A strain capable of synchronously degrading DEHP and removing heavy metals and its application in the treatment of complex pollution environment

Abstract

This invention discloses a bacterial strain capable of simultaneously degrading DEHP and removing heavy metals, and its application in the remediation of environments with complex pollution. This invention provides a strain of *Serratia marcescens* (…). Serratia decaying The strain 25S0741-2 was deposited at the Guangdong Provincial Microbial Culture Collection Center on June 16, 2025, with accession number GDMCC NO: 66522. This strain exhibits good salt and alkali tolerance, high tolerance to DEHP and heavy metal ions, and can efficiently degrade di(2-ethylhexyl) phthalate (DEHP) while simultaneously removing heavy metal ions. It can also degrade DEHP in heavy metal stress environments, making it suitable for adsorbing and removing heavy metal ions and degrading DEHP in coastal saline-alkali environments. This provides a new strain resource for the bioremediation of complex polluted environments in saline-alkali soils and offers a new approach for the synergistic treatment of heavy metals and organic pollutants.

Description

A strain capable of simultaneously degrading DEHP and removing heavy metals and its application in the remediation of complex polluted environments. Technical Field

[0001] This invention relates to the field of microbial and environmental pollution control technology, and more specifically, to a strain capable of simultaneously degrading DEHP and removing heavy metals and its application in the treatment of complex polluted environments. Background Technology

[0002] In agriculture, plastic film is widely used due to its significant advantages such as increasing crop yield, drought resistance, water conservation, and soil moisture retention. However, because plastic products are difficult to degrade naturally, coupled with improper use and recycling management, large amounts of residues accumulate in farmland soil. This not only leads to soil compaction, secondary salinization, and weakened soil fertility, but also causes crop yield reduction and affects grain production. Di(2-ethylhexyl) phthalate (DEHP), as a plasticizer in plastic products such as polyvinyl chloride (PVC), increases the elasticity and toughness of plastics and is widely used in the plastics industry. DEHP, as an environmental pollutant, can be widely detected in surface water, groundwater, drinking water, air, soil, and in the bodies of animals and plants, entering the body through the gastrointestinal tract, respiratory tract, and skin absorption. In recent years, DEHP has been detected as a major PAE (paraben) pollutant in soil-plant systems. With the increasing production of DEHP year by year, its concentration in the environment is also gradually increasing, and its toxic side effects are receiving increasing attention.

[0003] Meanwhile, heavy metal pollution is a prominent and highly concerning issue in soil environmental problems. Its sources are widespread, encompassing industrial emissions, pesticide application, agricultural use of sewage sludge, and mining activities. Because heavy metals are difficult to degrade and bioaccumulate, they remain in the soil for extended periods, accumulating through the food chain and severely threatening the balance and stability of the soil ecosystem. Microplastics, as carriers of heavy metals, further exacerbate the threats to soil quality and crop growth. For example, the combined pollution of microplastics and copper ions significantly impacts the physicochemical properties of the soil, plant growth, soil flora, and microbial communities. Currently, this combined pollution formed by heavy metals and plastic residues has a serious impact on the soil ecosystem. On the one hand, it damages the original soil structure, exacerbates soil acidification and salinization, leading to soil compaction and decreased fertility; on the other hand, plant growth is significantly inhibited, root development is hindered, photosynthesis is weakened, and both crop yield and quality are affected.

[0004] Coastal polluted environments not only face pollution from heavy metals and plastic residues, but also face significant challenges in pollution control due to their specific saline-alkaline conditions. Current treatment methods, such as chemical precipitation for heavy metals, consume large amounts of reagents and are prone to secondary pollution; membrane separation is costly and prone to clogging in high-salt environments; and the chemical degradation of DEHP pollutants requires harsh conditions such as high temperature and strong acid, resulting in enormous energy consumption. Existing biodegradation methods mostly utilize single-function strains (degrading only DEHP or adsorbing only heavy metals), which cannot survive in environments with combined pollution. The high salinity and alkalinity of coastal areas place extremely high demands on the tolerance of microorganisms, making existing degradation strains unsuitable for treating environments with combined pollution. Currently, there are few microbial strains that can efficiently degrade DEHP and simultaneously remove heavy metal pollution, and there is still a lack of microbial strains that can be used simultaneously for the treatment of environments with combined DEHP and heavy metal pollution, especially in coastal environments with combined pollution. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing microbial strains that can be used for the treatment of combined DEHP and heavy metal pollution. The present invention provides a strain that can simultaneously degrade DEHP and remove heavy metals and its application in the treatment of combined pollution.

[0006] The first objective of this invention is to provide a strain of Serratia marcescens 25S0741-2.

[0007] A second objective of this invention is to provide the application of strain 25S0741-2 or its bacterial culture.

[0008] A third objective of this invention is to provide a product.

[0009] The fourth objective of this invention is to provide a method for simultaneously degrading DEHP and removing heavy metal ions.

[0010] The fifth objective of this invention is to provide a method for treating environmental pollution caused by salinization and alkalinity.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention provides a strain of Serratia marcescens 25S0741-2, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 16, 2025, with accession number GDMCC NO: 66522.

[0013] This invention isolates a strain of *Serratia marcescens*, 25S0741-2, from the intestine of *Eisenia fetida*. The colonies are round or nearly round, pink, opaque, with a smooth, raised surface, a certain degree of stickiness, and intact edges. Gram staining is negative, and reactions with catalase and oxidase are also negative. Studies show that the optimal culture conditions for strain 25S0741-2 are: pH range 6-9, temperature range 20-40℃. It exhibits certain salt and alkali tolerance, high tolerance to DEHP, and can rapidly degrade DEHP. Furthermore, strain 25S0741-2 has good tolerance to heavy metal ions, maintaining normal growth even at high concentrations of copper, cadmium, and chromium ions, and can adsorb copper, cadmium, and chromium ions from the environment, making it suitable for removing these heavy metals from heavily contaminated environments. Meanwhile, strain 25S0741-2 can also efficiently degrade DEHP under heavy metal ion stress, and can be better applied to the degradation of DEHP in soil environments contaminated with heavy metal ions, as well as the removal of DEHP pollution and heavy metal pollution in environments with combined pollution. It is of great significance for improving the soil quality of heavy metal ion pollution and degrading DEHP pollutants.

[0014] Therefore, the present invention provides the following applications of Serratia marcescens strain 25S0741-2 or its bacterial culture:

[0015] Application in the simultaneous degradation of DEHP and removal of heavy metal ions.

[0016] Application in the preparation of products that simultaneously degrade DEHP and remove heavy metal ions.

[0017] Application in the removal of heavy metal ions in environments with complex pollution.

[0018] Application in the treatment of complex pollution caused by salinization and alkalinity.

[0019] Application in the degradation of DEHP in complex polluted environments.

[0020] Preferably, the heavy metal ions are copper, cadmium, or chromium ions.

[0021] Preferably, the compound polluted environment refers to an environment polluted by saline-alkali stress, heavy metals, and / or DEHP.

[0022] More preferably, the compound polluted environment refers to an environment polluted by saline-alkali stress and heavy metals, saline-alkali stress and DEHP, heavy metals and DEHP, or saline-alkali stress and heavy metals and DEHP.

[0023] Preferably, the salinization and alkalinity complex pollution environment refers to a complex pollution environment of heavy metals and / or DEHP.

[0024] The present invention provides a product containing Serratia marcescens strain 25S0741-2 or its bacterial culture.

[0025] Preferably, the product is a DEHP degrading agent or a heavy metal ion adsorbent.

[0026] More preferably, the present invention also provides a method for preparing Serratia marcescens 25S0741-2 bacterial agent: 25S0741-2 bacterial suspension is added to LB liquid medium and cultured at 200 r / min and 28℃ for 12 h. 2% inoculum is added to LB medium and cultured at 30℃ and 180 rpm with constant temperature shaking for 1 day. Then, the culture solution is centrifuged at 8000 r / min for 10 min, the bacterial cells are resuspended in sterile water, and the medium is washed away by centrifugation twice. The bacterial agent is then prepared again with sterile water.

[0027] More preferably, the concentration of *Serratia marcescens* strain 25S0741-2 in the bacterial agent is not less than 1×10⁻⁶. 8 cfu / mL.

[0028] This invention provides a method for simultaneously degrading DEHP and removing heavy metal ions, using Serratia marcescens strain 25S0741-2 or the above-mentioned products for treatment.

[0029] This invention provides a method for treating environmental pollution caused by salinization and alkalinity, which uses Serratia marcescens strain 25S0741-2 or the above-mentioned products to treat the polluted environment.

[0030] Preferably, the salinization and alkalinity pollution refers to the heavy metal and / or plastic pollution in wastewater or farmland in coastal areas.

[0031] More preferably, the plastic refers to DEHP contamination.

[0032] More preferably, the heavy metals refer to copper, cadmium, and chromium ion pollution.

[0033] The present invention has the following beneficial effects:

[0034] This invention provides a *Serratia marcescens* strain 25S0741-2 that can simultaneously remove DEHP and heavy metal ion pollution in a combined pollution environment. This strain exhibits good salt and alkali tolerance and is tolerant of DEHP, enabling it to efficiently degrade DEHP. Furthermore, strain 25S0741-2 demonstrates good tolerance to heavy metals, adsorbing copper, cadmium, and chromium ions from the environment, making it suitable for the remediation of heavy metal-contaminated environments and removing these ions. Simultaneously, strain 25S0741-2 can also efficiently degrade DEHP under heavy metal ion stress, demonstrating its ability to efficiently degrade DEHP in heavy metal-stressed environments. Moreover, it can degrade DEHP and remove copper, cadmium, and chromium ions in saline-alkali environments contaminated with both heavy metals and DEHP. This allows for better application in the degradation of DEHP in heavy metal-contaminated soil environments and the removal of DEHP and heavy metal pollution in combined pollution environments, which is of significant importance for improving soil quality and degrading DEHP pollutants.

[0035] The salt- and alkali-tolerant Serratia marcescens strain 25S0741-2 provided by this invention can simultaneously remove DEHP and copper, cadmium, and chromium ions in a saline-alkali environment, filling the gap in the field of efficient treatment of complex pollution in existing technologies and providing a green and low-cost solution for coastal ecological restoration. Attached Figure Description

[0036] Figure 1 shows a colony photograph of strain 25S0741-2.

[0037] Figure 2 shows the growth curve of strain 25S0741-2.

[0038] Figure 3 is a phylogenetic tree diagram of strain 25S0741-2.

[0039] Figure 4 shows the growth bar graph of strain 25S0741-2 under different temperature conditions.

[0040] Figure 5 shows the growth bar graph of strain 25S0741-2 under different pH conditions.

[0041] Figure 6 shows the growth bar graph of strain 25S0741-2 under different salt concentrations.

[0042] Figure 7 shows the growth of strain 25S0741-2 and the DEHP degradation rate under different DEHP concentrations.

[0043] Figure 8 shows the results of copper ion removal rate by strain 25S0741-2 under different copper ion concentrations.

[0044] Figure 9 shows the cadmium ion removal rate of strain 25S0741-2 under different cadmium ion concentrations.

[0045] Figure 10 shows the chromium ion removal rate of strain 25S0741-2 under different chromium ion concentrations.

[0046] Figure 11 shows the degradation rate and copper ion removal rate of DEHP by strain 25S0741-2 at different copper ion concentrations.

[0047] Figure 12 shows the degradation rate and cadmium ion removal rate of DEHP by strain 25S0741-2 at different cadmium ion concentrations.

[0048] Figure 13 shows the degradation rate and chromium ion removal rate of DEHP by strain 25S0741-2 at different chromium ion concentrations.

[0049] Figure 14 shows the degradation and removal rates of DEHP and heavy metals by strain 25S0741-2 under the combined pollution environment of coastal saline-alkali soil. Detailed Implementation

[0050] 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.

[0051] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0052] The culture medium formulation used in the examples is as follows:

[0053] (1) Solid culture medium:

[0054] LB medium (g / L): yeast extract 5g, peptone 10g, NaCl 5g, H2O 1000mL, pH 7.4~7.6, agar powder: 20g.

[0055] (2) Liquid culture medium:

[0056] Inorganic salt culture medium (g / L): NH4Cl 2.5g, CaCl2·2H2O 0.05g, Na2HPO4 2.5g, MgSO4·7H2O 0.05g, KH2PO4 2.5g, MnCl2·4H2O 0.05g, H2O 1000mL, pH 7.5.

[0057] NA medium (g / L): 3g beef extract, 5g peptone, 1000mL water, adjust pH to 6.8~7.2 with 1mol / L NaOH or HCl.

[0058] LB liquid medium (g / L): yeast extract 5g, peptone 10g, NaCl 5g, H2O 1000mL, pH 7.4~7.6.

[0059] Example 1: Isolation and Characterization of Strains

[0060] 1. Isolation and purification of strains

[0061] (1) Enrichment of bacterial strains: Accurately weigh 2g of sample (derived from earthworm intestines) and place it in a 250mL Erlenmeyer flask containing 90mL of sterile water. Shake for 30min to thoroughly mix the sample with the water and disperse the cells to form a uniform bacterial suspension. Let stand for 20-30s, then aspirate 2mL of the supernatant into a liquid culture medium containing 100mg / L DEHP (using DEHP as the sole nitrogen source). Incubate at 30℃ and 180rpm in the dark with shaking. After 5 days of incubation, inoculate again at a 2% inoculum into a liquid culture medium containing 200mg / L DEHP and incubate under the same conditions. Repeat this process until the DEHP concentration in the liquid culture medium reaches 900mg / L. Each enrichment culture includes a blank control without bacterial suspension.

[0062] (2) Separation and purification: The enriched culture medium was purified by passing it through sterile water at a ratio of 10:10. -1 10 -2 10 -3 The bacteria were serially diluted, and then each dilution was evenly spread on solid medium containing DEHP (100 mg / L). The medium was then incubated at 30°C in the dark, and colony morphology was observed. Single bacteria with different morphologies were picked and further isolated and purified on solid medium. After multiple streak purification cultures, six strains were obtained. The strains were then divided according to their dilutions of 1-10... -1 / 10 -2 / 10 -3 -X is used for numbering, where 1 represents its origin and X represents the numerical code.

[0063] 2. Screening of degrading bacteria

[0064] A certain amount of suspensions of six acclimatized single bacterial strains were inoculated into an inorganic salt culture medium with a single carbon source and cultured at 30℃ and 200 r / min for 24 h with shaking. The DEHP content before and after culture was determined by ultra-high performance liquid chromatography (UPLC), and its degradation amount was calculated. A blank control was set up without inoculation, i.e., the inorganic salt culture medium was not inoculated with acclimatized strains, to eliminate the influence of physicochemical factors. The results showed that among the six obtained single bacterial strains, those numbered 2-10... -2 The strain with a temperature of -4 showed the best degradation effect on DEHP, with a degradation rate of 86.6% after 48 hours and a degradation rate of 43.3 mg / (L·d).

[0065] 3. Characterization of strains

[0066] (1) Morphological characteristics of the strains: The purified strains numbered 2-10 -2 Strains of strain -4 were streaked on LB solid medium and agar blank solid medium supplemented with 100 mg / L DEHP, respectively, and cultured at 30°C for 24 h. The colony growth was observed and a growth curve of the strain was plotted. Then the colony morphology was observed.

[0067] The colony morphology is shown in Figure 1. The colonies are round, with a raised surface, pink in color, opaque, and have clear edges and a smooth surface. The strain is Gram-negative. Numbered 2-10 -2 The growth curve of strain 2-10 under 100 mg / L DEHP is shown in Figure 2. -2 The -4 strain reached an OD value of 1.48 on day 3.

[0068] (2) Physiological and biochemical characteristics of the strain: After physiological and biochemical characteristic testing, it was found that the strain numbered 2-10 -2 The strain with a temperature of -4 was negative for both oxidase and catalase, could utilize glucose and maltose, but could not hydrolyze starch.

[0069] (3) Molecular biological characteristics: Total bacterial DNA was extracted using a kit method. PCR amplification of bacterial 16S rDNA was performed using universal primers 27F and 1492R. The amplification results showed a distinct band around 1478 bp. The PCR amplification products were recovered and sequenced. The obtained DNA sequences were then subjected to BLAST alignment analysis on the NCBI website (http: / / www.ncbi.nlm.nih.gov), and a phylogenetic tree was constructed using MEGA software (version 11.0).

[0070] The phylogenetic tree results of the strains are shown in Figure 3, displaying strains numbered 2-10. -2 The 16S rDNA sequence of strain -4 shows high homology with that of *Serratia marcescens* strain AFS051532 (OP986799.1). The nucleotide sequence of the 16S rDNA of this strain is shown in SEQ ID NO.1.

[0071] Based on the identification results of the above morphological characteristics, physiological and biochemical characteristics, and molecular biological characteristics, numbers 2-10 were selected. -2The strain with -4 is taxonomically classified as *Serratia marcescens*, named strain 25S0741-2, and deposited on June 16, 2025, at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No:66522. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0072] Example 2: Growth condition test of strain 25S0741-2

[0073] 1. The effect of temperature on the growth of bacterial strains

[0074] Add 50 μL of bacterial suspension 25S0741-2 to 150 mL of sterilized LB liquid medium and incubate at 200 rpm and 30 °C for 12 h. Inoculate 2% of the culture into LB solid medium at temperatures of 24, 26, 28, 30, 32, 34, 36, 38, and 40 °C (3 replicates). Incubate at 200 rpm and different temperatures with constant temperature shaking. After 24 h of incubation, take 200 μL of the bacterial suspension for OD (dose dispersive precipitate). 600 ), and plot the growth curve.

[0075] The test results are shown in Figure 4. The results show that the OD value of strain 25S0741-2 increases with increasing temperature, and reaches its maximum at 30℃. This indicates that the suitable temperature conditions for strain 25S0741-2 are 24-40℃.

[0076] 2. Effect of pH on bacterial growth

[0077] Add 50 μL of the 25S0741-2 bacterial culture to 150 mL of sterilized LB liquid medium and incubate at 200 rpm and 30 °C for 24 h. Inoculate 2% of the culture into LB medium, setting the pH to 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9, with three replicates. Incubate at 200 rpm and 28 °C with constant temperature shaking for 24 h. After each incubation, take 200 μL of the bacterial culture for OD (October Spectrophotometry). 600 ), and plot the growth curve.

[0078] The results are shown in Figure 5, which show that strain 25S0741-2 can still survive under pH 4.5-9 conditions, and its OD value reaches the maximum at pH 7, indicating that the suitable pH culture conditions for strain 25S0741-2 are pH 6-8.

[0079] 3. Effects of salinity on bacterial growth

[0080] Add 50 μL of bacterial suspension 25S0741-2 to 150 mL of sterilized LB liquid medium and incubate at 200 rpm and 30 °C for 24 h. Inoculate 2% of the culture into LB solid medium at salinities of 0%, 3%, 6%, 9%, 12%, and 15%, with three replicates. Incubate at 200 rpm and 30 °C with constant temperature shaking. After 24 h of incubation, collect 200 μL of the bacterial suspension for OD (dose dispersive precipitate). 600 ), and plot the growth curve.

[0081] The test results are shown in Figure 6. The OD value of strain 25S0741-2 peaks at 3%, and it can tolerate a maximum salinity of 15%, indicating that the suitable salt concentration range for strain 25S0741-2 is 0%-15%. It can be seen that a salt concentration of 3% promotes the growth of strain 25S0741-2, making it suitable for growth in coastal saline-alkali environments.

[0082] 4. Tolerance of the strain to different DEHP concentrations

[0083] Add 50 μL of the 25S0741-2 bacterial culture to 150 mL of sterilized LB liquid medium and incubate at 200 rpm and 30 °C for 24 h. Inoculate 2% of the culture into LB solid medium at DEHP concentrations of 100, 200, 300, 400, 500, 600, 700, 800, and 900 mg / L, with three replicates. Incubate at 200 rpm and 30 °C with constant temperature shaking. After 24 h of incubation, collect 200 μL of the bacterial culture for OD (dose dispersive precipitate). 600 ), and plot the growth curve.

[0084] The test results are shown in Figure 7. The OD value of strain 25S0741-2 decreases with increasing DEHP concentration, indicating that strain 25S0741-2 can tolerate DEHP concentrations up to 900 mg / L, with a degradation rate of 8.2%-86.3%.

[0085] Based on the statistical results of the growth conditions of the above strains, the suitable culture conditions for strain 25S0741-2 are: pH range 4.5-9, temperature range 24-40℃, salt concentration range 0%-15%, and the strain can tolerate DEHP up to 900mg / L.

[0086] Example 3: Degradation test of strain 25S0741-2 on different concentrations of DEHP

[0087] Using an inoculation loop, 25S0741-2 bacterial strain was inoculated into liquid culture medium with DEHP concentrations of 100, 200, 300, 400, 500, 600, 700, 800, and 900 mg / L, respectively, and incubated at 30℃ and 180 rpm for 48 h. The residual DEHP content in the culture was determined by ultra-high performance liquid chromatography (UPLC), and its degradation amount was calculated.

[0088] Degradation rate = (initial DEHP concentration - residual DEHP concentration) / initial DEHP concentration × 100%;

[0089] The test results are shown in Figure 7. The results indicate that strain 25S0741-2 can tolerate and degrade DEHP up to 900 mg / L, with the highest degradation rate (86.6%) at a DEHP concentration of 100 mg / L. As the DEHP concentration increases, the degradation rate gradually decreases, dropping to 9% at 900 mg / L. High concentrations of DEHP have an inhibitory effect on the strain, leading to a decrease in its degradation rate.

[0090] Example 4: Adsorption test of heavy metal ions by strain 25S0741-2

[0091] Using an inoculation loop, pick up the 25S0741-2 bacterial strain, streak it on LB solid medium, and incubate it in an incubator for 24 hours to obtain a single colony. Pick a single colony and inoculate it into a test tube containing 4 mL of NA liquid medium, and incubate it in a shaker at 30°C and 180 rpm for 24 hours. Take 1 mL of the culture medium containing the bacterial strain and transfer it to an Erlenmeyer flask containing 100 mL of NA liquid medium. Incubate with shaking for 24 h, then centrifuge. Discard the supernatant and resuspend the culture in 100 mL of sterile physiological saline (pH 7.0 ± 0.2) containing different concentrations of heavy metals copper, cadmium, and chromium ions. This yields bacterial suspensions of different concentrations of heavy metals copper (10, 20, 50, 100, 150, 200, 400, 600 mg / L), cadmium (10, 20, 30, 50, 70, 100, 150, 200 mg / L), and chromium ions (10, 20, 30, 50, 70, 100, 150, 200 mg / L). Place these suspensions on a shaker at 30°C and 180°C. After oscillating at rpm for the same amount of time, samples were taken to test the concentrations of heavy metal copper, cadmium, and chromium ions. The concentrations of copper, cadmium, and chromium in the supernatant were determined using an AA.800 atomic absorption spectrometer, and a bar chart of the removal of heavy metal copper, cadmium, and chromium ions was plotted.

[0092] The formula for calculating the removal rate is:

[0093] Heavy metal removal rate = (initial heavy metal concentration - final heavy metal concentration) / initial heavy metal concentration × 100%;

[0094] The test results are shown in Figures 8-10. They indicate that strain 25S0741-2 can tolerate heavy metals copper, cadmium, and chromium. However, the adsorption rate of these heavy metals gradually decreases with increasing concentrations. The maximum tolerated concentration of copper by strain 25S0741-2 is 600 mg / L, with a degradation rate of 34.2%; the maximum removal rate is 94.2% at 10 mg / L. The maximum tolerated concentration of cadmium and chromium by strain 25S0741-2 is 200 mg / L. The maximum removal rate of cadmium is 91.5% at 10 mg / L, and the maximum removal rate of chromium is 90.6% at 10 mg / L. These results demonstrate that strain 25S0741-2 has the ability to adsorb heavy metals copper, cadmium, and chromium ions, and can be better applied to the adsorption and removal of these heavy metals in the environment, which is of great significance for the remediation of heavy metal pollution.

[0095] Example 5 Degradation of DEHP by strain 25S0741-2 under heavy metal stress

[0096] Laboratory simulation of DEHP degradation under different heavy metal ion concentrations: The obtained *Serratia marcescens* strain 25S0741-2 was cultured in LB broth at 30℃ for 24 h, then centrifuged at 12000 rpm for 10 min to obtain bacterial cells. The cells were resuspended in an equal volume of CuS broth (simulating a field environment with heavy metal contamination) and centrifuged twice. The obtained bacterial cells were then prepared into a 1×10⁻⁶ CuS solution. 8 25S0741-2 bacterial suspension at cfu / mL.

[0097] DEHP at a concentration of 200 mg / L and bacterial culture of 25S0741-2 were added to modified inorganic salt medium (liquid medium) containing copper (10, 20, 50, 100, 150, 200, 400, 600 mg / L), cadmium (10, 20, 30, 50, 70, 100, 150, 200 mg / L), and chromium (10, 20, 30, 50, 70, 100, 150, 200 mg / L), respectively, and cultured for 24 hours. The DEHP content after culture was determined by ultra-high performance liquid chromatography (UPLC), and its degradation amount was calculated. Simultaneously, the concentrations of copper, cadmium, and chromium in the supernatant were determined by an AA.800 atomic absorption spectrometer, and the removal rates were calculated.

[0098] The results, shown in Figures 11-13, indicate that strain 25S0741-2 can efficiently degrade DEHP under heavy metal stress. Specifically, under copper ion stress, DEHP degradation occurred simultaneously with copper ion removal. As the copper ion concentration increased, both the degradation efficiency and copper ion removal rate gradually decreased, with the copper ion removal rate decreasing from 94.2% to 34.2% and the DEHP degradation rate decreasing from 68.7% to 18.4%. Similarly, it can efficiently degrade DEHP under cadmium ion stress. With increasing cadmium ion concentration, both the cadmium ion removal rate and DEHP degradation initially stabilized relatively, then gradually decreased, with the cadmium ion removal rate decreasing from 86.1% to 20.4% and the DEHP degradation rate decreasing from 73.2% to 40.8%. It can also efficiently degrade DEHP under chromium ion stress, exhibiting high degradation and removal rates at chromium ion concentrations of 10-50 mg / L. The cadmium ion removal rate decreased from 93.2% to 13.4%, and the DEHP degradation rate decreased from 76.7% to 39.6%. Therefore, the results show that strain 25S0741-2 can efficiently degrade DEHP under the stress of heavy metals copper, cadmium, and chromium ions, and can be better applied to the efficient degradation of DEHP in environments with combined heavy metal pollution. Strain 25S0741-2's tolerance to cadmium and chromium ions is significantly stronger than its tolerance to copper ions.

[0099] Example 6: Effects of strain 25S0741-2 in a complexly polluted environment

[0100] 1. Preparation of 25S0741-2 microbial agent

[0101] Add 50 μL of 25S0741-2 bacterial suspension to 150 mL of sterile LB broth and incubate at 200 rpm and 30 °C for 24 h. Inoculate 2% of the culture into 150 mL of LB broth and incubate at 30 °C and 180 rpm with constant temperature shaking for 1 day. Then, centrifuge the culture at 8000 rpm for 10 min, resuspend the bacterial cells in sterile water, centrifuge twice to wash away the culture medium, and then prepare a 1×10⁻⁶ solution with sterile water. 8 CFU / mL bacterial agent.

[0102] 2. Degradation effect of the strain on DEHP in polluted coastal waters

[0103] The polluted water used in this embodiment was taken from the sea area near the estuary of the Nanliu River in Zhanjiang City, Guangdong Province, which is a saline-alkali polluted water area. The properties of the polluted water were measured as follows: pH 7.5 and salinity 3.1%. The effect of strain 25S0741-2 in a combined polluted environment (marine saline-alkali environment polluted by heavy metals and DEHP) was simulated. The DEHP concentration was set at 100, 200, 300, 500, 700, and 900 mg / L, with 3 replicates for each treatment. Copper, cadmium, and chromium ions were added at concentrations of 10, 20, 30, 50, 70, and 100 mg / L, respectively. The removal capacity of strain 25S0741-2 of DEHP in the coastal polluted water area was evaluated by the changes in DEHP concentration and the changes in the concentrations of heavy metals such as copper, cadmium, and chromium ions. The residual content of DEHP in the cultured water was determined by ultra-high performance liquid chromatography (UPLC), and its degradation amount was calculated. The DEHP degradation rate was calculated using the same method as in Example 3, and the heavy metal removal rate was calculated using the same method as in Example 4.

[0104] The results, shown in Figure 14, indicate that in the mixed-polluted coastal saline-alkali environment, strain 25S0741-2 can simultaneously remove heavy metals (Cu, Cd, and Cr ions) and degrade DEHP. At low concentrations (100-300 mg / L), the degradation rate is high, and the differences between different heavy metal groups are relatively small. At high DEHP concentrations (700-900 mg / L), the degradation rate significantly decreases, indicating that strain 25S0741-2 can tolerate up to 900 mg / L of DEHP in the coastal saline-alkali environment while still being able to simultaneously remove heavy metals (Cu, Cd, and Cr ions). The DEHP degradation rate decreases with increasing concentration, with high concentrations of DEHP showing a significant inhibitory effect. The heavy metal degradation rate is negatively correlated with DEHP concentration, partly due to inhibited bacterial growth and partly due to saturation of adsorption sites on the bacterial surface, leading to a decrease in both adsorption and degradation rates. This demonstrates that strain 25S0741-2 can be used for the simultaneous removal of heavy metals and DEHP in environments with combined pollution, and is suitable for the bioremediation of environments with combined pollution such as saline-alkali soil, providing a new approach for the synergistic treatment of heavy metals and organic pollutants.

[0105] 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 Serratia marcescens 25S0741-2, characterized in that, The strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 16, 2025, with accession number GDMCC NO: 66522.

2. The application of the 25S0741-2 strain or its bacterial culture as described in claim 1 in the simultaneous degradation of DEHP and removal of heavy metal ions, characterized in that, The heavy metal ions are copper, cadmium, and chromium ions.

3. The application of the 25S0741-2 strain or its bacterial culture as described in claim 1 in the preparation of a product that simultaneously degrades DEHP and removes heavy metal ions, characterized in that, The heavy metal ions are copper, cadmium, and chromium ions.

4. The application of the 25S0741-2 strain or its bacterial solution as described in claim 1 in the removal of heavy metal ions in a complexly polluted environment, characterized in that, The heavy metal ions are copper, cadmium, and chromium ions.

5. The application of the 25S0741-2 strain or its bacterial solution as described in claim 1 in the degradation of DEHP in a complex polluted environment.

6. The application of the 25S0741-2 strain or its bacterial solution as described in claim 1 in the treatment of saline-alkali compound pollution environments, characterized in that, The aforementioned salinization and alkalinity compound pollution environment refers to an environment polluted by heavy metals and / or DEHP; the heavy metals are copper, cadmium, and chromium ions.

7. A product characterized in that, Contains the 25S0741-2 strain or its bacterial culture as described in claim 1.

8. A method for simultaneously degrading DEHP and removing heavy metal ions, characterized in that, The product is treated with the strain 25S0741-2 described in claim 1 or the product described in claim 7; the heavy metal ions are copper, cadmium, and chromium ions.

9. A method for treating environmental pollution caused by salinization and alkali formation, characterized in that, The polluted environment is treated using the strain 25S0741-2 described in claim 1 or the product described in claim 7; the salinized compound polluted environment refers to an environment polluted by heavy metals and / or DEHP; the heavy metals are copper, cadmium, and chromium ions.

Images