A group of microbial flora for degradation of short side chain phthalate and repair of plant damage

Abstract

This invention provides a group of microbial flora for the degradation of short-chain phthalates and the repair of plant damage, belonging to the field of microbial technology. The aforementioned microbial flora includes *Arthrobacter* (…). Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas Paenarthrobacter sp. 60231 and Paenarthrobacter sp. PAEs3-4 were deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2026, with accession number CCTCC M 2026142. These bacterial groups can achieve plant damage repair during growth while ensuring the degradation of short-chain PAEs.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a group of bacteria used for the degradation of short-chain phthalates and the repair of plant damage. Background Technology

[0002] Short-chain phthalate esters (SC-PAEs), such as dimethyl phthalate (DMP) and diethyl phthalate (DEP), are important industrial plasticizers and solvents, widely used in agricultural films, packaging materials, and personal care products. Compared to long-chain PAEs, SC-PAEs have higher water solubility and mobility, making them commonly detected in agricultural soils, groundwater, and greenhouse soils, forming persistent pollution. SC-PAEs and their metabolic intermediates not only disrupt soil microbial community structure and inhibit plant seed germination and root development, but may also accumulate through the food chain, posing a direct threat to ecological security and agricultural product quality.

[0003] Currently, remediation methods for PAE-contaminated soils mainly include physical soil replacement, chemical oxidation, and bioremediation. Physical methods are costly and damage soil structure; chemical methods may introduce secondary pollution and adversely affect soil ecological functions. Bioremediation technology, with microorganisms at its core, has become a research hotspot due to its low cost and good environmental compatibility. Existing technologies mainly focus on screening single, highly efficient degrading strains (such as certain Pseudomonas and Bacillus strains) or using commercial microbial agents for bioaugmentation. However, these methods often face bottlenecks in practical applications: single strains have limited functions and are unable to cope with the complex spectrum of pollutants and dynamically changing intermediate products in the soil; after being introduced into the soil, exogenous functional bacteria are easily affected by factors such as competition from native microorganisms, nutrient deficiency, and environmental stress, leading to difficulties in colonization, unstable activity, and poor sustainability of remediation effects. Therefore, developing a complex functional microbial community that can be self-sustaining, synergistic, and adapted to the actual soil habitat has become the key to improving the effectiveness of bioremediation.

[0004] In recent years, "plant growth-promoting and pollution-eliminating" microbial communities that combine pollutant degradation and plant growth promotion functions have attracted attention. These communities can not only directly metabolize target pollutants but also promote plant growth by secreting plant hormones, dissolving phosphates, and producing siderophores, thereby enhancing the stability and efficiency of the "plant-microbe" co-remediation system through a "rhizosphere synergy" mechanism. However, the market and research currently lack specialized, standardized composite microbial community products that are specifically designed for the characteristics of SC-PAE-contaminated soils and possess both highly efficient degradation and stable plant growth-promoting capabilities. Meanwhile, existing microbial communities typically exert their respective abilities to degrade PAE-contaminated soil and promote plant growth. However, for plants in PAE-contaminated soil, existing strain remediation methods are insufficient to effectively address the complex PAE pollutant spectrum and dynamically changing intermediate products in the soil environment. This results in plant roots being under long-term stress, and their own antioxidant defense system is insufficient to completely remove excessive reactive oxygen species, leading to membrane lipid peroxidation, growth inhibition, and even plant death. This not only seriously threatens crop yield and quality but also poses an urgent risk to farmland ecological security and food supply.

[0005] Therefore, there is an urgent need for a complex functional microbial community that can stably colonize the rhizosphere, efficiently degrade PAEs, and continuously enhance the antioxidant and repair capabilities of plants. Through synergistic effects, it can directly eliminate pollutant stress and activate the plant's intrinsic defense mechanisms, thereby achieving root remediation of plants in contaminated sites. Summary of the Invention

[0006] This invention proposes a group of microbial communities for the degradation of short-chain phthalates and the repair of plant damage. These microbial communities have high degradation capacity and can repair plant growth damage.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a group of microbial communities for the degradation of short-chain phthalates and the repair of plant damage, the microbial community including Arthroblasts (… Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas sp.) 60231; the bacterial community contains Arthroblasts ( Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas The viable count ratio of sp.) 60231 is 1~2:1~2:1~2; the Arthroblastus ( Paenarthrobacter sp.) PAEs3-4 was deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2026. The described Arthroblastus ( Paenarthrobacter The accession number for sp.) PAEs3-4 is CCTCC M 2026142.

[0008] Among them, the above-mentioned Corynebacterium glutamicum ( Glutamicibacter sp.) A4 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC M20221850, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. *Pseudomonas aeruginosa* (sp.) PseudomonasSP.) 60231 was purchased from the China Agricultural Microbial Culture Collection Center (ACCC), with accession number ACCC 60231. 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.

[0009] In one implementation of the first aspect, Arthroblastus ( Paenarthrobacter The 16S rRNA sequence of PAEs3-4 (sp.) is shown in SEQ ID NO.1.

[0010] In a second aspect, the present invention provides a microbial agent made from a group of bacteria as described in the first aspect.

[0011] In one implementation of the second aspect, the bacterial agent is a bacterial suspension or an immobilized bacterial agent.

[0012] In one implementation of the second aspect, the method for preparing the bacterial suspension includes: activating Arthroblasts (… Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas sp.) 60231; Activated Arthroblasts ( Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas Sp.) 60231, mixed at a live bacteria count ratio of 1~2:1~2:1~2; and prepared into OD 600nm Bacterial suspension with a value of 1.

[0013] In one implementation of the second aspect, the method for preparing the immobilized bacterial agent includes: Rice straw or corn straw is pyrolyzed at 800~1200℃ for 12~24 hours to obtain activated carbon carrier; The bacterial suspension and the activated carbon carrier were mixed at a bacterial substrate ratio of 20-30 mL: 1.5 g to obtain a mixture; The mixture was placed in a shaking incubator and solidified at 25-30°C for 0.5-4 days. Then, it was centrifuged to collect the solid fraction, thus obtaining the immobilized bacterial agent.

[0014] Thirdly, the present invention provides the application of the microbial community described in the first aspect or the microbial agent of the second aspect, using the microbial community or microbial agent in the short-chain phthalates and / or in the repair of plant growth damage. The short-chain phthalates include dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate.

[0015] Fourthly, the present invention provides a method for degrading phthalates, based on the microbial agent described in the second aspect above; comprising the following steps: The bacterial suspension or the immobilized bacterial agent is uniformly mixed into the contaminated soil at an inoculation rate of 5-10% of the mass of the farmland soil contaminated with phthalates. The soil moisture content was controlled at 60-80% of field capacity, the temperature was 25-30℃, and the cultivation period was 30-90 days. The soil was tilled every 7 days, and the phthalate concentration in the soil was tested every 14 days.

[0016] Fifthly, the present invention provides a method for repairing plant damage, based on the microbial agent described in the second aspect above; comprising the following steps: The bacterial suspension was inoculated at a rate of 3-5% of the farmland soil mass and colonized using a root irrigation method. Surface-sterilized plant seeds (Shanghai bok choy seeds) were placed in a constant temperature incubator and cultured until they sprouted. They were then sown in PAE-contaminated soil. On the 15th day after emergence, the functional endophytic bacterial suspension was irrigated into the soil around the plant roots. The control group underwent the same procedure with an equal volume of sterile water. Alternatively, the immobilized bacterial agent can be added to the PAE-contaminated soil at a ratio of 3% by mass and mixed evenly, and then the surface-sterilized plant seeds (Shanghai bok choy seeds) can be sown in the soil.

[0017] Apply the fertilizer every 10 days, and take samples at 25, 35, 45 and 55 days of growth of Shanghai bok choy.

[0018] Compared with the prior art, the present invention has the following beneficial effects.

[0019] This invention provides the application of L1, a combination of degrading and growth-promoting bacteria, in the remediation of phthalate-contaminated soil. The strains *Paenarthrobacter* sp. *PAEs3-4* and *Glutamicibacter* sp. *A4* can utilize dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate as their sole carbon and energy sources for growth and reproduction. Under pure culture conditions, these bacteria can degrade over 90% of a 20 mg / L mixture of PAEs (containing 20 mg / L DMP, DEP, DBP, and BBP) in an inorganic salt medium within 7 days. The application described in this invention shows great promise in the bioreduction of environmental pollutants and the remediation of phytoremediation. Attached Figure Description

[0020] Figure 1 An antagonistic experiment diagram of three bacterial strains in bacterial community L1 provided in the embodiments of this application; Figure 2The degradation effect of bacterial community L1 on six mixed PAEs provided in the embodiments of this application is shown in the figure. Figure 3 This is a diagram illustrating the growth-promoting effect of microbial community L1 in a pot experiment, as provided in an embodiment of this application. Figure 4 The growth-promoting effect of the bacterial community L1 provided in the embodiments of this application on plants; Figure 5 The effect of bacterial community L1 provided in the embodiments of this application on the activity of peroxidase in plants. Detailed Implementation

[0021] In the specification and claims of this invention, the terms "first" and "second," etc., are used to distinguish different objects, rather than to describe a specific order of objects.

[0022] In the embodiments of this application, "and / or" indicates a relationship between objects. For example, A and / or B can represent the following three situations: A exists alone, B exists alone, and A and B exist simultaneously.

[0023] 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.

[0024] In the embodiments of the present invention, DEP is an abbreviation for diethyl phthalate, DBP is an abbreviation for dibutyl phthalate, BBP is an abbreviation for butyl benzyl phthalate, and DMP is an abbreviation for dimethyl phthalate; the tested leafy green vegetable is Shanghai bok choy.

[0025] Example 1: This example describes a group of microbial communities used for the degradation of short-chain phthalates and the repair of plant damage. These microbial communities include *Arthrobacter* (…). Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas sp.) 60231; the bacterial community contains Arthroblasts ( Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas The live bacteria count ratio of sp.) 60231 was 1:1:1; the Arthrobacterium ( Paenarthrobacter sp.) PAEs3-4 was deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2026. The described Arthroblastus ( PaenarthrobacterThe accession number for sp.) PAEs3-4 is CCTCC M 2026142.

[0026] In some other specific embodiments, the bacterial flora contains *Arthrobacter* (…). Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas The live bacteria ratio of sp.)60231 can be 2:1:1 or 1:2:2. The embodiments of this application do not limit the value of the above live bacteria ratio.

[0027] Specifically, the aforementioned Corynebacterium glutamicum ( Glutamicibacter sp.) A4 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC M20221850, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. *Pseudomonas aeruginosa* (sp.) Pseudomonas SP.) 60231 was purchased from the China Agricultural Microbial Culture Collection Center (ACCC), with accession number ACCC 60231. 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.

[0028] The above-mentioned arthrobacteria ( Paenarthrobacter The 16S rRNA sequence of PAEs3-4 (sp.) is shown in SEQ ID NO.1.

[0029] SEQ ID NO.1: (I) Arthroblasts ( Paenarthrobacter New species of sp.) PAEs3-4 have been identified; By using the above-mentioned Arthroblasts ( Paenarthrobacter The 16S rRNA sequence of sp.) PAEs3-4 was compared with that of existing PAEs-degrading strains. The comparison showed that the sequence of this strain was similar to that of known degrading strains. Rhodococcus The sequence similarity of sp. AH-ZY2 is 89.41% (<98.65%). Combined with the differences between the two in terms of PAEs degradation substrate preference and physiological and biochemical characteristics, it can be clearly determined that the strain of this invention is not the same species as the previously reported strain of the same genus, and has novelty at the species level, and can be identified as a potential new species. 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, it can be confirmed that the aforementioned *Arthrobacter* (… Paenarthrobacter sp.) PAEs3-4 is a new strain.

[0030] (II) Arthroblasts ( Paenarthrobacter Identification of PAEs3-4 (sp.); 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; for solid medium, add 1.5% (w / v) agar powder.

[0031] 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, and sterilize at 121℃ for 20 minutes; For solid medium, add 1.5% (w / v) agar powder.

[0032] 2. Separation and identification; Weigh about 5g of the test soil and add it to a 250mL Erlenmeyer flask. Add 100mL of ultrapure water and place it in a shaker at 30℃ and 150rpm in the dark for 8 hours. After 8 hours, remove the flask and let it stand for about two hours. The supernatant is the enriched initial indigenous microorganism.

[0033] Take 5 mL of supernatant and transfer it to 95 mL of inorganic salt medium containing PAEs (5 mg / L DEP, 5 mg / L DMP, 5 mg / L DBP, and 5 mg / L BBP). Incubate at 30 °C and 150 rpm for 5 days using a shaker. Then, continuously enrich the medium with 5 mL of the inoculum, subculturing 5 times, increasing the PAE concentration in the medium to 80 mg / L (i.e., containing 80 mg / L DEP, 80 mg / L DMP, 80 mg / L DBP, and 80 mg / L BBP; the PAE concentration after the first subculture is 5 mg / L; after the second subculture, it is 10 mg / L; after the third subculture, it is 20 mg / L; after the fourth subculture, it is 40 mg / L; and after the fifth subculture, it is 80 mg / L). The culture medium, after five acclimatization cycles, was diluted 10³ to 10⁴ and spread onto MSM solid medium containing 20 mg / L PAEs contaminants. It was then incubated upside down at 30°C for 1 to 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 labeled PAEs3-4. This strain was then inoculated onto LB solid plates and incubated upside down at 30°C for 5 days, during which colony morphology was observed.

[0034] This strain was streaked onto LB agar for 5 days. Colonies were round, pale yellow, opaque, smooth, moist, and raised, without a halo. See details for morphological information. Figure 1 .

[0035] 3. Observation and identification by transmission electron microscopy; The purified bacterial strain PAEs3-4 was inoculated into LB liquid medium and activated overnight. 1 mL of the bacterial culture was centrifuged at 8000 rpm for 3–5 min, the supernatant was discarded, and the bacteria 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 bacteria were washed three times with 0.1 M, pH 7.0 PBS for 15 min each time. The sample was fixed with 1% osmium tetroxide solution for 1–2 h. The osmium tetroxide waste solution was carefully removed, and the sample was rinsed three times with 0.1 M, pH 7.0 PBS for 15 min each time. The sample was 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 sample was treated with pure acetone for 20 min. The sample was treated with a mixture of embedding agent and acetone (V / V=1 / 1) for 1 h; then with a mixture of embedding agent and acetone (V / V=3 / 1) for 3 h; and finally with pure embedding agent overnight. The infiltrated sample was then embedded and heated at 70 °C overnight to obtain the embedded sample. The sample was sectioned using an ultramicrotome to obtain sections of 70–90 nm. The sections were stained with lead citrate solution and 50% ethanol-based uranium acetate solution for 5–10 min each, and then air-dried for observation under a transmission electron microscope. Scanning electron microscopy revealed that the morphology was elliptical. Figure 2 .

[0036] 4. Identification of the strain's 16S rRNA molecules; Total bacterial DNA was extracted, and the bacterial genome was amplified by PCR using universal primers for bacterial 16S rDNA. The PCR products were sequenced (by Shanghai Sangon Biotech), and the sequencing results were compared for homology with previously reported 16S rDNA sequences in GenBank. Phylogenetic analysis was then performed on relevant bacterial species. The results are as follows Figure 3 As shown, the 16S rDNA gene sequence of the strain PAEs3-4 isolated and purified in this invention is similar to that of Arthrobacter spp. ( Paenarthrobacter sp. (Genbank accession number MW578881.1) has the highest homology.

[0037] Therefore, the strains obtained by screening in this invention were identified as belonging to the genus Arthrobacter ( ). Paenarthrobacter sp.), named Paenarthrobacter sp. PAEs3-4.

[0038] Arthroblasts were isolated. Paenarthrobacter After PAEs3-4, the following is given: Arthroblastus (sp.) Paenarthrobacter (sp.) PAEs3-4: Verification process of PAEs' degradation ability.

[0039] 1. Preparation of bacterial suspension The bacterial strain was inoculated into 100 ml LB medium and incubated at 30 °C and 150 rpm for 24 h, followed by centrifugation at 8000 rpm for 5 min. The bacteria were then washed twice with MSM, and the OD values ​​were calculated. 600nm The value was adjusted to 1.0 to prepare the bacterial suspension, and it was temporarily stored at 4 ℃ for later use.

[0040] 2. Arthroblasts ( Paenarthrobacter Degradation performance determination of sp.) PAEs3-4; 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 DMP, 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 7 days. Samples were taken on days 1, 3, 5, and 7. 40 mL of chromatographically pure methanol was added to the collected Erlenmeyer flasks, and the mixture was 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).

[0041] 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.

[0042] Arthroblasts ( Paenarthrobacter The degradation effect of sp.) PAEs3-4 on the two mixed PAEs is as follows: Figure 4 As shown, this bacterium exhibits a significant degradation effect on both PAEs under 7 days of shaking culture. (Arthrobacterium tumefaciens) Paenarthrobacter sp.) PAEs3-4 showed a degradation rate of approximately 90% for both PAEs on day 1; by day 7, Arthroblastus ( Paenarthrobacter Paenarthrobacter sp. PAEs3-4 can almost completely remove DEP, DMP, DBP, and BBP from the system. This indicates that Paenarthrobacter sp. PAEs3-4 has a highly efficient degradation ability for both types of PAEs.

[0043] The following section discusses the arthropathogenic bacteria (Arthrobacter) in the above bacterial groups.Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas We need to verify whether there is an antagonistic effect between sp.)60231.

[0044] The three selected strains were streaked in pairs onto LB solid medium and incubated upside down in a 30°C constant temperature incubator for 2-3 days. After the incubation, the presence of a sterile area was observed at the streaked area of ​​the three strains. Figure 1 This demonstrates that there is no antagonistic reaction among the three strains.

[0045] Given that the above-mentioned *Glutamicinus A4* has the ability to degrade PAEs and the above-mentioned *Pseudomonas aeruginosa* (… Pseudomonas Given that sp.) 60231 has crop growth-promoting capabilities, the above-mentioned content of this embodiment further corroborates the above-mentioned Arthrobacterium (sp.) Paenarthrobacter sp.) PAEs3-4 not only possesses PAEs degradation ability, but also verifies the above-mentioned arthrobacteria (sp.) Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas There is no antagonistic reaction between sp.) 60231. It is conceivable that the above-mentioned bacterial community composed of the three strains can simultaneously possess the ability to degrade phthalates and repair plant growth damage.

[0046] Example 2: This example describes a bacterial agent prepared based on the bacterial community provided in Example 1.

[0047] 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.

[0048] Step 1.1, Arthroblastus ( Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas sp.)60231 was inoculated into 100 ml LB medium and cultured at 30 ℃ and 150 rpm for 24 h. After centrifugation at 8000 rpm for 5 min, three activated strains were obtained.

[0049] Step 1.2: After washing the three activated bacterial strains twice with MSM, the three strains were then recombined at a live bacteria ratio of 1:1:1 to obtain OD. 600nm The bacterial suspension with a value of 1 was stored at 4 °C for later use.

[0050] In some other specific embodiments, the above-mentioned live bacteria ratio can be 2:1:1 or 1:2:2. The embodiments of this application do not limit the value of the above-mentioned live bacteria ratio.

[0051] 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.

[0052] Step 2.1: Pyrolyze rice straw or corn straw at 800~1200℃ for 12~24 hours to obtain activated carbon carrier; Step 2.2: Mix the bacterial suspension obtained in step 1.2 with the activated carbon carrier at a bacterial substrate ratio of 20 mL: 1.5 g to obtain a mixture; The mixture was placed in a shaking incubator and solidified at 25-30°C for 0.5-4 days. Then, it was centrifuged to collect the solid fraction, thus obtaining the immobilized bacterial agent.

[0053] 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.

[0054] Example 3: This example describes the application of a group of bacteria provided in Example 1 in the degradation of phthalates and the promotion of crop growth.

[0055] 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 achieve phthalate degradation and crop growth promotion.

[0056] 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.

[0057] 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 microbial community to achieve phthalate degradation and crop growth promotion 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.

[0058] The PAEs degradation ability of the bacterial agent prepared from the above-mentioned bacterial community will be verified below.

[0059] (1) Preparation of bacterial suspension Three bacterial strains 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 then washed twice with MSM, and the OD values ​​were... 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 mixed to form a bacterial community and stored at 4 ℃ for later use.

[0060] (2) Degradation performance of the microbial community One mL of the bacterial suspension containing the above-mentioned bacterial groups was inoculated into 19 mL of MSM culture medium containing 20 mg / L 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 DnOP). No inoculation was used as a control. The pH was adjusted to 7.0, and each group was repeated in triplicate. The culture was carried out at 30°C and 150 rpm in a constant-temperature shaker until day 7. 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 degradation effect of bacterial community L1 on four mixed PAEs is as follows: Figure 2As shown, this bacterium exhibits significant degradation effects on four types of PAEs under 7-day shaking culture, with a degradation rate exceeding 90% on day 7, indicating that L1 has a highly efficient degradation capacity for the four types of PAEs.

[0063] The following section verifies the ability of the aforementioned microbial community to repair plant growth damage.

[0064] (1) Determination of growth-promoting indicators of gut microbiota ① Qualitative detection of nitrogen fixation capacity. The bacterial community was streaked on nitrogen-free solid plates and incubated at 30°C for 7 days. The growth was observed, and the culture was then subcultured three times. If growth was observed, the bacteria had nitrogen fixation capacity.

[0065] ② Determination of potassium-solubilizing capacity. 100 mL of potassium-solubilizing culture medium was placed in 250 mL Erlenmeyer flasks, with 5 mL of bacterial suspension (approximately 1 × 10⁸ CFU·mL⁻¹) added to each flask. An equal volume of sterile water was added to the control group. Each group was replicated in triplicate and cultured at 28℃ and 160 r·min⁻¹ for 7 days. The culture medium was then centrifuged at 1000 r·min⁻¹ for 10 min to remove coarse residue. 10 mL of the culture was then centrifuged at 10000 r·min⁻¹ for 10 min. The available potassium content in the fermentation broth was determined by atomic absorption spectrometry and compared with the control group to obtain the potassium-solubilizing efficiency. Increase in available potassium (%) = (Available potassium content in experimental group (mg·L⁻¹) - Available potassium content in control group (mg·L⁻¹)) × 0.1 L / (Amount of potassium feldspar powder (g) × Total potassium content (%)) × 100.

[0066] 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 L-tryptophan stock solution, v:v). Incubate at 30℃ and 180 r·min⁻¹ for 48 h on a shaker. Centrifuge the culture at 8000 r·min⁻¹ for 10 min. Discard the precipitate, add 1 mL of the supernatant to a 5 mL centrifuge tube, and simultaneously add 2 mL of Salkowski's colorimetric reagent and mix thoroughly. Place in the dark and incubate at 25℃ for 30 min. Measure the color at 530 nm using a UV spectrophotometer. A control group without inoculation was used.

[0067] ④ Siderophore determination. Inoculate 1 mL of bacterial suspension into 20 mL of nitrogenous medium, incubate at 30℃ with shaking at 150 rpm for 48 h, centrifuge at 8000 rpm for 10 min, and transfer 3 mL of the supernatant to a 10 mL centrifuge tube. Add an equal volume of CAS detection solution, mix thoroughly, and allow to stand for 1-2 h. Measure the absorbance at 630 nm using a UV spectrophotometer. Separately, mix 3 mL of CAS detection solution with 3 mL of uninoculated nitrogenous medium supernatant as a blank control group (CK). The ratio of the experimental group to the CK indicates the siderophore-producing capacity of the bacterial community.

[0068] 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. It should be noted that the Monkina and PKO media are commonly used media in this technical field, and the formulations of these two media will not be further described in this embodiment.

[0069] The results of the above five measurements are shown in Table 1 below.

[0070] Table 1 Results of the Measurement of Growth-Promoting Indicators

[0071] As shown in Table 1 above, the bacterial flora described in this embodiment has strong potassium solubilizing, iron-producing, and phosphorus-solubilizing abilities, thus having a better ability to promote crop growth.

[0072] (2) To verify the crop growth-promoting ability of the microbial community; Functional Microbial Community Pot Experiment: The functional microbial community pot experiment used a root irrigation method to colonize the growth-promoting and pollution-eliminating microbial community L1. The specific procedure was as follows: Surface-sterilized lettuce seeds were placed in a constant temperature incubator and cultured. After emergence, they were sown in PAE-free soil. On day 15 after emergence, the bacterial solution was irrigated into the soil around the plant roots. The control group underwent the same procedure with an equal volume of sterile water. Growth status and other indicators of Shanghai bok choy were measured on days 25, 35, 45, and 55. Results are as follows: Figure 4 .Depend on Figure 4 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 bacterial suspension are better than those of Shanghai bok choy irrigated without bacterial suspension.

[0073] This verifies that the microbial community provided in this embodiment can promote crop growth while ensuring the degradation effect of PAEs. It is conceivable that the aforementioned microbial community, while ensuring the degradation effect of PAEs and promoting crop growth, can also degrade phthalates in farmland soil without crop cultivation, or promote crop growth in farmland soil without phthalate contamination.

[0074] (3) Verify the ability of the microbial community to repair plant damage; Determination of POD activity in plants; determination of peroxidase in vegetables: A certain amount of tissue was weighed, and 1 mL of extraction buffer was added for homogenization in an ice bath. The mixture was centrifuged at 8000 rpm and 4℃ for 10 min, and the supernatant was collected and placed on ice for testing. Reagents 1, 2, and 3 were mixed in a ratio of 11 μL:4 μL:4 μL, and prepared immediately. 10 μL of sample and 190 μL of working solution were added sequentially to a 96-well plate, and the mixture was gently shaken to mix. The absorbance value A1 at 470 nm for 30 s and A2 after 5 min 30 s were recorded. ΔA = A2 - A1 was calculated. The formula for calculating peroxidase activity in vegetables is as follows: In the formula, V 反总 V is the total volume of the reaction system; 样 The volume of the sample to be added. Unit definition: One unit of enzyme activity is defined as the change in absorbance at 470 nm of 0.005 per minute per gram of tissue in each mL of reaction system.

[0075] The determination was carried out in accordance with Table 2 below; Table 2. Procedure for determining POD activity in vegetables

[0076] ① Early activation of the plant's antioxidant system by the microbial community; At the initial stage of cultivation (day 5), there was a significant difference in POD activity between the test tubes and the control tubes (p<0.05). Notably, in the early stages after the addition of microbial communities (days 3-5), the concentration of PAEs in the soil had not yet decreased significantly, but the antioxidant enzyme activity of the treated group plants was already significantly higher than that of the control group.

[0077] This phenomenon indicates that the microbial community described in this invention does not merely alleviate plant stress indirectly by reducing pollutant concentrations in the later stages; rather, it directly stimulates the plant's antioxidant defense system early in the colonization process by secreting signaling substances such as extracellular polysaccharides, small molecule peptides, or plant hormone analogs. This mechanism puts the plant in a "warning" or "enhanced" physiological state, thereby giving it a stronger intrinsic ability to resist subsequent pollution stress.

[0078] ②The microbial community continuously enhances the plant's restorative ability; Combination Figure 5 As shown, with the increase of culture days (day 15 to day 55), the POD activity in the test tube plants showed a gradual increasing trend, and was significantly higher than that in the control group throughout the entire growth period. This further confirms that the addition of functional microbiota not only triggered a transient stress response, but also continuously improved the activity of antioxidant enzymes in plants through long-term stable rhizosphere colonization, effectively clearing the excessive reactive oxygen species induced by pollutants and alleviating membrane lipid peroxidation damage.

[0079] In summary, the composite functional microbial community described in this invention significantly enhances the antioxidant and repair capabilities of plants through a dual pathway of early signal activation and long-term pollution reduction, thus achieving effective repair of plant damage under PAE pollution stress.

Claims

1. A group of microbial communities for the degradation of short-chain phthalates and the repair of plant damage, characterized in that, The bacterial flora includes Arthrobacterium ( Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas sp.) 60231; the bacterial community contains Arthroblasts ( Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas The viable count ratio of sp.) 60231 is 1~2:1~2:1~2; the Arthroblastus ( Paenarthrobacter sp.) PAEs3-4 was deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2026. The described Arthroblastus ( Paenarthrobacter The accession number for sp.) PAEs3-4 is CCTCC M 2026142.

2. A microbial inoculant, characterized in that, It includes the microbial community as described in claim 1 and an agriculturally acceptable carrier.

3. The microbial agent as described in claim 2, characterized in that, The bacterial agent is in the form of a bacterial suspension or an immobilized bacterial agent.

4. The microbial agent as described in claim 3, characterized in that, The OD of the microbial community in the bacterial suspension 600nm The value is 1, and the immobilized bacterial agent comprises an activated carbon carrier and the microbial community.

5. A method for preparing the bacterial suspension as described in claim 3, characterized in that, Includes the following steps: Activate Arthroblasts respectively ( Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas sp.) 60231; Activated Arthroblasts ( Paenarthrobacter sp.) PAEs3-4, Corynebacterium glutamicum (sp.) Glutamicibacter sp.) A4 and Pseudomonas aeruginosa (sp.) A4 and Pseudomonas aeruginosa ( Pseudomonas sp.)60231, mixed according to a live bacteria ratio of 1~2:1~2:1~2; And formulated into OD 600nm Bacterial suspension with a value of 1.

6. A method for preparing the immobilized bacterial agent as described in claim 3, characterized in that, Includes the following steps: Rice straw or corn straw is pyrolyzed at 800~1200℃ for 12~24 hours to obtain activated carbon carrier; The bacterial suspension and the activated carbon carrier were mixed at a bacterial substrate ratio of 20-30 mL: 1.5 g to obtain a mixture; The mixture was placed in a shaking incubator and solidified at 25-30°C for 0.5-4 days. Then, it was centrifuged to collect the solid fraction, thus obtaining the immobilized bacterial agent.

7. The microbial community as described in claim 1, characterized in that, The short-chain phthalates include dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate.

8. A method for degrading phthalates, based on the microbial agent of claim 3, characterized in that, Includes the following steps: The bacterial suspension or the immobilized bacterial agent is uniformly mixed into the contaminated soil at an inoculation rate of 5-10% of the mass of the farmland soil contaminated with phthalates. The soil moisture content was controlled at 60-80% of field capacity, the temperature was 25-30℃, and the cultivation period was 30-90 days. The soil was tilled every 7 days, and the phthalate concentration in the soil was tested every 14 days.

9. A method for repairing plant damage, based on the microbial agent according to claim 3, characterized in that, Includes the following steps: The bacterial suspension of the microbial community was inoculated at a rate of 3-5% of the quality of PAE-contaminated soil and colonized by root irrigation. Alternatively, the immobilized bacterial agent was added to the PAE-contaminated soil at a rate of 3% of the farmland soil quality and mixed evenly. Then, surface-sterilized plant seeds were sown in the soil. Then, the plant was fertilized every 10 days, and the peroxidase activity of the plant was measured at 25, 35, 45 and 55 days of plant growth. The peroxidase activity was used to characterize the degree of relief of plant stress caused by PAE pollution and the effect of damage repair.

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