Bacillus X16-3 strain and application thereof

Abstract

The invention belongs to the technical field of remediation of contaminated soil by microbial agents, and particularly relates to a bacillus X16-3 strain and application thereof. The bacillus strain X16-3 is classified and named as Heynickia oleronia, the bacillus strain X16-3 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35230, and the preservation date is July 17, 2025. The bacillus strain X16-3 has the advantages that the bacillus strain X16-3 can be used for preparing the bacillus strain X16-3; the bacillus X16-3 strain screened by the invention can efficiently degrade phthalic acid esters in various environments, so that the bacillus X16-3 strain can be used for bioremediation of polluted soil, and the bacillus X16-3 strain is adaptive to actual farmland multi-component pollution scenes. The bacillus X16-3 strain provided by the invention is high in degradation efficiency, the remediation period is remarkably shortened, and a technical support is provided for efficient and green remediation of PAEs pollution of farmland soil.

Description

Technical Field

[0001] This invention belongs to the technical field of microbial agents for the remediation of contaminated soil, specifically relating to a Bacillus strain X16-3 and its application. Background Technology

[0002] As the world's largest area covered by agricultural film, my country used 2.358 million tons of agricultural plastic film in 2021, including 1.32 million tons of mulch film, but the recycling rate was less than 60%. When residual mulch film degrades and breaks down in the soil, it continuously releases phthalates (PAEs)—endocrine disruptors with carcinogenic, mutagenic, and teratogenic effects. These substances are chemically stable, degrade slowly naturally, and can accumulate in the soil for a long time. They can enter the food chain through crop absorption and threaten human health (e.g., pregnant mice exposed to DBP are prone to reproductive system developmental abnormalities in their offspring; long-term human exposure may damage liver and kidney function and induce chronic diseases such as hyperlipidemia).

[0003] For soil PAEs pollution, the mainstream remediation technologies are currently divided into three categories: physical, chemical, and ecological remediation. However, all three methods have limitations.

[0004] 1. Physical remediation: Physical adsorption can only fix PAEs but cannot completely degrade them. Adsorbents (such as activated carbon) need to be replaced regularly. Long-term use can easily clog soil pores and reduce soil aeration. Excavation and landfill cause great disturbance to soil structure, making them unsuitable for large areas of farmland, and may cause secondary pollution due to PAE leakage.

[0005] 2. Chemical remediation techniques: Techniques such as Fenton oxidation and soil leaching require the addition of chemical reagents, which can damage the soil microbial community structure. For example, when the concentration of PAEs exceeds 10 mg / kg, the soil enzyme activity inhibition rate exceeds 40%. Chemical reagent residues may also affect crop quality and pose long-term environmental risks.

[0006] 3. Microbial Remediation Technology: Existing reported strains (such as DEHP-S2 screened by Gan Deping and Rhodococcus WJ4 used by Wang et al.) are mostly effective against single PAEs, with limited degradation capacity for multi-component PAEs (such as mixed pollution of DMP, DEP, and DBP), and the degradation efficiency is low. For example, DEHP-S2 only achieved a degradation rate of 52.47% for 500 mg / L DEHP at 10℃ within 72 hours, and Rhodococcus WJ4 only achieved a degradation rate of over 55% for 1.0 g / kg DEHP after 21 days, which is insufficient to meet the remediation needs of multi-component pollution in actual farmland. Furthermore, the adaptability to remediation conditions is poor: most strains have not clearly defined optimal degradation conditions, and the pH and temperature of actual farmland soils fluctuate greatly (e.g., soil pH in Northwest my country is mostly between 7-9, and the diurnal temperature range exceeds 10℃), which easily leads to a decrease in the degradation activity of the strains and makes it impossible to stably exert the remediation effect.

[0007] In summary, physical remediation (such as activated carbon adsorption and excavation and landfill) relies on adsorbent fixation or ex-situ treatment; chemical remediation (such as Fenton oxidation and photolysis) degrades PAEs using chemical reagents; and in ecological remediation, microbial remediation has become a research hotspot due to its low cost and environmental friendliness, with degrading strains such as Pseudomonas and Rhodococcus already screened, but these are mostly targeted at single PAEs (such as DEHP). In actual farmland soils, PAEs often coexist in multiple components. For example, in typical agricultural areas of Shaanxi Province, the average DBP concentration in the soil reaches 6750.73 μg / kg, and DEHP reaches 118.26 μg / kg. Moreover, DMP and DEP have similar sources, while DBP has an independent source, making it difficult for existing remediation technologies to adapt to such complex pollution scenarios. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Bacillus X16-3 strain and its application.

[0009] The objective of this invention is achieved through the following technical solution: A Bacillus strain X16-3, classified and named Heyndrickxia oleronia, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35230, deposited on July 17, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The above-mentioned Bacillus strains are used in the degradation of phthalate contaminants.

[0011] As a preferred technical solution, the phthalate includes dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DNOP).

[0012] The application of the aforementioned Bacillus strain X16-3 in the bioremediation of contaminated soil.

[0013] As a preferred technical solution, the contaminated soil contains phthalates.

[0014] As a more preferred technical solution, the amount of Bacillus inoculation in the contaminated soil is 20 mL of bacterial solution with an absorbance value of 1.0 at a wavelength of 600 nm per 200 g of contaminated soil.

[0015] As a preferred technical solution, the optimal degradation conditions for the Bacillus strain X16-3 are a pH of 8 and a temperature of 30°C.

[0016] A soil remediation agent containing at least the aforementioned Bacillus strain X16-3.

[0017] As a preferred technical solution, the effective viable count of the Bacillus strain X16-3 is ≥1.0×10⁻⁶. 9 CFU / g.

[0018] This invention offers the following advantages: It screens a Bacillus strain X16-3 that can efficiently degrade phthalates (PAEs) in various environments, including DMP, DEP, DBP, BBP, DEHP, and DNOP. Therefore, it can be used for bioremediation of contaminated soil. The Bacillus strain X16-3 disclosed in this invention is suitable for real-world multi-component pollution scenarios in farmland. Experiments have shown that the optimal degradation conditions for Bacillus strain X16-3 are pH 8 and temperature 30℃, thus enhancing its adaptability to different soil environments. The Bacillus strain X16-3 provided by this invention exhibits high degradation efficiency, significantly shortening the remediation cycle and providing technical support for the efficient and green remediation of PAEs contaminated farmland soil. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited to the following description:

[0020] This invention provides a Bacillus strain X16-3, classified as Heyndrickxia oleronia, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35230 and deposit date of July 17, 2025.

[0021] This strain has the following properties:

[0022] Morphological characteristics: Colonies are milky white, moist, 1 mm to 2 mm in diameter, and cell diameter ≤ 2 μm.

[0023] Physiological and biochemical characteristics: Gram-negative bacteria.

[0024] Example 1: Screening of strains

[0025] Step 1: Soil Collection and Pretreatment

[0026] Soil samples were collected from typical mulched farming areas in eleven study regions within Shaanxi Province. The soil samples were placed in sterile bags. After impurity removal and homogenization, the soil samples were divided into two parts. One part was placed in 50mL sterile centrifuge tubes, flash-frozen in liquid nitrogen, temporarily stored in an incubator, and then transported back to the laboratory for storage at -80℃ for determining soil microbial community diversity. The other part of the soil was air-dried, homogenized, ground, and sieved through 1mm and 0.15mm sieves.

[0027] 2. Step Two: Stress Culture

[0028] A gradient pressure acclimatization method was adopted: 100 mL of MSM medium was sterilized in a 250 mL Erlenmeyer flask. After cooling, under aseptic conditions, a mixed sample stock solution of 6 PAEs (DEP, DMP, DBP, BBP, DEHP, DNOP) (ethyl acetate as solvent) was added to achieve a final concentration of 50 mg / L. At the same time, 5 g of the collected treated phthalate-contaminated soil sample was added and cultured in the dark on a shaker at 30℃ and 150 r / min for 7 days. Then, it was inoculated into the same medium at a 5% inoculum, and the concentration of PAEs was gradually increased to acclimatize the sample. This process was repeated 6 times, with concentrations set at 50 mg / L, 150 mg / L, 250 mg / L, 350 mg / L, 450 mg / L, 600 mg / L, and 750 mg / L, and finally the concentration was increased to 1200 mg / L.

[0029] 3. Step Three: Screening and Purification:

[0030] The culture medium for gradient pressure acclimatization was used. 0.2 mL of the basal culture medium fermentation broth was evenly spread onto a nutrient agar plate and repeatedly streaked for separation. The plate was then incubated at 37±1℃ for 48 h. Dominant single colonies of different colors and morphologies were selected and repeatedly streaked on beef extract peptone medium to purify the strains with consistent morphology. The purified strains were then stored on beef extract peptone solid medium for further screening and activation to obtain the strains for later use. The composition of the nutrient agar plate culture medium was as follows: the core components and proportions of the nutrient agar medium were: 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 1.5%-2% agar, and 100% water. All of the above are mass percentages. The pH of the culture medium was 7.0-7.5, and the medium was sterilized at 105 kPa for 20-25 min. The composition of the beef extract peptone plate medium is as follows: 0.5% beef extract, 1% peptone, 0.5% sodium chloride, 1.5%-2% agar (for solid culture medium), and 100% water. All of the above are mass percentages. The pH value is usually adjusted to 7.2-7.4, and sterilized at 105 kPa for 20-25 minutes.

[0031] 4. Step Four: Secondary Screening

[0032] (1) The purified bacterial strains were inoculated into beef extract peptone medium. The culture flasks containing the strains were placed in a constant temperature shaking incubator and cultured for 1 day at a suitable temperature and a rotation speed of 150 r / min. After 1 day of culture, the bacterial cells were centrifuged to precipitate. The bacterial solution was diluted with sterile water, and the absorbance of the bacterial solution was measured at OD600 nm using a UV spectrophotometer. The bacterial solution concentration was then adjusted to OD600 = 1. The adjusted bacterial solution was then inoculated into culture flasks containing 50 mL of liquid culture medium. Phthalate esters (PAEs) were added to the culture medium of each culture flask to a mass concentration of 200 mg / L. The culture was continued under the same conditions as above, and the degradation time was set to 48 hours for this determination.

[0033] (2) Add 30 mL of ethyl acetate extract to the supernatant, shake at a constant temperature for 30 min, then transfer to a separatory funnel. After standing for 10 min, collect the lower organic phase (ethyl acetate) and pour the upper aqueous phase back into the original culture flask. Extract the aqueous phase twice more with ethyl acetate in the same manner. Combine the three collections of organic phase (ethyl acetate). Dry the final organic phase (ethyl acetate) by passing it through a 15 cm column of anhydrous Na2SO4 (dried at 400 °C) to remove residual water. Transfer all the filtrate to a pistol flask and evaporate it to near dryness using a rotary evaporator. Transfer all the concentrate to a 10 mL volumetric flask and wash the pistol flask three times with 6 mL of chromatographically pure methanol. Make up the final volume to 25 mL, filter 2 mL through a 0.45 μm microporous membrane, collect the filtrate in a sample vial, and determine the residual amount using gas chromatography (GC). Analyze the PAEs standard curve obtained in the previous experiment to calculate the degradation rate. GC detection conditions: Column: HP-50.25μm×0.25mm×30m; Carrier gas: High-purity helium, initial pressure: 33.6kPa, flow rate: 1.0mL·min -1 Splitless injection was used, with an injection volume of 1.0 μL. Temperature program: initial temperature 100℃, held for 2 min; then increased at 15℃ / min. -1 The temperature rose to 129°C at a rate of 40°C / min; thereafter, it increased at a rate of 40°C / min. -1 The temperature was increased to 280℃ and maintained for 5 minutes. The injection port temperature was 250℃, and the ion source temperature was 220℃. The solvent delay time was 1 minute, and the run time was 11.0 minutes.

[0034] 5. Step Five: Contaminated Soil Remediation Test

[0035] (1) Place 200g of soil in a 500mL Erlenmeyer flask, add 6 PAEs solutions (DEP, DMP, DBP, BBP, DEHP, DNOP), and adjust the final concentration to approximately 200mg / kg to prepare artificially contaminated soil. Inoculate the soil with the seed liquid of the dominant strain at a mass concentration of 10%, using uninoculated soil as a blank control group, and make the moisture content reach 20%. Place the Erlenmeyer flask in a 30℃ constant temperature incubator and incubate in the dark. Take samples at 0, 3, 7, 14, and 21 days to determine the remaining PAEs content.

[0036] (2) Extraction: Accurately weigh 2.0 g of the soil sample into a 50 mL glass centrifuge tube, add 4 mL of ultrapure water, vortex for 30 s, add 5 mL of chromatographically pure dichloromethane, vortex vigorously for 10 min in a multi-tube vortex mixer, then sonicate at 40 Hz for 15 min, centrifuge at 3500 pm for 5 min, take 1 mL of the upper organic phase, vortex, and filter through a 0.22 μm organic filter membrane. Determination of PAEs content: Measure and calculate the PAEs content in the extract using gas chromatography, calculate the degradation rate, and retain strains with high degradation rates.

[0037] Example 2: Identification of the strain

[0038] Step 1: Extract total DNA.

[0039] (1) Extract bacterial genomic DNA using the Omega Genomic DNA Extraction Kit. Take 1-5 ml of bacterial culture medium, centrifuge at 10,000 rpm (~11,500 x g) for 1 min, and aspirate the supernatant as thoroughly as possible. Add 200 μl of buffer GA to the bacterial pellet and vortex until the bacterial cells are completely resuspended. Add 20 μl of proteinase K solution to the tube and mix well. Add 220 μl of buffer GB, vortex for 15 sec, incubate at 70°C for 10 min, and the solution should become clear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap. Add 220 μl of anhydrous ethanol, vortex thoroughly for 15 sec, at which point flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0040] (2) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, centrifuge at 12,000 rpm (~13,400 x g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into a collection tube. Add 500 μl of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400 x g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into a collection tube. Add 600 μl of wash buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400 x g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into a collection tube. Repeat the above steps. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400 x g) for 2 minutes, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any remaining wash liquid in the adsorption material.

[0041] (3) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200pl of elution buffer TE to the middle of the adsorption membrane, place at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400xg) for 2 min, and collect the solution into the centrifuge tube.

[0042] 2. Step Two: PCR Amplification of 16S rDNA:

[0043] Using the total DNA extracted in step one as a template, PCR amplification was performed. The PCR reaction system was prepared as follows: 25 μl Mixture, 1 μl 27F primer, 1 μl 1492R primer, 1 μl extracted genomic DNA, and 22 μl dd H2O were added sequentially to a centrifuge tube, for a total volume of 50 μl. The prepared reaction system was placed in a PCR instrument and amplified under the following conditions: 95℃ for 5 min (pre-denaturation), followed by 32 cycles, each cycle consisting of 94℃ for 45 s (denaturation), 55℃ for 45 s (annealing), 72℃ for 1 min 15 s (extension), and finally 72℃ for 10 min (extension). The primer sequences for the PCR amplification were:

[0044] Upstream primer (27F): 5'-AGAGTTTGATCMTGGCTCAG-3'; SEQ ID NO.1

[0045] Downstream primer (1492R): 5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO.2.

[0046] 3. Step Three: 16S rDNA sequence analysis and alignment of the strain:

[0047] The PCR amplification product (fragment size approximately 1.4kb) from step two was sequenced (assisted by Sangon Biotech (Shanghai) Co., Ltd.). The obtained 16S rDNA partial gene sequence was compared with the 16S rDNA sequence of Heyndrickxiaoleronia (Heyndrickxia oleronia M1 / 25) (NR_119157.1) published in GenBank, and the similarity reached 100.0%.

[0048] The sequence of the strain's 16S rDNA is shown in SEQ ID NO.3:

[0049]

[0050] Based on the combined physiological and biochemical identification and 16S rDNA sequence analysis results, the strain of this invention was identified as Heyndrickxia oleronia.

[0051] Example 3: Optimal Degradation Conditions Test

[0052] Step 1: Pick the isolated and purified X16-3 colonies and inoculate them into LB medium and culture for 16 hours. After centrifuging the obtained bacterial solution at 4600× for 5 minutes, discard the supernatant.

[0053] Step 2: Setting Degradation Conditions: pH Gradient Setting: Use a pH meter to adjust the pH of 50 mL of sterile culture medium in a 250 mL culture flask to 5, 6, 7, 8, 9, and 10 respectively. Simultaneously add six types of PAEs as growth substrates, bringing the final concentration to 200 mg / L. Inoculate at a rate of 0.6 (OD200). 600 Inoculation was performed using nm), with the uninoculated treatment serving as a control.

[0054] Temperature gradient setup: In a 250 mL Erlenmeyer flask containing 50 mL of sterile culture medium (pH 7.0), PAEs were added as the growth substrate to achieve a final concentration of 200 mg / L and an inoculum size of 0.6 (OD). 600 The Erlenmeyer flasks were then placed in shakers at 20℃, 25℃, 30℃, 35℃, and 40℃ and continuously shaken (150 r / min) for 3 days, with the uninoculated treatment serving as a control.

[0055] Microbial inoculum gradient settings: Inoculum amounts were set at 0.2, 0.4, 0.6, 0.8, and 1.0 (OD) respectively. 600 (nm) was inoculated into a 250mL culture flask containing 50mL of sterile culture medium (pH 7.0). PAEs were added simultaneously as a growth substrate to a final concentration of 200mg / L. The culture was continuously shaken at 30℃ and 150r / min for 3 days, with the uninoculated treatment serving as a control.

[0056] Step 3: Determination of the supernatant after centrifugation of the culture medium: Add 30 mL of ethyl acetate extract to the supernatant, shake at a constant temperature for 30 min, then transfer to a separatory funnel. Shake thoroughly and allow to stand for separation. Wash with the extract and transfer all the solution to a flat-bottomed flask. Add anhydrous sodium sulfate, Florisil, and anhydrous sodium sulfate in a 2:4:2 (mass ratio) sequentially to the purification column. Prewash and elute the purification column with 10 mL and 50 mL of extract, respectively. Collect all the eluent in a pistol flask, concentrate by rotary evaporation to dryness, and dilute to 25 mL with chromatographic grade ethyl acetate for gas chromatography analysis.

[0057] GC detection conditions: Column: HP-50.25μm×0.25mm×30m; Carrier gas: High-purity helium, initial pressure: 33.6kPa, flow rate: 1.0mL·min -1 Splitless injection was used, with an injection volume of 1.0 μL. Temperature program: initial temperature 100℃, held for 2 min; then increased at 15℃ / min. -1 The temperature rose to 129°C at a rate of 40°C / min; thereafter, it increased at a rate of 40°C / min. -1 The temperature is increased to 280℃ and maintained for 5 minutes.

[0058] The experimental results are shown in Tables 1, 2, and 3:

[0059] Table 1. Gas chromatographic determination results of PAEs in liquid culture medium at different pH values.

[0060]

[0061]

[0062] Table 2. Gas chromatography results of PAEs in liquid culture medium at different temperatures.

[0063]

[0064]

[0065] Table 3. Gas chromatographic determination results of PAEs in liquid culture medium under different inoculum amounts.

[0066]

[0067]

[0068] Analysis of experimental results:

[0069] ① Effect of pH on degradation efficiency: Comparing the degradation rates of PAEs under different pH conditions, the degradation rate of each PAE was relatively high at pH 8. Taking DMP as an example, the degradation rate reached 60.2% (X16-3 samples). This indicates that this specific strain has a better degradation effect on PAEs in a weakly alkaline environment, while excessively acidic or alkaline environments may inhibit strain activity and affect degradation.

[0070] ② Effect of temperature on degradation efficiency: In the temperature gradient experiment of 20℃-40℃, the degradation rate of each PAE was better at 30℃. For example, the degradation rate of DMP in sample X16-3 was 59.4% at 30℃. This indicates that around 30℃ is the more suitable temperature for this strain to degrade PAEs. Temperatures that are too high or too low will affect the metabolic activity of the strain and thus affect the degradation efficiency.

[0071] ③ Effect of inoculum size on degradation efficiency: 0.2-1.0 OD 600 Under an inoculum density gradient of nm, the overall degradation rate increases with increasing inoculum density. Taking DMP as an example, when the inoculum density is 1.0 OD600 nm, the degradation rate of sample X16-3 is 58.6%, and that of sample X24-3 is 51.9%. This indicates that within a certain range, increasing the inoculum density is beneficial to improving the degradation rate of PAEs, and at higher inoculum densities, the lag period can be shortened, accelerating the decomposition of PAEs.

[0072] Example 4: Application Effect Test

[0073] Step 1: Place 200g of soil in a 500mL Erlenmeyer flask, add six PAE solutions (DEP, DMP, DBP, BBP, DEHP, DNOP), and adjust the final concentration to approximately 200mg / kg to prepare artificially contaminated soil. Inoculate the soil with the seed culture of the dominant bacterial strain at a concentration of 10% by mass. Use uninoculated soil as a blank control group. Ensure the moisture content reaches 20%. Place the Erlenmeyer flask in a 30℃ constant temperature incubator in the dark. Take samples at 0, 3, and 7 days to determine the remaining PAE content.

[0074] Step 2: Extraction: Accurately weigh 2.0 g of the soil sample into a 50 mL glass centrifuge tube, add 4 mL of ultrapure water, vortex for 30 s, add 5 mL of chromatographically pure dichloromethane, vortex vigorously for 10 min in a multi-tube vortex mixer, then sonicate at 40 Hz for 15 min, centrifuge at 3500 pm for 5 min, take 1 mL of the upper organic phase, vortex, and filter through a 0.22 μm organic filter membrane. Determination of PAEs content: Measure and calculate the PAEs content in the extract using gas chromatography, calculate the degradation rate, and retain strains with high degradation rates.

[0075] Step 3, Degradation rate calculation formula:

[0076] Degradation rate (%) = (1 - A / A0) * 100

[0077] In the formula: A is the residual concentration of PAEs after treatment with degrading bacteria; A0 is the residual concentration of PAEs in the control treatment.

[0078] The raw data for the degradation rate of PAE-contaminated soil from 0 to 7 days are shown in Table 4. The degradation rate is shown in Table 5.

[0079] Table 4. Peak area results (fA*s) of PAEs in contaminated soil determined by gas chromatography.

[0080]

[0081] Table 5. Degradation rate of PAEs in contaminated soil by degrading bacteria after 3 days and 7 days.

[0082]

[0083] As can be seen from Tables 4 and 5, under the 7-day degradation conditions, the degradation rate data shows that x16-3 has a superior degradation ability for the six PAEs (DEP, DMP, DBP, BBP, DEHP, DNOP), with a degradation rate of up to 92.5% for DEP.

[0084] In summary, the Bacillus strain X16-3 of the present invention has the following advantages:

[0085] 1. The X16-3 strain (Heyndrickxia oleronia M1 / 25) of this invention was obtained from phthalic acid ester (PAE) contaminated farmland soils that had been continuously cropped for 2, 7, and 8 years. It was obtained through gradient acclimatization and screening with 50-1200 mg / LPAEs. Its native growth environment is highly matched with the physicochemical properties of the target soil to be remediated. It also has physiological and biochemical characteristics such as Gram-negative staining, positive catalase and oxidase, and starch hydrolysis ability. Therefore, after entering the soil, this bacterium quickly becomes the dominant bacterial group, occupies a certain ecological niche, effectively promotes the degradation of PAEs compounds, optimizes the soil microbial system, improves soil aggregate structure and repairs compaction problems, and does not damage soil organic matter, thereby enhancing the biological activity of the soil.

[0086] 2. The X16-3 strain of this invention uses phthalate (PAE) compounds as its sole carbon source. During its growth and reproduction, this strain can alter the chemical properties of PAEs, causing a directional transformation of their complex ester structures. Through enzymatic hydrolysis, it breaks the ester bonds of PAEs, gradually decomposing previously recalcitrant long-chain or medium-to-short-chain PAEs (such as DBP, DEHP, DMP, etc.) into intermediate products such as phthalic acid, which are ultimately mineralized into CO2 and H2O. Compared to existing remediation technologies, the microbial degradation pathway of X16-3 is more ecologically safe. Compared to chemical methods, it does not produce secondary pollution, and compared to physical methods, it reduces disturbance to the soil ecosystem.

[0087] 3. The X16-3 strain of the present invention can simultaneously degrade 6 common PAEs.

[0088] 4. The X16-3 strain of the present invention can degrade PAEs pollutants in soil in a short time.

[0089] 5. The optimal degradation conditions for X16-3 of the present invention are pH=8, temperature 30℃, and inoculation amount of 1.0 OD600nm (i.e., 20 mL of bacterial solution with an absorbance of 1.0 at 600 nm wavelength is added to every 200 g of artificially contaminated soil containing phthalates, ensuring that the effective viable count of Bacillus strain X16-3 in the bacterial solution is ≥1.0 × 10⁻⁶). 9The conditions (CFU / mL) are highly compatible with the natural pH (6.5-8.5) and temperature (25-35℃) of farmland soil in northern my country. There is no need for artificial adjustment of extreme acidity, alkalinity or high temperature. Degradation can be initiated simply by inoculating with conventional microbial agents.

[0090] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A Bacillus strain X16-3, characterized in that, The specimen is classified and named Heyndrickxia oleronia, and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35230 on July 17, 2025.

2. The application of the Bacillus strain of claim 1 in the degradation of phthalic acid ester contaminants.

3. The application according to claim 2, characterized in that, The phthalates include dimethyl phthalate, diethyl phthalate, dibutyl phthalate, butyl benzyl phthalate, di(2-ethylhexyl) phthalate, and di-n-octyl phthalate.

4. The application of the Bacillus strain X16-3 according to claim 1 in the bioremediation of contaminated soil.

5. The application according to claim 4, characterized in that, The contaminated soil contains phthalates.

6. The application according to claim 5, characterized in that, The amount of Bacillus inoculation in the contaminated soil was 20 mL of bacterial solution with an absorbance of 1.0 at a wavelength of 600 nm per 200 g of contaminated soil.

7. The application according to claim 5, characterized in that, The optimal degradation conditions for the Bacillus strain X16-3 are pH 8 and temperature 30℃.

8. A soil remediation microbial agent, characterized in that, It contains at least the Bacillus strain X16-3 as described in claim 1.

9. The soil remediation microbial agent according to claim 8, characterized in that, The effective viable count of the Bacillus strain X16-3 is ≥1.0 × 10⁻⁶. 9 CFU / g.

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