Pseudomonas cerumen and application thereof in polluted environment treatment

Abstract

The invention discloses pseudoumen and an application of the pseudoumen in polluted environment treatment. According to the invention, a strain of pseudoumen pseudomonas 25S0261-4 is separated from soil and is gram negative bacillus, the optimal growth temperature of the strain 25S0261-4 is 20-40 DEG C, and the optimal pH range is 5-9. Researches show that the strain 25S0261-4 has a good phosphorus-solubilizing characteristic, and can be used as a phosphorus-solubilizing bacterium for increasing the phosphorus content in soil and improving the soil fertility. The 25S0261-4 strain has an efficient degradation effect on PBAT, the degradation effect of PBAT can be further improved when the 25S0261-4 strain is combined with earthworms for treatment, and it is shown that the earthworms can cooperate with the 25S0261-4 strain to degrade PBAT, be used for treating plastic polluted environments and degrading plastic pollutants in soil and have important significance on improving soil quality and degrading the plastic pollutants.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial degradation, and more specifically relates to a strain of Pseudomonas cerumenensis and its application in the treatment of polluted environments. Background Art

[0002] Biodegradable mulch is considered a promising alternative to traditional plastic mulch because it has similar positive effects on crop growth and quality while degrading into carbon dioxide, water, and minerals in the soil. Polybutylene adipate terephthalate (PBAT) is a biodegradable plastic alternative to traditional plastics. PBAT mulch has good degradation properties in the environment and is expected to become a major component in mulch production. Although biodegradable mulch has a shorter shelf life than traditional mulch, it also produces microplastics, which can still cause soil pollution. The degradation efficiency of biodegradable mulch in saline-alkali environments and its impact on soil nutrient cycling are still unclear.

[0003] Microbial degradation is the primary pathway for PBAT degradation in soil, which also restricts its widespread application in agricultural production. Microbial activity is inhibited, further limiting PBAT degradation. Studies have found that currently, there are relatively few strains capable of degrading PBAT, resulting in a low degradation rate of approximately 10%. This efficiency needs to be improved.

[0004] Earthworms, as a representative representative of soil fauna, contribute a significant portion of the biomass of terrestrial ecosystems and are known as "ecosystem engineers." They play a vital role in soil fertility, as well as microbial metabolism, structure, and function. Earthworms promote the cycling of soil elements by influencing the growth of soil microorganisms. Earthworms are widely distributed worldwide, possess strong adaptability and reproductive capacity, and exhibit high tolerance, bioaccumulation, and resistance to pollution stress. The impact of earthworm activity on soil contributes to the deactivation of soil pollutants such as heavy metals and the removal of pesticides, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls. However, earthworms alone have a limited effect and the degradation cycle is long, typically requiring months or longer. Existing research has found that earthworms can promote soil nitrification by stimulating the growth of ammonia-oxidizing bacteria and archaea in the soil. However, reports on the role of earthworms in the remediation of microplastics, particularly PBAT, are very limited, and there are few studies combining earthworms with microbial strains for environmental remediation.

[0005] Therefore, developing more microbial strains that can efficiently degrade PBAT and can be used in combination with earthworms will help improve soil quality and increase land use efficiency, and will have important economic value and practical significance for farmland soil and the ecological environment. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the existing problem of rarely combining earthworms with microbial strains for polluted environment treatment, and the low efficiency of microbial strains in PBAT degradation, and to provide a strain of Pseudomonas cerumen and its application in polluted environment treatment.

[0007] The first object of the present invention is to provide a strain of Pseudomonas cerumensis.

[0008] The second object of the present invention is to provide the use of Pseudomonas cerumensis.

[0009] The third object of the present invention is to provide an application of Pseudomonas cerumensis in combination with earthworms in the degradation of PBAT.

[0010] A fourth object of the present invention is to provide a preparation.

[0011] A fifth object of the present invention is to provide a method for degrading plastic products containing polybutylene terephthalate-adipate.

[0012] The sixth object of the present invention is to provide a method for controlling plastic pollution in the environment.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides a strain of Pseudomonas cerumensis ( Pseudomonas ceruminis ) 25S0261-4 strain, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 7, 2025, with the collection number GDMCC No: 66103.

[0014] The present invention isolates and obtains a Pseudomonas cerumenicus 25S0261-4 strain in soil. The strain is a Gram-negative bacillus, and its colonies are white and opaque, with a white and opaque surface, a smooth and raised surface, and complete edges, and a diameter of 4 mm to 10 mm. The strain 25S0261-4 has an optimum growth temperature of 20 to 40 DEG C and an optimum pH range of 5 to 9. The strain 25S0261-4 also has good phosphate-solubilizing properties and can decompose tricalcium phosphate, iron phosphate, aluminum phosphate and lecithin. The strain has a maximum decomposition capacity of 75.33 mg / L for tricalcium phosphate, 79.53 mg / L for iron phosphate, 44.24 mg / L for aluminum phosphate, and 7.58 mg / L for lecithin. As a phosphate-solubilizing bacterium, the strain 25S0261-4 can be used to increase the phosphorus content in the soil and improve soil fertility.

[0015] Research in this paper demonstrates that the strain 25S0261-4 can grow on PBAT mulch as its sole carbon source and is highly effective at degrading poly(butylene terephthalate-adipate) (PBAT). Combining the 25S0261-4 strain with earthworms further enhances PBAT degradation, demonstrating that earthworms can synergize with the 25S0261-4 strain to promote PBAT degradation. This suggests that the strain could be used to better manage plastic-contaminated environments and degrade plastic pollutants in soil, potentially contributing to improving soil quality and degrading plastic pollutants.

[0016] The present invention provides the use of the 25S0261-4 strain or its bacterial liquid in the biodegradation of polybutylene terephthalate-adipate or in the degradation of plastic products containing polybutylene terephthalate-adipate.

[0017] The present invention provides application of the 25S0261-4 strain or its bacterial liquid in the preparation of a polybutylene terephthalate-adipate degradation preparation.

[0018] The present invention provides application of the 25S0261-4 strain or its bacterial liquid in environmental treatment of polybutylene terephthalate-adipate plastic pollution.

[0019] The present invention provides the use of a 25S0261-4 strain in combination with earthworms in degrading polybutylene terephthalate-adipate plastic pollution.

[0020] The present invention provides the application of the 25S0261-4 strain as phosphate-solubilizing bacteria.

[0021] Preferably, the 25S0261-4 strain can solubilize soluble phosphorus.

[0022] More preferably, the specifically dissolved soluble phosphorus is tricalcium phosphate, iron phosphate, aluminum phosphate and lecithin.

[0023] The present invention provides use of the 25S0261-4 strain or its bacterial solution in preparing a preparation for increasing soil phosphorus content.

[0024] The invention provides a preparation containing the 25S0261-4 strain.

[0025] Preferably, the degradation agent contains a culture, a culture suspension or a fermentation broth of the 25S0261-4 strain.

[0026] The present invention provides a method for degrading plastic products containing polybutylene terephthalate-adipate, which uses the above-mentioned degradation agent for treatment.

[0027] Preferably, the above-mentioned degradation agent is used together with earthworms for treatment.

[0028] The present invention also provides a method for treating a plastic-polluted environment, which uses the 25S0261-4 strain and its bacterial liquid, or the above-mentioned degradation agent for treatment.

[0029] Preferably, earthworms and 25S0261-4 strain are used to synergistically control plastic-polluted environments.

[0030] Preferably, the plastic pollution refers to pollution from plastic products containing polybutylene terephthalate-adipate.

[0031] Preferably, the polluted environment is contaminated soil, specifically seaside saline soil.

[0032] The present invention has the following beneficial effects: The isolated Pseudomonas cerumenicola strain 25S0261-4 is a Gram-negative bacillus with excellent phosphate-solubilizing properties. It can decompose tricalcium phosphate, iron phosphate, aluminum phosphate, and lecithin. Its maximum decomposition capacity for tricalcium phosphate is 75.33 mg / L, for iron phosphate is 79.53 mg / L, for aluminum phosphate is 44.24 mg / L, and for lecithin is 7.58 mg / L. As a phosphate-solubilizing bacterium, it can be used to increase soil phosphorus content and improve soil fertility. Furthermore, the 25S0261-4 strain has been shown to be able to grow using PBAT mulch as its sole carbon source and to be highly effective in degrading PBAT. Combining the 25S0261-4 strain with earthworms further enhanced its PBAT degradation efficiency, demonstrating that earthworms can synergize with the 25S0261-4 strain to degrade PBAT. This study is useful for the management of plastic-contaminated environments, effectively degrading plastic contaminants in soil, and is of great significance for improving soil quality and degrading plastic contaminants. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the colony morphology of the strain.

[0034] Figure 2 This is the phylogenetic tree analysis diagram of the strain.

[0035] Figure 3 These are the optimal growth conditions for strain 25S0261-4 (a is temperature; b is pH).

[0036] Figure 4 This is the phosphate solubility test result of strain 25S0261-4.

[0037] Figure 5 It is a graph showing the growth status of the strain under different conditions.

[0038] Figure 6 It is a bar chart of PBAT degradation rate under different treatments.

[0039] Figure 7These are SEM comparison pictures of PBAT degradation (a is CK; b is earthworm + strain 25S0261-4; c is strain 25S0261-4). DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples 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 the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0042] Modified SM medium (carbon-free): 1.0 g / L NH4NO3, 0.2 g / L MgSO4·7H2O, 1.0 g / L K2HPO4, 0.1 g / L CaCl2·2H2O, 0.15 g / L KCl, 0.001 g / L FeSO4·6H2O, 0.001 g / L ZnSO4·7H2O, 0.001 g / L MnSO4, pH adjusted to 7.4 ± 0.2, sterilized at 121°C for 20 min; solid modified SM medium with 15-20 g / L agar powder added.

[0043] LB medium: 5 g yeast extract, 10 g peptone, 5 g NaCl, 1000 mL water, pH 7.4-7.6; LB solid medium with 18-20 g agar powder added.

[0044] Tricalcium phosphate medium: glucose 10 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, Ca3(PO4)25.0 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 30 mg, MnSO4·H2O 30 mg, pH = 7±0.2.

[0045] Example 1 Screening and Isolation of Strain Soil samples were collected from the vicinity of Nanshan Village, Nanshan Town, Xuwen County, Zhanjiang City. 1.0 g of soil sample was accurately weighed, air-dried, ground and dispersed evenly, dissolved in sterile water at a mass ratio of 1:10, and the filtrate was filtered and used as 10 -1 Initial solution, obtain 10 -2 and 10 -3 Soil gradient dilutions were diluted and spread onto solid modified SM medium and covered with PBAT mulch (purchased from Hengrun Plastic Co., Ltd., Gaomi City, Shandong Province). Culture was performed at 28°C for 5–7 days until a single colony grew. Single colonies were then picked and purified on LB medium until a single strain was obtained.

[0046] The strains screened above were inoculated into LB medium and cultured at 30°C with shaking at 180 rpm for 24 h. -4 , 10 -5 , 10 -6 , 10 -7 Spread 100 μL of each dilution onto tricalcium phosphate medium in triplicate for each of the four gradients. Incubate the cells upside down at 30°C for 3 days, observing for clear zones on the medium. Select colonies with clear zones and repeatedly streak and purify them. Isolate and purify bacteria with excellent phosphate-solubilizing ability. The resulting strain, designated 25S0261-4, was stored in a refrigerator at 4°C.

[0047] Example 2 Identification of strain characteristics 1. Morphological characteristics of strains The colony morphology of 25S0261-4 was observed by plate streaking method, Gram staining of activated strains was performed, and the bacterial and spore morphology was observed by optical microscope. Figure 1 As shown, the colonies are white, opaque, with a smooth and raised surface, complete edges, and a diameter of 4 mm to 10 mm; Gram staining identified the 25S0261-4 strain as a Gram-negative bacterium.

[0048] 2. Molecular Biology Identification Total bacterial DNA was extracted using a kit, and the bacterial 16S rDNA universal primers 27F and 1492R were used to amplify the bacterial 16S rDNA by PCR. The PCR amplification product was recovered and sequenced. The sequencing results showed that the nucleotide sequence of strain 25S0261-4 was shown in SEQ ID NO: 1. The obtained DNA sequence was then subjected to Blast analysis on the NCBI website (http: / / www.ncbi.nlm.nih.gov), and a phylogenetic tree was constructed using MEGA6.0 software.

[0049] The constructed phylogenetic tree is as follows Figure 2 As shown, the 16S rDNA sequence of the strain 25S0261-4 isolated and identified by the present invention is shown. Pseudomonas ceruminis strain BML-PP028 has a high homology. Based on the above strain morphological characteristics and molecular biological identification results, strain 25S0261-4 was taxonomically assigned to the genus Pseudomonas ( Pseudomonas cerumen in Pseudomonas cerumen ( Pseudomonas ceruminis) strain, named as strain 25S0261-4, and deposited in Guangdong Provincial Microbiological Culture Collection Center on April 7, 2025, with the deposit number GDMCC No: 66103, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0050] Example 3 Growth conditions of strains 1. Effect of temperature on strain growth 50 μL of 25S0261-4 bacterial suspension was added to 150 mL of sterilized LB liquid medium and cultured at 200 rpm and 28°C for 24 h. 1% inoculum was inoculated into LB solid medium and the temperature was set at 10, 20, 30, 40, and 50°C for three replicates. Culture was performed at 200 rpm and 28°C under constant temperature shaking. 200 μL of bacterial suspension was collected every 6 h for detection (OD 600 ), draw a growth curve.

[0051] The results are as follows Figure 3 As shown in (a), the optimum temperature of strain 25S0261-4 is 20-40°C. The growth curve of strain 25S0261-4 is shown in Figure 5 As shown, the strain can reach the optimal growth state in 20 minutes.

[0052] 2. Effect of pH on strain growth The bacterial liquid of strain 25S0261-4 was inoculated into LB medium at a 1% inoculum volume, with 3 replicates. The pH values ​​were set at 4, 5, 6, 7, 8, 9, and 10, respectively. The culture was cultured at 30°C and 180 rpm under constant temperature shaking conditions. The growth of the strain was measured after 3 days (OD 600 nm).

[0053] The results are as follows Figure 3 As shown in (b), the optimal pH range of strain 25S0261-4 is 5-9.

[0054] Example 4 Phosphate solubilization performance test of strains Strain 25S0261-4 was inoculated at 1% inoculum into tricalcium phosphate, iron phosphate, aluminum phosphate, and lecithin media, respectively. Three replicates were cultured at 30°C, 180 rpm, and constant temperature shaking for 5 days. Daily, 2 mL of culture medium was centrifuged at 8000 rpm for 10 minutes. The supernatant of the uninoculated culture medium was used as a blank control. The absorbance at 700 nm was measured using the molybdenum antimony colorimetric method to calculate the soluble phosphorus increment.

[0055] The results of the test are as follows Figure 4As shown, the maximum decomposition ability of the 25S0261-4 strain on tricalcium phosphate was 75.33 mg / L after 5 days, the maximum decomposition ability on iron phosphate was 79.53 mg / L, the maximum decomposition ability on aluminum phosphate was 44.24 mg / L, and the maximum decomposition ability on lecithin was 7.58 mg / L, indicating that the 25S0261-4 strain has good phosphate solubilization performance.

[0056] Example 5 PBAT degradation ability Laboratory simulation of PBAT mulch degradation: On a sterile workbench, cut the PBAT mulch into 2.0 cm x 2.0 cm sheets. Weigh the initial mass, recorded as M0. Soak the sheets in 3% KCl solution for 1 hour, then rinse them three to four times with 100% anhydrous ethanol. Rinse with sterile water, dry them in a sterile Petri dish, and sterilize them under UV light for 4 hours.

[0057] The purified strain 25S0261-4 was cultured in LB liquid at 28°C for 18 h, and then centrifuged at 12000 rpm for 10 min to obtain the bacterial cells. After adding an equal volume of PBS liquid to resuspend and centrifuge twice, the obtained bacterial cells were prepared with PBS to a concentration of 1×10 8 cfu / mL of 25S0261-4 bacterial solution.

[0058] The sterilized PBAT mulch and 25S0261-4 bacterial solution were added to 100 mL of modified SM medium (liquid medium), and a blank control group (CK) was set up. The OD values ​​of the bacterial solution were recorded on the 10th, 20th, 30th, and 40th days. 600 nm, the PBAT film was taken out on the 40th day, and the remaining mass M1 of the PBAT film was weighed after drying. 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.

[0059] The calculation formula of the weight loss method is: Degradation efficiency ŋ = (M0-M1) / M0×100% The PBAT degradation rate of strain 25S0261-4 was calculated to be 27% after 40 days using the weight loss method.

[0060] Example 6 Degradation of PBAT by earthworms Earthworms of roughly uniform size and with adult rings were selected and placed on a culture dish lined with moistened filter paper. The dish was then covered with plastic wrap, punctured, and placed in an incubator. The dish was incubated at 25°C in the dark for 24 hours to allow for the elimination of intestinal contents. Eight earthworms were placed. The soil depth of the incubator was set to 30 cm. PBAT was mixed and stirred in the top 2 cm of soil and incubated at 25°C. The soil concentration was maintained at 60%. On the 40th day, the PBAT film was removed, dried, and the remaining mass of the PBAT film was weighed and calculated using the weight loss method formula from Example 5.

[0061] The earthworms broke up the PBAT film through feeding and intestinal peristalsis. The results showed that the degradation rate of PBAT by earthworms was 21% after 40 days.

[0062] Example 7 Degradation of PBAT by earthworm combined with bacterial strain Earthworms of roughly the same size and with adult rings were selected and placed on a culture dish lined with moistened filter paper. The top was covered with plastic wrap, punctured, and placed in an incubator. The dish was incubated at 25°C in the dark for 24 hours to allow the intestinal contents to be eliminated. Sterilized soil was used, and eight earthworms were placed. The sterilized soil in the culture box was set to a depth of 30 cm. PBAT was mixed and stirred in the top 2 cm of sterilized soil. 1×10 8 cfu / mL of strain 25S0261-4, then added earthworms and incubated at 25°C. The soil fertility was maintained at 60%. On the 40th day, the PBAT film was removed, dried, and the remaining mass of the PBAT film was weighed. A control group, CK, was treated with only PBAT. The PBAT degradation rate was calculated using the weight loss method described in Example 5, and the degradation effect at different times was statistically analyzed.

[0063] The degradation rates of different treatment groups were as follows Figure 6 As shown in the data, it was calculated that the degradation rate of PBAT in the combined treatment group of earthworms and bacterial strains was 35%. Compared with the treatment of PBAT with 25S0261-4 or earthworms alone, the combined use of 25S0261-4 and earthworms can improve the degradation rate of PBAT, indicating that earthworms and bacterial strains have a synergistic effect, and their combined use can promote the degradation of PBAT.

[0064] Scanning electron microscopy observations were performed to compare the SEM images of PBAT films treated with CK, 25S0261-4, and earthworm combined strain 25S0261-4 for 40 days. Figure 7 As shown, the surface of the control group CK and PBAT membranes was relatively smooth and without cracks ( Figure 7 a) The PBAT membrane treated with strain 25S0261-4 became rough and uneven, with obvious erosion holes and clear cracks ( Figure 7b) The PBAT membrane treated with earthworm combined with strain 25S0261-4 also showed erosion and cracks ( Figure 7 c).

[0065] In summary, the present invention isolated a strain of Pseudomonas cerumenicus that has the ability to solubilize phosphate and degrade PBAT microplastics ( Pseudomonas ceruminis ) 25S0261-4 strain, 25S0261-4 strain is an efficient degrading bacterium of polybutylene terephthalate adipate (PBAT), which can use PBAT mulch as the only carbon source for growth and has a good degradation effect on PBAT mulch; at the same time, 25S0261-4 strain combined with earthworms can also degrade PBAT well and promote the degradation of PBAT. It can be used for the degradation of plastic pollutants in the soil, which is of great significance to improving soil quality and degrading plastic pollutants.

[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Pseudomonas cerumenicus ( Pseudomonas ceruminis ) 25S0261-4 strain, characterized in that This strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 7, 2025, with the collection number GDMCC No: 66103.

2. Use of the 25S0261-4 strain or its bacterial solution according to claim 1 in the biodegradation of polybutylene terephthalate-adipate or in the degradation of plastic products containing polybutylene terephthalate-adipate.

3. Use of the 25S0261-4 strain or its bacterial solution according to claim 1 in environmental remediation of polybutylene terephthalate-adipate plastic pollution.

4. Use of the 25S0261-4 strain described in claim 1 in combination with earthworms in degrading polybutylene terephthalate-adipate plastic pollution.

5. Use of the 25S0261-4 strain according to claim 1 as a phosphate-solubilizing bacterium.

6. Use of the 25S0261-4 strain or its bacterial solution according to claim 1 in preparing a preparation for increasing soil phosphorus content.

7. A preparation, characterized in that Contains the 25S0261-4 strain according to claim 1.

8. A method for degrading a plastic product containing polybutylene terephthalate-adipate, characterized in that: The method comprises using the preparation according to claim 7 for treatment.

9. The method according to claim 8, characterized in that The preparation according to claim 7 is used for treatment together with earthworms.

10. A method for treating plastic pollution environment, characterized in that: The treatment is carried out using the 25S0261-4 strain or its bacterial liquid described in claim 1, or using the preparation described in claim 7.

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