A functional bacterial community J-3 and its application in the degradation of phthalates

Abstract

This invention provides a functional bacterial community J-3 and its application in the degradation of phthalates, belonging to the field of microbial technology. The aforementioned functional bacterial community J-3 includes Agrobacterium (…). Agrobacterium sp. W221 and Delft ( Delftia sp. W141, Agrobacterium ( Agrobacterium sp. W221 was deposited on December 17, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20252942, 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 aforementioned functional bacterial group J-3 can ensure the simultaneous degradation of four PAEs, and the degradation effect is significantly higher than the degradation effect of any one of the strains alone.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a functional bacterial community J-3 and its application in the degradation of phthalates. Background Technology

[0002] Phthalate esters (PAEs) are a common type of environmental pollutant. As endocrine disruptors, they have estrogenic effects, and some PAEs also possess teratogenic, carcinogenic, and mutagenic potential. Due to their excellent plasticizing and adhesive properties, PAEs are widely used in plastic softening, building materials, furniture, medical equipment, paints, and cosmetics. In agricultural production, the extensive use of plastic agricultural films and wastewater irrigation activities lead to PAEs seeping into the soil, causing a series of environmental pollution problems.

[0003] Bioremediation has become an important research direction in the field of pollution control, and in recent years, a large number of microorganisms capable of degrading PAEs and living in various environments such as soil and water have been obtained. However, most current research focuses on the screening and characterization of single degrading bacteria. The degradation activity of these strains for PAEs is highly dependent on specific environmental conditions, with a relatively narrow substrate range and limited degradation efficiency. Therefore, when facing actual contaminated sites with complex compositions and the coexistence of multiple PAEs (including dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), and butyl benzyl phthalate (BBP)), single strains often have insufficient remediation efficacy.

[0004] In soils contaminated with persistent organic pollutants (POPs) over a long period, stable and fully functional indigenous microbial communities develop through natural adaptation and domestication. These communities collectively possess the ability to efficiently degrade toxic organic matter, providing new resources and ideas for developing highly adaptable and robust bioremediation strategies. Summary of the Invention

[0005] This invention proposes a functional bacterial community J-3 and its application in the degradation of phthalates, which can achieve the bio-treatment effect of simultaneously degrading four types of PAEs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a functional bacterial community J-3, which includes Agrobacterium (…). Agrobacterium sp. W221 and Delft ( Delftia sp. W141; Agrobacterium in functional bacterial group J-3 ( Agrobacterium sp. W221 and Delft ( Delftia sp. The viable cell ratio of W141 is 1:1~2; The above-mentioned Agrobacterium ( Agrobacterium sp. W221 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252942; The above Delft bacteria ( Delftia sp. W141 was deposited at the China Center for Type Culture Collection on December 17, 2022, with accession number CCTCC M20252940.

[0007] Secondly, the present invention provides the application of the functional bacterial community J-3 described in the first aspect in the degradation of phthalates.

[0008] In one application scenario of the second aspect, the aforementioned phthalates include: dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate.

[0009] In one application method of the second aspect, the bacterial agent prepared from the functional bacterial group J-3 is inoculated into a medium contaminated with phthalic acid esters, wherein the bacterial agent is a bacterial suspension or an immobilized bacterial agent.

[0010] In one implementation of the second aspect, the method for preparing the bacterial suspension is as follows: First, Agrobacterium ( Agrobacterium sp. W221 and Delft ( Delftia sp. W141 was inoculated into LB medium and activated until both strains reached the logarithmic growth phase. Then, according to the Agrobacterium (… Agrobacterium sp. W221 and Delft ( Delftia sp. W141 was compounded with live bacteria at a ratio of 1:1~2 to obtain OD. 600nm Bacterial suspension with a value of 1.

[0011] In one implementation of the second aspect, the method for preparing the immobilized bacterial agent is as follows: The bacterial suspension was loaded onto an activated carbon carrier at a bacterial material ratio of 20-25 mL: 1.5 g and then solidified to obtain an immobilized bacterial agent.

[0012] In one application method of the second aspect, the medium is water or soil.

[0013] Compared with the prior art, the present invention has the following beneficial effects.

[0014] The functional bacterial community J-3 provided by this invention 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 functional bacterial community J-3 can almost completely degrade the four PAEs in an inorganic salt culture medium with a total concentration of 80 mg / L, with a degradation rate of over 99%. Therefore, the application of this functional bacterial community J-3 in the biological treatment of environmental pollutants has great potential. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the growth morphology of Agrobacterium W221 cultured on LB solid medium according to the embodiments of this application; Figure 2 This is a scanning electron microscope image of Agrobacterium W221 provided in the embodiments of this application; Figure 3 This is a schematic diagram of the phylogenetic tree of 16S rRNA of Agrobacterium W221 provided in the embodiments of this application; Figure 4 This is a statistical chart showing the degradation effect of Agrobacterium W221 on six PAEs simultaneously, as provided in the embodiments of this application.

[0016] Figure 5 This is a schematic diagram of the growth morphology of Delft bacillus W141 cultured on LB solid medium, as provided in the embodiments of this application. Figure 6 This is a scanning electron microscope image of Delft bacillus W141 provided in the embodiments of this application; Figure 7 This is a schematic diagram of the phylogenetic tree of 16S rRNA of Delftibella W141 provided in the embodiments of this application; Figure 8 This is a statistical chart showing the degradation effect of Delft bacterium W141 on four types of PAEs simultaneously, as provided in the embodiments of this application.

[0017] Figure 9 This is a statistical chart showing the degradation effect of functional bacterial community J-3 provided in this application on four types of PAEs simultaneously.

[0018] Figure 10 This is a statistical graph showing the degradation effect of functional microbial community J-3 on the simultaneous degradation of four PAEs as the concentration of a single PAE changes.

[0019] Figure 11 This is a statistical chart showing the degradation effect of functional bacterial community J-3 on four types of PAEs simultaneously as temperature changes.

[0020] Figure 12This is a statistical graph showing the degradation effect of functional bacterial community J-3 on four types of PAEs simultaneously as pH changes.

[0021] Figure 13 This is a statistical graph showing the degradation effect of functional bacterial community J-3 on four types of PAEs simultaneously as the NaCl concentration changes. Detailed Implementation

[0022] 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.

[0023] Example 1: This example describes a functional bacterial group J-3, which includes Agrobacterium (…). Agrobacterium sp. W221 and Delft ( Delftia sp. W141; Agrobacterium in functional bacterial group J-3 ( Agrobacterium sp. W221 and Delft ( Delftia sp. The viable count ratio of W141 was 1:1.5.

[0024] In some other specific embodiments, Agrobacterium (J-3) is a functional microbial community. Agrobacterium sp. W221 and Delft ( Delftia sp. The viable count ratio of W141 can be 1:1, Agrobacterium ( Agrobacterium sp. W221 and Delft ( Delftia sp. The live bacteria ratio of W141 can also be 1:2. This application does not limit the value of the above live bacteria ratio in the embodiments.

[0025] Specifically, the above-mentioned Agrobacterium ( Agrobacterium sp. W221 and Delft ( Delftia sp. All W141 strains are new. Agrobacterium ( Agrobacterium sp. W221 was deposited at the China Center for Type Culture Collection on December 17, 2025, with accession number CCTCC M20252942; the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0026] The above Delft bacteria ( Delftia sp. W141 was deposited at the China Center for Type Culture Collection on December 17, 2022, with accession number CCTCC M20252940; the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0027] The above-mentioned Agrobacterium ( Agrobacterium sp The 16S rRNA gene sequence of W221 is shown in SEQ ID NO.1.

[0028] SEQ ID NO.1:

[0029] The above-mentioned Agrobacterium ( Agrobacterium sp The 16S rRNA gene sequence of W221 was compared with that of existing PAE-degrading Agrobacterium strains. The comparison showed that the sequence of this strain is similar to that of known PAE-degrading Agrobacterium strains. Agrobacterium tumefaciens The D-1 sequence similarity is 81.95% (<98.65%), which indicates that it is a potential new species.

[0030] The following is an introduction to Agrobacterium ( Agrobacterium sp The process of separating and identifying W221 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.

[0031] 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.

[0032] 2. Separation and Identification Weigh 5 g of yellow-brown 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 six PAEs, with a total concentration of 30 mg / L (i.e., the six PAEs included 5 mg / L DMP, 5 mg / L DEP, 5 mg / L DBP, 5 mg / L BBP, 5 mg / L DEHP, and 5 mg / L DOP). After incubation at 30℃ and 150 rpm in the dark for 5 days, the bacterial culture was obtained. 5 μL of the culture was taken directly each time. mL of bacterial culture was transferred to new MSM liquid medium containing 6 types of PAEs, and the process was repeated 4 times for enrichment culture to obtain the culture medium. The total concentration of the 6 types of PAEs in the MSM liquid medium after the first transfer was 60 mg / L; the total concentration of the 6 types of PAEs in the MSM liquid medium after the second transfer was 120 mg / L; the total concentration of the 6 types of PAEs in the MSM liquid medium after the third transfer was 240 mg / L; and the total concentration of the 6 types of PAEs in the MSM liquid medium after the fourth transfer was 360 mg / L, and the proportion of the single PAE among the 6 types of PAEs remained unchanged. Dilute the culture medium by 10 5 The culture medium was then plated onto MSM solid medium containing a total concentration of 30 mg / L of six PAEs (i.e., 5 mg / L DMP, 5 mg / L DEP, 5 mg / L DBP, 5 mg / L BBP, 5 mg / L DEHP, and 5 mg / L DOP) and incubated upside down at 30°C for 3 days. After single colonies grew on the MSM solid medium, a single colony was picked and streaked four times for purification, resulting in a bacterial strain, designated W221. Strain W221 was then inoculated onto 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.

[0033] like Figure 1 As shown, the colonies of strain W221 are milky white, round, opaque, with a raised, moist, sticky, and smooth surface.

[0034] 3. Identification by transmission electron microscopy The purified strain W221 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 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] like Figure 2 As shown, strain W221, as observed by transmission electron microscopy, is morphologically round and lacks flagella.

[0036] 4. Molecular identification of 16S rRNA in strain W221 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 reported 16S rRNA sequences in GenBank. Specifically, the 16S rRNA sequence of strain W221 was compared with other registered bacterial strains' 16S rRNA sequences using the BLAST program on the NCBI website.

[0037] Phylogenetic analysis was performed on relevant bacterial species, and the results for strain W221 are as follows:Figure 3 As shown, the 16S rRNA gene sequence of strain W221 is similar to that of Agrobacterium (…). Agrobacterium sp. The strain W221 obtained by screening in this invention showed the highest similarity (Genbank accession number AB247586.1), with a homology rate of 100%. Therefore, the strain W221 obtained by screening in this invention was identified as Agrobacterium (Genbank accession number AB247586.1). Agrobacterium sp. ), named Agrobacterium ( Agrobacterium sp. W221.

[0038] The above Agrobacterium ( ) are given below. Agrobacterium sp. The verification process of W221's ability to degrade PAEs.

[0039] 1) Preparation of bacterial suspension Agrobacterium ( Agrobacterium sp. A single colony of W221 was inoculated into 100 mL of LB liquid medium and cultured at 30 °C and 150 rpm for 24 h. The colony was then centrifuged at 8000 rpm for 5 min to obtain a bacterial pellet. The pellet was washed twice with MSM liquid medium before the bacterial OD was measured. 600nm The value was adjusted to 1.0, and Agrobacterium ( Agrobacterium sp. W221 bacterial suspension, store temporarily at 4℃ for later use.

[0040] 2) Agrobacterium ( Agrobacterium sp. Degradation performance determination of W221 1 mL of Agrobacterium was inoculated into 19 mL of MSM liquid culture medium containing a total concentration of 120 mg / L of six PAEs (i.e., 20 mg / L DMP, 20 mg / L DEP, 20 mg / L DBP, 20 mg / L BBP, 20 mg / L DEHP, and 20 mg / L DOP). Agrobacterium sp. W221 bacterial suspension was used as a control, with no inoculation, and the pH was adjusted to 7.0. Three replicates were performed for each group. The whole-bottle extraction method was used (the samples were incubated at 30℃ and 150 rpm for 48 h on a constant temperature shaker; on days 1, 3, 5, and 7, the conical flasks were removed, 40 mL of chromatographically pure methanol was added, and the samples were sonicated in a water bath for 1 h; after sonication, the samples were vortexed, and the supernatant was filtered through a 0.22 μm organic phase filter membrane and transferred to a 2 mL brown liquid chromatography vial). The residual concentrations of six PAEs in the brown liquid chromatography vials were detected using high performance liquid chromatography.

[0041] The detection conditions for the high performance liquid chromatograph (the detection conditions for the high performance liquid chromatographs used in this article are the same): LC-20AT high performance liquid chromatograph (equipped with SPD-2A ultraviolet detector) was used. The detection time was 40 min, and the injection volume of the injection system 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 method 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 then the gradient elution ended. The chromatographic column was a Φ4.6×250 mm Inertsil ODS-P liquid chromatography column, and the column temperature was 40℃. The detection system used an ultraviolet detector with dual-wavelength detection mode enabled, at 225 nm and 290 nm.

[0042] The results are as follows Figure 4 As shown, Agrobacterium ( Agrobacterium sp. W221 achieved a degradation rate of over 90% for all four PAEs (DMP, DEP, DBP, and BBP) on day 1. After day 5, it was able to completely degrade DMP and DEP, and the degradation rates for DEHP and DOP reached 37.4% and 30.9% respectively on day 5.

[0043] The above Delft bacteria ( Delftia The 16S rRNA gene sequence of sp.) W141 is shown in SEQ ID NO.2.

[0044] SEQ ID NO.2:

[0045] The above Delft bacteria ( Delftia The 16S rRNA gene sequence of sp. W141 was compared with that of existing PAE-degrading Delft strains. The comparison showed that this strain's sequence was similar to that of known PAE-degrading strains. Delftia The sp. L3 sequence similarity is 97.12% (<98.65%), which can be identified as a potential new species.

[0046] The following is a list of the above-mentioned Delft bacteria ( Delftia sp. The process of separating and identifying W141.

[0047] 1. Separation process Weigh 5 g of yellow-brown 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., 5 mg / L DMP, 5 mg / L DEP, 5 mg / L DBP, and 5 mg / L BBP). After incubation at 30℃ and 150 rpm in the dark for 5 days, the bacterial culture was obtained. Each time, 5 mL of the bacterial culture was directly transferred to a new MSM liquid medium containing the four PAEs, and this 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, with the proportion of each individual PAE remaining unchanged. Dilute the culture medium by 10 5 The culture medium was then spread onto MSM solid medium containing a total concentration of 20 mg / L of four PAEs (i.e., 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, a single colony was picked and streaked five times for purification to isolate a single bacterium, designated W141. Strain W141 was then inoculated onto LB solid medium and incubated upside down at 30°C for 3 days, followed by streaking for another 2 days, and its colony morphology was observed.

[0048] like Figure 5As shown, the colonies of strain W141 are milky white, round and opaque, with irregular edges, and are moist, sticky and smooth.

[0049] 2. Identification process The identification method for strain W141 is the same as that for strain W221, which was identified by transmission electron microscopy, and will not be repeated here.

[0050] like Figure 6 As shown, strain W141, as observed by transmission electron microscopy, is elliptical in shape and lacks flagella.

[0051] 3. Molecular identification of 16S rRNA in strain W141 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 sequence of strain W141 was compared with other registered bacterial strains' 16S rRNA sequences using the BLAST program on the NCBI website.

[0052] Phylogenetic analysis was performed on relevant bacterial species, and the results for strain W141 are as follows: Figure 7 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.

[0053] The following is a list of the above-mentioned Delft bacteria ( Delftia sp. The verification process of W141's ability to degrade PAEs.

[0054] 1) Preparation of bacterial suspension Delft ( Delftia sp. A single colony of W141 was inoculated into 100 mL of LB liquid medium and cultured at 30°C and 150 rpm for 24 h. The colony was then centrifuged at 8000 rpm for 5 min to obtain a bacterial pellet. The pellet was washed twice with MSM liquid medium before the bacterial OD was measured. 600nm Adjusting the value to 1.0 yields Delft ( Delftia sp. W141 bacterial suspension, store temporarily at 4 ℃ for later use.

[0055] 2) Delft ( Delftia sp. Degradation performance determination of W141 1 mL of Delftobacterium was inoculated into 19 mL of MSM liquid culture medium containing a total concentration of 80 mg / L of four PAEs (i.e., 20 mg / L DMP, 20 mg / L DEP, 20 mg / L DBP, and 20 mg / L BBP). Delftia sp. W141 bacterial suspension was used, with no inoculation as a control, and the pH was adjusted to 7.0. Three replicates were performed for each group. The whole-bottle extraction method was used to process the samples, and the residual concentrations of the four PAEs in the brown liquid chromatographs were detected by high performance liquid chromatography.

[0056] The results are as follows Figure 8 As shown, Delft ( Delftia sp. W141 achieved a degradation rate of over 99% for DMP and DEP on day 3 and over 93% for DBP and DEP on day 5.

[0057] The following will verify whether there is an antagonistic effect between Agrobacterium W221 and Delftella W141 in the above-mentioned functional bacterial group J-3.

[0058] Agrobacterium W221 and Delftella W141 were used to conduct an antagonistic experiment between the strains. The filter paper method was used in this experiment. One bacterial culture that had been cultured to the logarithmic phase was spread on a plate, and the other bacterial culture was placed on the plate by dipping filter paper in it. The plates were incubated at 30°C for 2 days, and the presence or absence 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).

[0059] Table 1. Antagonistic response results of functional bacterial group J-3

[0060] As shown in Table 1, there is no antagonistic reaction between Agrobacterium W221 and Delft Bacillus W141, and they can be combined to obtain functional bacterial group J-3.

[0061] Example 2: This example describes the application of the functional bacterial group J-3 provided in Example 1 in the degradation of phthalic acid esters, including: dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate.

[0062] The above applications include: inoculating a bacterial agent prepared from functional bacterial group J-3 into a medium contaminated with phthalates, wherein the bacterial agent is a bacterial suspension or an immobilized bacterial agent, and adjusting the pH of the medium to 7.0 and controlling the temperature to 30°C, so as to achieve the degradation of DMP, DEP, DBP and DEP.

[0063] In one implementation, when the bacterial agent obtained from the functional bacterial group J-3 is a bacterial suspension, the preparation process of the bacterial suspension is as follows: Step 1: Inoculate Agrobacterium W221 and Delftella W141 into 100 mL LB medium respectively, and activate them at 30°C and 150 rpm until both strains reach the logarithmic growth phase, i.e., OD. 600nm =1.0, then centrifuged at 8000 rpm for 5 min to obtain activated Agrobacterium W221 and activated Delft bacteria W141.

[0064] Optionally, the centrifugation speed can also be 7500 or 8500 r / min, and the centrifugation time can also be 10 min. This embodiment does not limit the centrifugation speed and time.

[0065] Step 2: After washing the activated Agrobacterium W221 and activated Delft bacteria W141 with MSM liquid medium, then according to Agrobacterium ( Agrobacterium sp. W221 and Delft ( Delftia sp. The viable count ratio of W141 was 1:1.5 when compounded to obtain OD. 600nm Bacterial suspension with a value of 1.

[0066] In some other specific embodiments, the ratio of viable counts of Agrobacterium W221 to Delft W141 is 1:1, and the ratio of viable counts of Agrobacterium W221 to Delft W141 can also be 1:2.

[0067] In another implementation method, when the bacterial agent prepared from functional bacterial group J-3 is an immobilized bacterial agent, the preparation process of the immobilized bacterial agent is as follows: Activated carbon carriers were obtained by carbonizing rice straw at 500℃ for 12 hours.

[0068] Optionally, the pyrolysis temperature can be 400°C and the pyrolysis time can be 24 hours. This embodiment does not limit the pyrolysis temperature and time.

[0069] The bacterial suspension prepared above was added to an activated carbon carrier at a bacterial substrate ratio of 20 mL: 1.5 g, and then solidified in a shaking incubator at 30 °C and 150 r / min for 2 days. After that, it was centrifuged using a disc centrifuge to obtain the immobilized bacterial agent.

[0070] In some other specific embodiments, the above-mentioned substrate ratio can be 25mL:1.5g or 22mL:1.5g. The present application does not limit the value of the above-mentioned substrate ratio.

[0071] Specifically, when the bacterial agent prepared from functional microbial community J-3 is a bacterial suspension, the above application specifically includes: inoculating the bacterial suspension into farmland soil contaminated with phthalates to achieve phthalate degradation. When the bacterial agent prepared from functional microbial community J-3 is an immobilized bacterial agent, the above application specifically includes: inoculating the immobilized bacterial agent into farmland soil contaminated with phthalates to achieve phthalate degradation. The inoculation amount of the bacterial suspension and the immobilized bacterial agent can be 5% of the farmland soil mass, 10% of the farmland soil mass, or other values ​​within a reasonable range; this embodiment does not limit this.

[0072] Furthermore, the soil mentioned above can be farmland soil contaminated with phthalates, or industrial soil or urban soil contaminated with phthalates; the method of treating the soil with the microbial agent prepared by the functional microbial community J-3 to achieve phthalate degradation can be in-situ remediation, and the remediation temperature range can be 10-20℃. The embodiments of this application do not further limit the type of soil and the treatment method mentioned above.

[0073] For example, when the medium is water, the method may involve inoculating the bacterial agent prepared from functional bacterial group J-3 into water contaminated with PAEs to degrade the PAEs in the water. The inoculation amount of the bacterial agent prepared from functional bacterial group J-3 may be 5% or 10%; this embodiment does not limit this.

[0074] The aforementioned water body can be water contaminated with phthalates, or it can be wastewater contaminated with phthalates in a wastewater treatment plant (such as wastewater in a biological reactor and a secondary sedimentation tank). This application embodiment does not limit the type and scenario of the aforementioned water body.

[0075] The PAEs degradation ability of functional bacterial community J-3 will be verified below.

[0076] One mL of bacterial suspension of functional bacterial group J-3 was inoculated into 100 mL of LB liquid medium and cultured at 30°C with shaking at 150 rpm for 24 h. The culture was then centrifuged at 4°C and 8000 rpm for 5 min to collect the bacterial cells and discard the supernatant. The OD of the bacterial cells was measured using MSM liquid medium. 600nm The value was adjusted to 1.0 to prepare the bacterial suspension, and it was temporarily stored at 4 ℃ for later use.

[0077] The bacterial suspension was added to MSM liquid medium with a total concentration of 120 mg / L for six PAEs (all six PAEs had the same individual concentration) at 30℃, 5% inoculum, and pH 7.0. The culture was incubated for 7 days. After incubation, the samples were processed using the whole-bottle extraction method, and the residual concentrations of the six PAEs in the brown liquid chromatographs were detected using high-performance liquid chromatography (HPLC). The degradation rates were calculated and compared. like Figure 9 As shown, the functional bacterial community J-3 achieved degradation rates exceeding 80% for DEP, DBP, and BBP on day 3 and exceeding 95% on day 5. However, its degradation rate for DMP was relatively slow, only reaching over 99% after day 7. Therefore, after 7 days of cultivation, the degradation rate for any of the four PAEs (DMP, DEP, DBP, and BBP) was above 99%. Furthermore, functional bacterial community J-3 exhibited some degradation effect on DEHP and DOP (not shown in the figure), with degradation rates below 20% after day 7. This indicates that functional bacterial community J-3 possesses highly efficient simultaneous degradation capabilities for the four PAEs (DMP, DEP, DBP, and BBP).

[0078] Comparing this functional bacterial community J-3 with the PAE-degrading bacterial community in the prior art, it can be seen that: in the prior art, the bacterial community degraded by PAEs... Pseudomonas putidastrain ShA, Gordonia alkanivorans Sh6 The compounded bacterial community An6 is a reportedly highly efficient bacterial community capable of degrading mixed PAEs. This community is characterized by degradation rates of 100%, 100%, 90.57%, and 58.73% for DMP, DEP, DBP, and DEHP at 2000 mg / L, respectively. However, compared to the functional bacterial community J-3 of this application, this community has significant limitations: Firstly, functional bacterial community J-3 was tested with a total concentration of 80 mg / L for four PAEs, a concentration closer to the actual pollution levels in the natural environment. Its research conclusions (degradation rates of over 99% for all four PAEs within 7 days) have higher reference value and guiding significance for practical remediation projects. In contrast, the experimental concentration of bacterial community An6 is as high as 2000 mg / L, which is severely out of sync with actual pollution scenarios and far exceeds the actual pollution levels of PAES in the natural environment, making it impossible to verify its degradation efficiency under low-concentration pollution scenarios. Secondly, the functional bacterial group J-3 achieved a degradation rate of 99% for DEP, DBP, BBP and DMP in 7 days, while the bacterial group An6 achieved a degradation rate of 90.57% for DBP, failing to achieve simultaneous and efficient degradation.

[0079] Since the functional microbial community J-3 has a low degradation rate of DEHP and DOP, the following statistics only show the degradation effect on four PAEs (DMP, DEP, DBP and BBP).

[0080] The degradation effect of functional microbial community J-3 on the simultaneous degradation of four PAEs at different total concentrations.

[0081] The bacterial suspension was added to MSM liquid medium at total concentrations of 40, 80, 160, 240, and 320 mg / L (corresponding to individual PAE concentrations of 10, 20, 40, 60, and 80 mg / L) of four PAEs at 30℃, 5% inoculum, and pH=7.0, and cultured for 7 days. After culture, 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 (HPLC). The degradation rates were calculated and compared. like Figure 10 As shown, as the concentration of a single PAE increased from 10 mg / L to 20 mg / L, the degradation rate of the functional microbial community J-3 for the simultaneous degradation of the four PAEs remained almost constant, all reaching over 95%. However, when the concentration increased from 20 mg / L to 80 mg / L, the degradation rate of the functional microbial community J-3 for the simultaneous degradation of the four PAEs gradually decreased to about 68%, showing an overall downward trend. Therefore, the degradation effect of the functional microbial community J-3 was optimal when the total concentration of the four PAEs was 80 mg / L (the concentration of a single PAE was 20 mg / L). Even when the total concentration of the four PAEs reached 320 mg / L (the concentration of a single PAE reached 80 mg / L), the degradation rate of the functional microbial community J-3 for the simultaneous degradation of the four PAEs was still about 68%, indicating that the functional microbial community J-3 has strong tolerance and degradation ability for PAEs at a wide range of concentrations.

[0082] The following shows the degradation effect of functional bacterial community J-3 on four types of PAEs simultaneously at different temperatures.

[0083] The bacterial suspension was added to MSM liquid medium with a total concentration of 80 mg / L for four PAEs at an inoculum volume of 5% and pH=7.0. The culture was incubated at 20, 25, 30, 35, and 40℃ for 7 days, respectively. After incubation, the samples were processed using the whole-bottle extraction method, and the residual concentrations of the four PAEs in the brown liquid chromatography vials were detected using high-performance liquid chromatography (HPLC). The degradation rates were calculated and compared. like Figure 11 As shown, as the temperature increased from 20℃ to 30℃, the degradation rate of the four PAEs by functional microbial community J-3 rapidly increased from approximately 82.8% to approximately 98%, indicating that within this temperature range, heating significantly promoted the degradation efficiency of functional microbial community J-3. However, when the temperature increased from 30℃ to 35℃, the degradation rate plummeted to 48.6%; and further decreased when the temperature continued to rise to 40℃. This suggests that functional microbial community J-3 is most suitable for application in environments with normal or slightly lower temperatures (around 30℃).

[0084] The following shows the degradation effect of functional bacterial community J-3 on four types of PAEs at different pH levels.

[0085] The pH of MSM liquid medium was adjusted to 3, 5, 7, 9, and 11. Then, bacterial suspensions were added to MSM liquid medium at pH 3, 5, 7, 9, and 11 (total concentration of four PAEs: 80 mg / L) at 30℃ and an inoculum size of 5%, respectively. The samples were cultured for 7 days. After culture, whole-bottle extraction was used to process the samples, and the residual concentrations of the four PAEs in the brown liquid chromatographs were detected using high-performance liquid chromatography (HPLC). The degradation rates were calculated and compared. like Figure 12 As shown, in acidic environments (pH=3, pH=5), the degradation rate of the four PAEs by functional bacteria J-3 was less than 10%. When the pH rose to 7, the degradation rate of the four PAEs by functional bacteria J-3 reached about 96%. When the pH continued to increase to alkaline environments (pH=9, pH=11), the degradation rate began to decrease. At pH=9 (weakly alkaline), the degradation rate could still be maintained at about 85%, showing a certain degree of tolerance. At pH=11 (strongly alkaline), the degradation rate dropped to about 64%, indicating that functional bacteria J-3 was more effective in simultaneously degrading the four PAEs in neutral and weakly alkaline environments.

[0086] The following shows the degradation effect of functional bacterial community J-3 on four types of PAEs simultaneously under different NaCl concentrations.

[0087] The NaCl concentration in MSM liquid medium was adjusted to 0, 5, 10, 15, and 20 g / L. The bacterial suspension was then added to MSM liquid medium with NaCl concentrations of 0, 5, 10, 15, and 20 g / L, respectively, at a total PAE concentration of 80 mg / L, under conditions of 30℃, 5% inoculum, and pH=7. The samples were cultured for 7 days. After culture, the whole-bottle extraction method was used to process the samples, and the residual concentrations of the four PAEs in the brown liquid chromatographs were detected using high-performance liquid chromatography (HPLC). The degradation rates were calculated and compared. like Figure 13 As shown, when the NaCl concentration increases from 0 to 10 g / L, the degradation rate of the four PAEs by functional bacterial group J-3 gradually increases, but the impact is small. The degradation rate reaches about 99% at 10 g / L. However, when the concentration is further increased to 20 g / L, the degradation rate of the four PAEs by functional bacterial group J-3 gradually decreases, but still remains above 90%, indicating that functional bacterial group J-3 has good acid and alkali resistance.

[0088] The following shows the degradation effect of functional microbial community J-3 on the simultaneous degradation of four PAEs when the total concentration of the four PAEs is 80 mg / L and the proportion of each individual PAE is different.

[0089] The mixture of four PAEs in MSM liquid medium was adjusted to either 22 mg / L DMP, 26 mg / L DEP, 18 mg / L DBP, and 14 mg / L BBP, or 18 mg / L DMP, 22 mg / L DEP, 14 mg / L DBP, and 26 mg / L BBP. Then, bacterial suspensions were added to the corresponding two MSM liquid media with different proportions of individual PAEs at 30℃ and an inoculum size of 5%. After 7 days of 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 using high-performance liquid chromatography (HPLC). The degradation rates were calculated and compared. When the mixture of four PAEs was 22 mg / L DMP, 26 mg / L DEP, 18 mg / L DBP, and 14 mg / L BBP, the functional microbial community J-3 achieved a degradation rate of over 60% for all four PAEs on day 3 and 95% on day 7. Similarly, when the mixture was 18 mg / L DMP, 22 mg / L DEP, 14 mg / L DBP, and 26 mg / L BBP, the degradation rate of the functional microbial community J-3 also reached over 60% on day 3 and 95% on day 7. This indicates that the functional microbial community J-3 exhibits the best degradation rate for each of the four PAEs in equal proportions compared to a single PAE, but also demonstrates good simultaneous degradation performance in various mixtures, making it suitable for practical application in PAE pollution remediation.

[0090] Thus, it can be verified that the functional bacterial community J-3 provided in this embodiment can simultaneously degrade four types of PAEs, and the degradation effect can reach more than 99%.

Claims

1. A functional bacterial community J-3, characterized in that, Including Agrobacterium ( Agrobacterium sp. W221 and Delft ( Delftia sp. W141, Agrobacterium in functional bacterial group J-3 ( Agrobacterium sp. W221 and Delft ( Delftia sp. The viable cell ratio of W141 is 1:1~2; Agrobacterium ( Agrobacterium sp. W221 was deposited on December 24, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20252942, belonging to Delft (…). Delftia sp. W141 was deposited at the China Center for Type Culture Collection on December 24, 2025, with accession number CCTCC M20252940.

2. The application of the functional microbial community J-3 as described in claim 1 in the degradation of phthalates.

3. The application as described in claim 2, characterized in that, The phthalates include: dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate.

4. The application as described in claim 3, characterized in that, The bacterial agent prepared from the functional bacterial group J-3 was inoculated into a medium contaminated with phthalic acid esters. The bacterial agent was a bacterial suspension or an immobilized bacterial agent.

5. The application as described in claim 4, characterized in that, The method for preparing the bacterial suspension is as follows: First, Agrobacterium ( Agrobacterium sp. W221 and Delft ( Delftia sp. W141 was inoculated into LB medium and activated until both strains reached the logarithmic growth phase. Then, according to the Agrobacterium (… Agrobacterium sp. W221 and Delft ( Delftia sp. W141 was compounded with live bacteria at a ratio of 1:1~2 to obtain OD. 600nm Bacterial suspension with a value of 1.

6. The application as described in claim 5, characterized in that, The method for preparing the immobilized bacterial agent is as follows: The bacterial suspension was loaded onto an activated carbon carrier at a bacterial material ratio of 20-25 mL: 1.5 g and then solidified to obtain an immobilized bacterial agent.

7. The application as described in claim 4, characterized in that, The medium is water or soil.

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