Nitrobenzene high-efficiency degrading bacterial agent, preparation and application thereof

Abstract

The application discloses a kind of nitrobenzene high-efficiency degradation bacterial agent, preparation and application, belong to biotechnology field, the microbial inoculum of nitrobenzene wastewater treated by the present application mainly consists of lysinibacillus (Lysinibacillus sp.LBQ22-1), and is identified as Lysinibacillus sp. by 16S rDNA sequence sequencing, has been preserved in China General Microbiological Culture Collection Center, preservation number is CGMCC NO.26095, and preservation date is November 10, 2022.The nitrobenzene degradation bacterial agent of the present application can grow with nitrobenzene as sole carbon source and nitrogen source, and 1600mg / L nitrobenzene can be degraded to 2mg / L or less in 36h, and the removal rate of nitrobenzene is more than 99.5%.The bacterial preparation prepared by the present application is added to wastewater at a mass ratio of 0.5~5 ‰, and has good degradation effect on nitrobenzene contaminated water body, solves the problem of difficult biodegradation of high-concentration nitrobenzene.

Description

Technical Field

[0001] This invention belongs to the field of biological wastewater treatment, specifically relating to the preparation and application of a composite bacterial agent for treating nitrobenzene wastewater. Background Technology

[0002] Nitrobenzene, as a monocyclic aromatic nitro-substituted compound, has long been widely used as an important chemical raw material in industrial production. Therefore, it leaves large amounts of residues in wastewater from industries such as pharmaceuticals, aniline, pesticides, and dyes. Nitrobenzene exhibits extremely high stability in water. Due to its density being greater than water, nitrobenzene entering water bodies will sink to the bottom and remain unchanged for a long time. Furthermore, because it has a certain solubility in water, the resulting water pollution can persist for a considerable period. The Classification and Marking of Commonly Used Hazardous Chemicals (GB13690-92) classifies this substance as a Class 6.1 toxic substance. Due to its biotoxicity, nitrobenzene is listed high on the world's list of priority toxic organic pollutants. my country also has strict controls on the concentration of nitrobenzene compounds in wastewater, stipulating the maximum allowable discharge concentrations as follows: Grade I standard 2.0 mg / L, Grade II standard 3.0 mg / L, and Grade III standard 5.0 mg / L.

[0003] Currently, the treatment of nitrobenzene wastewater mainly employs physicochemical methods. Physical methods include solvent extraction, adsorption, and air stripping, while chemical methods include ozone oxidation, hydrogen peroxide oxidation, supercritical water oxidation, and ultrasonic oxidation. Although physicochemical methods for treating nitrobenzene wastewater are highly efficient and relatively simple, they generally have specific requirements for reaction conditions, high treatment costs, and are prone to causing secondary pollution, making them difficult to apply on a large scale in practical wastewater treatment projects. Biological methods for treating nitrobenzene wastewater have lower treatment costs, simpler operation and management, and no secondary pollution. Furthermore, microorganisms have strong variability and adaptability, making them suitable for various types and conditions of wastewater, and thus easily applied on a large scale in practical wastewater treatment projects.

[0004] Under anaerobic conditions, microbial degradation of nitrobenzene primarily occurs through a reduction pathway, reducing nitrobenzene to aniline. Aniline then undergoes aerobic degradation, where, under the action of dioxygenases, it is converted into catechol or hydroquinone, further undergoing ring-opening degradation of the benzene ring, ultimately resulting in mineralization. In contrast to anaerobic conditions, many bacteria and fungi can completely mineralize or transform nitrobenzene under aerobic conditions. The aerobic degradation pathways of nitrobenzene mainly consist of two routes: aerobic oxidation and aerobic partial reduction.

[0005] Li Zhanjiang et al. isolated and domesticated two highly efficient anaerobic bacteria for degrading nitrobenzene, *Bacteroides distasonis* and *Bacteroides merdae*, from sewer sediment. These bacteria can co-metabolize and reduce nitrobenzene with glucose, achieving a maximum degradation rate of 95 mg / (L·d), with 1g of glucose capable of co-metabolizing and reducing 200–260 mg of nitrobenzene. Li Yi et al. screened a *Pseudomonas putida* strain capable of mineralizing 20 mg / L of nitrobenzene and rapidly degrading it; nitrobenzene concentrations not exceeding 100 mg / L can be completely degraded by this bacterium. Liu Hongguo et al. isolated *Staphylococcus*, which, after 3 days of cultivation in a medium containing 1000 mg / L nitrobenzene, degraded 44% of the nitrobenzene. Xu Chengbin et al. isolated *Bacillus cereus*, achieving a 72.70% degradation rate of 200 mg / L nitrobenzene after 72 hours. The Acinetobacter baumannii strain screened by Ma Xiping et al. showed a degradation rate of 66.84% for 200 mg / L nitrobenzene in 48 hours. Patent application CN113801828A, which describes a highly efficient nitrobenzene-degrading bacterium and its preparation and application, mentions that *Pseudomonas schlegelii* can degrade over 99.5% of 900 mg / L nitrobenzene within 24 hours and over 99.5% of 1000 mg / L nitrobenzene within 32 hours. In its application in nitrobenzene wastewater treatment, it can be continuously cultured and stably degrades 500 mg / L nitrobenzene within 24 hours.

[0006] Multiple studies have shown that most nitrobenzene-degrading bacteria exhibit slow degradation rates, low degradation rates at high concentrations of nitrobenzene, and a limited number of species capable of degrading nitrobenzene at concentrations of 500–1000 mg / L and higher. Therefore, isolating and cultivating strains with high tolerance and efficient degradation capabilities for high concentrations of nitrobenzene for the biological treatment of nitrobenzene wastewater is particularly important. Summary of the Invention

[0007] This invention addresses the problems existing in the prior art by disclosing a highly efficient nitrobenzene-degrading bacterial agent, its preparation, and its application. The microbial agent prepared using Lysinibacillus sp. LBQ22-1 can reduce the nitrobenzene content in nitrobenzene wastewater with a nitrobenzene concentration of 200-1600 mg / L by more than 99.5% through aerobic biodegradation.

[0008] This invention is implemented as follows:

[0009] A method for preparing a highly efficient nitrobenzene-degrading bacterial agent, characterized in that the method comprises the following steps:

[0010] Step 1: Solid plate culture: Transfer Lysinibacillus sp. LBQ22-1 onto a solid culture medium and incubate at 25-35℃ for 36 hours to activate the strain;

[0011] Step 2, Seed culture: Inoculate the strain activated in Step 1 into liquid culture medium and culture at 25-35℃ and 150-200 rpm for 24-36 hours until the strain is in the logarithmic growth phase.

[0012] Step 3: Fermentation and scale-up of the strain: Inoculate the culture medium obtained in Step 2 with 10% of the culture solution and carry out fermentation culture; obtain a concentration of 1×10⁻⁶. 9 ~1×10 11 CFU / mL fermentation product;

[0013] Step 4: Add 20% protectant to the fermentation product obtained in Step 4 to prepare a dry powder inoculum.

[0014] Furthermore, the solid culture medium in step one is prepared as follows: 3.0 g / L beef extract; 10.0 g / L peptone; 5.0 g / L NaCl; dissolved in deionized water, pH 7-8, sterilized at 115-121℃ for 15-30 min.

[0015] Furthermore, the preparation method of the liquid culture medium in step two is as follows: yeast extract 1-10 g / L, peptone 5-10 g / L, glucose 5-10 g / L, NH4Cl 1-10 g / L, K2HPO4 1-10 g / L, dissolved in deionized water, pH=7.0-8.0, sterilized at 115-121℃ for 15-30 min.

[0016] Furthermore, the fermentation culture medium for step three is as follows: yeast extract 1-5 g / L, peptone 5-10 g / L, glucose 5-10 g / L, NH4Cl 1-5 g / L, K2HPO4 1-5 g / L, KH2PO4 1-5 g / L, trace element solution 1-5 mL, antifoaming agent 0.2%, dissolved in distilled water, pH = 7.0-8.0, sterilized at 115-121℃ for 15-30 min.

[0017] Furthermore, the fermentation conditions in step three are as follows: tank temperature 37℃, tank pressure 0.01~0.1MPa, aeration rate 1:0.6~1.2, stirring speed 150rpm, and fermentation time 48~72h.

[0018] Furthermore, the protective agent formulation in step five is as follows: 35% yeast powder; 35% fish protein; and 30% trace elements.

[0019] Furthermore, the formula of the trace element solution is as follows: MgSO4 0.5 g / L; MnSO4 0.1 g / L; H3BO3 0.1 g / L; ZnSO4·7H2O 0.1 g / L; FeSO4·7H2O 0.3 g / L; pH = 7.

[0020] This invention also discloses the application of a highly efficient nitrobenzene-degrading bacterial agent, which is achieved through the following methods:

[0021] The nitrobenzene-degrading bacteria produced in the above steps are added at a mass ratio of 0.5–5‰, along with 0.5–1.5 g / L glucose (or 0.5 g / L glucose and 0.2 g / L nitrobenzene), tap water, and pH 7–8. The bacteria are then cultured in a culture tank at 25–35°C with aeration (DO 2–4 mg / L) for 48 hours to activate the bacterial strain. After activation, the bacterial density is measured; microscopic examination shows a density of 1 × 10⁻⁶. 9 CFU / mL indicates that the activation culture has been completed, forming a suspension of nitrobenzene-efficient degrading bacteria.

[0022] Furthermore, the activated bacterial suspension of the nitrobenzene-efficient degrading agent was added to an inorganic salt culture medium containing 1600 mg / L nitrobenzene at a mass ratio of 5-10‰. After 36 hours of aerobic degradation (25-35℃, DO 2-4 mg / L), the nitrobenzene concentration could be reduced to below 1 mg / L, and the nitrobenzene degradation rate was greater than 99.5%.

[0023] Adding a suspension of nitrobenzene-efficient degrading bacteria at a rate of 5-10‰ to a dye wastewater containing 1528 mg / L nitrobenzene, aerobic degradation (25-35℃, DO 2-4 mg / L) for 36 hours can reduce the nitrobenzene concentration to below 2 mg / L, with a nitrobenzene degradation rate greater than 99.5%.

[0024] The advantages of this invention compared to the prior art are as follows:

[0025] 1. The nitrobenzene-degrading bacterial agent provided by this invention can treat high-concentration nitrobenzene wastewater by a fully biological method, with good effect, low cost and no secondary pollution.

[0026] 2. The present invention provides a highly efficient nitrobenzene-degrading bacterial agent with strong tolerance to nitrobenzene and rapid degradation of high concentrations of nitrobenzene. When the nitrobenzene concentration is ≤1600mg / L, the nitrobenzene degradation rate is above 99.5%.

[0027] Microbial preservation for patent procedures:

[0028] Deposit date: November 10, 2022

[0029] Name of depositary institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0030] Accession number: CGMCC NO.26095

[0031] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0032] Classification and nomenclature: Lysinibacillus sp. LBQ22-1 Attached Figure Description

[0033] Figure 1 The colony morphology of Lysinibacillus sp. LBQ22-1 in Example 1 of this invention is shown.

[0034] Figure 2 This shows the degradation of nitrobenzene at different concentrations over time in Example 2 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following examples provide a more detailed description of the invention. It should be noted that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0036] The main component of the nitrobenzene-efficient degrading bacterial agent of this invention is Lysinibacillus sp. LBQ22-1, and the effective viable count of the microbial preparation is ≥10. 10 The preparation method of the CFU / g nitrobenzene-efficient degrading bacterial agent includes the following sequence and steps:

[0037] Step 1: Solid plate culture: Transfer Lysinibacillus sp. LBQ22-1 onto a solid culture medium and incubate at 25-35℃ for 36 hours to activate the strain;

[0038] Step 2, Seed culture: Inoculate the strain activated in Step 1 into liquid culture medium and culture at 25-35℃ and 150-200 rpm for 24-36 hours until the strain is in the logarithmic growth phase.

[0039] Step 3: Fermentation and scale-up of the strain: Inoculate the culture medium obtained in Step 2 with 10% of the culture solution and carry out fermentation culture; obtain a concentration of 1×10⁻⁶. 9 ~1×10 11 CFU / mL fermentation product;

[0040] Step 4: Add 20% protectant to the fermentation product obtained in Step 4 to prepare a dry powder inoculum. The protectant formula is: yeast powder 35%; fish protein 35%; trace elements 30%. The trace element solution formula is: MgSO4 0.5g / L; MnSO4 0.1g / L; H3BO3 0.1g / L; ZnSO4·7H2O 0.1g / L;

[0041] FeSO4·7H2O 0.3g / L; pH=7.

[0042] An application of a highly efficient nitrobenzene-degrading bacterial agent, characterized in that the treatment method involves wastewater sequentially undergoing activation and cultivation with the highly efficient nitrobenzene-degrading bacterial agent → wastewater treatment. The specific implementation scheme is as follows:

[0043] Add 0.3-1‰ of nitrobenzene-efficient degrading bacterial agent, 0.5-1.5 g / L glucose, tap water, pH 7-8, and culture in a bacterial culture tank at 25-35℃ with aeration (DO 2-4 mg / L) for 48 h to activate the bacterial strain. Further, after activation, measure the bacterial density; if it reaches 1×10⁻⁶, microscopic examination reveals a density of 1×10⁻⁶. 9 CFU / mL indicates that the activation culture has been completed, forming a suspension of nitrobenzene-efficient degrading bacteria.

[0044] Adjust the wastewater to pH 7-8.5, and ensure the influent nitrobenzene concentration is less than 500 mg / L (or COD less than 1500 mg / L, with COD taking priority). You can add 500 mg / L COD equivalent carbon source.

[0045] The implementation method of using nitrobenzene high-efficiency degrading bacteria and polyurethane packing is as follows: The activated bacterial suspension of the nitrobenzene high-efficiency degrading bacteria is added to a reaction tank containing 70% polyurethane packing in 2-3 batches at a mass ratio of 10%. The residence time is controlled at 24-72 hours. Samples are taken to measure indicators such as COD and nitrobenzene index. After the nitrobenzene degradation rate is greater than 90%, the removal effect is stable for 5-10 days. The concentration of nitrobenzene in the influent is gradually increased in a nitrobenzene concentration gradient of 200-500 mg / L. The water exchange volume is 50-80% for each batch until the influent load is the target influent nitrobenzene concentration.

[0046] The treatment of nitrobenzene wastewater using the highly efficient nitrobenzene-degrading bacterial agent provided by this invention allows for the selection of a suitable biochemical treatment process based on the nitrobenzene concentration in the wastewater and the actual site conditions: for nitrobenzene concentrations ≤1500 mg / L, an aerobic biochemical process can be used; for nitrobenzene concentrations >1500 mg / L, an A / O process can be used. Both the aerobic and anaerobic reaction tanks are inoculated with the highly efficient nitrobenzene-degrading bacterial agent and 70% polyurethane packing material. The hydraulic retention time (HRT) is: 24 h for influent nitrobenzene concentrations of 200-1000 mg / L; and 48 h for influent nitrobenzene concentrations of 1000-1600 mg / L.

[0047] The following are specific examples illustrating the application of the aforementioned nitrobenzene-efficient degrading bacterial agent:

[0048] Example 1

[0049] Strain acquisition and identification. The steps for obtaining and identifying strains are as follows:

[0050] Step 1: Sample Collection and Strain Acclimation

[0051] Activated sludge from the biological treatment pond of a dye production company in Jiangsu Province was added to an inorganic salt culture medium containing 200 mg / L nitrobenzene as the sole carbon and nitrogen source. The inoculum size of the activated sludge was 10% (V / V). The culture was acclimated and cultured for 5 days at 30℃ and 150 r / min in a constant temperature shaker to obtain acclimation culture solution 1. After the culture was completed, acclimation culture solution 1 was inoculated into an inorganic salt culture medium containing 200 mg / L nitrobenzene. The inoculum size of acclimation culture solution 1 was 10% (V / V). The culture was then cultured for 5 days to obtain acclimation culture solution 2. The above acclimation and culture process was repeated continuously until the nitrobenzene degradation rate was greater than 90%, thus obtaining the nitrobenzene-degrading bacteria acclimation culture solution.

[0052] The inorganic salt culture medium formula is as follows: KH2PO4 0.5g / L, MgSO4·7H2O 0.2g / L, FeSO4·7H2O 0.02g / L, MnSO4·H2O 0.02g / L, CaCl2·6H2O 0.01g / L, pH 7.0.

[0053] Step 2: Strains Isolation and Purification

[0054] Take 1 mL of the nitrobenzene-degrading bacteria acclimatization culture and dilute it with autoclaved deionized water to a concentration of 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 The bacterial suspensions at a concentration of 1:1 were spread onto inorganic salt solid medium containing 500 mg / L nitrobenzene and incubated at 30°C for 2-3 days. After single colonies grew on the medium, the single colonies with the largest number and the same morphology were picked and purified by streak plating three times to obtain the target strain LBQ22-1, which was then stored at 4°C.

[0055] Step 3: Identification of strain LBQ22-1

[0056] The strain LBQ22-1 was inoculated onto beef extract peptone agar and incubated at 30°C for 48 hours. The colonies of strain LBQ22-1 were observed to be opaque, pale yellow, and exhibited the following morphology: [details omitted]. Figure 1 After Gram staining, it appears as a Gram-positive rod-shaped organism under a microscope. The 16S rDNA of strain LBQ22-1 was sequenced by PCR, and the 16S rDNA gene sequence of strain LBQ22-1 is shown in the sequence listing. BLAST comparison analysis using the NBCI gene library showed that strain LBQ22-1 had 99% homology with Lysinibacillus sp. strain in the database. Combined with physiological and biochemical identification of strain LBQ22-1, it was determined that strain LBQ22-1 belongs to the Lysinibacillus genus and was named Lysinibacillus sp. LBQ22-1.

[0057] The beef extract peptone solid medium is made by adding 20 g / L agar to the beef extract peptone medium; the beef extract peptone medium is made by adding 3 g of beef extract, 10 g of peptone, 5 g of NaCl, 1 L of distilled water, adjusting the pH to 7.5 ± 0.2, and autoclaving at 121 °C for 20 min.

[0058] Step 4: Preservation of strain Lysinibacillus sp. LBQ22-1

[0059] The strain Lysinibacillus sp. LBQ22-1 was deposited at the China General Microbiological Culture Collection Center on November 10, 2022, with accession number CGMCC NO.26095.

[0060] Example 2

[0061] The nitrobenzene-degrading bacterial agent provided by this invention was inoculated at a mass ratio of 1‰ into an inorganic salt culture medium containing 200 mg / L nitrobenzene and 1 g / L glucose to activate the bacterial strain. After inoculation, the culture was placed in a shaker at 30℃ and 150 r / min for 24–48 h until the bacterial density reached 1 × 10⁻⁶. 9 CFU / mL, forming a suspension of nitrobenzene-efficient degrading bacterial agent.

[0062] The aforementioned nitrobenzene-efficient degrading bacterial agent activation suspension was inoculated at a 5% (v / v) inoculation rate into inorganic salt culture media containing nitrobenzene concentrations of 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L, 1200 mg / L, 1400 mg / L, and 1600 mg / L, respectively. The media were incubated at 37°C in a shaker, and samples were taken at different time points to determine the residual nitrobenzene content. The results are as follows: Figure 2As shown, at concentrations of 200–600 mg / L nitrobenzene, it can be degraded to below 1 mg / L in 12 hours; at concentrations of 800–1200 mg / L nitrobenzene, it can be degraded to below 1 mg / L in 24 hours; and at concentrations of 1400–1600 mg / L nitrobenzene, it can be degraded to below 1 mg / L in 36 hours. Specific results are illustrated in the figure below. Figure 1 As shown.

[0063] In some embodiments, the inorganic salt culture medium may include any one or a combination of (NH4)2SO4, NH4Cl, K2HPO4, KH2PO4, NaCl, Na2SO4, MgSO4·7H2O, MnSO4·H2O, and FeSO4·7H2O. In Example 2, the inorganic salt culture medium formulation is: NH4Cl 0.3 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.02 g / L, MnSO4·H2O 0.02 g / L, CaCl2·6H2O 0.01 g / L, pH 7.0.

[0064] Example 3

[0065] The nitrobenzene-degrading high-efficiency bacterial agent provided by this invention was added to wastewater containing nitrobenzene. The wastewater was taken from a dye production company in Jiangsu Province. The raw wastewater had a COD of 4328 mg / L and a nitrobenzene concentration of 1529 mg / L. The initial influent nitrobenzene concentration was 400 mg / L. The pH was adjusted to 7.5, and 70% polyurethane packing was added. The nitrobenzene-degrading high-efficiency bacterial agent with a mass ratio of 1‰ was added. The wastewater was aerobically aerated at 30℃ (DO 2-4 mg / L). Samples were taken to measure indicators such as COD and nitrobenzene. The batch water exchange rate was 80%, and the HRT was 24-48 h. After the nitrobenzene degradation rate was greater than 90%, the removal effect was stable for 4-6 days. The influent nitrobenzene concentration was gradually increased from 400 mg / L to 1529 mg / L of the raw water. After three batches of treatment with 1529 mg / L of raw water nitrobenzene stabilized, 95% of the water was exchanged. Samples were taken at different time points after the water exchange to measure the remaining nitrobenzene content. The results showed that the nitrobenzene concentration in the raw water was 1529 mg / L, and the concentration could be reduced to below 2 mg / L after 36 hours, with a removal rate of over 99.5%. The COD in the effluent was reduced to 180-200 mg / L, with a COD removal rate of approximately 95%.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A Lysinibacillus sp. LBQ22-1, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC NO.26095, and the deposit date is November 10, 2022.

2. A method for preparing a highly efficient nitrobenzene-degrading bacterial agent, characterized in that, The method is as follows: the nitrobenzene-efficient degrading bacterial agent contains Lysinibacillus sp. LBQ22-1, and the preparation method is as follows: Step 1: Solid plate culture: Transfer Lysinibacillus sp. LBQ22-1 onto a solid culture medium and incubate at 25-35℃ for 36 hours to activate the strain; Step 2, Seed culture: Inoculate the strain activated in Step 1 into liquid culture medium and culture at 25-35℃ and 150-200 rpm for 24-36 hours until the strain is in the logarithmic growth phase. Step 3: Fermentation and scale-up of the strain: Inoculate the culture medium obtained in Step 2 with 10% of the culture solution and carry out fermentation culture; obtain a concentration of 1×10 9 ~1×10 11 CFU / mL fermentation product; Step 4: Add 20% protectant to the fermentation product obtained in Step 4 to prepare a dry powder inoculum.

3. The preparation method of the nitrobenzene high-efficiency degrading bacterial agent according to claim 1, characterized in that, The solid culture medium in step one is prepared as follows: 3.0 g / L beef extract; 10.0 g / L peptone; 5.0 g / L NaCl; dissolved in deionized water, pH 7-8, sterilized at 115-121℃ for 15-30 min.

4. The method for preparing the nitrobenzene-efficient degrading bacterial agent according to claim 1, characterized in that, The preparation method of the liquid culture medium in step two is as follows: yeast extract 1-10 g / L, peptone 5-10 g / L, glucose 5-10 g / L, NH4Cl 1-10 g / L, K2HPO4 1-10 g / L, dissolved in deionized water, pH=7.0-8.0, sterilized at 115-121℃ for 15-30 min.

5. The method for preparing the nitrobenzene-efficient degrading bacterial agent according to claim 1, characterized in that, The fermentation culture medium for step three is as follows: yeast extract 1-5 g / L, peptone 5-10 g / L, glucose 5-10 g / L, NH4Cl 1-5 g / L, K2HPO4 1-5 g / L, KH2PO4 1-5 g / L, trace element solution 1-5 mL, antifoaming agent 0.2%, dissolved in distilled water, pH = 7.0-8.0, sterilized at 115-121℃ for 15-30 min; the formula of the trace element solution is: MgSO4 0.5 g / L; MnSO4 0.1 g / L; H3BO3 0.1 g / L; ZnSO4·7H2O 0.1 g / L; FeSO4·7H2O 0.3 g / L; pH = 7; The fermentation conditions in step three are as follows: tank temperature 37℃, tank pressure 0.01~0.1MPa, aeration rate 1:0.6~1.2, stirring speed 150rpm, and fermentation time 48~72h.

6. The method for preparing the nitrobenzene-efficient degrading bacterial agent according to claim 1, characterized in that, The protective agent formulation in step five is as follows: yeast powder 35%; fish peptone 35%; trace elements 30%; the trace element solution formulation is as follows: MgSO4 0.5g / L; MnSO4 0.1g / L; H3BO3 0.1g / L; ZnSO4·7H2O 0.1g / L; FeSO4·7H2O 0.3g / L; pH=7.

7. A highly efficient nitrobenzene-degrading bacterial agent prepared by the preparation method according to any one of claims 1 to 6.

8. The nitrobenzene high-efficiency degrading bacterial agent according to claim 7, characterized in that, The number of effective live bacteria in the microbial preparation of the active microorganisms is ≥10. 10 CFU / g.

9. An application of the nitrobenzene-degrading bacterial agent as described in claim 7 in chemical wastewater, wherein the nitrobenzene-degrading bacterial agent is added at a ratio of 0.5-5‰ to an inorganic salt culture medium containing 200-1600 mg / L nitrobenzene; at a nitrobenzene concentration of 200-600 mg / L, it can be degraded to below 1 mg / L in 12 hours; at a nitrobenzene concentration of 800-1200 mg / L, it can be degraded to below 1 mg / L in 24 hours; at a nitrobenzene concentration of 1400-1600 mg / L, it can be degraded to below 1 mg / L in 36 hours; when the nitrobenzene-degrading bacterial agent is added at a ratio of 0.5-5‰ to a dye production wastewater containing 1529 mg / L nitrobenzene, it can degrade nitrobenzene to below 2 mg / L in 48 hours, with a nitrobenzene removal rate exceeding 99.5%.

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