Sphingomonas sp. capable of degrading phthalate, bacterial combination and complex microbial agent and application thereof

Abstract

The application provides a strain of sphingomonas which can degrade phthalate, a bacterial combination and a composite microbial agent and application thereof, and belongs to the technical field of microorganisms. The application provides a strain of sphingomonas which can degrade phthalate, WXX-05, which can simultaneously degrade multiple phthalates. The application also provides a bacterial combination, which comprises the sphingomonas WXX-05 and a new species of rhizomucor WXX-04. The new species of rhizomucor WXX_04 strain and the sphingomonas WXX_05 strain can simultaneously degrade multiple phthalates. The bacterial combination of the application has strong adaptability to the environment, can effectively degrade multiple phthalates, and can improve the phthalate pollution remediation efficiency of soil by being applied to phthalate-polluted farmland soil.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and particularly relates to a sphingosine box bacterium that degrades phthalates, a bacterial combination and compound bacterial agent and their applications. Background Technology

[0002] Phthalate esters (PAEs), as a new class of pollutants with endocrine-disrupting effects, are widely used in agricultural fields such as plastic films, packaging materials, and pesticide adjuvants. PAEs are linked to plastic molecules by hydrogen bonds or van der Waals forces, retaining relatively independent chemical properties. Therefore, they are extremely prone to aging and decomposition into the soil during the use of various plastic products, and are subsequently absorbed by crops, threatening human health.

[0003] Microbial degradation is the mainstream technology for soil degradation of PAEs. Microorganisms can completely mineralize PAEs into harmless small molecules through specific enzyme systems, avoiding pollutant residues and offering advantages such as environmental friendliness, low cost, and high safety. However, current PAE-degrading strains lack environmental adaptability in contaminated soil remediation scenarios. When degradation bacteria screened from non-arable land soil media are transplanted into PAE-contaminated arable land, their colonization efficiency is low, making it difficult to achieve stable and efficient pollutant degradation. Summary of the Invention

[0004] The purpose of this invention is to provide a sphingosine box bacterium that degrades phthalates, a bacterial combination and compound bacterial agent, and their applications. The strain, bacterial combination and compound bacterial agent of this invention have strong adaptability and can effectively degrade phthalates in arable soil.

[0005] This invention provides a sphingosine box bacterium that degrades phthalates ( Sphingopyxis sp .) WXX-05, the preservation number of the sphingosine box bacterium WXX-05 is: CGMCC No.36358.

[0006] The present invention also provides a bacterial agent comprising the sphingosine box bacteria WXX-05 described in the above-described scheme.

[0007] The present invention also provides a bacterial composition including Sphingosine Box Bacteria (Sphingosine Box Bacteria) Sphingopyxis sp WXX-05 and a new species of the genus *Pseudomonas* ( Agromyces sp .) WXX-04; the preservation number of the sphingosine clade fungus WXX-05 is: CGMCC No. 36358; the preservation number of the new species of the genus *Pseudomonas* WXX-04 is: CGMCC No. 36357.

[0008] This invention provides a compound bacterial agent comprising the bacterial combination described in the above-described scheme.

[0009] Preferably, the compound microbial agent comprises a bacterial suspension of *Sphingosporium sphingolipidae* WXX-05 and a bacterial suspension of a new species of *Pseudomonas* WXX-04; the volume ratio of the bacterial suspension of *Sphingosporium sphingolipidae* WXX-05 to that of *Pseudomonas* WXX-04 is (1~2):(1~2); and the OD values ​​of both the bacterial suspensions of *Sphingosporium sphingolipidae* WXX-05 and *Pseudomonas* WXX-04 are OD. 600nm =0.8.

[0010] Preferably, the volume ratio of the bacterial suspension of *Sphingosine Trichophyton* WXX-05 to the bacterial suspension of *Pseudomonas aeruginosa* WXX-04 is 1:1.

[0011] The present invention also provides the application of the above-described Sphingosine Box Bacterium WXX-05, the bacterial agent, the bacterial combination, or the compound bacterial agent in the degradation of phthalates or the remediation of phthalate-contaminated media.

[0012] Preferably, the phthalate comprises one or more of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di(2-ethylhexyl) phthalate, dibutyl phthalate, and di-n-octyl phthalate.

[0013] The present invention also provides a method for degrading phthalates, comprising the following steps: The above-described sphingosine-containing bacteria WXX-05, the bacterial agent, the bacterial combination, or the compound bacterial agent are inoculated or applied to the phthalate-containing sample to be degraded for degradation.

[0014] Preferably, the effective viable bacterial concentration inoculated into the sample to be degraded is 10. 6 ~10 8 CFU / g.

[0015] This invention provides a sphingosine box bacterium that degrades phthalates ( Sphingopyxis sp WXX-05, the preservation number of the *Sphingosine Box Bacterium* WXX-05, is CGMCC No. 36358. *Sphingosine Box Bacterium* WXX_05 was screened from peanut rhizosphere soil covered with film for over ten years in Shandong Province and can simultaneously degrade multiple phthalates. Verification showed that WXX_05 achieved a 98.55% degradation rate of DBP and a 97.94% degradation rate of DEHP in MSM medium.

[0016] The present invention also provides a bacterial composition including Sphingosine Box Bacteria (Sphingosine Box Bacteria) Sphingopyxis sp WXX-05 and a new species of the genus *Pseudomonas* ( Agromyces spThe preservation number of *Sphingosine Boxerella* WXX-04 is CGMCC No. 36358; the preservation number of the new species *Pterygomyces* WXX-04 is CGMCC No. 36357. Both *Pterygomyces* species WXX_04 and *Sphingosine Boxerella* strain WXX_05 were screened from peanut rhizosphere soil covered with plastic film for over ten years in Shandong Province. Both strains can simultaneously degrade multiple phthalates. The bacterial combination culture method of this invention is simple, the strains grow rapidly, are not prone to mutation, have strong environmental adaptability, and can effectively degrade multiple phthalates. By applying them to phthalate-contaminated soil, the remediation efficiency of phthalate pollution in soil can be improved, and the accumulation of PAEs in peanuts can be significantly reduced. In particular, the remediation efficiency for dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) is over 70%, showing great application potential. This invention is simple to operate, has high degradation efficiency, can be industrially produced, and is environmentally friendly. It is of great significance for ensuring the safety of peanut production in soil contaminated with low to medium concentrations of PAEs. Furthermore, the compound microbial agent of this invention can efficiently degrade phthalates, and can reduce the accumulation of intermediate products through synergistic effects between strains. The degradation efficiency in soil can be increased from 44% to 64% compared to single-strain degradation, and the degradation efficiency in peanuts can be increased from 26% to 42% compared to single-strain degradation. It has strong environmental adaptability, effectively degrades phthalates in the soil-peanut system, and ensures the safety of the soil environment in peanut production areas.

[0017] Biological Preservation Instructions Sphingosine box bacteria ( Sphingopyxis sp WXX-05 was deposited on October 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36358.

[0018] New species of the genus *Pseudomonas* ( Agromyces sp WXX-04 was deposited on October 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36357. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a colony morphology diagram of strain WXX_04 on LB medium; Figure 2 This is a colony morphology diagram of strain WXX_05 on LB medium; Figure 3 A scanning electron microscope image of strain WXX_04; Figure 4 A scanning electron microscope image of strain WXX_05; Figure 5 A 16S phylogenetic tree of strain WXX_04; Figure 6 A 16S phylogenetic tree of strain WXX_05; Figure 7 A statistical chart of COG annotations for strain WXX_05; Figure 8 GO annotation classification statistics for strain WXX_05; Figure 9 KEGG annotation classification statistics for strain WXX_05; Figure 10 This is a genomic circle diagram of strain WXX_05; Figure 11 The degradation effect of strain WXX_04 on DBP and DEHP in MSM; Figure 12 The degradation effect of strain WXX_05 on DBP and DEHP in MSM; Figure 13 The effects of different ratios of compound microbial agents on the degradation of DBP and DEHP in MSM; Figure 14 The degradation effects of strains WXX_04, WXX_05 and compound bacterial agent on different PAEs in soil were studied. Figure 15 The effects of strains WXX_04, WXX_05 and compound inoculum on the accumulation of different PAEs in the underground parts of peanut; Figure 16 The effects of strains WXX_04, WXX_05 and compound inoculant on the accumulation of different PAEs in the aboveground parts of peanut. Detailed Implementation

[0021] This invention provides a sphingosine box bacterium that degrades phthalates ( Sphingopyxis sp .) WXX-05, the preservation number of the sphingosine box bacterium WXX-05 is: CGMCC No.36358.

[0022] In this invention, *Sphingosine Box* WXX-05 is a Gram-negative bacterium. On LB agar, its colonies are yellowish-brown, smooth, raised, opaque, and have regular edges. Under a scanning electron microscope, it appears as rod-shaped bacteria, 0.5–1 μm in length, with a rough surface. Based on a combination of morphological characteristics, growth conditions, and physiological and biochemical identification results, WXX_05 is determined to belong to the genus *Sphingosine Box*. Sphingopyxis sp .).

[0023] In this invention, the nucleotide sequence of the 16S rDNA of *Sphingosine Box Bacteria WXX-05* is as shown in SEQ ID NO.1, specifically: cttgagagtt tgatcctggc tcagaacgaa cgctggcggc atgcctaaca catgcaagtc 60 gaacgaactc ttcggagtta gtggcgcacg ggtgcgtaac gcgtgggaat ctgcccttgg 120 gtacggaata actcagagaa atttgtgcta ataccgtata atgacttcgg tccaaagatt 180 tatcgcccaa ggatgagccc gcgtaagatt agctagttgg tggggtaaaa gcctaccaag 240 gcgacgatct ttagctggtc tgagaggatg atcagccaca ctgggactga gacacggccc 300 agactcctac gggaggcagc agtggggaat attggacaat gggcgaaagc ctgatccagc 360 aatgccgcgt gagtgatgaa ggccctaggg ttgtaaagct cttttacccg ggatgataat 420 gacagtaccg ggagaataag ctccggctaa ctccgtgcca gcagccgcgg taatacggag 480 ggagctagcg ttattcggaa ttactgggcg taaagcgcgc gtaggcggtt ttttaagtca 540 ggggtgaaag cccggagctc aactccggaa tagcctttga aactggaaaa cttgaatctt 600 ggagaggtca gtggaattcc gagtgtagag gtgaaattcg tatattcg gaagaacacc 660 agtggcgaag gcgactgact ggacaagtat tgactctgag gtgcgaaagc gtggggagca 720 aacaggatta gataccctgg tagtccacgc cgtaaacgat gataactagc tgtccggact 780 catagagttt gggtggcgca gctaacgcat taagttgtcc gcctggggag tacggtcgca 840 agattaaaac tcaaaggaat tgacggggc ctgcacaagc ggtggagcat gtggtttaat 900 tcgaagcaac gcgcagaacc ttaccagcgt ttgacatcct gatcgcggat tagagagatc 960 ttttccttca gttcggctgg atcagtgaca ggtgctgcat ggctctcgtc agctcgtgtc1020 gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctc atccctagtt gccatcattc1080 agttgggcac tctaaggaaa ctgccggtga taagccggag gaaggtgggg atgacgtcaa1140 gtcctcatgg cccttacgcg ctgggctaca cacgtgctac aatggcggtg acagtgggca1200 gcaacccgc gaggggtagc taatctccaa aagccgtctc agttcggatt gttctctgca1260 actcgagagc atgaaggcgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac1320 gttcccaggc cttgtacaca ccgcccgtca caccatggga gttggtttca cccgaaggca1380 gtgctctaac ccgcaaggga ggaagctgac cacggtggga tcagcgactg gggtgaagtc1440 gtaacaaggt agccgtaggg gaacctgcgg ctggatcacc tccttt 1486.

[0024] The present invention also provides a bacterial agent comprising the sphingosine box bacteria WXX-05 described in the above-described scheme.

[0025] The present invention also provides a bacterial composition including Sphingosine Box Bacteria (Sphingosine Box Bacteria) Sphingopyxis sp WXX-05 and a new species of the genus *Pseudomonas* ( Agromyces sp .) WXX-04; the preservation number of the sphingosine clade fungus WXX-05 is: CGMCC No. 36358; the preservation number of the new species of the genus *Pseudomonas* WXX-04 is: CGMCC No. 36357.

[0026] In one embodiment, the ratio of viable counts of the new species of *Pseudomonas* WXX-04 and *Sphingosine Box* WXX-05 in the bacterial combination is (0.5:1) to (1:0.5), and more specifically 1:1.

[0027] In this invention, the novel species of *Pseudomonas* WXX-04 was cultured on LB agar at 30°C for 3 days. The colonies were round, pale yellow, smooth, moist, opaque, and had regular edges. After four days, they almost completely covered the plate. Under a scanning electron microscope, they appeared as rod-shaped with a smooth surface. Based on the morphological characteristics, growth conditions, and physiological and biochemical identification results, WXX_04 was determined to belong to the genus *Pseudomonas* (…). Agromyces sp .) New species.

[0028] In this invention, the nucleotide sequence of the 16S rDNA of the new species WXX-04 of the genus *Pseudomonas* is shown in SEQ ID NO.2, specifically as follows: tttggagagt ttgatcctgg ctcaggacga acgctggcgg cgtgcttaac acatgcaagt 60 cgaacgatga acctggagct tgctctgggg gattagtggc gaacgggtga gtaacacgtg 120 agtaacctgc cctggactct gggataactt cgagaaatcg gagctaatac cggataggac 180 cttgcaccgc atggtgtggg gtggaaagtt tttcggtttg ggatggactc gcggcctatc 240 agcttgttgg tgaggtaatg gctcaccaag gcgtcgacgg gtagccggcc tgagagggtg 300 accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 360 attgcacaat gggcgcaagc ctgatgcagc aacgccgcgt gcgggatgac ggccttcggg 420 ttgtaaaccg cttttagtag ggaagaagcc ttcgggtgac ggtacctgca gaaaaaggac 480 cggctaacta cgtgccagca gccgcggtaa tacgtagggt ccgagcgttg tccggaatta 540 ttgggcgtaa agagctcgta ggcggtttgt cgcgtctgct gtgaaaacta gaggctcaac 600 ctctagcctg cagtgggtac gggcagactt gagtggtgta ggggagactg gaattcctgg 660 tgtagcggtg gaatgcgcag atatcaggag gaacaccgat ggcgaaggca ggtctctggg 720 cacttactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag 780 tccacgccgt aaacgttggg cgctagatgt ggggaccttt ccacggtttc cgtgtcgtag 840 ctaacgcatt aagcgccccg cctggggagt acggccgcaa ggctaaaact caaaggaatt 900 gacgggggcc cgcgcaagcg gcggagcatg cggattaatt cgatgcaacg cgaagaacct 960 taccaaggct tgacatacac gagaacgggc cagaaatggt caactctttg gacactcgtg1020 aacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa1080 cgagcgcaac cctcgtcgca tgttgccagc acgttatggt ggggactcat gtgagactgc1140 cggggtcaac tcggaggaag gtggggatga cgtcaaatca tcatgcccct tatgtcttgg1200 gcttcacgca tgctacaatg gccggtacaa agggctgcga tgtcgtaagg cggagcgaat1260 cccaaaaagc cggtctcagt tcggattgag gtctgcaact cgacctcatg aagtcggagt1320 cgctagtaat cgcagatcag caacgctgcg gtgaatacgt tcccgggcct tgtacacacc1380 gcccgtcaag tcatgaaagt cggtaacacc cgaagccggt ggcctaaccc ttgtggaggg1440 agccgtcgaa ggtgggatcg gtgattagga ctaagtcgta acaaggtagc cgtaccggaa1500 ggtgcggctggatcacctccttt1523.

[0029] In this invention, the new species of *Pterocarya* WXX-04 and *Sphingosine Boxerella* WXX-05 were both isolated from peanut rhizosphere soil in farmland contaminated with PAEs for a long period of time (coverage period ≥ 10 years). They were obtained through domestication with phthalic acid ester concentration gradients (5 mg / L → 10 mg / L → 20 mg / L → 40 mg / L → 80 mg / L → 160 mg / L) and have high efficiency in degrading phthalic acid esters: in an inorganic salt culture medium system, the degradation rate of the target substrate by *Pterocarya* WXX-04 and *Sphingosine Boxerella* WXX-05 can reach more than 90%.

[0030] The present invention also provides a compound bacterial agent comprising the bacterial combination described in the above-described scheme.

[0031] In this invention, the composite microbial agent achieves a degradation rate of over 99% for the target substrate. The composite microbial agent of this invention has a simple cultivation method, grows rapidly, is not prone to mutation, and has strong environmental adaptability. It can effectively degrade various phthalates. By applying it to phthalate-contaminated soil, it can improve the remediation efficiency of phthalate pollution in soil and significantly reduce the accumulation of PAEs in peanuts. In particular, the remediation efficiency for dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) both reach over 70%, demonstrating excellent application potential. This invention is simple to operate, has high degradation efficiency, can be industrially produced, and is environmentally friendly. It is of great significance for the safety of peanut production in soils contaminated with low to medium concentrations of PAEs.

[0032] In the soil-peanut system, the degradation rates of phthalates in the soil treated with strains WXX_05 and WXX-04 were significantly increased compared to the control, with increases ranging from 9 to 44 percentage points. The highest degradation rates were for dibutyl phthalate (DBP) and dimethyl phthalate (DMP), reaching 54.23% and 69.16%, respectively. The phthalate content in peanuts treated with strains WXX_05 and WXX-04 was significantly lower than the control. The degradation rate of phthalates in the underground parts ranged from 22.37% to 58.02%, while the degradation rate in the aboveground parts was significantly lower. The degradation rate of phthalates reached 21.16%~35.97%; the degradation rate of phthalates in the soil under the compound microbial agent treatment was significantly improved compared with the control, with an increase of 29~55 percentage points. Among them, dibutyl phthalate (DBP) and dimethyl phthalate (DMP) had the highest degradation rates, reaching 64.71% and 78.41%, respectively; the degradation rate of phthalates in the underground parts of peanut reached 27.45%~69.00%, and the degradation rate of phthalates in the aboveground parts reached 35.02%~46.00%, both of which were significantly better than those of single microbial agents.

[0033] The compound microbial agent of this invention has strong adaptability, high phthalate degradation efficiency, and is suitable for complex PAE mixed pollution scenarios in actual environments. It can simultaneously reduce multiple PAE pollutions in the soil-peanut system without adverse environmental impact. It can specifically solve the problem of phthalate plasticizer pollution in long-term mulched peanut production, and provide efficient functional strain resources for the bioremediation of phthalate pollution in farmland soil. It has significant practical value and broad application prospects.

[0034] In one embodiment, the compound microbial agent comprises a bacterial suspension of *Sphingosporium sphingolipidae* WXX-05 and a bacterial suspension of a new species of *Pseudomonas* WXX-04; the volume ratio of the bacterial suspension of *Sphingosporium sphingolipidae* WXX-05 to that of *Pseudomonas* WXX-04 is (1~2):(1~2), further comprising 1:2, 1:1, or 2:1, and even further comprising 1:1; the OD values ​​of both the bacterial suspensions of *Sphingosporium sphingolipidae* WXX-05 and *Pseudomonas* WXX-04 are OD. 600nm =0.8.

[0035] The present invention also provides a method for preparing the compound microbial agent described above, comprising the following steps: New species of *Pseudomonas* WXX-04 and *Sphingosine Box* WXX-05 were inoculated into LB solid medium and cultured. Single colonies were picked and inoculated into LB liquid medium to obtain seed culture of new species of *Pseudomonas* WXX-04 and *Sphingosine Box* WXX-05, respectively. Seed cultures of new species of *Pseudomonas* WXX-04 and *Sphingosine Box* WXX-05 were inoculated into new LB liquid medium at an inoculation rate of 2% to 5% by volume, and cultured to the logarithmic phase. After centrifugation, the mycelial cells of new species of *Pseudomonas* WXX-04 and *Sphingosine Box* WXX-05 were collected separately. After washing the cells of the new species of *Pseudomonas* WXX-04 and *Sphingosine Box* WXX-05 with PBS buffer 2-3 times, they were resuspended in PBS to obtain bacterial suspensions of *Pseudomonas* WXX-04 and *Sphingosine Box* WXX-05, respectively. The OD values ​​were then adjusted. 600 To 0.8~1.0; A compound bacterial agent was obtained by mixing bacterial suspensions of a new species of *Pseudomonas*, WXX-04, and *Sphingosine Box Bacteria*, WXX-05.

[0036] The present invention also provides the application of the above-described Sphingosine Box Bacterium WXX-05, the bacterial agent, the bacterial combination, or the compound bacterial agent in the degradation of phthalates or the remediation of phthalate-contaminated media.

[0037] In one embodiment, the medium is soil or water; the concentration of phthalates in the medium is 5-160 mg / L or 5-160 mg / kg, more preferably 10-80 mg / L or 10-80 mg / kg, and even more preferably 20-40 mg / L or 20-40 mg / kg; the soil includes peanut rhizosphere soil. This invention is particularly suitable for the remediation of plasticizer pollution in flower production areas under long-term mulching agriculture scenarios.

[0038] In one embodiment, the phthalate includes one or more of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di(2-ethylhexyl) phthalate, dibutyl phthalate, and di-n-octyl phthalate.

[0039] In this invention, both the new species of *Pseudomonas* WXX-04 and *Sphingosine Box* WXX-05 can use the above-mentioned phthalate compounds as carbon and energy sources to grow, thereby increasing biomass accumulation and degrading these phthalate compounds.

[0040] The present invention also provides a method for degrading phthalates, comprising the following steps: The above-described sphingosine-containing bacteria WXX-05, the bacterial agent, the bacterial combination, or the compound bacterial agent are inoculated or applied to the phthalate-containing sample to be degraded for degradation.

[0041] As one implementation method, the effective viable bacteria concentration inoculated into the sample to be degraded is 10. 6 ~10 8 CFU / g.

[0042] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a sphingosine box bacterium that degrades phthalates, a bacterial combination and compound bacterial agent, and their applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0043] In the embodiments of the present invention, the inorganic salt liquid culture medium using dibutyl phthalate and di(2-ethylhexyl) phthalate as carbon sources comprises: sodium dihydrogen phosphate dodecahydrate 1.5 g / L, dipotassium hydrogen phosphate 1.5 g / L, ammonium sulfate 2.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium chloride dihydrate 0.01 g / L, ferrous sulfate heptahydrate 0.001 g / L, trace element solution 1 mL / L, DBP and DEHP concentrations of 100 mg / L each, pH 7.2, and inoculum size of degrading bacteria 2%. The trace element solution comprises: disodium ethylenediaminetetraacetate 500 mg / L, zinc sulfate heptahydrate 10 mg / L, manganese sulfate 5 mg / L, boric acid 30 mg / L, cobalt sulfate heptahydrate 24 mg / L, copper sulfate pentahydrate 5 mg / L, sodium molybdate dihydrate 30 mg / L, calcium hydroxide 50 mg / L, and pH 7.2-7.5.

[0044] In the embodiments of the present invention, the LB medium consisted of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with 1.5% agar powder added to the solid medium; pH 7.2~7.5.

[0045] In an embodiment of the present invention, the preparation method of the compound microbial agent is as follows: a loopful of purified WXX_04 and WXX_05 from the plate is inoculated into 150 mL of sterile LB medium and cultured in a shaker at 30℃ and 180 r / min for 24 h to prepare an inoculum solution. Then, the inoculum solution is inoculated into 500 mL of LB liquid medium at a volume ratio of 2% to 5% to obtain a bacterial suspension. The bacterial suspension is dispensed into 100 mL sterile centrifuge tubes, centrifuged at 8000 rpm for 5 min, the supernatant is discarded, and the suspension is washed twice with PBS and resuspended in PBS to prepare a bacterial suspension. This suspension is inoculated into phthalate-contaminated soil and peanuts are planted and cultured until the seedling stage to degrade phthalates in the soil-peanut system.

[0046] In embodiments of the present invention, the absorbance (OD) of the bacterial suspension at a wavelength of 600 nm is... 600 The inoculation amount is 0.8~1.0, and the inoculation volume is 10. 6 ~10 8 CFU / g soil.

[0047] In an embodiment of the present invention, the purified WXX_04 and WXX_05 bacterial cells from the plate were prepared into a bacterial suspension and inoculated into MSM liquid medium containing 100 mg / L phthalate for shaking culture. During the growth process, WXX_04 and WXX_05 degraded phthalate.

[0048] Example 1: Isolation and Identification of Strains (1) Strains isolation and screening Rhizosphere soil samples of peanuts that have been covered with plastic film for more than ten years were collected from Shandong Province. 200 g of the samples were stored in a pre-washed and dried brown glass bottle (250 mL) in the dark and transported to the laboratory at 4℃. Following the procedure reported in Yang Ting (2018), 10 g of the sample from the sampling site was weighed and added to 100 mL of MSM medium (PAE concentration of 0 / 5 / 20 mg / L, the same below). The samples were then placed in a shaker (30℃, 180 rpm) and cultured for 7 days. Then, 2% enriched culture solution was inoculated into 100 mL of fresh MSM medium. Gradient pressure acclimatization method was used, and the transfer was carried out every 7 days. The amount of substrate PAE added during the transfer was gradually increased, i.e., 5, 10, 20, 40, 80, and 160 mg / L. This operation was repeated.

[0049] After acclimatization, a small amount of cultured bacterial solution was streaked onto a solid MSM medium plate containing 5 / 20 mg / L PAES using an inoculation needle. The plate was then incubated upside down in a constant temperature (30℃) incubator in the dark. Colony growth was observed periodically (every 24 hours), and single colonies with good growth were selected. The streaking process was repeated until purification. A purified single colony was inoculated into 100 mL of LB liquid medium, with uninoculated LB liquid medium as a control. Once the bacterial concentration reached OD600 = 0.8 (absorbance at 600 nm, without centrifugation), 1 mL of bacterial suspension (resuspended in PBS buffer) was taken and inoculated into 100 mL of MSM liquid medium containing 5 / 20 mg / L PAES, with an uninoculated control under the same conditions. The plates were incubated in a constant temperature shaking incubator (30℃, 180 rpm) for 7 days. Substrate residue was then measured, and degrading bacteria were screened. All experiments were performed in quadruplicate.

[0050] Single colonies showing degradation effect were streaked again onto solid MSM agar plates (5 / 20 mg / L PAES), repeating the above steps until pure colonies with a uniform colony morphology were obtained. Following the purification standards in microbiological experiments, single colonies repeatedly streaked onto plates were picked up with an inoculation needle and transferred to a glass slide in a clean bench for microscopic examination to confirm their uniform bacterial shape. The purified strain was inoculated into LB liquid medium for overnight activation. 1.5 mL of the bacterial solution was centrifuged at 8000 rpm for 3 min, and then transferred to fresh, identical medium at a 1% inoculation rate (v / v). This enrichment and subculturing was performed four times. The degradation effect of the fifth-generation enrichment solution was assessed using GC-MS. The bacterial suspension effectively degrading PAEs was diluted and spread on LB solid medium containing 5 / 20 mg / L PAEs (DBP+DEHP), and incubated at 30°C for 5 days. Single colonies from the plates were picked and transferred to 10 mL of liquid LB test tube medium, then stored and transferred to 100 mL of MSM containing 5 / 20 mg / L PAEs (DBP+DEHP), and incubated at 30°C for 5 days. Finally, PAEs-degrading strains WXX_04 and WXX_05 were obtained.

[0051] Morphological observation: WXX_04 and WXX_05 were spread onto solid LB agar and incubated upside down at 30°C for 3 days. WXX_04 colonies were round, pale yellow, smooth, moist, opaque, and had regular edges; after four days, they almost completely covered the plate. WXX_05 colonies were round, yellowish-brown, smooth, raised, opaque, and had regular edges; after four days, they almost completely covered the plate. See details below. Figure 1 and Figure 2 .

[0052] Sample preparation and observation for electron microscopy: Step 1: Collect bacterial cells: Centrifuge the culture medium at 8000 rpm for 3-5 min, discard the supernatant, and add 2.5% glutaraldehyde fixative; Step 2: Fixation and washing: Fix with 2.5% glutaraldehyde for 4 h or overnight at 4℃, washing three times with 0.2 M PBS buffer (pH 6.8) for 15-20 min each time; Step 3: Dehydration and drying: Treat with 30%, 50%, 70%, 85%, and 95% ethanol sequentially for 15-20 min each time, and finally treat twice with 100% ethanol for 10-15 min each time, followed by freeze-drying; Step 4: Conductivity treatment and measurement: First, firmly attach the sample to conductive adhesive, ensuring good grounding. Then, use an ion sputtering or vapor deposition apparatus to uniformly spray a layer of gold or platinum conductive film several nanometers to tens of nanometers thick onto the sample surface. The purpose is to eliminate the sample charging effect, enhance the secondary electron emission signal, and obtain high-quality images; After processing, perform measurements using an SU8220 microscope. Figure 3 and Figure 4As shown, under a scanning electron microscope, WXX_04 cells are rod-shaped and about 1-2 μm long, while WXX_05 cells are rod-shaped and about 1 μm long.

[0053] (2) Physiological and biochemical identification Physiological and biochemical characteristics of WXX_04 and WXX_05 were identified, and the results are shown in Table 1.

[0054] Table 1. Identification of physiological and biochemical characteristics of WXX_04 and WXX_05

[0055] Note: "+" represents positive; "-" represents negative.

[0056] (3) Molecular identification 16S rDNA molecular identification of strains WXX_04 and WXX_05: Total bacterial DNA was extracted, and the bacterial genome was amplified by PCR using universal primers for bacterial 16S rDNA. After sequencing of the PCR products (performed by Shanghai Sangon Biotech), the 20 strains most closely related at the species level were selected based on the 16S rRNA sequence, and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA 6.0 software. WXX_04 was found to be most similar to Agromyces sp. (…). Figure 5 Based on the morphological characteristics of WXX_04 and the phylogenetic tree comparison analysis of the 16S rRNA gene, WXX_04 was finally identified as belonging to the genus *Agromyces*. WXX_05 is most similar to *Sphingopyxis* sp. Figure 6 Based on the morphological characteristics of WXX_05 and the phylogenetic tree comparison analysis of 16S rRNA genes, WXX_05 was finally identified as Sphingopyxis sp.

[0057] (4) Preservation Therefore, strains WXX_04 (Agromyces sp.) and WXX_05 (Sphingopyxis sp.) were deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 3, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession numbers CGMCC No. 36357 and CGMCC No. 36358.

[0058] Example 2: Genomic study of degrading bacterium WXX_05 WXX_05 cells were inoculated into liquid LB medium and cultured at 30°C and 180 rpm for 24 h on a shaker. The cells were then collected by centrifugation and sent to Shanghai Meiji Biotechnology Co., Ltd. on dry ice for genome sequencing to obtain the genomic information of WXX_05. The specific process is as follows: (1) DNA extraction The WXX_05 strain was inoculated onto LB agar plates and cultured overnight at 37°C. Single colonies were picked using an inoculation needle and inoculated into 200 mL of LB liquid medium. The culture was incubated at 37°C on a shaker until the logarithmic growth phase at 180 rpm, followed by centrifugation at 12000 rpm for 10 min at 4°C. The supernatant was discarded, and the bacterial cells were collected. Simultaneously, the bacterial cells were washed with sterile water to remove any remaining culture medium components. Genomic DNA was extracted according to the bacterial DNA extraction kit (magnetic bead method) (T07-100, China). The purified genomic DNA was quantified, and high-quality DNA was used for subsequent library construction and sequencing.

[0059] (2) Whole genome sequencing Whole-genome sequencing of strain WXX_05 was performed using PacBio Sequel IIe and an Illumina sequencer (NovaSeq 6000). For Illumina library construction, genomic DNA was fragmented into 400 bp fragments, fragment size distribution was identified by agarose gel electrophoresis, and the library was prepared using the NEXTFLEX RapidDNA-Seq Kit. Bioinformatics analysis was performed using data generated by the PacBio Sequel IIe and Illumina sequencing platforms. For Illumina sequencing, the prepared libraries were sequenced at both ends (2 × 150 bp) on an Illumina sequencer (NovaSeq 6000). For PacBio sequencing, the single-stranded library was annealed and attached to the polymerase at the bottom of the ZMW (zero-mode waveguide) of the PacBiosequel IIe sequencer. Sequencing reaction reagents were then added. Each base pairing and synthesis resulted in the emission of corresponding light, which was detected. Each synthesized base was displayed as a pulse peak, which was detected in real time using a high-resolution optical detection system. All analyses were performed on the Shanghai MajorBio Cloud Platform (http: / / cloud.majorbio.com).

[0060] The WXX_05 genome is 4,045,229 bp in length and consists of one chromosome. The chromosome's G+C content is 66.90%. The total gene base length is 3,693,939 bp, with 3,716 predicted protein-coding sequences (CDS) identified. The average length of the coding genes is 994.06 bp, and all genes are estimated to cover 91.32% of the entire genome. This chromosome also encodes 50 tRNAs and 3 rRNAs, including 5S rRNA, 16S rRNA, and 23S rRNA. Detailed information is shown in Table 2.

[0061] Table 2. Statistical Table of Coding Gene Prediction

[0062] (3) Whole genome annotation This sample was assembled using Unicycler v0.4.8 (Wick RR, Judd LM, Gorrie CL, et al. Unicycler: resolving bacterial genome assemblies from short and longsequencing reads[J]. PLoS computational biology, 2017, 13(6), e1005595.) after quality control of Illumina data and HiFi reads. Glimmer or Prodigal v2.6.3 (Hyatt D, Chen GL, LoCascio PF, et al. Prodigal: prokaryotic gene recognition and translation initiation site identification[J]. BMC bioinformatics, 2010, 11(1), 1-11) was used to predict coding sequences (CDS) in the genome. Plasmid genes were predicted using GeneMarkS software (Besemer J, Borodovsky M. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses[J]. Nucleic Acids Research, 2005, 33(Web Server):W451-W454.). tRNAscan-SEv2.0 software (Chan, PP and Lowe, TM (2019) tRNAscan-SE: Searching for tRNA Genes in GenomicSequences. Methods Mol Biol.) was used. tRNA prediction was performed using 1962:1-14., and rRNA prediction was performed using Barrnapv0.9 (https: / / github.com / tseemann / barrnap). Sequence alignment tools such as BLASTP, Diamond, and HMMER were used to functionally annotate the predicted CDS from databases such as GO, COG, and KEGG. Secondary metabolite synthesis gene clusters were predicted using antiSMASHv5.1.2.

[0063] 1) COG annotation Strain WXX_05 has 3079 functional genes assigned to the COG database, accounting for 82.86% of the total genes. For example... Figure 7 It was found that a large number of proteins were involved in lipid transport and metabolism (I) and general function prediction only (R), accounting for 10.17% and 9.42% of the predicted functional genes, respectively. Transcription (K) accounted for 8.28%, and 228 proteins were classified as amino acid transport and metabolism (E), accounting for 7.41%. Relatively fewer proteins were involved in translation, ribosomal structure and biogenesis (J), inorganic ion transport and metabolism (P), and cell wall / membrane / envelope biogenesis, accounting for 7.11%, 6.89%, and 6.46%, respectively.

[0064] 2) GO comments After summarizing and statistically analyzing the functions of the CIP gene in strain WXX_05 from three aspects—cellular component, biological process, and molecular function—a total of 2242 GO classification annotations were obtained. Among them, 1320 annotations were related to cellular components, 1244 to biological processes, and 1845 to molecular functions. The GO annotation information was simplified to obtain GOslim classifications. The top 20 GOslim subclasses with the most annotations under each category were selected for plotting, as shown below. Figure 8 As shown.

[0065] In the annotation of cellular component genes, the largest number of genes are related to the membrane (337); the number of genes related to the plasma membrane is also relatively large (271); the number of genes related to cytosol is 258; and the number of functional genes related to cytoplasm is 209. The remaining annotated genes mainly involve cellular components such as the outer membrane, the large subunit of cytoplasmic ribosomes, extracellular regions, and the periplasmic space surrounded by the outer membrane. In the annotation of biological process genes, the largest number is for proteolysis (60); followed by translation (52); and the regulation of DNA-templated transcription (48). In addition, biological processes also include annotated genes for methylation, peptidoglycan biosynthesis, carbohydrate metabolism, cell wall organization, and cell morphology regulation. Among the molecular functional annotations, the most annotated genes are those related to ATP binding (200), followed by those related to metal ion binding (167), and DNA binding (140). The remaining annotated genes mainly include DNA binding transcription factor activity, hydrolase activity, oxidoreductase activity, and ATP hydrolase activity.

[0066] 3) KEGG annotations The KEGG database annotated 3716 functional genes in the genome of strain WXX_05. Figure 9 The annotation results are categorized into six types: cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organic systems. Metabolism-related genes are the most numerous, totaling 2531 (67.30%), followed by cellular processes (such as cell community-prokaryotes, cell movement, transport, and catabolism) and environmental information processing functions (such as signal transduction and membrane transport), with 267 and 225 genes respectively. Among metabolic function genes, global and overview maps (986), amino acid metabolism (305), and carbohydrate metabolism (277) are the three main secondary categories.

[0067] (4) Bacterial genome circle map Genome circummaps can comprehensively display the characteristics of the genome, such as the distribution of genes on the sense and antisense strands, the COG functional classification of genes, GC content, genome islands, homologous genes, etc. Various information (referring to GC content, GC bias, tRNA / rRNA, COG annotation, base modifications, and restriction modification system-related enzymes) are integrated and plotted on a single genome circummap using Circos software.

[0068] Figure 10 The genome diagram of bacterium WXX_05 is shown. The outermost circle indicates the genome size; the second and third circles represent the CDS on the positive and negative strands, with different colors indicating different COG functional classifications of the CDS; the fourth circle represents rRNA and tRNA; the fifth circle represents GC content; the red areas on the outside indicate that the GC content of the region is higher than the average GC content of the whole genome, and the higher the peak, the greater the difference from the average GC content; the blue areas on the inside indicate that the GC content of the region is lower than the average GC content of the whole genome, and the higher the peak, the greater the difference from the average GC content; the innermost circle represents the GC-Skew value, specifically the GC / G+C algorithm, which can help determine the leading and lagging strands. Generally, the GC skew of the leading strand is >0, and the GC skew of the lagging strand is <0. It can also help determine the origin of replication (minimum cumulative offset) and the endpoint (maximum cumulative offset), which is especially important for circular genomes.

[0069] The genome of strain WXX_05 has a total length of 4,045,229 bp, a GC content of 66.90%, and contains 3,716 CDS. 3,079 CDS were annotated by COG; 2,242 CDS were annotated by GO; and 2,729 CDS were annotated by KEGG.

[0070] In addition, strain WXX_05 is involved in the metabolic degradation pathways of various types of compounds, with a total of 171 genes involved in 19 metabolic pathways of exogenous compounds. It has abundant genes for the degradation of exogenous substances. The specific degradation information is shown in Table 3. Therefore, it can be inferred that there are a large number of unknown functional genes in strain WXX_05 that can be further analyzed and explored.

[0071] Table 3. Statistical Table of Coding Gene Prediction

[0072] Example 3: Degradation effect of strains WXX_04 and WXX_05 on phthalates in inorganic salt medium (MSM) (1) Preparation of bacterial suspension: The purified WXX_04 and WXX_05 were inoculated into 10 mL of LB liquid medium and cultured overnight to the logarithmic phase. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min, washed twice with PBS and resuspended. The OD600 was adjusted to 0.8 to obtain the bacterial suspension.

[0073] (2) Degradation performance of the strain: 2 mL of bacteria were inoculated into 100 mL of MSM culture medium containing 100 mg / L phthalates (100 mg / L dibutyl phthalate and 100 mg / L di(2-ethylhexyl) phthalate), with no inoculation as a control. Each group was replicated four times. The culture was carried out at 30℃ and 180 rpm in a constant temperature shaker for 7 days. The substrate concentration and strain growth (OD600) were measured by GC-MS every 1 day until 7 days.

[0074] (3) Detection of dibutyl phthalate and di(2-ethylhexyl) phthalate by gas chromatography-mass spectrometry (GC-MS): Take 6 mL of the sample to be tested, add 3 mL of n-hexane + acetone (9:1) for extraction twice, combine the organic phases, dehydrate with anhydrous Na2SO4, filter paper and concentrate to 0.5-1 mL by nitrogen blowing, and detect by gas chromatography-mass spectrometry. The chromatographic conditions are: injection port temperature 280℃, injection mode is splitless injection, carrier gas is helium, injection volume is 1 uL, temperature program: initial column temperature 50℃, hold for 1 min, then increase the temperature to 200℃ at a rate of 15℃ / min, hold for 1 min, then increase the temperature to 280℃ at a rate of 8℃ / min and hold for 3 min. The mass spectrometry conditions were as follows: ionization mode was electron impact source (EI), ion source temperature was 230℃, ionization energy was 70eV, chromatograph-mass spectrometer interface temperature was 280℃, quadrupole temperature was 150℃, mass scan range was 35U-450U, and data acquisition mode was SIM.

[0075] GC-MS analysis was performed on samples taken at regular intervals. The degradation results of dibutyl phthalate and di(2-ethylhexyl) phthalate by WXX_04 and WXX_05 are as follows: Figure 11 and Figure 12 As shown, by day 7, WXX_04 and WXX_05 achieved degradation rates of 98.11% and 98.55% for DBP in MSM medium, respectively, and degradation rates of 97.44% and 97.94% for DEHP.

[0076] Example 4: Degradation effect of different ratios of compound bacterial agents on PAEs (1) Preparation of bacterial suspension: as described above.

[0077] (2) Different proportions of bacterial agents were compounded: The prepared bacterial suspensions were mixed with WXX_04 and WXX_05 in volume ratios of 1:1, 2:1, and 1:2 to form three compound bacterial agents, named bacterial agent A, bacterial agent B, and bacterial agent C. The OD values ​​of the bacterial suspensions of WXX_04 and WXX_05 were both OD. 600nm =0.8.

[0078] (3) Degradation performance of the bacterial agent: 2 mL of bacteria were inoculated into 100 mL of MSM culture medium containing 100 mg / L phthalates (100 mg / L dibutyl phthalate and 100 mg / L di(2-ethylhexyl) phthalate), with no inoculation as a control. Each group was replicated four times. The culture was carried out at 30℃ and 180 rpm in a constant temperature shaker for 7 days. The substrate concentration and bacterial growth (OD600) were measured by GC / MS every 1 day until 7 days.

[0079] (4) Detection of dibutyl phthalate and di(2-ethylhexyl) phthalate by gas chromatography-mass spectrometry (GC-MS): as described above.

[0080] GC-MS analysis was performed on samples taken at regular intervals. The degradation results of dibutyl phthalate and di(2-ethylhexyl) phthalate by the three compound bacterial agents are as follows: Figure 13 As shown, in MSM medium containing 100 mg / L PAES (100 mg / L dibutyl phthalate and 100 mg / L di(2-ethylhexyl) phthalate), bacterial agent A achieved a degradation rate of over 87% for both dibutyl phthalate and di(2-ethylhexyl) phthalate after 48 h, which is shorter and higher than that of bacterial agents B and C. Therefore, bacterial agent A showed the best degradation effect on compound PAES contamination. Thus, a compound bacterial agent was prepared using bacterial agent A with a WXX_04:WXX_05 ratio of 1:1.

[0081] Example 5: Degradation effect of WXX_04 and WXX_05 on phthalates in soil-peanut system (1) Soil sample preparation: The PAE-contaminated soil sample was sieved (sieve mesh size 2 mm) to remove large particles and ensure that the soil was uniform. It was then divided into terracotta flower pots (bottom 12cm, top 18cm, height 17cm), with 1.5 kg of soil (dry soil) in each pot. The soil moisture was adjusted to between 50% and 60% to ensure that the soil was not dry.

[0082] (2) Preparation of bacterial suspension: The purified WXX_04 and WXX_05 were inoculated into 10 mL of LB liquid medium and cultured overnight to the logarithmic phase. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min, washed twice with PBS and resuspended. The OD600 was adjusted to 0.8 to obtain the bacterial suspension.

[0083] (3) Inoculation: WXX_04, WXX_05 and the compound inoculant selected in Example 3 were applied to the terracotta flowerpots respectively, according to a ratio of 10 6 -10 8 Soil samples were inoculated with CFU / g bacteria, with uninoculated soil serving as a control. Peanuts were planted in each group, with four replicates per group. After 30 days of cultivation, the samples were harvested, cleaned, freeze-dried, ground, and passed through a 60-mesh sieve. Phthalate concentrations were then determined by GC-MS.

[0084] (4) Detection of phthalates in soil by gas chromatography-mass spectrometry (GC-MS): Take 5 g of soil sample and add 30 mL of hexane + acetone (1:1) mixed solvent. After vortexing, let stand for 12 h. At water temperature of 25℃, extract by ultrasonication at 100 kHz for 30 min. Centrifuge at 3000 r / min for 5 min. Filter into a round-bottom flask. Add 15 mL of hexane + acetone (1:1) mixed solvent and sonicate for 15 min. Repeat twice. Combine the supernatant in a round-bottom flask and concentrate to about 1 mL. Add 3 mL of hexane and mix well. Concentrate to about 1 mL under the same conditions. GC-MS identification.

[0085] (5) Detection of phthalates in plants by gas chromatography-mass spectrometry (GC-MS): Take 5g of plant sample, add 30mL of dichloromethane, vortex and let stand for 12 h, extract by ultrasonication at 100 kHz for 30 min at 25℃, centrifuge at 3000 r / min for 5 min, filter into a round-bottom flask, wash several times with 10 mL of dichloromethane, combine the filtrates into a round-bottom flask, concentrate to about 1 mL, and identify by GC-MS.

[0086] The chromatographic conditions were as follows: injection port temperature 280℃, splitless injection, helium as carrier gas, injection volume 1 μL, and temperature program: initial column temperature 50℃, hold for 1 min, then increase to 200℃ at a rate of 15℃ / min, hold for 1 min, then increase to 280℃ at a rate of 8℃ / min, hold for 3 min. The mass spectrometry conditions were as follows: ionization method electron impact (EI), ion source temperature 230℃, ionization energy 70 eV, chromatograph-mass spectrometer interface temperature 280℃, quadrupole temperature 150℃, mass scan range 35 U–450 U, and data acquisition method SIM.

[0087] Phthalate levels in soil and peanut samples were determined by GC-MS after 30 days. The degradation rate of phthalates in the soil was as follows: Figure 14 As shown. Compared with uninoculated soil samples, inoculation treatment significantly reduced phthalate content, and the degradation rate was significantly improved compared with the control. Specifically, WXX_04, WXX_05, and the compound bacterial agent selected in Example 3 increased the degradation rate of dibutyl phthalate in soil by 44 percentage points (from 10% to 44%), 44 percentage points (from 10% to 44%), and 55 percentage points (from 10% to 55%), respectively, and the degradation rate of di(2-ethylhexyl) phthalate increased by 27 percentage points (from 15% to 42%), 22 percentage points (from 15% to 37%), and 35 percentage points (from 15%), respectively. The percentages for dimethyl phthalate (DMT) increased by 39 percentage points (from 29% to 68%), 40 percentage points (from 29% to 69%), and 49 percentage points (from 29% to 78%), respectively; for diisobutyl phthalate (DIP), DMT increased by 21 percentage points (from 35% to 56%), 10 percentage points (from 35% to 45%), and 29 percentage points (from 35% to 64%), respectively; and for di-n-octyl phthalate (DIP), DMT increased by 26 percentage points (from 27% to 53%), 27 percentage points (from 27% to 54%), and 36 percentage points (from 27% to 63%), respectively.

[0088] The degradation rate of phthalates in the underground and aboveground parts of peanuts is as follows: Figure 15 and Figure 16As shown. Compared with untreated peanut samples, inoculation treatment significantly reduced the phthalate content in peanuts. The content of various phthalates in peanuts was significantly reduced compared with the control. Specifically, compared with the control, the content of dibutyl phthalate in the underground parts of peanuts treated with WXX_04, WXX_05, and the compound inoculant selected in Example 3 decreased by 21.28%, 22.37%, and 27.45%, respectively; the content of di(2-ethylhexyl) phthalate decreased by 44.04%, 37.64%, and 55.71%, respectively; the content of dimethyl phthalate decreased by 57.09%, 58.02%, and 69.00%, respectively; and the content of diisobutyl phthalate decreased by [missing information]. The levels of di-n-octyl phthalate decreased by 36.04%, 36.67%, and 42.77%, respectively, while the levels of di-n-octyl phthalate decreased by 37.19%, 34.74%, and 51.19%, respectively. In peanut aboveground parts, the levels of dibutyl phthalate decreased by 32.55%, 34.68%, and 40.55%, respectively; di(2-ethylhexyl) phthalate decreased by 26.01%, 31.79%, and 41.92%, respectively; dimethyl phthalate decreased by 34.66%, 35.97%, and 46.00%, respectively; diisobutyl phthalate was not detected; and di-n-octyl phthalate decreased by 20.87%, 21.16%, and 35.02%, respectively.

[0089] It is evident that the preferred compound microbial agent A can significantly improve the degradation rate of phthalates in the soil and reduce the phthalate content in peanuts, with a significantly better effect than a single microbial agent.

[0090] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of sphingosine-box bacteria that degrades phthalates ( Sphingopyxis sp WXX-05, characterized in that, The preservation number of the sphingosine box bacterium WXX-05 is: CGMCC No.36358.

2. A microbial agent, characterized in that, It includes the sphingosine box bacteria WXX-05 as described in claim 1.

3. A bacterial assemblage, characterized in that, Including Sphingosine Box Bacteria ( Sphingopyxis sp WXX-05 and a new species of the genus *Pseudomonas* ( Agromyces sp .) WXX-04; The preservation number of the sphingosine box bacterium WXX-05 is: CGMCC No. 36358; The preservation number of the new species of the genus *Pseudomonas* WXX-04 is: CGMCC No. 36357.

4. A compound microbial agent, characterized in that, It comprises the bacterial combination described in claim 3.

5. The compound microbial agent according to claim 4, characterized in that, The compound microbial agent comprises a bacterial suspension of *Sphingosporium sphingolipidae* WXX-05 and a bacterial suspension of a new species of *Pseudomonas* WXX-04; the volume ratio of the bacterial suspension of *Sphingosporium sphingolipidae* WXX-05 to that of *Pseudomonas* WXX-04 is (1~2):(1~2); the OD values ​​of both the bacterial suspensions of *Sphingosporium sphingolipidae* WXX-05 and *Pseudomonas* WXX-04 are OD. 600nm =0.

8.

6. The compound microbial agent according to claim 5, characterized in that, The volume ratio of the bacterial suspension of *Sphingosine Boxer* WXX-05 to that of *Pseudomonas aeruginosa* WXX-04 is 1:

1.

7. The application of the Sphingosine Box Bacterium WXX-05 of claim 1, the bacterial agent of claim 2, the bacterial combination of claim 3, or the compound bacterial agent of any one of claims 4 to 6 in the degradation of phthalates or the remediation of phthalate-contaminated media.

8. The application according to claim 7, characterized in that, The phthalate esters include one or more of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di(2-ethylhexyl) phthalate, dibutyl phthalate, and di-n-octyl phthalate.

9. A method for degrading phthalates, characterized in that, Includes the following steps: The sphingosine box bacteria WXX-05 of claim 1, the bacterial agent of claim 2, the bacterial combination of claim 3, or the compound bacterial agent of any one of claims 4 to 6 are inoculated or applied to the sample containing phthalates to be degraded for degradation.

10. The method according to claim 8, characterized in that, The effective viable bacterial concentration inoculated into the sample to be degraded was 10. 6 ~10 8 CFU / g.

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