A bacterium, a bacterial agent, a preparation method and an application for degrading chlorobenzene compounds

Abstract

The application discloses bacteria for degrading chlorobenzene compounds, a bacterial agent, a preparation method and application. The bacteria are Pseudomonas sp. CBD1, belong to Pseudomonas, and are preserved in Guangdong Microbial Culture Collection Center on March 21, 2025, and are located at No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100, with a preservation number of GDMCC NO. 66041. The bacteria have good degradation performance on chlorobenzene compounds such as chlorobenzene and 1,2-dichlorobenzene.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a bacterium, a bacterial agent, a preparation method, and an application for degrading chlorobenzene compounds. Background Technology

[0002] With the increasing emphasis placed on soil pollution control by the government, initial progress has been made in soil environmental remediation. However, persistent organic pollutants left in the soil due to their stable chemical properties make them difficult to degrade through various pathways, thus allowing them to persist in the environment. These pollutants can lead to long-term land abandonment, posing a serious threat to human health and socio-economic development.

[0003] Chlorobenzenes (CBs) are recalcitrant organic compounds used as raw materials and intermediates in chemical synthesis and widely applied across various industrial sectors. They are prone to leakage into the environment during production, transportation, and use, and remain as major pollutants in sites left behind after factory relocation. Due to their high toxicity, recalcitrant nature, and chemical stability, chlorobenzenes easily accumulate in the environment, causing persistent pollution. They also readily spread with groundwater flows, increasing the area of ​​the pollution plume and affecting surrounding potential sensitive receptors. Therefore, many countries have included them in their priority pollutant lists, setting strict standards to control emissions and focusing on the remediation of sites contaminated with chlorobenzenes.

[0004] Mainstream remediation methods for sites contaminated with chlorobenzene compounds include physical remediation, chemical remediation, and microbial remediation. Microbial remediation offers low operating costs, high pollutant reduction rates, and no secondary pollution, better meeting the safety and sustainability requirements of remediation at relocated chemical plant sites. However, current research focuses on a limited number of degrading microorganisms for complex contamination systems, and the introduction of exogenous functional microorganisms is susceptible to local environmental factors, leading to degradation of their degradation capabilities and limiting their practical engineering applications. Therefore, it is necessary to continuously research and optimize biological treatment methods, enriching, screening, and acclimatizing other species of degrading microorganisms from contaminated sites, expanding the existing resource pool of chlorobenzene-degrading microbial strains, and obtaining more efficient, sustainable, and adaptable microbial remediation methods for chemically contaminated sites with complex compositions.

[0005] It should be noted that the information disclosed in the background section above is only for understanding the background of this application, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a bacterium, a bacterial agent, a preparation method, and an application for degrading chlorobenzene compounds.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Firstly, a bacterium for degrading chlorobenzene compounds is provided, said bacterium being *Pseudomonas fluorescens*, belonging to the genus *Pseudomonas* (…). Pseudomonas ), named Pseudomonas sp. CBD1 was deposited on March 21, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNO. 66041.

[0009] Furthermore, the bacteria appear white in inorganic salt culture medium, indicating that it is an opaque strain.

[0010] Furthermore, the bacteria were obtained by enrichment, domestication, and screening from soil contaminated with chlorobenzene compounds in the contaminated site.

[0011] In a second aspect, a microbial agent for degrading chlorobenzene compounds is provided, the microbial agent comprising the bacteria described in the first aspect.

[0012] Thirdly, a method for preparing the bacteria for degrading chlorobenzene compounds as described in the first aspect is provided, comprising the following steps:

[0013] (1) Prepare solutions containing chlorobenzene compounds;

[0014] (2) Add soil samples contaminated with chlorobenzene compounds from the contaminated site to multiple first containers, and then add sterile beef extract peptone liquid culture medium, sterile glass beads and a predetermined volume of the solution prepared in step (1) to each first container, and shake well to prepare a soil suspension.

[0015] (3) After the soil suspension is cultured at constant temperature and shaken for several days, it is taken out and left to stand. The supernatant in each of the first containers is transferred to multiple second containers. In the second containers, inorganic salt culture medium and the solution prepared in step (1) are added. After sealing, the mixture is cultured at constant temperature and shaken for several days to obtain microbial culture medium.

[0016] (4) Using an acclimatization culture medium, the microbial culture solution obtained in step (3) is acclimatized by concentration gradient method and cultured at constant temperature and shaking for several days to obtain a mixed bacterial solution; wherein, the acclimatization culture medium includes chlorobenzene compounds, the inorganic salt culture medium in step (3) and nonionic surfactants, and the amount of nonionic surfactants added is sufficient to ensure that the chlorobenzene compounds are completely dissolved and inhibit their volatilization;

[0017] (5) The mixed bacterial solution obtained in step (4) is subjected to bacterial separation to obtain the bacteria described in the first aspect for degrading chlorobenzene compounds.

[0018] Further, the sterilized beef extract peptone liquid culture medium in step (2) is prepared as follows: 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride and 1 L of distilled water are mixed and stirred evenly, the pH value is adjusted to 7.0, and sterilized in a high temperature and high pressure autoclave at 121℃ for 20 min; the inorganic salt culture medium in step (3) is prepared as follows: 0.1 g of CaCl2, 0.2 g of MgSO4, 1 g of NaCl, 2.02 g of NH4Cl, 3 g of KH2PO4, 2 g of Na2HPO4 are added to 1 L of distilled water, and the pH is adjusted to 6.5 with NaOH solution; the nonionic surfactant in step (4) is polysorbate 80.

[0019] Further, in the concentration gradient acclimatization method in step (4), the concentration gradient of chlorobenzene compounds is 80, 120, 160, 200, and 300 mg / L; in step (3), the soil suspension is cultured for 5 days in a constant temperature shaking incubator at 28℃ and 140 r / min; in step (3), inorganic salt culture medium and the solution prepared in step (1) are added to the second container and sealed, and then cultured for 6 days in a constant temperature shaking incubator at 28℃ and 140 r / min; in step (4), the mixture is cultured for 6 days in a constant temperature shaking incubator at 28℃ and 140 r / min.

[0020] Fourthly, the application of the bacteria described in the first aspect for degrading chlorobenzene compounds in the degradation of chlorobenzene compounds is provided, wherein the bacteria described in the first aspect for degrading chlorobenzene compounds are reacted with a sample contaminated with chlorobenzene compounds to degrade the chlorobenzene compounds in the sample.

[0021] Furthermore, the chlorobenzene compound is at least one of chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene.

[0022] The beneficial effects of this invention include: the bacteria of this invention have good degradation performance on chlorobenzene, 1,2-dichlorobenzene, 1,4-dichlorobenzene and other refractory chlorobenzene compounds. It has high practical application value and good development prospects in the in-situ remediation of chlorobenzene compound-contaminated soil and treatment of groundwater. It also has broad application prospects in the field of in-situ remediation of soil and groundwater at the site of relocation of related chemical enterprises. Attached Figure Description

[0023] Figure 1 Optical photographs taken immediately after inoculation and 6 days after inoculation during concentration gradient acclimatization in step (4) of Example 1 of the present invention.

[0024] Figure 2 This is a bar chart showing the species and relative proportions of various genera of the bacterial community in Example 2 of the present invention before the isolation of pure bacteria.

[0025] Figure 3a and Figure 3b The images are bar graphs showing the changes in the relative abundance of functional genes at the L2 and L3 taxonomic levels, respectively, after 16S rDNA sequencing of the bacterial community during domestication in Example 2 of this invention and functional prediction using PICRUSt2.

[0026] Figure 4 This is a graph showing the degradation characteristics of 1,2-dichlorobenzene by bacteria in Example 3 of the present invention.

[0027] Figure 5 This is a graph showing the pollutant degradation characteristics of bacteria in Example 3 of the present invention at the highest pollutant concentration they can tolerate.

[0028] Figure 6 This is a graph showing the changes in pollutant degradation effect of bacteria during the simulated contaminated soil remediation process in Example 4 of the present invention.

[0029] Figure 7a , Figure 7b and Figure 7c The degradation capabilities of the bacteria in Example 5 of this invention for chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene were evaluated. Detailed Implementation

[0030] The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary and not intended to limit the scope and application of the present invention. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0031] 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.

[0032] In the following embodiments, unless otherwise specified, the raw materials or processing techniques are all conventional commercially available raw materials or conventional processing techniques in the art.

[0033] In this invention, bacteria capable of degrading chlorobenzene compounds were isolated, enriched, and domesticated from contaminated soil at the original site of a chemical plant in Jiangsu Province that was contaminated with chlorobenzene compounds (CBs). These bacteria simultaneously and efficiently complete the biodegradation of chlorobenzene, 1,2-dichlorobenzene, and other chlorobenzene compounds. Specific embodiments of this invention are further described below.

[0034] Example 1: Preparation method of bacteria for degrading chlorobenzene compounds

[0035] (1) Prepare a solution containing chlorobenzene compounds (preferably 1,2-dichlorobenzene).

[0036] Specifically, a methanol solution of 1,2-dichlorobenzene with a concentration of 80 g / L is prepared to form a mother liquor.

[0037] (2) Soil samples contaminated with chlorobenzene compounds from the contaminated site are added to multiple first containers respectively. Then, sterile beef extract peptone liquid culture medium, sterile glass beads and a predetermined volume of the solution prepared in step (1) are added to each first container and shaken to prepare a soil suspension.

[0038] Specifically, 10 g of fresh soil samples (from fresh soil samples in the study area that are severely contaminated with CBs) were weighed and placed in 250 mL Erlenmeyer flasks. 150 mL of sterile beef extract peptone liquid culture medium, sterile glass beads and a certain volume of the mother liquor from step (1) were added to each flask and shaken to prepare a soil suspension.

[0039] The sterilized beef extract peptone culture medium is prepared by mixing 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride and 1 L of distilled water, stirring evenly, adjusting the pH to 7.0, and then sterilizing in a high-temperature and high-pressure autoclave at 121℃ for 20 min.

[0040] (3) After the soil suspension is cultured at a constant temperature and shaken for several days, it is taken out and left to stand. The supernatant in each of the first containers is transferred to multiple second containers. In the second containers, inorganic salt culture medium and the solution prepared in step (1) are added. After sealing, the mixture is cultured at a constant temperature and shaken for several days to obtain microbial culture medium.

[0041] Specifically, the conical flask from step (2) was cultured in a constant temperature shaking incubator at 28℃ and 140 r / min for 5 days, then removed and allowed to stand for 1 hour. Then, 10 mL of the supernatant of the soil suspension after standing was transferred to a 250 mL conical flask, and inorganic salt culture medium and the mother liquor from step (1) were added to make the total volume of the system 150 mL. The mouth of the conical flask was sealed with a sealing film and aluminum foil, and the flask was continued to be shaken and cultured in a constant temperature shaking incubator at 28℃ and 140 r / min for 6 days to obtain the microbial culture medium (i.e., the microbial liquid enriched from the original contaminated soil).

[0042] The inorganic salt culture medium is prepared by adding 0.1g CaCl2, 0.2g MgSO4, 1g NaCl, 2.02g NH4Cl, 3g KH2PO4, and 2g Na2HPO4 to 1L of distilled water, and adjusting the pH to 6.5 with NaOH solution. All references to this inorganic salt culture medium in the following text refer to this specific inorganic salt culture medium.

[0043] (4) Using an acclimatization culture medium, the microbial culture solution obtained in step (3) is acclimatized by concentration gradient and cultured at constant temperature for several days to obtain a mixed bacterial solution; wherein, the acclimatization culture medium includes chlorobenzene compounds, the inorganic salt culture medium in step (3) and nonionic surfactants, and the amount of nonionic surfactants added is sufficient to ensure that the chlorobenzene compounds are completely dissolved and inhibit their volatilization.

[0044] Specifically, the microbial culture obtained in step (3) is inoculated at a volume ratio of 8% into another acclimatization medium containing a higher concentration of chlorobenzene compounds (in this example, 1,2-dichlorobenzene). The concentration gradient acclimatization method is adopted. The acclimatization medium includes the above-mentioned inorganic salt medium, 1,2-dichlorobenzene, and a corresponding volume of nonionic surfactant polysorbate 80. The concentration of polysorbate 80 in the solution is 4 CMC (critical micelle concentration) to ensure that 1,2-dichlorobenzene is completely dissolved and volatilization is inhibited. The mouth of the conical flask is sealed with a sealing film and aluminum foil. The concentration gradient of 1,2-dichlorobenzene is 80, 120, 160, 200, and 300 mg / L. After shaking, the conical flasks were placed in a constant temperature shaking incubator at 28℃ and 140 rpm for 6 days. A continuous concentration gradient was used to acclimate the bacteria to a substrate concentration of 300 mg / L. During this process, the degradation amount and rate of the substrate by the bacterial community were monitored to observe the enhancement of degradation ability. Figure 1 As shown, Figure a) on the left and Figure b) on the right correspond to the growth of the bacterial community immediately after inoculation and 6 days after inoculation when the concentration gradient acclimatization is carried out in step (4). It can be seen that after 6 days of inoculation, the liquid culture medium becomes turbid, indicating that the bacterial community can grow and reproduce with 1,2-dichlorobenzene as a carbon source.

[0045] (5) The mixed bacterial solution obtained in step (4) is subjected to bacterial isolation to obtain the bacteria used to degrade chlorobenzene compounds. Specifically, in this example, the streak plate method is used for bacterial isolation.

[0046] Example 2: Identification of bacteria used to degrade chlorobenzene compounds

[0047] The target fragment was obtained by extracting the strain's genome, and primers were designed for PCR amplification. The amplified product was then sent to Universal Bio for sequencing. Based on the 16S rRNA sequence provided by Universal Bio, homology comparison was performed in the NCBI database, which allowed for the identification of the species of bacteria selected for degrading chlorobenzene compounds.

[0048] A small amount of the mixed bacterial culture obtained in step (4) of Example 1 was taken, and total microbial DNA was extracted using the EZNA® Soil DNA Kit (OmegaBio-tek, Norcross, GA, US). Microbial species were analyzed by 16S rDNA amplification and sequencing. The names and sequences of the universal 16S rDNA primers are as follows:

[0049] Forward primer 27F—5'-AGRGTTYGATYMTGGCTCAG-3' (SEQ ID NO:1)

[0050] Reverse primer 1492R—5'-RGYTACCTTGTTACGACTT-3' (SEQ ID NO:2).

[0051] The PCR amplification reaction system consisted of: 10 ng template DNA, 4 μL 5x FastPfu buffer, 2 μL 2.5 mM dNTPs, 0.8 μL each of 5 μM forward and reverse primers, and 0.4 μL FastPfu polymerase. The PCR reaction program was as follows: pre-denaturation at 95°C for 2 minutes, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, repeated for 25 cycles, followed by a final extension at 72°C for 5 minutes. The PCR amplification products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, US) after 2% agarose gel electrophoresis. After purification and detection, SMRTbell libraries were prepared by flat ligation. Each sample was ligated with a specific barcode sequence and then mixed in equal mass. The amplicon mixture was analyzed using Pacific Biosciences SMRTbell. TM The Template Prep kit 1.0 was used to construct sequencing libraries and perform sequencing on PacBio Sequel II.

[0052] The 16S rDNA sequence was compared with the BLAST sequence. The comparison results showed that the nucleotide sequence of the 16S rDNA of the target strain in the bacterial culture was highly homologous with the nucleotide sequence of the Pseudomonas strain. It can be considered that the strain with the main ecological niche is Pseudomonas.

[0053] like Figure 2 As shown, the specific species composition of the bacterial community in the mixed bacterial solution obtained in step (4) of Example 1 includes: *Pseudomonas fluorescens* (… Pseudomonas fluorescens The relative abundance was 65.0%, and *Pseudomonas mifepristone* ( Pseudomonas migulae The relative abundance was 11.8%, and the concentration of *Rhizobium* sp. 002294985 was [missing information]. Mesorhizobium sp. 002294985) relative abundance 8.0%, Pseudomonas wrinklis ( Pseudomonas corrugata The relative abundance was 3.64%, and *Pseudomonas sevivar* ( Pseudomonas thivervalensis The relative abundance was 2.30%, and the microbacterium sp. 004794315 ( Microbacterium The relative abundance of sp.004794315 was 2%, and the relative abundance of other bacteria (unidentified species) was 7.26%.

[0054] like Figure 3a As shown, the comparison results with the PICRUSt2 database indicate that, at the L2 functional level, with the increase of pollutant acclimation concentration gradient, while the overall abundance level of carbohydrate metabolism genes remained stable, the abundance of genes related to exogenous compound biodegradation, biofilm transport, and amino acid metabolism in the 400 mg / L group increased by 90.0%, 97.9%, and 13.3%, respectively, compared to 50 mg / L. This suggests that the ability of the microbial community to metabolize exogenous compounds was significantly enhanced, achieving a good balance between pollutant detoxification and basal metabolism. Figure 3b As shown, delving into the L3 classification level, it can be found that the relative abundance of metabolic pathway genes related to chlorobenzene compounds and their metabolic intermediates, such as polycyclic aromatic hydrocarbon degradation, chlorocyclohexane and chlorobenzene degradation, chlorohydrocarbon degradation and bisphenol degradation, increased by 131.5%, 71.2%, 70.0% and 58.0%, respectively.

[0055] Based on the results of the above embodiments, the target strain (i.e., the bacterium of the present invention for degrading chlorobenzene compounds) obtained by separating it from the mixed bacterial solution obtained in step (4) through step (5) is a new species of Pseudomonas (specifically, *Pseudomonas fluorescens*). Pseudomonas fluorescens In inorganic salt culture medium, the bacteria appear white and are opaque; this strain is named […]. Pseudomonas sp. CBD1 was deposited on March 21, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO. 66041. Its taxonomic name is... Pseudomonas sp.

[0056] Example 3: Preparation of bacterial culture and degradation characteristics and carrying capacity limit of the bacteria

[0057] The bacteria obtained in Example 1 for degrading chlorobenzene compounds were mixed with an inorganic culture medium to prepare a bacterial solution. This bacterial solution reacted with a sample contaminated with chlorobenzene compounds to degrade the chlorobenzene compounds in the sample.

[0058] Degradation characteristics of the bacteria: After activating and culturing the bacterial solution for a certain period of time, the bacteria were inoculated into an inorganic salt medium containing 1,2-dichlorobenzene (initial concentration C0 = 600 mg / L) at a ratio of 6% of the total volume in the experimental group. The absorbance value (OD) at 600 nm was measured using a UV-Vis spectrophotometer. 600 =0.11. To prevent the volatilization of 1,2-dichlorobenzene and improve the biodegradability of pollutants, 4 CMC of polysorbate 80 was added to the system. After sealing the conical flask with sealing film, it was placed in a constant temperature shaking incubator at 28℃ and 140 r / min for incubation. The blank group without bacterial culture was used as a control. Parallel samples were set up for all cases, and the changes in the concentration of 1,2-dichlorobenzene were measured.

[0059] Bacterial carrying capacity limit: The carrying capacity limit of the bacteria was determined by setting a contaminant concentration gradient. After activating and culturing the bacterial culture for a certain period of time, the bacteria were inoculated into an inorganic salt medium containing a certain concentration of 1,2-dichlorobenzene at a ratio of 6% of the total system volume in the experimental group. The absorbance value (OD) at 600 nm was measured using a UV-Vis spectrophotometer. 600 =0.11. To prevent the volatilization of 1,2-dichlorobenzene and improve the biodegradability of pollutants, 4 CMC of polysorbate 80 was added to the system. A blank control group without bacterial culture was used. Parallel samples were prepared for each case. The conical flasks were sealed with sealing film and then incubated in a constant temperature shaking incubator at 28℃ and 140 r / min. OD was measured every 1 day. 600 The changes in 1,2-dichlorobenzene concentration were studied. Using a 100 mg / L concentration gradient of 1,2-dichlorobenzene, the substrate concentration was continuously increased until the degradation of bacteria and contaminants was completely inhibited.

[0060] Because chlorobenzene compounds are highly volatile and easily evaporate into a gaseous state, the following formula is used to calculate the degradation amount: Degradation amount = Initial concentration of experimental group - Current concentration of experimental group - (Initial concentration of blank control group - Current concentration of blank control group).

[0061] like Figure 4 As shown, the degradation rate of 1,2-dichlorobenzene was high within the first 48 hours, exceeding 3%. With continued culturing, the concentration of 1,2-dichlorobenzene decreased, the bacterial growth rate slowed, and the degradation rate decreased accordingly. By 144 hours, the degradation rate of 1,2-dichlorobenzene was 10.9%. Figure 5 As shown, when the concentration of 1,2-dichlorobenzene (CO) reaches 1600 mg / L, the degradation rate is less than 1%, indicating that at this concentration, the pollutant greatly inhibits the growth of bacteria, and the highest pollutant concentration that the bacteria can withstand is determined to be 1600 mg / L.

[0062] Example 4: Small-scale simulated remediation of chlorobenzene-contaminated soil

[0063] Soil properties at the contaminated site: The contaminated site is located along the lower reaches of the Yangtze River. Exploration revealed that within a depth of 40 m below the surface, the soil mainly consists of clay, silt, and silty sand, with moderate permeability. The main surface water system adjacent to the site is the Yangtze River to the north, with well-developed surface water. Groundwater is primarily buried in fill, silt interbedded with silty clay, silty clay interbedded with silt, and silty clay interbedded with silt layers. Its main recharge sources are atmospheric precipitation, artificial water use, and surface runoff, and it is mainly discharged through evapotranspiration. The measured groundwater level depth is 0.9–2.8 m, with an annual variation of approximately ±0.5 m, and a moisture content of approximately 40%. The highest water level is close to the surface. Currently, the soil and groundwater within this site are in a state of cross-contamination. Soil pollution investigation indicates that the main pollutant exceeding the screening value in the soil is 1,2-dichlorobenzene.

[0064] Small-scale simulated remediation test of contaminated soil: To verify the performance of bacteria in degrading chlorobenzene compounds in soil media, a small-scale simulated remediation test was conducted in the laboratory. 3 g of soil from the original contaminated site, after sufficient volatilization of pollutants, was placed in a 10 mL gas chromatograph with a cap. A certain volume of 1,2-dichlorobenzene solution (to achieve a pollutant concentration of 1200 mg / kg in the soil), 100 μL of bacterial suspension, 50 μL of 60 g / L polysorbate 80 solution, 200 μL of inorganic salt culture medium, and 800 μL of water were added sequentially, simulating a soil moisture content of 40%. No bacterial suspension was added to the control group. Five control experiments were also set up, placed in a cool, dark place, and the changes in pollutant concentration within the system were tested on days 0, 4, 9, 16, 30, and 45.

[0065] The results are as follows Figure 6 As shown, monitoring of pollutant concentration changes after injection of the bacterial agent (i.e., bacterial solution) revealed that in the initial stage of injection, the bacteria adapted to the soil environment quickly, resulting in a rapid degradation rate. However, as the monitoring time increased, the biodegradability of pollutants decreased due to the depletion of biostimulants in the soil, hindering bacterial reproduction and growth, thus slowing down the pollutant degradation rate. Overall, the bacterial solution can degrade chlorobenzene compounds in simulated contaminated soil remediation; however, in actual remediation, regular replenishment of the bacterial solution over time is necessary to enhance the remediation effect.

[0066] Example 5: Broad-spectrum degradation of chlorobenzene compounds

[0067] An experimental group and a blank control group were set up. In the experimental group, a certain concentration or volume of inorganic salt culture medium, a solution of a chlorobenzene compound, polysorbate 80 solution, and bacterial suspension were added to a 250 mL Erlenmeyer flask, making the total volume of the culture system 100 mL. The initial concentration of the chlorobenzene compound is shown in Table 1. The inoculum size was 8%, and the pH of the culture system was adjusted to 7. No bacterial suspension was added to the blank control group. The mouths of the Erlenmeyer flasks were sealed with sealing film and aluminum foil. A blank control group or experimental group was set up for mutual control, and parallel experiments were conducted for each group. The Erlenmeyer flasks were placed in a constant temperature shaking incubator at 28℃ and 140 r / min for 6 days, and the changes in the concentration of chlorobenzene compounds in the system were measured.

[0068] Table 1: Single CBs concentration settings for broad-spectrum pollutant degradation experiments

[0069]

[0070] The degradation amount and degradation rate of bacteria in the presence of a single chlorobenzene compound are as follows: Figures 7a-7c As shown in the figure, the bacteria can grow and metabolically degrade chlorobenzene in the presence of high concentrations of chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene. Overall, the degradation ability of the bacteria for the three chlorobenzene compounds is ranked as follows: 1,2-dichlorobenzene > chlorobenzene > 1,4-dichlorobenzene, with degradation amounts of 46.4 ppm, 27.0 ppm, and 12.7 ppm, and degradation rates of 4.5%, 5.4%, and 12.5%, respectively.

[0071] In summary, the bacteria in the embodiments of this invention can effectively degrade high concentrations of chlorobenzene compounds, and have broad application prospects in the field of in-situ remediation of soil and groundwater at sites after the relocation of relevant chemical enterprises.

[0072] The above description provides a further detailed explanation of the present invention in conjunction with specific / preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the concept of the present invention, and all such substitutions or modifications should be considered within the scope of protection of the present invention. In the description of this specification, the reference to terms such as "an embodiment," "some embodiments," "preferred embodiment," "example," "specific example," or "some examples," etc., indicates that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. A bacterium for degrading 1,2-dichlorobenzene, characterized in that, The bacteria described were *Pseudomonas fluorescens*, belonging to the genus *Pseudomonas*, and named... Pseudomonas sp. CBD1 was deposited on March 21, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO.66041.

2. A bacterial agent for degrading 1,2-dichlorobenzene, characterized in that, The bacterial agent contains the bacteria described in claim 1.

3. The application of the bacterium for degrading 1,2-dichlorobenzene as described in claim 1 in the degradation of 1,2-dichlorobenzene, characterized in that, The bacteria for degrading 1,2-dichlorobenzene as described in claim 1 are used to degrade the 1,2-dichlorobenzene in the sample by reacting with the sample contaminated with 1,2-dichlorobenzene.

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