A di(2-ethylhexyl) phthalate degrading bacterium, bacterial composition and application thereof

Abstract

The application discloses a di(2-ethylhexyl) phthalate degrading bacterium, a bacterial composition and application thereof. The degrading bacterium is named Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31. Research finds that GHQ28 and GHQ31 have chemotaxis to DEHP and downstream metabolites thereof, and there is a synergistic interaction between the two bacteria; the growth and DEHP degradation speed of the two strains under co-culture conditions are faster than that under single culture conditions. The bacterial composition composed of the two strains has a highest degradation rate of 99.2±0.59% to 400mg / L DEHP within 72h, compared with single culture, the total biomass is increased by 1.4-50.3 times, and the DEHP degradation rate is increased by 1.07-58.82 times. The application provides a new technical means for remediation of DEHP contaminated soil or water.

Description

Technical Field

[0001] This invention relates to a bacterial composition for degrading di(2-ethylhexyl) phthalate, and also to the application of said bacterial composition in the synergistic degradation of di(2-ethylhexyl) phthalate. This invention belongs to the field of environmental remediation technology. Background Technology

[0002] Phthalate esters (PAEs) are a class of organic compounds formed by the esterification reaction of phthalic anhydride and corresponding alcohols under acid catalysis. Di(2-ethylhexyl) phthalate (DEHP), a low-cost, high-performance plasticizer, is widely used in production and daily life; its chemical formula is C2. 24 H 38 O4, the structural formula is shown below:

[0003]

[0004] DEHP, a commonly used plasticizer, is easily released and migrates into the environment during plastic manufacturing, use, and disposal, severely impacting key ecological functions such as soil nutrient cycling and microbial activity. DEHP present in environmental media can be absorbed by plants in contaminated soil and, through bioaccumulation, spread to humans via the food chain. Excessive exposure to PAEs can lead to teratogenic, carcinogenic, and mutagenic effects, causing dysfunction in reproductive and immune systems. Therefore, the US Environmental Protection Agency (USEPA), the European Union (EU), and the China National Environmental Monitoring Center (CNEMC) have listed six PAE compounds, including DEHP, as priority pollutants. Microbial degradation, as a highly efficient and pollution-free remediation method, is widely used in the treatment of contaminated environments. Soil microorganisms play a crucial role in organic matter mineralization, element cycling, environmental stabilization, and the degradation of residual pollutants in the soil. Despite the numerous benefits of microbial remediation, single-microbial degradation has weaknesses such as the accumulation of intermediate metabolites and a limited substrate scope. Furthermore, some single bacteria that exhibit good degradation performance under laboratory conditions may not be fully adaptable to harsh natural environments. Synthetic microbial communities exhibit high adaptability under natural conditions. They not only resist environmental disturbances but also rapidly occupy ecological niches in competition with indigenous microorganisms, thereby playing a degradative role. This explains why synthetic microbial communities demonstrate superior degradation effects compared to single microorganisms. Therefore, synthetic microbial communities have broad application prospects in bioremediation agents and will also have a significant impact on my country's environmental governance development.

[0005] The inventors of this invention isolated and purified two DEHP-degrading bacteria, named Sphingomonasparapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31, respectively. The two strains were combined into a bacterial composition for the degradation of DEHP, and it was found that the two strains had a synergistic promoting effect on the degradation of DEHP. Summary of the Invention

[0006] One of the objectives of this invention is to provide two newly isolated bacteria with DEHP degradation capabilities.

[0007] The second objective of this invention is to provide a bacterial composition consisting of the two degrading bacteria and its application in promoting DEHP degradation.

[0008] To achieve the above objectives, the present invention employs the following technical means:

[0009] The two DEHP-degrading bacteria isolated in this invention are named *Sphingomonas parapaucimobilis* GHQ28 and *Stenotrophomonas maltophilia* GHQ31, respectively. *Sphingomonas parapaucimobilis* GHQ28 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNo. M 20242193, deposited on October 14, 2024. *Stenotrophomonas maltophilia* GHQ31 is also deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNo. M 20242181, deposited on October 14, 2024.

[0010] Furthermore, the present invention also proposes the application of the aforementioned DEHP-degrading bacteria in promoting DEHP degradation.

[0011] Preferably, the DEHP-degrading bacteria can accelerate the degradation of DEHP through inter-strain interaction without producing the accumulation of intermediate metabolites, thereby achieving complete degradation without residue.

[0012] Furthermore, the present invention also proposes a bacterial composition for degrading DEHP, wherein the bacterial composition comprises Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31, both of which are deposited at the China Center for Type Culture Collection (CCTCC) with accession numbers CCTCC No. M20242193 and CCTCC No. M 20242181, respectively.

[0013] Preferably, Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 are mixed in an equal volume ratio.

[0014] Furthermore, the present invention also proposes the application of the bacterial composition described herein in promoting the degradation of di(2-ethylhexyl) phthalate (DEHP).

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The inventors of this invention isolated and purified two DEHP-degrading bacteria, named *Sphingomonas parapaucimobilis* GHQ28 and *Stenotrophomonas maltophilia* GHQ31, respectively. Whole-genome sequencing results showed that *Sphingomonas parapaucimobilis* GHQ28 had 108 genes detected in carbon metabolism, 14 genes in aromatic compound metabolism, 9 genes in benzoate degradation, 47 genes in bacterial chemotaxis, and 35 genes in the ABC transport system. *Stenotrophomonas maltophilia* GHQ31 had 106 genes detected in carbon metabolism, 13 genes in aromatic compound metabolism, 13 genes in benzoate degradation, and 36 genes in bacterial chemotaxis. This study investigated the chemotaxis of *Sphingomonas parapaucimobilis* GHQ28 and *Stenotrophomonas maltophilia* GHQ31 towards DEHP and its downstream metabolites, as well as their degradation effect on DEHP. The results showed that both strains exhibited chemotaxis towards DEHP and its downstream metabolites, and the bacterial composition (A) formed by the two strains significantly increased biomass and the DEHP degradation rate. A metabolite-feeding nutrient relationship existed between the strains; co-culturing provided GHQ31 with a more suitable growth environment and allowed it to utilize carbon sources other than DEHP for its own growth, thus participating in the DEHP degradation process and accelerating the degradation of DEHP by the bacterial community. The bacterial community provided by this invention achieved complete DEHP degradation 68 hours faster than *Burkholderia pyrrocinia* B1213 (accession number CGMCC, No. 12806, Li et al., 2019) and 24 hours faster than *Achromobacter sp. RX* (Wang et al., 2021). Secondly, the study of the synergistic interaction characteristics between the strains showed that the two strains can coexist stably within the bacterial community. Finally, this invention proposes a method to promote DEHP degradation, which achieves complete degradation in a short time. This invention provides high-quality microbial resources for the preparation of bioremediation materials and offers a new technical means for the remediation of DEHP-contaminated soil or water. Attached Figure Description

[0017] Figure 1 Sequence alignment results and safranin staining results for Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31;

[0018] Among them, a is the sequence alignment result of Sphingomonas parapaucimobilis GHQ28; b is the morphological characteristics of Sphingomonas parapaucimobilis GHQ28; c is the sequence alignment result of Stenotrophomonas maltophilia GHQ31; and d is the morphological characteristics of Stenotrophomonas maltophilia GHQ31.

[0019] Figure 2 The chemotactic ability of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 strains to DEHP and its downstream metabolites was evaluated.

[0020] In the figure, a represents Sphingomonasparapaucimobilis GHQ28 and b represents Stenotrophomonasmaltophilia GHQ31. Different letters in the figure represent significant differences.

[0021] Figure 3 Growth and degradation characteristics of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 strains in single and co-culture.

[0022] Wherein, a represents the growth characteristics of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 in monoculture and coculture; b represents the degradation effect of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 on DEHP in monoculture and coculture; A represents the bacterial composition consisting of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31.

[0023] Figure 4 Quantitative analysis of metabolites of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 during feeding and co-culture.

[0024] Wherein, a represents the growth of GHQ28 after the addition of its own metabolites; b represents the growth of GHQ31 after the addition of its own metabolites; c represents the growth of GHQ31 after the addition of GHQ28 metabolites; d represents the growth of GHQ28 after the addition of GHQ31 metabolites; e represents the growth of the two strains over time under co-culture conditions as detected by qPCR.

[0025] Figure 5 Intermediate metabolites detected during the degradation of DEHP by the bacterial composition.

[0026] Where a represents LC-MS detection; b represents GC-MS detection;

[0027] Figure 6 The results are inferred from the degradation pathway of bacterial composition (A).

[0028] Where A represents β-oxidation; B represents deesterification; C represents decarboxylation; and D represents hydrolysis. Detailed Implementation

[0029] The present invention will be further illustrated below through experiments and embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention.

[0030] Example 1: Isolation and identification of DEHP-degrading bacteria Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31

[0031] 1. Test materials

[0032] The original microbial community used was a naturally enriched microbial community screened by Qiqihar University in four black soil plots based on the microcosm experiment (soil samples were collected from four regions in China: Hailun City, Heilongjiang Province; Keshan County, Heilongjiang Province; Hongxinglong Farm, Heilongjiang Province; and Ewenki Autonomous Banner, Inner Mongolia Autonomous Region).

[0033] 2. Isolation of bacteria

[0034] After activation of the natural bacterial flora, serial dilutions were performed, and DEHP-degrading single bacteria were isolated using inorganic salt medium coated with 400 mg / L DEHP. Bacterial 16S RNA sequencing was performed to identify the strains. The two isolated and purified DEHP-degrading single bacteria were named *Sphingomonas parapaucimobilis* GHQ28 and *Stenotrophomonas maltophilia* GHQ31, respectively, and deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, with accession numbers CCTCC No. M 20242193 and CCTCC No. M20242181, respectively, on October 14, 2024.

[0035] 3. Data Analysis

[0036] The sequencing results of strains GHQ28 and GHQ31 were compared with genes by BLAST in the NCBI (National Center of Biotechnology Information) database, and a phylogenetic tree was constructed.

[0037] 4. Results and Analysis

[0038] The sequence alignment results of strain GHQ28 are as follows: Figure 1 As shown in a, the isolated strain is genetically closest to *Sphingomonasparapaucimobilis* NBRC 15100, combined with the strain's morphological characteristics ( Figure 1 b) Strain GHQ28 was identified as *Sphingomonas parapaucimobilis*. The sequence alignment results for strain GHQ31 are as follows: Figure 1 As shown in c, its genetic evolutionary distance is closest to Stenotrophomonas maltophilia IAM 12423, combined with cell morphology ( Figure 1 d) GHQ31 was identified as Stenotrophomonas maltophilia.

[0039] Example 2: Chemotaxis of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 strains to DEHP and downstream metabolites.

[0040] 1. Materials and Methods

[0041] 1.1 Test strains: Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31, isolated and identified in Example 1.

[0042] Inorganic salt medium (MSM): 5.8g K2HPO4, 4.5g KH2PO4, 0.16g MgCl2, 2.0g (NH4)2SO4, 0.02g CaCl2, 0.024g Na2MoO4·2H2O, 0.015g MnCl2·2H2O, 0.018g FeCl3, add water to a final volume of 1L, pH 7.0±0.2.

[0043] Beef extract peptone liquid culture medium: 10.0g peptone, 3.0g beef extract, 5.0g sodium chloride, add water to a final volume of 1L, pH 7.0.

[0044] Substrate chemotaxis solutions: 400 mg / L DEHP, dibutyl phthalate (DBP), diethyl phthalate (DEP), dimethyl phthalate (DMP), diisobutyl phthalate (DIBP), phthalic acid (PA), benzoic acid (BA), and protocatechuic acid (PCA) were prepared as the sole carbon sources and added to the inorganic salt culture medium.

[0045] 1.2 Chemotaxis detection of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31

[0046] GHQ28 and GHQ31 were pre-cultured in beef extract peptone medium to the exponential phase (OD). 600nm =1.0), resuspend in MSM as a bacterial suspension for later use. Dissolve DEHP, DBP, DMP, DEP, DIBP, PA, BA, and PCA in MSM to prepare a chemotactic solution of 400 mg / L. Add 30 μL of bacterial suspension to the left side of a sterile glass slide and 30 μL of chemotactic solution to the right side, 1 cm apart. Connect the slides with an inoculation loop and let them stand for 15 min in a clean bench. Use sterile water and MSM solution as negative and positive controls, respectively. Then, transfer the chemotactic solution to a hemocytometer for counting. If the bacterial concentration in the chemotactic solution is significantly higher than that in the negative and positive controls, it indicates that the bacteria have a chemotactic effect on this metabolite.

[0047] 1.3 Data Processing

[0048] All raw data in this experiment were initially processed using Microsoft Excel Office 2021 software. Experimental results were plotted using R.

[0049] 2. Results and Analysis

[0050] The chemotactic abilities of strains *Sphingomonas parapaucimobilis* GHQ28 and *Stenotrophomonas maltophilia* GHQ31 under different carbon sources are as follows: Figure 2 As shown. Both strains exhibited chemotaxis towards DEHP and its downstream metabolites, with the highest chemotaxis towards DEHP.

[0051] Example 3: Characteristic analysis of the synergistic degradation of DEHP by Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31

[0052] 1. Materials and Methods

[0053] 1.1 Test materials

[0054] 1.1.1 Test strains

[0055] Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 were isolated and identified by Example 1.

[0056] 1.1.2 Culture medium

[0057] Inorganic salt medium (MSM): 5.8g K₂HPO₄, 4.5g KH₂PO₄, 0.16g MgCl₂, 2.0g (NH₄)₂SO₄, 0.02g CaCl₂, 0.024g Na₂MoO₄·2H₂O, 0.015g MnCl₂·2H₂O, 0.018g FeCl₃, diluted with water to 1L, pH 7.0±0.2. 400mg / L DEHP was prepared as the sole carbon source and added to the inorganic salt medium.

[0058] Beef extract peptone liquid culture medium: 10.0g peptone, 5.0g beef extract, 5.0g sodium chloride, add water to a final volume of 1L, pH 7.0.

[0059] 1.2 Study on the growth and degradation characteristics of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 in monoculture and co-culture

[0060] Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 were pre-cultured in beef extract peptone medium to the exponential phase (OD200). 600nm =1.0), then the cells were washed with MSM and cultured individually and in combination. For individual culture, GHQ28 and GHQ31 were seeded at a 2% v / v in 50 mL MSM containing 400 mg / L DEHP and cultured in the dark at 30°C and 150 rpm for 72 h. For co-culture, GHQ28 and GHQ31 were mixed at a 1:1 volume ratio and simultaneously seeded at a 2% v / v in 50 mL MSM containing 400 mg / L DEHP and cultured in the dark at 30°C and 150 rpm for 72 h. The OD of the samples was measured every 3 h using a UV spectrophotometer. 600 Samples were collected every 12 hours, and the residual DEHP content and its downstream metabolites were determined by gas chromatography.

[0061] 1.3 Cross-feeding study of metabolites from *Sphingomonas parapaucimobilis* GHQ28 and *Stenotrophomonas maltophilia* GHQ31

[0062] Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 were inoculated into inorganic salt medium with DEHP as the sole carbon source and cultured in the dark at 30°C and 150 rpm for 72 h until the death phase. The bacterial culture was centrifuged at 8000 rpm for 5 min, and the supernatant was passed through a 0.22 μm sterile membrane to obtain the metabolites produced during the culture. GHQ28 and GHQ31 were pre-cultured in beef extract peptone medium until the exponential growth phase (OD200). 600nm =1.0), resuspended in MSM as a bacterial suspension for later use. The two bacterial strains were inoculated into inorganic salt medium, and either their own or the supernatant of the other strain was added. The culture system was carried out in 96-well plates, each well containing 180 μL of MSM medium and 10% supernatant, and inoculated with 20 μL of bacterial suspension. The plates were incubated statically at 30°C for 108 h, and the growth of the strains (OD) was recorded every 6 h using a microplate reader. 600nm ).

[0063] 1.4 Quantitative analysis of co-cultured cell numbers

[0064] Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 were pre-cultured and then inoculated into an inorganic salt medium with DEHP as the sole carbon source at a 1:1 initial inoculation volume ratio. Samples were taken at 24h, 36h, 48h, and 72h of culture, and the number of cells in the co-culture was quantitatively detected by qPCR. Specific primers based on the 16S sequences of GHQ28 and GHQ31 were designed using Premier 5.0 software (Table 1). The reaction mixture was carried out in 10μL volumes, containing 5μL SybrGreen qPCR Master Mix (2×), 0.2μL (10μmol / L) of each primer, 1μL of template DNA, and 3.6μL of ddH2O. A standard curve was generated using the 2692bp pMD18-T plasmid vector, and gene copy number is expressed as log10.

[0065] Table 1. Specific primers for each strain

[0066]

[0067] 1.5 GC-MS and LC-MS detection of intermediate metabolites of DEHP co-degradation by GHQ28 and GHQ31

[0068] Both strains were pre-cultured in beef extract peptone medium to the exponential growth phase (OD). 600nm =1.0), then the cells were washed with inorganic salt medium. The strain was co-cultured at an inoculation volume ratio of 1:1, and 2% was inoculated into 50 mL of MSM containing 400 mg / L DEHP. The cells were incubated in the dark at 30 °C and 150 rpm for 72 h, with samples taken every 12 h. An equal volume of ethyl acetate was added to the sample vial to extract all the liquid. After thorough mixing, the organic phase was collected by centrifugation. The remaining aqueous phase was extracted three times using the same method. All organic phases were combined and evaporated to dryness. The sample was reconstituted in 5 mL of n-hexane and filtered through a sterile membrane (0.22 μm). The metabolites of DEHP degradation were detected using gas chromatography-mass spectrometry (GC-MS).

[0069] GC-MS parameter settings: The system was operated in electron collision and selective ion monitoring (SCM) mode, using an HP-5MS (30m × 0.25mm × 0.25mm) quartz capillary column and high-purity helium (99.999%) as the carrier gas. Chromatographic separation was performed using a constant flow rate of 1.0 mL / min, an initial filament current of 0.1 ppm, an initial ion source temperature of 220°C, and an MS1 ​​quadrupole temperature of 150°C. The programmed temperature oven was initially set at 60°C, held for 1 min, then increased to 220°C at a rate of 20°C / min, held for 1 min, and finally increased to 260°C at a rate of 5°C / min. The injection volume was 1 μL, and the injection port temperature was 250°C.

[0070] Samples co-cultured for 36 h were subjected to metabolomics analysis, with 8 replicates. After pre-culturing the bacterial strain, it was inoculated into an inorganic salt medium with DEHP as the sole carbon source and cultured with shaking for 36 h. The bacterial culture was centrifuged at 8000 rpm for 8 min, and the supernatant was filtered through a 0.22 μm sterile membrane and then lyophilized. The dried sample was placed in a 2 mL centrifuge tube, and a 6 mm diameter grinding bead and 400 μL of extraction buffer (methanol:water, volume ratio 4:1) containing 0.02 mg / mL internal standard (L-2-chlorophenylalanine) were added for metabolite extraction. The samples were treated with a 50 Hz cryo-tissue homogenizer at -10 °C for 6 min, followed by low-temperature ultrasonic extraction for 30 min (5 °C, 40 kHz). The samples were incubated at -20 °C for 30 min, then centrifuged at 13000 rpm for 15 min at 4 °C, and the supernatant was used for non-target metabolomics analysis. The analysis was performed using a UHPLC-Q Exactive system (Thermo Fisher).

[0071] LC-MS parameter settings: 2 μL samples were separated using an HSS T3 column (100 mm × 2.1 mm id, 1.8 μm) before being analyzed by mass spectrometry. Mobile phase A consisted of 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B consisted of 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). Separation gradient: 0–0.1 min, mobile phase B linearly increased from 0% to 5%; 0.1–2 min, mobile phase B linearly increased from 5% to 25%; 2–9 min, mobile phase B linearly increased from 25% to 100%; 9–13 min, mobile phase B linearly maintained at 100%; 13.0–13.1 min, mobile phase B linearly decreased from 100% to 0%; 13.1–16 min, mobile phase B linearly maintained at 0%. The flow rate was 0.40 mL / min, and the column temperature was 40 °C. Sample mass spectrometry signal acquisition employed positive and negative ion scanning modes, with a mass scan range of m / z: 70-1050. Ion spray voltages were set at 3500V for positive ions and 2800V for negative ions, with a sheath gas pressure of 40psi and an auxiliary heating gas pressure of 10psi. The ion source heating temperature was 400℃, with a cyclic collision energy of 20-40-60V. MS1 resolution was 70000, and MS2 resolution was 17500.

[0072] 1.6 Data Processing

[0073] The initial organization was done using Microsoft Excel Office 2021 software. Graphs were created using R.

[0074] 2. Results and Analysis

[0075] 2.1 Study on the growth and DEHP degradation characteristics of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 in monoculture and co-culture

[0076] Strains *Sphingomonas parapaucimobilis* GHQ28 and *Stenotrophomonas maltophilia* GHQ31 were cultured individually and in combination, and inoculated separately or simultaneously into inorganic salt medium with DEHP as the sole carbon source. The growth of the strains was observed, and the OD values ​​of the bacterial culture were measured. 600nm The value was measured. *Sphingomonas parapaucimobilis* GHQ28 grows well in an inorganic salt environment with DEHP as the sole carbon source, reaching its highest OD value within 72 hours. 600nmThe value was 1.486±0.010, while Stenotrophomonas maltophilia GHQ31 showed poor adaptability. However, both strains significantly increased biomass during co-culture, with the highest OD value within 72 hours. 600nm The value was 2.051 ± 0.010, indicating that co-culturing the two strains significantly increased biomass compared to monoculture. Figure 3 a). The degradation effects of single and co-culture of strains Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 on DEHP are as follows: Figure 3 As shown in b, the degradation curve of the co-cultured strains exhibits a continuous upward trend followed by a plateau, preferentially reaching a peak degradation rate of 90.72 ± 1.74% at 48 h, with the highest degradation rate reaching 99.20 ± 0.59% within 72 h. Compared to single-strain culture, the DEHP degradation rate of co-culture was increased by 1.07–58.82 times. In conclusion, the bacterial composition (A) consisting of two strains demonstrates stronger growth adaptability to DEHP and improves the DEHP degradation rate.

[0077] 2.2 Study on the synergistic interaction between Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31

[0078] We conducted a cross-feeding experiment with metabolites from two strains. The results showed that, under static culture conditions, supplementing with GHQ28 metabolites could promote its own growth. Figure 4 a), but GHQ31 is not sensitive to its own metabolites. Figure 4 b). Supplementation with GHQ31 metabolites significantly enhanced the growth of GHQ28, demonstrating a powerful metabolic promoting effect. Figure 4 c) However, supplementing with GHQ31 metabolites did not have a significant promoting or inhibiting effect on the growth of GHQ28. Figure 4 d). To further investigate the symbiotic state of the two strains under co-culture conditions, the growth of the two strains over time was quantitatively detected by qPCR. Strain GHQ31 adapted to an inorganic salt environment with DEHP as the sole carbon source under co-culture conditions. Figure 4 e) It is possible that the strain maintained its growth by ingesting metabolites produced by GHQ28, thereby playing a role in the degradation of DEHP. This process further confirms that the strains degrade DEHP through synergistic cooperation.

[0079] 2.3 Degradation and metabolic pathway prediction of bacterial composition (A)

[0080] Based on the substances detected by GC-MS and LC-MS ( Figure 5 ), and speculate on the metabolic pathway by which bacterial composition (A) degrades DEHP ( Figure 6 In the upstream degradation phase, DEHP undergoes β-oxidation, sequentially removing ethyl groups to form short-chain PAEs, which are then hydrolyzed to phthalic acid. Simultaneously, DEHP undergoes deesterification to form single-chain mono-diethylhexyl phthalate (MEHP). MEHP can further undergo deesterification to form PA, or it can undergo decarboxylation to form 2-ethylhexyl benzoate, followed by deesterification to form BA. Multiple pathways occur simultaneously, completing the upstream degradation. In the downstream degradation phase, PA undergoes double oxidation at the 4,5 positions of the benzene ring to produce cis-4,5-dihydro-4,5-dihydroxyphthalate, which is then dehydrogenated to form 4,5-dihydroxyphthalate, and further decarboxylated to form PCA. PCA can undergo ortho-ring unraveling to form β-keto adipic acid, thus entering the TCA cycle. Simultaneously, PA can decarboxylate to form BA, which can then undergo p-hydroxybenzoate to form β-keto adipic acid, entering the TCA cycle. Alternatively, it can be converted from 1-cyclohexene carboxylic acid to 2-hydroxycyclohexane carboxylic acid, entering the TCA cycle via the adipic acid pathway. Based on the metabolites detected above, it can be concluded that the bacterial composition (A) has the ability to completely metabolize DEHP into H2O and CO2.

[0081] in conclusion:

[0082] Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 exhibit chemotaxis towards DEHP and its downstream metabolites. Through synergistic action, they enhance their own growth while simultaneously accelerating DEHP degradation. A nutrient relationship exists between the two bacteria, with GHQ28 supplying GHQ31 with growth-required substances, thereby enhancing the growth and degradation capacity of the bacterial composition (A). These findings suggest that this functional microbial consortium can synergistically accelerate the complete degradation of DEHP, offering a significant practical solution for addressing soil DEHP pollution.

Claims

1. A bacterial composition for degrading di(2-ethylhexyl) phthalate (DEHP), characterized in that, The bacterial composition is composed of Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 composition, the aforementioned Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia All GHQ31 samples are deposited at the China Center for Type Culture Collection (CCTCC), with accession numbers CCTCC No. M 20242193 and CCTCC No. M 20242181, respectively.

2. The bacterial composition according to claim 1, characterized in that, The aforementioned Sphingomonas parapaucimobilis GHQ28 and Stenotrophomonas maltophilia GHQ31 is mixed in equal volume ratios.

3. The use of the bacterial composition of claim 1 in promoting the degradation of di(2-ethylhexyl) phthalate (DEHP).

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