Agrobacterium tumefaciens with ability of degrading 4-nitrochlorobenzene and application thereof

Abstract

The application discloses a strain of Agrobacterium tumefaciens with the ability of degrading 4-nitrochlorobenzene and application thereof. The Agrobacterium tumefaciens (Agrobacterium pusense) is named as Agrobacterium tumefaciens GDUTXIONG6, and the strain has been preserved in the China Center for Type Culture Collection on November 23, 2023, with a preservation number of CCTCC NO: M 20232309. The Agrobacterium tumefaciens GDUTXIONG6 strain has excellent degrading ability for 4-nitrochlorobenzene, and the degrading rate can reach more than 85%, belongs to a high-efficiency degrading bacterium, and can be used for preparing a degrading bacterium agent. The degrading bacterium agent containing the Agrobacterium tumefaciens GDUTXIONG6 strain not only has high degrading efficiency, but also will not cause pollution and public hazards to the environment in the use process, and has great application value in repairing and treating 4-nitrochlorobenzene-polluted environments.

Description

Technical Field

[0001] This invention relates to the field of microbial degradation technology, and more specifically, to an Agrobacterium rhizogenes strain capable of degrading 4-nitrochlorobenzene and its applications. Background Technology

[0002] 4-Nitrochlorobenzene (4-CNB) is a commonly used chemical raw material in industrial production. It is widely used in pharmaceuticals, agriculture, printing and dyeing, and chemical manufacturing, possessing significant industrial value. However, it is also one of the most significant sources of pollution during its production. In recent years, the use of 4-nitrochlorobenzene has increased annually. Due to its complex structure and stable chemical properties, it is difficult to degrade in the natural environment, thus causing pollution and damage to the ecological environment. Studies have shown that even small doses from skin contact, inhalation, or ingestion can directly lead to lesions in the nervous system, blood system, and organs such as the liver and spleen. 4-Nitrochlorobenzene is listed as a Group 3 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization, indicating that 4-nitrochlorobenzene in the natural environment poses a potentially significant threat to human health.

[0003] Biological treatment of 4-nitrochlorobenzene is a method that utilizes microorganisms to degrade 4-nitrochlorobenzene in the environment. This method has many advantages, such as no secondary pollution, high treatment capacity, low operating costs, and good purification effect. Currently, the number of microbial strains that can be used for biological treatment of 4-nitrochlorobenzene is relatively limited; existing technologies have only reported a few strains, including Lysinibacillus sp. and Bacillus. To date, no other microbial studies have reported that can be used for the degradation of 4-nitrochlorobenzene.

[0004] Therefore, discovering more 4-nitrochlorobenzene-degrading bacteria with better degradation effects and applying them to contaminated environments will effectively reduce the content of harmful pollutants in the environment and provide more options for the biological degradation of 4-nitrochlorobenzene. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of Agrobacterium tumefaciens with the ability to degrade 4-nitrochlorobenzene and its applications.

[0006] The first objective of this invention is to provide a strain of Agrobacterium tumefaciens GDUTXIO NG6 that degrades 4-nitrochlorobenzene.

[0007] A second objective of this invention is to provide an agent for degrading 4-nitrochlorobenzene.

[0008] A third objective of this invention is to provide a fermentation broth for degrading 4-nitrochlorobenzene.

[0009] A fourth objective of this invention is to provide a method for degrading 4-nitrochlorobenzene.

[0010] The fifth objective of this invention is to provide a method for culturing Agrobacterium tumefaciens GDUTXIONG6.

[0011] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0012] This invention studies and isolates a strain of Agrobacterium pusense GDUTXIONG6 with highly efficient ability to degrade 4-nitrochlorobenzene. Agrobacterium pusense GDUTXIONG6 was deposited at the China Center for Type Culture Collection on November 23, 2023, with accession number CCTCC NO: M 20232309.

[0013] Therefore, the application of Agrobacterium tumefaciens GDUTXIONG6 in the degradation of 4-nitrochlorobenzene should also be within the scope of protection of this invention.

[0014] The application of Agrobacterium tumefaciens GDUTXIONG6 in the preparation of formulations that degrade 4-nitrochlorobenzene should also be within the scope of protection of this invention.

[0015] Based on this, the present invention also provides a formulation for degrading 4-nitrochlorobenzene, wherein the formulation contains the Agrobacterium tumefaciens GDUTXIONG6.

[0016] The present invention also provides a fermentation broth for degrading 4-nitrochlorobenzene, wherein the fermentation broth is obtained by fermentation culture of Agrobacterium rhizogenes GDUTXIONG6 or an inoculum containing Agrobacterium rhizogenes GDUTXIONG6.

[0017] The application of the aforementioned formulation or fermentation broth in the degradation of 4-nitrochlorobenzene should also be within the scope of protection of this invention.

[0018] The present invention also provides a method for degrading 4-nitrochlorobenzene, wherein the method comprises applying the aforementioned Agrobacterium tumefaciens GDUTXIONG6, or the aforementioned preparation, or the aforementioned fermentation broth.

[0019] As a reference indicator for specific implementation, the recommended application rate of Agrobacterium tumefaciens GDUTXIONG6 is: after application, the concentration of Agrobacterium tumefaciens GDUTXIONG6 in the water or soil environment should be 1–9 × 10⁻⁶. 6 CFU / L or 1~9×10 6 CFU / g.

[0020] The method for culturing Agrobacterium rhizogenes GDUTXIONG6 includes the following steps:

[0021] S1. Agrobacterium rhizogenes GDUTXIONG6 was inoculated into a solid activation medium and activated to obtain the activated strain.

[0022] S2. Take the activated strain obtained in step S1 and inoculate it into liquid culture medium to prepare seed culture;

[0023] S3. Take the seed liquid obtained in step S2 and transfer it to the expansion culture medium at a volume percentage of 1% to 10% for expansion culture to obtain Agrobacterium rhizogenes GDUTXIONG6 bacterial solution.

[0024] Preferably, the culture temperature is 30–40°C.

[0025] More preferably, the activation culture conditions in step S1 are: culture at 30-40°C for 12-18 hours.

[0026] Most preferably, the activation culture conditions in step S1 are: 36.5-37.5℃ for 15 hours.

[0027] More preferably, the conditions for the expanded culture in step S3 are: 30-40°C, 150-200 rpm for 12-18 hours.

[0028] Most preferably, the conditions for the expanded culture in step S3 are: 36.5-37.5℃, 150 rpm for 15 hours.

[0029] Preferably, the solid activated culture medium in step S1 uses 4-nitrochlorobenzene as the sole carbon source.

[0030] Preferably, the culture method of Agrobacterium rhizogenes GDUTXIONG6 satisfies one or more of the following conditions:

[0031] i. The Agrobacterium tumefaciens GDUTXIONG6 described in step S1 is streaked onto a solid activated culture medium;

[0032] ii. The components of the solid activation culture medium mentioned in step S1 are as follows:

[0033] 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L NaCl, 20 mg / L 4-nitrochlorobenzene, 15 g / L agar and ultrapure water, pH 7.0–7.4;

[0034] iii. The components of the liquid culture medium in step S2 and the expansion culture medium in step S3 are as follows:

[0035] 5.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L NaCl, 5.0 g / L yeast extract powder and ultrapure water, pH 6.8–7.2;

[0036] iv. The culture conditions described in step S2 are: 30-40℃, 150-200rpm for 12-18 hours.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention provides a strain of *Agrobacterium rhizobium* GDUTXIONG6, classified as *Agrobacterium pusense*, capable of degrading 4-nitrochlorobenzene. This strain achieves a degradation rate of over 85% for 4-nitrochlorobenzene, classifying it as a highly efficient degrading bacterium. Furthermore, using *Agrobacterium rhizobium* GDUTXIONG6 for 4-nitrochlorobenzene degradation does not cause secondary pollution or environmental harm. Therefore, this strain can be widely used for the remediation and treatment of environments contaminated with 4-nitrochlorobenzene. This research has significant practical implications and value for the degradation of 4-nitrochlorobenzene and the remediation of environments contaminated with it. Attached Figure Description

[0039] Figure 1 This is a colony morphology diagram of Agrobacterium tumefaciens GDUTXIONG6.

[0040] Figure 2 This is a morphological image of Agrobacterium tumefaciens strain GDUTXIONG6 under a scanning electron microscope.

[0041] Figure 3 This is a phylogenetic tree diagram of Agrobacterium tumefaciens GDUTXIONG6.

[0042] Figure 4 The graph shows the degradation rate of Agrobacterium tumefaciens GDUTXIONG6 under different initial 4-nitrochlorobenzene concentrations.

[0043] Figure 5 The graph shows the degradation rate of 4-nitrochlorobenzene by Agrobacterium tumefaciens GDUTXIONG6 in wastewater.

[0044] Figure 6 The graph shows the degradation rate of 4-nitrochlorobenzene by Agrobacterium tumefaciens GDUTXIONG6 in soil. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0046] Example 1: Isolation of strains

[0047] The inorganic salt culture medium formula used in this embodiment is as follows: 1.75 g / L K₂HPO₄·H₂O, 3.40 g / L NaHPO₄·12H₂O, 1.5 g / L KH₂HPO₄, 1.5 g / L MgSO₄, 1.4 g / L CaCl₂, 0.25 g / L FeCl₃·6H₂O, 0.9 mg / L MnSO₄·H₂O, 0.8 mg / L ZnSO₄·H₂O, and 0.7 mg / L (NH₄)₆Mo₇O. 24 ·4H2O.

[0048] The formula for a solid culture medium containing 4-nitrochlorobenzene is: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L NaCl, 20 mg / L 4-nitrochlorobenzene, and 15 g / L agar.

[0049] The LB medium (pH = 6.8–7.2) is formulated with 5.0 g / L glucose, 10.0 g / L peptone, 5.0 g / L NaCl and 5.0 g / L yeast extract.

[0050] Soil from a farmland in Tangshan City, Hebei Province, was selected as a sample. 4-Nitrochlorobenzene was added to the inorganic salt culture medium as a carbon and energy source to prepare inorganic salt culture media with substrate concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L, for strain screening and acclimatization.

[0051] The steps for strain domestication are as follows:

[0052] S1. Take 10 grams of soil and add it to an inorganic salt culture medium containing 1 mg / L 4-nitrochlorobenzene. Then, acclimate the soil at 37℃ and 150 rpm for 5 days. The first acclimatization is then complete.

[0053] S2. Take the upper culture medium obtained after the first acclimatization in step S1 and transfer it into 3 ml of inorganic salt medium with a concentration of 5 mg / L 4-nitrochlorobenzene at an inoculation rate of 10%. Acclimatize for 5 days at 37°C and 150 rpm. The second acclimatization is then complete.

[0054] S3. Take the upper culture medium obtained after the second acclimatization in step S2, transfer it into 3 ml of inorganic salt medium with a concentration of 10 mg / L 4-nitrochlorobenzene at an inoculation rate of 10%, and acclimatize it for 5 days at 37℃ and 150 rpm. The third acclimatization is then completed.

[0055] S4. Take the upper culture medium obtained after the third acclimatization in step S3, transfer it into 3 ml of inorganic salt medium with a concentration of 15 mg / L 4-nitrochlorobenzene at an inoculation rate of 10%, and acclimatize it for 5 days at 37℃ and 150 rpm. The fourth acclimatization is then completed.

[0056] S5. Take the upper culture medium obtained after the third acclimatization in step S4, transfer it into 3 ml of inorganic salt medium with a concentration of 20 mg / L 4-nitrochlorobenzene at an inoculation rate of 10%, and acclimatize it for 5 days at 37℃ and 150 rpm. The acclimatization process is then complete.

[0057] The bacterial cultures obtained after the domestication process were diluted to 10 μL. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Seven gradient dilutions were prepared, and 0.2 mL of each was evenly spread onto solid culture medium containing 4-nitrochlorobenzene and incubated at 36±0.5℃. After 18–24 h, single colonies with good morphology and uniform distribution were selected and inoculated into LB medium for proliferation. The obtained single colony suspensions were used for subsequent strain identification.

[0058] Example 2: Identification of the strain

[0059] The solid culture medium formula is: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L NaCl and 15 g / L agar.

[0060] (1) Morphological identification

[0061] 100 μL of the single-colony bacterial suspension described in Example 1 was spread onto a solid culture medium and incubated at 36 ± 0.5 °C for 24 h. The morphological characteristics of the colonies were then observed under an electron microscope. The colony morphology is as follows: Figure 1 As shown, the colony diameter is 0.5–1.0 mm, the colony morphology is round, pale yellow, and semi-transparent; the strain morphology is as follows. Figure 2 As shown, the strain has a size of (0.8–1.0) × (0.2–0.3) μm, is rod-shaped, and has no flagella.

[0062] (2) Physiological and biochemical characteristics

[0063] The physiological and biochemical identification process was referenced in Bergey's Manual of Bacterial Identification, 8th Edition. The identification results are shown in Table 1.

[0064] Table 1 Physiological and biochemical characteristics of bacterial cells

[0065]

[0066] Note: The reaction status is divided into positive and negative. Positive shape is coded as "+" and negative shape is coded as "-".

[0067] (3) Identification of 16S rDNA gene sequence

[0068] The strain isolated in Example 1 was molecularly identified using 16S rDNA gene sequence analysis. The specific steps are as follows:

[0069] S1. Extract genomic DNA from the test strain;

[0070] S2. PCR amplification was performed using the universal 16S rDNA primers 27F (SEQ ID NO: 1) and 1492R (SEQ ID NO: 2). The PCR amplification system is shown in Table 1.

[0071] Table 1 PCR amplification system

[0072]

[0073] The sequencing results of the amplified products are shown in SEQ ID NO: 3. Nucleotide sequence homology analysis was performed between the sequencing results of the amplified products and related sequences already registered in GenBank, and a phylogenetic tree was constructed. The results are as follows: Figure 3 As shown, the test strain in Figure 3 The Chinese character is represented by "F". Figure 3 It can be seen that the tested strain and Agrobacterium pusense NRCPB10 clustered on the same branch in the phylogenetic tree, which indicates that the tested strain and Agrobacterium pusense NRCPB10 are closely related in evolution.

[0074] The sequencing results of the amplified products were compared with the relevant sequences already registered in GenBank for nucleotide sequence homology analysis. The homology analysis results showed that the strain isolated in this invention was most similar to Agrobacterium pusense NRCPB10, with a similarity of 99.49%.

[0075] Based on the above morphological characteristics, physiological and biochemical properties, and 16S rDNA gene sequence identification results, the strain isolated in this invention is classified as Agrobacterium pusense and named Agrobacterium pusense GDUTXIONG6. It was deposited at the China Center for Type Culture Collection (CCTCC) on November 23, 2023, with accession number CCTCC NO: M 20232309.

[0076] Example 3: Degradation of 4-nitrochlorobenzene by Agrobacterium tumefaciens GDUTXIONG6

[0077] The inorganic salt culture medium and LB culture medium used in this embodiment are the same as those described in Example 1.

[0078] I. Experimental Methods

[0079] Prepare 100 mL of inorganic salt culture medium in an Erlenmeyer flask, and autoclave the prepared inorganic salt culture medium at 121 °C for 30 min for later use.

[0080] Agrobacterium rhizogenes GDUTXIONG6 was proliferated in LB medium until the bacterial culture OD... 600 After reaching a pH of 1.0–1.2, centrifuge at 8000 × g for 5 min, remove the supernatant, collect the bacterial cells, and wash the collected bacterial cells three times with phosphate buffer.

[0081] Subsequently, the collected Agrobacterium rhizogenes GDUTXIONG6 cells were suspended at a 10% inoculum in four Erlenmeyer flasks containing 100 mL of inorganic salt medium, and appropriate amounts of 4-nitrochlorobenzene solution were added to each flask to make the 4-nitrochlorobenzene concentrations 1 mg / L, 5 mg / L, 25 mg / L and 50 mg / L, respectively, to obtain culture media containing Agrobacterium rhizogenes GDUTXIONG6 and 4-nitrochlorobenzene.

[0082] The culture medium was incubated at 37℃ and 200 rpm for 168 hours. During the incubation process, 1 mL of culture medium was collected at 24, 48, 72, 96, 120, 144, and 168 hours, and centrifuged at 10000g for 5 min to collect the supernatant. The supernatant was then filtered through a 0.22 μm aqueous filter membrane, and the concentration of 4-nitrochlorobenzene in the solution was determined using a high-performance liquid chromatograph (HPLC, Agilent, USA, model 1260).

[0083] The high-performance liquid chromatography (HPLC) column was a C18 reverse-flow column, measuring 150 mm × 4.6 mm, with a standard injection volume of 20 μL. The mobile phase for HPLC detection was methanol and water at a volume ratio of 75:25, with a flow rate of 1 ml / min, and the UV detector wavelength was 279 nm.

[0084] II. Experimental Results

[0085] The degradation results of 4-nitrochlorobenzene (4-CNB) by Agrobacterium tumefaciens strain GDUTXIONG6 at different initial concentrations are as follows: Figure 4 As shown. In natural water bodies, the content of 4-nitrochlorobenzene ranges from 0.08 to 5.14 μg / L, and in natural water body sediments, the content ranges from 0.284 to 3.5523 μg / L. Figure 4 It can be seen that the Agrobacterium tumefaciens strain GDUTXIONG6 can achieve a degradation rate of over 85% for 1 mg / L 4-CNB within 168 h; according to my country's GB8978-1996 "Integrated Wastewater Discharge Standard", the national discharge standards for 4-chloronitrobenzene in industrial wastewater are shown in Table 2. Figure 4 It can be seen that the Agrobacterium tumefaciens strain GDUTXIONG6 has a degradation capacity of over 75% for 5 mg / L 4-CNB in ​​168 h, and further, the Agrobacterium tumefaciens strain GDUTXIONG6 has a degradation capacity of over 65% for 50 mg / L 4-CNB in ​​168 h.

[0086] Table 2 National Emission Standards for 4-Chloronitrobenzene in Industrial Wastewater

[0087]

[0088] Therefore, it can be seen that the Agrobacterium tumefaciens strain GDUTXIONG6 has a good degradation effect on 4-nitrochlorobenzene and can be widely used in the remediation and purification of 4-nitrochlorobenzene pollution. Moreover, since the Agrobacterium tumefaciens strain GDUTXIONG6 was screened in the soil environment, it will not introduce foreign chemicals or cause further pollution to the environment. It has good eco-friendliness, will not cause secondary pollution to the environment, and will not produce public hazards.

[0089] Example 4: Degradation of 4-nitrochlorobenzene in wastewater by Agrobacterium rhizogenes GDUTXIONG6

[0090] I. Experimental Methods

[0091] Wastewater samples were taken from the University Town Campus of Guangdong University of Technology (No. 100, Waihuan West Road, Guangzhou University Town, Panyu District, Guangzhou). The experiment was divided into control group A and experimental group B, as designed below:

[0092] Control group A: Wastewater and 30 mg / L 4-nitrochlorobenzene were added;

[0093] Experimental group B: Wastewater, 30 mg / L 4-nitrochlorobenzene, and 1.0 × 10⁻⁶ mg / L were added. 6 CFU / L Agrobacterium tumefaciens strain GDUTXIONG6.

[0094] Control group A and experimental group B were cultured at 37℃ and 200 r / min for 168 hours. During the culture, samples from both groups were collected at 24, 48, 72, 96, 120, 144, and 168 hours. Both groups were centrifuged at 10000g for 5 min, and the supernatant was collected. 1 ml of the supernatant was filtered through a 0.22 μm aqueous filter membrane to obtain the filtrate. The concentration of 4-nitrochlorobenzene in the filtrate was determined using high-performance liquid chromatography (HPLC, Agilent, USA, 1260).

[0095] The high-performance liquid chromatography (HPLC) column was a C18 reverse-flow column, measuring 150 mm × 4.6 mm, with a standard injection volume of 20 μL. The mobile phase for HPLC detection was methanol and water at a volume ratio of 75:25, with a flow rate of 1 ml / min, and the UV detector wavelength was 279 nm.

[0096] II. Experimental Results

[0097] The degradation results of 4-nitrochlorobenzene (4-CNB) in wastewater by Agrobacterium tumefaciens strain GDUTXIONG6 are as follows: Figure 5 As shown, by Figure 5 It is known that the Agrobacterium tumefaciens strain GDUTXIONG6 can degrade 4-nitrochlorobenzene in sewage at a rate of over 60%, effectively degrading 4-nitrochlorobenzene in sewage. Moreover, since Agrobacterium tumefaciens strain GDUTXIONG6 is isolated from soil, its application in sewage treatment will not cause secondary pollution to the environment or generate public hazards, and it has broad application prospects in the treatment of 4-nitrochlorobenzene pollution in sewage.

[0098] Example 5: Degradation of 4-nitrochlorobenzene in soil by Agrobacterium rhizogenes GDUTXIONG6

[0099] I. Experimental Methods

[0100] The experimental soil was taken from the University Town Campus of Guangdong University of Technology (No. 100, Waihuan West Road, Guangzhou University Town, Panyu District, Guangzhou), and was air-dried before being sieved through a 2mm sieve. The experimental groups are as follows:

[0101] Control group A: Soil + 30 mg / kg 4-nitrochlorobenzene;

[0102] Experimental Group B: Soil + 30 mg / kg 4-nitrochlorobenzene + 1.0 × 10⁻⁶ 6 CFU / g Agrobacterium rhizogenes GDU TXIONG6 strain.

[0103] The soil moisture content of control group A and experimental group B was adjusted to 30% with deionized water. After adding 30 mg / kg 4-nitrochlorobenzene, experimental group B was sprayed with Agrobacterium tumefaciens strain GDUTXIONG6 to achieve a concentration of Agrobacterium tumefaciens strain GDUTXIONG6 in the soil of 1.0 × 10⁻⁶. 6 CFU / g. Control group A and experimental group B were incubated at 37℃ and 200 r / min for 168 hours. During the incubation period, 5 g samples from control group A and experimental group B were taken at 24, 48, 72, 96, 120, 144, and 168 hours. 10 mL of dichloromethane was added to each sample, and the mixture was vigorously shaken in a shaker for 2 hours, then allowed to stand for 1 hour, followed by another 2 hours of shaking. The mixture was then centrifuged at 8000×g for 10 minutes, and 5 mL of the supernatant was collected. The supernatant was concentrated using a rotary evaporator, and the remaining 4-nitrochlorobenzene was dissolved in 5 mL of chromatographic grade methanol to obtain the test solution. The test solution was filtered through a 0.22 μm organic phase filter membrane to obtain the filtrate. The concentration of 4-nitrochlorobenzene in the filtrate was determined using high-performance liquid chromatography (HPLC, Agilent, USA, 1260).

[0104] The high-performance liquid chromatography (HPLC) column was a C18 reverse-flow column, measuring 150 mm × 4.6 mm, with a standard injection volume of 20 μL. The mobile phase for HPLC detection was methanol and water at a volume ratio of 75:25, with a flow rate of 1 ml / min, and the UV detector wavelength was 279 nm.

[0105] II. Experimental Results

[0106] The degradation results of 4-nitrochlorobenzene (4-CNB) in soil by Agrobacterium tumefaciens strain GDUTXIONG6 are as follows: Figure 6 As shown, by Figure 6 It is known that the *Agrobacterium tumefaciens* strain GDUTXIONG6 can degrade 4-nitrochlorobenzene in soil at a rate exceeding 55%, effectively degrading 4-nitrochlorobenzene in soil. Furthermore, *Agrobacterium tumefaciens* strain GDUTXIONG6 was screened from soil environments, thus exhibiting strong adaptability and the ability to survive and reproduce under various soil types and environmental conditions. It also does not introduce external chemicals or cause secondary pollution to the environment, demonstrating its eco-friendliness. Therefore, *Agrobacterium tumefaciens* strain GDUTXIONG6 has significant application value in the remediation of 4-nitrochlorobenzene pollution in soil.

[0107] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An Agrobacterium tumefaciens strain that degrades 4-nitrochlorobenzene ( Agrobacterium pusense GDUTXIONG6, characterized in that, Agrobacterium rhizogenes GDUTXIONG6 was deposited at the China Center for Type Culture Collection on November 23, 2023, with accession number CCTCC NO: M 20232309.

2. The application of Agrobacterium tumefaciens GDUTXIONG6 as described in claim 1 in the degradation of 4-nitrochlorobenzene.

3. The use of Agrobacterium tumefaciens GDUTXIONG6 as described in claim 1 in the preparation of formulations that degrade 4-nitrochlorobenzene.

4. A formulation for degrading 4-nitrochlorobenzene, characterized in that, The formulation contains Agrobacterium rhizogenes GDUTXIONG6 as described in claim 1.

5. A fermentation broth for degrading 4-nitrochlorobenzene, characterized in that, It is obtained by fermentation culture of Agrobacterium rhizogenes GDUTXIONG6 as described in claim 1 or an inoculum containing Agrobacterium rhizogenes GDUTXIONG6 as described in claim 1.

6. The application of the formulation of claim 4 or the fermentation broth of claim 5 in the degradation of 4-nitrochlorobenzene.

7. A method for degrading 4-nitrochlorobenzene, characterized in that, The method involves applying Agrobacterium rhizogenes GDUTXIONG6 as described in claim 1, or the formulation as described in claim 4, or the fermentation broth as described in claim 5.

8. The method for culturing Agrobacterium rhizogenes GDUTXIONG6 according to claim 1, characterized in that, Includes the following steps: S1. Agrobacterium rhizogenes GDUTXIONG6 was inoculated into a solid activation medium and activated to obtain the activated strain. S2. Take the activated strain obtained in step S1 and inoculate it into liquid culture medium to prepare seed culture; S3. Take the seed liquid obtained in step S2 and transfer it to the expansion culture medium at a volume percentage of 1% to 10% for expansion culture to obtain the fermentation broth of Agrobacterium rhizogenes GDUTXIONG6.

9. The cultivation method according to claim 8, characterized in that, The culture temperature was 37℃.

10. The cultivation method according to claim 8, characterized in that, The solid activation culture medium described in step S1 uses 4-nitrochlorobenzene as the sole carbon source.

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