Acidophilic, high-efficiency dichloroethylene-degrading soil bacillus FHY-13 and application thereof

Abstract

The application discloses a soil Brevibacillus agri FHY-13 which is acidophilic and can efficiently degrade dichloroethylene, and an application thereof. The soil Brevibacillus agri FHY-13 is preserved in the China Center for Type Culture Collection, located in Wuhan University, Wuhan, China, and has a postal code of 430072, a preservation number of CCTCC NO: M 2023420 and a preservation date of March 28, 2023. The discovery of the strain is of great significance for efficient purification of chlorinated organic pollutants such as DCE in industrial wastewater and waste gas.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more particularly to an acidophilic, highly efficient soil brevicorbacterium FHY-13 that degrades dichloroethylene and its applications. Background Technology

[0002] Dichloroethylene (DCE) is a common organic compound that is volatile and soluble, and is widely used in chemical, pharmaceutical, coating, and solvent industries. However, due to its high toxicity, long-term exposure or contact can harm human health, and DCE has been listed as an environmental pollutant. Therefore, DCE remediation technologies have gradually attracted the attention of scholars.

[0003] Biological purification technology is widely used in pollution control due to its high removal efficiency, low treatment cost, and minimal secondary pollution. However, due to the extremely poor solubility of dichloroisocyanuric acid (DCE) and the high bond energy between carbon and chlorine atoms, only a small number of DCE-degrading bacteria have been isolated, mainly including Pseudomonas, Bacillus, Fusobacterium, and Pandoraea, and their degradation efficiency still needs further improvement. Currently, isolating and screening highly efficient DCE-degrading bacteria from the environment remains one of the important methods for eliminating chlorinated organic pollutants in the environment. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing an acidophilic, highly efficient soil brevicorbacterium FHY-13 that degrades dichloroethylene and its applications.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention provides an acidophilic, highly efficient soil breviculobacillus strain that degrades dichloroethylene, microbially named *Brevibacillus agri* FHY-13, which was deposited on March 28, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, postal code 430072, China; accession number CCTCCNO:M 2023420; the 16S rRNA sequence of FHY-13 is shown in SEQ ID NO.1.

[0007] Secondly, the present invention provides a bacterial suspension containing an acidophilic, highly efficient dichloroethylene-degrading soil brevicorbacterium FHY-13 as an active ingredient and a method for preparing the bacterial suspension.

[0008] The bacterial suspension was prepared by solid culture, seed culture, and liquid culture of soil-borne Bacillus hygroscopicus FHY-13, which is acidophilic and highly efficient at degrading dichloroethylene.

[0009] The method for preparing the bacterial suspension includes the following specific steps:

[0010] (1) Solid culture: The acidophilic, highly efficient dichloroethylene-degrading soil spore bacillus FHY-13 was inoculated onto an agar slant culture medium and cultured at 20-40℃ for 2-4 days to obtain slant cells; the final concentration composition of the agar slant culture medium was: K2HPO4 1500mg / L, KH2PO4 500mg / L, NaCl 1000mg / L, dichloroethylene 50mg / L, MgSO4·7H2O 200mg / L, water as solvent, pH 6.0-8.0, and agar 18-20g / L;

[0011] (2) Seed culture: Colonies were picked from the slant culture and inoculated into the seed culture medium. The culture was carried out at 20-40℃ for 12-24h to obtain the seed liquid. The final concentration of the seed culture medium was: NaCl 10g / L, yeast extract 5g / L, peptone 10g / L, water as solvent, and pH 6.0-8.0.

[0012] (3) Liquid culture: The seed culture is inoculated into the fermentation medium at a volume concentration of 1% and cultured at 20-40℃ for 12-24h to obtain the fermentation culture medium, which is the bacterial suspension; the final concentration composition of the fermentation medium is: K2HPO4 1500mg / L, KH2PO4 500mg / L, NaCl 1000mg / L, dichloroethylene 50mg / L, MgSO4·7H2O 200mg / L, water as solvent, and pH value of 6.0-8.0.

[0013] Thirdly, the present invention provides the application of a bacterial suspension containing FHY-13, an acidophilic and highly efficient dichlorodegrading soil brevicorbacterium, as an active ingredient in the degradation of dichlorodegrading, and a specific method thereof.

[0014] The specific method is as follows: the bacterial suspension is inoculated into a first DCE liquid selective medium with a salt concentration of 0-1%, using dichloroethylene as the sole carbon source, and cultured at 25-35°C to achieve the degradation of dichloroethylene; the final concentration composition of the first DCE liquid selective medium is: DCE 1-25 mg / L, KH2PO4 376 mg / L, K2HPO4 456 mg / L, (NH4)2SO4 480 mg / L, NaNO3 680 mg / L, Mg(NO3)2 250 mg / L, CaCl2·2H2O 11 mg / L, trace elements, water as solvent, and pH value of 6.0-8.0; the final concentration composition of the trace elements is: MnCl2·H2O 60 mg / L, ZnCl2 88 mg / L, KI 10 mg / L, NaMoO4·2H2O 100 mg / L, H3BO3 50 mg / L, solvent is water.

[0015] Further, the specific method is as follows: the bacterial suspension is inoculated into a first DCE liquid selective medium with a salt concentration of 0.89%, using dichloroethylene as the sole carbon source, and cultured at 35°C to achieve the degradation of dichloroethylene; the final concentration composition of the first DCE liquid selective medium is: DCE 1-25 mg / L, KH2PO4 376 mg / L, K2HPO4 456 mg / L, (NH4)2SO4 480 mg / L, NaNO3 680 mg / L, Mg(NO3)2 250 mg / L, CaCl2·2H2O 11 mg / L, trace elements, with water as the solvent and a pH value of 6.0; the final concentration composition of the trace elements is: MnCl2·H2O 60 mg / L, ZnCl2 88 mg / L, KI 10 mg / L, NaMoO4·2H2O 100 mg / L, H3BO3 50 mg / L, solvent is water.

[0016] Fourthly, the present invention also provides the application of a bacterial suspension containing the acidophilic and highly efficient dichloroethylene-degrading soil brevicorbacterium FHY-13 as the active ingredient in the degradation of dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene or hexachlorobenzene.

[0017] The beneficial effects of this invention are as follows: Strain FHY-13 can degrade DCE at an initial concentration of 0-25 mg / L, with the final degradation products being CO2, H2O, and cell biomass. The average mineralization rate is 61.12%, the average yield coefficient is 0.1904 mg cells / mgDCE, and the average chloride ion release rate is 84.70%. The discovery of this degrading bacterium is of great significance for the efficient purification of DCE in wastewater and exhaust gas from chemical synthesis and pharmaceutical industries. This strain can also degrade other common industrial organic pollutants, especially dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene, and hexachlorobenzene. Strain FHY-13, taken from an exhaust gas treatment unit, has a good degradation effect on chlorinated hydrocarbons, especially DCE, and can relatively completely convert DCE into harmless substances such as CO2, H2O, and cell biomass. At the same time, this strain can also degrade common industrial pollutants such as benzene and chlorobenzene to varying degrees, thus having broad application prospects in the biological purification of industrial waste gas and wastewater. Attached Figure Description

[0018] Figure 1 Plate image of soil brevicorbacterium FHY-13;

[0019] Figure 2 Scanning electron microscope image of soil brevicorbacterium FHY-13;

[0020] Figure 3 Phylogenetic tree diagram of soil brevicorbacterium FHY-13;

[0021] Figure 4 The graph shows the changes in DCE concentration and cell biomass of *Bacillus brevis* FHY-13 under different DCE concentrations over time. Figure 4 (a) is a graph showing the change of DCE concentration in soil-borne Bacillus brevis FHY-13 over time at different DCE concentrations. Figure 4 (b) is a graph showing the change in cell biomass of soil brevicorbacterium FHY-13 with different concentrations of DCE over time;

[0022] Figure 5 The CO2 and Cl- production of soil spore-forming bacteria FHY-13 under different concentrations of DCE were measured. - Graph showing changes in release amount and cell biomass;

[0023] Figure 6 The figures show the degradation rate and cell growth of common industrial organic pollutants by *Bacillus brevis* FHY-13. Figure 6 (a) is an experimental graph showing the degradation rate and cell growth of common industrial organic pollutants after 24 hours. Figure 6 (b) is an experimental graph showing the degradation rate and cell growth of common industrial organic pollutants at 72 h. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0025] For test methods not specifying specific experimental conditions, the following methods are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0026] This invention provides an acidophilic, highly efficient soil brevicorbacterium that degrades dichloroethylene, microbially named *Brevibacillus agri* FHY-13, which was deposited at the China Center for Type Culture Collection on March 28, 2023, with accession number CCTCC NO: M 2023420; the 16S rRNA sequence of FHY-13 is shown in SEQ ID NO. 1.

[0027] *Bacillus spp.* is a common bacillus. A search of patents and other relevant literature has not revealed any reports of its use in degrading DCE. The discovery of this degrading bacterium is of great significance for the efficient purification of DCE and other chlorinated hydrocarbon pollutants, as well as other hydrocarbon pollutants, from industrial wastewater and exhaust gases.

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0029] Example 1: Isolation, purification and identification of soil brevibacillus agri FHY-13

[0030] 1. Isolation and purification of *Brevibacillus agri* FHY-13

[0031] Brevibacillus agri. FHY-13 is a Gram-positive bacterium that was domesticated and isolated from activated sludge in the waste gas treatment unit of a rubber factory in Taizhou, Zhejiang Province. The specific steps are as follows:

[0032] (1) Sampling: Multiple samples were taken from the activated sludge of the waste gas treatment device of a rubber factory in Taizhou, Zhejiang Province, as raw materials for screening soil short-spore Bacillus FHY-13, which is acidophilic and highly efficient at degrading dichloroethylene;

[0033] (2) Strain isolation: Take an appropriate amount of activated sludge from the waste gas treatment device, rinse it 5 times with distilled water, and then aerate it for 24 hours to remove residual organic matter; prepare a second DCE liquid selective medium to acclimate the activated sludge. Add 5% by volume of acclimated sludge to the second DCE liquid selective medium and shake it on a constant temperature shaker at 30℃ and 160rpm. After 3 days, transfer it to a new second DCE liquid selective medium at a volume of 10% and shake it until a single colony appears. Pick a single colony and inoculate it into the second DCE solid selective medium to obtain a single colony that grows quickly, has regular colony structure, and stable characteristics. This single colony is recorded as strain FHY-13.

[0034] In this embodiment, the final concentration composition of the second DCE liquid selective medium is as follows: DCE 50 mg / L, KH2PO4 376 mg / L, K2HPO4 456 mg / L, (NH4)2SO4 480 mg / L, NaNO3 680 mg / L, Mg(NO3)2 250 mg / L, CaCl2·2H2O 11 mg / L, trace elements, water as solvent, and pH 6.0. The final concentration composition of the trace elements is as follows: MnCl2·H2O 60 mg / L, ZnCl2 88 mg / L, KI 10 mg / L, NaMoO4·2H2O 100 mg / L, H3BO3 50 mg / L, water as solvent.

[0035] The final concentration composition of the second DCE fixation selective medium is as follows: DCE 50 mg / L, KH2PO4 376 mg / L, K2HPO4 456 mg / L, (NH4)2SO4 480 mg / L, NaNO3 680 mg / L, Mg(NO3)2 250 mg / L, CaCl2·2H2O 11 mg / L, agar 20 g / L, trace elements, water as solvent, and pH 6.0.

[0036] 2. Identification of strain FHY-13

[0037] a. Physiological and biochemical characteristics of strain FHY-13

[0038] The obtained strain FHY-13 was subjected to morphological observation and physiological and biochemical identification. The colonies were yellow, with regular, smooth, and moist edges. Scanning electron microscopy revealed that the bacteria were long rods, non-motile, Gram-negative, oxidase-positive, and positive for starch hydrolysis and indole tests. It could utilize sucrose and lactose. Strain FHY-13... Figure 1 As shown; under a scanning electron microscope, the morphology of this bacterium is that of a long rod-shaped organism, such as... Figure 2 As shown.

[0039] b. 16S rRNA sequence analysis of strain FHY-13

[0040] Based on 16S rRNA sequence analysis and physiological and biochemical experiments, strain FHY-13 was identified as Brevibacillusagri.

[0041] The sequencing results are as follows:

[0042]

[0043] DNA was extracted and purified from the bacterial strain using a bacterial DNA extraction kit (OMEGA, catalog number D3350) and stored at 4°C. 16S rRNA was amplified by PCR using universal bacterial primers 27F (forward primer 27F, 5AGAGTTTGA TCC TGG CTC AG-3') and 1492R (reverse primer 1492R, 5-GGT TAC CTT GTT ACG ACT T-3').

[0044] PCR experiments were performed using TOYOBO's high-fidelity PCR polymerase product, KOD One™ PCR Master Mix (catalog number KMM-101). The reaction system is shown in Table 1.

[0045] Table 1: Reaction System

[0046]

[0047]

[0048] The 16S rRNA sequence of strain FHY-13 was tested, and the 16S rRNA sequence of strain FHY-13 is shown in SEQ ID NO.1.

[0049] The 16S rRNA sequence of strain FHY-13 was uploaded to GenBank for homology comparison, and it was found that it belongs to Brevibacillus agri. It has the highest homology with Brevibacillus agri DSM 6348, reaching 99.72%. Figure 3 This is a phylogenetic tree diagram of strain FHY-13.

[0050] Based on sequencing results and physiological and biochemical assays, strain FHY-13 was identified as belonging to *Brevibacillus agri*. Therefore, strain FHY-13 was named *Brevibacillus agri* FHY-13 and deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2023420, deposit date March 28, 2023, address: Wuhan University, Wuhan, China, postal code: 430072.

[0051] Example 2: Preparation process of bacterial suspension using acidophilic, highly efficient dichloroethylene-degrading soil brevicorbacterium FHY-13 as active ingredient

[0052] The preparation process of the bacterial suspension:

[0053] (1) Solid culture: Acidophilic, highly efficient dichloroethylene-degrading soil spores FHY-13 were inoculated onto slant culture medium and cultured at 30℃ for 3 days to obtain slant cells; the final concentration composition of the slant culture medium was: K2HPO4 1500mg / L, KH2PO4 500mg / L, NaCl 1000mg / L, dichloroethylene 50mg / L, MgSO4·7H2O 200mg / L, water as solvent, pH 7.0, and agar 18g / L;

[0054] (2) Seed culture: Colonies were picked from the slant culture and inoculated into seed culture medium. The culture was carried out at 30°C for 18 hours to obtain seed liquid. The final concentration of the seed culture medium was: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L, water as solvent, and pH 7.0.

[0055] (3) Liquid culture: The seed culture was inoculated into the fermentation medium at a volume concentration of 1% and cultured at 30℃ for 18h to obtain the fermentation culture medium as the bacterial suspension; the final concentration composition of the fermentation medium was: K2HPO4 1500mg / L, KH2PO4 500mg / L, NaCl 1000mg / L, dichloroethylene 50mg / L, MgSO4·7H2O 200mg / L, with water as the solvent and pH value of 7.0.

[0056] Example 3: Response surface optimization experiment on the degradation of DCE environmental factors by soil brevibacillus agri FHY-13

[0057] 1. Response surface methodology and the acquisition of a model for predicting the degradation rate of DCE.

[0058] The effects of three factors—culture medium pH, culture temperature, and culture medium salt concentration—on the biodegradation of DCE were investigated. A three-factor, three-level response surface methodology was designed using Design Expert software to predict the degradation rate of DCE by *Brevibacillus agri* FHY-13 under different culture conditions.

[0059] A three-factor, three-level experimental design was conducted using Design Expert software. The three factors were X1: pH of the culture medium, X2: culture temperature, and X3: salt concentration of the culture medium. The code values ​​-1, 0, and 1 corresponded to culture medium pH=6, pH=7, pH=8, or culture temperature=20℃, 30℃, 40℃, or culture medium salt concentration=0%, 0.5%, and 1.0%, respectively.

[0060] The experimental response values ​​and predicted values ​​are shown in Table 2.

[0061] Table 2: Experimental Response Values ​​and Predicted Values

[0062]

[0063] Prepare the first DCE liquid selective medium with different culture medium pH, different culture temperature, and different culture medium salt concentration according to Table 2. Dispense the medium into 250mL serum bottles, with a volume of 50mL, and sterilize at 121℃ for 20min. After the first DCE liquid selective medium has cooled, add the bacterial suspension with an initial biomass of 0.75mg / L prepared according to the method in Example 2 to the first DCE liquid selective medium with different culture medium pH, different culture temperature, and different culture medium salt concentration.

[0064] In this embodiment, the first DCE liquid selective medium consists of DCE 20 mg / L, KH2PO4 376 mg / L, K2HPO4 456 mg / L, (NH4)2SO4 480 mg / L, NaNO3 680 mg / L, Mg(NO3)2 250 mg / L, CaCl2·2H2O 11 mg / L, trace elements, and water as the solvent; the final concentration of the trace elements is: MnCl2·H2O 60 mg / L, ZnCl2 88 mg / L, KI 10 mg / L, NaMoO4·2H2O 100 mg / L, H3BO3 50 mg / L, and water as the solvent.

[0065] DCE was used as the sole carbon source at an initial concentration of 20 mg / L. After sealing, the culture was placed in shakers at different temperatures for incubation. Separately, serum bottles containing the same culture medium were sterilized and DCE was added, but no FHY-13 bacterial suspension was added, serving as a blank control. After 24 hours of incubation, the residual DCE concentration in the culture medium was analyzed, and biomass was measured. Based on the measured degradation rate, a quadratic multiple regression was performed using Design Expert software, and the resulting prediction model was:

[0066] Y=-73.008+14.117*X1+2.973*X2+1.817*X3-0.491*X1*X1

[0067] -0.092*X1*X2-0.383*X1*X3-0.440*X2*X2+0.194*X2*X3;

[0068] -5.167*X3*X3

[0069] Where Y is the degradation rate of DCE, in mg / (L*h).

[0070] The correlation coefficient R of the prediction model 2=0.9937, indicating that the degradation rate predicted by the prediction model has a good correlation with the actual degradation rate, and can be used to predict the degradation rate of DCE by strains under different culture conditions.

[0071] 2. Optimal environmental factors for the degradation of DCE by *Brevibacillus agri* FHY-13.

[0072] The predicted model was analyzed using Design Expert software. By setting its first-order partial derivative to zero, the environmental factor combination at which the degradation rate of DCE by *Brevibacillus agri* FHY-13 reached its maximum was obtained: a culture temperature of 35℃, a culture medium pH of 6.0, and a culture medium salt concentration of 0.89%. Under these conditions, the predicted degradation rate was 0.579 mg / (L*h). The actual degradation rate of DCE by strain FHY-13 under the above culture conditions was 0.568 mg / (L*h), which is quite close to the predicted value.

[0073] The specific experimental procedure is as follows:

[0074] Take 800 mL of the first DCE liquid selective medium with a pH of 6.0, add NaCl to bring the salt concentration of the medium to 0.89%, adjust the pH of the medium to 6.0, and dispense into four 250 mL serum bottles. Sterilize at 121°C for 20 min. After the medium cools, add bacterial suspension with an initial biomass of 0.75 mg / L prepared according to the method in Example 2 to three of the serum bottles, using DCE with an initial concentration of 20 mg / L as the sole carbon source. Add only 20 mg / L DCE to the remaining serum bottle as a blank control. After incubating on a shaker at 35°C for 24 h, analyze the residual DCE concentration and determine the biomass simultaneously.

[0075] Under the conditions of a culture temperature of 35℃, a culture medium pH of 6.0, and a culture medium salt concentration of 0.89%, the actual degradation rates of DCE by *Brevibacillus agri* FHY-13 were 0.577, 0.563, and 0.564 mg / (L*h), with an average degradation rate of 0.568 mg / (L*h), which is close to the model prediction value. This indicates that this combination of environmental factors represents the optimal environmental conditions for the degradation of DCE by *Brevibacillus agri* FHY-13.

[0076] Example 4: Degradation performance of soil breviculobacillus agri FHY-13 on different concentrations of DCE

[0077] The degradation performance of *Brevibacillus agri* FHY-13 on DCE at concentrations of 1–35 mg / L was investigated under optimal environmental conditions: a culture temperature of 35℃, a culture medium pH of 6.0, and a culture medium salt concentration of 0.89%. The results showed that strain FHY-13 could completely degrade DCE at concentrations of 1–25 mg / L, but could not degrade DCE at initial concentrations greater than 25 mg / L.

[0078] The specific experimental procedure is as follows:

[0079] A third DCE liquid selective medium with a pH of 6.0 and a salt concentration of 0.89% was prepared. 200 mL of this medium was added to each 250 mL serum bottle, and the bottles were sterilized at 121°C for 20 min. After cooling, eight serum bottles were given a bacterial suspension with an initial biomass of 0.75 mg / L prepared according to the method in Example 2. Simultaneously, DCE at concentrations of 1, 5, 10, 15, 20, 25, 30, and 35 g / L was added as the sole carbon source. Eight other serum bottles served as blank controls (only DCE was added, without the bacterial suspension). After deoxygenation and sealing, the serum bottles were cultured at 35°C with shaking. The residual DCE concentration and biomass in the culture medium were measured periodically, and degradation curves and biomass change curves of strain FHY-13 at different initial DCE concentrations were plotted.

[0080] In this embodiment, the third DCE liquid selective medium consists of DCE 1-35 mg / L, KH2PO4 376 mg / L, K2HPO4 456 mg / L, (NH4)2SO4 480 mg / L, NaNO3 680 mg / L, Mg(NO3)2 250 mg / L, CaCl2·2H2O 11 mg / L, trace elements, and water as the solvent; the final concentration of the trace elements is: MnCl2·H2O 60 mg / L, ZnCl2 88 mg / L, KI 10 mg / L, NaMoO4·2H2O 100 mg / L, H3BO3 50 mg / L, and water as the solvent.

[0081] Experimental results are as follows Figure 4 (a) and Figure 4 As shown in (b), strain FHY-13 can degrade initial concentrations below 25 mg·L⁻¹. -1 The DCE concentration increased, and a relatively significant hysteresis period appeared within this range as the concentration increased. When the initial DCE concentration was 30 mg·L⁻¹ -1At that time, strain FHY-13 only degraded DCE within the first 72 hours, and the concentration of residual DCE in the liquid phase did not change significantly thereafter. This may be because some toxic metabolites produced by the strain under high concentration of DCE conditions severely inhibited the activity of the strain.

[0082] Example 5: Analysis of mineralization rate, chloride ion release rate and yield coefficient of DCE by soil brevibacillus agri FHY-13

[0083] Under optimal environmental conditions—a culture temperature of 35℃, a culture medium pH of 6.0, and a culture medium salt concentration of 0.89%—the mineralization rate, chloride ion release rate, and yield coefficient of *Brevibacillus agri* FHY-13 for DCE concentrations ranging from 1 to 25 mg / L were investigated. The results showed that strain FHY-13 could convert DCE into CO2, H2O, and cell biomass, with an average mineralization rate of 61.12%, an average chloride ion release rate of 84.70%, and an average yield coefficient of 0.1904 mg cells / mg DCE.

[0084] The specific experimental procedure is as follows:

[0085] Prepare a first DCE liquid selective medium with a pH of 6.0 and a salt concentration of 0.89%. Add 50 mL of the first DCE liquid selective medium to each 250 mL serum bottle and sterilize at 121 °C for 20 min. After the culture medium cools, add a bacterial suspension with an initial biomass of 0.75 mg / L prepared according to the method in Example 2 to 12 serum bottles. Simultaneously, add DCE at concentrations of 1, 5, 10, 15, 20, and 25 mg / L as the sole carbon source (two serum bottles for each concentration). Use six serum bottles as blank controls (only DCE is added, without bacterial suspension). After deoxygenation and sealing, degauss and culture at 35 °C with shaking. Measure DCE concentration, chloride ion concentration, cell biomass, and CO2 concentration periodically. Mineralization and dechlorination curves of strain FHY-13 at different DCE concentrations were plotted, and the average mineralization rate and average chloride ion release rate were obtained by fitting. Combined with cell proliferation, the relationship curves between cell biomass, chloride ion release, and CO2 generation were plotted, and the average cell yield was calculated.

[0086] Experimental results are as follows Figure 5 As shown, the amount of chloride ions removed is linearly related to the concentration of DCE, and the fitted linear equation is y = 0.6021x, with a correlation coefficient R. 2=0.9910, indicating that strain FHY-13 can produce 0.6021 mg of chloride ions for every 1 mg of DCE used. Theoretically, it can produce 0.7107 mg of chloride ions for every 1 mg of DCE used. Therefore, the average chloride ion release rate is 84.70%.

[0087] There is also a linear relationship between CO2 generation and DCE degradation, with the fitted linear equation being y = 0.5549x and a correlation coefficient R0. 2 =0.9946, indicating that strain FHY-13 can produce 0.5549 mg of CO2 by completely mineralizing 1 mg of DCE; theoretically, complete oxidation of DCE produces H2O and CO2, and complete mineralization of 1 mg of DCE can produce 0.9078 mg of CO2; therefore, the average mineralization rate of strain FHY-13 is 61.12%.

[0088] During the degradation of DCE, strain FHY-13 can utilize organic carbon to synthesize its own cellular substances. The linear relationship between cell proliferation and DCE degradation is y = 0.1904x, with a correlation coefficient R0. 2 =0.9886, indicating that strain FHY-13 can synthesize 0.1904 mg of its own cell biomass for every 1 mg of DCE degraded.

[0089] Example 6: Degradation performance analysis of common industrial organic pollutants by soil brevibacillus agri FHY-13

[0090] The degradation capacity of *Brevibacillus agri* FHY-13 of common industrial organic pollutants—dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene, and hexachlorobenzene—was investigated using these as the sole carbon source. The results showed that strain FHY-13 could degrade these organic pollutants to varying degrees. It could relatively completely degrade chlorobenzene, pentachlorobenzene, and hexachlorobenzene within 24 hours, and could degrade dichloromethane, dichloropropane, and benzene within 72 hours.

[0091] The specific experimental procedure is as follows:

[0092] Prepare a first DCE liquid selective medium with a pH of 6.0 and a salt concentration of 0.89%. Add 50 mL of the first DCE liquid selective medium to each 250 mL serum bottle and sterilize at 121°C for 20 min. Add a bacterial suspension with an initial biomass of 0.75 mg / L prepared according to the method in Example 2 to six of these serum bottles. Use dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene, or hexachlorobenzene at an initial concentration of 10 mg / L as the sole carbon source, respectively. Use another six serum bottles as blank controls (adding only DCE without bacterial suspension). After deoxygenation and sealing, decompose the serum bottles and culture with shaking at 35°C. Analyze the residual organic matter concentration in the liquid phase at 24 h and 72 h, determine the corresponding biomass, and plot the degradation rate of different organic carbon sources against the cell growth of strain FHY-13.

[0093] like Figure 6 As shown in (a), when the culture time was 24 h, strain FHY-13 could degrade chlorobenzene, pentachlorobenzene, and hexachlorobenzene to a large extent, with degradation rates reaching 91.5%, 83.3%, and 76.7%, respectively. However, at this time, the degradation rates of dichloromethane, dichloropropane, and benzene by strain FHY-13 were only 14.4%, 17.3%, and 32.8%, respectively. Figure 6 As shown in (b), when the culture time was 72 h, the degradation rates of dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene, and hexachlorobenzene by strain FHY-13 reached 78.9%, 45.7%, 59.5%, 100%, 100%, and 100%, respectively. These experimental results demonstrate that strain (Brevibacillus agri) FHY-13 can degrade a variety of common organic pollutants, and this provides certain guidance for the application of this strain in practical industrial pollution control.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A highly efficient soil-borne short-spore bacillus strain that degrades dichloroethylene ( Brevibacillus agri FHY-13, characterized in that, It was deposited at the China Center for Type Culture Collection on March 28, 2023, with accession number CCTCC NO:M2023420; the 16S rRNA sequence of the FHY-13 is shown in SEQ ID NO.

1.

2. A bacterial suspension using *Bacillus brevis* as the active ingredient according to claim 1, characterized in that, The bacterial suspension was prepared by solid culture, seed culture, and liquid culture of soil-borne Bacillus brevis FHY-13, which is highly efficient at degrading dichloroethylene.

3. A method for preparing the bacterial suspension according to claim 2, characterized in that, The specific steps include the following: (1) Solid culture: The soil styrax FHY-13, which is highly efficient at degrading dichloroethylene, was inoculated onto an agar slant culture medium and cultured at 20-40℃ for 2-4 days to obtain slant cells; the final concentration composition of the agar slant culture medium was: K2HPO4 1500 mg / L, KH2PO4 500 mg / L, NaCl 1000 mg / L, dichloroethylene 50 mg / L, MgSO4·7H2O 200 mg / L, water as solvent, pH 6.0-8.0, and agar 18-20 g / L; (2) Seed culture: Colonies were picked from the slant culture and inoculated into the seed culture medium. The culture was carried out at 20-40℃ for 12-24 h to obtain the seed liquid. The final concentration of the seed culture medium was: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L, water as solvent, and pH 6.0-8.

0. (3) Liquid culture: The seed culture is inoculated into the fermentation medium at a volume concentration of 1% and cultured at 20~40℃ for 12~24 h to obtain the fermentation culture medium as the bacterial suspension; the final concentration composition of the fermentation medium is: K2HPO4 1500 mg / L, KH2PO4 500 mg / L, NaCl 1000 mg / L, dichloroethylene 50 mg / L, MgSO4·7H2O 200 mg / L, the solvent is water, and the pH value is 6.0~8.

0.

4. The application of the bacterial suspension of claim 2 in the degradation of dichloroethylene.

5. The application according to claim 4, characterized in that, Specifically, the bacterial suspension is inoculated into a first DCE liquid selective medium with a salt concentration of 0-1%, using dichloroethylene as the sole carbon source, and cultured at 25-35°C to achieve the degradation of dichloroethylene. The final concentration composition of the first DCE liquid selective medium is: DCE 1-25 mg / L, KH2PO4 376 mg / L, K2HPO4 456 mg / L, (NH4)2SO4 480 mg / L, NaNO3 680 mg / L, Mg(NO3)2 250 mg / L, CaCl2·2H2O 11 mg / L, trace elements, with water as the solvent and a pH value of 6.0-8.

0. The final concentration composition of the trace elements is: MnCl2·H2O 60 mg / L, ZnCl2 88 mg / L, KI 10 mg / L, NaMoO4·2H2O 100 mg / L, H3BO3 50 mg / L. mg / L, solvent is water.

6. The application according to claim 4, characterized in that, Specifically, the bacterial suspension was inoculated into a first DCE liquid selective medium with a salt concentration of 0.89%, using dichloroethylene as the sole carbon source, and cultured at 35°C to achieve the degradation of dichloroethylene. The final concentration composition of the first DCE liquid selective medium was: DCE 1~25 mg / L, KH2PO4 376 mg / L, K2HPO4 456 mg / L, (NH4)2SO4 480 mg / L, NaNO3 680 mg / L, Mg(NO3)2 250 mg / L, CaCl2·2H2O 11 mg / L, trace elements, water as solvent, and pH 6.

0. The final concentration composition of the trace elements was: MnCl2·H2O 60 mg / L, ZnCl2 88 mg / L, KI 10 mg / L, NaMoO4·2H2O 100 mg / L, H3BO3 50 mg / L, water as solvent.

7. The use of the bacterial suspension of claim 2 in the degradation of dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene or hexachlorobenzene.

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