Microbial synthetic flora with degradation of phthalate and growth promotion and application thereof

Abstract

This invention discloses a microbial synthetic flora with both phthalate (PAE) degradation and growth-promoting effects, and its applications. The synthetic flora provided by this invention contains *Gordonella isopyrene-eating*, *Rhodococcus pyridostigmine*, and *Azotobacter brasiliensis*. Inoculation with this synthetic flora improves soil physicochemical properties, promotes the decomposition of soil organic matter, and increases nitrogen, phosphorus, and potassium content. Furthermore, the three strains work synergistically to promote rice growth. Simultaneously, the synthetic flora reduces PAE residues in the soil, rapidly degrades long-chain di(2-ethylhexyl) phthalate and dibutyl phthalate, shortens their half-life, and significantly accelerates the removal of PAEs from the soil. In PAE-contaminated environments, the synthetic flora increases rice chlorophyll content, enhances photosynthetic capacity, and promotes rice biomass accumulation, while simultaneously reducing the absorption and accumulation of PAEs within the rice plant, thus achieving a dual effect of PAE degradation and rice growth promotion.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology. More specifically, it relates to a microbial synthetic flora that has both phthalate (PAE) degradation and growth-promoting effects, and its applications. Background Technology

[0002] PAEs are widely used as plasticizers and additives in various products. They easily migrate from various plastic products and additives into environmental media such as the atmosphere, water, and soil, causing pollution. PAEs belong to the endocrine disrupting compounds and pose serious threats to human health and the ecological environment. They are a key new pollutant of concern in the "New Pollutant Control Action Plan" promulgated by the State in 2022. Because PAEs easily accumulate in soil, when farmland soil is severely polluted by PAEs, it not only affects the soil's ecological function and crop growth and quality, but also affects the soil's physicochemical properties. Furthermore, PAEs are easily absorbed and accumulated by crops, thus posing a threat to human health through the food chain.

[0003] PAEs can be degraded in various ways, including natural photolysis, chemical catalytic oxidation, and biodegradation. Among these, microbial degradation of PAEs offers advantages such as high efficiency, stability, environmental friendliness, and economic feasibility. Current research shows that different strains of bacteria capable of degrading PAEs have been identified in the environment or plants, which can alleviate the stress of PAEs on plants and reduce PAE residues in soil and the accumulation of PAEs by plants. However, these studies have focused on individual strains specifically for the degradation of organic pollutants, with a greater emphasis on the degradation effect itself.

[0004] Because actual soil environments are complex and diverse, they not only contain various PAE contaminants but also many different indigenous microorganisms. Experimental single bacteria may have a much lower ecological niche than indigenous microorganisms, and when using degrading bacteria alone to remediate PAE-contaminated soil in the field, they may not coexist well with indigenous microorganisms, lacking an advantage in resource competition and leading to the elimination of functional bacteria, resulting in a significant reduction in degradation efficiency when applied to soil. Most PAE-degrading strains are studied under laboratory conditions, exhibiting limited degradation effects and facing difficulties in practical application. Therefore, for practical applications of bioremediation, currently studied single-degrading bacteria struggle to adapt to environmental conditions and achieve optimal degradation. Artificially synthesized microbial communities, on the other hand, offer more diverse metabolic pathways and a broader range of growth environments than single bacteria. Their reproducibility and controllability in addressing complex natural environmental microbial community issues make them a popular approach for soil remediation. However, there are currently few reports on optimizing different functional strains through synthetic microbial technology to prepare synthetic microbial communities with multiple functions. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned defects and deficiencies, and to provide a microbial synthetic flora that has both the function of degrading PAEs and promoting growth, and its application.

[0006] The first objective of this invention is to provide a microbial synthetic flora that has both the function of degrading PAEs and promoting growth.

[0007] The second objective of this invention is to provide applications for synthetic microbial communities.

[0008] The third objective of this invention is to provide a microbial agent that can both degrade PAEs and promote rice growth.

[0009] The fourth objective of this invention is to provide a method for degrading PAEs and / or promoting rice growth.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a microbial biosynthesis group that combines the functions of degrading phthalates and promoting growth, composed of *Gordonella foenum-graecum* (…). Gordonia polyisoprenivorans ), Pyridine-loving Rhodococcus ( Rhodococcus pyridinivorans ) and Brazilian azospira ( Azospirillam brasilense )composition.

[0011] This invention employs synthetic microbial technology to degrade PAEs. Gordonia polyisoprenivorans and Rhodococcus pyridinivorans And has growth-promoting function for rice Azospirillam brasilense Three different bacterial species were successfully used to construct a functional microbial community (JQ). Studies showed that the species exhibited no antagonistic effects and were able to stably colonize, survive, and occupy a certain proportion in the soil. Inoculation with JQ improved soil physicochemical properties, promoted the decomposition of organic matter, and increased soil nitrogen, phosphorus, and potassium content, with alkaline-available nitrogen, available potassium, and available phosphorus increasing by 11%–39.2%. Simultaneously, the three bacteria showed synergistic effects, with JQ promoting rice growth and significantly increasing plant height, fresh weight, root length, root surface area, and root tip number. Inoculation with JQ also helped rice resist PAE stress, significantly increasing photosynthetic rate, transpiration rate, stomatal conductance, and intercellular carbon dioxide concentration, while reducing the absorption and accumulation of PAEs by plants. Inoculation with JQ reduced the accumulation of PAEs in the aboveground and belowground parts of rice by 57.6%–73.5% and 52.2%–62.2%, respectively, indicating that JQ can promote rice growth under PAE pollution conditions.

[0012] Furthermore, this invention demonstrates that the microbial community JQ can grow in soil using dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) as the sole carbon source. Within 5 days, it can degrade 90.1% and 86.2% of DBP and DEHP at an initial concentration of 200 mg / L, respectively, which is significantly more effective than individual degrading bacteria, achieving better results while effectively reducing the inoculation dosage. Moreover, the microbial community JQ significantly reduces the half-life, enabling faster pollutant removal with a 53%–63% reduction in time. Inoculation with the microbial community JQ significantly reduces PAE residues in the rice rhizosphere microdomain, and the microbial community JQ continues to exert its effect, still degrading PAEs up to day 120.

[0013] Therefore, the following applications of the synthetic microbial community provided by this invention are all within the scope of protection of this invention: Application of synthetic microbial communities in promoting rice growth and degrading soil PAEs, or in promoting rice growth under PAE pollution.

[0014] Application of synthetic microbial communities in the preparation of PAE-degrading bacterial agents and / or rice growth-promoting bacterial agents.

[0015] Preferably, the PAEs are dibutyl phthalate and / or di(2-ethylhexyl) phthalate.

[0016] The application of synthetic microbial communities as microbial fertilizers or in the preparation of microbial remediation agents, and the application of said synthetic microbial communities in promoting the decomposition of soil organic matter and increasing the nitrogen, phosphorus and potassium content of soil.

[0017] Preferably, the OD values ​​of *Gordonella pyrenoidosa*, *Rhodococcus pyridostigmine*, and *Azotobacter brasiliensis* in the synthetic microbial community are... 600 =0.5±0.05, volume ratio is 1:(0.8~1.5):(0.8~2).

[0018] More preferably, the volume ratio of *Gordonella pyrenoidosa*, *Rhodococcus pyridostigmine*, and *Azotobacter brasiliensis* is 1:1:1.

[0019] In particular, the synthetic microbial community used in this invention includes *Gordonella foenum-graecum* (Gordonella foenum-graecum). Gordonia polyisoprenivorans ) and Rhodococcus pyridostigmine ( Rhodococcus pyridinivorans Both have the function of degrading PAEs contamination, and *Azotobacter brasiliensis* (Braziliana) Azospirillam brasilenseThese bacteria can produce plant hormones and secrete siderophores, thereby promoting plant growth. While the effects may vary between different strains, the effects are significantly enhanced and consistent when constructed into a synthetic bacterial community. As the most preferred embodiment, this invention provides a synthetic bacterial community constructed from *Gordonella glutinis* L191, *Rhodococcus pyridostigmine* XB, and *Azotomyces brasiliensis* L25, or a synthetic bacterial community constructed from *Gordonella glutinis* L191, *Rhodococcus pyridostigmine* XB, and *Azotomyces brasiliensis* 1.1899. *Azotomyces brasiliensis* 1.1899 and L25 both have growth-promoting effects, and verification has shown that their synthetic bacterial communities have consistent and insignificant effects, making them interchangeable.

[0020] Specific strain information is as follows: *Gordonella oryzae* strain L191, deposited at the Guangdong Provincial Microbiological Culture Collection Center on September 24, 2021, accession number GDMCC NO. 61949; *Rhodococcus pyridococcus* strain XB, deposited at the Guangdong Provincial Microbiological Culture Collection Center on September 28, 2016, accession number GDMCC NO. 60054; *Azospirillum brasiliensis*... Azospirillam brasilense L25 (Liu Lihui. Genetic diversity and identification of new species of endophytic fungi in wild rice and Kalanchoe pinnatifida in southern China [D]. South China Agricultural University, 2018. DOI:10.27152 / d.cnki.ghanu.2018.000016.); Azotobacter brasiliensis 1.1899 was deposited at Guangdong Provincial Microbial Culture Collection Center on September 16, 2020, with accession number GDMCC1.1899.

[0021] This invention provides a microbial agent for degrading PAEs and / or promoting rice growth, containing the above-mentioned synthetic microbial groups or their bacterial solutions.

[0022] The present invention also provides a method for degrading PAEs and / or promoting rice growth, wherein the rice is treated with the above-mentioned synthetic microbial community or its bacterial solution.

[0023] Preferably, the inoculum size of the bacterial solution is 1-5%, and the OD value is... 600 =0.5±0.05.

[0024] Preferably, the bacterial solution of the synthetic microbial community is applied to the near-root zone of rice.

[0025] The present invention has the following beneficial effects: This invention provides a biosynthetic microbial community that can degrade PAEs and promote growth. The community contains *Gordonella isopyramidalis*, *Rhodococcus pyridostigmine*, and *Azotobacter brasiliensis*. Studies show that inoculation with this community can improve soil physicochemical properties, promote the decomposition of soil organic matter, and increase nitrogen, phosphorus, and potassium content. The three bacteria work synergistically to promote rice growth. Simultaneously, the community can reduce PAE residues in the soil and rapidly degrade DEHP and DBP, with degradation rates of 90.1% and 86.2%, respectively. Its effect is significantly greater than that of individual degrading bacteria, demonstrating… At the same dosage, the microbial community JQ showed better efficacy than single-strain microorganisms, achieving better results while effectively reducing the dosage. Furthermore, the microbial community JQ significantly reduced the degradation half-life, enabling faster removal of pollutants by 53%–63% and increasing the degradation rate of soil PAEs by 62.56%–73.17%. In PAE-contaminated environments, it increased rice biomass, relative chlorophyll content, and photosynthetic capacity, while reducing the absorption and accumulation of PAEs in rice, thus jointly achieving the degradation of PAEs and promoting growth. Attached Figure Description

[0026] Figure 1 The figures show the growth of colonies at 0.6 cm and 1.2 cm of strains L191 and L25(a), and XB, respectively, with L191 and L25(b). Figure 2 The relative proportions of strains XB, L191, and L25 in LB and sterilized soil, respectively (L represents LB, S represents soil, and the numbers represent parallel samples). Figure 3 Photographs of rice plants and roots (from left to right, the concentration treatments are: 0, 100, 100+JQ, unit: mg / L). Figure 4 These are scanned images of rice roots (concentration treatments are as follows: a: F0, b: F0+JQ, c: F20, d: F20+JQ, e: F100, f: F100+JQ. F represents different rice treatment groups, and 0, 20, and 100 represent PAE concentrations in mg / L). Figure 5 The graph shows the comparison of degradation rates of two PAEs by single bacteria and synthetic bacterial groups (significant differences were compared between DBP and DEHP in different bacterial cultures, with lowercase letters used to indicate significant differences in DBP treatment and uppercase letters used to indicate significant differences in DEHP treatment (P<0.05)). Figure 6 Degradation kinetics of DBP and DEHP in soil under different treatments; Figure 7 The trend of total concentration of two PAEs in the root growth chamber of rice over time; Figure 8 Changes in PAE residue levels in soil of different rhizosphere zones of rice under different treatments; Figure 9 The PAE content in different parts of rice under different PAE concentrations. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] LB broth medium (g / L): 10g peptone, 5g sodium chloride, 5g yeast extract, 20g agar (for solid culture medium).

[0030] Basic salt medium (g / L): K2HPO4 5.8g, KH2PO4 4.5g, (NH4)2SO4 2.0g, MgCl2 0.16g, CaCl2 0.02g, Na2MoO4·2H2O 0.0024g, FeCl3 0.0018g, MnCl2·2H2O 0.0015g.

[0031] After preparing the culture medium, adjust the pH value to 7.0, and sterilize it in a high-temperature and high-pressure autoclave at 121 ℃ for 20 min before use.

[0032] The DBP and DEHP used in this invention were purchased from Aladdin Chemical Co., Ltd. in Shanghai, with a purity of >98%; the standards DBP, DEHP, MBP, MEHP, and PA were purchased from Shanghai Anpu Experimental Technology Co., Ltd.; the chromatographically pure methanol, dichloromethane, and acetone were purchased from Sigma-Aldrich, USA; and the remaining reagents were of analytical grade and were purchased from Tianjin Damao Reagent Co., Ltd.

[0033] The rice seeds used in this invention were all purchased from the Guangdong Academy of Agricultural Sciences. The test soil was collected from the 0-20 mm tillage layer of a farm, air-dried, and sieved before use. The physicochemical properties of the control soil (uninoculated, uncontaminated, and unplanted) were as follows: pH 5.81, available nitrogen content 41.19 mg / kg, available potassium content 110.72 mg / kg, organic matter content 27.62 g / kg, and available phosphorus content 40.46 mg / kg.

[0034] All experimental data were processed using Excel 2015 (Microsoft, USA) and analyzed using SPSS 26.0 (IBM, USA) in a one-way ANOVA. A p-value < 0.05 was considered statistically significant. GraphPad Prism 8.0 was used to create the graphs.

[0035] Example 1: Construction of Functional Microbial Community JQ 1. Test strains The strain used in this invention is *Rhodococcus pyridostigmine*, which our research group previously screened from activated sludge and found to have a degrading effect on DEHP. Rhodococcus pyridinivorans XB, this strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on September 28, 2016, with accession number GDMCC NO. 60054; *Gordonella edodes*, an endophytic bacterium that degrades DBP, isolated from wild rice in southern China. Gordonia polyisoprenivorans L191, this strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on September 24, 2021, with accession number GDMCC NO. 61949; and *Azotobacter brasiliensis*, which has a growth-promoting function on rice (…). Azospirillam brasilense L25, this strain is preserved by the research team of this invention and has been isolated and identified, as recorded in the prior art: Liu Lihui. Genetic diversity and identification of new species of endophytic fungi in wild rice and Kalanchoe pinnatifida in southern China [D]. South China Agricultural University, 2018. DOI:10.27152 / d.cnki.ghanu.2018.000016.

[0036] 2. Preparation of seed fungal suspension Single colonies of strains XB, L191, and L25 were picked from purified plates and placed in sterile LB broth. The Erlenmeyer flasks were sealed and incubated overnight at 30°C and 150 rpm in a shaker. The bacteria were then separated from the culture medium using a high-speed centrifuge (5000 rpm, 5 min). The bacteria were washed three times with 0.9% sterile saline, and the suspension was resuspended. The OD value was adjusted using a UV spectrophotometer. 600 The value was 0.5. In all experiments of the invention, the inoculum size (OD) of the strain used was [value missing]. 600 =0.5) are all 2% (v / v), unless otherwise stated.

[0037] 3. Plate antagonism test Seed culture of three bacterial strains (XB, L191, and L25) cultured overnight was pipetted at 1.5 μL onto labeled LB agar plates. For each pair of strains, bacterial colony markers were placed at distances of 0.6 cm and 1.2 cm (1.2 cm served as a control for normal colony growth, while 0.6 cm was used to observe whether close proximity between the two colonies caused antagonism). The inoculated plates were then incubated at 30 °C in the dark for 48 h, with each treatment repeated three times. Colony morphology was observed to analyze whether antagonistic or symbiotic relationships existed among the three strains.

[0038] Strains XB, L191, and L25 were inoculated at different distances on LB agar plates to observe the pairwise interactions. The results are as follows: Figure 1 As shown, at a spacing of 1.2 cm, XB, L191, and L25 all exhibited normal growth morphology. When the sampling distance was 0.6 cm, although the colonies were close together, they all maintained normal morphology, indicating that they did not affect each other's growth. This shows that strains XB, L191, and L25 are not antagonistic to each other in terms of growth.

[0039] 4. Artificial construction of functional microbial communities JQ The three strains of bacteria that showed no antagonistic relationship after verification in the above steps were mixed in equal volumes to construct a functional bacterial community JQ. Then, equal volumes of the seed bacterial suspensions (OD) of the three strains were taken separately. 600 =0.5) mixed together to form a synthetic bacterial suspension. When inoculating into the culture medium or soil, the bacterial group is used by simultaneous inoculation, that is, three strains (OD) are always inoculated simultaneously in equal volumes. 600 =0.5).

[0040] Example 2: Population structure and stability of functional bacterial community JQ The bacterial community JQ was inoculated into LB liquid medium and sterilized, uncontaminated soil, respectively. The soil was cultured in LB medium for 24 h, and the soil inoculated with JQ was sealed with newspaper and placed in a biochemical constant temperature incubator for 7 days. Each sample was tested in triplicate. Samples were then sent to OE Biomedical Technology Co., Ltd. for high-throughput sequencing analysis of the microbial composition of the JQ community. Soil bacterial DNA was extracted using the MagPure Soil DNA Kit (OE, Shanghai). The concentration and quality of the extracted DNA were then detected using agarose gel electrophoresis and NanoDrop 2000. Library construction, sequencing, and data analysis were also performed by Shanghai OE Biomedical Technology Co., Ltd.

[0041] After preprocessing the sequencing data to generate high-quality sequences, Vsearch software was used to align and annotate all representative sequences with databases. 16S sequences were aligned using the Silva (version 132) database, retaining annotation results with confidence intervals greater than 0.7.

[0042] The results are as follows Figure 2 As shown, strains XB, L191, and L25 coexisted in both liquid culture medium and soil matrix, each accounting for a certain proportion. The genus containing strain L25 had the highest relative abundance, at 90% and 67%, respectively, followed by strain XB at approximately 6% and 10%, while the genus containing strain L19 had a relatively low proportion, at approximately 2% and 5%. Sequencing results from LB liquid medium and soil confirmed that strains XB, L191, and L25 remained stable in both LB medium and soil after 7 days, indicating the successful construction of the bacterial community JQ.

[0043] In soil without added pollutants, strains L25, L191, and XB all grew, with L25 exhibiting a higher proportion, indicating that L25 occupies an ecological niche and plays a growth-promoting role in the soil. High-throughput sequencing results demonstrated that even after equal volumes of bacterial solutions from each strain were added to the soil, they still grew rapidly, indicating that the bacterial community could successfully colonize the soil.

[0044] Furthermore, this embodiment also verified the use of different volume ratios of the bacterial community JQ, such as 1:0.8:0.8 or 1:1.5:2. The results showed that only at OD... 600 When the concentration of the synthetic microbial community is 0.5 ± 0.05 and the volume ratio is 1:1:1, the synthetic microbial community can stably colonize and grow in the soil without affecting its functional effects.

[0045] Example 3: Effects of functional microbial community JQ on soil physicochemical properties Sterilized, contaminant-free rhizosphere soil samples were collected, and five indicators—pH, available nitrogen, available phosphorus, available potassium, and organic matter—were measured before and after inoculation. Pretreatment: Before measuring the soil's physicochemical properties, the soil underwent air drying, pulverization, and sieving. The prepared samples were then sealed and stored in a cool, dry place. The bacterial suspension of the JQ bacterial community was mixed with the soil at an inoculation rate of 5% of the soil volume. Sampling was performed 30 days after inoculation, with three replicates for each treatment. Soil pH was determined using the potentiostatic method; available phosphorus was determined using the sodium bicarbonate extraction-molybdenum antimony spectrophotometric method; available nitrogen was determined using the ferrous sulfate method; available potassium was determined using neutral ammonium acetate solution; and soil organic matter was determined using the potassium dichromate oxidation with external heating method. Each treatment was measured three times. The obtained data were calculated, classified, and organized. Significance analysis was performed on the data for each physicochemical indicator, with a 5% confidence interval.

[0046] Table 1 shows the soil fertility and pH values ​​of different treatments in different rhizosphere zones. The pH values ​​ranged from 5.89 to 6.08 in both the near-rhizosphere and non-rhizosphere zones. A significant correlation was found between available nitrogen in rice and JQ inoculation (P<0.05), with JQ inoculation increasing the content of available nitrogen by 29.96%–33.60%. JQ inoculation also significantly increased the content of available potassium (P<0.05), with a 15.05% increase in the non-rhizosphere zone and a 33.82% increase in the near-rhizosphere zone. After JQ inoculation, the organic matter content decreased significantly (P<0.05), and the contents of organic matter and available potassium were significantly higher in the non-rhizosphere zone than in the near-rhizosphere zone (P<0.05). Available phosphorus was significantly higher in the non-rhizosphere zone than in the near-rhizosphere zone. This indicates that the microbial community JQ can promote the decomposition of soil organic matter and increase nitrogen, phosphorus, and potassium content.

[0047] Table 1. Soil physicochemical properties in different rhizosphere zones under different treatments

[0048] Note: Different letters in each column indicate significant differences (P<0.05); inoculation refers to the inoculated bacterial group JQ.

[0049] Example 4: Growth-promoting effect of functional microbial community JQ on rice under PAE pollution stress 1. Hydroponic rice experiment Nutrient solutions for rice growth were prepared using the international Hogland nutrient solution formula. Rice plants were planted in clean beakers containing 2L of nutrient solution, with the beakers wrapped in aluminum foil (to prevent photodegradation by PAEs and algae growth). The beakers were then covered with circular linden wood boards with five small holes. Five uniformly growing rice plants were selected for each hole as a cluster, with five clusters of plants per beaker. To avoid burning the seedlings, the plants were cultured with a 1 / 2 concentration of nutrient solution for the first 5 days, then switched to a full-concentration nutrient solution containing PAEs. PAE concentrations were set at 0, 20, and 100 mg / L, and the methanol content as a solvent did not exceed 0.1% (to avoid plant damage). Rice samples were collected 30, 60, and 120 days after transplanting. All parts of the rice were washed with deionized water, dried, and then the corresponding indicators were measured.

[0050] 2. Rice biomass measurement When collecting potted samples, the above-ground parts (stems and leaves) and underground parts of rice in each pot were weighed (fresh weight) and the plant height was measured. Each sample was measured three times and the average value was taken. After the measurement, the samples were put into kraft paper bags, freeze-dried in a freeze dryer, and then weighed as the rice biomass index.

[0051] 3. Rice root system scan After washing the rice with deionized water, the above-ground parts and roots were cut apart. The roots were placed in a root scanner with water added to ensure that each root did not overlap. The root scanner scanned root parameters such as root length (cm) and root surface area (cm²). 2 ), Root diameter (mm), Root volume (cm³) 3 The number of root tips, etc., were analyzed and exported using WinRHIZO software.

[0052] 4. Determination of photosynthesis and relative chlorophyll content On sunny days, between 9:00 AM and 11:00 AM, photosynthetic parameters of the second-to-last functional leaves of rice plants treated as described above were measured using a portable photosynthesis meter. Six plants were measured for each treatment, with four replicates per plant. The measured parameters were intercellular CO2 concentration (Ci), stomatal conductance (Gs), net photosynthetic rate (Pn), and transpiration rate (E). A portable chlorophyll content meter (SPAD502, Japan) was used to measure the transmittance of rice leaves in two wavelength ranges to determine the relative amount of chlorophyll in the leaves.

[0053] 5. Results The biomass results of rice under different treatments are shown in Table 2 below. Inoculation with JQ significantly increased the biomass of rice under normal, pollution-free conditions and promoted rice growth. Simultaneously, under different concentrations of PAE pollutants, the plant height of rice treated with JQ was significantly increased compared to the untreated group (P<0.05), increasing by 25.9%. Figure 3 It is known that high concentrations of PAEs inhibit rice growth, while inoculation with JQ can alleviate this stress.

[0054] Table 2 Biomass of hydroponic rice under different treatments

[0055] Note: ① 0, 20, and 100 are the PAEs treatment concentrations, in mg / L; ② Inoculation refers to the inoculated bacterial group JQ; ③ Different letters in each column indicate significant differences (P<0.05).

[0056] The root morphology of rice changes under different concentrations of PAEs stress, such as Figure 4 As shown, inoculation with JQ promotes root growth. Figure 4 (a and b) The effect of low concentrations of PAEs (20 mg / L) on root growth was not significant. Figure 4 c and d); high concentrations of low concentrations of PAEs (100 mg / L) significantly inhibited root growth, and the inhibition was alleviated after inoculation with JQ. Figure 4The differences in the changes of different indicators of rice root system with different treatments are shown in Table 3 below. Under the absence of PAEs stress, the root area, root volume and root tip number of rice in the inoculated group were significantly greater than those in the uninoculated group. With increasing PAE concentration, root parameters such as root length, root surface area, root diameter, root volume, and root tip number in rice decreased by 29.1%–86.1%, respectively. However, after inoculation with JQ, the inhibitory effect of PAE on root length, root surface area, root volume, and root tip number in rice was significantly reduced (P<0.05). Under 100 ppm stress, the root length, root surface area, root volume, and root tip number in rice inoculated with JQ were 2.14, 1.81, 1.45, and 1.79 times that of uninoculated rice, respectively.

[0057] Table 3. Rice root system indicators under different treatments

[0058] Note: ① 0, 20, and 100 are the PAEs treatment concentrations, in mg / L; ② Inoculation refers to the inoculated bacterial group JQ; ③ Different letters in each column indicate significant differences (P<0.05).

[0059] The effects of inoculating microbial community JQ on rice photosynthesis and relative chlorophyll content are shown in Table 4. With increasing PAE concentration, the stomatal conductance (Ci), intercellular carbon dioxide concentration (Gs), net photosynthetic rate (Pn), and transpiration rate (E) of rice all decreased significantly (P<0.05). Specifically, the decreases in Ci, Gs, Pn, and E were 12.8%, 22.4%, 38.4%, and 22.8%, respectively. This indicates that rice has good tolerance to high concentrations of PAE stress, and the specific numerical changes reflect its ability to absorb and accumulate organic matter.

[0060] Table 4 Gas exchange and chlorophyll content in rice leaves

[0061] Note: ① 0, 20, and 100 are the PAEs treatment concentrations, in mg / L; ② Inoculation refers to the inoculated bacterial group JQ; ③ Different letters in each column indicate significant differences (P<0.05).

[0062] Example 5: The degradation capacity of functional microbial community JQ on PAEs in soil 1. Functional microbial community JQ degrades PAEs in soil Artificial preparation of PAE-contaminated soil: Using acetone as a solvent, working stock solutions of DBP and DEHP at certain concentrations were prepared and mixed with soil samples that had passed through 1 mm and 5 mm sieves, respectively, to prepare soils containing DBP and DEHP at concentrations of 0, 20, and 100 mg / kg. After thorough mixing with a mixer, the soils were left to age naturally in the dark for 15 days.

[0063] PAEs-contaminated soil remediation experiment: 4 kg of soil with different concentrations of contaminants was placed in each clean beaker, and bacterial suspensions of single bacteria XB, L191, and bacterial community JQ were inoculated and mixed thoroughly with the soil. Sampling was conducted every 2 days starting from day 10, and then at days 30, 60, and 120 from day 10 onwards. Each treatment was replicated in triplicate.

[0064] A first-order degradation kinetic model was used to fit the residual concentration (C) and time (t) of PAEs degraded by JQ at different time points. The calculation formulas for the first-order degradation kinetic model and the degradation half-life model are shown below: Ct=C0×e -kt Where Ct is the residual concentration of PAEs at time t (g / kg), C0 is the initial concentration of DEHP, and k is the degradation rate constant (t-1 or d-1). t1 / 2 = (ln2) / k Degradation rate (%) = (1 - AB / ACK) × 100 Where AB represents the residual concentration of PAEs in the culture medium of degrading bacteria, and ACK represents the initial concentration of PAEs in the aseptic control treatment.

[0065] The results are as follows Figure 5 As shown, compared with the control group without bacterial inoculation, XB, L191, and JQ all exhibited high degradation rates of DBP, and the degradation capacity of different bacterial cultures for DBP differed significantly (P<0.05). XB's degradation rates for DBP and DEHP were 67.35% and 79.73%, respectively; L191's degradation rates for DBP and DEHP were 82.17% and 56.76%, respectively; and JQ's degradation rates for DBP and DEHP were 90.1% and 86.2%, respectively. The degradation rate of DBP by JQ was significantly higher than the other two strains, by 33.8% and 9.70%, respectively, and its degradation rate of DEHP was 8.08% and 51.8% higher, respectively. This indicates that the functional bacterial culture JQ can better degrade both PAEs simultaneously, with a significantly greater effect than individual degrading bacteria. This demonstrates that at the same dosage, JQ has a better effect than single bacteria, effectively reducing the dosage while achieving better results.

[0066] 2. Degradation efficiency of microbial communities for PAEs The preparation method for the mixed standard curve of DBP and DEHP required for the bacterial degradation rate and soil degradation kinetics experiments is as follows: Accurately weigh and measure DBP and DEHP on a balance, dilute to volume with chromatographic grade methanol, and then perform serial dilutions to prepare working stock solutions with DBP and DEHP concentrations of 0.8 g / L each, as well as mixed standard solutions with concentrations of 8, 4, 2, 1, and 0.5 μg / L. Filter the solutions through a 0.45 µm filter membrane and store them in brown volumetric flasks at 4°C in the dark. To prevent the evaporation of organic solvents, all samples should be analyzed along with the standard curve as soon as possible after extraction.

[0067] PAEs degradation experiment: A quantitative amount of the above working stock solution was taken into an Erlenmeyer flask containing sterilized MSM, and an MSM medium with a concentration of 200 mg / kg and DBP and DEHP as mixed carbon sources was prepared. Then, an equal volume of seed bacterial suspension (OD) was taken. 600 =1.0) was added, and the Erlenmeyer flask was sealed and placed in a constant temperature shaker at 30℃ and 150 r / min for 5 days in the dark (the degradation rate was measured later). The culture medium was then used for liquid-liquid extraction.

[0068] The detection methods and conditions for PAEs determined by GC-MS are based on existing technologies: (1) Cai QY, Xiao PY, Chen T., Lü HX, Zhao HM, Zeng QY, Li YW, Li H., Xiang L., Mo CH. Genotypic variation in the uptake, accumulation, and translocation of di-(2ethylhexyl) phthalate by twenty cultivars of rice ( Oryza sativa L.).Ecotox. Environ. Safe. 2015, 116:50-58. (2) Zhao HM, Hu RW, Chen XX, Chen XB, Lü H, Li YW, Li H, Cai QY, Wong MH. Biodegradation pathway of di-(2-ethylhexyl) phthalate by a novel Rhodococcus pyridinivoransXB and its bioaugmentation for remediation of DEHP contaminated soil. Science of TheTotal Environment, 2018a, 640-641: 1121-1131. The standard curves were prepared using standard solutions of PAEs at concentrations of 0.5, 1, 2, 4, and 8 μg / mL.

[0069] Quality Assurance and Quality Control: To avoid potential PAE contamination, plastic containers were not used during the experiment. The chromatography column packing and all glassware were soaked overnight in 5% alkali solution, rinsed with deionized water, dried, and then calcined in a muffle furnace at 400 °C for 4 h. All processing, procedural blanks, and blank spikes were analyzed simultaneously. The recoveries of PAE compounds in the blank spikes were determined, with recoveries ranging from 90.1% to 108.7%. To evaluate the remediation potential of the functional microbial community JQ on PAE-contaminated soil, degradation kinetics experiments were designed for two types of PAEs at different concentrations of JQ, as shown in Table 5 below. The degradation results are as follows: Figure 6 As shown, the degradation process of PAEs conforms to the first-order kinetic equation (R0). 2 >0.9).

[0070] The half-lives of DBP and DEHP differed significantly under different treatments (P<0.05), with DEHP having a longer half-life than DBP. The degradation half-life increased with increasing PAE concentration. Furthermore, in soil contaminated with 20 ppm of microorganisms, inoculation reduced the degradation half-life of DBP by 40.22% and DEHP by 48.81%. In soil contaminated with 100 ppm of microorganisms, the degradation half-life of DBP decreased by 37.25% and DEHP by 47.42%. This indicates that inoculating with the microbial community JQ can reduce the degradation half-life of PAEs in the soil environment, allowing DBP and DEHP to be removed more quickly.

[0071] Table 5. Kinetic parameters of the descending solution under different treatments

[0072] Note: ① Inoculation refers to the inoculation of bacterial flora JQ; ② Different letters indicate significant differences between different treatments (P<0.05).

[0073] Example 6: Functional microbial community JQ reduces PAE residues in contaminated soil 1. Seed germination and cultivation Select relatively uniform rice seeds and disinfect them with 15% H2O2. The disinfection time increases with the number of times (a total of 5 times). After disinfection, place them on petri dishes (with sponges and gauze placed on the petri dishes in sequence to maintain a certain water retention). Then place them in a climate incubator and cultivate them at a temperature of 30 ℃ and a humidity of 30% until the seeds show white sprouts. Then evenly scatter them on a seedling board with a 1:1 volume ratio of organic matter and paddy soil. After the seedlings have grown 4-6 leaves, transplant them into root boxes.

[0074] The root box experiment followed existing techniques: Wang GH, Jin J, Pan XW, et al. Effect of different rotation systems on soil pH and N nutrition distribution across soybean rhizosphere[J]. Chinese Journal of Oil Crop Sciences, 2004, 26:55-59. Seedlings were transplanted into root growth chambers. Soil (1 kg total) that had passed through a 1 mm sieve was placed in the rhizosphere zone, while soil in other rhizosphere zones was placed in 5 mm sieves. A total of 4.5 kg of soil was added to each root box, with 6 g of urea mixed in each kg of soil, maintaining 60% field capacity. Rice samples were numbered and collected at 30, 60, and 120 days.

[0075] 2. Addition of synthetic bacterial culture and setting of PAE concentration Since the newly transplanted seedlings are relatively weak, the bacterial solution should be added in the evening a few days later when the rice has "turned green again." The same applies to hydroponic treatment. The method for artificially preparing contaminated soil, the preparation of the bacterial solution, the amount added, and the method of addition are the same as in Example 5.

[0076] 3. Determination of PAEs in soil from different parts and root chambers of rice plants Soil and plant samples were collected after rice had grown for 30, 60, and 120 days. Plant samples were washed with tap water and deionized water, then separated into underground and aboveground parts, and freeze-dried for PAE content analysis.

[0077] 4. Results The results are as follows Figure 7 As shown, the higher the concentration of added PAEs, the higher the residual amount in the soil, and the residual amount of DEHP is significantly higher than that of DBP. Figure 8The results showed that the lowest DEHP residue in the uninoculated group occurred in the near-rhizosphere region under low-concentration PAEs treatment, at 10.2 mg / kg, while the highest residue was in the non-rhizosphere region of rice under high-concentration PAEs treatment, reaching 66.8 mg / kg. In contrast, the DEHP residue in the JQ-inoculated groups decreased by 56.9% and 54.2%, respectively. JQ showed a greater reduction rate of DBP (62%–77%) than of DEHP (51%–56%), and its removal capacity for low-concentration PAEs was higher than that for high-concentration PAEs. Meanwhile, the differences in PAE absorption and accumulation in different parts of rice at different concentrations were compared, and the results are as follows: Figure 9 As shown, with the increase of PAE concentration, the accumulation in both the aboveground and underground parts increased accordingly, and most PAEs were retained in the roots. The accumulation of DEHP in rice was greater than that of DBP. The accumulation of PAEs in the aboveground and underground parts of rice decreased by 57.3%–67.4% and 34.8%–61.8%, respectively.

[0078] This invention also investigated the construction of synthetic microbial communities using other different strains. Based on the above research, it verified that *Gordonella oryzae* L191 and *Rhodococcus pyridostigmine* XB, along with *Azotobacter brasiliensis*, which also has a growth-promoting effect, were compatible. Azospirillam brasilense The function of 1.1899 ( Azospirillam brasilense Strain 1.1899 is a commercially available strain, obtainable from the Guangdong Academy of Sciences Institute of Microbiology (Guangdong Provincial Microbiology Analysis and Testing Center), with the strain accession number GDMCC 1.1899. The study was conducted after replacing L25 with 1.1899 in the bacterial community JQ, following the same methods and procedures as in the previous examples. The results showed... Azospirillam brasilense The effects of 1.1899 and L25 are consistent, with no significant difference. Azospirillam brasilense 1.1899 can also be used to construct JQ bacterial communities.

[0079] In conclusion, from Rhodococcus pyridinivorans , Gordonia polyisoprenivorans and Azospirillam brasilenseThe constructed microbial community JQ can improve soil physicochemical properties, promote the decomposition of soil organic matter, and increase nitrogen, phosphorus, and potassium content. The three bacteria work together synergistically to promote rice growth. At the same time, the microbial community can reduce the residue of PAEs in the soil and rapidly degrade DEHP and DBP, with degradation rates of 90.1% and 86.2%, respectively. Its effect is significantly better than that of individual degrading bacteria, indicating that at the same dosage, microbial community JQ has a better effect than single bacteria, and can effectively reduce the dosage while achieving better results. Furthermore, microbial community JQ significantly reduces the degradation half-life, enabling faster removal of pollutants, shortening the time by 53% to 63%, and increasing the soil PAEs degradation rate by 62.56% to 73.17%. In the PAEs-contaminated environment, it can increase rice biomass, increase relative chlorophyll content, enhance rice photosynthetic capacity, and reduce the absorption and accumulation of PAEs by rice, thus jointly achieving the degradation of PAEs and promoting growth.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A microbial synthetic flora with both phthalate degradation and growth-promoting effects, characterized in that, Contains Gordon's bacteria (which eats isoprene) Gordonia polyisoprenivorans ), Pyridine-loving Rhodococcus ( Rhodococcus pyridinivorans ) and Brazilian azospira ( Azospirillam brasilense The volume ratio of *Gordonella pyrenoidosa*, *Rhodococcus pyridostigmine*, and *Azotobacter brasiliensis* in the synthetic microbial community is 1:1:

1.

2. The application of the synthetic microbial community according to claim 1 in promoting rice growth and degrading soil PAEs, or promoting rice growth under PAE pollution, is characterized in that, The PAEs are dibutyl phthalate and / or di(2-ethylhexyl) phthalate.

3. The application of the synthetic microbial community according to claim 1 in the preparation of PAEs-degrading bacterial agents and / or rice growth-promoting bacterial agents, characterized in that, The PAEs are dibutyl phthalate and / or di(2-ethylhexyl) phthalate.

4. The application of the synthetic microbial community as described in claim 1 in microbial fertilizer products.

5. A microbial agent that combines the functions of degrading PAEs and promoting rice growth, characterized in that, Contains the synthetic bacterial flora or its bacterial solution as described in claim 1.

6. A method for degrading PAEs and / or promoting rice growth, characterized in that, The synthetic microbial community or its bacterial solution described in claim 1 is used to irrigate rice.

7. The method according to claim 6, characterized in that, The inoculum size of the bacterial suspension is 1%–5%, OD 600 =0.5±0.05.

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