Bacterial strain capable of synchronously degrading DEHP and removing heavy metal and application of bacterial strain in treatment of combined polluted environment

Abstract

The invention discloses a bacterial strain capable of synchronously degrading DEHP and removing heavy metals and application of the bacterial strain in treatment of a combined polluted environment. The invention provides a serratia marcescens 25S0741-2 strain which is preserved in the Guangdong Microbial Culture Collection Center on June 16, 2025, and the preservation number of the serratia marcescens 25S0741-2 strain is GDMCC (China General Microbiological Culture Collection Center) NO: 66522. The strain has good salt and alkali resistance and high tolerance to DEHP and heavy metal ions, can efficiently degrade di (2-ethylhexyl) phthalate (DEHP) and synchronously remove the heavy metal ions, can also degrade DEHP in a heavy metal stress environment, is suitable for adsorbing and removing the heavy metal ions and degrading DEHP in a coastal saline-alkali environment, and has a wide application prospect. A new strain resource is provided for bioremediation of a combined pollution environment of saline-alkali soil, and a new way is provided for cooperative treatment of heavy metals and organic pollutants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms and contaminated environment remediation technology, in particular, to a strain capable of synchronously degrading DEHP and removing heavy metals and its application in the remediation of complex contaminated environment. BACKGROUND

[0002] In the field of agriculture, mulching film is widely used due to its advantages of improving crop yield, drought resistance, water saving, temperature increase, and soil conservation. However, due to the difficulty of natural degradation of plastic products, combined with improper use and recycling management, a large amount of plastic products are accumulated in farmland soil. This not only causes soil compaction, secondary salinization, and soil fertility decline, but also causes crop yield reduction and affects food production. Among them, di(2-ethylhexyl) phthalate (DEHP) is a plasticizer for plastic products such as polyvinyl chloride (PVC), which can increase the elasticity and toughness of plastics and is widely used in the plastic industry. DEHP, as an environmental pollutant, can be detected in surface water, groundwater, drinking water, air, soil, and in the bodies of animals and plants, and can enter the body through the gastrointestinal tract, respiratory tract, and skin. In recent years, DEHP has been detected as the main PAEs pollution compound in soil-plant systems. With the increasing production of DEHP, its concentration in the environment has gradually increased, and its toxic side effects have attracted more and more attention.

[0003] At the same time, in the problem of soil environment, heavy metal pollution is also a prominent problem that has attracted much attention. Its pollution sources are widespread, covering industrial emissions, pesticide application, sludge farming, mining activities, etc. Due to the difficulty of degradation and bioaccumulation of heavy metals, they can remain in the soil for a long time, accumulate through the food chain, and seriously threaten the balance and stability of the soil ecosystem. Microplastics, as carriers of heavy metals, can further threaten soil quality and crop growth and development. For example, the combined pollution of microplastics and copper ions has a significant impact on the physical and chemical properties of soil, plant growth, soil animals, and microbial communities. At present, this combined pollution of heavy metals and plastic product residues has a serious impact on the soil ecosystem. On the one hand, it can destroy the original structure of the soil, accelerate the process of soil acidification and salinization, and cause soil compaction and fertility decline; on the other hand, plant growth is significantly inhibited, root development is hindered, photosynthesis is weakened, and crop yield and quality are affected.

[0004] In the coastal pollution environment, not only heavy metals and plastic product residues are faced, but also the specific saline-alkali environmental conditions make the pollution treatment more difficult. The current treatment methods, such as chemical precipitation method for treating heavy metals, consume a large amount of reagents and easily cause secondary pollution; membrane separation method has high cost and the membrane is easy to block in high-salt environment; chemical degradation of DEHP pollutants requires harsh conditions such as high temperature and strong acid, which consumes a large amount of energy. The existing biological degradation method uses most of the single functional strains (only degrading DEHP or only adsorbing heavy metals), and the strains cannot survive in the compound pollution environment. The high salt and alkali environment in the coastal area has high requirements for the tolerance of microorganisms, and the existing degradation strains are difficult to be used for the treatment of compound pollution environment. At present, 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 for the treatment of DEHP and heavy metal compound pollution environment, especially in the treatment of compound pollution in coastal environment. SUMMARY

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

[0006] The first object of the present application is to provide a strain of Serratia marcescens (Serratia marcescens) 25S0741-2. Serratia marcescens )25S0741-2 strain.

[0007] The second object of the present application is to provide the application of 25S0741-2 strain or its bacterial liquid.

[0008] The third object of the present application is to provide a product.

[0009] The fourth object of the present application is to provide a method for simultaneously degrading DEHP and removing heavy metal ions.

[0010] The fifth object of the present application is to provide a method for treating saline-alkali compound pollution environment.

[0011] The above objects of the present application are achieved by the following technical solutions: The present application provides a strain of Serratia marcescens (Serratia marcescens) 25S0741-2 strain, which has been preserved in Guangdong Microbial Culture Collection Center on June 16, 2025, and the preservation number is GDMCC NO: 66522. Serratia marcescens

[0012] ​The present application isolates a strain of Serratia marcescans 25S0741-2 from the intestinal tract of Eisenia foetida, which has a colony in a round or nearly round shape, pink, opaque, smooth and raised surface, certain adhesion, and complete edge; and is negative after Gram staining, and negative in reactions with peroxidase and oxidase. Research shows that the suitable culture conditions of the 25S0741-2 strain are as follows: pH range 6-9, temperature range 20-40 DEG C, certain salt and alkali tolerance, high tolerance to DEHP, and rapid degradation of DEHP. In addition, the 25S0741-2 strain has good heavy metal ion tolerance, can grow normally in a high copper, cadmium and chromium ion concentration range, and can adsorb heavy metal copper, cadmium and chromium ions in the environment to remove heavy metal copper, cadmium and chromium ions in the heavy metal pollution environment. Meanwhile, the 25S0741-2 strain can also efficiently degrade DEHP under heavy metal ion stress, can be better applied to the degradation of DEHP in the heavy metal ion pollution soil environment, and the removal of DEHP pollution and heavy metal pollution in the compound pollution environment, and has important significance for improving the soil quality of heavy metal ion pollution and degrading DEHP pollutants.

[0013] Therefore, the present application provides the following applications of the Serratia marcescans 25S0741-2 strain or its bacterial liquid: Application in simultaneous degradation of DEHP and removal of heavy metal ions.

[0014] Application in preparation of products for simultaneous degradation of DEHP and removal of heavy metal ions.

[0015] Application in removal of heavy metal ions in a compound pollution environment.

[0016] Application in treatment of a salinization compound pollution environment.

[0017] Application in degradation of DEHP in a compound pollution environment.

[0018] Preferably, the heavy metal ions are copper, cadmium and chromium ions.

[0019] Preferably, the compound pollution environment refers to a salt and alkali stress, heavy metal and / or DEHP pollution environment.

[0020] More preferably, the compound pollution environment refers to a salt and alkali stress and heavy metal pollution, salt and alkali stress and DEHP pollution, heavy metal and DEHP pollution, and salt and alkali stress and heavy metal and DEHP pollution environment.

[0021] Preferably, the salinization compound pollution environment refers to a heavy metal and / or DEHP compound pollution environment.

[0022] The present application provides a product containing the Serratia marcescans 25S0741-2 strain or its bacterial liquid.

[0023] Preferably, the product is a DEHP degrading agent or a heavy metal ion adsorbent.

[0024] More preferably, the application further provides a preparation method of the Serratia marcescens 25S0741-2 bacterial agent: take the Serratia marcescens 25S0741-2 bacterial liquid and add it into LB liquid medium, and culture at 200 r / min and 28℃ for 12 hours. Inoculate into LB medium at 2% inoculation amount, and culture at 30℃ and 180 rpm / min in constant temperature shaking for 1 day, then centrifuge the culture liquid at 8000 r / min for 10 minutes, suspend the bacteria with sterile water, centrifuge twice to wash away the culture medium, and then prepare with sterile water to obtain the bacterial agent.

[0025] Further preferably, the concentration of the Serratia marcescens 25S0741-2 strain in the bacterial agent is not less than 1×10 8 cfu / mL.

[0026] The application provides a method for simultaneously degrading DEHP and removing heavy metal ions, which adopts the Serratia marcescens 25S0741-2 strain or the above product for treatment.

[0027] The application provides a method for treating saline-alkali compound contaminated environment, which adopts the Serratia marcescens 25S0741-2 strain or the above product for treatment.

[0028] Preferably, the saline-alkali compound contaminated environment refers to heavy metal and / or plastic compound pollution of coastal area wastewater or farmland.

[0029] More preferably, the plastic refers to DEHP pollution.

[0030] More preferably, the heavy metal refers to copper, cadmium, chromium ion pollution.

[0031] The application has the following beneficial effects: The application provides a Serratia marcescens 25S0741-2 strain which can simultaneously remove DEHP pollution and heavy metal ion pollution in a complex pollution environment, the strain has good salt and alkali tolerance, and can efficiently degrade DEHP. In addition, the 25S0741-2 strain has good heavy metal tolerance, can adsorb heavy metal copper, cadmium and chromium ions in the environment, and is used for the treatment of heavy metal pollution environment to remove heavy metal copper, cadmium and chromium ions; meanwhile, the 25S0741-2 strain can also efficiently degrade DEHP under heavy metal ion stress, can efficiently degrade DEHP in a heavy metal stress environment, and can degrade DEHP and remove heavy metal copper, cadmium and chromium ions in a salinization and heavy metal and DEHP combined pollution environment, can be better applied to the degradation of DEHP in a heavy metal pollution soil environment and the removal of DEHP pollution and heavy metal pollution in a combined pollution environment, and has important significance for improving the soil quality of heavy metal pollution and degrading DEHP pollutants.

[0032] The salt and alkali tolerant Serratia marcescens 25S0741-2 strain provided by the application can simultaneously remove DEHP and copper, cadmium and chromium ions in a salt and alkali environment, fills the blank of the prior art in the field of efficient treatment of combined pollution, and provides a green and low-cost solution for coastal ecological restoration. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is a colony photograph of the strain 25S0741-2.

[0034] Figure 2 It is a growth curve graph of the strain 25S0741-2.

[0035] Figure 3 It is a phylogenetic tree graph of the strain 25S0741-2.

[0036] Figure 4 It is a growth column graph of the strain 25S0741-2 in different temperature environments.

[0037] Figure 5 It is a growth column graph of the strain 25S0741-2 in different pH environments.

[0038] Figure 6 It is a growth column graph of the strain 25S0741-2 in different salt concentration environments.

[0039] Figure 7 It is a growth condition and DEHP degradation rate column graph of the strain 25S0741-2 in different DEHP concentration environments.

[0040] Figure 8 It is a copper ion removal rate result graph of the strain 25S0741-2 in different copper ion concentration environments.

[0041] Figure 9 Figure for the removal rate of cadmium ions by strain 25S0741-2 under different cadmium ion concentrations.

[0042] Figure 10 Figure for the removal rate of chromium ions by strain 25S0741-2 under different chromium ion concentrations.

[0043] Figure 11 Figure for the degradation rate of DEHP and the removal rate of copper ions by strain 25S0741-2 under different copper ion concentrations.

[0044] Figure 12 Figure for the degradation rate of DEHP and the removal rate of cadmium ions by strain 25S0741-2 under different cadmium ion concentrations.

[0045] Figure 13 Figure for the degradation rate of DEHP and the removal rate of chromium ions by strain 25S0741-2 under different chromium ion concentrations.

[0046] Figure 14 Figure for the degradation rate and removal rate of DEHP and heavy metals of different concentrations by strain 25S0741-2 under the compound pollution environment of coastal saline-alkali. DETAILED DESCRIPTION

[0047] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0048] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0049] The medium formula used in the examples is as follows: (1) Solid medium: LB medium (g / L): yeast extract 5 g, protein peptone 10 g, NaCl 5 g, H2O 1000 mL, pH 7.4~7.6, agar powder: 20 g.

[0050] (2) Liquid medium: Inorganic salt medium (g / L): NH4Cl 2.5 g, CaCl2·2H2O 0.05 g, Na2HPO4 2.5 g, MgSO4·7H2O 0.05 g, KH2PO4 2.5 g, MnCl2·4H2O 0.05 g, H2O 1000 mL, pH 7.5.

[0051] NA medium (g / L): beef extract 3 g, peptone 5 g, H2O 1000 mL, adjust pH to 6.8-7.2 with 1 mol / L NaOH or HC1.

[0052] LB liquid medium (g / L): yeast extract 5 g, peptone 10 g, NaCl 5 g, H2O 1000 mL, pH 7.4-7.6.

[0053] Example 1 Isolation and identification of strains 1. Isolation and purification of strains (1) Enrichment of strains: accurately weigh 2 g of sample (from earthworm gut) into a 250 mL flask containing 90 mL of sterile water, and shake for 30 min to mix the sample with water and disperse the cells to form a uniform bacterial suspension. Let stand for 20-30 s, then take 2 mL of supernatant and inoculate into 100 mg / L DEHP inorganic salt liquid medium (DEHP as the sole nitrogen source), and incubate at 30°C, 180 rpm, in the dark. After 5 days of incubation, inoculate 2% of the culture into 200 mg / L DEHP inorganic salt liquid medium, and incubate under the same conditions. Repeat the process until the DEHP concentration in the inorganic salt medium reaches 900 mg / L. Each enrichment culture has a blank control without inoculation.

[0054] (2) Isolation and purification: dilute the enrichment culture with sterile water at gradients of 10 -1 , 10 -2 , and 10 -3 , respectively, and then evenly spread the different dilutions on solid medium containing DEHP (100 mg / L), and incubate in a 30°C constant temperature incubator in the dark. Observe the colony morphology, and pick single colonies with different morphologies for further isolation and purification on solid medium. After multiple streaking and purification, 6 strains are obtained. The strains are numbered according to their dilution ratios 1-10 -1 / 10 -2 / 10 -3 -X, where the number 1 represents the source, and X represents the number.

[0055] 2. Screening of degrading bacteria Inoculate a certain amount of suspension of the 6 strains after domestication into inorganic salt medium with a single carbon source, and incubate at 30°C, 200 r / min for 24 h. Measure the DEHP content before and after incubation using ultra-performance liquid chromatography (UPLC) method, and calculate the degradation amount. At the same time, set a blank control without inoculation of domesticated strains in the inorganic salt medium to eliminate the influence of physical and chemical factors. The results show that among the 6 strains obtained, the strain numbered 2-10 -2The 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).

[0056] 3. Characterization of strains (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.

[0057] Colony morphology as Figure 1 As shown, the colonies are round, raised, pink, opaque, with clear edges and a smooth surface. The strain is Gram-negative. Numbered 2-10 -2 The growth curve of strain -4 is shown below. Figure 2 As shown, the results are displayed under a 100 mg / L DEHP environment, numbered 2-10. -2 The -4 strain reached an OD value of 1.48 on day 3.

[0058] (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.

[0059] (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).

[0060] The phylogenetic tree results of the strains are as follows Figure 3 As shown, display number 2-10 -2 The 16S rDNA sequence of strain -4 is similar to that of Serratia marcescens. Serratia marcescens strain AFS051532 (OP986799.1) exhibits high homology. The nucleotide sequence of the 16S rDNA of this strain is shown in SEQ ID NO.1.

[0061] Based on the identification results of the above morphological characteristics, physiological and biochemical characteristics, and molecular biological characteristics, numbers 2-10 were selected.-2 The taxonomy of the strain 4 belongs to Serratia marcescens Serratia marcescens ), named 25S0741-2 strain, and was preserved in Guangdong Microbial Culture Collection Center on June 16, 2025, with the strain preservation number GDMCC No: 66522 and the preservation address being No. 59 Building, 5th Floor, Guangzhou Institute, 100 Middle Liujie Road, Guangzhou.

[0062] Example 2: Test of growth conditions of 25S0741-2 strain 1. Effect of temperature on growth of the strain 50 μL of 25S0741-2 bacterial solution was added to 150 mL of sterilized LB liquid medium, and was cultured at 200 r / min and 30°C for 12 h. The inoculation amount was 2%, and the temperature was set to 24, 26, 28, 30, 32, 34, 36, 38, and 40°C, respectively, with 3 repeats. The culture was incubated at 200 r / min and different temperatures, and 200 μL of bacterial solution was taken for detection (OD 600 ) at 24 h after culture, and the growth curve was drawn.

[0063] The determination results are shown in Figure 4 , which show that the OD value of the 25S0741-2 strain increases with the increase of temperature, and the OD value reaches the maximum at 30°C, indicating that the suitable temperature condition for the 25S0741-2 strain is 24-40°C.

[0064] 2. Effect of pH on growth of the strain 50 μL of 25S0741-2 bacterial solution was added to 150 mL of sterilized LB liquid medium, and was cultured at 200 r / min and 30°C for 24 h. The inoculation amount was 2%, and the pH was set to 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9, respectively, with 3 repeats. The culture was incubated at 200 r / min and 28°C, and 200 μL of bacterial solution was taken for detection (OD 600 ) at 24 h after culture, and the growth curve was drawn.

[0065] The determination results are shown in Figure 5 , which show that the 25S0741-2 strain can still survive under the conditions of pH 4.5-9, and the OD value reaches the maximum at pH 7, indicating that the suitable pH culture condition for the 25S0741-2 strain is pH 6-8.

[0066] 3. Effect of salinity on growth of the strain 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.

[0067] The measurement results are as follows Figure 6 As shown, 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.

[0068] 4. Tolerance of the strain to different DEHP concentrations 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.

[0069] The measurement results are as follows Figure 7 As shown, 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%.

[0070] 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.

[0071] Example 3: Degradation test of strain 25S0741-2 on different concentrations of DEHP The 25S0741-2 strain was inoculated with a loop, and was inoculated into liquid medium with DEHP concentrations of 100, 200, 300, 400, 500, 600, 700, 800, and 900 mg / L, respectively, and was placed in a 30°C, 180 rpm shaker for 48 h. The residual content of the cultured DEHP was determined by ultra-high performance liquid chromatography (UPLC) method, and the degradation amount was calculated.

[0072] The degradation rate = (initial concentration of DEHP - residual concentration of DEHP) / initial concentration of DEHP x 100%; The results are shown in Table 1. Figure 7 As shown in Table 1, the 25S0741-2 strain can tolerate and degrade DEHP at a concentration of up to 900 mg / L, and the highest degradation rate is 86.6% when the concentration of DEHP is 100 mg / L. As the concentration of DEHP increases, the degradation rate gradually decreases, and the degradation rate decreases to 9% at a concentration of 900 mg / L. High concentrations of DEHP can inhibit the strain, and the degradation rate also decreases.

[0073] Example 4: Test of adsorption of heavy metal ions by 25S0741-2 strain The 25S0741-2 strain was inoculated with a loop, and was inoculated into LB solid medium for plate streaking, and was placed in an incubator for 24 h to obtain single colonies. The single colonies were picked and inoculated into a test tube containing 4 mL of NA liquid medium, and were placed in a 30°C, 180 rpm shaker for 24 h. 1 mL of the strain-containing culture was taken and added to a conical flask containing 100 mL of NA liquid medium, and was shaken for 24 h. The supernatant was then poured out, and 100 mL of sterile physiological saline (pH 7.0±0.2) containing different concentrations of heavy metal copper, cadmium, and chromium ions was added to resuspend the bacteria, to obtain bacterial suspensions containing different concentrations of heavy metal 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). The conical flask was placed in a shaker at 30°C and 180 rpm for the same treatment time, and the heavy metal copper, cadmium, and chromium ion concentrations were determined by AA.800 atomic absorption spectrophotometer, and a bar chart of the removal of heavy metal copper, cadmium, and chromium ions was drawn.

[0074] The calculation formula of the removal rate is: The heavy metal removal rate = (initial concentration of heavy metal - final concentration of heavy metal) / initial concentration of heavy metal x 100%; The results are shown in Table 2. Figure 8~Figure 10As shown, it is shown that the 25S0741-2 strain can tolerate heavy metals copper, cadmium, chromium, and the adsorption rate of copper, cadmium, chromium ions gradually decreases with the increase of heavy metal copper, cadmium, chromium ion concentration. The maximum tolerance concentration of 25S0741-2 strain to copper reaches 600 mg / L, and the degradation rate is 34.2%; the maximum removal rate reaches 94.2% at 10 mg / L. The tolerance concentration of 25S0741-2 strain to cadmium and chromium 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. It is shown that the 25S0741-2 strain has the effect of adsorbing heavy metal copper, cadmium, chromium ions, and can better be applied to the adsorption and removal of heavy metal copper, cadmium, chromium ions in the environment, and has important significance for the treatment of heavy metal contaminated environment.

[0075] Example 5 Determination of DEHP degradation by 25S0741-2 strain under heavy metal stress Laboratory simulation of DEHP degradation under different heavy metal ion concentrations: After the obtained Serratia marcescens 25S0741-2 strain was cultured in LB liquid at 30°C for 24 h, the bacterial cells were obtained by centrifugation at 12000 rpm for 10 min, resuspended with an equal volume of CuS liquid (simulating field heavy metal contaminated environment) and centrifuged twice, and then the obtained bacterial cells were prepared into 1×10 8 cfu / mL of 25S0741-2 bacterial solution with CuS.

[0076] 200 mg / L of DEHP and 25S0741-2 bacterial solution were added to modified inorganic salt medium (liquid medium) containing 10, 20, 50, 100, 150, 200, 400, 600 mg / L of heavy metal copper, 10, 20, 30, 50, 70, 100, 150, 200 mg / L of cadmium, and 10, 20, 30, 50, 70, 100, 150, 200 mg / L of chromium ion concentration and cultured for 24 hours. The content of DEHP after culture was determined by ultra performance liquid chromatography (UPLC) method and the degradation amount was calculated, and the concentration of copper, cadmium, chromium in the supernatant was determined by AA.800 atomic absorption spectrometer and the removal rate was calculated.

[0077] The determination results are as follows: Figure 11~Figure 13As shown, it is shown that the 25S0741-2 strain can also efficiently degrade DEHP under heavy metal stress; among them, under the stress of copper ions, DEHP is degraded while copper ions are removed synchronously, and with the increase of copper ion concentration, the degradation efficiency and the removal rate of copper ions gradually decrease, the removal rate of copper ions decreases from 94.2% to 34.2%, and the DEHP degradation rate decreases from 68.7% to 18.4%. Similarly, under the stress of cadmium ions, DEHP can also be efficiently degraded, and with the increase of cadmium ion concentration, the removal rate of cadmium ions and the DEHP degradation rate are relatively stable at first and then gradually decrease, the removal rate of cadmium ions decreases from 86.1% to 20.4%, and the DEHP degradation rate decreases from 73.2% to 40.8%. Under the stress of chromium ions, DEHP can also be efficiently degraded, and under the concentration of 10-50 mg / L of chromium ions, the degradation rate and the removal rate are relatively high, the removal rate of cadmium ions decreases from 93.2% to 13.4%, and the DEHP degradation rate decreases from 76.7% to 39.6%. Therefore, it is shown that the 25S0741-2 strain can efficiently degrade DEHP under the stress of heavy metals copper, cadmium and chromium ions, and can be better applied to the environment of heavy metal complex pollution to efficiently degrade DEHP. The ability of the 25S0741-2 strain to resist cadmium ions and chromium ions is obviously stronger than the ability to resist copper ions.

[0078] Example 6 Effect of 25S0741-2 strain in complex pollution environment 1. Preparation of 25S0741-2 bacterial agent 50 μL of 25S0741-2 bacterial solution was added to 150 mL of sterilized LB liquid medium, and cultured at 200 r / min and 30°C for 24 h. 2% inoculation amount was inoculated into 150 mL of LB medium, and cultured at 30°C and 180 rpm / min in a constant temperature shaker for 1 d, then the culture solution was centrifuged at 8000 r / min for 10 min, the bacterial body was suspended with sterile water, centrifuged twice to wash away the culture medium, and then the bacterial agent with a concentration of 1×10 8 cfu / mL was prepared with sterile water.

[0079] 2. Degradation effect of strain on DEHP in coastal polluted water area The contaminated water used in the present embodiment is taken from the sea area near the estuary of Nanliu River in Zhanjiang City, Guangdong Province, which is a contaminated water area in a saline-alkali environment. The properties of the contaminated water determined are: pH is 7.5, and salt content is 3.1%. The effect of 25S0741-2 strain in the complex pollution environment (heavy metal + DEHP polluted coastal saline-alkali environment) is simulated. The DEHP concentration is set to 100, 200, 300, 500, 700, and 900 mg / L, respectively, and each treatment has 3 replicates. Heavy metals copper, cadmium, and chromium ions are added at 10, 20, 30, 50, 70, and 100 mg / L, respectively. The removal ability of strain 25S0741-2 to DEHP in the coastal contaminated water area is evaluated by changing the concentration of DEHP and the concentration of heavy metals copper, cadmium, and chromium ions. The residual content of DEHP in the culture is determined by ultra-high performance liquid chromatography (UPLC) method, and the degradation amount is calculated. The DEHP degradation rate calculation method is the same as that in Example 3, and the heavy metal removal rate calculation method is the same as that in Example 4.

[0080] The determination results are shown in Table 3. Figure 14 As shown in Table 3, in the complex pollution of coastal saline-alkali environment, 25S0741-2 strain can simultaneously remove heavy metals (Cu, Cd, Cr ions) and degrade DEHP, and at low concentration (100-300 mg / L), the degradation rate is higher, and the difference between different concentrations of heavy metal groups is relatively small; at high DEHP concentration (700-900 mg / L), the degradation rate is significantly reduced, showing that 25S0741-2 strain can tolerate up to 900 mg / L of DEHP in the coastal saline-alkali environment and still can simultaneously remove heavy metals (Cu, Cd, Cr ions). The degradation rate of DEHP decreases with increasing concentration, and the inhibition effect of high concentration DEHP is significant. The degradation rate of heavy metals is negatively correlated with the concentration of DEHP. On the one hand, the growth of bacteria is inhibited, and on the other hand, the adsorption sites on the surface of the bacteria are saturated, and the adsorption rate and degradation rate will decrease. Therefore, it is shown that 25S0741-2 strain can be used for the simultaneous removal of heavy metals and DEHP in the complex pollution environment, and is suitable for the bioremediation of complex pollution environment such as saline-alkali soil, and provides a new way for the coordinated management of heavy metals and organic pollutants.

[0081] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. A strain of Serratia marcescens ( Serratia marcescens strain 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 use of the 25S0741-2 strain or its bacterial culture as described in claim 1 in the preparation of products that simultaneously degrade DEHP and remove heavy metal 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 according to claim 4 or 5, characterized in that, The term "complex polluted environment" refers to an environment polluted by saline-alkali stress, heavy metals, and / or DEHP.

7. 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 term "complex polluted environment" refers to an environment polluted by heavy metals and / or DEHP.

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

9. 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 8.

10. A method for treating environmental pollution caused by salinization and alkalinity, characterized in that, The 25S0741-2 strain described in claim 1 or the product described in claim 8 is used to treat polluted environments.

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