Rhodococcus ruber WM-5 strain and application thereof

Abstract

The invention discloses a rhodococcus ruber WM-5 strain, and the preservation number of the rhodococcus ruber WM-5 strain is CCTCC (China Center for Type Culture Collection) NO.M2025874. Research shows that the rhodococcus ruber WM-5 strain has the functions of resisting cadmium and degrading phthalic acid ester, has extremely high degradation efficiency on DBP (dibutyl phthalate), and is relatively wide in suitable degradation condition range; the Rhodococcus ruber WM-5 strain has strong tolerance to Cd < 2 + > and PAEs, when the Rhodococcus ruber WM-5 strain is applied to PAEs polluted solution systems under the condition of different initial concentrations of PAEs and Cd-PAEs composite polluted solution systems with different initial concentrations of Cd < 2 + >, high PAEs degradation efficiency can be maintained, and technical support is provided for remediation of Cd-PAEs composite pollution. In a word, the invention makes up the technical blank of Cd-resistant PAEs degrading strains, and the Rhodococcus ruber WM-5 strain or the microbial agent thereof has great application value in the aspects of degrading phthalate and repairing Cd-PAEs composite pollutants.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, and in particular relates to a Rhodococcus rhamnosus strain WM-5 and its application. Background Technology

[0002] Phthalate esters (PAEs) are indispensable plasticizers in the plastics manufacturing process, primarily enhancing the plasticity of plastic products during processing. Therefore, PAEs are widely used worldwide. However, as organic substances with strong endocrine disrupting properties, they have been increasingly identified as a new type of pollutant in recent years. Their characteristics of being difficult to degrade, highly stable, and highly toxic make them posing significant environmental hazards and potential risks. Among them, six types of PAEs—dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DOP)—have been identified as priority pollutants by the EPA. DEHP is also classified as a Group 2B carcinogen by the IARC.

[0003] The bonding process between PAEs and plastic products typically occurs based on physical interactions such as van der Waals forces. This makes it easy for PAEs to be released from plastic products and migrate into the environment during actual use. They can then enter the human body through the food chain, causing serious harm such as cancer and endocrine disorders. Currently, PAEs have been detected in various environmental media in China, including water bodies, air, soil, and sediments. The detected PAEs are predominantly DBP and DEHP, indicating that PAE pollution in the environment often exists as a DBP-DEHP complex, highlighting the urgent need for a remediation method for PAE pollution.

[0004] Remediation technologies for treating PAEs contamination in the environment include physical, chemical, and microbial methods. Physical methods mainly use adsorbents such as activated carbon to adsorb PAEs in the contaminated environment, thereby reducing the mobility of PAEs and thus reducing their environmental risk. While this method has the advantages of low cost and simple operation, it does not remove PAEs from the contaminated environment at the source. Chemical methods mainly include chemical oxidation or leaching, which mainly remove PAEs by oxidizing them with oxidants or by using surfactants to enhance the apparent water solubility of PAEs, allowing them to be leached from the environment. While this method can remove PAEs from the contaminated environment with high efficiency, it is relatively expensive, and leaching carries a certain risk of secondary pollution. Compared with the methods mentioned above, the microbial method is one of the most mainstream remediation methods for PAE pollution in the environment. It mainly involves inoculating or applying functional strains with high PAE degradation performance in the polluted area. Through the microbial degradation reaction process, the PAE pollutants in the environment are gradually mineralized into CO2 and H2O as the energy required for their growth and metabolism. This method has the advantages of low cost, strong environmental friendliness, and high removal efficiency, and has great potential for application and promotion.

[0005] Furthermore, multiple types of pollutants often exist in the real environment, and cadmium (Cd), as a heavy metal pollutant, poses a significant threat to the environment. In fact, numerous studies have shown that Cd and PAEs (paraffinic acid esters) coexist in the environment, and Cd-PAE co-pollution has become one of the more typical types of environmental co-pollution. When Cd and PAEs coexist, the activity of PAE-degrading strains is greatly affected by the presence of coexisting Cd as a heavy metal. 2+ The effects of Cd on the environment reduce its activity and decrease its degradation function. However, to date, most research on functional PAE strains has focused on the degradation of single PAE pollutants. This contradicts the trend of multiple PAE pollutants, mainly DBP and DEHP, coexisting in the actual environment. Furthermore, it has not addressed the strains' tolerance to Cd or Cd-related pollutants. 2+ The application effects under stress conditions need to be studied in depth. Therefore, it is necessary to further screen Cd-resistant PAE-degrading strains from the natural environment to provide a technical basis and theoretical support for the targeted remediation of Cd-PAEs co-polluted environments. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a Rhodococcus rhamnosus strain WM-5 and its application, which has good PAE degradation performance and Cd degradation performance. 2+ It has strong resilience.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] Rhodococcus WM-5 strain, preservation number CCTCC NO.M2025874.

[0009] The microbial agent uses the aforementioned Rhodococcus WM-5 strain as its active ingredient.

[0010] The application of the aforementioned Rhodococcus WM-5 strain or microbial agent in the degradation of phthalates.

[0011] Phthalate esters (PAEs) are one or more of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DOP).

[0012] Degradation occurs under cadmium stress.

[0013] A method for remediating phthalate contamination involves adding the Rhodococcus erythrococcus WM-5 strain of claim 1 or the microbial agent of claim 2 to the contaminant.

[0014] The pollutants contain both cadmium and phthalate esters.

[0015] Cd in pollutants 2+ The concentration of phthalates is 0–50 mg / L, and the concentration of phthalates is 0–3000 mg / L.

[0016] The pollutants were located in an environment with a temperature of 30-35℃, a pH of 6-9, and a salinity of 0.75g / L-20g / L.

[0017] The inventors discovered the Rhodococcus rubrum strain WM-5, with accession number CCTCC NO. M2025874. Studies have shown that Rhodococcus rubrum strain WM-5 possesses both cadmium tolerance and phthalate degradation capabilities, exhibiting extremely high degradation efficiency for DBP, with a relatively wide range of suitable degradation conditions; Rhodococcus rubrum strain WM-5 also shows good resistance to Cd. 2+ It exhibits strong resistance to PAEs. The Rhodococcus rhamnosus strain WM-5 was applied to PAE-contaminated solution systems with different initial concentrations of PAEs and different initial concentrations of Cd. 2+ In Cd-PAEs complex pollution solution systems, this invention maintains high PAEs degradation efficiency, providing technical support for the remediation of Cd-PAEs complex pollution. In summary, this invention fills the technical gap in Cd-resistant PAEs-degrading strains, and Rhodococcus rhamnosus strain WM-5 or its microbial agents have significant application value in degrading phthalates and remediating Cd-PAEs complex pollutants. Attached Figure Description

[0018] Figure 1 This is the morphological characteristics and phylogenetic tree of Rhodococcus WM-5 strain. In the figure: a. Morphological characteristics of strain WM-5 (physical observation), b. Morphological characteristics of strain WM-5 (electron microscopy observation), c. Phylogenetic tree of strain WM-5.

[0019] Figure 2 This is a graph showing the broad-spectrum test results of Rhodococcus rhamnosus strain WM-5 in degrading PAEs.

[0020] Figure 3 This is the growth and degradation curve of Rhodococcus rhamnosus strain WM-5.

[0021] Figure 4 This is a graph showing the effect of inoculum quantity on the degradation of PAEs by Rhodococcus rhamnosus strain WM-5.

[0022] Figure 5 This is a graph showing the effect of pH on the degradation of PAEs by Rhodococcus rhamnosus strain WM-5.

[0023] Figure 6 This is a graph showing the effect of temperature on the degradation of PAEs by Rhodococcus rhamnosus strain WM-5.

[0024] Figure 7 This is a graph showing the effect of salinity on the degradation of PAEs by Rhodococcus rhamnosus strain WM-5.

[0025] Figure 8 It is the Rhodococcus WM-5 strain that is effective against PAEs and Cd. 2+ The tolerance results are shown in the figure, where: a Cd 2+ Tolerance results, b PAEs tolerance results.

[0026] Figure 9 This is a graph showing the degradation effect of Rhodococcus rhamnosus strain WM-5 in PAE-contaminated solution systems of different concentrations.

[0027] Figure 10 This is a diagram showing the degradation effect of Rhodococcus rhamnosus strain WM-5 in a Cd-PAEs complex contaminated solution system.

[0028] Preservation Information

[0029] Rhodococcus ruber strain WM-5, accession number CCTCC NO.M2025874, accession date: April 24, 2025, accession address: Wuhan University, Wuhan, China, 430072, China, depositary institution: China Center for Type Culture Collection.

[0030] Storage conditions: Store at -70°C in 25% glycerol tubes. Detailed Implementation

[0031] Example 1: Screening, Identification, and Broad-spectrum Degradation Test of Strains

[0032] (1) Screening and identification of strains

[0033] A gradient acclimatization method was used to screen Cd-tolerant PAE (pesophyll-eating) highly efficient degrading strains from soil samples. In a sterile environment, 5.0 g of soil sample (collected from a greenhouse grape plantation in Wuming County, Nanning City) was placed in a solution containing 10 mg / L Cd. 2+ The PAEs were added to 100 mL of Cd-PAEs liquid MSM medium (K2HPO4 5.8 g / L, KH2PO4 4.5 g / L, (NH4)2SO4 2 g / L, NaCl 0.75 g / L, MgCl2 0.16 g / L, CaCl2 0.02 g / L, FeCl3 0.002 g / L, CdCl2 0.01 g / L, pH = 7.0) containing 200 mg / L PAEs (100 mg / L DBP + 100 mg / L DEHP; considering that PAEs pollution in the actual environment is mostly a combination of DBP and DEHP, unless otherwise specified, PAEs in this invention specifically refer to a mixture of DBP and DEHP at equal mass concentrations).

[0034] The culture was incubated at 30℃ with light-shielded shaking at 180 rpm for 7 days. If obvious colony growth was observed in the culture medium after 7 days, 0.1 mL of the culture was transferred to a Cd-PAEs liquid MSM medium with the next PAE concentration for further enrichment and acclimatization, and incubated for another 7 days under the same conditions. This acclimatization process was repeated for three cycles, with the PAE contaminant concentrations adjusted to 400 mg / L, 600 mg / L, and 800 mg / L for subsequent cycles. Afterward, 1 mL of the culture from the final acclimatization cycle was diluted to 10 times its original volume with sterile physiological saline (0.9% NaCl). -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Then, 100 μL of culture medium at different dilution ratios was taken and spread onto a substrate containing 10 mg / L Cd using the dilution-spreading method. 2+Cd-PAEs were cultured on solid MSM medium (K2HPO4 5.8g / L, KH2PO4 4.5g / L, (NH4)2SO4 2g / L, NaCl 0.75g / L, MgCl2 0.16g / L, CaCl2 0.02g / L, FeCl3 0.002g / L, CdCl2 0.01g / L, agar 16g / L, pH=7.0) at 30℃ for 3–5 days until obvious single colonies appeared on the medium. The dominant strain was isolated and purified by the four-zone streak method to obtain the Cd-resistant PAEs highly efficient degrading strain. The bacteria were inoculated onto LB solid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 16 g / L agar, pH=7) to observe their macroscopic morphological characteristics, and their microscopic morphological characteristics were observed by scanning electron microscopy. Finally, 16S rRNA sequencing analysis was performed to identify the bacterial species and a phylogenetic tree was constructed.

[0035] (2) Broad-spectrum test of the strain's ability to degrade PAEs

[0036] Single colonies of strain WM-5 were picked using an inoculation loop and transferred to 20 mL of LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7). The culture was activated at 30°C with light-shielded shaking at 180 rpm for 24 h. The cells were then collected by centrifugation at 8000 rpm for 5 min and resuspended in physiological saline to the OD value of the bacterial culture. 600 A value of 1.0 yields the seed culture of strain WM-5 (with a viable count of approximately 3.4 × 10⁻⁶). 9 (cfu / mL).

[0037] Subsequently, the seed culture of strain WM-5 was inoculated at a rate of 5% (v / v) into MSM liquid medium (K2HPO4 5.8 g / L, KH2PO4 4.5 g / L, (NH4)2SO4 2 g / L, NaCl 0.75 g / L, MgCl2 0.16 g / L, CaCl2 0.02 g / L, FeCl3 0.002 g / L, pH = 7.0) containing 200 mg / L DMP, 200 mg / L DEP, 200 mg / L DBP, 200 mg / L BBP, 200 mg / L DEHP, and 200 mg / L DOP, respectively. The medium was then cultured at 30°C with shaking at 180 rpm in the dark for 2 days. Samples were taken after 2 days to determine the OD of the bacterial culture. 600 .

[0038] Results: A highly efficient PAE-degrading strain, WM-5, tolerant to Cd, was screened from soil samples using a gradient concentration acclimatization method. Its macroscopic and microscopic morphological characteristics are as follows: Figure 1 (a) and Figure 1 As shown in (b), the macroscopic morphology of a single colony of strain WM-5 on LB solid medium is round, pink, with a raised center, smooth surface, and rounded edges. The microscopic morphology of strain WM-5 under scanning electron microscopy is that of a thin rod-like structure, approximately 1.8–2.0 μm in length and 0.3–0.4 μm in width. Furthermore, 16S rRNA sequencing analysis confirmed that the gene sequence of strain WM-5 is highly similar to that of *Rhodococcus ruber*, and its phylogenetic tree is shown below. Figure 1 As shown in (c), it was therefore identified and named Rhodococcus ruber strain WM-5, and has been deposited at the China Center for Type Culture Collection. On the other hand, the broad-spectrum degradation of PAEs by Rhodococcus ruber strain WM-5 was tested as follows: Figure 2 As shown, the Rhodococcus ruber WM-5 strain can grow using six PAEs (polyester esters) as the sole carbon source, including DMP, DEP, DBP, BBP, DEHP, and DOP. This preliminarily confirms that the Rhodococcus ruber WM-5 strain has the ability to degrade PAEs, which has significant research value.

[0039] Example 2: Degradation efficiency of WM-5 strain on PAEs

[0040] Methods: Single colonies of Rhodococcus ruber WM-5 strain were picked using an inoculation loop and transferred to 20 mL of LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7). The medium was activated and cultured at 30°C with shaking at 180 rpm in the dark for 24 h. The cells were then collected by centrifugation at 8000 rpm for 5 min and resuspended in physiological saline to the OD value of the bacterial culture. 600 A value of 1.0 yields a seed culture of Rhodococcus ruber strain WM-5 (with a viable count of approximately 3.4 × 10⁻⁶). 9 (cfu / mL).

[0041] Subsequently, the seed culture of Rhodococcus ruber WM-5 strain was inoculated at a rate of 5% (v / v) into MSM liquid medium (K2HPO4 5.8 g / L, KH2PO4 4.5 g / L, (NH4)2SO4 2 g / L, NaCl 0.75 g / L, MgCl2 0.16 g / L, CaCl2 0.02 g / L, FeCl3 0.002 g / L, pH = 7.0) containing 200 mg / L PAEs contaminants (100 mg / L DBP + 100 mg / L DEHP). The same medium without inoculation served as a control group. The culture was incubated at 30°C with light-shielded shaking at 180 rpm for 7 days. Destructive sampling was performed every day to determine the OD of the culture medium at each time point. 600 The content of residual PAEs contaminants was determined, and the PAEs degradation rate was calculated according to Equation (1). Three replicates were set for each treatment, and the growth and degradation curves of Rhodococcus ruber WM-5 strain were finally plotted.

[0042]

[0043] Research results: The growth and degradation curve of Rhodococcus ruber strain WM-5 using PAE contaminants (100 mg / L DBP + 100 mg / L DEHP) as the sole carbon source is shown in the figure below. Figure 3 As shown. From the perspective of the growth of Rhodococcus ruber WM-5 strain, the strain rapidly utilizes PAEs as a carbon source to accelerate its reproduction from 0 to 2 days, enters a stable phase from 2 to 5 days, and enters a decline phase from 5 to 7 days. From the perspective of the degradation of PAEs (100 mg / L DBP + 100 mg / L DEHP) by Rhodococcus ruber WM-5 strain, under the condition of using 200 mg / L PAEs (100 mg / L DBP + 100 mg / L DEHP) as the sole carbon source, Rhodococcus ruber WM-5 strain exhibits extremely high degradation efficiency for DBP, reaching 97.91% at 2 days. However, its degradation efficiency for DEHP is lower, remaining below 20.00% within 7 days.

[0044] Example 3: Suitable conditions for PAE degradation by strain WM-5

[0045] Methods: Single colonies of Rhodococcus ruber WM-5 strain were picked using an inoculation loop and transferred to 20 mL of LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7). The medium was activated and cultured at 30°C with shaking at 180 rpm in the dark for 24 h. The cells were then collected by centrifugation at 8000 rpm for 5 min and resuspended in physiological saline to the OD value of the bacterial culture. 600 A value of 1.0 yields a seed culture of Rhodococcus ruber strain WM-5 (with a viable count of approximately 3.4 × 10⁻⁶). 9 (cfu / mL).

[0046] Subsequently, the seed culture of Rhodococcus ruber WM-5 was inoculated into MSM liquid medium (K2HPO4 5.8 g / L, KH2PO4 4.5 g / L, (NH4)2SO4 2 g / L, NaCl 0.75 g / L, MgCl2 0.16 g / L, CaCl2 0.02 g / L, FeCl3 0.002 g / L, pH = 7.0) containing 200 mg / L PAEs contaminants (100 mg / L DBP + 100 mg / L DEHP). The same medium without inoculation was used as a control group. The medium was cultured at 180 rpm with shaking in the dark for 2 days. On day 2, samples were taken to measure the OD of the culture medium. 600 The content of residual PAEs pollutants was determined, and the PAEs degradation rate was calculated according to Equation (1). Three replicates were set up for each treatment.

[0047] The initial culture conditions were set as follows: inoculum concentration 5%, pH = 7, temperature 30℃, and salinity 0.75 g / L. Using a single-factor experimental method, inoculum concentration gradients of 1%, 2%, 3%, 4%, and 5% were set; pH gradients of 5, 6, 7, 8, and 9 were set; temperature gradients of 25℃, 30℃, and 35℃ were set; and salinity gradients of 0.75 g / L, 1.5 g / L, 5 g / L, 10 g / L, 20 g / L, and 30 g / L were set to investigate the influence of environmental factors on the degradation of PAEs by the strain and determine the suitable conditions for its degradation.

[0048] Results: The effects of environmental factors on the degradation of PAEs (100 mg / L DBP + 100 mg / L DEHP) by Rhodococcus ruber WM-5 strain are as follows: Figures 4 to 7 As shown. The optimal conditions for the degradation of DBP and DEHP by Rhodococcus ruber strain WM-5 were determined to be: inoculum concentration of 3%–5%, pH 6–9, temperature of 30℃–35℃, and salinity of 0.75 g / L–20 g / L.

[0049] Example 4: Degradation effect of strain WM-5 in solution system

[0050] Methods: Single colonies of Rhodococcus ruber WM-5 strain were picked using an inoculation loop and transferred to 20 mL of LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7). The medium was activated and cultured at 30°C with shaking at 180 rpm in the dark for 24 h. The cells were then collected by centrifugation at 8000 rpm for 5 min and resuspended in physiological saline to the OD value of the bacterial culture. 600 A value of 1.0 yields a seed culture of Rhodococcus ruber strain WM-5 (with a viable count of approximately 3.4 × 10⁻⁶). 9 (cfu / mL).

[0051] Seed culture of Rhodococcus ruber WM-5 strain was inoculated at a 2% (v / v) inoculation rate into PAEs contaminants and Cd contaminants at different initial concentrations (0 mg / L DBP + 0 mg / L DEHP, 100 mg / L DBP + 100 mg / L DEHP, 200 mg / L DBP + 200 mg / L DEHP, 500 mg / L DBP + 500 mg / L DEHP, 800 mg / L DBP + 800 mg / L DEHP, 1000 mg / L DBP + 1000 mg / L DEHP, 1500 mg / L DBP + 1500 mg / L DEHP) and Cd contaminants at different initial concentrations (0, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100 mg / L). 2+ The culture medium was incubated in LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7) at 30 °C with light-shielded shaking at 180 r / min for 24 h. The OD of the culture medium was measured at 24 h. 600 To investigate the effects of Rhodococcus ruber strain WM-5 on PAEs and Cd. 2+ Tolerance.

[0052] Seed culture of Rhodococcus ruber WM-5 was inoculated at a 5% (v / v) in MSM liquid medium containing different initial concentrations of PAEs (painful effervescent particles) (100 mg / L DBP + 100 mg / L DEHP, 200 mg / L DBP + 200 mg / L DEHP, 300 mg / L DBP + 300 mg / L DEHP, 400 mg / L DBP + 400 mg / L DEHP, 500 mg / L DBP + 500 mg / L DEHP). The medium was incubated at 30°C with shaking at 180 rpm in the dark for 2 days. The OD of the culture medium was measured after 2 days. 600 The residual PAE content was determined, and the PAE degradation rate was calculated according to formula (1). Three replicates were set up for each treatment to explore the application effect of Rhodococcus ruber WM-5 strain in PAE-contaminated solution systems with different initial concentrations.

[0053] Seed culture was inoculated at a 5% (v / v) inoculum concentration into containers containing different initial concentrations (0, 5, 10, 15, 20, 30, 40, 50 mg / L) of Cd. 2+ The culture medium was infused with 200 mg / L PAEs contaminants (100 mg / L DBP + 100 mg / L DEHP) in MSM liquid medium and cultured at 30°C with light-shielded shaking at 180 rpm for 2 days. The OD of the culture medium was measured at 2 days. 600 The content of residual PAEs pollutants was determined, and the PAEs degradation rate was calculated according to formula (1). Three replicates were set up for each treatment to explore the application effect of Rhodococcus ruber WM-5 strain in Cd-PAEs complex pollutant solution system.

[0054] Research results: Rhodococcus ruber strain WM-5 showed resistance to PAEs contaminants and Cd. 2+ Tolerance such as Figure 8 As shown, the Rhodococcus ruber WM-5 strain exhibits resistance to PAE contaminants and Cd. 2+ The maximum tolerable concentrations are 3000 mg / L and 50 mg / L, respectively, indicating high application potential.

[0055] The degradation effect of Rhodococcus ruber WM-5 strain in PAE-contaminated solution systems of different concentrations is as follows: Figure 9As shown, when the initial concentration of PAEs is 200 mg / L to 800 mg / L (100 mg / L DBP + 100 mg / L DEHP, 200 mg / L DBP + 200 mg / L DEHP, 300 mg / L DBP + 300 mg / L DEHP, 400 mg / L DBP + 400 mg / L DEHP), the degradation rate of DBP by Rhodococcus ruber WM-5 strain can reach 98.29% to 98.79% after 2 days, while the degradation rate of DEHP can reach 3.20% to 3.69%. When the initial concentration of PAEs exceeds 800 mg / L (400 mg / L DBP + 400 mg / L DEHP), its degradation effect decreases rapidly.

[0056] The degradation effect of Rhodococcus ruber strain WM-5 in Cd-PAEs co-polluting solution system is as follows: Figure 10 As shown, when Cd 2+ When the initial concentration was 0–10 mg / L (0, 5, 10 mg / L) and the initial concentration of PAEs was 200 mg / L (100 mg / L DBP + 100 mg / L DEHP), the degradation rate of DBP by Rhodococcus ruber WM-5 strain reached 95.26%–99.42% after 2 days, while the degradation rate of DEHP reached 7.52%–8.35%; when Cd 2+ When the initial concentration exceeds 10 mg / L, its degradation effect decreases rapidly. Rhodococcus ruber strain WM-5 has significant application value in both PAE contamination systems and Cd-PAE combined contamination systems.

[0057] In conclusion,

[0058] This invention is the first to discover that the Rhodococcus ruber strain WM-5 can survive using six PAEs—DMP, DEP, DBP, BBP, DEHP, and DOP—as the sole carbon source, and thus has great application potential.

[0059] This invention is the first to discover that Rhodococcus ruber strain WM-5, when cultured for 7 days with 200 mg / L PAEs contaminants (100 mg / L DBP + 100 mg / L DEHP) as the sole carbon source, exhibits extremely high degradation efficiency for DBP, reaching 97.91% after 2 days. However, its degradation efficiency for DEHP is relatively low, remaining below 20.00% within 7 days.

[0060] This invention is the first to discover that the optimal conditions for the degradation of DBP and DEHP by Rhodococcus ruber strain WM-5, using 200 mg / L PAEs contaminants (100 mg / L DBP + 100 mg / L DEHP) as the sole carbon source, are: inoculum amount 3%–5%, temperature 30℃–35℃, pH 6–9, and salinity 0.75–20 g / L.

[0061] This invention is the first to discover that Rhodococcus ruber strain WM-5, under total nutrient conditions (LB medium), exhibits resistance to Cd. 2+ The maximum tolerable concentrations for PAEs are 50 mg / L and 3000 mg / L, respectively.

[0062] This invention is the first to discover that when seed culture of Rhodococcus ruber WM-5 strain is inoculated at a 5% inoculum concentration into a solution system using PAEs (equal concentrations of DBP + equal concentrations of DEHP) as the sole carbon source, and when the initial PAE concentrations are 200–800 mg / L (100 mg / L DBP + 100 mg / L DEHP, 200 mg / L DBP + 200 mg / L DEHP, 300 mg / L DBP + 300 mg / L DEHP, 400 mg / L DBP + 400 mg / L DEHP), the degradation rates of DBP and DEHP by Rhodococcus ruber WM-5 strain can reach 98.29%–98.79% and 3.20%–3.69%, respectively, after 2 days. Furthermore, when Rhodococcus ruber WM-5 strain is inoculated at a 5% inoculum concentration... Seed culture of strain WM-5 (ruber) was inoculated in a solution system with 200 mg / L PAEs contaminants (100 mg / L DBP + 100 mg / L DEHP) as the sole carbon source. When Cd 2+ When the initial concentration was 0–10 mg / L (0, 5, 10 mg / L), the degradation rates of DBP and DEHP by Rhodococcus ruber strain WM-5 reached 95.26%–99.42% and 7.52%–8.35% after 2 days.

[0063] Therefore, the Rhodococcus ruber WM-5 strain has a high efficiency in PAE degradation, and its suitable degradation conditions are relatively wide. It also has strong tolerance to both Cd and PAE pollutants, and has great application value and potential. This invention can provide certain technical support and foundation for the future application of microbial remediation technology in the remediation of PAE pollution or Cd-PAEs combined pollution environments.

Claims

1. Rhodococcus rubrum strain WM-5, preservation number CCTCC NO.M2025874.

2. A microbial inoculant, characterized in that... The active ingredient is the Rhodococcus WM-5 strain described in claim 1.

3. The application of the Rhodococcus WM-5 strain of claim 1 or the microbial agent of claim 2 in the degradation of phthalates.

4. The application according to claim 3, characterized in that: The phthalate is one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, butyl benzyl phthalate, di(2-ethylhexyl) phthalate, and di-n-octyl phthalate.

5. The application according to claim 3, characterized in that: The degradation occurred under cadmium stress.

6. A method for remediating phthalate contamination, characterized in that... Add the Rhodococcus WM-5 strain of claim 1 or the microbial agent of claim 2 to the contaminant.

7. The method according to claim 6, characterized in that: The pollutants include both cadmium and phthalate esters.

8. The method according to claim 7, characterized in that: The pollutant contains Cd 2+ The concentration of phthalates is 0–50 mg / L, and the concentration of phthalates is 0–3000 mg / L.

9. The method according to claim 7, characterized in that: The pollutants are located in an environment with a temperature of 30-35℃, a pH of 6-9, and a salinity of 0.75g / L-20g / L.

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