Degradation bacteria for alkane and polycyclic aromatic hydrocarbon in high-salinity water body and application thereof

Abstract

The application provides a degrading bacteria agent for alkane and polycyclic aromatic hydrocarbon in high-salt water body and application thereof. The bacteria agent contains Bacillus thuringiensis NH26 strain, and is prepared by activating, fermentation culture, bacteria body collection, suspension preparation and freeze-drying of the NH26 strain. The Bacillus thuringiensis NH26 bacteria agent prepared by the application has excellent degradation capacity for alkane and polycyclic aromatic hydrocarbon, for example, the total degradation rate of 20 g / L diesel oil is 90.2%, and the total degradation rate of 50 g / L diesel oil is 84.8%. In addition, the degrading bacteria agent of the application also has strong degradation capacity for polycyclic aromatic hydrocarbon in seawater, for example, the removal rate of 50 mg / L phenanthrene in seawater is 68.56%, and the removal rate of 100 mg / L phenanthrene is 63.47%, so that the bacteria agent of the application can be applied to repair high-salt water body polluted by alkane and polycyclic aromatic hydrocarbon.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a degrading microbial agent for alkane and polycyclic aromatic hydrocarbon in high-salinity water and application thereof. BACKGROUND

[0002] Alkane and polycyclic aromatic hydrocarbon are common toxic and harmful and difficult-to-degrade organic pollutants, which seriously endanger human health and ecological environment health. The incomplete combustion of fossil fuels such as gasoline, coal and petroleum, petroleum leakage, farmland sewage irrigation, atmospheric deposition, etc. are all main sources thereof.

[0003] The existing degradation and remediation methods for alkane and polycyclic aromatic hydrocarbon are mostly physical and chemical methods, such as isolation method, chemical oxidation method, photocatalysis method, thermodynamic remediation method, etc., which can treat alkane and polycyclic aromatic hydrocarbon to achieve remediation effect. However, these existing methods have limited effect on one hand, and high cost and easy to cause secondary pollution on the other hand, which limits the application.

[0004] Microbial remediation method shows more and more advantages. For example, the invention patent "Alkane and polycyclic aromatic hydrocarbon degrading composite microbial agent and preparation method thereof" provides a composite microbial agent prepared from Gordonia amicalis and Pseudomonas stutzeri, which can degrade alkane and polycyclic aromatic hydrocarbon. For another example, Sun W et al. (2019) provides a Bacillus thuringiensis CQ8-1 strain with degradation function for petroleum hydrocarbon in freshwater [Sun W et al., 2019. Isolation, identification, and characterization of diesel-oil-degrading bacterial strains indigenous to Changqing oil field, China. Journal of Basic Microbiology, 59: 723-734], but the highest degradation rate of the strain for petroleum hydrocarbon is only 66%, and the strain can only survive in freshwater conditions and cannot be applied to the removal of high-salinity water pollution.

[0005] In addition, the activity of microbial strains is difficult to maintain for a long time, and even degradation may occur. Therefore, it is of great significance to explore more microbial strains, especially microbial strains capable of degrading alkane and polycyclic aromatic hydrocarbon in high-salinity water, and to explore the production process of microbial agent products, for the remediation of high-salinity water polluted by alkane and polycyclic aromatic hydrocarbon. SUMMARY

[0006] The present application aims at the insufficient remediation technology of pollutants such as alkane and / or polycyclic aromatic hydrocarbon in the prior art, and aims to provide a degradation bacterium agent of alkane and polycyclic aromatic hydrocarbon in high-salinity water and application thereof, so as to realize microbial remediation of the environment such as marine oil pollution.

[0007] The primary object of the present application is to provide a Bacillus thuringiensis NH26 bacterium agent.

[0008] Another object of the present application is to provide the application of the above-mentioned Bacillus thuringiensis NH26 bacterium agent in degrading alkane and / or polycyclic aromatic hydrocarbon, or in removing alkane and / or polycyclic aromatic hydrocarbon pollution, or in remediation of the environment polluted by alkane and / or polycyclic aromatic hydrocarbon.

[0009] Another object of the present application is to provide the application of the above-mentioned bacterium agent in degrading pollutants rich in alkane and / or polycyclic aromatic hydrocarbon, or in removing pollutants rich in alkane and / or polycyclic aromatic hydrocarbon, or in remediation of the environment polluted by pollutants rich in alkane and / or polycyclic aromatic hydrocarbon.

[0010] The present application realizes the above-mentioned application objects through the following technical solutions:

[0011] The present application establishes a preparation method of the Bacillus thuringiensis NH26 bacterium agent, and optimizes the preparation process of the Bacillus thuringiensis NH26 bacterium agent.

[0012] The degradation rate of the prepared Bacillus thuringiensis NH26 bacterium agent to various components of n-dodecane to n-heptacosane in refined diesel oil with a concentration of 20 g / L in natural seawater is all above 88.4%, and the total degradation rate is 90.2%; the degradation rate of the prepared Bacillus thuringiensis NH26 bacterium agent to various components of n-dodecane to n-heptacosane in refined diesel oil with a concentration of 50 g / L in natural seawater is all above 88%, and the total degradation rate is 84.8%; the removal rates of anthracene, pyrene and phenanthrene with a concentration of 50 mg / L in natural seawater are 43.54%, 44.82% and 68.56% respectively; the removal rates of anthracene, pyrene and phenanthrene with a concentration of 100 mg / L in natural seawater are 39.02%, 41.69% and 63.47% respectively.

[0013] Therefore, the following technical solutions should be within the protection scope of the present application:

[0014] The present application provides a Bacillus thuringiensis NH26 bacterium agent, which comprises a Bacillus thuringiensis NH26 (Bacillus thuringiensis NH26) strain; the Bacillus thuringiensis NH26 strain is preserved in the China Center for Type Culture Collection (CCTCC) on July 21, 2022, with a preservation number of CCTCC NO: M20221149 and a preservation address of China. Wuhan. Wuhan University.

[0015] Preferably, the degradation bacteria agent is prepared by fermentation of a fermentation bacteria comprising Bacillus thuringiensis NH26 strain.

[0016] Preferably, the fermentation preparation method is: Bacillus thuringiensis NH26 strain is activated, fermented, cultured, collected, suspended, and freeze-dried.

[0017] Preferably, the activation time is 6-10 hours. Most preferably, 8 hours.

[0018] Preferably, the activation temperature is 25-35℃. Most preferably, 30℃.

[0019] Preferably, the activation is carried out at a rotation speed of 150-210 rpm. Most preferably, 180 rpm.

[0020] Preferably, the culture medium used in the activation comprises glucose 15-25 g / L, yeast extract 4-15 g / L, ammonium sulfate 2-8 g / L, KH2PO4 1-5 g / L, MgSO4·7H2O 0.1-1.0 g / L, and NaCl 0.1-1.0 g / L. Most preferably, glucose 20 g / L, yeast extract 10 g / L, ammonium sulfate 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, and NaCl 0.5 g / L.

[0021] Preferably, the pH value of the culture medium used in the activation is adjusted to 6.5-7.5. Most preferably, the pH value is adjusted to 7.0.

[0022] Preferably, the culture medium used in the fermentation culture comprises soluble starch 37.5-62.5 g / L, yeast extract 2.5-7.5 g / L, MnSO4·H2O 2-8 mg / L, FeSO4·7H2O 0.2-0.8 mg / L, NaCl 1.0-3.0 g / L, and sodium glutamate 5-15 g / L. Most preferably, soluble starch 50 g / L, yeast extract 5 g / L, MnSO4·H2O 5 mg / L, FeSO4·7H2O 0.5 mg / L, NaCl 2 g / L, and sodium glutamate 10 g / L.

[0023] Preferably, the pH value of the culture medium used in the fermentation culture is adjusted to 6.5-7.5. Most preferably, the pH value is adjusted to 7.0.

[0024] Preferably, the fermentation culture temperature is 25-35℃. Most preferably, 30℃.

[0025] Preferably, the rotation speed of the fermentation culture is 150-180 rpm. Most preferably, 160 rpm.

[0026] Preferably, the inoculation amount of the fermentation culture is 1-15%. Most preferably, it is 5%.

[0027] Preferably, the time of the fermentation culture is 14-18 hours. Most preferably, it is 16 hours.

[0028] Preferably, the method of the bacteria collection is centrifugation, washing, and centrifugation again.

[0029] More preferably, the centrifugation condition is 3000-8000 x g, 0-10 °C for 15-25 minutes. Most preferably, it is 5000 x g, 4 °C for 10 minutes.

[0030] More preferably, the washing is with normal saline.

[0031] More preferably, the washing is with sterile normal saline.

[0032] More preferably, the washing is 2-4 times. Most preferably, it is 3 times.

[0033] Preferably, the suspension preparation is mixing the collected bacteria with a protective agent solution.

[0034] More preferably, the protective agent solution comprises the following mass percentage components: skimmed milk powder 10-20%, sucrose 15-25%, polyvinylpyrrolidone-K30 5-9%, and glutathione 1.0-2.0%. Most preferably, it is skimmed milk powder 15%, sucrose 20%, polyvinylpyrrolidone-K30 7%, and glutathione 1.3%.

[0035] More preferably, the pH value of the protective agent solution is 6.5-7.5. Most preferably, it is 7.0.

[0036] More preferably, the amount of the compound protective agent added during the suspension preparation is 1-2 times the volume of the fermentation liquid. Most preferably, it is 1.5 times.

[0037] Preferably, the pre-freezing condition during the freeze-drying is -196 to -60 °C for 0.20-2 hours. Most preferably, it is -196 °C for 15 minutes.

[0038] Preferably, the freeze-drying thickness during the freeze-drying is 0.4-0.6 cm. Most preferably, it is 0.5 cm.

[0039] Preferably, the cold trap temperature during the freeze-drying is -80 to -40 °C. Most preferably, it is -60 °C.

[0040] Preferably, the vacuum degree during the freeze-drying is 0.1-0.2 Mbar. Most preferably, it is 0.15 Mbar.

[0041] Preferably, the freeze-drying time is 24 to 48 hours. Most preferably, it is 36 hours.

[0042] The present invention also provides the application of the described degrading microbial agent in the degradation of alkanes and / or polycyclic aromatic hydrocarbons, or in the removal of alkane and / or polycyclic aromatic hydrocarbon contamination, or in the remediation of environments contaminated by alkane and / or polycyclic aromatic hydrocarbons.

[0043] The present invention also provides the application of the described degrading microbial agent in degrading pollutants rich in alkanes and / or polycyclic aromatic hydrocarbons, or in removing pollutants rich in alkanes and / or polycyclic aromatic hydrocarbons, or in remediating environments contaminated by pollutants rich in alkanes and / or polycyclic aromatic hydrocarbons.

[0044] More preferably, the alkane includes, but is not limited to, n-dodecane to n-heptadecane.

[0045] More preferably, the polycyclic aromatic hydrocarbons include, but are not limited to, anthracene, phenanthrene, and pyrene.

[0046] The technical solution of the present invention has the following beneficial effects:

[0047] This invention provides a Bacillus thuringiensis NH26 bacterial agent and specifies the optimal conditions for its preparation. The Bacillus thuringiensis NH26 bacterial agent prepared by this invention exhibits excellent degradation capabilities for alkanes and polycyclic aromatic hydrocarbons (PAHs), thus demonstrating excellent removal effects on pollutants rich in alkanes and PAHs. For example, it exhibits excellent degradation capabilities for petroleum hydrocarbons in seawater. When the petroleum hydrocarbon concentration is 20 g / L, the degradation rate for various petroleum hydrocarbon molecules reaches over 88.4%, with a total degradation rate of 90.2%; when the petroleum hydrocarbon concentration is 50 g / L, the degradation rate for various petroleum hydrocarbon molecules reaches over 88%, with a total degradation rate of 84.8%. Furthermore, the Bacillus thuringiensis NH26 bacterial agent of this invention also exhibits strong degradation capabilities for PAHs in seawater. For example, its removal rate for phenanthrene in seawater is 68.56% at a concentration of 50 mg / L and 63.47% at a concentration of 100 mg / L. Attached Figure Description

[0048] Figure 1 The growth curve of Bacillus thuringiensis NH26 strain before condition optimization.

[0049] Figure 2 The fermentation curve of Bacillus thuringiensis NH26 strain before condition optimization is shown.

[0050] Figure 3 The fermentation curve of a 200 mL system of Bacillus thuringiensis NH26 strain after optimization is shown.

[0051] Figure 4To optimize the fermentation curve of Bacillus thuringiensis NH26 strain 5L system after conditions.

[0052] Figure 5 To optimize the fermentation curve of Bacillus thuringiensis NH26 strain 30L system after conditions. DETAILED DESCRIPTION

[0053] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0054] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0055] The Bacillus thuringiensis NH26 used in the present application is Bacillus thuringiensis NH26 strain, which is isolated from the surface soil / sediment of coastal wetlands contaminated by crude oil in Huizhou, Guangdong, and is preserved in China Center for Type Culture Collection (CCTCC) on July 21, 2022, with the preservation number CCTCC NO: M20221149 and the preservation address China. Wuhan. Wuhan University.

[0056] All the media in the following examples are sterilized at 121°C for 20 minutes before use.

[0057] Example 1 Preparation of Bacillus thuringiensis NH26 inoculant Activation, fermentation process parameter optimization

[0058] In this example, the activation and fermentation process of Bacillus thuringiensis NH26 inoculant preparation are optimized.

[0059] I. Activation condition optimization

[0060] (1) Activation time optimization (growth curve of Bacillus thuringiensis NH26 strain)

[0061] Bacillus thuringiensis NH26 strain cultured in Luria-Bertani (LB) agar medium (its components are: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L and agar 20 g / L) was inoculated into seed culture medium (its composition is: glucose 20 g / L, yeast extract 10 g / L, ammonium sulfate 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L and NaCl 0.5 g / L; adjust the pH value to 7.0, sterilize at 121°C for 20 minutes). Take 3 parallel samples and cultivate at 30°C, 180 rpm. Measure the absorbance OD of the culture at wavelength 610 nm every 2 hours with a microplate reader.610 OD over time 610 The curve is the growth curve of Bacillus thuringiensis NH26 strain, and the activation time of Bacillus thuringiensis NH26 strain is determined according to the growth curve.

[0062] The growth curve determination results are shown in Figure 1 From Figure 1 the results, it can be seen that the logarithmic growth phase of Bacillus thuringiensis NH26 strain is 2-10 hours, and then enters the stable growth phase, and the cells begin to decline after 34 hours, reflecting that Bacillus thuringiensis NH26 strain is easy to cultivate and grows fast in the seed culture medium.

[0063] According to the experimental results, 6-10 hours (optimum 8 hours) are selected as the cultivation time of Bacillus thuringiensis NH26 strain activated in the seed culture medium.

[0064] II. Optimization of fermentation conditions

[0065] 1. Fermentation curve of Bacillus thuringiensis NH26 strain before optimization of fermentation conditions

[0066] The seed culture solution of Bacillus thuringiensis NH26 strain (cultured in the seed culture medium for 8 hours) was transferred to 200 mL liquid fermentation medium (its composition is: soybean oil 20 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, (NH4)2SO4 5 g / L, NaCl 0.5 g / L, FeSO4·7H2O 1.5 mg / L, MnSO4·H2O 0.005 g / L and sodium glutamate 5 g / L; the pH value was adjusted to 7.0, and sterilized at 121°C for 20 minutes) at a 10% inoculation amount, and cultured at 30°C and 180 rpm for 4 days. During the fermentation, samples were taken at regular intervals, and after centrifugation of the fermentation broth sample, the dry weight of the precipitated cells was measured after vacuum freeze-drying to obtain the dry weight of biomass of Bacillus thuringiensis NH26 strain. The dry weight of biomass, surface tension (measured by a surface tension meter) and pH value (measured by an acidity meter) of the above obtained Bacillus thuringiensis NH26 strain fermentation broth were plotted against time to obtain the fermentation curve of Bacillus thuringiensis NH26 strain. From the figure, the growth of Bacillus thuringiensis NH26 strain and the yield change of surfactant during the fermentation process can be seen, and the fermentation time of the strain is determined according to the fermentation curve.

[0067] The fermentation curve determination results are shown in Figure 2 From Figure 2It is found that the biomass of Bacillus thuringiensis NH26 strain reaches the maximum after 12 hours of fermentation, and tends to be stable after 40 hours, reflecting that Bacillus thuringiensis NH26 strain is easy to culture and grows fast in the liquid fermentation medium. The cell enters the stable phase, and great changes occur in its structure and physiology, including the expression level of stress proteins, membrane composition and cell wall structure, so as to increase the resistance of the cell. Therefore, 40 hours is the optimal fermentation time of the bacterial agent before the optimization of fermentation conditions.

[0068] 2. Optimization of fermentation medium

[0069] (1) Effect of carbon source on Bacillus thuringiensis NH26 strain

[0070] Carbon source and nitrogen source are the most important nutritional components for the growth of Bacillus thuringiensis NH26 strain.

[0071] On the basis of the original liquid fermentation medium formula, the effect of carbon source on Bacillus thuringiensis NH26 strain is studied by taking carbon source as a single variable. 20 g / L of glucose, soluble starch, soybean oil, coconut oil, palm oil or camellia oil is used as the only carbon source for the fermentation medium of Bacillus thuringiensis NH26 strain, and Bacillus thuringiensis NH26 strain is cultured at 30°C and 180 rpm. The surface tension, emulsifying rate and biomass of the fermentation broth before and after culture are used as indexes to comprehensively evaluate the carbon source utilization rate of Bacillus thuringiensis NH26 strain. The results show that the order of carbon source utilization of Bacillus thuringiensis NH26 strain is: soluble starch > glucose > palm oil > soybean oil > camellia oil > coconut oil. Soluble starch is the best carbon source.

[0072] (2) Effect of nitrogen source on Bacillus thuringiensis NH26 strain

[0073] On the basis of the original liquid fermentation medium formula, soluble starch is selected as the best carbon source, and 5 g / L of yeast extract, (NH4)2SO4, NH4NO3 or NH4Cl is used as the only nitrogen source for the liquid fermentation medium. Bacillus thuringiensis NH26 strain is cultured at 30°C and 180 rpm. The surface tension, emulsifying rate and biomass of the fermentation broth before and after culture are used as indexes to comprehensively evaluate the nitrogen source utilization rate of Bacillus thuringiensis NH26 strain. The results show that the order of nitrogen source utilization of Bacillus thuringiensis NH26 strain is: yeast extract > NH4Cl > (NH4)2SO4 > NH4NO3. Yeast extract is the best nitrogen source.

[0074] (3) Effect of mass ratio of carbon source to nitrogen source on Bacillus thuringiensis NH26 strain

[0075] After the best carbon source and nitrogen source were obtained, liquid fermentation medium was prepared with the mass ratio of carbon source to nitrogen source being 20:1, 20:2, 20:3, 20:4, 20:5, and 20:10. The surface tension, emulsification rate and biomass of the fermentation broth before and after cultivation were used as indexes to comprehensively evaluate the influence of the mass ratio of carbon source to nitrogen source on the strain NH26. The results showed that the mass ratio of carbon source to nitrogen source was preferably 20:1-3, and the optimal mass ratio of carbon source to nitrogen source for the strain NH26 was 20:2.

[0076] (4) Influence of trace elements iron and manganese on Bacillus thuringiensis NH26 strain

[0077] Liquid fermentation medium was prepared with the optimal carbon source, nitrogen source and mass ratio of carbon source to nitrogen source, and the surface tension, emulsification rate and biomass of the fermentation broth before and after cultivation were used as indexes to comprehensively evaluate the influence of the concentration of trace elements iron and manganese on the Bacillus thuringiensis NH26 strain. The results showed that the concentration of trace elements iron and manganese was preferably 0.2-0.8 mg / L for FeSO4·7H2O and 2-8 mg / L for MnSO4·H2O, the optimal concentration of FeSO4·7H2O was 0.5 mg / L, and the optimal concentration of MnSO4·H2O was 5 mg / L.

[0078] (5) Influence of NaCl concentration on Bacillus thuringiensis NH26 strain

[0079] Liquid fermentation medium was prepared with the optimal carbon source, nitrogen source, mass ratio of carbon source to nitrogen source and concentration of trace elements, and the surface tension, emulsification rate and biomass of the fermentation broth before and after cultivation were used as indexes to comprehensively evaluate the influence of NaCl concentration on the strain NH26. The results showed that the concentration of NaCl was preferably 1.0-3.0 g / L, and the optimal concentration of NaCl was 2.0 g / L.

[0080] (6) Influence of sodium glutamate concentration on Bacillus thuringiensis NH26 strain

[0081] The concentration of sodium glutamate was set as a single variable in the liquid fermentation medium, and six concentrations of 1, 2, 5, 10, 15 and 20 g / L were set. The surface tension, emulsification rate and biomass of the fermentation broth before and after cultivation were used as indexes to comprehensively evaluate the influence of sodium glutamate on the Bacillus thuringiensis NH26 strain. The results showed that the concentration of sodium glutamate was preferably 5-15 g / L, and the optimal concentration of sodium glutamate was 10 g / L.

[0082] In summary, the optimal formula of the fermentation medium for the Bacillus thuringiensis NH26 is as follows:

[0083] Soluble starch 37.5-62.5 g / L, yeast extract 2.5-7.5 g / L, MnSO4-H2O 2-8 mg / L, FeSO4 7H2O 0.2-0.8 mg / L, NaCl 1.0-3.0 g / L, sodium glutamate 5-15 g / L.

[0084] The optimal selection is:

[0085] Soluble starch 50 g / L, yeast extract 5 g / L, MnSO4-H2O 5 mg / L, FeSO4 7H2O 0.5 mg / L, NaCl 2.0 g / L, sodium glutamate 10 g / L.

[0086] 3. Optimization of culture conditions

[0087] Effect of culture conditions on Bacillus thuringiensis NH26 strain

[0088] The Bacillus thuringiensis NH26 strain uses the above-mentioned optimized fermentation medium formula (medium components: soluble starch 50 g / L, yeast extract 5 g / L, MnSO4-H2O 5 mg / L, FeSO4 7H2O 0.5 mg / L, NaCl 2.0 g / L, sodium glutamate 10 g / L), with biomass and surfactant yield as indicators, four culture conditions of culture temperature, rotation speed, medium pH value and inoculum size are selected to carry out single factor test, and the optimal fermentation culture conditions are comprehensively evaluated. The optimal fermentation culture conditions of Bacillus thuringiensis NH26 strain are 25-35°C, pH 6.5-7.5, 150-180 rpm and 1-15% inoculum size; the optimal fermentation culture conditions are 30°C, pH 7.0, 160 rpm and 5% inoculum size.

[0089] On the basis of the above-mentioned single factor test, four culture conditions of culture temperature, rotation speed, medium pH value and inoculum size are selected as variables (Table 1), and L9(3 4 ) four-factor three-level orthogonal experiment (Table 2) is set up, the results show that the optimal fermentation conditions of strain NH26 are 30°C, 160 rpm and pH 7.0, with 5% inoculum size for fermentation culture.

[0090] Table 1 Culture condition variables

[0091]

[0092] Table 2 L9(3 4 ) four-factor three-level orthogonal experiment table

[0093]

[0094] 4. Effect of fermentation scale on fermentation time of the strain

[0095] Under the optimized fermentation medium and fermentation conditions (inoculation amount of 5%, medium composition: soluble starch 50 g / L, yeast extract 5 g / L, MnS04H20 5 mg / L, FeS04·7H20 0.5 mg / L, NaCl 2.0 g / L, sodium glutamate 10 g / L, pH 7.0, sterilization at 121°C for 20 minutes, culture conditions: 30°C, 160 rpm), Bacillus thuringiensis NH26 strain was subjected to fermentation culture in a 200 mL system, a 5 L system and a 30 L system by using a shake flask and a fermenter with different volumes. Biomass and surface tension of the fermentation broth were used as indexes to comprehensively evaluate the influence of fermentation scale on Bacillus thuringiensis NH26 strain.

[0096] The results show that, for the 200 mL shake flask system, high biomass appears at 16-24 hours ( Figure 3 ); for the 5 L fermenter system, high biomass appears at 15-25 hours ( Figure 4 ); and for the 30 L fermentation system, high biomass appears at 12-16 hours ( Figure 5 ). Therefore, in actual work, for a fermentation scale less than 30 L, fermentation time of 16-24 hours is selected; and for a fermentation scale equal to or greater than 30 L, fermentation time of 12-16 hours is selected. Therefore, for any fermentation scale, fermentation time of 14-18 hours is selected as the preferred fermentation time (the best selection is 16 hours).

[0097] Example 2 Optimization of freeze-drying process parameters for preparing Bacillus thuringiensis NH26 microbial agent

[0098] 1. During the experiment, the determination method of viable cell count and freeze-drying survival factor of Bacillus thuringiensis NH26 is as follows:

[0099] The viable cell count of the sample before freeze-drying and the sample after rehydration of the freeze-dried microbial agent is determined by the above plate counting method, and the freeze-drying survival factor of the bacterial cells is calculated. The viable cell count before freeze-drying is the viable cell count in the bacterial liquid; and the viable cell count after freeze-drying is the viable cell count after rehydration at 30°C for 30 minutes to recover the activity of the freeze-dried microbial agent suspended in 10% sucrose solution.

[0100] The determination method of viable cell count is as follows: the sample of Bacillus thuringiensis NH26 before freeze-drying and the sample after rehydration of the freeze-dried microbial agent are diluted by 10 times in gradient, 10 -6 , 10 -7 , 10 -8 times dilution gradient is used for plate coating, 30°C culture for 16 hours, and the culture dishes with colony number between 30-300 CFU / mL are selected for counting to obtain the viable cell count of Bacillus thuringiensis NH26.

[0101] Survival factor of freeze-drying = [1-(lg viable cell number before freeze-drying-lg viable cell number after freeze-drying) / lg viable cell number before freeze-drying]

[0102] Viable cell number before freeze-drying (CFU) = bacterial concentration before freeze-drying (CFU / mL) x v

[0103] Viable cell number after freeze-drying (CFU) = bacterial concentration after freeze-drying (CFU / g) x m

[0104] Wherein v represents the volume of bacterial solution before freeze-drying (mL); m represents the mass of bacterial powder after freeze-drying (g).

[0105] 2. The optimized freeze-drying process and parameters of Bacillus thuringiensis NH26 bacterial agent are as follows:

[0106] Before the optimization of freeze-drying conditions, one of the macromolecular protectants, sugars, polymers or antioxidants is usually selected to be dissolved in distilled water, stirred uniformly, sterilized at 121°C for 15 minutes, and then a single-component freeze-drying protectant solution is prepared and stored in a 4°C refrigerator for standby.

[0107] The bacterial precipitate produced by fermentation of Bacillus thuringiensis NH26 strain is collected by centrifuging the bacterial solution at 5000 x g and 4°C for 10 minutes; the freeze-drying protectant is added to the bacterial suspension at a ratio of 1:1 (v / v); after calculating the initial viable cell number, the bacterial suspension is placed in a -80°C refrigerator for pre-freezing for 2 hours; after complete freezing, the bacterial suspension sample is placed in a cold trap with a temperature of -50°C and a vacuum degree of 0.20 Mbar for freeze-drying in a vacuum freeze-drying machine for 24 hours; finally, the freeze-dried sample is stored in a -20°C refrigerator.

[0108] 3. The following is the parameter optimization test of the freeze-drying process:

[0109] (1) Optimization of freeze-drying protectant type

[0110] Single-factor freeze-drying protectant optimization tests are carried out by selecting macromolecular materials (skimmed milk powder, yeast powder, soluble starch and malt dextrin), sugar materials (trehalose, sucrose and lactose), polymer materials (gelatin and polyvinylpyrrolidone-K30) and antioxidant materials (vitamin C and glutathione).

[0111] (a) Selection of freeze-drying protectant type

[0112] The following protectants are respectively selected and mixed with the bacterial bodies to observe the survival of the bacterial bodies:

[0113] Macromolecular protectants: skimmed milk powder, yeast powder, soluble starch and malt dextrin were selected for the test, with a concentration of 10%. Sugar protectants: trehalose, sucrose and lactose were selected for the test, with a concentration of 10%. Polymer protectants: polyvinylpyrrolidone-K30 and gelatin were selected for the test, with a concentration of 5%. Antioxidant protectants: vitamin C and glutathione were selected for the test, with a concentration of 1%.

[0114] The above protectants were dissolved in distilled water according to the concentration, stirred uniformly, sterilized at 121°C for 15 minutes, and stored in a 4°C refrigerator for standby; vitamin C and glutathione were filtered with a 0.22μm filter to remove bacteria. The survival factor of the bacterial body was used as an index to evaluate the protection effect of the freeze-drying protectant, and the type of freeze-drying protectant was optimized.

[0115] The test showed that the protection effect of skimmed milk powder was the best, significantly higher than that of yeast powder, soluble starch and malt dextrin. Therefore, skimmed milk powder was selected as the macromolecular protectant; sugar could also significantly improve the survival factor of the bacterial body, among which sucrose had the best protection effect; among the polymer protectants, polyvinylpyrrolidone-K30 had better protection effect; among the antioxidant protectants, glutathione had better protection effect.

[0116] (b) Selection of freeze-drying protectant concentration

[0117] According to the test results of (a), the survival factor of the freeze-dried bacterial powder was used as an index to select skimmed milk powder, sucrose, polyvinylpyrrolidone-K30 and glutathione as protectants, and different concentration gradients were set for the test to determine the optimal concentration of their addition. The concentration gradients of skimmed milk powder and sucrose were set as: 0%, 5%, 10%, 15%, 20%; the concentration gradients of polyvinylpyrrolidone-K30 were set as: 0%, 1%, 3%, 5%, 7%, 9%; the concentration gradients of glutathione were set as: 0%, 0.4%, 0.7%, 1.0%, 1.3%, 1.6%.

[0118] The single-factor test comprehensive evaluation showed that the concentration of skimmed milk powder was preferably 10-20%, and the best was 15%; the concentration of sucrose was preferably 15-25%, and the best was 15%; the concentration of polyvinylpyrrolidone-K30 was preferably 5-9%, and the best was 7%; the concentration of glutathione was preferably 1.0-2.0, and the best was 1.0%.

[0119] (c) Optimization of the mass percentage concentration of the composite freeze-drying protectant

[0120] Based on the above single-factor test results, the survival factor of the bacterial strain was used as an index to carry out orthogonal test of the composite freeze-drying protectant. Through the orthogonal test L9(34) 4The optimal freeze-drying protectant formulation was selected. The factor levels for the orthogonal experiment are shown in Table 3. The orthogonal experiment for the freeze-drying protectant is shown in Table 4.

[0121] The results showed that the best protective agent combination was: 15% skim milk powder, 20% sucrose, 7% polyvinylpyrrolidone-K30, and 1.3% glutathione.

[0122] Table 3. Orthogonal factor levels of lyophilization protectants

[0123]

[0124] Table 4. Results of the orthogonal experiment on freeze-drying protectants

[0125] Number A B C D 1 1 1 1 1 2 1 2 2 2 3 1 3 3 3 4 2 1 2 3 5 2 2 3 1 6 2 3 1 2 7 3 1 3 2 8 3 2 1 3 9 3 3 2 1

[0126] (3) pH optimization of freeze-drying protectant

[0127] The selected composite protectants were adjusted to pH values ​​of 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 respectively using sterilized acetic acid or ammonia. After equilibration with the bacterial cells at 30°C for 20 minutes, they were freeze-dried under vacuum, and the survival factor of the bacterial cells was calculated. Optimization experiments showed that the preferred pH value was 6.5–7.5, with 7.0 being the optimal value.

[0128] (4) Determination of the ratio of bacterial culture to protectant

[0129] Using the bacterial survival factor as an indicator, the optimal ratio of bacterial culture to protectant was evaluated. Freeze-drying experiments were conducted at bacterial culture / protectant ratios of 1:1, 1:1.5, 1:2, 1:2.5, and 1:3 (v / v). The results showed that the preferred ratio of bacterial culture to protectant was 1:1 to 2, with 1:1.5 being the optimal ratio.

[0130] (5) Optimal freeze-drying thickness

[0131] After the bacterial cells and the cryoprotectant were shaken to mix thoroughly, they were loaded into glass petri dishes at different thicknesses (0.3, 0.4, 0.5, 0.6, and 0.7 cm) and freeze-dried in a vacuum freeze dryer. The optimal freeze-drying thickness was evaluated using the bacterial cell survival factor as an indicator. Optimization experiments showed that the preferred freeze-drying thickness was 0.4–0.6 cm, with 0.5 cm being the optimal value.

[0132] (6) Pre-freezing method optimization

[0133] The bacterial cells were frozen at -20℃ for 2 hours, -80℃ for 2 hours, and -196℃ (liquid nitrogen) for 15 minutes, respectively, and then freeze-dried under vacuum for 24 hours. The survival factor of the bacterial cells was then measured. The experimental results showed that the preferred pre-freezing method was freezing at -196℃ to -80℃ for 0.20 to 2 hours, with the optimal method being freezing at -196℃ (liquid nitrogen) for 15 minutes.

[0134] In summary, the optimized preparation method of Bacillus thuringiensis NH26 inoculant (i.e. the fermentation method of the strain and the freeze-drying process) is shown in Example 3.

[0135] Example 3: Preparation method of Bacillus thuringiensis NH26 inoculant

[0136] (1) Activation of Bacillus thuringiensis NH26 strain

[0137] According to the optimization results of Example 1, the activation method of Bacillus thuringiensis NH26 strain is determined as follows:

[0138] Inoculate Bacillus thuringiensis NH26 strain into seed culture medium and cultivate at 25-35°C and 150-210 rpm for 6-10 hours (preferably at 30°C and 180 rpm for 8 hours) to obtain seed culture solution.

[0139] The composition of the seed culture medium is as follows: glucose 15-25 g / L, yeast extract 4-15 g / L, ammonium sulfate 2-8 g / L, KH2PO4 1-5 g / L, MgSO4·7H2O 0.1-1.0 g / L, and NaCl 0.1-1.0 g / L; and the pH value is adjusted to 6.5-7.5.

[0140] The optimal composition of the seed culture medium is as follows: glucose 20 g / L, yeast extract 10 g / L, ammonium sulfate 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, and NaCl 0.5 g / L; and the pH value is adjusted to 7.0.

[0141] (2) Fermentation culture of Bacillus thuringiensis NH26 strain

[0142] Transfer the activated Bacillus thuringiensis NH26 strain seed culture solution at an inoculation amount of 1-15% (preferably 5%) into liquid fermentation medium and cultivate at 25-35°C and 150-180 rpm (preferably at 30°C and 160 rpm) for 14-18 hours (preferably 16 hours) to obtain fermentation broth.

[0143] The liquid fermentation medium ingredients are: soluble starch 37.5-62.5 g / L, yeast extract 2.5-7.5 g / L, MnSO4H2O 2-8 mg / L, FeSO4 7H2O 0.2-0.8 mg / L, NaCl 1.0-3.0 g / L, sodium glutamate 5-15 g / L; the pH value is adjusted to 6.5-7.5 (preferably: soluble starch 50 g / L; nitrogen source is yeast extract 5 g / L; trace elements are MnSO4H2O 5 mg / L, FeSO4 7H2O 0.5 mg / L, NaCl 2 g / L, sodium glutamate 10 g / L; the pH value is adjusted to 7.0).

[0144] (3) Collecting Bacillus thuringiensis NH26 strain cell bodies

[0145] The fermentation broth after fermentation is placed in a centrifuge, centrifuged at 3000-8000 x g, 0-10°C for 15-25 minutes (preferably 5000 x g, 4°C for 10 minutes), and the supernatant is discarded; the cell bodies are washed with sterile normal saline for 2-4 times (preferably 3 times), and then centrifuged under the same conditions as above to obtain Bacillus thuringiensis NH26 cell body precipitate.

[0146] (4) Preparation of Bacillus thuringiensis NH26 cell body suspension

[0147] The Bacillus thuringiensis NH26 cell body precipitate is added with a compound protective agent solution in a ratio (v / v) of 1:1.0-2.0 (preferably 1:1.5) of the cell body (the volume of the cell body before centrifugation) to the protective agent to prepare a Bacillus thuringiensis NH26 cell body suspension;

[0148] The composition (mass percentage) of the compound protective agent solution is skimmed milk powder 10-20%, sucrose 15-25%, polyvinylpyrrolidone-K30 5-9%, and glutathione 1.0-2.0% (preferably skimmed milk powder 15%, sucrose 20%, polyvinylpyrrolidone-K30 7%, and glutathione 1.3%; pH 7.0).

[0149] (5) Freeze-drying of Bacillus thuringiensis NH26 cell bodies

[0150] The initial cell number of Bacillus thuringiensis NH26 cell suspension was calculated, and the sample was then frozen at -196 to -60°C for 0.2 to 2 hours (preferably, pre-frozen at -196°C for 15 minutes); after complete freezing, the pre-frozen sample (lyophilization thickness of 0.4 to 0.6 cm, preferably 0.5 cm) was placed in a vacuum freeze dryer with a cold trap temperature of -80 to -40°C and a vacuum degree of 0.1 to 0.2 Mbar, and lyophilized for 24 to 48 hours (preferably, at -60°C, a vacuum degree of 0.15 Mbar, and lyophilized for 36 hours). The Bacillus thuringiensis NH26 lyophilized agent was obtained after lyophilization.

[0151] The viable cell number of Bacillus thuringiensis NH26 strain before lyophilization and the viable cell number in the lyophilized agent were determined by the method described in Example 2, and the survival factor was calculated.

[0152] The detection results show that the survival factor of the NH26 lyophilized agent prepared by the above method is 0.993 to 0.997 ± 0.02, and the viable cell number is (2.14 to 3.23 ± 0.03) x 10 11 CFU / g.

[0153] The following Examples 4 to 5 are degradation experiments of the Bacillus thuringiensis NH26 agent prepared under the optimal conditions in Example 3.

[0154] Example 4 Degradation of alkanes in seawater by Bacillus thuringiensis NH26 agent

[0155] I. Degradation ability of Bacillus thuringiensis NH26 agent on dodecane to heptacosane in seawater with a refined diesel concentration of 20 g / L

[0156] 1 g of Bacillus thuringiensis NH26 lyophilized powder was added to a seed culture solution (the components of which were: glucose 20 g / L, yeast extract 10 g / L, ammonium sulfate 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, and NaCl 0.5 g / L; the pH value was adjusted to 7.0, and sterilized at 121°C for 20 minutes), and after 8 hours of culture, the solution was diluted 500 times with seawater (natural seawater collected from the South China Sea was filtered with a 0.22 μm filter to obtain seawater with a salinity of 35‰); then, 10% of the inoculum was transferred to a refined diesel enrichment culture medium (the components of which were: refined diesel 20 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, (NH4)2SO4 5 g / L, FeSO4·7H2O 1.5 mg / L, MnSO4·H2O 0.005 g / L, and sodium glutamate 5 g / L; the pH value was adjusted to 7.0, and sterilized at 121°C for 20 minutes), and cultured at 30°C and 160 rpm for 10 days at a pH value of 7.0.

[0157] After degradation by Bacillus thuringiensis NH26 inoculum, the residual alkanes in the culture medium were first extracted with dichloromethane, and then the alkane composition was analyzed by gas chromatography-mass spectrometry (GC-MS) and compared with the refined diesel fuel composition before the degradation experiment. The degradation rates of various alkane components from dodecane to heptadecane were calculated by comparing the peak areas of each component before and after the degradation experiment. The results are shown in Table 5.

[0158] Table 5. Degradation rates of various components of dodecane to heptadecane by Bacillus thuringiensis NH26 inoculum at a seawater refined diesel concentration of 20 g / L.

[0159] Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) C12 92.0 C18 90.2 C24 89.6 C13 90.2 C19 89.5 C25 90.1 C14 91.4 C20 89.9 C26 89.2 C15 90.8 C21 89.0 C27 90.1 C16 91.3 C22 88.7 C17 90.5 C23 88.4

[0160] As shown in Table 5, the degradation rate of various components of dodecane to heptadecane by NH26 bacterial agent reached over 88.4%, with a total degradation rate of 90.2%, indicating that NH26 bacterial agent has excellent degradation ability for various components of dodecane to heptadecane in seawater.

[0161] II. Degradation capacity of Bacillus thuringiensis NH26 inoculum on various components of dodecane to heptadecane at a seawater refined diesel concentration of 50 g / L.

[0162] 1g of Bacillus thuringiensis NH26 lyophilized inoculum powder was added to the seed culture medium (its components are: glucose 20g / L, yeast extract 10g / L, ammonium sulfate 5g / L, KH2PO4 2g / L, MgSO4·7H2O 0.5g / L and NaCl 0.5g / L; the pH was adjusted to 7.0 and sterilized at 121℃ for 20 minutes). After 8 hours of cultivation, the medium was diluted 500 times with seawater (obtained by filtering natural seawater from the South China Sea through a 0.22μm filter membrane, with a salinity of 35‰). Then, at a 10% inoculum, the medium was transferred to refined diesel enrichment medium (its components are: refined diesel 50g / L, yeast extract 5g / L, KH2PO4 2g / L, MgSO4·7H2O 0.5g / L, (NH4)2SO4 5g / L, FeSO4·7H2O). 1.5 mg / L, MnSO4·H2O 0.005 g / L and sodium glutamate 5 g / L; adjust pH to 7.0 and sterilize at 121℃ for 20 minutes). Incubate for 10 days at pH 7.0, 30℃ and 160 rpm.

[0163] After degradation by NH26 inoculant, the residual alkanes in the culture medium were first extracted with dichloromethane, and then the alkane composition was analyzed by GC-MS and compared with the refined diesel fuel composition before the degradation experiment. By comparing the peak areas of various alkane components before and after the degradation experiment, the degradation rates of various components from dodecane to heptadecane were calculated, and the results are shown in Table 6.

[0164] Table 6 Degradation rate of dodecane to heptacosane by Bacillus thuringiensis NH26 inoculum in seawater with 50 g / L refined diesel oil

[0165] Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) C12 89.7 C18 86.0 C24 87.6 C13 89.2 C19 87.2 C25 87.2 C14 88.8 C20 85.9 C26 86.5 C15 87.7 C21 85.4 C27 86.2 C16 86.4 C22 85.1 C17 86.1 C23 84.8

[0166] As shown in Table 6, the degradation rate of dodecane to heptacosane by NH26 inoculum was more than 88%, and the total degradation rate was 84.8%, indicating that NH26 inoculum still had good degradation ability for dodecane to heptacosane in natural seawater with high concentration (50 g / L) of refined diesel oil.

[0167] Example 5 Degradation of polycyclic aromatic hydrocarbons by Bacillus thuringiensis NH26 inoculum in seawater

[0168] I. Degradation rate of anthracene, phenanthrene and pyrene by Bacillus thuringiensis NH26 inoculum in seawater with 50 mg / L of each of the three compounds

[0169] One gram of Bacillus thuringiensis NH26 freeze-dried powder was added to a seed culture solution (the components of which were: glucose 20 g / L, yeast extract 10 g / L, ammonium sulfate 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L and NaCl 0.5 g / L; the pH value was adjusted to 7.0 and sterilized at 121°C for 20 minutes), and after 8 hours of culture, the solution was diluted 500 times with seawater (natural seawater collected from the South China Sea was filtered with a 0.22 μm filter to obtain the seawater, which had a salinity of 35‰); then, 10% of the inoculum was transferred to a polycyclic aromatic hydrocarbon-rich culture medium (the components of which were: anthracene 50 mg / L, phenanthrene 50 mg / L, pyrene 50 mg / L, yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, (NH4)2SO4 5 g / L, FeSO4·7H2O 1.5 mg / L, MnSO4·H2O 0.005 g / L and sodium glutamate 5 g / L; the pH value was adjusted to 7.0 and sterilized at 121°C for 20 minutes), and the culture was carried out at 30°C and 160 rpm for 30 days at a pH value of 7.0. The residual amounts of anthracene, phenanthrene and pyrene in the culture medium were detected every 5 days, the degradation rate was calculated, and the specific detection method was as follows: dichloromethane was used for extraction, and GC-MS was used for analysis of the residual amounts of anthracene, phenanthrene and pyrene, which were compared with those before the degradation experiment. The degradation rate was calculated by comparing the peak areas of anthracene, phenanthrene and pyrene before and after the degradation experiment, and the results are shown in Table 7.

[0170] Table 7 Degradation rate of anthracene, phenanthrene and pyrene by Bacillus thuringiensis NH26 inoculum in seawater with 50 mg / L of each of the three compounds

[0171] Time (days) Degradation rate of anthracene (%) Degradation rate of pyrene (%) Degradation rate of phenanthrene (%) 5 3.32 6.17 9.45 10 12.15 15.89 20.09 15 20.11 27.53 43.26 20 32.43 37.42 54.07 25 38.72 41.77 62.16 30 43.54 44.82 68.56

[0172] As shown in Table 7, the removal rates of anthracene, phenanthrene, and pyrene by Bacillus thuringiensis NH26 inoculant all showed an increasing trend with the extension of time; after 30 days of degradation, the removal rates of anthracene, pyrene, and phenanthrene by Bacillus thuringiensis NH26 inoculant at a concentration of 50 mg / L were 43.54%, 44.82%, and 68.56%, respectively.

[0173] II. Degradation rates of anthracene, phenanthrene, and pyrene by Bacillus thuringiensis NH26 inoculum in seawater at a concentration of 100 mg / L.

[0174] 1g of Bacillus thuringiensis NH26 lyophilized inoculum powder was added to the seed culture medium (its components are: glucose 20g / L, yeast extract 10g / L, ammonium sulfate 5g / L, KH2PO4 2g / L, MgSO4·7H2O 0.5g / L and NaCl 0.5g / L; the pH was adjusted to 7.0, and sterilized at 121℃ for 20 minutes). After 8 hours of cultivation, the medium was diluted 500 times with seawater (obtained by filtering natural seawater from the South China Sea through a 0.22μm filter membrane, with a salinity of 35‰). Then, at a 10% inoculum, the medium was transferred to a polycyclic aromatic hydrocarbon (PAH) enrichment medium (its components are: anthracene 100mg / L, phenanthrene 100mg / L, pyrene 100mg / L, yeast extract 5g / L, KH2PO4 2g / L, MgSO4·7H2O 0.5g / L, (NH4)2SO4 5g / L). The medium contained 1.5 mg / L FeSO4·7H2O, 0.005 g / L MnSO4·H2O, and 5 g / L sodium glutamate; the pH was adjusted to 7.0, and the medium was sterilized at 121℃ for 20 minutes. The medium was cultured for 30 days at pH 7.0, 30℃, and 160 rpm. The residual amounts of anthracene, phenanthrene, and pyrene in the medium were measured every 5 days, and their degradation rates were calculated. For residual anthracene, phenanthrene, and pyrene in the medium, extraction with dichloromethane was performed first, followed by GC-MS analysis of the residual amounts of anthracene, phenanthrene, and pyrene, and the results were compared with those before the degradation experiment. The degradation rates were calculated by comparing the peak areas of anthracene, phenanthrene, and pyrene before and after the degradation experiment, and the results are shown in Table 8.

[0175] Table 8. Degradation rates of anthracene, phenanthrene, and pyrene by Bacillus thuringiensis NH26 inoculum in seawater at a concentration of 100 mg / L.

[0176] Time (days) Degradation rate of anthracene (%) Degradation rate of pyrene (%) Degradation rate of phenanthrene (%) 5 2.21 4.05 8.98 10 11.06 13.14 18.86 15 18.52 24.71 40.89 20 30.23 34.83 52.81 25 36.45 38.59 58.65 30 39.02 41.69 63.47

[0177] As shown in Table 8, the removal rates of anthracene, phenanthrene, and pyrene by Bacillus thuringiensis NH26 inoculant all showed an increasing trend with the extension of time; after 30 days of degradation, the removal rates of anthracene, pyrene, and phenanthrene by Bacillus thuringiensis NH26 inoculant at a concentration of 100 mg / L were 39.02%, 41.69%, and 63.47%, respectively.

[0178] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A bacterial agent for degrading alkanes and polycyclic aromatic hydrocarbons in a high-salinity water body, characterized by, Contains Bacillus thuringiensis (Bt) Bacillus thuringiensis The Bacillus thuringiensis NH26 strain was deposited at the China Center for Type Culture Collection on July 21, 2022, with accession number CCTCC NO: M20221149. The bacterial agent was prepared by fermentation of Bacillus thuringiensis NH26 strain. The fermentation preparation method was as follows: Bacillus thuringiensis NH26 strain was activated, fermented, collected, suspended, and freeze-dried. The suspension is prepared by mixing the collected bacterial bodies with a protective agent solution; The protective agent solution is composed of the following mass percentage components: skimmed milk powder 10-20%, sucrose 15-25%, polyvinylpyrrolidone-K30 5-9%, and glutathione 1.0-2.0%, with the rest being made up with distilled water.

2. The degrading agent according to claim 1, wherein The activation time is 6-10 hours.

3. The degrading agent according to claim 1, wherein The fermentation culture medium comprises the following components: soluble starch 37.5-62.5 g / L, yeast extract 2.5-7.5 g / L, MnSO4·H2O 2-8 mg / L, FeSO4·7H2O 0.2-0.8 mg / L, NaCl 1.0-3.0 g / L, and sodium glutamate 5-15 g / L.

4. The degrading agent of claim 1, wherein The inoculation amount of the fermentation culture is 1-15%.

5. The degrading agent of claim 1, wherein The pre-freezing condition during freeze-drying is freezing at -196 to -60℃ for 0.20-2 hours.

6. Use of the degrading bacterial agent according to claim 1 for degrading alkanes and / or polycyclic aromatic hydrocarbons, or for cleaning up contamination by alkanes and / or polycyclic aromatic hydrocarbons, or for remediating an environment contaminated by alkanes and / or polycyclic aromatic hydrocarbons, characterized in that, The alkane is n-dodecane-n-heptacosane; the polycyclic aromatic hydrocarbon is anthracene, phenanthrene, and pyrene.

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