A floating composite microbial preparation for removing marine oil pollution and a preparation method thereof

Abstract

The application discloses a floating composite microbial preparation for removing marine oil pollution and a preparation method thereof. The floating composite microbial preparation contains bacillus thuringiensis NH26 strain, hollow glass microbeads and a lipopeptide surfactant purified from a fermentation liquor of the NH26 strain, has floating characteristics, can rapidly activate and efficiently degrade oil hydrocarbon, has good degradation capacity for alkanes and polycyclic aromatic hydrocarbons in seawater, and still has good degradation capacity for alkanes in seawater containing high-concentration oil hydrocarbon. The floating composite microbial preparation can be used for removing marine oil pollution and repairing marine environment. In addition, the application further provides a preparation method of the floating composite microbial preparation. The preparation method has simple preparation process, the obtained floating composite microbial preparation has uniform particle size, has high embedding rates for nitrogen and phosphorus, and can be prepared on a large scale.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial remediation of marine oil pollution. More specifically, it relates to a floating composite microbial preparation for removing marine oil pollution and a preparation method thereof. BACKGROUND

[0002] Petroleum is an important energy and industrial raw material essential for human production and life. However, with the development of the oil industry and the development of port oil transportation, the discharge of ship oil during ship navigation, anchoring and loading and unloading, as well as oil spill accidents caused by ship collisions, capsizing and leakage of oil storage and transportation facilities of offshore oil platforms, have caused the marine environment to deteriorate. At the same time, the toxic substances such as polycyclic aromatic hydrocarbons and benzene series commonly contained in petroleum and its products have also posed a serious threat to marine ecology and human health.

[0003] The main methods for treating marine oil pollution are physical treatment, chemical treatment and microbial remediation. Among them, physical and chemical treatment methods mainly recover the leaked oil in the sea to reduce the pollution load caused by the leaked oil to achieve the purpose of treating marine oil pollution. However, since the leaked oil cannot be degraded or completely removed by physical and chemical methods, the use of physical and chemical treatment methods cannot truly remediate the marine environment contaminated by oil. Although microbial remediation can overcome the shortcoming of not being able to degrade oil and can remediate the marine environment contaminated by oil, the lack of microbial agents that can efficiently remove marine oil pollution limits the popularization and application of microbial remediation of marine oil pollution.

[0004] Many strains that can degrade petroleum hydrocarbons have been reported. For example, Bacillus paramycoides A4-1 strain can degrade petroleum hydrocarbons in simulated wastewater under high temperature conditions. In addition, there are Bacillus aerophilus, Alcanivorax jadensis and Alcanivorax venustensis, etc. However, most of the existing research on petroleum hydrocarbon-degrading bacteria or microbial agents is in freshwater environments, and it is unknown whether they can be applied to marine environments and whether they can degrade oil in high-salinity water. In addition, some petroleum hydrocarbon-degrading bacteria also have the shortcoming of low petroleum hydrocarbon degradation efficiency. Therefore, it is necessary to develop a floating composite microbial preparation with high degradation efficiency for removing marine oil pollution and provide a method for large-scale preparation of the composite microbial preparation. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the defects and shortcomings of the prior art and provide a floating composite microbial preparation for removing marine oil pollution and a preparation method thereof.

[0006] The first object of the present application is to provide a floating composite microbial preparation for removing marine oil pollution.

[0007] The second object of the present application is to provide a preparation method of the floating composite microbial preparation for removing marine oil pollution.

[0008] The third object of the present application is to provide the use of the floating composite microbial preparation.

[0009] The above objects of the present application are achieved by the following technical solutions.

[0010] The present application has obtained a Bacillus thuringiensis NH26 strain capable of degrading petroleum hydrocarbons in high-salinity water. On the basis of the strain, the present application has prepared a floating composite microbial preparation with floating properties and capable of rapidly activating and efficiently degrading petroleum hydrocarbons by using a sharp-hole coagulation bath method, adding the NH26 strain, hollow glass microbeads and a lipopeptide surfactant separated and purified from the fermentation broth of the NH26 strain in the core material. The floating composite microbial preparation can be used for removing marine oil pollution. Therefore, the present application claims protection of the floating composite microbial preparation and the preparation method thereof.

[0011] The present application provides a floating composite microbial preparation for removing marine oil pollution, which contains hollow glass microbeads, Bacillus thuringiensis NH26 strain and lipopeptide surfactant secreted by itself; the NH26 strain was deposited with the China Center for Type Culture Collection on July 21, 2022, and the deposit number is CCTCC NO: M20221149; the chemical structural formula of the lipopeptide surfactant is shown as formula (I):

[0012]

[0013] Specifically, the main component of the hollow glass microbeads is micron-sized hollow glass microspheres of borosilicate; the particle size of the hollow glass microspheres is 30-70 μm, and the density is 0.4-0.6 g / mL.

[0014] Specifically, the lipopeptide surfactant is obtained by acid precipitation, organic solvent extraction and column chromatography purification from the fermentation broth of the Bacillus thuringiensis NH26 strain; the preparation method of the lipopeptide surfactant comprises the following steps:

[0015] S1. Culturing the Bacillus thuringiensis NH26 strain in a liquid fermentation medium to obtain a fermentation broth;

[0016] S2. Acid precipitation; the pH of the fermentation broth obtained in step S1 is adjusted to 7.8-8.2, centrifuged, the supernatant is taken, the pH is adjusted to 1.8-2.2 by adding hydrochloric acid, and it is allowed to stand overnight at 4°C, centrifuged and the precipitate is taken; the precipitate is dissolved in a small amount of ultrapure water, the pH is adjusted to 6.8-7.2 with NaOH solution, frozen at -20°C, and the frozen precipitate is vacuum freeze-dried to obtain the initial precipitate;

[0017] S3. Organic solvent extraction; the initial precipitate obtained in step S2 is ultrasonically extracted with petroleum ether, ethyl acetate and methanol in sequence, filtered with an organic filter membrane and the extract is rotary evaporated to dryness to obtain a fractionated crude product;

[0018] S4. Column chromatography; a petroleum ether / ethyl acetate solution system with a volume ratio of 9:1 is configured as the equilibrium liquid, and a wet-packed chromatography column is used; a small amount of silica gel and the fractionated crude product obtained in step S3 are dissolved in ethyl acetate, mixed well, rotary evaporated under reduced pressure, dried, mixed with a small amount of equilibrium liquid, and added to the chromatography column to complete the sample loading; petroleum ether / ethyl acetate with volume ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 100% ethyl acetate are used as the mobile phase in sequence for elution, and the eluate is blown dry with nitrogen to obtain the lipopeptide surfactant.

[0019] Before column chromatography, the obtained fractionated crude product can be diluted into a solution with ultrapure water for emulsification experiment, the emulsification activities of the obtained fractionated crude products are compared, and the crude product with significant emulsification capacity is selected for column chromatography separation.

[0020] The application also provides a preparation method of the floating composite microbial preparation for removing marine oil pollution, comprising the following steps:

[0021] S1. Preparing an OD 600 of 0.45-0.65 of a bacterial suspension of Bacillus thuringiensis NH26 strain;

[0022] S2. Preparing a sodium alginate colloid with a mass percentage concentration of 2-4%;

[0023] S3. Adding the bacterial suspension obtained in step S1 into the sodium alginate colloid prepared in step S2 at a volume ratio of 1:10-20, and mixing well;

[0024] S4. Adding a composite freeze-drying protective agent solution into the colloid obtained in step S3; in the composite freeze-drying protective agent solution, the mass percentage concentration of skimmed milk powder is 10-20%, the mass percentage concentration of sucrose is 15-25%, the mass percentage concentration of polyvinylpyrrolidone-K30 is 5-9%, and the mass percentage concentration of glutathione is 1.0-2.0%; 0.5-1.5 mL of the composite freeze-drying protective agent solution is added into every 200 mL of the colloid, and mixed well;

[0025] S5. Adding yeast powder, glycerophosphate sodium, hollow glass microbeads and lipopeptide surfactant into the colloidal body obtained in step S4; 5-9 g of yeast powder, 0.5-1.5 g of glycerophosphate sodium, 0.4-0.8 g of lipopeptide surfactant and 0.8-1.2 g of hollow glass microbeads are added into 200 mL of the colloidal body, and mixed thoroughly;

[0026] S6. Preparing a CaCl2 solution with a mass percentage concentration of 2.5-3.5%; 30-40 g of yeast powder and 2-4 g of glycerophosphate sodium are added into 1000 mL of the CaCl2 solution, and mixed thoroughly to obtain a CaCl2 crosslinking agent containing nitrogen and phosphorus nutrients;

[0027] S7. Slowly dropping the colloidal body prepared in step S5 into the CaCl2 crosslinking agent containing nitrogen and phosphorus nutrients prepared in step S6 using a syringe under normal pressure to solidify into microspheres with a diameter of 2.5-4.5 mm;

[0028] S8. After standing for 1.5-2.5 hours, the microspheres obtained in step S7 are subjected to suction filtration to obtain a wet buoyant composite microbial preparation;

[0029] S9. The wet buoyant composite microbial preparation is pre-frozen at -196 to -60℃ for 0.20-2 hours after being divided into portions, and the pre-frozen buoyant composite microbial preparation is vacuum freeze-dried to obtain the buoyant composite microbial preparation.

[0030] Specifically, the hollow glass microbeads in step S4 are micron-sized hollow glass microspheres mainly composed of borosilicate; the particle size of the hollow glass microspheres is 30-70 μm, and the density is 0.4-0.6 g / mL.

[0031] Specifically, when the colloidal body prepared in step S5 is slowly dropped into the CaCl2 crosslinking agent containing nitrogen and phosphorus nutrients prepared in step S6 using a syringe, the diameter of the needle of the syringe is 0.8-1.6 mm, and the height of the needle from the surface of the crosslinking agent is 10-20 cm.

[0032] Specifically, the vacuum freeze-drying in step S9 is as follows: the pre-frozen buoyant composite microbial preparation is placed in a vacuum freeze-drying machine with a cold trap temperature of -80 to -40℃ and a vacuum degree of 0.1-0.2 Mbar, and freeze-dried for 24-48 hours.

[0033] Specifically, the bacterial suspension in step S1 is obtained by fermentation culture of Bacillus thuringiensis NH26 strain with a preservation number of CCTCC NO: M20221149, and the preparation process includes the following steps:

[0034] S11. Take the preserved bacteria liquid of Bacillus thuringiensis NH26 strain, inoculate it into liquid fermentation medium, and culture at 25-35 DEG C and 140-180 rpm to make the culture liquid turbid to obtain the seed culture liquid of NH26 strain;

[0035] S12. The seed culture liquid prepared in step S11 is inoculated into liquid fermentation medium at an inoculation amount of 5%, and cultured at 25-35 DEG C and 140-180 rpm for 16-24 hours;

[0036] S13. The fermentation culture liquid is centrifuged at 4 DEG C, and the supernatant is discarded; the precipitate is washed with sterile normal saline for 3 times, and then centrifuged under the same conditions to obtain the NH26 bacterial precipitate;

[0037] S14. The NH26 bacterial precipitate prepared in step S13 is added with a composite freeze-drying protective agent solution in a ratio of 1:1.0-2.0 (bacterial liquid volume before centrifugation: composite freeze-drying protective agent solution volume) to prepare a Bacillus thuringiensis NH26 bacterial suspension.

[0038] Specifically, the liquid fermentation medium contains soluble starch (carbon source) 50 g / L, yeast extract (nitrogen source) 5 g / L, MnSO4·H2O 5 mg / L, FeSO4·7H2O 0.5 mg / L, NaCl 2 g / L, and sodium glutamate 10 g / L; and the pH value is 7.0.

[0039] Specifically, in the composite freeze-drying protective agent solution, the mass percentage concentration of skimmed milk powder is 10-20%, the mass percentage concentration of sucrose is 15-25%, the mass percentage concentration of polyvinylpyrrolidone-K30 is 5-9%, and the mass percentage concentration of glutathione is 1.0-2.0%.

[0040] Preferably, the fermentation culture conditions of NH26 strain are as follows: 30 DEG C, 160 rpm.

[0041] The application also protects the floating composite microbial preparation for removing marine oil pollution prepared by the preparation method.

[0042] The application also protects the application of the floating composite microbial preparation in degrading alkanes and / or polycyclic aromatic hydrocarbons.

[0043] The application also protects the application of the floating composite microbial preparation in preparing products for degrading alkanes and / or polycyclic aromatic hydrocarbons.

[0044] The application also protects the application of the floating composite microbial preparation in repairing the environment polluted by alkanes and / or polycyclic aromatic hydrocarbons, or in repairing the environment polluted by pollutants containing alkanes and / or polycyclic aromatic hydrocarbons.

[0045] The application also claims the use of the floating composite microbial preparation in the preparation of a product for repairing an environment contaminated by alkanes and / or polycyclic aromatic hydrocarbons, or in the preparation of a product for repairing an environment contaminated by a contaminant containing alkanes and / or polycyclic aromatic hydrocarbons.

[0046] Specifically, the environment is a high-salt water body.

[0047] More specifically, the high-salt water body is seawater.

[0048] The application has the following beneficial effects:

[0049] The application provides a floating composite microbial preparation for removing marine oil pollution and a preparation method thereof. The floating composite microbial preparation contains Bacillus thuringiensis NH26 strain, hollow glass microbeads and a lipopeptide biosurfactant purified from a fermentation broth of the NH26 strain, has floating properties and can quickly activate and efficiently degrade petroleum hydrocarbons. The floating composite microbial preparation has excellent degradation capacity for alkanes in seawater, and the total degradation rate thereof is 92.3% in 10 days. The floating composite microbial preparation still has good degradation capacity for alkanes in seawater containing high-concentration petroleum hydrocarbons (the concentration of refined diesel is as high as 50 g / L), and the total degradation rate thereof is 87.9% in 10 days. The floating composite microbial preparation also has good degradation capacity for polycyclic aromatic hydrocarbons such as anthracene, pyrene and phenanthrene in seawater, and the total degradation rates thereof for 100 mg / L of anthracene, pyrene and phenanthrene are 42.01%, 44.62% and 68.14% respectively in 30 days, so the floating composite microbial preparation can be used for removing marine oil pollution.

[0050] In addition, the application also provides a preparation method of the floating composite microbial preparation, and the preparation method has a simple preparation process, the size of the microparticles in the obtained floating composite microbial preparation is uniform, the embedding rate of nitrogen and phosphorus is high, and the floating composite microbial preparation can be prepared on a large scale. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 A Fourier transform infrared spectrum of the lipopeptide biosurfactant purified from the fermentation broth of the NH26 strain.

[0052] Figure 2 A carbon nuclear magnetic resonance spectrum of the lipopeptide biosurfactant purified from the fermentation broth of the NH26 strain; wherein, Fig. A is a carbon spectrum of the lipopeptide biosurfactant in the range of 0-180 ppm; and Fig. B is a carbon spectrum of the lipopeptide biosurfactant in the range of 172-173 ppm.

[0053] Figure 3 A hydrogen nuclear magnetic resonance spectrum of the lipopeptide biosurfactant purified from the fermentation broth of the NH26 strain.

[0054] Figure 4 DEPT 90 spectrum of the lipopeptide biosurfactant purified from the fermentation broth of NH26 strain.

[0055] Figure 5 DEPT 135 spectrum of the biosurfactant purified from the fermentation broth of NH26 strain.

[0056] Figure 6 GC-MS spectrum of the fatty acids in the lipopeptide biosurfactant purified from the fermentation broth of NH26 strain.

[0057] Figure 7 Mass spectrum of the lipopeptide biosurfactant purified from the fermentation broth of NH26 strain.

[0058] Figure 8 Particle state of the buoyant composite microbial preparation prepared in Example 1.

[0059] Figure 9 Results of the buoyancy test of the buoyant composite microbial preparation prepared in Example 1 using natural seawater. DETAILED DESCRIPTION

[0060] 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.

[0061] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0062] The Bacillus thuringiensis NH26 used in the present application is a Bacillus thuringiensis NH26 strain isolated from surface soil / sediment collected from a coastal wetland contaminated with crude oil, and was preserved in the China Center for Type Culture Collection (CCTCC) on July 21, 2022, with the preservation number CCTCC NO: M20221149 and the preservation address being Room 299, Baoyi Road, Wuhan University, Wuhan, Hubei Province, China.

[0063] All the culture media used in the following examples were sterilized at 121℃ for 20 minutes before use, unless otherwise specified.

[0064] Preparation of the buoyant composite microbial preparation in Example 1

[0065] The present application provides a buoyant composite microbial preparation for removing marine oil pollution, which is prepared by a sharp-hole solidification bath method. The specific steps are as follows:

[0066] S1. Preparation of OD 600 Bacillus thuringiensis NH26 strain bacterial suspension 20 mL, wherein the concentration of the bacterial suspension is 0.55;

[0067] S2. Preparation of sodium alginate colloid; 6 g of sodium alginate was slowly added into 200 mL of distilled water, and stirred for 10 minutes to prepare 3% sodium alginate colloid;

[0068] S3. The bacterial suspension was quickly added into the sodium alginate colloid prepared in step S2 at a volume ratio of 1:15, and stirred for 10 minutes to fully mix;

[0069] S4. The composite freeze-drying protective agent solution was added into the colloid obtained in step S3; the composite freeze-drying protective agent solution contained skimmed milk powder, sucrose, polyvinylpyrrolidone-K30 and glutathione; the mass percentage concentration of the skimmed milk powder was 15%, the mass percentage concentration of the sucrose was 20%, the mass percentage concentration of the polyvinylpyrrolidone-K30 was 7%, and the mass percentage concentration of the glutathione was 1.3%; 1 mL of the composite freeze-drying protective agent solution was added into 200 mL of the colloid, and stirred for 10 minutes to fully mix;

[0070] S5. Yeast powder, glycerophosphate sodium, lipopeptide surfactant and hollow glass microbeads were added into the colloid obtained in step S4; 7 g of yeast powder, 1 g of glycerophosphate sodium, 0.6 g of lipopeptide surfactant and 1 g of hollow glass microbeads were added into 200 mL of the colloid, and stirred for 10 minutes to fully mix;

[0071] The hollow glass microbeads were micron-sized borosilicate hollow glass microspheres; the hollow glass microspheres used in this embodiment had a particle size of 50 μm and a density of 0.5 g / mL;

[0072] S6. 1000 mL of 3% CaCl2 solution was prepared, and 35 g of yeast powder and 3 g of glycerophosphate sodium were added and mixed to obtain nitrogen and phosphorus nutrient-containing CaCl2 crosslinking agent;

[0073] S7. The colloid prepared in step S5 was slowly dropped into the nitrogen and phosphorus nutrient-containing CaCl2 crosslinking agent prepared in step S6 using a 100 mL syringe (needle diameter 1.2 mm) (the needle was 15 cm above the liquid level of the crosslinking agent), so as to be solidified into microspheres with a diameter of about 3 mm;

[0074] S8. After standing for 2 hours, the microspheres obtained in step S7 were subjected to suction filtration to obtain wet floating composite microbial agent;

[0075] S9. The wet floating composite microbial agent is pre-frozen for 15 minutes at -196℃, and then is placed in a vacuum freeze dryer with a cold trap temperature of -60℃ and a vacuum degree of 0.15 Mbar for 36 hours to obtain the floating composite microbial agent of the present application. The bacterial suspension in step S1 is obtained by fermentation of Bacillus thuringiensis NH26 strain, and the preparation process comprises the following steps:

[0076] S11. 100 μL of the preserved bacterial solution of Bacillus thuringiensis NH26 strain is inoculated into 50 mL of liquid fermentation medium, and is cultured at 25-35℃ and 140-180 rpm (preferably at 30℃ and 160 rpm) until the culture solution becomes turbid to obtain the seed culture solution of NH26 strain;

[0077] S12. The seed culture solution obtained in step S11 is inoculated into 200 mL of liquid fermentation medium at an inoculation amount of 5%, and is cultured at 30℃ and 160 rpm for 16-24 hours;

[0078] The liquid fermentation medium contains soluble starch (carbon source) 50 g / L, yeast extract (nitrogen source) 5 g / L, MnSO4·H2O 5 mg / L, FeSO4·7H2O 0.5 mg / L, NaCl 2 g / L, and sodium glutamate 10 g / L, and has a pH value of 7.0;

[0079] S13. The fermented bacterial solution is centrifuged at 5000 x g and 4℃ for 10 minutes, and the supernatant is discarded; the precipitate is washed with sterile normal saline for 3 times, and then is centrifuged under the same conditions to obtain the NH26 bacterial precipitate;

[0080] S14. The NH26 bacterial precipitate obtained in step S13 is mixed with the composite freeze-drying protective agent solution at a ratio of 1:1.0-2.0 (preferably 1:1.5) to prepare the Bacillus thuringiensis NH26 bacterial suspension.

[0081] The mass percentage concentration of each component in the composite freeze-drying protective agent solution is: skimmed milk powder 15%, sucrose 20%, polyvinylpyrrolidone-K30 7%, and glutathione 1.3%.

[0082] The lipopeptide surfactant in step S5 is obtained by acid precipitation, organic solvent extraction and column chromatography from the fermentation broth of Bacillus thuringiensis NH26 strain (the preparation process of the fermentation broth is the same as that of S11-S12), and the specific process is as follows:

[0083] (1) Acid precipitation: the pH of the fermentation broth was adjusted to 8.0 with NaOH to ensure that the biosurfactant would not precipitate; the broth was centrifuged at 10,000 rpm for 10 minutes at 4°C to remove all the bacterial cells; 6 mol / L hydrochloric acid was added to the supernatant to adjust the pH to 2.0, and the obtained supernatant was allowed to stand overnight in a refrigerator at 4°C to make the biosurfactant form flocculent precipitate; the mixture was taken out of the refrigerator and centrifuged at 10,000 rpm for 10 minutes at 4°C, and the supernatant was discarded; the precipitate was dissolved in a small amount of ultrapure water, the pH was adjusted to 7.0 with NaOH solution, and the frozen precipitate was pre-frozen at -20°C for 2 hours, then the frozen precipitate was placed in a vacuum freeze dryer and freeze-dried at -60°C to obtain the initial precipitate of the biosurfactant;

[0084] (2) Organic solvent extraction: the obtained initial precipitate of the biosurfactant was further extracted and purified with organic solvents, specifically, the initial precipitate was ultrasonically extracted with petroleum ether, ethyl acetate and methanol in sequence according to the polarity of the solvents from small to large, and the extract was filtered through a 0.22 μm organic filter membrane, and then the extract was rotary evaporated at 50°C to obtain a fractionated crude product of the biosurfactant; the fractionated crude product was diluted with ultrapure water to a 1 g / L solution for emulsification experiment, the emulsification activity of the fractionated crude product was compared, and the crude product with significant emulsifying capacity was selected for column chromatography separation;

[0085] (3) Column chromatography: a 9:1 (v / v) petroleum ether / ethyl acetate solution system was configured as an equilibration liquid, and a wet-packed chromatography column was used; 200-300 mesh silica gel was mixed with the petroleum ether / ethyl acetate solution system (9:1, v / v) and packed into a glass chromatography column at a packing rate of 80% of the glass column; after the silica gel was settled, the petroleum ether / ethyl acetate (9:1, v / v) was added at a flow rate of 2 mL / min to equilibrate the chromatography column;

[0086] A small amount of silica gel and the fractionated crude product with significant emulsifying capacity were dissolved in ethyl acetate, mixed well, and then rotary evaporated at 50°C under reduced pressure; after drying, the mixture was mixed with a small amount of equilibration liquid and added to the chromatography column to complete the sample loading; the mobile phase was a petroleum ether / ethyl acetate system (v / v), and the elution was performed in sequence with 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 petroleum ether / ethyl acetate systems (v / v) and 100% ethyl acetate; the elution volume of each mobile phase was 2 times the column volume, and the flow rate was 2 mL / min; the eluate was collected in a bottle with a capacity of 20 mL, and the process was continued until the elution was completed; finally, the eluate was blown dry with nitrogen at 50°C to obtain the lipopeptide surfactant.

[0087] The lipopeptide surfactant was subjected to the following systematic structural characterization:

[0088] Characteristic functional group analysis: Fourier transform infrared spectroscopy was used to analyze the characteristic functional groups of the surfactant sample produced by strain NH26 (i.e., the sample prepared by the purification method described above in this invention). The Fourier transform infrared spectrum of the lipopeptide surfactant purified from the fermentation broth of strain NH26 is shown below. Figure 1 As shown. From Figure 1 It can be seen that at 3425.011cm -1 There is a relatively broad absorption peak at 2923.602 cm⁻¹, which indicates the stretching vibrations of -OH and -NH; -1 and 2856.104cm -1 The presence of distinct -CH3 and -CH2 stretching vibration absorption peaks at 1735.647 cm⁻¹ indicates the presence of saturated aliphatic chains in the compound; -1 The presence of an absorption peak at 1656.579 cm⁻¹ indicates the presence of an amide bond (CO-N); -1 A characteristic CN absorption peak appears at [location missing]. Combined with the infrared spectra of previously reported lipopeptide compounds, it can be confirmed that the characteristic peaks of this surfactant sample's infrared spectrum match those of lipopeptide compounds, leading to the conclusion that it is a lipopeptide biosurfactant.

[0089] Nuclear magnetic resonance (NMR) spectroscopy analysis: The carbon (C), proton (H), DEPT90, and DEPT135 NMR spectra of the surfactant sample produced by strain NH26 are shown below. Figures 2 to 5 As shown. From Figure 2 and Figure 3 It can be seen that the sample has characteristic peaks of long carbon chains in the range of 10–30 ppm; and three characteristic peaks of C=O bonds in the range of 172–173 ppm, with the area of ​​the second peak being about twice that of the other two peaks, indicating the presence of four peptide bonds. Figure 4 Observe the upward CH peak; from Figure 5 The NMR spectroscopy revealed upward-pointing peaks for CH and CH3, while CH2 was an inverted peak. Numerous characteristic inverted CH2 peaks were observed in the 20–50 ppm range, indicating the presence of an alkyl chain in the compound. This NMR spectroscopy analysis further confirmed that the surfactant is a lipopeptide compound.

[0090] Chemical structure analysis: The surfactant sample produced by strain NH26 was hydrolyzed, and the amino acid composition was analyzed using an amino acid analyzer. The results are shown in Table 1. The fatty acid composition was detected using gas chromatography-mass spectrometry (GC-MS), and the results are shown in Table 1. Figure 6 As shown in Table 1, the amino acid composition ratio of this surfactant is approximately Ala:Phe:Tyr:Pro:Thr = 1:1:1:1:1. Figure 6 The results suggest that the structure of the fatty acid methyl ester in this surfactant is CH3(CH2). 20COOCH3, i.e. the fatty acid chain of the surfactant is CH3(CH2) 20 COOH. The surfactant produced by the NH26 strain was also detected by thermospray mass spectrometry, and the results are shown in Table 1. Figure 7 The molecular weight of the surfactant was determined to be 919 according to the detection results of thermospray mass spectrometry.

[0091] Table 1 Amino acid composition in the biosurfactant

[0092]

[0093] Based on the above comprehensive analysis, it is shown that the chemical structural formula of the surfactant produced by the NH26 strain is shown as formula (I), i.e. the chemical structural formula of the lipopeptide surfactant is shown as formula (I):

[0094]

[0095] wherein Phe, Pro, Thr, Ala and Tyr represent phenylalanine, proline, threonine, alanine and tyrosine, respectively.

[0096] The critical micelle concentration (CMC) and stability of the purified lipopeptide surfactant were also detected, and the results show that the critical micelle concentration of the obtained lipopeptide surfactant is 120 mg / L; the lipopeptide surfactant can reduce the surface tension of water to 29.69 mN / m at the CMC concentration; and the lipopeptide surfactant has strong emulsifying ability and stability under alkaline conditions, and the emulsifying ability is the strongest at 30°C.

[0097] The first step of microbial degradation of petroleum is to emulsify petroleum. Under the premise of no addition of exogenous surfactant, the oil-degrading bacteria first secretes biosurfactant after being applied, which not only consumes a large amount of nutrients such as nitrogen and phosphorus, but also significantly reduces the hydrocarbon-degrading ability of the bacteria, and the activation of the secretion of the surfactant by the bacteria needs time, which results in a significant prolongation of the time required for the removal of petroleum hydrocarbons. The prior art is to add chemical surfactants. However, the matching degree and fusion of the chemical surfactants with the microbial strains are poor. The present application overcomes the technical problem of poor matching degree and fusion between the strains and the chemical surfactants by adding the biosurfactant (i.e. the lipopeptide surfactant) with excellent performance, which is produced by the oil-degrading bacteria (NH26 strain) itself, and significantly improves the hydrocarbon-degrading ability of the oil-degrading bacteria.

[0098] Example 2 Performance evaluation of the floating composite microbial preparation obtained in Example 1

[0099] The performance of the microbial preparation obtained in Example 1 was evaluated in this invention, as detailed below:

[0100] 1. Determination of bacterial cell encapsulation rate:

[0101] (1) Weigh 1g of the prepared floating composite microbial preparation and dissolve it in 10mL of phosphate buffer solution (phosphate concentration of 1% and pH of solution of 7.0); crush it with a glass rod and vortex for 10 minutes.

[0102] (2) Take 1 mL of the dissolution obtained in (1) and dilute it stepwise; take 0.2 mL of the diluted solution and spread it on beef extract peptone plate medium, and incubate it in a constant temperature incubator at 30℃ for 24 hours, and calculate the number of viable bacteria;

[0103] (3) Before embedding, take 1 mL of bacterial suspension and dilute it stepwise and spread it on beef extract protein plate medium, and then count the bacteria in the plate culture.

[0104] (4) The bacterial count and encapsulation rate of the obtained unit mass of floating composite microbial preparation are calculated using the following formula:

[0105] Viable bacteria count (CFU / g) = (Number of bacteria after dilution × Dilution factor × Total volume) / Sample volume (a)

[0106] Encapsulation rate (%) = (Number of viable bacteria in the product / Number of viable bacteria in the bacterial suspension at the time of encapsulation) × 100% (b)

[0107] Based on stepwise dilution testing and calculations according to formulas (a) and (b), the encapsulation rate of the floating composite microbial preparation prepared in this embodiment of the invention was 86.9%–90.9%, with an average value of 88.9%; the viable cell count was (1.37–2.19) × 10⁻⁶. 11 CFU / g, with an average value of 1.78 × 10⁻⁶. 11 CFU / g.

[0108] 2. Observation of floating compound microbial preparation particles:

[0109] The floating composite microbial preparation was observed under an optical microscope. The particle state of the floating composite microbial preparation prepared in Example 1 is as follows. Figure 8 As shown, by Figure 8 It can be seen that the obtained microbial inoculant particles are relatively uniform spherical. The particle size read from the built-in micrometer of the microscope is 2.6-4.5 mm, with the vast majority being 3-4 mm.

[0110] 3. Density measurement:

[0111] The average density of the floating compound microbial preparation was measured using an ET-320 solid density meter. The specific measurement steps are as follows:

[0112] (1) Use a spoon to randomly scoop 5 samples of floating compound microbial preparations;

[0113] (2) Place the spherical stainless steel filter screen into the basket in the water tank of the solid density meter, so that it is completely submerged in the water. Then place the small glass cup on the weighing platform and press the [ZERO] button to tare.

[0114] (3) When M1 flashes on the screen, pour one of the samples to be tested into the glass on the weighing platform; when the stability symbol “O” is displayed in the upper left corner of the screen, press the [ENTER] key. At this time, the screen changes from flashing M1 to flashing M2, indicating that the mass (m1) of the sample in the air has been recorded.

[0115] (4) Open the spherical stainless steel filter screen and pour all the sample to be tested from the glass into it, then close the spherical stainless steel filter screen; in order to avoid the generation of air bubbles when the sample to be tested enters the water and affect the accuracy of the measurement results, first rinse the sample to be tested with alcohol and then put the spherical stainless steel filter screen into the basket in the water tank, and put the empty glass back on the weighing platform.

[0116] (5) When the stable symbol “O” is displayed in the upper left corner of the screen, press the [ENTER] key. The instrument will record the weight (m2) of the sample in water. At the same time, M2 on the screen will disappear and the density value (ρ) of this floating composite microbial preparation sample measured by the instrument will be displayed.

[0117] (6) After recording the data, remove the sample; press the [ENTER] key to return to the test mode and measure the next sample;

[0118] (7) After completing the measurement of 5 samples, calculate the average density of the samples.

[0119] The average density of the five floating compound microbial agents ranged from 0.95 to 0.98 g / cm³. 3 Its density is slightly less than that of seawater (1.02–1.07 g / cm³). 3 It has buoyancy properties.

[0120] In addition, the present invention tested the prepared floating composite microbial preparation with natural seawater, and the results are as follows: Figure 9 As shown, this further confirms that the composite microbial preparation obtained in Example 1 of the present invention has buoyancy properties and can float on the sea surface.

[0121] 4. Determination of nitrogen / phosphorus encapsulation rate of floating compound microbial preparations:

[0122] A certain amount of floating composite microbial preparation was weighed and placed in a small beaker containing 50 mL of phosphate buffer solution. It was first crushed with a glass rod, then ultrasonically crushed for 10 minutes in an ultrasonic cleaner. After shaking well, 5 mL was diluted to 50 mL and nitrified in a sterilizer at 120℃ for 30 minutes. The total nitrogen and total phosphorus contents of the floating composite microbial preparations prepared by each method were determined. Total nitrogen was determined using the alkaline potassium persulfate oxidation-cadmium column reduction method, and total phosphorus was determined using the alkaline potassium persulfate oxidation-phosphomolybdic blue method. The nitrogen / phosphorus encapsulation ratio of the floating composite microbial preparation was measured and calculated according to formula (c).

[0123] Nitrogen / phosphorus encapsulation rate = (mass of nitrogen or phosphorus in the compound microbial preparation / total mass of the compound microbial preparation) × 100% (c)

[0124] The nitrogen and phosphorus encapsulation rates in the floating composite microbial preparation prepared in Example 1 were 98% and 78%, respectively.

[0125] 5. Determination of nitrogen / phosphorus dissolution in floating compound microbial preparations:

[0126] The nitrogen / phosphorus leaching amount in floating compound microbial preparations was measured using seawater as the leaching medium and the conical flask immersion method. The specific operating steps are as follows:

[0127] Twenty samples of the prepared floating composite microbial preparation, each weighing 0.5 g, were added to twenty conical flasks, each containing 50 mL of seawater. The flasks were then kept at 25°C and 120 rpm with constant temperature and repeated shaking. At the measurement times listed in Table 2, one conical flask was removed, and 10 mL of the dissolution solution was filtered through a 0.45 μm filter membrane. This solution was then diluted to 50 mL with Milli Q and nitrified in a sterilizer at 120°C for 30 minutes. The total nitrogen and total phosphorus contents of the 20 floating composite microbial preparation samples at different dissolution times were determined. Total nitrogen was determined using the alkaline potassium persulfate oxidation-cadmium column reduction method; total phosphorus was determined using the alkaline potassium persulfate oxidation-phosphomolybdic blue method.

[0128] Based on the cumulative release percentages (%) of total nitrogen and total phosphorus in the floating compound microbial preparation, in seawater culture medium with pH=7.3, both total nitrogen and total phosphorus were released rapidly (over 50%) within 24 hours; the release slowed down after 24 hours; and were basically completed after 72 hours. The release rate of total phosphorus was slightly faster than that of total nitrogen. Specific data are shown in Tables 2 and 3.

[0129] Because there are a large number of floating petroleum hydrocarbons (i.e. carbon source excess) in the oil-polluted seawater, in order to remove the petroleum hydrocarbons, the floating composite microbial agent must be added, because only the floating composite microbial agent can meet the basic condition for removing the petroleum hydrocarbons, i.e. the floating property can ensure that the microbial agent can be in contact with the petroleum; the embedded slow-release nitrogen and phosphorus can ensure that the nutrient source is continuously supplied to the petroleum-degrading bacteria, and promote the rapid growth of the hydrocarbon-degrading bacteria; the supply of a large number of high-efficiency petroleum-degrading bacteria can significantly improve the ability of removing the petroleum hydrocarbons and shorten the removal time. From the detection data of this example, it can be seen that the floating composite microbial agent prepared by the present application has the characteristics of floating property, slow-release of nitrogen and phosphorus and supply of high-density and high-efficiency petroleum-degrading bacteria, and therefore can be used for removing the marine oil pollution.

[0130] Table 2 Cumulative release proportion of total nitrogen in seawater culture solution with pH = 7.3

[0131]

[0132] Table 3 Cumulative release proportion of total phosphorus in seawater culture solution with pH = 7.3

[0133]

[0134]

[0135] Example 3 Preparation of floating composite microbial agent

[0136] The floating composite microbial agent described in this example is prepared by the sharp-hole solidification bath method; the preparation methods of the bacterial suspension of Bacillus thuringiensis NH26 strain and the lipopeptide surfactant are the same as those in Example 1, and the difference lies in that the mass percentage concentrations of the components in the composite freeze-drying protective agent used are different. In this example, the mass percentage concentration of skimmed milk powder in the composite freeze-drying protective agent solution is 10%, the mass percentage concentration of sucrose is 15%, the mass percentage concentration of polyvinylpyrrolidone-K30 is 5%, and the mass percentage concentration of glutathione is 1%.

[0137] The specific steps for preparing the floating composite microbial agent in this example are as follows:

[0138] S1. Prepare the bacterial suspension of Bacillus thuringiensis NH26 strain with OD 600 of 0.45, 20 mL;

[0139] S2. Prepare the sodium alginate colloid; slowly add 4 g of sodium alginate in 200 mL of distilled water, stir for 5 minutes, and prepare the sodium alginate colloid with a concentration of 2%;

[0140] S3. Add the bacterial suspension into the sodium alginate colloid prepared in step S2 as soon as possible at a volume ratio of 1:10, stir for 5 minutes, and mix thoroughly;

[0141] S4. Adding a complex freeze-drying protective agent solution to the colloidal body obtained in step S3; in the complex freeze-drying protective agent solution, the mass percentage concentration of skimmed milk powder is 10%, the mass percentage concentration of sucrose is 15%, the mass percentage concentration of polyvinylpyrrolidone-K30 is 5%, and the mass percentage concentration of glutathione is 1%; 0.5 mL of the complex freeze-drying protective agent solution is added to 200 mL of the colloidal body, and stirring is performed for 5 minutes to allow sufficient mixing;

[0142] S5. Adding yeast powder, glycerophosphate sodium, lipopeptide surfactant, and hollow glass microbeads to the colloidal body obtained in step S4; 5 g of yeast powder, 0.5 g of glycerophosphate sodium, 0.4 g of lipopeptide surfactant, and 0.8 g of hollow glass microbeads are added to 200 mL of the colloidal body; stirring is performed for 5 minutes to allow sufficient mixing;

[0143] The hollow glass microspheres used in this example have a particle size of 30 μm and a density of 0.6 g / mL;

[0144] S6. Preparing 1000 mL of a 2.5% CaCl2 solution, and adding 30 g of yeast powder and 2 g of glycerophosphate sodium after mixing to obtain a nitrogen and phosphorus nutrient-containing CaCl2 crosslinking agent;

[0145] S7. Slowly dropping the colloidal body prepared in step S5 into the nitrogen and phosphorus nutrient-containing CaCl2 crosslinking agent prepared in step S6 using a 100 mL syringe (needle diameter 0.8 mm) at normal pressure (the needle is 10 cm above the liquid level of the crosslinking agent), so that the colloidal body is solidified into microspheres with a diameter of about 2.5 mm;

[0146] S8. After standing for 1.5 hours, the microspheres obtained in step S7 are subjected to suction filtration to obtain a wet floating composite microbial agent;

[0147] S9. The wet floating composite microbial agent is sub-packed and pre-frozen at -196°C for 0.20 hours; the pre-frozen sample (freeze-drying thickness is 0.4 cm) is placed in a vacuum freeze-drying machine with a cold trap temperature of -80°C and a vacuum degree of 0.1 Mbar, and freeze-drying is performed for 24 hours to obtain the floating composite microbial agent of the present application.

[0148] The performance of the obtained floating composite microbial agent is evaluated according to the method in Example 2, and the results show that the performance of the floating composite microbial agent prepared in this example is slightly lower than that of the microbial agent prepared in Example 1, but there is no significant difference in performance evaluation between the microbial agents prepared in Examples 1 and 4.

[0149] Example 4 Preparation of a floating composite microbial agent

[0150] The buoyant composite microbial preparation described in this example is prepared by sharp hole coagulation bath method; the preparation methods of Bacillus thuringiensis NH26 strain bacterial suspension and lipopeptide surfactant used are the same as those of Example 1, the difference is that the mass percentage concentrations of the components in the composite freeze-drying protective agent used are different. In this example, the mass percentage concentration of skimmed milk powder in the composite freeze-drying protective agent solution used is 20%, the mass percentage concentration of sucrose is 25%, the mass percentage concentration of polyvinylpyrrolidone-K30 is 9%, and the mass percentage concentration of glutathione is 2%.

[0151] The specific steps for preparing the buoyant composite microbial preparation in this example are as follows:

[0152] S1. Prepare 20 mL of Bacillus thuringiensis NH26 strain bacterial suspension with OD 600 of 0.65;

[0153] S2. Prepare sodium alginate colloid; slowly add 8 g of sodium alginate to 200 mL of distilled water, stir for 15 minutes, and prepare 4% sodium alginate colloid;

[0154] S3. Add the bacterial suspension to the sodium alginate colloid prepared in step S2 as soon as possible at a volume ratio of 1:20, stir for 15 minutes, and mix thoroughly;

[0155] S4. Add composite freeze-drying protective agent solution to the colloid obtained in step S3; the mass percentage concentration of skimmed milk powder in the composite freeze-drying protective agent solution is 20%, the mass percentage concentration of sucrose is 25%, the mass percentage concentration of polyvinylpyrrolidone-K30 is 9%, and the mass percentage concentration of glutathione is 2%; add 1.5 mL of the composite freeze-drying protective agent solution to every 200 mL of colloid, stir for 15 minutes, and mix thoroughly;

[0156] S5. Add yeast powder, glycerol phosphate sodium, lipopeptide surfactant, and hollow glass microspheres to the colloid obtained in step S4; add 9 g of yeast powder, 1.5 g of glycerol phosphate sodium, 0.8 g of lipopeptide surfactant, and 1.2 g of hollow glass microspheres to every 200 mL of colloid; stir for 15 minutes, and mix thoroughly;

[0157] The hollow glass microspheres used in this example have a particle size of 70 μm and a density of 0.4 g / mL;

[0158] S6. Prepare 1000 mL of 3.5% CaCl2 solution, add 40 g of yeast powder and 4 g of glycerol phosphate sodium, and mix well to obtain a CaCl2 cross-linking agent containing nitrogen and phosphorus nutrients;

[0159] S7. The prepared gelatinous body in step S5 was slowly dropped into the nitrogenous phosphorus nutrient-containing CaCl2 crosslinking agent prepared in step S6 under normal pressure using a 100 mL syringe (needle diameter 1.6 mm) (the needle was 20 cm above the liquid level of the crosslinking agent) to solidify into microspheres with a diameter of about 4.5 mm;

[0160] S8. After standing for 2.5 hours, the microspheres obtained in step S7 were suction filtered to obtain wet floating composite microbial agent;

[0161] S9. The wet floating composite microbial agent was sub-packed and pre-frozen at -60°C for 2 hours. The pre-frozen sample (lyophilization thickness 0.6 cm) was placed in a vacuum freeze dryer with a cold trap temperature of -40°C and a vacuum degree of 0.2 Mbar, and freeze-dried for 48 hours to obtain the floating composite microbial agent of the present application.

[0162] The performance of the obtained floating composite microbial agent was evaluated according to the method in Example 2, and the results showed that the performance of the floating composite microbial agent prepared in this example was slightly lower than that of the microbial agent prepared in Example 1, but there was no significant difference in performance evaluation between the microbial agents prepared in Examples 1 and 2.

[0163] Example 5 Degradation of alkanes in seawater by floating composite microbial agent

[0164] In this example, the degradation ability of the floating composite microbial agent prepared in Example 1 for alkanes in seawater was tested.

[0165] 4 g of refined diesel oil (obtained by distilling commercially available diesel oil at 100°C for 240 hours, with a concentration of 20 g / L) was added to 200 mL of seawater medium (obtained by filtering natural seawater collected from the South China Sea with a 0.22 μm filter, with a salinity of 35‰), shaken and mixed; then 5 g of floating composite microbial agent was added to the mixed solution, and the pH value was adjusted to 7.3. The mixture was incubated at 30°C and 160 rpm for 10 days. After degradation by the floating composite microbial agent, the residual alkanes in the seawater medium contaminated with refined diesel oil were extracted with dichloromethane, and the composition of the alkanes was detected by gas chromatography-mass spectrometry (GC-MS) and compared with that of the refined diesel oil before the degradation experiment. By comparing the peak areas of various alkanes before and after the degradation experiment, the degradation rates of various components of n-dodecane to n-heptacosane were calculated (Table 4). As can be seen from Table 4, the degradation rates of the floating composite microbial agent prepared in this example for various components of n-dodecane to n-heptacosane were all above 90.0%, and the total degradation rate was 92.3%, indicating that the floating composite microbial agent had excellent degradation ability for various components of n-dodecane to n-heptacosane in seawater medium.

[0166] In 200 mL of seawater culture solution, 10 g of refined diesel oil (50 g / L) was added and shaken well; 5 g of the floating composite microbial preparation was added to the mixture, and the pH was adjusted to 7.3. The mixture was incubated at 30°C and 160 rpm for 10 days. After degradation by the floating composite microbial preparation, the residual alkanes in the seawater culture solution contaminated by the refined diesel oil were extracted with dichloromethane, and the alkanes were detected by GC-MS. The degradation rates of n-dodecane to n-heptacosane were calculated by comparing the peak areas of the alkanes before and after the degradation experiment (Table 5). As shown in Table 5, the degradation rates of the floating composite microbial preparation for n-dodecane to n-heptacosane were all above 86.6%, and the total degradation rate was 87.9%, indicating that the floating composite microbial preparation still had good degradation ability for n-dodecane to n-heptacosane in the seawater culture solution with high concentration (50 g / L) of refined diesel oil, although the degradation rate decreased.

[0167] Table 4 Degradation rates of n-dodecane to n-heptacosane in seawater culture solution with 20 g / L of refined diesel oil by the floating composite microbial preparation

[0168] Carbon number of alkane Degradation rate (%) Carbon number of alkane Degradation rate (%) Carbon number of alkane Degradation rate (%) C12 94.1 C18 92.5 C24 91.4 C13 92.6 C19 91.2 C25 90.1 C14 93.3 C20 91.6 C26 91.5 C15 92.7 C21 90.4 C27 90.7 C16 93.0 C22 90.8 C17 92.3 C23 90.0

[0169] Table 5 Degradation rates of n-dodecane to n-heptacosane in seawater culture solution with 50 g / L of refined diesel oil by the floating composite microbial preparation

[0170] Carbon number of alkane Degradation rate (%) Carbon number of alkane Degradation rate (%) Carbon number of alkane Degradation rate (%) C12 92.0 C18 88.5 C24 87.3 C13 91.3 C19 88.0 C25 86.8 C14 91.7 C20 87.6 C26 87.2 C15 89.2 C21 87.1 C27 87.0 C16 88.7 C22 86.8 C17 88.2 C23 86.6

[0171] Example 6 Degradation of polycyclic aromatic hydrocarbons in seawater culture solution by the floating composite microbial preparation

[0172] In this example, the degradation ability of the floating composite microbial preparation prepared in Example 1 for polycyclic aromatic hydrocarbons in seawater was tested.

[0173] In 200 mL of seawater culture solution, 10 mg of anthracene, phenanthrene and pyrene (the final concentration of each was 50 mg / L) was added and shaken well; 5 g of the prepared floating composite microbial agent was then added to the mixture, and the pH value was adjusted to 7.3. The culture was incubated at 30°C and 160 rpm for 30 days. The residual amount of anthracene, phenanthrene and pyrene in the seawater culture solution was detected every 5 days, and the degradation rate was calculated. For the residual anthracene, phenanthrene and pyrene in the seawater culture solution, dichloromethane was used for extraction, and GC-MS was used to detect the residual amount of anthracene, phenanthrene and pyrene, respectively, and compared with that before the degradation experiment. By comparing the peak area of anthracene, phenanthrene and pyrene before and after the degradation experiment, the degradation rate was calculated, and the results are shown in Table 6. As can be seen from Table 6, with the extension of time, the removal rate of anthracene, phenanthrene and pyrene by the floating composite microbial agent showed an upward trend; after 30 days of degradation, the removal rates of anthracene, pyrene and phenanthrene by the floating composite microbial agent were 45.86%, 47.23% and 71.05%, respectively.

[0174] Table 6 Degradation rate of anthracene, phenanthrene and pyrene in seawater culture solution with the concentration of each being 50 mg / L by the agent

[0175] Time (days) Degradation rate of anthracene (%) Degradation rate of pyrene (%) Degradation rate of phenanthrene (%) 5 5.80 9.21 12.39 10 13.28 17.38 22.14 15 21.26 29.43 45.84 20 34.58 39.21 56.98 25 40.97 43.89 66.31 30 45.86 47.23 71.05

[0176] In 200 mL of seawater culture solution, 20 mg of anthracene, phenanthrene and pyrene (the final concentration of each was 100 mg / L) was added and shaken well; 5 g of the floating composite microbial agent was then added to the mixture, and the pH value was adjusted to 7.3. The culture was incubated at 30°C and 160 rpm for 30 days. The residual amount of anthracene, phenanthrene and pyrene in the seawater culture solution was detected every 5 days, and the degradation rate was calculated. For the residual anthracene, phenanthrene and pyrene in the seawater culture solution, dichloromethane was used for extraction, and GC-MS was used to detect the residual amount of anthracene, phenanthrene and pyrene, respectively, and compared with that before the degradation experiment. By comparing the peak area of anthracene, phenanthrene and pyrene before and after the degradation experiment, the degradation rate was calculated, and the results are shown in Table 7. As can be seen from Table 7, with the extension of time, the removal rate of anthracene, phenanthrene and pyrene by the floating composite microbial agent showed an upward trend; after 30 days of degradation, the removal rates of anthracene, pyrene and phenanthrene by the floating composite microbial agent were 42.01%, 44.62% and 68.14%, respectively.

[0177] To the best of the inventors' knowledge, there is no report on the use of the floating composite microbial agent embedding Bacillus thuringiensis to remove polycyclic aromatic hydrocarbons in seawater.

[0178] Table 7 Degradation rate of anthracene, phenanthrene and pyrene in seawater culture solution with the concentration of each being 100 mg / L by the agent

[0179] Time (days) Degradation rate of anthracene (%) Degradation rate of pyrene (%) Degradation rate of phenanthrene (%) 5 5.67 7.89 10.22 10 12.22 15.88 19.77 15 20.35 27.10 42.78 20 32.71 36.62 53.60 25 38.14 41.25 63.32 30 42.01 44.62 68.14

[0180] 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 floating composite microbial preparation for removing marine oil pollution, characterized in that, The floating composite microbial preparation contains hollow glass microspheres, Bacillus thuringiensis NH26 strain, and its own secreted lipopeptide surfactant. The hollow glass microspheres are micron-sized hollow glass microspheres with borosilicate as the main component, and the particle size of the hollow glass microspheres is 30-70 μm, with a density of 0.4-0.6 g / mL. The NH26 strain was deposited at the China Center for Type Culture Collection on July 21, 2022, with accession number CCTCC NO: M20221149. The chemical structural formula of the lipopeptide surfactant is shown in formula (I). Formula (I); The preparation method of the floating composite microbial preparation includes the following steps: S1. Preparation of OD 600 A bacterial suspension of Bacillus thuringiensis NH26 strain with a concentration of 0.45–0.65; S2. Prepare sodium alginate gel with a mass percentage concentration of 2-4%; S3. Add the bacterial suspension obtained in step S1 to the sodium alginate gel prepared in step S2 at a volume ratio of 1:10-20, and mix thoroughly. S4. Add a composite lyophilization protectant solution to the mixture obtained in step S3; the composite lyophilization protectant solution contains skim milk powder at a mass percentage concentration of 10-20%, sucrose at a mass percentage concentration of 15-25%, polyvinylpyrrolidone-K30 at a mass percentage concentration of 5-9%, and glutathione at a mass percentage concentration of 1.0-2.0%; add 0.5-1.5 mL of the composite lyophilization protectant solution to every 200 mL of the mixture obtained in S3, and mix thoroughly; S5. Add yeast powder, sodium glycerophosphate, hollow glass microspheres and lipopeptide surfactant to the mixture obtained in step S4; add 5-9 g yeast powder, 0.5-1.5 g sodium glycerophosphate, 0.4-0.8 g lipopeptide surfactant and 0.8-1.2 g hollow glass microspheres to every 200 mL of the mixture obtained in S4, and mix thoroughly. S6. Prepare a CaCl2 solution with a mass percentage concentration of 2.5-3.5%. Add 30-40g of yeast powder and 2-4g of sodium glycerophosphate to every 1000 mL of CaCl2 solution, mix thoroughly, and obtain a CaCl2 crosslinking agent containing nitrogen and phosphorus nutrients. S7. The mixture prepared in step S5 is slowly dripped into the CaCl2 crosslinking agent containing nitrogen and phosphorus nutrients prepared in step S6 under normal pressure using a syringe, so that it is solidified into microspheres with a diameter of 2.5 to 4.5 mm. S8. After standing for 1.5 to 2.5 hours, the microspheres obtained in step S7 are filtered to obtain a wet floating composite bacterial agent. S9. After dispensing the wet floating compound microbial agent, pre-freeze it at -196 to -60°C for 0.20 to 2 hours; vacuum freeze-dry the pre-frozen floating compound microbial agent to obtain the floating compound microbial preparation.

2. The floating composite microbial preparation according to claim 1, characterized in that, The preparation method of the lipopeptide surfactant includes the following steps: S1. Bacillus thuringiensis NH26 strain was placed in liquid fermentation medium and cultured to obtain fermentation broth; S2. Acid precipitation: Adjust the pH of the fermentation broth obtained in step S1 to 7.8-8.2, centrifuge, take the supernatant, add hydrochloric acid to adjust the pH to 1.8-2.2, let stand overnight at 4℃, centrifuge to take the precipitate; dissolve the precipitate with a small amount of ultrapure water, adjust the pH to 6.8-7.2 with NaOH solution, freeze at -20℃, freeze-dry the frozen precipitate under vacuum to obtain the initial precipitate; S3. Organic solvent extraction: The initial precipitate obtained in step S2 was extracted by ultrasonic extraction with petroleum ether, ethyl acetate and methanol in several steps, respectively. The extract was filtered with an organic filter membrane and evaporated by rotary evaporation to obtain crude products. S4. Column chromatography: Prepare a petroleum ether / ethyl acetate solution system with a volume ratio of 9:1 as the equilibration solution, and pack the chromatography column using a wet method; dissolve a small amount of silica gel and the fractionated crude product obtained in step S3 in ethyl acetate, mix well, evaporate under reduced pressure by rotary evaporation, dry, mix with a small amount of equilibration solution, add to the chromatography column, and complete the sample injection; use a mixture of petroleum ether / ethyl acetate with volume ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9 and 100% ethyl acetate as the mobile phase for sequential elution, and dry the eluent with nitrogen gas to obtain the lipopeptide surfactant.

3. The floating composite microbial preparation according to claim 1, characterized in that, The vacuum freeze-drying described in step S9 is as follows: the pre-frozen floating composite bacterial agent is 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 freeze-dried for 24 to 48 hours.

4. The use of the floating composite microbial preparation according to any one of claims 1 to 3 in the degradation of alkanes and / or polycyclic aromatic hydrocarbons.

5. The use of the floating composite microbial preparation according to any one of claims 1 to 3 in the preparation of products that degrade alkanes and / or polycyclic aromatic hydrocarbons.

6. The use of the floating composite microbial preparation according to any one of claims 1 to 3 in the remediation of environments contaminated with alkane and / or polycyclic aromatic hydrocarbons, or in the remediation of environments contaminated by pollutants containing alkane and / or polycyclic aromatic hydrocarbons.

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