylene terephthalate (PBAT), even under high cadmium stress. This makes it suitable for degrading plastic pollutants in cadmium-contaminated soil, which is of significant importance for improving soil quality and degrading plastic pollutants in cadmium-contaminated soil.

[0013] Therefore, the present invention provides the application of *Priestella megaterium* S261-3 strain or its bacterial culture in the degradation of polybutylene terephthalate (PET) under heavy metal contamination.

[0014] Preferably, the heavy metal polluted environment refers to an environment polluted by the heavy metal cadmium.

[0015] More preferably, the cadmium concentration in the cadmium-polluted environment is 50–300 mg / L.

[0016] This invention provides the use of *Priestella megaterium* S261-3 strain or its bacterial culture in the preparation of plastic products containing polybutylene terephthalate (PET) or in the preparation of heavy metal resistant agents.

[0017] This invention provides the application of *Priestella megaterium* S261-3 strain or its bacterial solution in the remediation of plastic and / or cadmium pollution environments.

[0018] Preferably, the plastic pollution refers to pollution from polybutylene terephthalate (PET) or plastic products containing PET.

[0019] This invention provides a degradation agent or heavy metal tolerance agent containing *Priscilla megaterium* strain S261-3.

[0020] Preferably, the degradation agent or heavy metal tolerance agent contains a culture, culture suspension or fermentation broth of strain S261-3.

[0021] More preferably, the concentration of the S261-3 bacterial solution is not less than 1×10⁻⁶. 8 cfu / mL.

[0022] This invention provides a method for degrading plastic products containing polybutylene terephthalate (PET), wherein the plastic products containing PET are treated with the aforementioned degrading agent or heavy metal resistant agent.

[0023] The present invention also provides a method for environmental remediation of plastic pollution and / or cadmium pollution, using *Priscilla megaterium* S261-3 strain or its bacterial culture, or using the above-mentioned degradation agent or heavy metal tolerance agent to treat the polluted environment.

[0024] The present invention has the following beneficial effects: This invention isolated a highly efficient PBAT-degrading bacterium, *Priscilla megaterium*, from the intestinal contents of *Eisenia fetida*. Priestia megaterium The S261-3 strain can grow at temperatures ranging from 10 to 50°C and exhibits certain salt and cadmium tolerance. It can still grow normally within high salt or cadmium concentration ranges, tolerating salt concentrations of 2-8% and cadmium concentrations of 50-300 mg / L. Furthermore, the S261-3 strain demonstrates good degradation of polybutylene terephthalate (PBAT), growing in environments where PBAT mulch film is the sole carbon source. It initiates hydrolysis on the PBAT film, leading to molecular chain breakage and thus degrading the PBAT film. The S261-3 strain also degrades PBAT effectively under cadmium stress, making it suitable for degrading plastic pollutants in cadmium-contaminated soils.

[0025] The microbial strains provided by this invention are more adaptable to polluted environments, can reduce biological treatment costs, meet the remediation needs of sites with complex pollution, and have the ability to resist cadmium and plastic degradation. They can be used for the comprehensive treatment of complex pollution of microplastics and heavy metals, help improve soil quality, increase land use efficiency, and have important practical significance for the remediation of cadmium pollution in soil. Attached Figure Description

[0026] Figure 1 This is a colony photograph of strain S261-3.

[0027] Figure 2 This is a phylogenetic tree diagram of strain S261-3.

[0028] Figure 3 This is a growth curve of strain S261-3.

[0029] Figure 4 This is a growth curve of strain S261-3 at different temperatures.

[0030] Figure 5 This is a growth curve of strain S261-3 at different salt concentrations.

[0031] Figure 6 The figure shows the effect of different cadmium ion concentrations on strain S261-2.

[0032] Figure 7 SEM comparison images of PBAT mulch film degraded by strain S261-3 ( Figure 7 In this context, A stands for CK. Figure 7 B in the text refers to S261-3 processing.

[0033] Figure 8 Fourier transform infrared spectrum of PBAT mulch film degraded by strain S261-3.

[0034] Figure 9 Bar chart showing the weight loss rate of PBAT mulch film degraded by strain S261-3 under cadmium stress. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] The culture medium used in the examples is as follows (pH adjusted with 1 mol / L NaOH and HCl): LB medium (g / L): yeast extract 5g, peptone 10g, NaCl 5g, water 1000mL, pH 7.4~7.6, agar powder: 2~2.5%.

[0038] NA medium (g / L): 3g beef extract, 5g peptone, 1000mL water, pH 6.6-7.0, agar powder: 2-2.5%.

[0039] Inorganic salt culture medium (g / L): NH4Cl 2.5g, CaCl2·2H2O 0.05g, Na2HPO4 2.5g, sulfuric acid heptahydrate 0.5g, KH2PO4 2.5g, MnCl2·4H2O 0.05g, H2O 1000mL, pH 7.5.

[0040] LB liquid medium (g / L): yeast extract 5g, peptone 10g, NaCl 5g, water 1000mL, pH 7.4~7.6.

[0041] Example 1: Isolation and Identification of Degrading Bacteria 1. Strain screening and isolation The samples were selected from healthy, vigorous adult Eisenia fetidae of the same generation, with similar weight and distinct clitellum. Before treatment, the Eisenia fetidae were placed in a greenhouse for 7 days to acclimatize, and then placed on moist filter paper for 48 hours for gut cleansing. After gut cleansing, nine earthworms were selected and immersed in 75% alcohol for 10 seconds. Under aseptic conditions, the earthworms were removed and disinfected in 95% alcohol for 2 minutes, rinsed in sterile water, and then aseptically dissected in a wax tray. The digestive tracts were removed and rapidly ground in a sterile mortar. The grinding solution was diluted with sterile water to a final concentration of 10. -2 10 -3 10 -4 10 -5 10 -6 Take 10 -3 and 10 -4 For each dilution, 50–100 μL of bacterial suspension from each group was transferred, and two plates were prepared for each dilution. Each dilution was repeated three times. The plates were incubated at 28°C for 5–7 days until a single colony grew. Colonies with different characteristics were picked, streaked on LB and NA plates, purified, and cultured until a single strain was finally obtained.

[0042] PBAT mulch film was uniformly cut into 2.5cm × 2.5cm pieces, soaked in 3% KCl solution for 1 hour, then washed 3-4 times with 100% anhydrous ethanol, and finally rinsed with sterile water. After drying in a dry, sterile petri dish, it was sterilized by irradiation under ultraviolet light for 4 hours. It was then placed in an inorganic salt medium (liquid medium) and inoculated with the single strain obtained above, with 3 replicates per group. The strains were placed in a 28℃ shaking incubator and the colony growth was observed. After repeated screening, the strain with the PBAT mulch film degradation effect was selected and named S261-3.

[0043] 2. Identification of strains (1) Morphological characteristics of the strain: The purified S261-3 strain was streaked on LB solid medium and cultured at 28℃ for 24 h. The results are as follows. Figure 1 As shown, the colonies are white, translucent, and glossy, with a raised center, a smooth, convex surface, and neat edges.

[0044] (2) Physiological and biochemical characteristics of the strain: Physiological and biochemical characteristics tests showed that strain S261-3 was Gram-positive, and reacted positively with catalase and catalase; and was negative with oxidase.

[0045] (3) Molecular biological characteristics: Total bacterial DNA was extracted using a kit method, and the bacterial 16S rDNA was amplified by PCR using universal primers 27F and 1492R. The PCR amplification products were recovered and sequenced. The sequencing results showed that the nucleotide sequence of strain S261-3 was as shown in SEQ ID NO:1. The obtained DNA sequence was then compared and analyzed by Blast on the NCB website, and a phylogenetic tree was constructed using MEGA6.0 software.

[0046] The constructed phylogenetic tree is as follows Figure 2 As shown, the 16S rDNA sequence of strain S261-3 isolated and identified in this invention is similar to... Bacillus megaterium strain IHB B4625 (KF475802.1) exhibits high homology (in the art). Bacillus megaterium Reclassified and renamed Priestia megaterium Based on the above morphological characteristics, physiological and biochemical characteristics, and molecular biological identification results, strain S261-3 is taxonomically classified as *Priscilla megaterium*. Priestia megaterium The strain was named S261-3 and deposited at the Guangdong Provincial Microbial Culture Collection Center on May 14, 2025, with accession number GDMCC No. 66327. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0047] Determination of growth conditions for strain S261-3 (Section 2) 1. Growth curve of strain S261-3 Add 50 μL of S261-3 bacterial culture to 150 mL of sterilized LB liquid medium and incubate at 200 rpm and 28 °C for 24 h. Inoculate 2% of the culture into LB solid medium and incubate at 200 rpm and 28 °C with constant temperature shaking. Take 200 μL of the bacterial culture every 10 min to detect the OD (dose dispersive precipitate). 600 ), and plot the growth curve.

[0048] The growth curve of strain S261-3 is shown in the figure. Figure 3 As shown, the strain reaches its optimal growth state in 30 minutes.

[0049] 2. The effect of temperature on strain growth Add 50 μL of S261-3 bacterial culture to 150 mL of sterilized LB liquid medium and incubate at 200 rpm and 28 °C for 24 h. Inoculate 2% of the culture into LB solid medium at temperatures of 10, 20, 30, 40, and 50 °C for three replicates, incubating at 200 rpm and 28 °C with constant temperature shaking. Take 200 μL of the bacterial culture every 6 h to analyze the OD (dose dispersive precipitate). 600 ), and plot the growth curve.

[0050] The growth curves of strain S261-3 at different temperatures are as follows: Figure 4 As shown, strain S261-3 can grow at temperatures ranging from 10 to 50°C, with the optimal growth temperature being 30°C.

[0051] 3. Effects of salinity on bacterial growth Add 50 μL of S261-3 bacterial culture to 150 mL of sterilized LB liquid medium and incubate at 200 rpm and 28 °C for 24 h. Inoculate 2% of the culture into LB solid medium at salinities of 0%, 2%, 4%, 6%, and 8%, with three replicates. Incubate at 200 rpm and 28 °C with constant temperature shaking. Take 200 μL of the bacterial culture every 6 h to analyze the OD (dose dispersive precipitate). 600 ), and plot the growth curve.

[0052] The growth curves of strain S261-3 under different salt stresses are as follows: Figure 5 As shown, strain S261-3 can grow under salinity conditions of 0–8%, and can also survive and grow in environments with higher salt concentrations, making it suitable for use in salt stress environments.

[0053] 4. Cd ion tolerance concentration Prepare the liquid nutrient medium required for the growth of strain S261-3, add a cadmium standard solution prepared with cadmium chloride, and adjust the Cd concentration in the medium. 2+ The concentrations were 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L, with a pH of 7.0. The activated strain S261-3 was inoculated into sterilized liquid nutrient medium as the experimental group, and uninoculated medium as the control group. Three replicates were set up, and the cultures were incubated at 200 rpm and 28℃ with constant temperature shaking. 200 μL of bacterial solution was collected every 6 hours to detect the OD (dose dispersive precipitate). 600 Growth curves were plotted. The strain's tolerance to different Cd ion concentrations was determined.

[0054] The growth curves of strain S261-3 at different cadmium concentrations are as follows: Figure 6 As shown, strain S261-3 can grow well under conditions of cadmium ion concentration of 50-300 mg / L, has a certain tolerance to Cd ions, and can grow and survive in heavy metal cadmium-polluted environments.

[0055] Example 3: Degradation effect of strain S261-3 on PBAT mulch film Laboratory simulation of PBAT mulch film degradation: On a sterile operating table, PBAT mulch film was uniformly cut into 2.5cm × 2.5cm pieces, and its initial mass was recorded as M0. First, it was soaked in 3% KCl solution for 1 hour, then washed 3-4 times with 100% anhydrous ethanol, then rinsed with sterile water, and finally dried in a dry, sterile petri dish and sterilized under ultraviolet light for 4 hours.

[0056] The purified strain S261-3 was cultured in LB broth at 28℃ for 18 h, and then centrifuged at 12000 rpm for 10 min to obtain bacterial cells. The cells were resuspended in an equal volume of PBS and centrifuged twice. The obtained bacterial cells were then prepared into 1×10⁻⁶ cells using PBS. 8 S261-3 bacterial suspension at cfu / mL.

[0057] Sterilized PBAT mulch film and S261-3 bacterial solution were added to 100 mL of inorganic salt medium (liquid medium), and a blank control group (CK) was set up. The OD of the bacterial solution was recorded on days 10, 20, 30 and 40. 600 nm, the PBAT film was removed on day 40, dried and the remaining mass M1 of the PBAT film was weighed. The degradation efficiency ŋ of the PBAT film was calculated by the weight loss method, and the degradation of the PBAT film was observed by scanning electron microscopy.

[0058] The formula for calculating degradation efficiency using the weight loss method is: ŋ = (M0) / (M0) M1) / M0×100%.

[0059] Scanning electron microscopy (SEM) was performed on PBAT membranes treated with CK and S261-3 for 40 days. The results are as follows: Figure 7 As shown, the control group CK and PBAT membrane surfaces were relatively smooth and without cracks. Figure 7 In strain A), PBAT membranes treated with strain S261-3 became rough and uneven, exhibiting obvious erosion holes and clear cracks, among other micromorphological features. Figure 7 (B in the middle).

[0060] PBAT mulch film at 500–4000 cm -1 Fourier infrared spectra between them are as follows Figure 8 As shown, at 2954cm -1 1721cm -1 and 1268 cm -1 The vibration peak showed significant weakening and positional shift, located at 1721 cm⁻¹. -1 One peak is the carbonyl absorption peak (C=O). As the degradation time increases, the carbonyl absorption peak is significantly enhanced. This may be because the ester bond in the biodegradable mulch film breaks down, resulting in a large number of carbonyl groups. The weakening of the C-O-C bond indicates that hydrolysis is initiated under the action of microbial strain S261-3, which eventually leads to the breakage of the molecular chain and the degradation of the PBAT mulch film. The degradation efficiency of PBAT was 42.37% as determined by the weight loss method.

[0061] Example 4: Degradation of PBAT by strain S261-3 under cadmium stress On a sterile operating table, PBAT mulch film was uniformly cut into 2.5cm × 2.5cm pieces, and its initial mass was recorded as M0. First, it was soaked in 3% KCl solution for 1 hour, then washed 3-4 times with 100% anhydrous ethanol, followed by rinsing with sterile water, and then dried in a dry, sterile petri dish and sterilized under ultraviolet light for 4 hours. The obtained Bacillus megaterium S261-3 strain was cultured in LB liquid at 28℃ for 18 hours, centrifuged at 12000 rpm for 10 minutes to obtain bacterial cells, resuspended in an equal volume of PBS, and centrifuged twice. The obtained bacterial cells were then prepared into 1×10⁻⁶ saturates using PBS. 8 CFU / mL Bacillus megaterium S261-3 bacterial suspension was used. Sterilized PBAT mulch film and S261-3 bacterial suspension were added to modified inorganic salt medium (liquid medium) with cadmium concentrations of 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L, respectively, for incubation. On day 40, the PBAT film was removed, dried, and the remaining mass M1 of the PBAT mulch film was weighed. The degradation efficiency ŋ of the PBAT mulch film at different salt concentrations was calculated using the weight loss method, the same as in Example 3.

[0062] Degradation rate statistics are as follows Figure 9 As shown, strain S261-3 exhibits good degradation effects on PBAT mulch film at cadmium concentrations of 50–100 mg / L. At 100 mg / L cadmium concentration, its degradation efficiency is comparable to that of PBAT alone, reaching 42.37%. This indicates that strain S261-3 can efficiently degrade PBAT in heavy metal contaminated environments, unaffected by cadmium. While the degradation efficiency of strain S261-3 on PBAT mulch film gradually decreases at cadmium concentrations of 200–300 mg / L, the degradation rate still reaches 15–22%. This suggests that strain S261-3 can be applied to environments with combined heavy metal and microplastic pollution, better degrading PBAT mulch film in cadmium-contaminated environments, contributing to improved soil quality and land use efficiency. The strain demonstrates strong adaptability to polluted environments and can meet the remediation needs of sites with combined pollution. These results provide a new microbial resource and technical approach for solving the problem of combined heavy metal and microplastic pollution in farmland soils. By applying strain S261-3 to soil environments contaminated with both cadmium and PBAT mulch film, it is hoped that the effective degradation of PBAT mulch film can be achieved while mitigating the harmful effects of cadmium on the soil ecosystem. This will provide a scientific basis and practical guidance for sustainable agricultural development and soil pollution remediation projects. Further research can optimize the strain's culture conditions and application methods to enhance its degradation efficiency and adaptability in complex real-world environments.

[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Priestella megaterium ( Priestia megaterium S261-3 strain, characterized in that, The strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 14, 2025, with accession number GDMCC No. 66327.

2. The application of the S261-3 strain or its bacterial culture as described in claim 1 in the degradation of polybutylene terephthalate (PET) under heavy metal contamination.

3. The application according to claim 2, characterized in that, The heavy metal pollution environment refers to the environment polluted by the heavy metal cadmium.

4. The S261-3 strain of claim 1 or its bacterial culture, or its use in the preparation of plastic products containing polybutylene terephthalate (PET) or in the preparation of heavy metal resistant agents.

5. The application of the S261-3 strain or its bacterial solution as described in claim 1 in the environmental remediation of plastic pollution and / or cadmium pollution.

6. The application according to claim 5, characterized in that, The plastic pollution refers to pollution from polybutylene terephthalate or plastic products containing polybutylene terephthalate.

7. A degradation agent or heavy metal tolerance agent, characterized in that, Contains the S261-3 strain as described in claim 1.

8. The degrading agent or heavy metal resistant agent according to claim 7, characterized in that, Cultures, culture suspensions, or fermentation broths containing strain S261-3.

9. A method for degrading plastic articles containing polybutylene terephthalate (PET), characterized in that, Plastic products containing polybutylene terephthalate are treated with the degrading agent or heavy metal resistant agent described in claim 7 or 8.

10. A method for environmental remediation of plastic pollution and / or cadmium pollution, characterized in that, The polluted environment may be treated by using the S261-3 strain or its bacterial culture as described in claim 1, or by using the degrading agent or heavy metal tolerance agent as described in claim 7 or 8.

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