Salt-tolerant microbacterium halophicum hsha1-2 and microbial agent and application thereof

Abstract

This invention relates to a salt-tolerant bacillus strain HSHA1-2, its inoculum, and its applications, belonging to the technical field of microbiology. The salt-tolerant bacillus (… Halalkalibacterium halodurans HSHA1-2 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38092, on April 9, 2026. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This invention also provides a bacterial agent containing salt- and alkali-tolerant *Bacillus halophilus* HSHA1-2 and its application in degrading benzene-containing wastewater. The salt- and alkali-tolerant *Bacillus halophilus* HSHA1-2 provided by this invention exhibits excellent salt and alkali tolerance, and can stably and efficiently degrade benzene compounds under extreme saline-alkali environments.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a salt-tolerant bacillus strain HSHA1-2, its inoculum, and its applications. Background Technology

[0002] Currently, the main methods for treating wastewater containing toluene, xylene, and nitrobenzene are divided into physical, chemical, and biological methods. Physical methods (such as adsorption, extraction, and membrane separation) can only transfer pollutants, not completely degrade them, and are costly and prone to secondary pollution. Chemical methods (such as oxidation and photocatalysis) have harsh reaction conditions, high reagent consumption, and high operating costs, making them unsuitable for large-scale industrial wastewater treatment scenarios. Biological methods, due to their advantages of being environmentally friendly, low-cost, completely degrading, and without secondary pollution, have become the mainstream technology for wastewater treatment. The core of this approach lies in screening and obtaining specific bacterial strains that are tolerant of extreme environments and possess highly efficient degradation capabilities.

[0003] While some microbial strains capable of degrading single aromatic pollutants have been reported in existing technologies, most strains can only function in neutral, low-salt environments and cannot adapt to complex wastewater environments with high salinity and alkalinity. Furthermore, strains capable of simultaneously tolerating saline-alkali environments and degrading multiple mixed pollutants such as toluene, xylene, and nitrobenzene are extremely rare. Existing strains suffer from low degradation efficiency, narrow substrate spectrum, and poor environmental adaptability, failing to meet the treatment needs of mixed high-salt and high-alkali wastewater in actual industrial applications. Therefore, developing a dedicated degrading strain with salt and alkali tolerance, a broad substrate spectrum, and high degradation efficiency, and constructing a biodegradation technology solution adapted to extreme environments, is of significant practical importance and application value for solving the problem of treating high-salt and high-alkali industrial wastewater and promoting the compliance of industrial wastewater discharge with standards. Summary of the Invention

[0004] To address the issue that existing biological methods for treating wastewater containing benzene compounds lack the application of salt-tolerant and alkali-tolerant bacilli, this invention provides a salt-tolerant and alkali-tolerant bacillus strain HSHA1-2, its agent, and its applications to solve the aforementioned problem. The salt-tolerant and alkali-tolerant bacillus HSHA1-2 provided by this invention can degrade benzene compounds in high-salt and high-alkali environments, and can degrade benzene compounds in wastewater even in extreme environments. Therefore, it can be directly applied to industrial wastewater, thereby achieving the goal of safe and efficient removal of benzene substances.

[0005] The technical solution of this invention is as follows: In a first aspect, the present invention provides a salt-tolerant bacillus strain HSHA1-2, wherein the salt-tolerant bacillus ( Halalkalibacterium haloduransHSHA1-2 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38092, on April 9, 2026. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] Secondly, the present invention provides a bacterial agent containing salt-tolerant bacillus HSHA1-2; the bacterial agent includes liquid bacterial agent and solid bacterial agent.

[0007] Furthermore, the viable count of salt-tolerant Microbacterium tumefaciens HSHA1-2 in the liquid bacterial agent is ≥3×10⁻⁶. 9 cfu / ml.

[0008] Furthermore, the viable count of salt-tolerant Microbacterium tumefaciens HSHA1-2 in the solid bacterial agent is ≥3.2 × 10⁻⁶. 10 CFU / g.

[0009] Furthermore, the preparation method of the liquid bacterial agent is as follows: the primary seed liquid is inoculated into the fermentation expansion medium at an inoculation rate of 10%, and the fermentation culture conditions are controlled as follows: temperature 40±1℃, stirring speed 200~250r / min, aeration rate 1.5~2.0vvm, tank pressure 0.05~0.1MPa, and cultured for 24~36h to obtain the liquid bacterial agent.

[0010] Furthermore, the solid microbial agent is prepared as follows: calcium carbonate and corn starch carrier are added at 6% of the fermentation broth to fix the microbial cells, and then dried by spray drying to obtain the solid microbial agent.

[0011] Furthermore, the fermentation expansion medium consists of: 15 g / L corn steep liquor, 10 g / L soybean meal powder, 5 g / L glucose, 5% NaCl, 1.2 g / L Na2CO3, 1 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.1 g / L CaCl2, 0.25 g / L MnSO4, and distilled water to a final volume of 1 L, with the pH adjusted to 9.0.

[0012] Thirdly, the present invention provides an application of salt-tolerant bacillus HSHA1-2 in the degradation of wastewater containing benzene compounds.

[0013] Furthermore, the benzene compounds are toluene, xylene, and nitrobenzene.

[0014] Furthermore, the salt concentration in the wastewater is ≤12%, and the pH is <11.0.

[0015] The beneficial effects of this invention are as follows: The salt- and alkali-tolerant bacillus HSHA1-2 provided by this invention exhibits excellent salt and alkali resistance, making it well-suited for treating high-salt and high-alkali wastewater in coastal saline-alkali areas and chemical plants. HSHA1-2 can stably and efficiently degrade benzene compounds under extreme saline-alkali environments, demonstrating a broad substrate degradation spectrum and the ability to simultaneously and synergistically degrade multiple pollutants in wastewater. HSHA1-2 achieves a 72-hour degradation rate of over 95% for toluene, xylene, and nitrobenzene in wastewater, demonstrating high degradation efficiency and stable and reliable treatment effects. Furthermore, the cultivation process for HSHA1-2 is simple and easy to implement, with strong feasibility for industrial-scale implementation. The entire wastewater treatment operation is simple, with low daily maintenance costs. Compared to traditional physical and chemical wastewater treatment methods, it can reduce treatment costs by more than 60%, demonstrating significant economic advantages and engineering application value. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a colony diagram of the salt-tolerant bacillus HSHA1-2 in Example 1 of the present invention.

[0018] Figure 2 This is a Gram staining image of the salt-tolerant bacillus HSHA1-2 in Example 1 of the present invention.

[0019] Figure 3 This is the growth curve (OD) of the salt-tolerant bacillus HSHA1-2 under different salt concentrations and pH conditions in Example 2 of this invention. 600 )picture.

[0020] Figure 4 This is a line graph showing the results of the wastewater degradation experiment simulating high-salt, high-alkali wastewater containing benzene compounds in Example 3 of this invention.

[0021] Figure 5 This is a line graph showing the degradation rate of benzene compounds under different salt concentrations and pH conditions in Example 3 of the present invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0023] Example 1 Isolation, screening and identification of salt-tolerant alkali-tolerant microbacterium HSHA1-2 1. Sampling On September 13, 2025, mixed samples of silt and soil were collected from the area surrounding the discharge outlet of saline-alkali chemical wastewater at Zhejiang Petrochemical Co., Ltd. A sterile sampler was used to collect the mixed samples of silt and topsoil at a depth of 5–15 cm. Three parallel samples were collected from each sampling point, each weighing 500 g. The samples were placed in sterile sealed sampling bags, and the sampling time, location, and environmental parameters were labeled. The bags were then quickly transported back to the laboratory under 4°C low-temperature refrigeration to prevent the attenuation of microbial activity.

[0024] 2. Separation (1) Sample pretreatment Take 100g of parallel samples from each sampling point, mix them thoroughly, and place them in a sterile Erlenmeyer flask. Add 500mL of sterile physiological saline (0.85% NaCl solution), add sterile glass beads, and place the flask in a constant temperature shaker at 40℃ and 150r / min for 30min to fully disperse the sample particles and release the microorganisms into the solution. After shaking, let it stand for 10min, and use a sterile pipette to aspirate the supernatant suspension. Filter the suspension through a 0.22μm sterile filter membrane to remove impurities and large particles, and obtain the bacterial source solution. Store the solution in a 4℃ refrigerator for no more than 24 hours to provide a pure and highly active bacterial source for subsequent gradient salt-alkali enrichment culture.

[0025] (2) Gradient salt-alkali enrichment culture Preparation of basal culture medium: Precisely prepare basal salt-alkali enrichment liquid culture medium with the following formula (per liter): 5.0g peptone, 2.0g yeast extract, 60g NaCl, 1.0g Na2CO3, 1.0g K2HPO4, 0.2g MgSO4·7H2O. Make up to 1L with distilled water, adjust the pH to 9.0±0.1 using a precision pH meter, dispense 200mL into 500mL sterile Erlenmeyer flasks, autoclave at 121℃ and 0.1MPa for 20min, cool to 40℃ for later use. After sterilization, test the sterility of the culture medium to ensure it is free from contamination.

[0026] First-generation enrichment culture: The pretreated bacterial culture was inoculated at a rate of 10% (v / v) into the basic saline-alkali enrichment liquid medium using a sterile pipette. Three replicates were set up for each treatment. The culture was placed in a constant temperature shaker at 40℃ and 180 rpm for 72 h. Samples were taken daily during the period to measure the OD of the bacterial culture. 600 Value, OD of bacterial culture 600 When the value reaches 1.0 or above, the first generation of enrichment culture is completed, the first generation of enriched bacterial solution is obtained, and the initial enrichment of salt-tolerant bacterial groups is achieved, laying the bacterial group foundation for subsequent gradient stress acclimatization.

[0027] Gradual salt-alkali stress acclimatization: Based on the first-generation enriched bacterial culture, four consecutive generations of gradual salt-alkali stress acclimatization were conducted. The NaCl concentration and pH value of the culture medium were gradually increased to construct progressively stronger environmental stress pressure, allowing for targeted screening of salt-alkali tolerant strains. Specific acclimatization parameters were as follows: Second-generation enrichment medium NaCl concentration increased to 8.0% (mass fraction), pH adjusted to 9.0±0.1; Third-generation NaCl concentration increased to 10% (mass fraction), pH adjusted to 9.5±0.1; Fourth-generation NaCl concentration increased to 12% (mass fraction), pH adjusted to 10.0±0.1; Fifth-generation NaCl concentration increased to 14% (mass fraction), pH adjusted to 11.0±0.1. For each generation of acclimatization, a precise 10% (v / v) inoculum was taken from the previously enriched bacterial culture and transferred to fresh culture medium of the corresponding concentration. The culture conditions were the same as the first generation (40℃, 180 rpm shaking culture for 72 h). OD of the bacterial culture was measured after each generation of acclimatization. 600 The values ​​are adjusted to ensure normal bacterial growth, eliminating strains with stagnant or slow growth, ultimately obtaining a fifth-generation enrichment bacterial solution with stable salt and alkali tolerance. This completes the gradient salt and alkali enrichment and acclimatization process, at which point the bacterial population possesses strong salt and alkali tolerance and can proceed to the subsequent targeted pollutant screening stage. Growth curves (OD) of bacterial strains under different salt concentrations and pH conditions are shown. 600 See details Figure 3 .

[0028] from Figure 3 The results show that strain HSHA1-2 can grow in salt concentrations of 0–15% and pH values ​​of 7–11.5, and its OD value is highest at salt concentrations of 6%–10% and pH values ​​of 8.5–9.5. 600 The maximum value was observed, and the strain was in a stagnant phase from 0 to 6 hours, entered the logarithmic growth phase from 6 to 24 hours, and the number of viable cells reached its peak at around 30 hours. As the cycle lengthened and nutrients were depleted, the number of viable cells began to decline slightly at 36 hours, thus providing a fermentation basis for subsequent expansion culture.

[0029] (3) Screening Preparation of selective culture medium: Prepare a salt-alkali selective solid culture medium using a mixture of toluene, xylene, and nitrobenzene (mass ratio 1:1:1) as the sole carbon and nitrogen source, controlling the total concentration of mixed contaminants to 200 mg / L (with each contaminant at a concentration of 66.7 mg / L). Other components of the culture medium are as follows (per liter): NaCl 70 g, Na2CO3 1.5 g, K2HPO4 1.0 g, MgSO4·7H2O 0.2 g, agar powder 18.0 g. Dilute to 1 L with distilled water, adjust the pH to 10.0 ± 0.1 using a precision pH meter, dispense approximately 20 mL into sterile petri dishes, autoclave at 121 °C and 0.1 MPa for 20 min, cool and solidify before use. After sterilization, a sterility test must be performed to ensure that the culture medium is free from microbial contamination.

[0030] Strains were isolated and initially screened: the fifth-generation enriched bacterial culture was serially diluted with sterile physiological saline at a dilution gradient of 10-10. -1 Up to 10 -6 Three replicates were set up for each dilution gradient. Using sterile pipettes, 0.1 mL of bacterial culture from each dilution gradient was accurately pipetted onto salt-alkali selective solid plates. The plates were spread evenly using a sterile spreader and incubated upside down at 30°C for 48–72 hours, with daily observation of colony growth. Colonies showing good growth, uniform morphology, and no contamination were selected and purified using the streak plate method. The "regional streak" principle was followed during streak culturing. After each streak, the plates were incubated upside down for 24–48 hours, and the streak purification was repeated 3–4 times until a single pure bacterial strain with uniform colony morphology and no contamination was obtained. These were the initial screening strains, numbered, recorded, and stored on slant plates at 4°C. This step, based on the previous salt-alkali tolerance acclimatization, uses mixed contaminant selection pressure to isolate strains capable of growing on the target contaminant, providing a candidate strain library for subsequent screening of highly efficient degrading strains.

[0031] Preparation of secondary screening medium: Prepare a saline-alkali liquid secondary screening medium with the same formula as the liquid medium used for pollutant-directed screening (with agar powder removed). The total concentration of mixed pollutants toluene, xylene, and nitrobenzene is increased to 300 mg / L (each pollutant concentration is 100 mg / L), the NaCl concentration is 7.0%, and the pH is 10.0 ± 0.1. Sterilize and use it for later use.

[0032] Secondary screening culture and detection: All initially screened strains were inoculated into secondary screening medium, with three replicates for each strain. A blank control group (without inoculation, all other conditions were the same) was also set up. The cultures were incubated at 40℃ and 180 rpm for 72 h. After incubation, the residual concentrations of toluene, xylene, and nitrobenzene in each strain's culture system were determined by gas chromatography (GB / T 11890-1989). The degradation rate of the three pollutants by each strain was calculated using the formula: Degradation rate (%) = (Initial pollutant concentration - Residual pollutant concentration) / Initial pollutant concentration × 100%. This step, by increasing the pollutant concentration and strictly controlling the detection standards, precisely screened the degradation performance of the initially screened strains, identifying strains with satisfactory degradation efficiency and stable performance.

[0033] Target strain identification: Pure strains with degradation rates of p-toluene, xylene, and nitrobenzene all above 70% and stable growth were screened out, and the strain number was HSHA1-2.

[0034] 4. Identification Morphological, physiological, biochemical, and molecular biological identification of strain HSHA1-2 was performed. The colony morphology of strain HSHA1-2 is shown in the figure below. Figure 1 As shown, the colonies are round, milky white to pale yellow, with a smooth and moist surface, neat edges, and a slightly raised center. The colony diameter is 2-3 mm. Under a microscope, they appear as short rods, Gram-positive, and produce spores (terminal). They are non-capsulated and approximately 0.5-0.8 μm × 2.0-4.0 μm in size.

[0035] After activation, strain HSHA1-2 was plated and sent to the Sanger sequencing laboratory of Beijing Nuosai Genome Research Center Co., Ltd. for identification. The gene sequence of strain HSHA1-2, shown in SEQ ID NO.1, was obtained. The identification result is a salt-tolerant *Microbacterium halophilum*. Halalkalibacterium halodurans Salt-tolerant bacillus HSHA1-2 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38092, deposited on April 9, 2026. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0036] Example 2 Preparation of a bacterium containing salt-tolerant and alkali-tolerant Microbacterium tumefaciens HSHA1-2 (1) Seed culture preparation: Prepare seed culture medium: glucose 10g / L, peptone 8g / L, yeast extract 3g / L, NaCl 5%, Na2CO3 1g / L, K2HPO4 1.5g / L, MgSO4·7H2O 0.3g / L, distilled water to a final volume of 1L, adjust pH to 9.0, and sterilize for later use. Inoculate the purified salt-tolerant bacillus HSHA1-2 onto the slant of the seed culture medium and incubate at 40℃ for 24h to obtain the slant culture; take one loopful of the slant culture with an inoculation loop and inoculate it into a 250mL Erlenmeyer flask containing 100mL of seed culture medium, and incubate at 40℃ and 200r / min for 18~24h until the OD of the culture is reached. 600 A value of 1.2 to 1.5 indicates that the seed solution is of grade one.

[0037] (2) Preparation of liquid inoculum: Prepare fermentation expansion medium: corn steep liquor 15g / L, soybean meal powder 10g / L, glucose 5g / L, NaCl 5%, Na2CO3 1.2g / L, K2HPO4 1g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.1g / L, MnSO4 0.25g / L, distilled water to a final volume of 100L, adjust pH to 9.0, sterilize and cool to 40℃ for later use. Inoculate the primary seed liquid into a 200L fermenter at an inoculation rate of 10%. The fermentation conditions are controlled as follows: temperature 40±1℃, stirring speed 200~250r / min, aeration rate 1.5~2.0vvm, tank pressure 0.05~0.1MPa, culture for 24~36h, during which time samples are taken regularly to determine the concentration of the inoculum until the viable count of the inoculum reaches 3×10 9 Once the cfu / ml concentration reaches a certain level, fermentation is stopped, yielding a high-density fermentation broth, i.e., a liquid bacterial agent containing salt-tolerant and alkali-tolerant Bacillus thuringiensis HSHA1-2. This liquid bacterial agent can be used directly for wastewater degradation treatment or further processed into a solid bacterial agent.

[0038] (3) Preparation of solid microbial agent: The fermentation broth was fixed with 6% calcium carbonate and 2% corn starch carrier. The feed pressure of the pressure spray drying tower (equipment manufacturer: Zhejiang Erle Drying Equipment Co., Ltd., model: YPG-5) was controlled at 25MPa, inlet air temperature of 175℃, outlet air temperature of 75℃~80℃, negative pressure of 50Pa, and moisture content controlled below 5%. 9.1kg of solid microbial agent was obtained and mixed evenly with a mixer (equipment manufacturer: Jiangsu Faster Machinery Co., Ltd., model: DZ005). After the sample was bathed in an 80℃ water bath for 20min, the spore count of the solid microbial agent was tested according to GB20287-2006 and found to be 3.2×10⁻¹⁰. 10 CFU / g.

[0039] Example 3 Degradation experiment of salt-tolerant bacillus HSHA1-2 in wastewater containing benzene compounds 1. Experiment simulating the degradation of wastewater containing benzene compounds with high salinity and alkalinity Simulated high-salt, high-alkali wastewater was prepared: each liter of wastewater contained the following components: NaCl concentration 8%, pH=9.0, toluene, xylene, and nitrobenzene concentrations of 100 mg / L each, total concentration 300 mg / L, (NH4)2SO4 0.19 g, KNO3 0.1 g, MgSO4·7H2O 0.05 g, K2HPO4·3H2O 0.2 g, NaCl 0.12 g, MnSO4·H2O 0.01 g, FeSO4·7H2O 0.01 g. 1 L of the above high-salt, high-alkali wastewater was taken, and 1 g of the solid bacterial agent prepared in Example 2 was added (dosage 1‰). The temperature was controlled at 40℃, and aeration was performed to achieve a dissolved oxygen concentration of 3-4 mg / L. The mixture was allowed to stand for 96 hours for degradation. Samples were taken every 24 hours to test the pollutant concentration. The test results are as follows: Figure 4 As shown.

[0040] Depend on Figure 4 The degradation curves show that at 24 hours, the degradation rates of toluene, xylene, and nitrobenzene were 47.5%, 44.8%, and 43.2%, respectively; at 48 hours, the degradation rates were 72.3%, 70.1%, and 69.2%, respectively; at 72 hours, the degradation rates were 95.6%, 92.9%, and 92.0%, respectively; and at 96 hours, the degradation rates of all three pollutants exceeded 95%.

[0041] 2. Degradation rate of benzene compounds under different salt concentrations and pH conditions Prepare culture media with different salt concentrations and pH values. The basic culture medium is as follows: 5.0 g peptone, 2.0 g yeast extract, 1.0 g Na2CO3, 1.0 g K2HPO4, 0.2 g MgSO4·7H2O, and bring the volume to 1 L with distilled water.

[0042] 8% NaCl, pH=9.0 medium: Add 80g NaCl to the above basal medium and adjust the pH to 9.0±0.1 using a precision pH meter. Then add mixed contaminants (100mg toluene, 100mg xylene, and 100mg nitrobenzene). Autoclave at 121℃ and 0.1MPa for 20min, cool to 40℃ for later use. After sterilization, the sterility of the medium must be tested to ensure it is free from contamination.

[0043] 10% NaCl, pH=9.5 medium: Add 100g NaCl to the above basal medium and adjust the pH to 9.5±0.1 using a precision pH meter. Then add mixed contaminants (100mg toluene, 100mg xylene, and 100mg nitrobenzene). Autoclave at 121℃ and 0.1MPa for 20min, cool to 40℃ for later use. After sterilization, the sterility of the medium must be tested to ensure it is free from contamination.

[0044] 12% NaCl, pH=10.0 medium: Add 120g NaCl to the above basal medium and adjust the pH to 10.0±0.1 using a precision pH meter. Then add mixed contaminants (100mg toluene, 100mg xylene, and 100mg nitrobenzene). Autoclave at 121℃ and 0.1MPa for 20min, cool to 40℃ for later use. After sterilization, the sterility of the medium must be tested to ensure it is free from contamination.

[0045] 14% NaCl, pH=11.0 medium: Add 140g NaCl to the above basal medium and adjust the pH to 11.0±0.1 using a precision pH meter. Then add mixed contaminants (100mg toluene, 100mg xylene, and 100mg nitrobenzene). Autoclave at 121℃ and 0.1MPa for 20min, cool to 40℃ for later use. After sterilization, the sterility of the medium must be tested to ensure it is free from contamination.

[0046] Take culture media with different salt concentrations and pH values ​​prepared above, and add 1% of the liquid bacterial agent prepared in Example 2 to each. Incubate at 40℃ for 72 h. For details of benzene degradation rates under different salt concentrations and pH conditions, see [link to relevant documentation]. Figure 5 .

[0047] from Figure 5 It can be seen that the salt-tolerant *Microbacterium halophilum* HSHA1-2 exhibits a degradation rate of 96.6% for toluene, 95.7% for xylene, and 95.3% for nitrobenzene under the conditions of 8% NaCl concentration, pH 9.0, and 40℃. Under the conditions of 10% NaCl concentration, pH 9.5, and 40℃, the degradation rates are 94.3% for toluene, 93.9% for xylene, and 93.7% for nitrobenzene; 12% NaCl concentration, pH 10.0, and 40℃, resulting in a degradation rate of 91.4% for toluene, 90.6% for xylene, and 90.2% for nitrobenzene; and 14% NaCl concentration, pH 11.0, and 40℃, leading to a degradation rate of 78.2% for toluene, 76.7% for xylene, and 75.3% for nitrobenzene. Therefore, it can be concluded that *Microbacterium halophilum* HSHA1-2 is a strain capable of efficiently degrading benzene compounds. From the degradation trend, as the salt concentration and pH increase, the degradation rate of toluene, xylene, and nitrobenzene by the salt-tolerant bacterium HSHA1-2 gradually decreases, especially when the NaCl concentration is 14% and the pH is 11.0, the degradation efficiency drops sharply. At a salt concentration of 12% and pH < 10, the average degradation rate is greater than 90%.

[0048] 3. Practical industrial wastewater treatment applications High-salt, high-alkali wastewater from a coastal chemical enterprise was collected. The wastewater composition was as follows: NaCl concentration 5.5%, pH 9.2, toluene concentration 88 mg / L, xylene concentration 104 mg / L, and nitrobenzene concentration 76 mg / L. 10 L of this wastewater was pretreated to remove suspended solids, and then 100 mL of the liquid bacterial agent prepared in Example 2 was added. The degradation temperature was controlled at 40°C, with continuous aeration and stirring at 2-hour intervals. 100 mL of the liquid bacterial agent was added at 24 and 48 hours. After 72 hours, the test results showed that the residual concentrations of toluene, xylene, and nitrobenzene were 3.2 mg / L, 5.5 mg / L, and 2.3 mg / L, respectively, with degradation rates of 96.36%, 94.71%, and 96.97%, achieving the pretreatment objective.

[0049] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A salt-tolerant bacillus strain HSHA1-2, characterized in that, The salt-tolerant microbacterium ( Halalkalibacterium halodurans HSHA1-2 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38092, on April 9, 2026. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The salt-tolerant bacillus HSHA1-2 as described in claim 1, characterized in that, The 16S rDNA sequence of the salt-tolerant bacterium HSHA1-2 is shown in SEQ ID NO.

1.

3. A bacterial agent containing the salt-tolerant microbacterium HSHA1-2 as described in claim 1, characterized in that, The microbial agents include liquid microbial agents and solid microbial agents.

4. The microbial agent as described in claim 3, characterized in that, The viable count of salt-tolerant Microbacterium tumefaciens HSHA1-2 in the liquid bacterial agent is ≥3×10⁻⁶. 9 cfu / ml.

5. The microbial agent as described in claim 3, characterized in that, The viable count of salt-tolerant Microbacterium tumefaciens HSHA1-2 in the solid bacterial agent is ≥3.2 × 10⁻⁶. 10 CFU / g.

6. The microbial agent as described in claim 4, characterized in that, The preparation method of the liquid bacterial agent is as follows: the primary seed liquid is inoculated into the fermentation expansion medium at an inoculation rate of 10%. The fermentation culture conditions are controlled as follows: temperature 40±1℃, stirring speed 200~250r / min, aeration rate 1.5~2.0vvm, tank pressure 0.05~0.1MPa, and culture for 24~36h to obtain the liquid bacterial agent.

7. The microbial agent as described in claim 5, characterized in that, The solid microbial agent is prepared as follows: calcium carbonate and corn starch carrier are added at 6% of the fermentation broth to fix the microbial cells, and then dried by spray drying to obtain the solid microbial agent.

8. The microbial agent as described in claim 6, characterized in that, The fermentation expansion medium consists of: 15 g / L corn steep liquor, 10 g / L soybean meal powder, 5 g / L glucose, 5% NaCl, 1.2 g / L Na2CO3, 1 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.1 g / L CaCl2, 0.25 g / L MnSO4, and distilled water to a final volume of 1 L, with the pH adjusted to 9.

0.

9. The application of the salt-tolerant bacillus HSHA1-2 as described in claim 1 in the degradation of wastewater containing benzene compounds.

10. The application as described in claim 9, characterized in that, The benzene compounds are toluene, xylene, and nitrobenzene.

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