A functional indigenous bacterial community J-17 and its products that simultaneously degrades four types of PAEs

Abstract

This invention provides a functional indigenous bacterial community J-17 and its products that simultaneously degrade four types of PAEs, belonging to the field of microbial technology. The functional indigenous bacterial community J-17 includes Acinetobacter (… Acinetobacter sp. W2-4 and Delft ( Delftia sp. W141, Acinetobacter ( Acinetobacter sp. W2-4 was deposited on December 17, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20252941, belonging to Delft (…). Delftia sp. W141 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252940. The functional indigenous bacterial group J-17 can be used to simultaneously degrade four types of PAEs, and its combined degradation efficiency is significantly better than any single strain, demonstrating good control efficacy against mixed PAEs.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a functional indigenous bacterial group J-17 that simultaneously degrades four types of PAEs and its products. Background Technology

[0002] Phthalate esters (PAEs) readily migrate from products and are released into the environment, leading to their widespread detection in the atmosphere, water bodies, soil, and sediments. They have become one of the most concerning environmental organic pollutants. Multiple studies have confirmed that some PAE compounds possess endocrine-disrupting toxicity, carcinogenicity, and reproductive toxicity, posing a potential threat to ecosystem safety and human health. Therefore, the development of efficient and green PAE pollution remediation technologies is urgently needed.

[0003] Currently, the main methods for removing PAEs from the environment include physical, chemical, and biological methods. Physical and chemical methods (such as adsorption and advanced oxidation) often have limitations such as high cost, potential for secondary pollution, or difficulty in completely mineralizing pollutants. In contrast, bioremediation technologies based on microbial degradation are considered the most promising direction for PAE pollution control due to their environmental friendliness, low cost, and potential to completely convert pollutants into harmless substances (such as CO2 and H2O).

[0004] Although several single microbial strains capable of degrading PAEs (such as *Pseudomonas* and *Gordonia*) have been isolated from different environments and their degradation effects have been confirmed under laboratory conditions, these existing technologies still face significant bottlenecks in practical applications. Firstly, most studies focus on the degradation of single high-concentration PAEs, which is severely out of sync with real-world scenarios of low-to-medium concentration complex pollution caused by the long-term coexistence of multiple PAEs, making it difficult to guarantee their degradation efficiency under such conditions. Secondly, the degradation spectrum of single strains is usually narrow, often exhibiting high degradation efficiency only for one or a few PAEs (such as short-chain DMP and DEP), while degrading other structurally similar homologues (such as DBP and BBP) slowly or incompletely, making them inadequate for handling complex mixed pollutant systems in the environment. Furthermore, while some reported complex microbial communities have attempted to broaden their degradation spectrum through strain combinations, their species composition remains relatively singular, their functional synergy is insufficient, or their actual remediation potential is questionable when assessed under non-environmentally relevant high-concentration conditions.

[0005] Based on this, the present invention provides a bacterial community to simultaneously degrade four PAEs (including dimethyl phthalate (hereinafter referred to as DMP), diethyl phthalate (hereinafter referred to as DEP), dibutyl phthalate (hereinafter referred to as DBP), and butyl benzyl phthalate (hereinafter referred to as BBP)). Summary of the Invention

[0006] This invention proposes a functional indigenous bacterial community J-17 and its products that can simultaneously degrade four types of PAEs, and has good treatment efficacy for mixed PAEs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a functional indigenous bacterial community J-17 that simultaneously degrades four types of PAEs, including: Acinetobacter ( Acinetobacter sp. W2-4 and Delft ( Delftia sp. W141, The Acinetobacter ( Acinetobacter sp. W2-4 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252941; the Delft bacterium ( Delftia sp. W141 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252940.

[0008] In one application scenario of the first aspect, the four PAEs are dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate.

[0009] In a second aspect, the present invention provides an article comprising the functional indigenous bacterial group J-17 provided in the first aspect.

[0010] In one implementation of the second aspect, the product is a bacterial suspension or an immobilized bacterial agent.

[0011] Thirdly, the present invention provides a method for simultaneously degrading four types of PAEs, wherein the functional indigenous microbial community J-17 provided in the first aspect or the product provided in the second aspect is inoculated into soil or water contaminated with PAEs to degrade the four types of PAEs.

[0012] In one method scenario of the third aspect, the inoculation amount of the functional indigenous microbial community J-17 or the product is 5-9% of the mass of the soil or water; during the degradation process, the pH of the soil or water is adjusted to 6-10, the degradation temperature is 20-40℃, and the degradation time is 7-9 days.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a functional indigenous bacterial community J-17 composed of two strains with no antagonistic effect between them. This functional indigenous bacterial community J-17 can utilize dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate as carbon and energy sources for growth and reproduction. Under pure culture conditions, this bacterium can almost completely degrade four PAEs (containing 20 mg / L DMP, DEP, DBP, and BBP respectively) in an inorganic salt culture medium with a total concentration of 80 mg / L in 7 days. The degradation rates of DEP, DBP, and BBP all exceed 95%. The application of this invention has great potential in the biological treatment of environmental pollutants. Attached Figure Description

[0014] Figure 1 The relative abundance of functional indigenous bacterial community J-17 at the bacterial phylum level; Figure 2 The relative abundance of functional indigenous bacterial community J-17 at the bacterial genus level; Figure 3 The Acinetobacter bacillus provided in the embodiments of this application ( Acinetobacter sp. A schematic diagram of the growth morphology of W2-4 cultured on LB solid medium; Figure 4 The Delft bacterium provided in the embodiments of this application ( Delftia sp. A schematic diagram of the growth morphology of W141 cultured on LB solid medium; Figure 5 The Acinetobacter bacillus provided in the embodiments of this application ( Acinetobacter sp. Scanning electron microscope image of W2-4; Figure 6 The Delft bacterium provided in the embodiments of this application ( Delftia sp. Scanning electron microscope image of W141; Figure 7 The Acinetobacter bacillus provided in the embodiments of this application ( Acinetobacter sp. A phylogenetic tree diagram of the 16S rRNA gene of W2-4; Figure 8 The Delft bacterium provided in the embodiments of this application ( Delftia sp. A phylogenetic tree diagram of the 16S rRNA of W141; Figure 9 Acinetobacter provided for the present invention ( Acinetobacter sp. W2-4 is a statistical chart showing the degradation effect of four PAEs during simultaneous degradation. Figure 10 Delftella provided for this invention ( Delftia sp. )Statistical chart of the degradation effect of W141 on the simultaneous degradation of 4 PAEs; Figure 11A statistical chart showing the degradation effect of the bacterial community J-17 on four PAEs simultaneously as the number of culture days changed, provided by the present invention. Figure 12 A statistical chart showing the degradation effect of the bacterial community J-17 provided by this invention on the simultaneous degradation of four PAEs after 7 days of culture, depending on the concentration of a single PAE. Figure 13 A statistical chart showing the degradation effect of the bacterial community J-17 provided by this invention on four PAEs simultaneously after 7 days of culture, depending on the change in NaCl concentration. Figure 14 A statistical chart showing the degradation effect of the bacterial community J-17 provided by this invention on four PAEs simultaneously after 7 days of culture, depending on pH changes. Figure 15 The graph shows the degradation effect of the bacterial community J-17 provided by this invention on four PAEs simultaneously after 7 days of culture, depending on temperature changes. Detailed Implementation

[0015] In the embodiments of this application, the terms "exemplary" or "for example" are used to indicate that something is an example, illustration, or description. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design. Specifically, the use of the terms "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0016] Example 1: This example describes a functional indigenous bacterial group J-17, including: Acinetobacter ( Acinetobacter sp. W2-4 and Delft ( Delftia sp. W141, among which Acinetobacter (J-17) of the functional indigenous flora was detected. Acinetobacter sp. The relative abundance of W2-4 was 31.97%, and that of Delft ( Delftia sp. The relative abundance of W141 was 13.33%. The Acinetobacter ( Acinetobacter sp. W2-4 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252941; the Delft bacterium ( Delftia sp. W141 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252940; The four PAEs are dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate.

[0017] Specifically, the aforementioned Acinetobacter ( Acinetobacter sp. W2-4 and Delft (Delftia sp. W141 is a novel strain provided by this invention; the above-mentioned Acinetobacter ( Acinetobacter sp. W2-4 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC NO: M20252941; the above-mentioned Delftobacterium ( Delftia sp. W141 was deposited on December 17, 2025, at the China Center for Type Culture Collection (CCTCCNO: M20252940), belonging to Acinetobacter bacillus (…). Acinetobacter sp. W2-4 and Delft ( Delftia sp. The depositary address of W141 is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0018] The above-mentioned Acinetobacter ( Acinetobacter sp. The 16S rRNA gene sequence of W2-4 is shown in SEQ ID NO.1.

[0019] SEQ ID NO.1:

[0020] The above Acinetobacter ( Acinetobacter The 16S rRNA sequence of sp. W2-4 was compared with the sequence similarity of existing Acinetobacter species that degrade PAEs. The results showed that the sequence of this strain is similar to that of known Acinetobacter species that degrade PAEs. Acinetobacter sp. strain The sequence similarity of A3 is only 93.13%, which is lower than 98.65%, indicating that the *Acinetobacter* strain in this application is not suitable for identification. Acinetobacter sp.) W2-4 is a potential new species.

[0021] The above Delft bacteria ( Delftia The 16S rRNA gene sequence of sp.) W141 is shown in SEQ ID NO.2.

[0022] SEQ ID NO.2:

[0023] The above Delft bacteria ( Delftia The 16S rRNA sequence of sp. W141 was compared with that of existing Arthroblasts that degrade PAEs, revealing that this strain's sequence is similar to that of Delftella, a known PAE-degrading bacterium. Delftia The sequence similarity of sp. L3 was only 97.12%, lower than 98.65%, which indicates that the strain in this application is Delft. Delftia sp.) W141 is a potential new species.

[0024] The culture method for functional indigenous bacterial group J-17 is given below.

[0025] 1. Preparation of culture medium Inorganic salt medium (MSM): (NH4)2SO4: 1.5 g / L; KH2PO4: 0.5 g / L; K2HPO4·3H2O: 1.91 g / L; NaCl: 0.5 g / L; MgSO4·7H2O: 0.2 g / L. Add ultrapure water to 1L based on this ratio, adjust the final pH to 7.0 with 1mol / L HCl or NaOH, and sterilize at 121℃ for 20 minutes to obtain MSM liquid medium. MSM solid medium is prepared by adding 1.5% (w / v) agar powder to the MSM liquid medium, and after sterilization and cooling to 45℃, pour plates under aseptic conditions.

[0026] LB medium: Yeast extract: 5.0 g; Tryptone: 10.0 g; Sodium chloride (NaCl): 10.0 g. Add ultrapure water to 1 L based on this ratio, adjust the final pH to 7.0 with 1 mol / L HCl or NaOH, and sterilize at 121℃ for 20 minutes to obtain LB liquid medium. The preparation of LB solid medium is to add 1.5% (w / v) agar powder to the LB liquid medium, and after sterilization and cooling to 45℃, pour plates under aseptic conditions.

[0027] 2. Enrichment culture Weigh 5 g of yellow soil sample contaminated with PAEs and add it to a 250 mL Erlenmeyer flask. Add 100 mL of ultrapure water and incubate in a shaker at 30 ℃ and 150 rpm in the dark for 8 h. After incubation, remove the sample and let it stand for 2 h to obtain the supernatant enriched with the initial indigenous microorganisms. The supernatant was transferred at a volume ratio of 5:95 to an inorganic salt liquid medium containing four PAEs, with a total concentration of 20 mg / L (i.e., the four PAEs included 5 mg / L DMP, 5 mg / L DEP, 5 mg / L DBP, and 5 mg / L BBP). After incubation at 30°C and 150 rpm in the dark for 5 days, the bacterial culture was obtained. 5 μL of the culture was taken directly each time. The bacterial culture was transferred to a new MSM liquid medium containing four types of PAEs, and the process was repeated four times for enrichment culture. The total concentration of the four PAEs in the MSM liquid medium after the first transfer was 40 mg / L; the total concentration of the four PAEs in the MSM liquid medium after the second transfer was 80 mg / L; the total concentration of the four PAEs in the MSM liquid medium after the third transfer was 160 mg / L; and the total concentration of the four PAEs in the MSM liquid medium after the fourth transfer was 320 mg / L. The proportion of each individual PAE remained unchanged. After enrichment, the bacterial culture of functional indigenous flora J-17 was obtained and preserved using the glycerol preservation method.

[0028] 3. Relative abundance test of functional indigenous bacterial group J-17 in bacterial phyla.

[0029] like Figure 1 As shown, the functional indigenous bacterial community J-17 is mainly composed of Pseudomonadota (Proteobacteria) at the phylum level, with a relative abundance of 99.71%. like Figure 2 As shown, at the genus level within the Proteobacteria phylum, the top three dominant genera of the functional indigenous bacterial group J-17 are Acinetobacter, Commonas, and Kosakonia, with relative abundances of 31.97%, 28.22%, and 22.78%, respectively. Among them, Acinetobacter (… Acinetobacter sp. The relative abundance of W2-4 was 31.97%, and that of Delft ( Delftia sp. The relative abundance of W141 was 13.33%; Acinetobacter and Commonas are both confirmed to be highly efficient PAE degraders in the literature, and their dominance indicates that the J-17 bacterial group has a strong PAE degradation potential.

[0030] The above-mentioned Acinetobacter ( ) are given below Acinetobacter sp. W2-4 and Delft ( Delftia sp. The process of separating and identifying W141.

[0031] 1. Dilute the bacterial suspension of the above-mentioned functional indigenous bacterial group J-17 by 10. 4The culture medium was then plated onto MSM solid medium containing four types of PAEs (the total concentration of the four PAEs was 20 mg / L, i.e., the four PAEs included 5 mg / L DMP, 5 mg / L DEP, 5 mg / L DBP, and 5 mg / L BBP) and incubated upside down at 30°C for 3 days. After single colonies grew on the MSM solid medium, single colonies were picked and streaked four times for purification, resulting in two bacterial strains, numbered W2-4 and W141, respectively. Strains W2-4 and W141 were then inoculated into their respective corresponding LB solid medium and incubated upside down at 30°C for 3 days, followed by streaking for another 2 days. The colony morphology was then observed.

[0032] like Figure 2 As shown, the colonies of strain W2-4 are pale yellow, round, opaque, with a raised, moist, sticky, and smooth surface.

[0033] like Figure 3 As shown, the colonies of strain W141 are milky white, round and opaque, with irregular edges, and are moist, sticky and smooth.

[0034] 2. Identification by transmission electron microscopy The purified strain W2-4 was inoculated into LB liquid medium and activated at 30 °C and 150 rpm for 24 h to obtain the activated solution. 1 mL of the activated solution was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the cells were washed three times with MSM liquid medium. 1 mL of 2.5% (v / v) glutaraldehyde was added to the harvested bacterial precipitate and mixed thoroughly. The mixture was incubated overnight (16 h) at 4 °C, and the glutaraldehyde was discarded. The bacterial precipitate was then washed three times with 0.1 M, pH 7.0 PBS for 15 min each time. 1 mL of 1% (v / v) osmium tetroxide solution was added to the bacterial precipitate and mixed thoroughly. The mixture was then fixed at 4 °C in the dark for 2 h. The osmium tetroxide waste liquid was removed, and the bacterial precipitate was washed three times with 0.1 M, pH 7.0 PBS for 15 min each time. The sample was dehydrated using ethanol solutions of varying concentrations (30%, 50%, 70%, 80%, 90%, and 95%) for 15 minutes each, followed by 20 minutes of dehydration with a 100% ethanol solution. Finally, the sample was dehydrated with pure acetone solution for 20 minutes to obtain a dehydrated sample. The dehydration process specifically involves immersing the sample completely in an ethanol solution or a pure acetone solution. After each dehydration process, the sample is centrifuged at 8000 rpm for 5 minutes and the supernatant is removed before the sample is subjected to the next dehydration process. Then, the copper mesh supporting the film, treated with glow discharge, was placed on a screen holder. 10 μL of dehydrated sample was pipetted and added to the center of the mesh. After standing for 1 minute, excess liquid was blotted from the edges with filter paper. Subsequently, 10 μL of phosphotungstic acid negative staining solution was immediately added and stained for 1 minute, then thoroughly blotted dry with filter paper. After the copper mesh dried at room temperature, it was placed in an HT7800 transmission electron microscope for observation and image acquisition at an accelerating voltage of 80 kV.

[0035] The transmission electron microscopy (TEM) identification method for strain W141 is the same as that for strain W2-4 described above, and will not be repeated here.

[0036] like Figure 5 As shown, strain W2-4, as observed by transmission electron microscopy, is elliptical in shape and lacks flagella. like Figure 6 As shown, strain W141, as observed by transmission electron microscopy, is elliptical in shape and lacks flagella.

[0037] 3. Molecular identification of the strain's 16S rRNA Total DNA was extracted from the bacteria, and the bacterial genome was amplified by PCR using universal primers for bacterial 16S rRNA. The PCR products were sequenced (by Shanghai Sangon Biotech), and the sequencing results were compared for homology with 16S rRNA sequences reported in GenBank. Specifically, the 16S rRNA sequences of strains W2-4 and W141 were compared with other registered bacterial strains' 16S rRNA sequences using the BLAST program on the NCBI website.

[0038] Phylogenetic analysis was performed on relevant bacterial species, and the results for strain W2-4 are as follows: Figure 7 As shown, the 16S rRNA gene sequence of strain W2-4 isolated and purified in this invention is similar to that of Acinetobacter (…). Acinetobacter sp. (Genbank accession number FJ848381.2) showed the highest homology, with a homology rate of 100%; therefore, strain W2-4 was identified as Acinetobacter (…). Acinetobacter sp. ), named Acinetobacter ( Acinetobacter sp. W2-4.

[0039] Results for strain W141 are as follows Figure 8 As shown, the 16S rRNA gene sequence of strain W141 is similar to that of Delft (…). Delftia sp. The strain W141 showed the highest homology (Genbank accession number PV259264.1), with a homology rate of 100%. Therefore, strain W141 was identified as Delft (…). Delftia sp. ), named Delft ( Delftia sp. W141.

[0040] The above-mentioned Acinetobacter ( ) are given below Acinetobacter sp. W2-4 and Delft ( Delftia sp. The verification process of W141's ability to degrade PAEs.

[0041] 1) Preparation of bacterial suspension Acinetobacter ( Acinetobacter sp. W2-4 and Delft ( Delftia sp. Single colonies of W141 were inoculated into 100 mL of their respective corresponding LB liquid medium and cultured at 30 °C and 150 rpm for 24 h. After centrifugation at 8000 rpm for 5 min, the bacteria were washed twice with MSM liquid medium before the OD values ​​were calculated. 600nm The value was adjusted to 1.0, and Acinetobacter bacilli were obtained respectively. Acinetobacter sp. W2-4 bacterial suspension and Delft bacteria ( Delftia sp. W141 bacterial suspension, store temporarily at 4 ℃ for later use.

[0042] 2) Acinetobacter ( Acinetobacter sp. W2-4 and Delft ( Delftia sp. Degradation performance determination of W141 1 mL of Acinetobacter bacillus was inoculated into 19 mL of MSM liquid culture medium containing a total concentration of 80 mg / L of four PAEs (i.e., containing 20 mg / L DMP, 20 mg / L DEP, 20 mg / L DBP, and 20 mg / L BBP). Acinetobacter sp. W2-4 bacterial suspension or Delft bacteria ( Delftia sp. W141 bacterial suspension was prepared in triplicate for each group, with no inoculation as a control. The pH was adjusted to 7.0. The suspension was incubated at 30°C and 150 rpm for 48 h in a constant-temperature shaker. On days 1, 3, 5, and 7, the conical flasks were removed, and 40 mL of chromatographically pure methanol was added. The flasks were then sonicated in a water bath for 1 h. After sonication, the mixture was vortexed, and the supernatant was filtered through a 0.22 μm organic phase filter membrane and transferred to a 2 mL amber liquid chromatography vial. The residual concentrations of the four PAEs were determined using high-performance liquid chromatography (HPLC).

[0043] HPLC detection conditions: An LC-20AT HPLC system (equipped with an SPD-2A UV detector) was used. The detection time was 40 min, and the injection volume was 20 μL. The separation system used acetonitrile-water as the mobile phase with an initial flow rate of 1.0 mL / min, employing gradient elution to separate PAEs. The gradient elution was as follows: at the beginning of elution, the mobile phase was 40% water and 60% acetonitrile; at 6 min, the water was reduced to 25% and the acetonitrile was increased to 75%; at 20 min, the water was reduced to 5% and the acetonitrile was increased to 95%; at 32 min, the water and acetonitrile were maintained at 5% and 95% respectively; at 35 min, the water was increased to 40% and the acetonitrile was reduced to 60%, maintained for 5 min, and the gradient elution ended. The chromatographic column was a Φ4.6×250 mm Inertsil ODS-P HPLC column, and the column temperature was 40℃. The detection system used a UV detector with dual-wavelength detection mode at 225 nm and 290 nm.

[0044] Acinetobacter ( Acinetobacter sp. The degradation effect of W2-4 on four types of PAEs is as follows: Figure 9 As shown, Acinetobacter ( Acinetobacter sp. W2-4 showed significant degradation effects on four types of PAEs under 3 days of shaking culture. Specifically, Acinetobacter (… Acinetobacter sp. W2-4 showed a degradation rate of over 96% for DMP, DEP, and BBP on day 3, and an 87% degradation rate for DBP on day 5. This indicates that Acinetobacter (…) Acinetobacter sp. W2-4 has a highly efficient ability to simultaneously degrade four types of PAEs.

[0045] Delft ( Delftia sp. The degradation effect of W141 on four types of PAEs is as follows: Figure 10 As shown, this bacterium exhibits significant degradation effects on four types of PAEs under 3 days of shaking culture. Specifically, Delftibacterium W141 achieved a degradation rate of over 99% for DMP and DEP on day 3, and a degradation rate of over 93% for DBP and DEP on day 5.

[0046] Experimental Example 1: Antagonistic experiment of functional indigenous bacterial group J-17 provided in Example 1 of this application.

[0047] Two bacterial strains, Acinetobacter W2-4 and Delftella W141, were used for an antagonistic experiment. The filter paper method was employed. One bacterial culture, cultured to the logarithmic growth phase, was spread onto a plate. A filter paper was then used to pick up the other bacterial culture and placed on the same plate. The plates were incubated at 30°C for 2 days, and the presence of a transparent antagonistic zone around the filter paper was observed. The presence of an antagonistic zone indicated an antagonistic reaction. The results are shown in Table 1 ("+" indicates an antagonistic reaction, and "-" indicates no antagonistic reaction). Table 1 shows that neither strain exhibited an antagonistic reaction.

[0048] Table 1. Antagonistic response results of functional indigenous bacterial group J-17 in Example 1

[0049] As shown in Table 1, there was no antagonistic reaction between Acinetobacter W2-4 and Delftus W141.

[0050] Example 2: This example describes a product containing the functional indigenous bacterial group J-17 described in Example 1. Specifically, the product is a bacterial suspension, and the preparation method includes the following steps: The functional indigenous bacterial group J-17 was inoculated into LB liquid medium for activation culture. The activation culture conditions were: temperature 30℃, rotation speed 150 rpm, initial pH 7.0, and culture in complete darkness for 12 hours. Afterwards, the bacterial cells were collected by centrifugation at 5000 rpm for 5 minutes at 4℃, and the supernatant was discarded. The cells were then washed with inorganic salt liquid medium, and centrifuged again under the same conditions, discarding the supernatant. This washing process was repeated twice. The bacterial OD was then... 600 Adjust the value to 1.0 to obtain the bacterial suspension.

[0051] In some other specific embodiments, the centrifugation speed may be 7500 or 8000 r / min, and the centrifugation time may be 5 or 10 min. The embodiments of this application do not limit the above centrifugation parameters.

[0052] In some other embodiments, the product is an immobilized microbial agent. The preparation method of the immobilized microbial agent is as follows: after pyrolyzing corn stalks at 800℃ for 12h, the above-mentioned microbial suspension is added to the pyrolyzed corn stalks at a microbial material ratio of 20mL:1g, and then solidified in a shaking incubator for 24h. After that, it is centrifuged at 4℃ and 6000r / min for 10min using a disc centrifuge, and then dried at -40℃ for 10h to obtain the immobilized microbial agent.

[0053] In other specific embodiments, the above-mentioned pyrolysis temperature can be 1000℃, the pyrolysis time can be 10h, the inoculum-to-substrate ratio can be 22mL:1g, or it can be 18mL:1g. The embodiments of this application do not limit the values ​​of the above-mentioned related parameters.

[0054] Example 3: This example describes a method for simultaneously degrading four types of PAEs. The functional indigenous microbial community J-17 provided in Example 1 or the product provided in Example 2 is inoculated into soil or water contaminated with PAEs to degrade the four types of PAEs.

[0055] Specifically, the bacterial community J-17 was inoculated into soil contaminated with PAEs to degrade the pollutants. The soil pH was adjusted to 7, the temperature was controlled at 35°C, and the degradation time was 7 days. The inoculation amount of bacterial community J-17 was 5% of the soil mass to achieve the degradation of DMP, DEP, DBP, and BBP.

[0056] Furthermore, depending on the type of product, the aforementioned bacterial suspension or immobilized bacterial agent can also be inoculated into contaminated soil in the same manner to achieve the degradation of the four PAEs. The inoculation amount can be determined according to the actual situation, for example, by adding 5% or 9% of the soil mass, or other proportions can be used within a reasonable range, which are not limited in this embodiment.

[0057] The above describes the use of microbial community J-17 for the degradation of PAEs in soil. Understandably, it can also be used to degrade PAEs in water bodies.

[0058] Furthermore, the aforementioned water body can be groundwater contaminated with PAEs, or surface water or industrial wastewater contaminated with PAEs; the aforementioned microbial community J-17 can also perform ex-situ remediation of contaminated soil. In ex-situ remediation projects, contaminated soil can be transferred to a controllable treatment facility, and the remediation temperature range can be 10~20℃. This application embodiment does not further limit the type of the aforementioned medium and the treatment method.

[0059] For example, the above bacterial suspension is inoculated into groundwater contaminated with PAEs to achieve the degradation of DMP, DEP, DBP and BBP.

[0060] Experiment 3: The degradation ability of the functional indigenous bacterial community J-17 provided in Example 1 of this application to simultaneously degrade 6 PAEs under different degradation conditions was verified, including a total of 6 experiments.

[0061] The bacterial suspension prepared from the functional indigenous bacterial group J-17 in Example 2 was used as the bacterial suspension for the experiment and temporarily stored at 4°C for later use.

[0062] Experiment 1: The above bacterial suspension was inoculated into MSM liquid medium with a total concentration of 80 mg / L for 7 days at 30℃, an inoculum size of 5%, and pH=7.0 (each of the four PAEs had the same proportion). After incubation, the samples were processed using the whole-bottle extraction method, and the residual concentrations of the four PAEs in the brown liquid chromatographs were detected by high performance liquid chromatography. The degradation rate was calculated and compared (the incubation days after inoculation, sample processing, and detection steps were the same in subsequent experiments and will not be repeated).

[0063] Experiment 2: Five MSM liquid culture media with total PAE concentrations of 40, 80, 160, 240, and 320 mg / L were prepared. The above-mentioned functional indigenous bacterial group J-17 was inoculated into the five MSM liquid culture media at 30℃, inoculum size of 5%, and pH=7.0 and cultured for 7 days. After the culture, the samples were processed by whole-bottle extraction method, and the residual concentrations of the four PAEs in the brown liquid chromatographs were detected by high performance liquid chromatography. The degradation rate was calculated and compared.

[0064] Experiment 3: MSM liquid culture media with a total PAE concentration of 80 mg / L and two different single PAE concentration ratios of DMP:DEP:DBP:BBP=11:13:9:7 and DMP:DEP:DBP:BBP=9:11:7:13 were prepared. The above-mentioned functional indigenous bacterial group J-17 was inoculated into the above two MSM liquid culture media at 30℃, inoculum size of 5%, and pH=7.0 and cultured for 7 days, and the results were compared and analyzed.

[0065] Experiment 4: Using NaCl-free MSM liquid medium as a baseline, 0, 5, 10, 15, and 20 g of NaCl were added to the adjusted volume of MSM liquid medium, respectively. After adjusting the pH, the medium was sterilized to obtain MSM liquid medium with salt concentrations of 0, 5, 10, 15, and 20 g / L. The above-mentioned functional indigenous bacterial group J-17 was inoculated into MSM liquid medium with different salt concentrations at 30℃, inoculum size of 5%, pH=7.0, and a total concentration of 80 mg / L of the four PAEs, and cultured for 7 days. The results were then compared and analyzed.

[0066] Experiment 5: MSM liquid culture medium was prepared at temperatures of 20, 25, 30, 35, and 40℃. The above-mentioned functional indigenous bacterial group J-17 was inoculated into the MSM culture medium at different temperatures under the conditions of pH=7.0, inoculum amount of 5%, and total concentration of 4 PAEs of 80mg / L, and cultured for 7 days and compared and analyzed.

[0067] Experiment 6: MSM liquid culture media with pH values ​​of 3.0, 5.0, 7.0, 9.0, and 11.0 were prepared. The above-mentioned functional indigenous bacterial group J-17 was inoculated into the above-mentioned MSM liquid culture media with different pH values ​​at 30℃, with an inoculum size of 5% and a total concentration of 80 mg / L of the four PAEs, and cultured for 7 days. The results were then compared and analyzed.

[0068] The results of Experiment 1 are as follows Figure 11 As shown, the functional indigenous bacterial community J-17 exhibited significant degradation effects on four PAEs under 7 days of shaking culture. Specifically, J-17 achieved a degradation rate of over 80% for BBP by day 3 and over 95% by day 5. The degradation rates for DMP, DEP, and DBP were relatively slower; the degradation rate of DMP reached 60% by day 7, while DEP and DBP were almost completely degraded by day 7, with degradation rates exceeding 95% for both. In summary, after 7 days of culture, the degradation rates of DEP, DBP, and BBP all exceeded 95%, and the degradation rate of DMP also exceeded 60%. This demonstrates that the functional indigenous bacterial community J-17 possesses a highly efficient simultaneous degradation capability for the four PAEs.

[0069] The results of Experiment 2 are as follows Figure 12 As shown, when the initial total concentrations of the four PAEs were 40, 80, 160, 240, and 320 mg / L (i.e., equal concentration ratios of individual PAEs with initial concentrations of 10, 20, 40, 60, and 80 mg / L), after 7 days of culture, the total degradation rates of the functional indigenous bacterial community J-17 for the simultaneous degradation of the four PAEs reached 91.5%, 91.6%, 78.6%, 51.3%, and 46.2%, respectively. Comparison shows that the substrate concentration of 80 mg / L (i.e., the initial concentration of individual PAEs was 20 mg / L) resulted in the optimal degradation effect of the functional indigenous bacterial community J-17 for the simultaneous degradation of the four PAEs.

[0070] The results of Experiment 3 are as follows: In a system with a total concentration of 80 mg / L for the four PAEs, when DMP:DEP:DBP:BBP = 11:13:9:7, the total degradation rate of the functional indigenous bacterial community J-17 for the four PAEs reached 88% on day 7. When DMP:DEP:DBP:BBP = 9:11:7:13, the total degradation rate of the functional indigenous bacterial community J-17 for the four PAEs also reached 90% on day 7. Therefore, it can be seen that when DMP:DEP:DBP:BBP = 1:1:1:1, the bacterial community J-17 provided by this invention has the best degradation effect on the simultaneous degradation of the four PAEs.

[0071] The results of Experiment 4 are as follows Figure 13 As shown, in a system with a total concentration of 80 mg / L for the four PAEs, after 7 days of cultivation, when the salt concentrations were 0, 5, 10, 15, and 20 g / L, the total degradation rates of the functional indigenous bacterial community J-17 provided by this invention for the simultaneous degradation of the four PAEs reached 91.2%, 91.3%, 94.6%, 95.8%, and 94.8%, respectively. This indicates that the degradation effect at a salt concentration of 15 g / L was significantly higher than that at other salt concentrations, suggesting that the functional indigenous bacterial community J-17 provided by this invention exhibited the best degradation effect on the simultaneous degradation of the four PAEs under this salt concentration condition. However, the overall degradation effect remained above 90%, indicating that the bacterial community J-17 possesses good salt and alkali tolerance.

[0072] The results of Experiment 5 are as follows Figure 14 As shown, in a system with a total concentration of 80 mg / L for the four PAEs, after 7 days of cultivation at temperatures of 20, 25, 30, 35, and 40 °C, the total degradation rates of the functional indigenous bacterial community J-17 provided by this invention for the simultaneous degradation of the four PAEs reached 82.7%, 87.6%, 88.7%, 90.3%, and 85.4%, respectively. This indicates that at 35 °C, the functional indigenous bacterial community J-17 provided by this invention has the best degradation effect on the simultaneous degradation of the four PAEs.

[0073] The results of Experiment 6 are as follows Figure 15 As shown, in a system with a total concentration of 80 mg / L of the four PAEs, after 7 days of cultivation at pH values ​​of 3.0, 5.0, 7.0, 9.0, and 11.0, the total degradation rates of the functional indigenous bacterial community J-17 provided by this invention for simultaneously degrading the four PAEs reached 21.5%, 35.2%, 95.5%, 91.6%, and 62.1%, respectively. Therefore, the functional indigenous bacterial community J-17 provided by this invention exhibits the best degradation effect on the simultaneous degradation of the four PAEs under the condition of pH=7.0.

[0074] The following is a comparison of the degradation effects of the functional indigenous bacterial community J-17 provided in Example 1 of this application and the existing reported PAEs-degrading bacterial communities on the simultaneous degradation of four PAEs under the optimal conditions selected in the above experiments.

[0075] Among the reported microbial communities, functional endophytic bacteria enriched and domesticated from PAE-contaminated vegetables (mainly composed of *Sphingobacterium* and *Delftibacterium*) have been reported to degrade multiple PAEs. This community achieved degradation rates exceeding 94% for DMP, DEP, and DBP within 7 days; however, its degradation rate for BBP was only 44.82%. Therefore, while this community can effectively degrade some PAEs, it has significant limitations: First, the degradation efficiency is highly uneven, with a clear weakness in BBP degradation, which may lead to BBP becoming a difficult-to-remove residual pollutant when treating mixed contamination. Second, the community's broad-spectrum degradation capacity is insufficient to meet the requirements for simultaneous and efficient degradation of multiple PAEs (especially mixed systems containing BBP) in real-world environments.

[0076] In summary, the functional indigenous microbial community J-17 provided in this application demonstrates significant degradation effects on four types of PAEs under 7 days of shaking culture. Specifically, the functional indigenous microbial community J-17 achieves a degradation rate of over 80% for BBP on day 3 and over 95% on day 5. For DEP and DBP, near-complete degradation is achieved by day 7, with degradation rates exceeding 95% for both. The degradation rate for DMP also exceeds 60%. Therefore, the functional indigenous microbial community J-17 provided in this application not only achieves efficient simultaneous degradation of four types of PAEs but also boasts advantages such as a short degradation cycle and rapid rate. More importantly, it exhibits significant removal capabilities for all four structurally diverse PAEs without any obvious degradation limitations, meeting the stringent requirements for simultaneous remediation of mixed PAE pollution in complex real-world environments.

[0077] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A functional indigenous bacterial community J-17 that simultaneously degrades four types of PAEs, characterized in that, include: Acinetobacter ( Acinetobacter sp. W2-4 and Delft ( Delftia sp. W141, The Acinetobacter ( Acinetobacter sp. W2-4 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252941; the Delft bacterium ( Delftia sp. W141 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252940.

2. The functional indigenous bacterial community J-17 as described in claim 1, characterized in that, The four PAEs are dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate.

3. An article, characterized in that, It includes the functional indigenous bacterial group J-17 as described in claim 1.

4. The article of claim 3, characterized in that, The product is a bacterial suspension or an immobilized bacterial agent.

5. A method for simultaneously degrading four types of PAEs, characterized in that, The functional indigenous bacterial group J-17 according to any one of claims 1 to 2 or the product according to any one of claims 3 to 4 is inoculated into soil or water contaminated with PAEs to degrade the four types of PAEs.

6. The method as described in claim 5, characterized in that, The inoculation amount of the functional indigenous microbial community J-17 or the product is 5-9% of the mass of the soil or water. During the degradation process, the pH of the soil or water is adjusted to 6-10, the degradation temperature is 20-40℃, and the degradation time is 7-9 days.

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