Bacterium for degrading chlorobenzene compounds, fungicide, preparation method and application

Abstract

The invention discloses a bacterium for degrading chlorobenzene compounds, a fungicide, a preparation method and application. The strain is pseudomonas fluorescens, belongs to Pseudomonas, is named as Pseudomonas sp.CBD1, is preserved in the Guangdong Microbial Culture Collection Center on March 21, 2025, is located on the 5th floor, No.59 building, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and has a preservation number of GDMCC NO.66041. The invention further discloses a preparation method of the pseudomonas fluorescens strain. The strain provided by the invention has good degradation performance on chlorobenzene compounds which are difficult to degrade, such as chlorobenzene, 1, 2-dichlorobenzene and the like.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to a bacterium, a bacterial agent, a preparation method and an application for degrading chlorobenzene compounds. Background Art

[0002] With the country's increasing attention to soil pollution control, preliminary results have been achieved in soil environmental remediation. However, the recalcitrant organic compounds left in the soil before are difficult to degrade through various means due to their stable chemical properties, and thus can persist in the environment. Such pollutants can cause long-term abandonment of land and pose a serious threat to human health and the development of the economic and social sectors.

[0003] Chlorobenzene compounds (CBs) are recalcitrant organic compounds, which can be used as raw materials and intermediates for chemical synthesis and are widely used in various industrial sectors. They are likely to leak into the environment during production, transportation and use, and remain as the main pollutants in the sites left after enterprise relocation. Due to the characteristics of strong toxicity, difficult degradation and stable chemical properties of chlorobenzene compounds, they are likely to accumulate in the environment and cause persistent pollution, and are also likely to spread with the flow of groundwater, resulting in an increase in the area of the pollution plume, thus affecting potential sensitive receptors in the vicinity. Therefore, many countries have included them in the list of priority control pollutants, set strict standards to control emissions, and focused on the remediation of sites contaminated with chlorobenzene substances.

[0004] The mainstream remediation methods for sites contaminated with chlorobenzene compounds include physical remediation, chemical remediation and microbial remediation. The microbial remediation method has low operating costs, high pollutant reduction rates, and does not produce secondary pollution, and can better meet the safety and sustainable technical requirements in the remediation of pollutant sites in relocated chemical industrial sites. However, there are currently few degrading microorganisms focusing on various types of complex pollution systems, and the introduction of exogenous functional microorganisms is easily affected by local environmental factors, and there are problems such as degradation function degradation, which limits the practical engineering application. Therefore, it is necessary to continuously research and optimize the biological treatment method, enrich, screen and domesticate other species of degrading functional microorganisms from polluted sites, expand the existing microbial strain resource library for degrading chlorobenzene compounds, and obtain a more efficient, sustainable and microbial remediation method that can adapt to chemical industrial polluted sites with complex components.

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

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

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] In the first aspect, a bacterium for degrading chlorobenzene compounds is provided, wherein the bacterium is Pseudomonas fluorescens, belonging to the genus Pseudomonas, named Pseudomonas sp.CBD1, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 21, 2025, with the address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCCNO.66041.

[0009] Furthermore, the bacteria appears white in an inorganic salt culture medium and is an opaque strain.

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

[0011] In a second aspect, a bacterial agent for degrading chlorobenzene compounds is provided, wherein the bacterial agent comprises the bacteria according to claim 1.

[0012] In a third aspect, a method for preparing the bacteria for degrading chlorobenzene compounds according to the first aspect is provided, comprising the following steps:

[0013] (1) preparing a solution containing chlorobenzene compounds;

[0014] (2) adding soil samples contaminated by chlorobenzene compounds from the contaminated site to a plurality of first containers respectively, then adding sterilized beef extract peptone liquid culture medium, sterilized glass beads and a predetermined volume of the solution prepared in step (1) to each first container, and shaking to prepare a soil suspension;

[0015] (3) culturing the soil suspension at a constant temperature with shaking for several days, then taking it out and letting it stand, taking the supernatant in each of the first containers and transferring it to a plurality of second containers, and adding an inorganic salt culture medium and the solution prepared in step (1) to each of the second containers, sealing and culturing at a constant temperature with shaking for several days, to obtain a microbial culture solution;

[0016] (4) using an acclimation medium, subjecting the microbial culture fluid obtained in step (3) to a concentration gradient acclimation method, and culturing the culture fluid under constant temperature shaking for several days to obtain a mixed bacterial fluid; wherein the acclimation medium comprises a chlorobenzene compound, the inorganic salt medium described in step (3), and a nonionic surfactant, and the amount of the nonionic surfactant added is sufficient to ensure that the chlorobenzene compound is completely dissolved and its volatilization is inhibited;

[0017] (5) Separating the mixed bacterial solution obtained in step (4) to obtain the bacteria for degrading chlorobenzene compounds as claimed in claim 1.

[0018] Further, the sterilized beef extract peptone liquid medium in step (2) is prepared as follows: Mix 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride and 1 L of distilled water, stir evenly, adjust the pH value to 7.0, and sterilize at 121 °C for 20 min in a high-temperature high-pressure sterilizer; the inorganic salt medium in step (3) is prepared as follows: Add 0.1 g of CaCl2, 0.2 g of MgSO4, 1 g of NaCl, 2.02 g of NH4Cl, 3 g of KH2PO4, and 2 g of Na2HPO4, add 1 L of distilled water, and adjust the pH to 6.5 with NaOH solution; the non-ionic surfactant in step (4) is polysorbate 80.

[0019] Further, in the concentration gradient acclimation method in step (4), the concentration gradients of chlorobenzene compounds are 80, 120, 160, 200, and 300 mg / L; in step (3), the soil suspension is cultured in a constant temperature shaking incubator at 28 °C and 140 r / min for 5 d; in step (3), after adding the inorganic salt medium and the solution prepared in step (1) to each second container and sealing, it is cultured in a constant temperature shaking incubator at 28 °C and 140 r / min for 6 d; in step (4), it is cultured in a constant temperature shaking incubator at 28 °C and 140 r / min for 6 d.

[0020] Fourthly, provided is an application of the bacterium for degrading chlorobenzene compounds described in the first aspect in degrading chlorobenzene compounds, wherein the bacterium for degrading chlorobenzene compounds described in the first aspect is allowed to act on a sample contaminated with chlorobenzene compounds to degrade the chlorobenzene compounds in the sample.

[0021] Further, the chlorobenzene compounds are at least one of chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene.

[0022] The beneficial effects of the present invention include: The bacteria of the present invention have good degradation performance for difficult-to-degrade chlorobenzene compounds such as chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene, and have high practical application value and good development prospects in the in-situ remediation of soil contaminated with chlorobenzene compounds and the treatment of groundwater, and have broad application prospects in the field of in-situ remediation of soil and groundwater at the sites after the relocation of relevant chemical enterprises. Description of the Drawings

[0023] Figure 1 It is the optical photograph at the time of just inoculation and 6 days after inoculation when performing concentration gradient acclimation in step (4) of Example 1 of the present invention.

[0024] Figure 2 It is the bar chart of the genus and the relative proportion occupied by various genera of the bacterial flora in Example 2 of the present invention before separating and purifying the bacteria.

[0025] Figure 3a andFigure 3b They are respectively bar graphs of the relative abundances of functional genes at the L2 and L3 classification levels after the 16S rDNA sequencing of the bacterial flora during domestication in Example 2 of the present invention and the use of PICRUSt2 for functional prediction.

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

[0027] Figure 5 It is a graph of the degradation characteristics of the bacteria for pollutants at the highest pollutant concentration that can be borne in Example 3 of the present invention.

[0028] Figure 6 It is a graph of the change in the degradation effect of pollutants during the simulated contaminated soil remediation process by the bacteria in Example 4 of the present invention.

[0029] Figure 7a 、 Figure 7b and Figure 7c They are respectively the evaluations of the degradation capabilities of the bacteria for chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene in Example 5 of the present invention. Detailed implementation manners

[0030] The following makes a detailed description of the implementation manners of the present invention. It should be emphasized that the following description is merely exemplary and not intended to limit the scope of the present invention and its applications. Without conflict, the embodiments and features in the present application can be combined with each other.

[0031] In particular, the endpoints and any values within the ranges disclosed in this article are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

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

[0033] In the embodiments of the present invention, bacteria capable of degrading chlorobenzene compounds are isolated, enriched, and domesticated from the original contaminated soil of a certain chemical industrial site in Jiangsu contaminated with chlorobenzene compounds (CBs), and the biodegradation of chlorobenzene compounds such as chlorobenzene and 1,2-dichlorobenzene is synchronously and efficiently completed. The following further describes the specific embodiments of the present invention.

[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 was prepared to make a stock solution.

[0037] (2) In multiple first containers, soil samples contaminated with chlorobenzene compounds in the contaminated site were respectively added. Then, in each of the first containers, a sterilized beef extract peptone liquid medium, sterilized glass beads, and a predetermined volume of the solution prepared in step (1) were added, and shaken well to make a soil suspension.

[0038] Specifically, 10 g of fresh soil samples (from fresh soil samples severely contaminated with CBs in the study area) were respectively weighed and placed in 250 mL conical flasks. 150 mL of sterilized beef extract peptone liquid medium, sterilized glass beads, and a certain volume of the stock solution of step (1) were added to each, and shaken well to make a soil suspension.

[0039] Among them, the sterilized beef extract peptone medium was: 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, and 1 L of distilled water were mixed and stirred evenly, then the pH value was adjusted to 7.0, and it was sterilized at 121 °C for 20 min in a high-temperature high-pressure sterilizer.

[0040] (3) After the soil suspension was incubated with constant shaking for several days and then taken out and allowed to stand, the supernatant in each of the first containers was respectively transferred to multiple second containers, and an inorganic salt medium and the solution prepared in step (1) were added to each of the second containers. After sealing, it was incubated with constant shaking for several days to obtain a microbial culture solution.

[0041] Specifically, the conical flasks in step (2) were cultured in a constant-temperature shaking incubator at 28 °C and 140 r / min for 5 d, then taken out and allowed to stand for 1 h. Then, 10 mL of the supernatant of the soil suspension after standing was respectively pipetted with a pipette gun and transferred to 250 mL conical flasks. An inorganic salt medium and the stock solution of step (1) were added to make the total volume of the system 150 mL. The mouths of the conical flasks were wrapped with a sealing film and tinfoil, and continued to be shaken and cultured in a constant-temperature shaking incubator at 28 °C and 140 r / min for 6 d to obtain a microbial culture solution (i.e., the microbial solution enriched from the original contaminated soil).

[0042] Among them, the inorganic salt medium was: 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, added with 1 L of distilled water, and the pH was adjusted to 6.5 with NaOH solution. The inorganic salt medium mentioned below all refers to this inorganic salt medium.

[0043] (4) Use a domestication medium. Subject the microbial culture solution obtained in step (3) to the concentration gradient domestication method and perform constant temperature shaking culture for several days to obtain a mixed bacterial solution. Among them, the domestication medium includes chlorobenzene compounds, the inorganic salt medium described in step (3), and a non-ionic surfactant. The addition amount of the non-ionic surfactant is sufficient to ensure the complete dissolution of chlorobenzene compounds and inhibit their volatilization.

[0044] Specifically, inoculate the microbial culture solution obtained in step (3) into another domestication medium containing a higher concentration of chlorobenzene compounds (in this example, 1,2-dichlorobenzene) at a volume ratio of 8%. Use the concentration gradient domestication method. The domestication medium includes the above-mentioned inorganic salt medium, 1,2-dichlorobenzene, and a corresponding volume of the non-ionic surfactant polysorbate 80. Polysorbate 80 makes the concentration in the solution 4 CMC (Critical Micelle Concentration, CMC) to ensure the complete dissolution and inhibit the volatilization of 1,2-dichlorobenzene. Wrap the mouth of the conical flask with a sealed sealing film and tin foil. The concentration gradient of 1,2-dichlorobenzene is 80, 120, 160, 200, 300 mg / L. After shaking well, place the conical flask in a constant temperature shaking incubator at 28 °C and 140 r / min for 6 d. Continuously perform concentration gradient domestication until the substrate concentration reaches 300 mg / L, and monitor the degradation amount and degradation rate of the bacterial flora on the substrate during this process, and observe the strengthening of the degradation ability. As Figure 1 shown, the left a) figure and the right b) figure respectively correspond to the growth conditions of the bacterial flora at the time of just inoculation and 6 days after inoculation when performing concentration gradient domestication in step (4). It can be seen that 6 days after inoculation, the liquid medium becomes turbid, indicating that the bacterial flora can grow and reproduce using 1,2-dichlorobenzene as a carbon source.

[0045] (5) Perform bacterial separation on the mixed bacterial solution obtained in step (4) to obtain the bacteria for degrading chlorobenzene compounds. Specifically, in this example, the plate streaking method is used for bacterial separation.

[0046] Example 2: Identification of bacteria for degrading chlorobenzene compounds

[0047] Extract the target fragment from the strain genome, design primers for PCR amplification, and send the amplification product to General Biology for sequencing. According to the 16S rRNA sequence provided by General Biology sequencing, perform a homologous comparison in the NCBI database to identify the genus and species of the selected bacteria for degrading chlorobenzene compounds.

[0048] Take a small amount of the mixed bacterial solution obtained in step (4) of Example 1 and use E.Z.N.A. Total microbial DNA was extracted using the Soil DNA Kit (OmegaBio-tek, Norcross, GA, U.S.). Microbial species were analyzed by 16S rDNA amplicon sequencing. The names and sequences of the 16S rDNA universal primers were 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 was: 10 ng of template DNA, 4 μL of 5x FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of 5 μM upstream and downstream primers each, and 0.4 μL of FastPfu polymerase. The PCR reaction program was: pre-denaturation at 95 °C for 2 minutes, annealing at 55 °C for 30 seconds, extension at 72 °C for 1 minute. These three steps were repeated for 25 cycles, and finally, extension at 72 °C was maintained for 5 minutes. After 2% agarose gel electrophoresis of the PCR amplification products, they were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.). After purification and detection of the PCR products, SMRTbell libraries were prepared by blunt ligation. After connecting specific barcode sequences to each sample and mixing them in equal amounts, the amplicon mixture was used with the Pacific Biosciences SMRTbell TM Template Prep kit 1.0 to construct sequencing libraries and sequenced on the PacBio SequelⅡ.

[0052] The obtained 16S rDNA sequences were subjected to BLAST alignment. The alignment results showed that the nucleotide sequence of the target strain 16S rDNA in the bacterial solution had a high degree of homology with the nucleotide sequence of the Pseudomonas strain. It can be considered that the strain in the main ecological niche is Pseudomonas.

[0053] Such as Figure 2As shown, the specific species composition of the flora in the mixed bacterial solution obtained in step (4) of Example 1 includes: Pseudomonas fluorescens with a relative abundance of 65.0%, Pseudomonas migulae with a relative abundance of 11.8%, Mesorhizobium sp.002294985 with a relative abundance of 8.0%, Pseudomonas corrugata with a relative abundance of 3.64%, Pseudomonas thivervalensis with a relative abundance of 2.30%, Microbacterium sp.004794315 with a relative abundance of 2%, and other bacteria (unidentified species) with a relative abundance of 7.26%.

[0054] As Figure 3a shown, the results of comparing with the PICRUSt2 database show that at the L2 functional level of analysis, with the increase of the pollutant domestication concentration gradient, when the overall level of the abundance of carbohydrate metabolism genes remains stable, the abundances of genes related to xenobiotic biodegradation, biofilm transport, and amino acid metabolism in the 400 mg / L concentration group are increased by 90.0%, 97.9%, and 13.3% respectively compared with 50 mg / L, indicating that the ability of the flora microorganisms to metabolize xenobiotics has been greatly enhanced, achieving a good balance between pollutant detoxification and basic metabolism; As Figure 3b shown, delving into the L3 classification level, it can be found that the relative abundances of metabolic pathway genes related to chlorobenzene compounds and their metabolic intermediates, such as polycyclic aromatic hydrocarbon degradation, chlorocyclohexane and chlorobenzene compound degradation, chlorohydrocarbon degradation, and bisphenol compound degradation, are increased by 131.5%, 71.2%, 70.0%, and 58.0% respectively.

[0055] Combining the results in the above examples, the target strain (i.e., the bacterium for degrading chlorobenzene compounds of the present invention) isolated from the mixed bacterial solution obtained in step (4) through step (5) is a new species of the genus Pseudomonas (Pseudomonas fluorescens). The bacterium is white and opaque in the inorganic salt medium, and is named Pseudomonas sp.CBD1 herein. It was deposited in the Guangdong Provincial Culture Collection Center of Microorganisms on March 21, 2025. The deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. The deposit number is GDMCC NO.66041, and the taxonomic name is Pseudomonas sp.

[0056] Example 3: Preparation of Bacterial Solution and Degradation Characteristics and Carrying Capacity Limit of Bacteria

[0057] The bacteria obtained in Example 1 for degrading chlorobenzene compounds were mixed with an inorganic medium to prepare a bacterial solution. This bacterial solution was allowed to act on the sample contaminated with chlorobenzene compounds, and the chlorobenzene compounds in the sample could be degraded.

[0058] Degradation characteristics of the bacteria: After the bacterial solution was activated and cultured for a certain period of time, in the experimental group, it was 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 of the system. The absorbance value OD at 600 nm was measured using a UV-visible spectrophotometer. 600 = 0.11. To prevent the volatilization of 1,2-dichlorobenzene and improve the biodegradability of the pollutant, 4 CMC of polysorbate 80 was added to the system, and the conical flask was sealed with a sealing film and then placed in a constant temperature shaking incubator at 28 °C and 140 r / min for cultivation. The blank group without the bacterial solution was used as a control, and parallel samples were set. The change in the concentration of 1,2-dichlorobenzene was measured.

[0059] Bearing capacity limit of the bacteria: The bearing capacity limit of the bacteria was determined by setting a pollutant concentration gradient. After the bacterial solution was activated and cultured for a certain period of time, in the experimental group, it was inoculated into an inorganic salt medium containing a certain concentration of 1,2-dichlorobenzene at a ratio of 6% of the total volume of the system. The absorbance value OD at 600 nm was measured using a UV-visible spectrophotometer. 600 = 0.11. To prevent the volatilization of 1,2-dichlorobenzene and improve the biodegradability of the pollutant, 4 CMC of polysorbate 80 was added to the system. The blank group without the bacterial solution was used as a control, and parallel samples were set. The conical flask was sealed with a sealing film and then placed in a constant temperature shaking incubator at 28 °C and 140 r / min for cultivation. Samples were taken every 1 d to measure OD 600 and the change in the concentration of 1,2-dichlorobenzene. With a 100 mg / L difference in the 1,2-dichlorobenzene concentration gradient, the substrate concentration was continuously increased until the degradation of the bacteria and the pollutant was completely inhibited.

[0060] Since chlorobenzene compounds have strong volatility and are easily volatilized and dispersed into a gaseous state, the following formula is used to calculate the degradation amount: Degradation amount = initial concentration of the experimental group - current concentration of the experimental group - (initial concentration of the blank control group - current concentration of the blank control group).

[0061] Such as Figure 4 shown, within the first 48 h, the degradation rate of 1,2-dichlorobenzene was relatively high, and the degradation rate exceeded 3%; when the cultivation continued, the concentration of 1,2-dichlorobenzene decreased, the growth rate of the bacteria slowed down, and the degradation rate decreased accordingly; by 144 h, the degradation rate of 1,2-dichlorobenzene was 10.9%. Such as Figure 5As shown, when the concentration C0 of 1,2-dichlorobenzene reaches 1600 mg / L, the degradation rate is less than 1%, indicating that the pollutant greatly inhibits the growth of bacteria at this concentration. It is determined that the maximum pollutant concentration that the bacteria can bear is 1600 mg / L.

[0062] Example 4: Pilot test on the simulated remediation of soil contaminated with chlorobenzene compounds

[0063] Properties of the contaminated site soil: The contaminated site area is located along the lower reaches of the Yangtze River. According to the exploration and disclosure, within a depth of 40 m below the ground surface, it is mainly composed of cohesive soil, silt, and silty sand, and the soil permeability is at a medium level. The main surface water system near the site is the Yangtze River water on the north side. The surface water is developed. The phreatic water is mainly buried in the fill soil, silt-clay interlayer, silty clay-silt interlayer, and clay-silt interlayer. Its main recharge sources are atmospheric precipitation, artificial water use, and surface runoff, and it is mainly discharged through transpiration. The measured phreatic water level is 0.9 - 2.8 m below the ground surface, with an annual variation range of about ±0.5 m, a moisture content of about 40%, and the highest water level is close to the ground surface. Currently, the soil and groundwater in this plot are in a cross-mixed pollution situation. The investigation of the soil pollution situation shows that the main pollutant exceeding the screening value in the plot soil is 1,2-dichlorobenzene.

[0064] Pilot test on the simulated remediation of contaminated soil: To verify the performance of the bacteria in degrading chlorobenzene compounds in the soil medium, a pilot test on simulated remediation was carried out in the laboratory. Take 3 g of the original contaminated site soil after fully volatilizing the pollutants and place it in a 10 mL capped gas chromatography vial. Then, add a certain volume of 1,2-dichlorobenzene solution (to make the pollutant concentration in the soil 1200 mg / kg), 100 μL of the bacterial solution, 50 μL of the polysorbate 80 solution with a concentration of 60 g / L, 200 μL of the inorganic salt medium, and 800 μL of water. The simulated soil moisture content is 40%, and the blank group does not add the bacterial solution. At the same time, set up 5 control tests and place them in a cool place away from light. Measure the changes in the pollutant concentration in the system at 0, 4, 9, 16, 30, and 45 days respectively.

[0065] The results are as Figure 6 shown. Monitoring the changes in the pollutant concentration after injecting the bactericide (i.e., the bacterial solution), it is found that in the initial stage of injection, the bacteria can quickly adapt to the soil environment and the degradation rate is relatively fast. As the monitoring time extends, due to the consumption of the content of the biostimulant in the soil, the biodegradability of the pollutant decreases and the growth of the bacteria is hindered, resulting in a slower pollutant degradation rate. Generally speaking, the bacterial solution can degrade chlorobenzene compounds during the process of simulating the remediation of contaminated soil, but in the actual remediation process, it is necessary to regularly supplement the bacterial solution over time to enhance the remediation effect.

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

[0067] The experimental group and the blank control group were set up. In the experimental group, a certain concentration or volume of inorganic salt medium, a solution of a certain chlorobenzene compound, a solution of polysorbate 80, and a bacterial solution were added to a 250 mL conical flask to make the total volume of the culture system 100 mL. The initial concentration of the chlorobenzene compound was as shown in Table 1. The inoculation amount of the bacterial solution was 8%, and the pH of the culture system was adjusted to 7. The blank control group did not add the bacterial solution. The mouths of the conical flasks were wrapped with a sealing film and tinfoil, and the blank control group or the experimental group was set for mutual comparison and parallel experiments were set for both. The conical flasks were placed in a constant temperature shaking incubator at 28 °C and 140 r / min for 6 days, and the change in the concentration of the chlorobenzene compound in the system was measured.

[0068] Table 1: Setting of single CBs concentration in the experiment on the broad-spectrum degradation of pollutants

[0069]

[0070]

[0071] The degradation amount and degradation rate of the bacteria in the presence of a single chlorobenzene compound are as Figure 7a - 7c shown. It can be seen from the figure that the bacteria can grow and metabolize and degrade in the presence of a single high concentration of chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene. Generally speaking, the order of the degradation ability of the bacteria for the three chlorobenzene compounds is: 1,2-dichlorobenzene > chlorobenzene > 1,4-dichlorobenzene. The degradation amounts are 46.4 ppm, 27.0 ppm, and 12.7 ppm respectively, and the degradation rates are 4.5%, 5.4%, and 12.5% respectively.

[0072] In summary, the bacteria in the embodiments of the present invention can effectively degrade high-concentration chlorobenzene compounds and have broad application prospects in the field of in-situ remediation of soil and groundwater at the sites after the relocation of relevant chemical enterprises.

[0073] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, they can make several substitutions or modifications to these described embodiments, and these substitution or modification methods should all be regarded as belonging to the protection scope of the present invention. In the description of this specification, the descriptions with reference to the terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic 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 a suitable manner in any one or more embodiments or examples. Without contradiction, those skilled in the art can combine and combine 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 alterations can be made herein without departing from the scope of protection of the patent application.

Claims

1. A bacterium for degrading chlorobenzene compounds, characterized in that: The bacteria is Pseudomonas fluorescens, belonging to the genus Pseudomonas, named Pseudomonas sp.CBD1, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 21, 2025, with the address on the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC NO.66041.

2. The bacteria for degrading chlorobenzene compounds according to claim 1, characterized in that: The bacteria appear white in an inorganic salt culture medium and are opaque strains.

3. The bacteria for degrading chlorobenzene compounds according to claim 1, characterized in that: The bacteria are obtained by enrichment, domestication and screening from soil contaminated by chlorobenzene compounds in a contaminated site.

4. A bacterial agent for degrading chlorobenzene compounds, characterized in that: The bacterial agent contains the bacteria according to claim 1.

5. A method for preparing bacteria for degrading chlorobenzene compounds according to claim 1, characterized in that: The steps include: (1) preparing a solution containing chlorobenzene compounds; (2) adding soil samples contaminated by chlorobenzene compounds from the contaminated site to a plurality of first containers respectively, then adding sterilized beef extract peptone liquid culture medium, sterilized glass beads and a predetermined volume of the solution prepared in step (1) to each first container, and shaking to prepare a soil suspension; (3) culturing the soil suspension at a constant temperature with shaking for several days, then taking it out and letting it stand, taking the supernatant in each of the first containers and transferring it to a plurality of second containers, and adding an inorganic salt culture medium and the solution prepared in step (1) to each of the second containers, sealing and culturing at a constant temperature with shaking for several days, to obtain a microbial culture solution; (4) using an acclimation medium, subjecting the microbial culture fluid obtained in step (3) to a concentration gradient acclimation method, and culturing the culture fluid under constant temperature shaking for several days to obtain a mixed bacterial fluid; wherein the acclimation medium comprises a chlorobenzene compound, the inorganic salt medium described in step (3), and a nonionic surfactant, and the amount of the nonionic surfactant added is sufficient to ensure that the chlorobenzene compound is completely dissolved and its volatilization is inhibited; (5) Separating the mixed bacterial solution obtained in step (4) to obtain the bacteria for degrading chlorobenzene compounds as claimed in claim 1.

6. The preparation method according to claim 5, characterized in that: 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 the mixture is sterilized in a high temperature and high pressure autoclave at 121° C. for 20 min; The inorganic salt 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, and 2 g of Na2HPO4 are added with 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.

7. The preparation method according to claim 5, characterized in that: In the concentration gradient acclimation method in step (4), the concentration gradient of the chlorobenzene compound is 80, 120, 160, 200, and 300 mg / L; In step (3), the soil suspension is cultured in a constant temperature shaking incubator at 28° C. and 140 rpm for 5 days; In step (3), the inorganic salt culture medium and the solution prepared in step (1) are added to the second container, sealed, and cultured in a constant temperature shaking incubator at 28° C. and 140 rpm for 6 days; In step (4), the culture is carried out in a constant temperature shaking incubator at 28° C. and 140 rpm for 6 days.

8. Use of the bacteria for degrading chlorobenzene compounds according to claim 1 in degrading chlorobenzene compounds, characterized in that: The bacteria for degrading chlorobenzene compounds according to claim 1 are allowed to act on a sample contaminated by chlorobenzene compounds to degrade the chlorobenzene compounds in the sample.

9. The use according to claim 8, characterized in that The chlorobenzene compound is at least one of chlorobenzene, 1,2-dichlorobenzene and 1,4-dichlorobenzene.

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