Bacillus subtilis and application thereof in degradation of phthalate

Abstract

The invention discloses a bacillus subtilis strain and an application of the bacillus subtilis strain in degradation of phthalate. According to the bacillus subtilis FQ7-2 for degrading the phthalic acid ester, the preservation number is GDMCC NO: 67356, the degradation rate of the bacillus subtilis FQ7-2 with the concentration of OD600 = 0.2 for degrading 200 ppm DBP for 4 h is as high as 96.18%, the degradation rate of the bacillus subtilis FQ7-2 for degrading 2000 DBP for 8 h is as high as 86.53%, and the bacillus subtilis FQ7-2 still has a good degradation characteristic on the DBP in the environment containing other carbon sources except the DBP; the strain has high degradation efficiency on DBP in a culture environment with a temperature of 30-45 DEG C and a pH value of 6-8, and shows a broad spectrum on PAEs degradation. The bacillus subtilis FQ7-2 provided by the invention has important application significance in repairing an environment which is seriously polluted by PAEs.

Description

Technical Field

[0001] This invention belongs to the field of microbial ecological governance technology, specifically involving a strain of Bacillus subtilis and its application in the degradation of phthalates. Background Technology

[0002] Phthalate esters (PAEs) are widely used in various industries (such as plastic products, cosmetics, medical devices, and toys), primarily to increase the flexibility and toughness of plastics. However, PAEs are also heavily criticized for their environmental residues and health risks. When released from plastics into the environment, PAEs pollute the atmosphere, water bodies, and soil. They can also enter the human body through the food chain and skin contact, affecting the endocrine, reproductive, and vascular systems, causing related illnesses. Furthermore, PAEs pose risks of carcinogenesis, teratogenesis, and mutagenesis after entering the human body. Currently, PAEs have been listed as priority environmental pollutants in developed countries and some developing countries. As the largest producer and user of PAEs, addressing the environmental residue problem of PAEs is a pressing issue.

[0003] Currently, there are three main methods for eliminating PAEs in the environment: photodegradation, chemical degradation, and biodegradation. Among these, biodegradation is currently the safest and most efficient degradation method. Several microorganisms have been reported to be able to degrade PAEs, such as... Arthrobacter sp. SF27, Sphingobium sp. SM42, Bacillus sp. K91、 Bacillus sp. LUNF1, Acinetobacter sp. LUNF3, Gordonia sp. GZ-YC7 and Rhodococcus sp. AH-ZY2, etc. However, the degradation of PAEs by these strains is all passive stress degradation, which requires PAEs as the sole carbon source. Since the environment is rich in nutrients, especially various carbon sources, this limits the application value of these strains.

[0004] Few bacterial strains can effectively degrade PAEs in environments where PAEs are not the sole carbon source. For example... Discovering strains that can adapt to environmental diversity and achieve efficient degradation of PAEs while growing and reproducing rapidly in nutrient-rich environments is an urgent task to be solved. Summary of the Invention

[0005] Based on the shortcomings and defects of existing technologies, this invention aims to provide a strain of Bacillus subtilis and its application in the degradation of phthalates (PAEs). This invention identifies a strain of Bacillus subtilis capable of efficiently degrading PAEs from activated sludge, and names it Bacillus subtilis (…). Bacillus subtilis Bacillus subtilis strain FQ7-2 (GDMCC NO: 67356, deposited on November 25, 2025, by GDMCC-Guangdong Provincial Microbial Culture Collection Center) differs from previously reported strains in that it exhibits highly efficient PAE degradation activity even under nutrient-rich (LB) conditions, demonstrating significant potential for environmental remediation.

[0006] The first objective of this invention is to provide a strain of Bacillus subtilis (B. subtilis) Bacillus subtilis )FQ7-2, its accession number is GDMCC NO:67356.

[0007] A second objective of this invention is to provide a formulation for degrading phthalates, wherein the active ingredient is a live cell of Bacillus subtilis FQ7-2 or a culture of the live cell of Bacillus subtilis FQ7-2.

[0008] Preferably, the culture of live Bacillus subtilis FQ7-2 in the formulation is a bacterial suspension of Bacillus subtilis FQ7-2.

[0009] Preferably, the concentration of Bacillus subtilis FQ7-2 bacterial suspension in the formulation is OD. 600 =0.2 or more.

[0010] A third object of the present invention is to provide the use of the aforementioned Bacillus subtilis FQ7-2 in the preparation of formulations that degrade phthalates.

[0011] A fourth object of the present invention is to provide the use of the aforementioned Bacillus subtilis FQ7-2 or the aforementioned formulation in the degradation of phthalates.

[0012] Preferably, the phthalate is dibutyl phthalate, dimethyl phthalate, diethyl phthalate, dipentyl phthalate, dicyclohexyl phthalate and / or dioctyl phthalate.

[0013] Preferably, the application shown includes the step of applying the Bacillus subtilis FQ7-2 or the preparation described above.

[0014] Preferably, the conditions for degrading phthalates are: 30℃~45℃ and pH 6~8.

[0015] A fifth object of the present invention is to provide the use of the Bacillus subtilis FQ7-2 or the preparations thereof in the remediation of environments contaminated with phthalates.

[0016] The beneficial effects of this invention are: The Bacillus subtilis FQ7-2 for degrading phthalates provided by this invention has the accession number GDMCCNO: 67356 and a concentration of OD. 600 Bacillus subtilis FQ7-2 at a concentration of 0.2 g / L achieved a degradation rate of 96.18% for 200 ppm DBP in 4 hours and 86.53% for 2000 ppm DBP in 8 hours. It also exhibited good degradation characteristics for DBP even in environments containing carbon sources other than DBP. Furthermore, it demonstrated high degradation efficiency for DBP under culture conditions of 30℃–45℃ and pH 6–8, and showed broad-spectrum degradation of PAEs. Compared to existing phthalate-degrading bacteria, the Bacillus subtilis FQ7-2 provided by this invention can achieve more efficient degradation of high concentrations of DBP in a shorter time, which is of significant application value for remediating environments severely contaminated with PAEs.

[0017] Preservation Instructions The Bacillus subtilis FQ7-2 provided by this invention ( Bacillus subtilis FQ7-2 was deposited on November 25, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO: 67356. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0018] Figure 1 This section presents the morphological characteristics and phylogenetic tree analysis of strain FQ7-2. A and B represent the colony and single-clone morphologies of strain FQ7-2 in an inorganic salt medium without an organic carbon source, respectively. C is the SEM image of strain FQ7-2, and D is the phylogenetic tree constructed for strain FQ7-2.

[0019] Figure 2 The degradation capacity of Bacillus subtilis FQ7-2 for DBP is shown in Figure 1. Specifically, A represents the degradation curve of 200 ppm DBP by Bacillus subtilis FQ7-2, B represents the degradation rate of different concentrations of DBP by Bacillus subtilis FQ7-2 after 3 hours, C represents the degradation curve of 2000 ppm DBP by Bacillus subtilis FQ7-2, and D represents the degradation capacity of the supernatant and cells of Bacillus subtilis FQ7-2 bacterial culture for DBP.

[0020] Figure 3The degradation characteristics of Bacillus subtilis FQ7-2 are shown in Figure 1. Among them, A represents the effect of different temperatures on the ability of Bacillus subtilis FQ7-2 to degrade DBP, B represents the effect of the same pH on the ability of Bacillus subtilis FQ7-2 to degrade DBP, and C represents the degradation rate of different PAEs by Bacillus subtilis FQ7-2. Detailed Implementation

[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0022] Example 1: Screening and identification of Bacillus subtilis FQ7-2 1. Enrichment and screening of strains This embodiment describes the screening and isolation of highly efficient DBP-degrading strains from activated sludge in a pesticide factory. An appropriate amount of activated sludge was taken and sterile physiological saline was added to prepare a bacterial suspension. An inorganic salt liquid culture medium (MSM liquid medium) without any organic carbon source was prepared with the following formula: (NH4)2SO4 1.0 g / L, K2HPO4 1.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.5 g / L, CaCl2 0.01 g / L, FeSO4·7H2O 0.001 g / L, with the remainder being water. The pH was adjusted to 7.0–7.2. The bacterial suspension was inoculated into MSM liquid medium at a volume fraction of 0.5%. After one week of incubation at 37°C and 200 rpm, it was transferred to fresh MSM liquid medium containing 200 ppm DBP at a volume fraction of 1%. Subsequently, every 7 days, it was transferred to fresh MSM liquid medium containing dibutyl phthalate (DBP) at a volume fraction of 1%, with the DBP concentration in the MSM liquid medium increased by 200 ppm each time, up to 2000 ppm. The medium was incubated at 37°C and 200 rpm to screen for and enrich microorganisms with strong degradation capabilities. After screening, the bacterial suspension cultured in MSM liquid medium with a DBP concentration of 2000 ppm for 7 days was diluted 1000 times, and 10 µL was plated onto MSM solid culture medium to obtain single colonies. Single colonies were then inoculated into 500 µL of LB liquid medium and cultured at 37°C and 200 rpm with shaking for 12 h to obtain the seed culture. Inoculate the seed culture into 20 mL of LB liquid medium at a volume fraction of 1%, and culture until OD500. 600 =0.8, diluted with LB liquid medium to OD 600=0.2, dispensed into 10 mL bottles, added 200 ppm DBP, and cultured at 37℃ and 200 rpm for 6 h with shaking. The degradation efficiency of DBP by the colonies was quantitatively analyzed by high performance liquid chromatography (HPLC). Finally, a strain that can degrade DBP with ultra-high efficiency in nutrient-rich liquid medium LB was selected and named FQ7-2.

[0023] 2. Isolation and identification of strains Identification of strain FQ7-2 was accomplished through DNA sequence alignment of the 16S rRNA gene and calculation of the ANI (mean nucleotide similarity) of the genome. Single clones were obtained by streaking on MSM solid medium supplemented with 200 ppm. The strains were irregularly edged, flat, relatively dry, and white and opaque. Figure 1 (A~B in the original text). Scanning electron microscopy (SEM) results showed that strain FQ7-2 was non-flagellated and rod-shaped. Figure 1 (C) The DNA sequence of 16S rRNA of strain FQ7-2 was obtained by bacterial PCR. The primers used for PCR were universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1; 1492R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). The full-length PCR product was 1511 bp. After electrophoresis on a 1% agarose gel, the product was recovered by gel cutting and tested with a UV spectrophotometer (nanodrop 2000). The product was then sent to the company for sequencing, and the obtained nucleotide sequence is shown in SEQ ID NO.3.

[0024] The nucleotide sequence obtained from sequencing, as shown in SEQ ID NO.3, was searched in NCBI's BLAST database. Thirteen representative, previously reported 16S rRNA sequences were selected, and a phylogenetic tree was constructed using MEGA 7.0. The results showed that strain FQ7-2 was most closely related to Bacillus subtilis. Figure 1 Therefore, strain FQ7-2 was preliminarily identified as Bacillus subtilis (D in the original text), and named Bacillus subtilis (D in the original text). Bacillus subtilis )FQ7-2.

[0025] Example 2: Degradation characteristics of DBP by Bacillus subtilis FQ7-2 1. Bacillus subtilis FQ7-2 can efficiently degrade DBP. Bacillus subtilis FQ7-2 was diluted 100-fold and spread onto MSM solid medium plates containing 500 ppm DBP. Single colonies were picked and transferred to LB liquid medium and cultured with shaking until the solution became turbid. Then, Bacillus subtilis FQ7-2 was inoculated into 100 mL of MSM liquid medium containing 200 ppm DBP until OD500 was reached. 600 After incubating overnight at 0.2 g / mL, the bacteria were cultured the next day. Once complete DBP degradation was confirmed by liquid chromatography, the cells were collected by centrifugation and resuspended in antibiotic-free LB broth to OD200. 600 The bacterial resuspension was obtained by setting the concentration to 0.2. The bacterial resuspension was dispensed into Erlenmeyer flasks, 5 mL per flask, and 200 ppm DBP was added. Samples were taken after incubation for 1 h, 2 h, 2.5 h, 3 h, 3.5 h, and 4 h to construct degradation curves and confirm the degradation ability of Bacillus subtilis FQ7-2 on DBP. The results showed that Bacillus subtilis FQ7-2 could efficiently degrade DBP in LB medium (containing carbon sources other than DBP). For 200 ppm DBP, 72.43% was degraded after 2.5 h, and 96.18% was degraded after 4 h. Figure 2 (A) The culture conditions in this experiment were 37℃ and 200 rpm.

[0026] Existing research indicates that most PAE biodegradation experiments are conducted in inorganic salt media with PAEs as the sole carbon source. The half-life of Bacillus PAE-degrading bacteria for degrading 200 ppm DBP ranges from 5 to 10 days. Few strains can degrade PAEs under nutrient-rich conditions. For example, Bacillus subtilis JF requires 24 hours to degrade 200 ppm DBP in LB medium. In LB medium at incubation temperatures of 30℃, 35℃, and 40℃, an inoculum concentration of 1.0 × 10⁻⁶ CFU / mL is used. 8 The half-lives of Bacillus subtilis JF at cfu / mL for degrading 200 ppm DBP were 6.62 h, 4.48 h, and 3.48 h, respectively. In LB medium with pH 6.0, 7.0, and 8.0, and at a culture temperature of 30℃, the half-lives of Bacillus subtilis JF for DBP degradation were 4.61 h, 4.66 h, and 5.33 h, respectively (Li Jianlong, Shao Xiangli, Liu Shuliang, Yao Kai, Zhao Qin, Hu Xinjie, Deng Weiqin, Screening and Identification of Dibutyl Phthalate Degrading Strains and Their Degradation Characteristics, Modern Food Science and Technology 30(10) (2014) 108-114+244). Therefore, Bacillus subtilis FQ7-2 can achieve efficient degradation of DBP in a nutrient-rich environment, which is superior to the effects of PAE-degrading bacteria disclosed in existing technologies, and has good application prospects for bioremediation of PAE-contaminated environments.

[0027] Given the high efficiency of Bacillus subtilis FQ7-2 in degrading DBP, we increased the concentration of DBP and explored the range of DBP that could be efficiently degraded. Therefore, the DBP concentration range was set at 200 ppm to 2000 ppm, and samples were collected after incubation at 37℃ and 200 rpm for 3 hours. The results showed that Bacillus subtilis FQ7-2 achieved a degradation rate of 81% for 200 ppm DBP. The degradation rate decreased with increasing concentration, reaching 12.98% for 2000 ppm DBP after 3 hours. Figure 2 (B in the text). Next, we plotted the degradation curve of Bacillus subtilis FQ7-2 on 2000 ppm DBP. The results showed that the degradation rate was as high as 86.53% after 8 h of shaking culture. Figure 2 In contrast, in existing technologies, when Gordon's bacillus GH-1 is inoculated at a 6% volume fraction in LB medium containing 2000 mg / L DBP and cultured at 30°C, it takes more than 5 days for Gordon's bacillus GH-1 to degrade 2000 ppm DBP (CNPeng, J. Tang, X. Yu, XR Zhou, MJ Wang, YY Zhang, H. Zhou, SQ Huang, Q. Wen, SQ Chen, WL Xiang, Q. Zhang, T. Cai, Biodegradation of various phthalic acid esters at high concentrations by GH-1 and its degradation mechanism, Environ Technol Inno 38 (2025).). This indicates that Bacillus subtilis FQ7-2 has high adaptability and degradation efficiency to high concentrations of DBP, and also has certain application potential for environments contaminated with high concentrations of DBP.

[0028] 2. Types of bioenzymes in Bacillus subtilis FQ7-2 that degrade DBP To determine whether the key enzyme in Bacillus subtilis FQ7-2 that degrades DBP is a secreted protein or an intracellular enzyme, Bacillus subtilis FQ7-2 cells obtained overnight in LB broth at 37°C and 200 rpm were separated by high-speed centrifugation. The bacterial cells and supernatant were collected separately. The bacterial cells were resuspended in LB broth and sonicated. The degradation of DBP by the bacterial cells (pellet) and the supernatant was then measured. 200 ppm DBP was added to the supernatant or the sonicated bacterial suspension, and the cells were incubated at 37°C and 200 rpm for 4 h before the DBP degradation rate was measured. The results showed that the supernatant had no effect on DBP degradation, while the degradation rate of DBP by the sonicated bacterial cells was 96.89%. Figure 2 (D in the text). Therefore, the bioenzyme that degrades DBP by FQ7-2 is an intracellular enzyme.

[0029] 3. Optimization of conditions for DBP degradation by Bacillus subtilis FQ7-2 Regarding the characteristics of Bacillus subtilis FQ7-2 in degrading DBP, we are particularly interested in its sensitivity to temperature and environmental pH in order to analyze its application value.

[0030] Regarding the temperature sensitivity of Bacillus subtilis FQ7-2, the following different temperatures were set in this experiment: 4℃, 16℃, 30℃, 37℃, 42℃, and 45℃. The DBP concentration in the LB liquid medium was 200 ppm, and the pH of the LB medium was 7. Bacillus subtilis FQ7-2 was inoculated to OD... 600 =0.2, and samples were collected after shaking and incubating at 200 rpm for 4 h. Experimental results showed that under low-temperature environments (4℃, 16℃), the degradation efficiency of DBP by Bacillus subtilis FQ7-2 was significantly inhibited, with a degradation rate of approximately 4%, indicating almost no degradation. As the temperature increased from 30℃ to 45℃, the DBP degradation rate slightly increased, both exceeding 80%, and the degradation rate reached as high as 96.26% at 45℃. Figure 3 (A in the original text). Therefore, Bacillus subtilis FQ7-2 is sensitive to low temperatures but resistant to high temperatures.

[0031] Regarding the sensitivity of Bacillus subtilis to pH, this experiment established LB liquid culture media with pH values ​​of 4, 5, 6, 7, 8, 9, and 10. The DBP concentration in the LB medium was 200 ppm. Bacillus subtilis FQ7-2 was inoculated to OD... 600 After the concentration reached 0.2, the sample was collected after shaking culture at 37℃ for 4 hours. Experimental results ( Figure 3B) indicates that the degradation efficiency of DBP by Bacillus subtilis FQ7-2 was significantly inhibited under extremely acidic (pH 4) or extremely alkaline (pH 9 or pH 10) conditions. After 4 h of shaking culture, only 10% to 20% of 200 ppm DBP was degraded. The more suitable pH range was pH 6 to pH 8, with a degradation rate of 87.9% to 95.75%.

[0032] 4. Degradation ability of Bacillus subtilis FQ7-2 against different PAEs This experiment analyzed the degradation effects of Bacillus subtilis FQ7-2 on different PAEs, including dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), diamyl phthalate (DPP), dicyclohexyl phthalate (DCHP), and dioctyl phthalate (DOP). The degradation was carried out at a temperature of 37℃, pH 7, and a concentration of OD. 600 Bacillus subtilis FQ7-1 bacterial culture (obtained by culturing on LB medium) with a concentration of 0.2 was used. The initial concentration of each PAE added was 200 ppm. Samples were collected and analyzed after 4 h and 9 h of shaking culture at 200 rpm. The results showed that Bacillus subtilis FQ7-2 exhibited certain degradation activity against all the aforementioned PAEs. The highest degradation rates were observed for DBP (95% degradation rate after 4 h and 99% after 9 h) and DPP (73% degradation rate after 4 h and 96% after 9 h). The other four PAEs showed approximately 50% degradation after 9 h of shaking culture. Figure 3 (C in the text). In summary, Bacillus subtilis exhibits broad-spectrum degradation of various PAEs, with higher degradation rates for DBP and DPP. We speculate that this may be due to differences in the binding strength between the active sites of the degrading enzymes and their substrates.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis FQ7-2, characterized in that, The preservation number of which is GDMCC NO: 67356.

2. A formulation for degrading phthalates, characterized by, The live Bacillus subtilis FQ7-2 or the culture of the live Bacillus subtilis FQ7-2 of claim 1 is the active ingredient.

3. The preparation according to claim 2, characterized in that, The culture of the live Bacillus subtilis FQ7-2 in the preparation is a bacterial suspension of the Bacillus subtilis FQ7-2.

4. The preparation according to claim 3, characterized in that, The concentration of the bacterial suspension of Bacillus subtilis FQ7-2 in the preparation is OD 600 = 0.2 or more.

5. The use of the Bacillus subtilis FQ7-2 of claim 1 in the preparation of a preparation for degrading phthalate.

6. The use of the Bacillus subtilis FQ7-2 of claim 1 or the preparation of claim 2 in degrading phthalate.

7. Use according to claim 6, characterized in that, The phthalate is dibutyl phthalate, dimethyl phthalate, diethyl phthalate, dipentyl phthalate, dicyclohexyl phthalate and / or dioctyl phthalate.

8. Use according to claim 6, characterized in that, The step of applying the Bacillus subtilis FQ7-2 or the preparation.

9. Use according to claim 6, characterized in that, The condition for degrading phthalate is 30-45℃ and pH 6-8.

10. The use of the Bacillus subtilis FQ7-2 of claim 1 or the preparation of claim 2 in repairing the environment contaminated by phthalate.

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