Growth promoting-decontamination flora and application thereof

Abstract

The invention provides a group of growth promoting-decontamination flora and application thereof, and belongs to the technical field of phthalate polluted soil remediation. The growth promoting-decontamination flora comprises the following components: Acinetobacter sp. Sb < 2-4 >, Gordonia phtheria 107, Gordonia phtheria 107, Gordonia phtheria 19743, Gordonia phtheria 109, Gordonia phtheria 109, Gordonia phtheria 109, Gordonia phtheria 109, Gordonia phtheria 109, Gordonia phtheria 109, Gordonia phtheria 109, Gordonia phtheria 109, Gordonia phtheria 109, Gordonia Wherein the endophytic acinetobacter Sb 2-4 is preserved in the China Center for Type Culture Collection on December 01, 2025, and the preservation number of the endophytic acinetobacter Sb 2-4 is CCTCC (China Center for Type Culture Collection) NO: M20252724. The growth promoting-decontamination flora can ensure the PAEs degradation effect and realize crop growth promotion at the same time.

Description

Technical Field

[0001] This invention relates to the field of phthalate contaminated soil remediation technology, and in particular to a group of growth-promoting and pollution-eliminating bacteria and their applications. Background Technology

[0002] Phthalate esters (PAEs) are widely present in environmental media such as water, soil, and atmosphere through various pathways including industrial wastewater, plastic waste degradation, and domestic sewage discharge. They have even been detected in remote areas such as polar regions, demonstrating their strong migration and diffusion capabilities. The environmental risks of PAEs stem not only from their pervasive presence but also from their serious threats to ecosystems and human health.

[0003] In existing technologies, PAE-degrading bacterial communities constructed from PAE-degrading strains are often used to remediate phthalate-contaminated soil. However, existing PAE-degrading bacterial communities are not effective in degrading PAEs and cannot promote crop growth. Summary of the Invention

[0004] This invention proposes a group of growth-promoting and pollution-eliminating bacteria and their applications, which can promote crop growth while ensuring the degradation effect of PAEs.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a group of growth-promoting and pollution-eliminating bacteria, including endophytic Acinetobacter (… Acinetobacter sp.) Sb 2-4, Gordon's phthalate ( Gordonia phthalatica )107 and Bacillus subtilis ( Bacillus subtilis The viable ratio of Acinetobacter endophyticus Sb 2-4, Gordon's phthalate 107, and Bacillus 19743 in the growth-promoting and decontamination bacteria group was 1-1.5:1-1.5:1-1.5.

[0006] The aforementioned Acinetobacter Sb 2-4 and Gordon's phthalate 107 are PAE-degrading bacteria, and the aforementioned Bacillus 19743 is a growth-promoting bacterium. The aforementioned Acinetobacter Sb 2-4 was deposited at the China Center for Type Culture Collection (CCTCC) on December 1, 2025, with accession number CCTCC NO: M20252724. The aforementioned Gordon's phthalate 107 was purchased from the China Industrial Microbial Culture Collection Center (CICC), with accession number CICC 24107, located at No. 50 Zhongling Street, Nanjing, Jiangsu Province. Bacillus 19743 was purchased from the China Agricultural Microbial Culture Collection Center (ACCC), with accession number ACCC 19743. The deposit address is Resource Building, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Haidian District, Beijing.

[0007] In one implementation of the first aspect, the 16S rRNA sequence of Acinetobacter endophyte Sb 2-4 is shown in SEQ ID NO. 1.

[0008] Secondly, the present invention provides the application of the group of growth-promoting and pollution-eliminating bacteria described in the first aspect in promoting crop growth and remediating phthalate-contaminated soil.

[0009] In one implementation of the second aspect, the above application includes: preparing a microbial agent using a growth-promoting and pollution-eliminating microbial community, and inoculating the microbial agent into farmland soil contaminated with phthalates to promote crop growth and degrade phthalates; wherein the inoculation amount of the microbial agent is 5-10% of the farmland soil mass.

[0010] In one implementation of the second aspect, the aforementioned growth-promoting and decontamination-eliminating bacterial agent is a bacterial suspension or an immobilized bacterial agent.

[0011] In one implementation of the second aspect, the method for preparing the bacterial suspension includes: Endophytic Acinetobacter Sb 2-4, Gordon's Phthalate 107, and Bacillus 19743 were activated to obtain activated endophytic Acinetobacter Sb 2-4, activated Gordon's Phthalate 107, and activated Bacillus 19743, respectively. Activated Acinetobacter endophyticus Sb 2-4, activated Gordon's phthalate 107, and activated Bacillus 19743 were mixed at a live bacteria ratio of 1-1.5:1-1.5:1-1.5 and prepared into OD. 600nm Bacterial suspension with a value of 1.

[0012] In one implementation of the second aspect, the method for preparing the immobilized bacterial agent includes: Activated carbon carriers are obtained by pyrolyzing rice straw or corn straw at 800-1200℃ for 12-24 hours. The bacterial suspension was added to the activated carbon carrier at a bacterial substrate ratio of 20-30 mL: 1.5 g, and then solidified in a shaking incubator for 0.5-4 days. After centrifugation using a disc centrifuge, the immobilized bacterial agent was obtained.

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

[0014] The growth-promoting and decontamination-eliminating bacterial community provided by this invention includes Acinetobacter endophyticus Sb 2-4 and Gordon's phthalate 107 as PAE-degrading bacteria, and Bacillus 19743 as a growth-promoting bacterium. When there is no antagonistic relationship between the three bacteria in the above-mentioned growth-promoting and decontamination-eliminating bacterial community, Acinetobacter endophyticus Sb 2-4 can utilize diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate as the sole carbon and energy source for growth and reproduction. Under pure culture conditions, this bacterium can degrade more than 60% of a 20 mg / L mixed PAE (containing 20 mg / L DEP, DBP, and BBP respectively) in an inorganic salt medium within 5 days. Building upon this, Acinetobacter endophyticus Sb 2-4 can synergistically enhance the degradation of PAEs with Gordon's phthalate 107. Simultaneously, under the action of Acinetobacter endophyticus Sb 2-4 and Gordon's phthalate 107, Bacillus 19743 can promote crop growth. Therefore, it is possible to promote crop growth while ensuring the degradation of PAEs, thus showing great promise in the biological reduction of environmental pollutants and the promotion of plant growth. Attached Figure Description

[0015] Figure 1 This is provided by the embodiments of this application. Acinetobacter A schematic diagram of the growth morphology of sp. Sb 2-4 cultured on LB medium for 2 days; Figure 2 This is provided by the embodiments of this application. Acinetobacter Scanning electron microscope image of sp. Sb 2-4; Figure 3 This is provided by the embodiments of this application. Acinetobacter A schematic diagram of the phylogenetic tree of 16S rRNA from sp. Sb 2-4; Figure 4 This is provided by the embodiments of this application. Acinetobacter Schematic diagram of the degradation effect of sp. Sb 2-4 on three mixed PAEs; Figure 5 This is an antagonistic experiment diagram of three strains in the growth-promoting and pollution-eliminating bacterial community provided in the embodiments of this application; Figure 6This is a diagram showing the degradation effect of the growth-promoting and pollution-eliminating bacterial community on mixed PAEs provided in the embodiments of this application; Figure 7 These are real-shot comparison images of the results of the potted plant experiment on the functional endophytic microbiota provided in the embodiments of this application; Figure 8 This is a comparison diagram of the root length of Shanghai bok choy in a pot experiment with functional endophytic flora provided in this application embodiment; Figure 9 This is a weight comparison chart of Shanghai bok choy in a pot experiment with functional endophytic microbiota provided in this application embodiment; Figure 10 This is a comparison chart of chlorophyll content in Shanghai bok choy during a pot experiment with functional endophytic flora provided in this application embodiment. Detailed Implementation

[0016] 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.

[0017] In the embodiments of this invention, DEP is an abbreviation for diethyl phthalate, DBP is an abbreviation for dibutyl phthalate, and BBP is an abbreviation for butyl benzyl phthalate. The tested leafy green vegetable was Shanghai bok choy.

[0018] Example 1: This example describes a group of growth-promoting and pollution-eliminating bacteria, including endophytic Acinetobacter (… Acinetobacter sp.) Sb 2-4, Gordon's phthalate ( Gordonia phthalatica )107 and Bacillus subtilis ( Bacillus subtilis The viable ratio of Acinetobacter endophyticus Sb 2-4, Gordon's phthalate 107, and Bacillus 19743 in the proliferative-decontamination bacterial community is 1:1:1. In some other specific embodiments, the viable ratio of Acinetobacter endophyticus Sb 2-4, Gordon's phthalate 107, and Bacillus 19743 in the bacterial community can be 1.5:1:1, or it can be 1:1.5:1.5. The embodiments of this application do not limit the value of the above viable ratio.

[0019] The aforementioned Acinetobacter endophyticus Sb 2-4 and Gordon's phthalate 107 are PAEs-degrading bacteria, and the aforementioned Bacillus 19743 is a growth-promoting bacterium.

[0020] The aforementioned Acinetobacter Sb 2-4 is a new strain. It was deposited at the China Center for Type Culture Collection (CCTCC) on December 1, 2025, with accession number CCTCC NO: M20252724 and deposit address: Wuhan University, Wuhan, China.

[0021] The aforementioned Acinetobacter endophyticus Sb 2-4 was deposited at the China Center for Type Culture Collection (CCTCC) on December 1, 2025, with accession number CCTCC NO: M20252724. The aforementioned Gordon's phthalate 107 was purchased from the China Industrial Microbial Culture Collection Center (CICC), with accession number CICC 24107, located at No. 50 Zhongling Street, Nanjing, Jiangsu Province. Bacillus 19743 was purchased from the China Agricultural Microbial Culture Collection Center (ACCC), with accession number ACCC 19743, located at Resource Building, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Haidian District, Beijing.

[0022] The 16S rRNA sequence of Acinetobacter endophyte Sb 2-4 is shown in SEQ ID NO.1. SEQ ID NO.1 includes the following.

[0023] The sequence similarity of the aforementioned endophytic Acinetobacter Sb 2-4 with existing PAE-degrading strains was compared. The comparison showed that the 16S rRNA sequence of the aforementioned endophytic Acinetobacter Sb 2-4 is similar to that of known degrading strains. Acinetobacter The sequence similarity of sp. M673 is 96.42% (<98.65%), which can be identified as a potential new species.

[0024] As is generally known in the field, the "Preliminary Identification of Bacterial Species Based on 16S rRNA Gene and Genomic Sequence" clearly states that when the similarity between the 16S rRNA gene sequences of two strains is less than 98.65%, they can be determined to belong to different species. Therefore, the aforementioned Acinetobacter endophyticus Sb 2-4 is a new strain.

[0025] The isolation and identification process of the aforementioned endophytic Acinetobacter Sb 2-4 is described below.

[0026] 1. Culture medium formulation 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; adjust the final pH of the inorganic salt medium to 7.0; add 1.5% (w / v) agar powder to the MSM solid medium.

[0027] LB medium: Yeast extract: 5.0 g; Tryptone: 10.0 g; Sodium chloride (NaCl): 10.0 g; Add ultrapure water to 1 L, adjust pH=7.0, sterilize at 121℃ for 20 min; For solid medium, add 1.5% (w / v) agar powder.

[0028] 2. Separation and Identification 2.1 Isolation and Culture of Strains The surface of Shanghai bok choy (grown in contaminated soil in Qixia District, Nanjing) was disinfected with 75% ethanol, followed by rinsing with sterile water to remove residual ethanol. The disinfected and cleaned Shanghai bok choy was transferred to a sterile mortar, chopped, and then ground thoroughly with sterile water. After standing for 5 min, 100 μL of the supernatant was transferred to an inorganic salt liquid medium containing PAEs (5 mg / L) and cultured in the dark at 30℃ and 150 rpm on a shaker. After 5 days, the medium was transferred to a new inorganic salt liquid medium containing PAEs (10 mg / L), and enriched for 5 days. The concentration of PAEs was then gradually increased, and the above steps were repeated. After 20 days of gradient concentration acclimatization, the culture medium was diluted 10...4 ~10 5 The culture was spread onto MSM solid medium containing 20 mg / L PAEs contaminants and incubated upside down at 30°C for 1–3 days. After single colonies grew on the plates, each colony was picked, streaked multiple times for purification, and a single bacterial strain was isolated and numbered Sb 2-4. The strain was then inoculated onto LB solid plates and incubated upside down at 30°C for 5 days, and its colony morphology was observed.

[0029] This strain was streaked on LB agar for 2 days. The colonies were pale yellow, round, opaque, with a raised, moist, sticky, and smooth surface. The specific morphology of this strain is shown below. Figure 1 As shown.

[0030] 2.2 Strain Identification Transmission electron microscopy (TEM) identification: The purified bacterial strain Sb 2-4 was inoculated into LB liquid medium and activated overnight. 1 mL of bacterial culture was centrifuged at 8000 rpm for 3–5 min, the supernatant was discarded, and the cells were washed three times with MSM. 1 mL of 2.5% (v / v) glutaraldehyde was added to the harvested bacterial precipitate and mixed thoroughly. The mixture was incubated overnight at 4°C. The fixative was discarded, and the cells were washed three times with 0.1 M, pH 7.0 PBS for 15 min each time. The samples were fixed with 1% osmium tetroxide solution for 1–2 h. The osmium tetroxide waste solution was carefully removed, and the samples were rinsed three times with 0.1 M, pH 7.0 PBS for 15 min each time. The samples were dehydrated with a gradient of concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 95%) for 15 min at each concentration, followed by treatment with 100% ethanol for 20 min. Finally, the samples were treated with pure acetone for 20 min.

[0031] Samples for transmission electron microscopy (TEM) were prepared using negative staining. First, a copper mesh with a support film treated with glow discharge was placed on a screen holder. 10 μL of sample suspension 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. Immediately afterwards, 10 μL of phosphotungstic acid (PTA) or uranium acetate-hydrogen oxide (URO) negative staining solution was 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 TEM and images were acquired and observed at an accelerating voltage of 80 kV. Figure 2 As shown, its morphological characteristics, as observed by transmission electron microscopy, are elliptical.

[0032] 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 (see SEQ ID NO.1 above) were compared for homology with 16S rRNA sequences reported in GenBank. Specifically, the 16S rRNA sequence of Acinetobacter Sb 2-4 was compared with the 16S rRNA sequences of other registered bacterial strains using the BLAST program on the NCBI website. The results showed that this strain was homologous to... Acinetobacter sp. It has the highest similarity, with a homology rate of 100%.

[0033] Phylogenetic analysis was performed on relevant bacterial species, and the results are as follows: Figure 3 As shown, the 16S rRNA gene sequence of the endophytic strain Sb2-4 isolated and purified in this invention is similar to that of Acinetobacter (…). Acinetobacter The strain obtained by screening in this invention showed the highest homology to *Acinetobacter sp.* (Genbank accession number MN515135.1). Therefore, the strain obtained by screening in this invention was identified as *Acinetobacter sp.* (Genbank accession number MN515135.1). Acinetobacter sp), named Acinetobacter sp Sb 2-4.

[0034] After isolating the above-mentioned Acinetobacter endophyte Sb 2-4, the verification process of the degradation effect of the above-mentioned Acinetobacter endophyte Sb 2-4 on PAEs is as follows.

[0035] 1. Preparation of bacterial suspension Endophytic Acinetobacter Sb 2-4 was inoculated into 100 ml LB medium and cultured at 30 °C and 150 rpm for 24 h. The culture was then centrifuged at 8000 rpm for 5 min. After washing twice with MSM, the bacterial OD values ​​were... 600nm The value was adjusted to 1.0 to prepare the bacterial suspension, and it was temporarily stored at 4 ℃ for later use.

[0036] 2. Acinetobacter Degradation performance determination of sp Sb 2-4 bacteria One mL of the above bacterial suspension was inoculated into 19 mL of MSM culture medium containing 20 mg / L PAEs (i.e., 20 mg / L DEP, 20 mg / L DBP, and 20 mg / L BBP). No inoculation was used as a control. The pH was adjusted to 7.0, with three replicates per group. The culture was incubated at 30°C and 150 rpm for 48 h in a constant-temperature shaker. Samples were taken every 6 h, and 40 mL of chromatographically pure methanol was added to the extracted conical flasks. 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 for analysis using high-performance liquid chromatography (HPLC).

[0037] Chromatographic conditions: An LC-20AT high-performance liquid chromatograph (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 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 in dual-wavelength detection mode at 225 nm and 290 nm.

[0038] Acinetobacter The degradation effect of sp Sb 2-4 bacteria on four mixed PAEs is as follows: Figure 4 As shown, this bacterium exhibits significant degradation effects on three types of PAEs (BBP, DEP, and DBP) under 3 days of shaking culture. Specifically, Acinetobacter spSb 2-4 showed a degradation rate of over 75% for BBP on day 5, and over 70% for DEP and DBP. (Note:) Acinetobacter sp Sb 2-4 has a highly efficient degradation ability for the three PAEs.

[0039] The following will verify whether there is an antagonistic effect among the endophytic Acinetobacter Sb 2-4, Gordon's phthalate 107, and Bacillus 19743 in the above-mentioned growth-promoting and pollution-eliminating bacterial group.

[0040] The above-mentioned endophytic Acinetobacter Sb 2-4, Gordon's phthalate 107, and Bacillus 19743 were streaked in pairs onto LB solid medium and incubated upside down in a 30°C incubator for 2-3 days. After incubation, the presence of a sterile zone at the streaked areas of the three strains was observed. The results are as follows: Figure 5 As shown, by Figure 5 It can be seen that there is no antagonistic reaction among the three strains mentioned above.

[0041] Given that *Gordonium phthalate* 107 possesses PAE degradation capabilities and *Bacillus thuringiensis* 19743 exhibits crop growth-promoting abilities, the above-described content of this embodiment further corroborates that *Acinetobacter endophyte* Sb2-4 possesses PAE degradation capabilities, while also verifying that there is no antagonistic reaction among *Acinetobacter endophyte* Sb2-4, *Gordonium phthalate* 107, and *Bacillus thuringiensis* 19743. It is conceivable that the aforementioned bacterial community composed of these three strains can simultaneously possess the ability to degrade phthalates and promote crop growth.

[0042] Example 2: This example describes a bacterial agent prepared based on the bacterial community provided in Example 1.

[0043] In one implementation, when the bacterial agent obtained from the preparation of the bacterial community is a bacterial suspension, the preparation process of the bacterial suspension includes the following steps.

[0044] Step 1: Inoculate endophytic Acinetobacter Sb 2-4, Gordon's phthalate 107, and Bacillus 19743 into 100 ml LB medium and incubate at 30 ℃ and 150 rpm for 24 h. Then centrifuge at 8000 rpm for 5 min to obtain activated endophytic Acinetobacter Sb 2-4, activated Gordon's phthalate 107, and activated Bacillus 19743.

[0045] Step 2: Wash the activated endophytic Acinetobacter Sb 2-4, activated Gordon's phthalate 107, and activated Bacillus aureus 19743 with inorganic salt culture medium. Combine the washed endophytic Acinetobacter Sb 2-4, Gordon's phthalate 107, and Bacillus aureus 19743 at a viable cell ratio of 1:1:1 to obtain OD. 600nm Bacterial suspension with a value of 1.

[0046] In some other specific embodiments, the above-mentioned live bacteria ratio can be 1.5:1:1 or 1:1.5:1.5. The embodiments of this application do not limit the value of the above-mentioned live bacteria ratio.

[0047] In another implementation, when the bacterial agent obtained from the bacterial community preparation is an immobilized bacterial agent, the preparation process of the immobilized bacterial agent includes the following steps.

[0048] Step 1: Inoculate endophytic Acinetobacter Sb 2-4, Gordon's phthalate 107, and Bacillus 19743 into 100 ml LB medium and incubate at 30 ℃ and 150 rpm for 24 h. Then centrifuge at 8000 rpm for 5 min to obtain activated endophytic Acinetobacter Sb 2-4, activated Gordon's phthalate 107, and activated Bacillus 19743.

[0049] Step 2: Wash the activated endophytic Acinetobacter Sb 2-4, activated Gordon's phthalate 107, and activated Bacillus aureus 19743 with inorganic salt culture medium. Combine the washed endophytic Acinetobacter Sb 2-4, Gordon's phthalate 107, and Bacillus aureus 19743 at a viable cell ratio of 1:1:1 to obtain OD. 600nm Bacterial suspension with a value of 1.

[0050] In some other specific embodiments, the above-mentioned live bacteria ratio can be 1.5:1:1 or 1:1.5:1.5. The embodiments of this application do not limit the value of the above-mentioned live bacteria ratio.

[0051] Step 3: After pyrolyzing rice straw or corn straw at 800℃ for 12 hours, activated carbon carrier is obtained.

[0052] Optionally, the pyrolysis temperature can also be 1200℃, and the pyrolysis time can also be 24h. This embodiment does not limit the pyrolysis temperature and time.

[0053] Step 4: Add the bacterial suspension to the activated carbon carrier at a bacterial substrate ratio of 20 mL: 1.5 g, and solidify it in a shaking incubator at 30 °C and 150 r / min for 0.5-4 days. Then, centrifuge it with a disc centrifuge to obtain the immobilized bacterial agent.

[0054] In some other specific embodiments, the above-mentioned substrate ratio can be 25mL:1.5g or 30mL:1.5g. The embodiments of this application do not limit the value of the above-mentioned substrate ratio.

[0055] Example 3: This example describes the application of growth-promoting and pollution-eliminating bacteria in promoting crop growth and remediating phthalate-contaminated soil, as provided in Example 1.

[0056] The above applications include using microbial agents prepared from microbial communities to achieve phthalate degradation and crop growth promotion. Specifically, the microbial agents prepared from microbial communities are added to phthalate-contaminated soil to promote crop growth and degrade phthalates.

[0057] Specifically, when the bacterial agent prepared from the microbial community is a bacterial suspension, the above application specifically includes: inoculating the bacterial suspension into farmland soil contaminated with phthalates to achieve phthalate degradation and promote crop growth. When the bacterial agent prepared from the microbial community 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 and promote crop growth. 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.

[0058] Furthermore, the aforementioned soil can be farmland soil contaminated with phthalates, or industrial or urban soil contaminated with phthalates; the method of treating soil with the microbial agent prepared from the microbial community to achieve phthalate degradation and crop growth promotion can be in-situ remediation, and the remediation temperature range can be 10-20℃. This application does not further limit the type of soil or the treatment method. The PAEs degradation ability of the microbial agent prepared from the above-mentioned growth-promoting and pollution-eliminating microbial community is verified below.

[0059] (1) Preparation of bacterial suspension Endophytic Acinetobacter Sb 2-4, Gordon's Phthalate 107, and Bacillus aureus 19743 were inoculated into 100 ml LB medium and cultured at 30°C and 150 rpm for 24 h, followed by centrifugation at 8000 rpm for 5 min. The bacteria were washed twice with MSM, and their OD values ​​were then... 600nm The value was adjusted to 1.0, and the three strains were combined in a 1:1:1 ratio to prepare a bacterial suspension, which was then compounded into a growth-promoting and pollution-eliminating bacterial group and stored at 4 ℃ for later use.

[0060] (2) Degradation performance of the microbial community One mL of the above bacterial suspension was inoculated into 19 mL of MSM culture medium containing 20 mg / L PAEs (i.e., 20 mg / L DEP, 20 mg / L DBP, and 20 mg / L BBP). No inoculation was used as a control. The pH was adjusted to 7.0, with three replicates per group. The culture was incubated at 30°C and 150 rpm on a shaker until day 5. Samples were taken, and 40 mL of chromatographically pure methanol was added to the extracted conical flask. The flask was then sonicated in a water bath for 1 h. After sonication, the sample 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 for analysis using high-performance liquid chromatography (HPLC).

[0061] Chromatographic conditions: An LC-20AT high-performance liquid chromatograph (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 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 in dual-wavelength detection mode at 225 nm and 290 nm.

[0062] The above-mentioned growth-promoting and pollution-eliminating bacterial groups have the following effects on the degradation of three mixed PAEs: Figure 6 As shown, by Figure 6 It can be seen that the growth-promoting and decontamination bacteria group has a significant degradation effect on three of the six mixed PAEs (DEP, DBP and BBP) within 5 days, with a degradation rate of over 60% on 5 days, indicating that the growth-promoting and decontamination bacteria group has a highly efficient degradation ability for the three PAEs.

[0063] Furthermore, comparing the degradation effect of the above-mentioned microbial community with the degradation effect of the microbial agent in the prior art application No. 202411049806.X entitled "A method for preparing biochar-supported synthetic microbial agent and its application in plant growth promotion and soil remediation", it can be seen that although the degradation rate of DBP by the above-mentioned microbial agent is higher than that of the microbial community provided in this embodiment, the microbial community provided in this embodiment has a clear broad-spectrum reduction effect on PAEs, that is, the above-mentioned microbial community has a good degradation effect on multiple PAEs at the same time.

[0064] The growth-promoting and pollution-eliminating bacterial community's growth-promoting ability will be verified below (same as the growth-promoting ability verification steps for single bacteria Sb 2-4).

[0065] (1) Determination of growth-promoting and pollution-eliminating bacterial flora growth-promoting indicators (1.1) Qualitative detection of nitrogen fixation capacity. The bacterial community was streaked on a nitrogen-free solid plate and cultured at 30°C for 7 days. The growth was observed and then subcultured three times. If the bacteria could grow, they had nitrogen fixation capacity.

[0066] (1.2) Determination of potassium-solubilizing capacity. Take 100 mL of potassium-solubilizing activity culture base and put it into a 250 mL Erlenmeyer flask. Inoculate each flask with 5 mL of bacterial suspension (the viable count is approximately 1 × 10⁻⁶). 8 CFU·mL -1 The control group was given an equal volume of sterile water. Each group had three replicates. The temperature was 28°C and the humidity was 160 r·min. -1 Cultured on a shaker for 7 days. Take the culture medium and incubate at 1000 r·min. -1 Centrifuge for 10 min, remove coarse residue, and then take 10 mL of bacterial culture at 10000 r·min. -1 Centrifuge for 10 min, and determine the available potassium content in the fermentation broth using atomic absorption spectrometry. Compare the result with a control group to determine the potassium solubilization efficiency. The potassium solubilization efficiency is described by the increase in available potassium, and the formula for calculating the increase in available potassium is shown below.

[0067] Increase in available potassium (%) = (available potassium content in the experimental group (mg·L)) -1 - Available potassium content in the control group (mg·L) -1 ))×0.1 L / (potassium feldspar powder content (g)×total potassium content (%))×100 (1.3) Quantitative determination of indoleacetic acid (IAA) production capacity. Collect the cultured bacterial cells, wash with sterile water, and resuspend. Inoculate the bacterial suspension at a 5% inoculum into 5 mL of nitrogenous medium containing L-tryptophan (20% 2.5 mg·mL⁻¹). -1 L-tryptophan stock solution, v:v), at 30°C, 180 r·min -1After 48 h of shaking culture, the culture medium was rotated at 8000 r·min -1 Centrifuge at high speed for 10 min; discard the precipitate, take 1 mL of supernatant and add it to a 5 mL centrifuge tube, and add 2 mL of Salkowski's colorimetric reagent and mix thoroughly. Place in the dark and incubate at 25°C for 30 min, then measure the color at 530 nm using a UV spectrophotometer. Set up the uninoculated group as the control.

[0068] (1.4) Siderophore determination. 1 mL of bacterial suspension was inoculated into 20 mL of nitrogenous medium and incubated at 30°C and 150 r·min. -1 Shaking culture for 48 h at 8000 r·min -1 Centrifuge at high speed for 10 min, take 3 mL of supernatant into a 10 mL centrifuge tube, add an equal volume of CAS detection solution, mix thoroughly, let stand for 1-2 h, and then measure the absorbance at 630 nm using a UV spectrophotometer. Separately, mix 3 mL of CAS detection solution with 3 mL of supernatant from uninoculated nitrogenous medium as a blank control group (CK). The ratio of the experimental group to the CK is used to determine the siderophore-producing capacity of the bacterial community.

[0069] (1.5) Phosphate solubility test. The strains were inoculated onto PKO inorganic phosphorus medium and Monkina organic phosphorus medium, respectively, and incubated at 30°C for 12 days. After incubation, the ratio of the diameter of the phosphorus-solubilizing transparent zone (D) to the colony diameter (d) was measured and calculated. The larger the ratio, the stronger the phosphorus-solubilizing ability; the smaller the ratio, the weaker the phosphorus-solubilizing ability. A ratio of 1 indicates that the colony has no phosphorus-solubilizing ability. Monkina medium was used for the organic phosphorus solubility test, and PKO medium was used for the inorganic phosphorus solubility test.

[0070] It should be noted that the above-mentioned Monkina medium and PKO medium are commonly used media in this technical field, and the formulations of the above two media will not be described in detail in this embodiment.

[0071] The results of the above-mentioned growth-promoting indicators are shown in Table 1 below.

[0072] Table 1 Results of the Measurement of Growth-Promoting Indicators

[0073] As shown in Table 1 above, the growth-promoting and pollution-eliminating bacterial group described in this embodiment has better potassium solubilization, iron carrier production, and inorganic phosphorus dissolution capabilities than the single bacterium Sb 2-4, thus exhibiting a more prominent ability to promote crop growth.

[0074] Furthermore, adopt Figure 4 and Figure 6The degradation abilities of the growth-promoting and pollution-eliminating bacterial community and the single-strain Sb 2-4 on PAEs (DEP, DBP, and BBP) were compared. For DEP, the degradation abilities of the growth-promoting and pollution-eliminating bacterial community and the single-strain Sb 2-4 were almost identical. For DBP, the degradation ability of the growth-promoting and pollution-eliminating bacterial community was stronger than that of the single-strain Sb 2-4. For BBP, the degradation abilities of the growth-promoting and pollution-eliminating bacterial community and the single-strain Sb 2-4 were almost identical. Therefore, the degradation ability of the growth-promoting and pollution-eliminating bacterial community provided in this embodiment is slightly higher than that of the single-strain Sb 2-4. Furthermore, the growth-promoting and pollution-eliminating bacterial community exhibits a more prominent crop growth-promoting ability compared to the single-strain Sb 2-4, indicating that the growth-promoting and pollution-eliminating bacterial community provided in this embodiment can achieve a strong crop growth-promoting effect while ensuring good PAE degradation.

[0075] (2) Verification of the crop growth-promoting ability of growth-promoting and pollution-eliminating bacteria Functional endophytic microbial community pot experiment: A root irrigation method was used to colonize growth-promoting and pollution-eliminating microbial communities. The specific procedure was as follows: Surface-sterilized Shanghai bok choy seeds were placed in a constant temperature incubator and cultured until they showed signs of emergence. Then, they were sown in PAE-free soil. On the 15th day after emergence, the soil around the plant roots was irrigated with a 5% (OD) bacterial solution (based on the total soil weight). 600 nm =1), and a blank control group CK was set up. The blank control group was treated with the same operation using an equal volume of sterile water. After 56 days, the growth status and other indicators of Shanghai bok choy were measured.

[0076] The comparison photos of Shanghai bok choy taken from the test results are as follows: Figure 7 As shown in the figure, the root length comparison diagram of Shanghai bok choy in the measurement results is as follows: Figure 8 As shown in the weight comparison chart... Figure 9 As shown in the figure, the comparison chart of chlorophyll content is as follows: Figure 10 As shown. By Figures 7 to 10 It can be seen that, regardless of the growth status of Shanghai bok choy, or the root length, weight, and chlorophyll content, the growth indicators of Shanghai bok choy irrigated with the growth-promoting and pollution-eliminating bacterial suspension are better than those of Shanghai bok choy irrigated without the growth-promoting and pollution-eliminating bacterial suspension.

[0077] This verifies that the growth-promoting and pollution-eliminating bacterial community provided in this embodiment can promote crop growth while ensuring the degradation effect of PAEs.

Claims

1. A group of growth-promoting and pollution-eliminating bacteria, characterized in that, The proliferative-disinfecting bacterial flora includes endophytic Acinetobacter (… Acinetobacter sp.) Sb 2-4, Gordon's phthalate ( Gordonia phthalatica )107 and Bacillus subtilis ( Bacillus subtilis The viable cell ratio of the endophytic Acinetobacter Sb 2-4, the phthalic acid Gordon's 107, and the growth-promoting Bacillus 19743 in the growth-promoting and decontamination bacterial group is 1-1.5:1-1.5:1-1.5; the endophytic Acinetobacter Sb 2-4 was deposited at the China Center for Type Culture Collection on December 1, 2025, and the accession number of the endophytic Acinetobacter Sb 2-4 is CCTCC NO: M20252724.

2. The application of the growth-promoting and pollution-eliminating bacterial community as described in claim 1 in promoting crop growth and remediating phthalate-contaminated soil.

3. The application as described in claim 2, characterized in that, The application includes: preparing a microbial agent using the growth-promoting and pollution-eliminating microbial community, and inoculating the microbial agent into farmland soil contaminated with phthalates to promote crop growth and degrade phthalates; wherein the inoculation amount of the microbial agent is 5-10% of the farmland soil mass.

4. The application as described in claim 3, characterized in that, The bacterial agent is 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 includes: Endophytic Acinetobacter Sb 2-4, Gordon's Phthalate 107, and Bacillus 19743 were activated to obtain activated endophytic Acinetobacter Sb 2-4, activated Gordon's Phthalate 107, and activated Bacillus 19743, respectively. Activated Acinetobacter endophyticus Sb 2-4, activated Gordon's phthalate 107, and activated Bacillus 19743 were mixed at a live bacteria ratio of 1-1.5:1-1.5:1-1.5 and prepared into 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 includes: Activated carbon carriers are obtained by pyrolyzing rice straw or corn straw at 800-1200℃ for 12-24 hours. The bacterial suspension was added to an activated carbon carrier at a bacterial substrate ratio of 20-30 mL: 1.5 g, and then solidified in a shaking incubator for 0.5-4 days. After centrifugation using a disc centrifuge, the immobilized bacterial agent was obtained.

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