Basic Salt Medium: Per liter, containing (NH₄)₂SO₄ 2.0 g, MgSO₄ 0.2 g, CaCl₂·2H₂O 0.01 g, FeSO₄ 0.001 g, Na₂HPO₄·12H₂O 1.5 g, KH₂PO₄ 1.5 g.

LB Medium: Per liter, containing NaCl 10 g, yeast extract 5 g, tryptone 10 g.

PDA Medium: Per liter, containing 200 g potato infusion, 20 g glucose.

Gause No. 1 Medium: Per liter, containing soluble starch 20 g, NaCl 0.5 g, KNO₃ 1 g, K₂HPO₄·3H₂O 0.5 g, MgSO₄·7H₂O 0.5 g, FeSO₄·7H₂O 0.01 g.

Beef Extract Peptone Medium: Per liter, containing beef extract 3 g, peptone 10 g, NaCl 5 g.

MacConkey Agar Medium: Per liter, containing peptone 5 g, glucose 10 g, KH₂PO₄ 1 g, MgSO₄ 0.5 g, crystal violet 0.03 g, chloramphenicol 0.1 g.

Soil samples were collected from Xuan’en County, Hubei Province, China, where tobacco had been continuously cultivated for over 15 years. Ten grams of soil samples were placed in sterilized Erlenmeyer flasks, and basic culture medium was added to a total volume of 100 ml. The flasks were then shaken overnight at 30˚C and 180 rpm to prepare bacterial suspensions. Ten milliliters of the bacterial suspensions were separately added to Gause No. 1 medium (for actinomycetes), MacConkey agar medium (for fungi), and beef extract peptone medium. The medium was pre-added with the allelochemicals phthalic acid to give a final concentration of 100 mg/l.

The gradient pressure domestication method was employed for cultivation, enriching the concentration of phthalic acid in the culture medium from 100 mg/l gradually to 700 mg/l. Solid culture medium containing 500 mg/l phthalic acid was prepared, and the tolerant degradation bacteria were initially isolated using the dilution spread plate method. Colonies showing good growth, clear morphology, and distinct characteristics were selected, and single colonies were isolated using the streak plate method, thus obtaining the phthalic acid-degrading bacteria.

Reference: “Bergey’s Manual of Systematic Bacteriology”, 9th edition.

Bacterial strains were cultured on LB agar medium for 8 hours. A volume of 100 μl of bacterial suspension was transferred to a sterilized 1.5 ml centrifuge tube. After boiling in a water bath for 15 minutes, the suspension was centrifuged at 12,000 rpm for 2 minutes, and the supernatant was collected as the template for 16S rRNA gene amplification by PCR.

The PCR reaction system comprised 5 μl of DNA template, 1 μl each of primers 27F/1492R, 1 μl of dNTP (10 mmol/l), 5 μl of Ex Taq buffer (10×), 1 μl of Ex Taq, and ddH₂O added to make up the volume to 50 μl.

Primer sequences:

27F: 5’-AGAGTTTGATCCTGGCTCAG-3’;

1492R: 5’-GGTTACCTTGTTACGACTT-3’.

The PCR amplification program included initial denaturation at 94˚C for 3 minutes, followed by denaturation at 94˚C for 1 minute, annealing at 61˚C for 1 minute, extension at 72˚C for 1 minute, for a total of 30 cycles, and final extension at 72˚C for 5 minutes. The reaction was terminated by cooling to 4˚C for 30 minutes. PCR products were analyzed by 1% agarose gel electrophoresis, and sequencing was performed by Wuhan Qingke Biotechnology Company. The obtained sequences were compared with the 16S rRNA sequences in the GenBank database.

Equal volumes of ethyl acetate were added to the bacterial suspension for liquid-liquid extraction. The ethyl acetate layer was then evaporated using a rotary evaporator, and the residue was dissolved in water. After filtration through a 0.22 μm membrane filter, the solution was subjected to liquid chromatography-mass spectrometry (LC-MS) to determine the concentration of phthalic acid.

Liquid chromatography conditions: Column: Agilent ZORBAX SB-Aq (250 mm × 4.6 mm); Mobile phase: 5 mmol/L H₂SO₄; Flow rate: 500 μL/min; Injection volume: 20 μL.

Single colonies of bacterial strains were inoculated into LB medium and cultured overnight. The bacterial cells were collected by centrifugation at 5000 rpm for 2 minutes, resuspended in inorganic salt medium, and adjusted to an OD600 of approximately 1 as a seed solution. The seed solution was inoculated into 50 mL of LB medium, and different cultivation conditions were set, including different temperatures (18˚C, 30˚C, 37˚C, 45˚C), pH levels (4, 5, 6, 7, 8, 9), concentrations of phthalic acid (100 - 1000 mg/L), and inoculation volumes of bacterial strain 3 (0.4%, 0.8%, 1.2%, 1.6%, and 2.0%). Samples were taken after 48 hours of cultivation for OD600 measurement (for the effect of inoculation volume on growth status, samples were taken every 8 hours). Each experiment was performed in triplicate.

Pot experiments were conducted in the greenhouse of Hubei University. Tobacco seeds with uniform size and appearance were selected, soaked in water for 12 hours, then disinfected with 70% alcohol for 1 minute, and rinsed repeatedly with sterile water. The treated tobacco seeds were sown in seedling pots, and after reaching the five-leaf stage, the tobacco seedlings were transplanted. Uniform-sized tobacco plants were selected and transplanted into flowerpots (9 × 9 × 12 cm) filled with sterilized soil. Degradation bacteria B3 were fermented under optimal conditions for later use. Three treatments were set up: PA, PA + B3, and CK. In the PA treatment, 50 mL of 2 g/L phthalic acid solution and 5 mL of water were added to each pot. In the PA + B3 treatment, 50 mL of 2 g/L phthalic acid solution and 5 mL of bacterial suspension of strain B3 (obtained by picking a single colony of strain B3 from an activated plate, overnight cultured in LB medium, centrifuged to remove the supernatant, and resuspended in an equal volume of basic salt medium) were added to each pot. The CK treatment involved adding 55 mL of water to each pot. After four weeks of cultivation in a constant temperature and humidity greenhouse, the plant height, stem diameter, fresh and dry weights of roots, stems, and leaves of tobacco seedlings were measured. Each treatment was replicated three times.

Soil samples from the tobacco seedling growth area were placed in sterile water and shaken overnight. After filtration to remove insoluble substances, the phthalic acid content in the water was detected using liquid chromatography. Liquid chromatography conditions were the same as described in Section 2.3.

A degradation bacterial strain, B3, exhibiting favorable degradation efficiency towards phthalic acid, was obtained through the dilution spread plate method. The degradation rate of phthalic acid by strain B3 was determined to be 70%. The colony morphology of strain B3 on agar plates was circular, milky white in color, and slightly elevated ( Figure 1 ). Further observation and study were conducted on the physiological and biochemical characteristics of strain B3, the results of which are presented in Table 1 .

The genomic DNA of strain B3 was utilized as a template for PCR amplification, and the resulting PCR product was sequenced to obtain the 16S rDNA sequence of strain B3, with a fragment length of 1466 bp. The obtained 16S rDNA gene sequence underwent a BLAST homology comparison on NCBI, revealing a 97% homology with bacteria of the Enterobacter hormaechei subsp. ( Figure 2 ).

The growth of strain B3 and its degradation of phthalic acid under temperature conditions ranging from 18˚C to 45˚C are illustrated in Figure 3 . The results indicate that with the increase in cultivation temperature, both the cell density of strain B3 and its degradation rate of phthalic acid initially increase and then decrease. The optimal growth temperature for strain B3 is 30˚C, and both too high

Note: + denotes positive, − denotes negative.

and too low temperatures inhibit the growth of the strain. At the optimal temperature, strain B3 exhibits the highest degradation rate of phthalic acid, indicating a positive correlation between the growth of strain B3 and its degradation efficiency.

The growth of strain B3 and its degradation of phthalic acid under pH conditions ranging from 4 to 9 are shown in Figure 4 . It is observed that the growth of strain B3 is optimal at pH values between 7 and 8. Moreover, at pH 8, strain B3 exhibits the highest degradation rate of phthalic acid. This suggests that strain B3 thrives in neutral to slightly alkaline environments, and alkaline conditions favor the degradation of phthalic acid by the strain.

The growth of strain B3 and its degradation rate of phthalic acid under initial concentrations of phthalic acid ranging from 100 to 1000 mg/L are illustrated in Figure 5 . The results indicate that with increasing initial concentrations of phthalic acid from 100 to 500 mg/L, the degradation rate of phthalic acid by strain B3 gradually increases. At an initial concentration of 500 mg/L, both the cell density of strain B3 and its degradation rate of phthalic acid reach their maximum. However, when the initial concentration ranges from 500 to 700 mg/L, both the cell density and degradation rate of strain B3 gradually decrease before stabilizing. Overall, when the initial concentration of phthalic acid exceeds 700 mg/L, the growth of strain B3 is inhibited, and its degradation rate decreases accordingly. This indicates that strain B3 exhibits more effective degradation of phthalic acid at lower concentrations.

In Figure 6 , it can be observed that when the inoculation volume is greater than 1.2%, there is no significant effect on the growth of strain B3. However, when the inoculation volume is less than 1.2%, there is a certain inhibitory effect on the growth of strain B3 before 72 hours, after which this inhibitory effect disappears.

Based on the results of the single-factor experiments, the optimal environmental conditions for strain B3 were determined to be pH 7, cultivation temperature

30˚C, initial concentration of phthalic acid 500 mg/L, and inoculation volume ≥ 1.2%. The above studies showed that under this optimum condition, the degradation rate of the strain was the highest. Under these optimal conditions, strain B3 was cultured in shake flasks for 7 days, with samples taken every 24 hours. The concentration of phthalic acid in the fermentation broth was measured using high-performance liquid chromatography (HPLC), and the results are shown in Figure 7 . As the cultivation time increased, the concentration of phthalic acid in the fermentation broth decreased continuously. Based on the calculations, strain B3 had a maximum degradation efficiency of 77% for phthalic acid in 7 days under the optimal condition.

To investigate the degradation of phthalic acid in soil by strain B3 in the natural environment, a pot experiment was conducted. Different treatments were applied to the soil of naturally growing tobacco plants. Subsequently, the heights, stem circumferences, and fresh and dry weights of roots, stems, and leaves of tobacco plants under different treatments (control, PA, PA + B3) were measured, and the results are presented in Table 2 . In the PA treatment, the heights and fresh weights of stems and leaves of tobacco plants were significantly lower than those in the control group. In contrast, compared to the PA treatment, the heights and fresh weights of roots and stems of tobacco plants were significantly increased in the PA + B3 treatment. These results indicate that phthalic acid has a significant inhibitory effect on the growth of tobacco plants, while strain B3 can effectively alleviate the growth inhibition caused by phthalic acid.

Based on liquid chromatography analysis, the content of phthalic acid in soil under different treatments was determined ( Figure 8 ). The results indicate that the content of phthalic acid in the soil of the PA + B3 group decreased by 45% compared to the PA soil (ANOVA, P < 0.01), suggesting that strain B3 has a degrading effect on phthalic acid in the soil.