A chlorobenzene-degrading bacterium and its application

Abstract

This invention relates to a bacterium that degrades chlorobenzene pollutants and its applications. The bacterium is a *Pseudomonas putida* strain, named *Pseudomonas putida* BS-1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2024441 on March 11, 2024. Compared with existing technologies, the strain provided by this invention can completely degrade a complex pollutant mixture of dichlorobenzene, chlorobenzene, and benzene within 48 hours. It can survive for a long time and maintain its degradation activity under actual pollution conditions of complex pollution and low temperatures. Furthermore, the functional genes related to the degradation of chlorobenzene pollutants are located on a plasmid, which helps the indigenous bacterium acquire the relevant functions for degrading chlorobenzene pollutants. This strain can be applied to in-situ bioremediation of groundwater with complex chlorobenzene pollution in chemical industrial parks, effectively overcoming the technical shortcomings of traditional reduction dechlorination remediation technologies, which often result in incomplete reduction of products, and achieving green, sustainable, and thorough purification without secondary pollutants.

Description

Technical Field

[0001] This invention belongs to the field of microbial remediation technology and relates to a chlorobenzene pollutant degrading bacterium and its application. Background Technology

[0002] Chlorobenzenes (CBs) have attracted widespread attention due to their high frequency of detection in sites, high toxicity, and tendency to bioaccumulate. In particular, dichlorobenzene isomers (DCB) and monochlorobenzene (MCB) are widely used not only in the manufacture of pesticides and dyes but also as industrial solvents, leading to their widespread release into the environment. Through rainfall and soil-water interactions, these pollutants form underground plumes and migrate beyond chemical industrial parks, seriously threatening sensitive receptors such as surrounding rivers.

[0003] The treatment of chlorobenzene contaminants has traditionally relied on chemical reduction and microbial degradation. Nano-zero-valent iron and bimetallic materials such as Pd / Fe and Ni / Fe have shown high efficiency in converting these contaminants, completing the dechlorination reaction of dichlorobenzene or compounds with more chlorine substituents within 1–4 hours. Furthermore, biodegradation by organohalogen-respiring bacteria (OHRB) under anaerobic conditions using aromatic organohalides as terminal electron acceptors has been extensively studied. Benzene is theoretically the final product of the reductive dechlorination of hexachlorobenzene, pentachlorobenzene, tetrachlorobenzene, and trichlorobenzene; in reality, the reaction pathway may stop at the DCB and MCB levels. Notably, under typical anaerobic conditions, the reductive dechlorination of p-dichlorobenzene (p-DCB) occurs very slowly; studies have reported that it can persist for over 700 days without degradation under simulated anaerobic dechlorination conditions. Therefore, p-dichlorobenzene, chlorobenzene, and benzene, as "stubborn" pollutants in chlorobenzene-contaminated sites, make reductive dechlorination remediation technologies difficult to address.

[0004] However, in aerobic environments, aerobic microorganisms can degrade p-DCB, CB, and B through pathways such as hydroxylation by oxygenases, ultimately leading to the metabolism of substrates into non-toxic products such as CO2. However, their degradation efficiency is often limited by factors such as ambient temperature and pH. The temperature of groundwater in contaminated sites is typically maintained between 10 and 20°C year-round, while the degradation capacity of existing dichlorobenzene-degrading bacteria at 20°C is usually 30%–50% lower than at the optimum temperature. For example, *Alishewanella aestuarii* DXL-1 (Liu Huihui, Jiangsu University, 2010) maintains a degradation rate of 70%–80% for 1,2-DCB within a temperature range of 25°C–35°C, but this rate decreases to 45%–55% when the temperature drops to 15°C–20°C. *Ralstoniapicketti* H2 (CN201010181332) exhibits a degradation rate of less than 15% for chlorobenzene at 15°C. Pandoraeasp.XJJ-1 (CN202211166232) can tolerate and degrade chlorobenzene up to 600 mg / L, but cannot degrade p-dichlorobenzene.

[0005] In addition, CN202311191119.7 discloses a low-temperature, high-efficiency aromatic hydrocarbon degrading bacterium belonging to the genus *Pseudomonas*, with the strain code BO3-4. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M 2023108, on February 13, 2023. This strain BO3-4 can independently degrade p-dichlorobenzene and chlorobenzene at an initial concentration of 10 mg / L within 96 hours. Its degradation performance is further slowed down under conditions of combined contamination or high concentrations. Furthermore, its metabolic pathways for p-dichlorobenzene and chlorobenzene are not yet clear. Summary of the Invention

[0006] The purpose of this invention is to provide a chlorobenzene-degrading bacterium and its application, so as to achieve efficient degradation of dichlorobenzene, chlorobenzene, benzene and other benzene series compounds at low temperatures.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In one aspect, this invention provides a chlorobenzene-degrading bacterium, *Pseudomonas putida*, strain named *Pseudomonas putida* BS-1, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, accession number CCTCC NO: M 2024441, on March 11, 2024.

[0009] Furthermore, it was Gram-negative, lacked a capsule, and exhibited regular colony morphology on Luria-Bertani medium, appearing pale yellow, raised, opaque, and smooth. Simultaneously, 16S rDNA sequencing was performed, and the obtained 16S rDNA sequence is shown in SEQ ID NO.1. BLAST alignment of the obtained 16S rDNA sequence showed that the nucleotide sequence of strain BS-1 had greater than 99% homology with the nucleotide sequences of different strains of *Pseudomonas putida*.

[0010] The 16S rRNA gene sequence of Pseudomonas putida BS-1 provided by this invention is as follows:

[0011]

[0012] Meanwhile, the degrading bacterium provided by this invention is Pseudomonas putida BS-1, with accession number CCTCC M2024441 and GenBank accession number PP478807.

[0013] In another aspect, the present invention also provides a method for screening bacteria that degrade chlorobenzene pollutants, comprising the following steps:

[0014] (1) Mix MSM medium (basal inorganic salt medium) and trace element mixture to obtain liquid medium;

[0015] (2) Take the contaminated soil from a chemical plant in East China and add it to a liquid culture medium to prepare the first mixed solution containing bacteria. Then, inject a certain amount of dichlorobenzene, chlorobenzene and benzene solution into the solution and mix them. After 3-5 days in a constant temperature shaking incubator at 20℃, transfer 10% of the first culture solution to another fresh liquid culture medium to obtain the second mixed solution. Continue to culture and inoculate under the same conditions until the nth mixed solution is obtained, where n = 5-8.

[0016] (3) The obtained sixth mixture was diluted and spread on an MSM solid culture plate and cultured in an incubator at 20℃~28℃ for 5~7 days. It was then streaked on Luria-Bertani medium plates for isolation and culture to obtain the dichlorobenzene, chlorobenzene and benzene degrading bacteria.

[0017] Preferably, in step (1), the inorganic salt components in the basic inorganic salt culture medium include: 0.112 g / L MgSO4·H2O, 0.34 g / L KH2PO4, 0.67 g / L Na2HPO4·7H2O, 0.017 g / L CaSO4, and 0.613 g / L (NH4)2SO4.

[0018] Preferably, in step (1), the solute components in the trace element mixture include: 5.0 g / L ZnSO4·7H2O, 0.26 g / L MnSO4·H2O, 0.22 g / L FeSO4·7H2O, 0.02 g / L CuSO4·5H2O, and the solvent component is deionized water.

[0019] Preferably, in step (2), the concentrations of dichlorobenzene, chlorobenzene, and benzene solutions are all 10 g / L, and the solvent is N,N-dimethylformamide.

[0020] Preferably, in step (2), the volume ratio of the inorganic salt culture medium and trace element solution in each liquid culture medium to the volume of the dichlorobenzene, chlorobenzene, and benzene solution added each time via syringe is 10. 3 :1:2:2:2.

[0021] Preferably, in step (2), when preparing the first mixture, the ratio of soil sample to liquid culture medium added is (4-6) g: 20 mL.

[0022] Preferably, in step (2), a serum bottle is used as a culture container, and the culture process is carried out under closed conditions with shaking culture at a temperature of 20-25℃, with each culture lasting about 5-7 days.

[0023] Preferably, in step (3), the MSM solid culture plate contains: 0.112 g / L MgSO4·H2O, 0.34 g / L KH2PO4, 0.67 g / L Na2HPO4·7H2O, 0.017 g / L CaSO4, 0.613 g / L (NH4)2SO4, and 15 g / L agar powder. The dilution, coating, and separation culture process is as follows: Take 0.1 mL of the nth transfer degradation bacterial solution and add it to 0.9 mL of sterile 0.9% NaCl solution, mix well, and the solution is 10 mL. -1 Gradually diluted to 10 -2 10 -3 10 -4 10 -5 The bacteria were spread on MSM solid agar plates, with three replicates for each gradient. A certain amount of p-dichlorobenzene, chlorobenzene, and benzene solutions were added to the bottom of the plates to utilize the volatility of these substances to provide the carbon source required for the growth of the strains on the plates. Single colonies were then picked up with an inoculation loop and isolated on Luria-Bertani agar plates. This process was repeated 2-3 times. The obtained single colonies were inoculated into the degradation system to verify their degradation ability. If it was observed that the bacteria could grow using p-dichlorobenzene, chlorobenzene, and benzene as the sole carbon source and that the concentrations of p-dichlorobenzene, chlorobenzene, and benzene decreased, then it was identified as a p-dichlorobenzene, chlorobenzene, and benzene degrading bacterium.

[0024] In another aspect, the present invention also provides the application of chlorobenzene-degrading bacteria for degrading p-dichlorobenzene, chlorobenzene or benzene.

[0025] Furthermore, this chlorobenzene-degrading bacteria can be used for in-situ remediation of groundwater in chlorobenzene-contaminated sites.

[0026] Furthermore, the working temperature for this chlorobenzene-degrading bacteria to degrade dichlorobenzene, chlorobenzene, and benzene is 15–28°C.

[0027] Furthermore, the pH value for the degradation of dichlorobenzene, chlorobenzene, and benzene by this chlorobenzene-degrading bacteria is 6-9.

[0028] Furthermore, when the chlorobenzene-degrading bacteria degrade dichlorobenzene, chlorobenzene, and benzene, the concentration of the target pollutant is 0 mg / L-60 mg / L, and is not 0.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) By taking contaminated soil from chlorobenzene contaminated sites in chemical industrial parks, and screening out the dichlorobenzene, chlorobenzene and benzene degrading bacteria BS-1, the bacteria can completely degrade the three pollutants within 48 hours under low temperature culture conditions of 20°C in a mixed substrate where dichlorobenzene, chlorobenzene and benzene coexist.

[0031] (2) This strain has high degradation efficiency for dichlorobenzene, chlorobenzene and benzene, and also has good degradation effect under different pH and initial substrate concentration conditions.

[0032] (3) The Pseudomonas putida BS-1 provided by this invention can degrade p-dichlorobenzene, chlorobenzene and benzene, and can achieve the purpose of removing pollutants. There is no accumulation of toxic intermediate products and no secondary pollutants are generated. The degradation effect on a variety of aromatic hydrocarbon pollutants is stable under low temperature conditions. It can be applied to a variety of application scenarios such as the remediation of polluted groundwater. It has little negative impact on the environment and has a good prospect for development and utilization.

[0033] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0034] Figure 1 The image provided by this invention shows the turbidity of the culture medium over time during the simultaneous degradation of dichlorobenzene, chlorobenzene, and benzene by the strain in a 150ml serum bottle.

[0035] Figure 2 The colony morphology and scanning electron microscope image of Pseudomonas putida BS-1 provided for this invention.

[0036] Figure 3 The phylogenetic tree of Pseudomonas putida BS-1 provided for this invention.

[0037] Figure 4 The degradation curves of dichlorobenzene, chlorobenzene, and benzene provided by Pseudomonas putida BS-1 for the present invention.

[0038] Figure 5 The diagram illustrates the degradation rates of p-dichlorobenzene, chlorobenzene, and benzene by Pseudomonas putida BS-1 at different temperatures, as provided by this invention.

[0039] Figure 6 This diagram illustrates the degradation rates of p-dichlorobenzene, chlorobenzene, and benzene by Pseudomonas putida BS-1 at different pH values, as provided by this invention.

[0040] Figure 7 The diagram illustrates the degradation rates of p-dichlorobenzene, chlorobenzene, and benzene by Pseudomonas putida BS-1 at different initial pollutant concentrations, as provided by this invention.

[0041] Figure 8 The diagram shows the degradation pathway of dichlorobenzene, chlorobenzene, and benzene by Pseudomonas putida BS-1 provided by this invention. Detailed Implementation

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0043] In particular, the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, which should be understood to include values ​​close to those ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] Meanwhile, regarding the attached diagram, Figure 5-7 In each group of bar charts, from left to right, they represent p-dichlorobenzene, chlorobenzene, benzene, and DO600. nm .

[0046] Example 1

[0047] Obtaining strains

[0048] This embodiment provides a highly efficient and stable strain of p-dichlorobenzene, chlorobenzene, and benzene-degrading bacteria, namely *Pseudomonas putida* BS-1. The strain is Gram-negative, has a regular morphology, and is pale yellow, raised, opaque, and smooth. 16S rDNA sequencing and whole-genome analysis were performed. BLAST alignment of the obtained 16S rDNA sequence showed that the nucleotide sequence of strain BS-1 had greater than 99% homology with that of *Pseudomonas putida*. *Pseudomonas putida* is a GRAS-certified microorganism, possessing functional genes such as monooxygenases and dioxygenases, oxidoreductases, ferrooxidoreductase proteins and cytochromes, glutathione transferase, sulfur metabolism proteins, and efflux pumps.

[0049] The strain provided in this embodiment is deposited at the China Center for Type Culture Collection, with accession number CCTCCM2024441, and the deposit date is March 11, 2024.

[0050] The above screening method for dichlorobenzene, chlorobenzene, and benzene-degrading bacteria includes the following steps:

[0051] (1) Mix MSM medium (basal inorganic salt medium) and trace element mixture to obtain liquid medium;

[0052] (2) Take the contaminated soil from a chemical plant in East China and add it to a liquid culture medium to prepare the first mixed solution containing bacteria. Then, inject a certain amount of dichlorobenzene, chlorobenzene and benzene solution into the solution and mix them. After 3-5 days in a constant temperature shaking incubator at 20℃, transfer 10% of the first culture solution to another fresh liquid culture medium to obtain the second mixed solution. Continue to culture and inoculate under the same conditions until the nth mixed solution is obtained, where n = 5-8.

[0053] (3) The obtained sixth mixture was diluted and spread on an MSM solid culture plate and cultured in an incubator at 20℃~28℃ for 5~7 days. It was then streaked on Luria-Bertani medium plates for isolation and culture to obtain the dichlorobenzene, chlorobenzene and benzene degrading bacteria.

[0054] In step (1), the inorganic salt components in the basic inorganic salt culture medium include: 0.112 g / L MgSO4·H2O, 0.34 g / L KH2PO4, 0.67 g / L Na2HPO4·7H2O, 0.017 g / L CaSO4, and 0.613 g / L (NH4)2SO4.

[0055] In step (1), the solute components in the trace element mixture include: 5.0 g / L ZnSO4·7H2O, 0.26 g / L MnSO4·H2O, 0.22 g / L FeSO4·7H2O, 0.02 g / L CuSO4·5H2O, and the solvent component is deionized water.

[0056] In step (2), the concentrations of dichlorobenzene, chlorobenzene, and benzene solutions are all 10 g / L, and the solvent is N,N-dimethylformamide.

[0057] In step (2), the volume ratio of the inorganic salt culture medium and trace element solution in each liquid culture medium to the volume of dichlorobenzene, chlorobenzene, and benzene solution added each time via syringe is 10. 3 :1:2:2:2.

[0058] In step (2), when the first mixture is prepared, the ratio of soil sample to liquid culture medium added is (4-6) g: 20 mL.

[0059] In step (2), serum bottles are used as culture containers. The culture process is carried out under closed conditions with shaking, at a temperature of 20-25℃, and each culture lasts for about 5-7 days.

[0060] In step (3), the MSM solid culture plate contains: 0.112 g / L MgSO4·H2O, 0.34 g / L KH2PO4, 0.67 g / L Na2HPO4·7H2O, 0.017 g / L CaSO4, 0.613 g / L (NH4)2SO4, and 15 g / L agar powder. The dilution, plating, and separation culture process is as follows: Take 0.1 mL of the nth transfer degradation bacterial solution and add it to 0.9 mL of sterile 0.9% NaCl solution, mix well, and the mixture is 10 mL. -1 Gradually diluted to 10 -2 10 -3 10 -4 10 -5 The culture was spread on MSM solid medium plates, with three replicates for each gradient. A certain amount of dichlorobenzene, chlorobenzene, and benzene solution was added to the bottom of the plate to utilize the volatility of these substances to provide the carbon source required for the growth of the strains on the plate.

[0061] Then, use an inoculation loop to pick single colonies and isolate them on Luria-Bertani agar plates. Repeat this process 2-3 times. Inoculate the obtained single colonies into the degradation system to verify their degradation ability. If it is observed that the bacteria can utilize p-dichlorobenzene, chlorobenzene, and benzene as the sole carbon source for growth and that the concentrations of p-dichlorobenzene, chlorobenzene, and benzene decrease, then it is a p-dichlorobenzene, chlorobenzene, and benzene-degrading bacterium. The colony morphology of BS-1 is as follows: Figure 2As shown, the morphology is regular, pale yellow, raised, opaque, and smooth. BLAST alignment of the measured 16S rDNA sequence showed that the nucleotide sequence of the 16S rDNA of strain BS-1 had greater than 99% homology with the nucleotide sequences of different strains of *Pseudomonas putida*. Figure 3 The genetic relationship between strain BS-1 and other Pseudomonas species is shown. It is in the same branch of the phylogenetic tree as Pseudomonas putida (AY332610.1), indicating that they are closely related.

[0062] Example 2

[0063] Study on the degradation of dichlorobenzene, chlorobenzene and benzene by the strain

[0064] 20 mL of inorganic salt culture medium containing 20 mg / L of p-dichlorobenzene, chlorobenzene, and benzene respectively was placed in a 150 mL serum bottle, and the inoculation procedure was the same as in Example 1. To prevent the volatilization of p-dichlorobenzene, chlorobenzene, and benzene, an aluminum cap with a polytetrafluoroethylene gasket was used for sealing. The culture was carried out at 20°C, 150 rpm, and in the dark with shaking. Samples were taken periodically to determine the residual concentrations of p-dichlorobenzene, chlorobenzene, and benzene.

[0065] Figure 1 The image shows the culture medium becoming turbid over time during the degradation of dichlorobenzene, chlorobenzene, and benzene by the strain in Example 2.

[0066] Figure 4 The results show that at a temperature of 20°C, Pseudomonas putida.BS-1 can completely degrade p-dichlorobenzene, chlorobenzene and benzene, all with an initial concentration of 20 mg / L, within 7 days, indicating that it can adapt to actual groundwater temperatures and maintain its degradation activity against the three target pollutants.

[0067] Example 3

[0068] Study on the degradation characteristics of the strain (at different temperatures, pH, and initial concentrations)

[0069] 100 mL of Luria-Bertani medium was transferred to a 250 mL Erlenmeyer flask, and BS-1 strain was inoculated into the flask, with specific amounts of p-dichlorobenzene, chlorobenzene, and benzene solutions added respectively. After 24 h of bacterial growth, the bacterial suspension was centrifuged at 8000 rpm for 5 min and washed with 0.9% NaCl solution, repeated twice. 20 mL of MSM medium was transferred to a 150 mL serum bottle. The washed cells were resuspended in 20 mL of carbon-free MSM medium, and the initial OD was determined. 600nmThe initial concentration was 0.1. The bottle was sealed, and para-dichlorobenzene, chlorobenzene, and benzene were added to the culture medium using a glass syringe. The final concentration of each of the three contaminants in the culture medium was 20 mg / L. To prevent the volatilization of para-dichlorobenzene, chlorobenzene, and benzene, an aluminum cap with a PTFE gasket was used for sealing. The serum bottle was placed in the dark and incubated with shaking at a speed of 150 rpm. Temperature, pH, and initial contaminant concentrations were varied, and samples were taken after 48 hours. The residual concentrations of para-dichlorobenzene, chlorobenzene, and benzene were determined using headspace gas chromatography.

[0070] 1. Different temperatures

[0071] Degradation conditions: pH 7.0, initial concentrations of dichlorobenzene, chlorobenzene, and benzene were all 20 mg / L; temperatures: 15℃, 20℃, and 28℃.

[0072] The results show that: Figure 5 This diagram illustrates the degradation effects of strain Pseudomonasputida.BS-1 on p-dichlorobenzene, chlorobenzene, and benzene at different temperatures. Strain BS-1 exhibits good degradation effects on p-dichlorobenzene, chlorobenzene, and benzene at temperatures ranging from 20℃ to 28℃, with degradation rates exceeding 95.2%. At a lower temperature of 15℃, it shows good degradation effects on chlorobenzene, with degradation rates reaching 93.3%–96.5%, and also exhibits some degradation effects on p-dichlorobenzene and benzene, with degradation rates of 51.9%–60.7% and 46.6%–53.5%, respectively.

[0073] 2. Different pH values

[0074] Degradation conditions: temperature 20℃, initial concentrations of p-dichlorobenzene, chlorobenzene and benzene all 20 mg / L; pH: 6, 7, 8, 9;

[0075] The results show that: Figure 6 This diagram illustrates the effect of different pH values ​​on the degradation rates of benzene compounds (dichlorobenzene, chlorobenzene, and benzene) by the *Pseudomonas putida* BS-1 strain provided in this invention. Strain BS-1 completely degrades all three pollutants within a pH range of 6.0-9.0, demonstrating excellent pH adaptability.

[0076] 3. Different initial pollutant concentrations

[0077] Degradation conditions: pH 7.0; temperature: 20℃; initial total concentrations of p-dichlorobenzene, chlorobenzene and benzene: 30 mg / L, 60 mg / L, 90 mg / L, 150 mg / L (p-DCB:CB:B=1:1:1).

[0078] The results show that: Figure 7This diagram illustrates the effect of different initial pollutant concentrations on the degradation rate of benzene compounds by the aromatic hydrocarbon-degrading strain Pseudomonas putida BS-1, as provided in this invention. Strain BS-1 can degrade a mixed system of dichlorobenzene, chlorobenzene, and benzene in the concentration range of 30 mg / L to 150 mg / L, with a degradation rate of 93.8% to 100% for the three target pollutants in the concentration range of 30 mg / L to 60 mg / L.

[0079] Similarly, strain BO3-4 (CN117402774A) achieved a degradation effect of 10 mg / L of single para-dichlorobenzene and chlorobenzene in 4 days. In contrast, strain BS-1 of this invention, as described above, can degrade 10 mg / L of single chlorobenzene within 1 day, 10 mg / L of single para-dichlorobenzene within 36 hours, and completely degrade a complex pollutant of 60 mg / L para-dichlorobenzene, chlorobenzene, and benzene (1:1:1) within 2 days. Therefore, it is evident that strain BS-1 of this invention has a superior degradation effect on chlorobenzene pollutants such as dichlorobenzene.

[0080] Example 4:

[0081] Study on the metabolic pathways of dichlorobenzene, chlorobenzene and benzene by strain BS-1

[0082] 20 mL of MSM medium was transferred into a 150 mL serum bottle. The washed cells were resuspended in 20 mL of carbon-free MSM medium. Initial OD... 600nm The concentration was 0.1. The bottle was sealed, and p-dichlorobenzene, chlorobenzene, and benzene were added to the culture medium using a glass syringe, resulting in a final concentration of 20 mg / L for each of the three contaminants. Samples were taken sequentially at 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h. Approximately 300 mL of bacterial culture was combined and centrifuged at 10,000 rpm to remove bacterial precipitate. The supernatant was extracted twice with n-hexane under alkaline conditions (pH = 10–11), followed by twice with dichloromethane under acidic conditions (pH = 4–5). The combined extracts were dried over anhydrous sodium sulfate and rotary evaporated to near dryness. The concentrated sample was then redissolved in acetone, filtered through a 0.22 μm organic solvent filter membrane, and derivatized using butylboronic acid in a 50°C water bath for 20 min. The degradation intermediates were qualitatively analyzed using a gas chromatograph-mass spectrometer (GC / MS) (7890A-5975C, Agilent, USA) equipped with an HP-5 capillary column (30m×250um×1.00um). A separate concentrated sample was dissolved in acetonitrile, added to N,O-bis(trimethylsilyl)trifluoroacetamide, filtered through a 0.22μm organic filter, and derivatized at 60°C for 1 h. Qualitative analysis was performed using GC / MS (GCNS-TQ8050, Shimadzu, Japan).

[0083] Mass spectrometry data of the intermediate products were compared with standard NIST 2020 library data. Five key intermediates were detected in the butylboronic acid derivatization system: phenol (9.543 min), o-chlorophenol (9.667 min), 2,5-dichlorophenol (15.095 min), 3-chlorocatechol (15.667 min), and p-dichlorobenzenedihydrodiol (26.917 min). Four key intermediates were detected in the N,O-bis(trimethylsilyl)trifluoroacetamide derivatization system: phenol (11.615 min), o-chlorophenol (16.110 min), catechol (19.250 min), and 2,5-dichlorophenol (20.165 min). Therefore, a total of six key intermediates were ultimately detected: phenol, o-chlorophenol, 2,5-dichlorophenol, 3-chlorocatechol, p-dichlorobenzenedihydrodiol, and catechol.

[0084] Whole genome analysis of strain BS-1

[0085] Strain BS-1 was cultured to the logarithmic growth phase in MSM medium containing 60 mg / L p-DCB, CB, and B. The bacterial culture was collected and centrifuged at 10,000 rpm for 5 min to collect the bacterial cells. DNA was extracted from the bacterial community according to the instructions of the MP BIO FastDNASpinKit for Soil. Genomic DNA was transported at low temperature to Shanghai Paisenno Biotechnology Co., Ltd. The total DNA amount was determined using a fluorescent dye (Quant-iT PicoGreen dsDNAAssay Kit). The concentration and purity of the genome were verified using 1% agarose gel electrophoresis and a micro-spectrophotometer to determine if the genome met the requirements for library experiments. The required genome library was constructed using the standard Illumina TruSeq Nano DNALT library preparation procedure (Illumina TruSeq DNA Sample Preparation Guide). A whole-genome shotgun (WGS) strategy was employed to construct libraries with different insert fragments. Next-generation sequencing (NGS) was used on the Illumina NovaSeq platform, and third-generation single-molecule sequencing (SMS) was used on the Oxford Nanopore ONT platform to sequence these libraries. FastP (https: / / github.com / OpenGene / fastp) was used for data quality control. The amino acid sequences of the coding genes were annotated and aligned in Non-Redundant (NR), Clusters of Orthologous Groups of Proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Swiss Prot databases to obtain functional prediction information for the coding genes.

[0086] Whole-genome analysis revealed that strain BS-1's genome comprises a circular chromosome and a circular plasmid. Degradation genes, located on the plasmid, encode the aromatic dioxygenase tod (RXFC1C2BADEGIHST), the cyclic hydrolase cmt (EFHG), and the chlorocatechol dioxygenase clc (RABCDE), converting p-dichlorobenzene, chlorobenzene, and benzene into non-toxic substances. Furthermore, genes located on the bacterial chromosome are considered non-mobile, while those on the plasmid are considered mobile genetic elements. Bacteria carrying the degradation plasmid in contaminated sites can transfer these mobile genetic elements to other strains. This mechanism facilitates the dissemination of catabolic genes into bacterial populations within contaminated sites; therefore, native microbial strains possessing this degradation plasmid are crucial for the bioremediation of exogenous contaminants.

[0087] Based on whole-genome analysis, the aromatic dioxygenase in strain BS-1 showed 99% similarity to that of Pseudomonas putida F1. P. putida F1's aromatic hydrocarbon dioxygenase (TOD) is a multi-component enzyme system that generates corresponding phenolic substances through two consecutive monooxygenation reactions. It can oxidize 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol, and then catalyze the monohydroxylation of phenols to the corresponding catechols. Therefore, it is speculated that strain BS-1 degrades p-dichlorobenzene through the action of dioxygenase to generate p-dichlorobenzene dihydrogen diol, followed by dehydrogenase to generate 3,6-dichlorocatechol, or through two consecutive monohydroxylation reactions by dioxygenase to generate 3,6-dichlorocatechol, which is then further ring-opened by chlorocatechol 1,2-dioxygenase to generate 2,5-dichloromucinic acid, which then enters the downstream pathway. Strain BS-1 degrades chlorobenzene and benzene via a similar process of sequential hydroxylation using dioxygenases to produce o-chlorophenol, 3-chlorocatechol, phenol, and catechol, followed by ring-opening and downstream pathways. The metabolic pathways for dichlorobenzene, chlorobenzene, and benzene degradation by the strain are as follows: Figure 8 As shown.

[0088] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A chlorobenzene-based pollutant-degrading bacterium, characterized in that, It is Pseudomonas putida, and the strain is named Pseudomonas putida BS-1 , and is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2024441 and the preservation time of March 11, 2024. The degrading bacteria can degrade p-dichlorobenzene, chlorobenzene and benzene at 15~28℃.

2. The chlorobenzene pollutant-degrading bacterium according to claim 1, characterized by, It is Gram-negative, the strain has no capsule, and the colony morphology on Luria-Bertani medium is regular, pale yellow, raised, opaque and smooth.

3. The use of the chlorobenzene-degrading bacterium according to claim 1 or 2, characterized in that, This chlorobenzene-degrading bacteria is used to degrade p-dichlorobenzene, chlorobenzene, or benzene.

4. The application of the chlorobenzene-degrading bacteria according to claim 3, characterized in that, This chlorobenzene-degrading bacteria can be used for in-situ remediation of groundwater in chlorobenzene-contaminated sites.

5. The application of the chlorobenzene-degrading bacteria according to claim 3, characterized in that, The working temperature for this chlorobenzene-degrading bacteria to degrade dichlorobenzene, chlorobenzene, and benzene is 15~28℃.

6. The application of the chlorobenzene-degrading bacteria according to claim 3, characterized in that, The pH value for the degradation of dichlorobenzene, chlorobenzene and benzene by this chlorobenzene-degrading bacteria is 6-9.

7. The application of the chlorobenzene-degrading bacteria according to claim 3, characterized in that, When the chlorobenzene-degrading bacteria degrade dichlorobenzene, chlorobenzene, and benzene, the concentration of the target pollutant is no higher than 60 mg / L.

Images