A method for remediating petroleum hydrocarbon contaminated soil based on a high-lipopeptide surfactant-producing strain

Abstract

The application discloses a kind of production lipopeptide surfactant and utilize the remediation method of the surfactant to oil hydrocarbon contaminated soil.The preparation method of the surfactant includes: to Bacillus amyloliquefaciens A3 strain is carried out fermentation culture, obtain the fermentation broth containing surfactant;Bacillus amyloliquefaciens A3 strain, preservation number is CGMCC No.6676.The optimal conditions for producing surfactant are: carbon source glycerol, nitrogen source urea, temperature is 25 DEG C, pH is 8.0, carbon source content is 40g / L.The maximum yield of surfactant can be obtained at 150rpm, 72h cultivation is 1.4114g / L.The product of the application has good emulsifying activity and surface activity, and the stability under harsh conditions is also very high, and has very good leaching effect in simulated in-situ remediation soil column leaching experiment compared with control group, and promotes the biodegradation of in-situ soil, and can be used for the remediation of oil hydrocarbon contaminated soil.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving the screening of a strain of Bacillus amyloliquefaciens A3 that produces high levels of lipopeptide surfactants, and the application of the surfactants produced by it in the remediation of petroleum hydrocarbon-contaminated soil, thereby achieving in-situ bioremediation of contaminated soil. Background Technology

[0002] Surfactants are a class of molecules that possess both hydrophilic and hydrophobic groups and can successfully reduce the surface tension of solutions. They can be broadly classified into biosurfactants and chemical surfactants. With the increasing use of surfactants, research has found that chemical surfactants generally have poor stability and are difficult to biodegrade, easily causing secondary environmental pollution. Biosurfactants, on the other hand, not only have high biocompatibility and low environmental hazard, but also possess antiviral and antibacterial capabilities, thus attracting widespread attention. Biosurfactants are produced from plants and microorganisms, such as soapberry, Bacillus sp., and Pseudomonas aeruginosa.

[0003] Biosurfactants have important applications in microbial enhanced oil recovery, pharmaceuticals, cosmetics, food, and environmental remediation. Among these, the remediation of petroleum hydrocarbon-contaminated soils with biosurfactants has attracted the most attention. With the gradual development of the petroleum industry, the environmental threat posed by oil spills during extraction, transportation, and use is increasing daily. Commonly used chemical and physical remediation methods cause serious secondary pollution, while phytoremediation is time-consuming and insufficient. Therefore, microbial remediation methods are widely used. Research has found that surfactants can reduce interfacial tension between two phases, increase the fluidity and bioavailability of petroleum hydrocarbons, and influence the extracellular polymeric substances (EPS) of bacteria, thereby altering the surface adsorption characteristics of bacteria. Furthermore, multiple experiments have demonstrated that the addition of biosurfactants can promote the solubilization, desorption, and in-situ degradation of petroleum hydrocarbons by microorganisms.

[0004] However, the difficulty in applying biosurfactants to actual contaminated soil lies in their low yield and extraction difficulty. Studies have shown that the surfactants produced by wild-type Bacillus are all below 1g / L, which has led to a significant increase in the cost of use. Therefore, there is an urgent need to find a strain that can produce high levels of surfactants for in-situ remediation of contaminated soil. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention screened a strain capable of producing high yields of lipopeptide surfactants. Using Bacillus amyloliquefaciens strain A3, a high production of lipopeptide biosurfactants can be obtained, thereby reducing the surface tension of the liquid phase. In soil column leaching experiments, this effectively promotes the solubilization, desorption, and leaching of petroleum hydrocarbons in the soil, facilitates the in-situ biodegradation of petroleum hydrocarbons in the soil, and simultaneously increases soil enzyme activity, thus achieving effective remediation of petroleum hydrocarbon-contaminated soil.

[0006] The purpose of this invention is to provide a method for preparing lipopeptide surfactants.

[0007] The method for preparing lipopeptide surfactants provided by the present invention includes the following steps: fermenting and culturing Bacillus amyloliquefaciens strain A3 to obtain a fermentation broth containing the lipopeptide surfactant.

[0008] The *Bacillus amyloliquefaciens* strain A3 used in this invention was deposited on October 15, 2012, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 6676. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, 100101, China. This strain has been described in ZL201110225699.8.

[0009] The fermentation culture conditions are: a temperature of 25-35℃, a pH of 6.0-8.0, and a rotation speed of 120-180 rpm for 72 hours; preferably, the fermentation culture conditions are: a temperature of 25℃, a pH of 8.0, and a rotation speed of 150 rpm for 72 hours.

[0010] The culture medium for fermentation contains a carbon source and a nitrogen source. The carbon source is selected from at least one of the following: glycerol, glucose, sodium citrate, sucrose, crude oil, and waste cooking oil, preferably glycerol. The nitrogen source is selected from at least one of the following: urea, ammonium sulfate, sodium nitrate, sodium nitrite, peptone, and ammonium nitrate, preferably urea.

[0011] Preferably, the carbon source content in the culture medium is 40 g / L.

[0012] The culture medium also contains the following inorganic salts: dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, and calcium chloride.

[0013] Furthermore, the culture medium is composed of the following: glycerol 40 g / L, urea 2 g / L, dipotassium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 1.1 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.01 g / L, calcium chloride 0.01 g / L, pH 7.2.

[0014] The optimal conditions for surfactant production by Bacillus amyloliquefaciens A3 are: glycerol as the carbon source, urea as the nitrogen source, a temperature of 25℃, a pH of 8.0, and a carbon source concentration of 40 g / L. The maximum surfactant yield of 1.4114 g / L can be obtained by culturing at 150 rpm for 72 h.

[0015] The fermentation culture involves inoculating a seed culture of Bacillus amyloliquefaciens A3 into a culture medium for fermentation culture; the inoculation amount of the seed culture of Bacillus amyloliquefaciens A3 can be 1-10%, specifically 5%.

[0016] The method for preparing the seed culture of Bacillus amyloliquefaciens A3 is as follows: Bacillus amyloliquefaciens A3 is transferred to LB medium for culture. After culturing at 25-35℃ and 120-180rpm for 24h, the bacterial culture is stored in a refrigerator at 4℃ as the seed culture.

[0017] The above-mentioned method for preparing lipopeptide surfactants further includes the step of separating the lipopeptide surfactant from the fermentation broth containing the lipopeptide surfactant, the specific method of which is as follows:

[0018] The fermentation broth containing the lipopeptide surfactant was centrifuged, and the pH of the supernatant was adjusted to 2-3 with hydrochloric acid. The supernatant was then refrigerated overnight at 4°C and centrifuged again. The centrifuged product was freeze-dried, and the resulting brownish-yellow product was the crude surfactant extracted.

[0019] In the above separation step, the centrifugation conditions can be: centrifugation at 8000-10000 rpm for 10 min.

[0020] In the above separation steps, the hydrochloric acid can be 6 mol / L hydrochloric acid.

[0021] The lipopeptide surfactants prepared by the above method are also within the scope of protection of this invention.

[0022] The present invention also protects the application of the lipopeptide surfactants.

[0023] The application of the lipopeptide surfactants described in this invention is selected from at least one of the following aspects:

[0024] 1) Applications in the emulsification, solubilization, and / or degradation of petroleum hydrocarbons;

[0025] 2) Application in the bioremediation of petroleum hydrocarbon contaminated soil.

[0026] The application of Bacillus amyloliquefaciens strain A3 in at least one of the following aspects is also within the scope of protection of this invention:

[0027] a) Application in the fermentation production of lipopeptide surfactants by bacterial strains;

[0028] b) Applications in the emulsification, solubilization and / or degradation of petroleum hydrocarbons;

[0029] c) Application in the bioremediation of petroleum hydrocarbon contaminated soil.

[0030] Metabolites of Bacillus amyloliquefaciens A3 and / or cultures of Bacillus amyloliquefaciens A3 are also within the scope of protection of this invention.

[0031] In the above text, the metabolites of Bacillus amyloliquefaciens A3 can be the fermentation broth of Bacillus amyloliquefaciens A3.

[0032] The fermentation broth of Bacillus amyloliquefaciens A3 can be prepared as follows: Bacillus amyloliquefaciens A3 is cultured in a liquid fermentation medium, and the fermentation broth (containing Bacillus amyloliquefaciens A3 and substances secreted into the liquid medium) is collected. This fermentation broth is the metabolite of Bacillus amyloliquefaciens A3.

[0033] In the above text, the culture of Bacillus amyloliquefaciens A3 is a substance obtained by culturing Bacillus amyloliquefaciens A3 in a microbial culture medium (such as fermentation broth containing Bacillus amyloliquefaciens A3 and substances secreted into the liquid culture medium).

[0034] The metabolites of Bacillus amyloliquefaciens A3 and / or the culture of Bacillus amyloliquefaciens A3 can be used for the bioremediation of petroleum hydrocarbon contaminated soil.

[0035] The petroleum hydrocarbons mentioned in this invention include diesel oil and engine oil, with a volume ratio of 1:1, both purchased from Sinopec Lubricating Oil Co., Ltd.

[0036] The fermentation broth of Bacillus amyloliquefaciens A3 can show obvious hemolysis zones on blood agar plates, exhibiting β-hemolysis. The surface tension of its culture medium can be reduced to below 28 mN / m by surface tension meter measurement, proving that strain A3 has the ability to produce surfactants.

[0037] The surfactant produced by *Bacillus amyloliquefaciens* A3 showed an orange-red color under ninhydrin chromatographic conditions after thin-layer chromatography, and was initially identified as a lipopeptide surfactant. Subsequent Fourier transform infrared spectroscopy confirmed the presence of characteristic peaks of lipopeptide surfactants. Furthermore, at the molecular level, *Bacillus amyloliquefaciens* A3 was found to contain key genes for the production of lipopeptide surfactants, comprehensively demonstrating that the biosurfactant produced by *Bacillus amyloliquefaciens* A3 belongs to the lipopeptide surfactant class.

[0038] The surfactant produced by Bacillus amyloliquefaciens A3 has high stability. The surface tension of its culture medium does not exceed 30 mN / m in the temperature range of 4-121℃, pH range of 4-12, and salinity range of 0-20%.

[0039] The surfactant produced by Bacillus amyloliquefaciens A3 has good emulsifying activity. The emulsification index for n-hexane can reach 51.20%, and the emulsification index for hydrophobic matrices such as n-dodecane, diesel, liquid paraffin, and petroleum ether is around 50%.

[0040] The critical micelle concentration of the surfactant produced by Bacillus amyloliquefaciens A3 was determined to be 498 mg / L, which can reduce the surface tension of deionized water from 71.90 mN / m to 27.1 mN / m, indicating good surface activity.

[0041] Soil column leaching experiments were conducted using Bacillus amyloliquefaciens A3 and its produced surfactants. After six repeated leaching cycles, the degradation rate of total petroleum hydrocarbons in the soil column reached 45.44%, accounting for 13.26% of the total leaching volume, which was 3.82 times that of the control group. The removal rate of C28-C24 n-alkanes was 100%, and it also showed excellent removal efficiency for long-chain alkanes. From the perspective of enzyme activity, the activities of soil catalase and lipase were increased by 44.86% and 261.11%, respectively, while soil dehydrogenase was inhibited to some extent.

[0042] Compared with existing results, the present invention has the following advantages:

[0043] The surfactant yield of Bacillus amyloliquefaciens A3 in this invention is high, reaching 1.4114 g / L under optimal conditions. Its product has excellent emulsifying and surface activity, and also exhibits high stability under harsh conditions. In the simulated in-situ remediation soil column leaching experiment, it also showed a better leaching effect than the control group and promoted the biodegradation of in-situ soil. Therefore, Bacillus amyloliquefaciens A3 can be used for the remediation of petroleum hydrocarbon contaminated soil. Attached Figure Description

[0044] Figure 1In the diagram, (A) represents the hemolytic zone of Bacillus amyloliquefaciens A3 on a blood agar plate; (B) represents the phylogenetic tree of Bacillus amyloliquefaciens BA.

[0045] Figure 2 (AC) represents the changes in OD600, surface tension, and surfactant yield of Bacillus amyloliquefaciens A3 in various carbon sources; (DF) represents the changes in OD600, surface tension, and surfactant yield of Bacillus amyloliquefaciens BA in various nitrogen sources.

[0046] Figure 3 In the diagram, A represents the response surface plot of maximum surfactant yield under the conditions of independent variables temperature and carbon source content; B represents the response surface plot of maximum surfactant yield under the conditions of independent variables temperature and pH; and C represents the response surface plot of maximum surfactant yield under the conditions of independent variables carbon source content and pH.

[0047] Figure 4 The chromatogram obtained by thin-layer chromatography after developing a surfactant produced by Bacillus amyloliquefaciens A3.

[0048] Figure 5 Fourier transform infrared spectrum of surfactant produced by Bacillus amyloliquefaciens A3.

[0049] Figure 6 The surface tension changes of surfactants produced by Bacillus amyloliquefaciens A3 under different pH (A), temperature (B), and salinity (C) conditions were investigated.

[0050] Figure 7 To investigate the emulsifying activity of surfactants produced by Bacillus amyloliquefaciens A3 on various hydrophobic matrices.

[0051] Figure 8 This is a schematic diagram of the apparatus for a soil column rinsing experiment.

[0052] Figure 9 The graph shows the leaching amount (A), leaching trend (B), removal rate of various n-alkanes (C) of Bacillus amyloliquefaciens A3 (BA) in a soil column leaching experiment; and the differences in the activities of catalase (D), lipase (E), and dehydrogenase (F) after leaching. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials are all available from publicly available commercial sources.

[0054] Example 1: Discovery of the surfactant-producing ability of Bacillus amyloliquefaciens A3

[0055] (1) Source of strain: The strain used in this invention is Bacillus amyloliquefaciens A3 strain, which was deposited on October 15, 2012, at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 6676. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, 100101, China.

[0056] (2) Culture medium:

[0057] Lysozyme broth (LB medium): yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, pH 7.2.

[0058] Lysozyme broth agar medium (LB solid medium): yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar 20 g / L, pH 7.2.

[0059] Inorganic salt culture medium (MSM medium): ammonium sulfate 2g / L, glucose 20g / L, dipotassium hydrogen phosphate 5g / L, potassium dihydrogen phosphate 2g / L, sodium chloride 1.1g / L, magnesium sulfate heptahydrate 0.2g / L, ferrous sulfate heptahydrate 0.01g / L, calcium chloride 0.01g / L, pH 7.2.

[0060] (3) Identification of the surfactant production ability of Bacillus amyloliquefaciens BA:

[0061] ① Activation of Bacillus amyloliquefaciens A3

[0062] Bacillus amyloliquefaciens A3, preserved in glycerol solution, was transferred to LB medium and cultured at 30°C and 150 rpm for 24 h. The bacterial culture was then stored in a refrigerator at 4°C as a seed culture for later use.

[0063] ② Hemolytic activity and surface tension determination of Bacillus amyloliquefaciens A3

[0064] The seed culture was serially diluted in sterile water to obtain 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Take 10 of the concentrated bacterial solution. -6 ~10 -8 100 μL of the concentrated bacterial suspension was spread onto blood agar plates (LB solid medium supplemented with 5% defibrinated sheep blood) and incubated at 30°C for 24 h. The size of the hemolysis zone was then observed. Figure 1 (A) shows a hemolytic zone exceeding 4 mm in diameter, indicating β-type hemolysis. Next, 15 mL of seed culture was centrifuged at 10000 rpm for 10 min, and the supernatant was collected. The surface tension of the supernatant was measured using a surface tension meter (QBZY-1, Fangrui), repeated three times. The results showed that *Bacillus amyloliquefaciens*, after cultivation, consistently reduced the surface tension of the culture medium supernatant to below 28 mN / m.

[0065] Example 2: Optimization of carbon and nitrogen sources for the production of surfactants using Bacillus amyloliquefaciens A3.

[0066] (1) Optimization of carbon source for surfactant production by Bacillus amyloliquefaciens A3

[0067] Glucose, sodium citrate, sodium acetate, sucrose, crude oil, glycerol, diesel oil, engine oil, n-hexadecane, waste cooking oil, liquid paraffin, and soybean oil were selected as the sole carbon source and added to the MSM culture medium (to replace the carbon source "glucose" in the MSM, at a dosage of 20 g / L). A 5% seed culture obtained in Example 1 was inoculated, and the mixture was cultured at 30°C and 150 rpm for 72 h. OD600, surface tension, and surfactant yield were measured before and after culture. All experiments were performed in triplicate. The experimental results are as follows: Figure 2 AC analysis showed that glycerol was the optimal carbon source for surfactant production by *Bacillus amyloliquefaciens* A3. Using glycerol as the carbon source, after 72 hours of cultivation, the surface tension of the supernatant decreased to 29.8 mN / m, the OD600 reached 2.055, and the highest surfactant yield was 0.7727 g / L. Furthermore, a certain amount of surfactant was also produced in glucose, sodium citrate, sucrose, crude oil, and waste cooking oil. Growth was weaker and no surfactant was produced in other carbon sources.

[0068] (2) Optimization of nitrogen source for surfactant production by Bacillus amyloliquefaciens A3

[0069] Using glycerol as the sole carbon source, ammonium sulfate, sodium nitrate, sodium nitrite, urea, peptone, and ammonium nitrate were selected as the sole nitrogen sources and added to the MSM culture medium. A 5% seed culture obtained in Example 1 was inoculated, and the medium was cultured at 30°C and 150 rpm for 72 h. OD600, surface tension, and surfactant yield were measured before and after culture. All experiments were performed in triplicate. The experimental results are as follows: Figure 2DF analysis showed that urea was the optimal nitrogen source for surfactant production by *Bacillus amyloliquefaciens* BA. Using urea as the nitrogen source, after 72 hours of cultivation, the surface tension of the supernatant decreased to 30.1 mN / m, the OD600 reached 1.838, and the highest surfactant yield was 1.0953 g / L. A certain amount of surfactant could also be produced from ammonium sulfate, sodium nitrate, sodium nitrite, peptone, and ammonium nitrate, while the surfactant yield was lower with other nitrogen sources.

[0070] Example 3: Optimization of the best conditions for surfactant production by Bacillus amyloliquefaciens A3 using response surface methodology-central complex design (RSM-CCD).

[0071] An experiment was designed using Design Expert 8.0 to investigate three independent variables affecting surfactant production by *Bacillus amyloliquefaciens* BA. Independent variables A were temperature (°C), B was carbon source content (g / L), and C was pH. Each independent variable had five levels: temperature ranges of 20°C, 25°C, 30°C, 35°C, and 40°C; carbon source content ranges of 10 g / L, 20 g / L, 30 g / L, 40 g / L, and 50 g / L; and pH ranges of 5.0, 6.0, 7.0, 8.0, and 9.0. Figure 3 As shown, a total of 20 control experiments were set up, and the samples were cultured for 72 h in MSM medium with glycerol as the carbon source and urea as the nitrogen source (at a concentration of 2 g / L). All treatments had three replicates.

[0072] The experimental design matrix for optimizing the production of surfactants from Bacillus amyloliquefaciens BA using response surface methodology-central composite design (RSM-CCD) and the actual and predicted surfactant yields are shown in Table 1.

[0073] Table 1

[0074]

[0075] Experimental results are as follows Figure 3As shown, after optimization of influencing factors and methodological analysis, the model obtained in this study has good significance (p < 0.0001), with R² and F values ​​of 0.9520 and 22.03, respectively, which also support the significance of the model. Among these parameters, carbon source content has the most significant impact on surfactant yield (p < 0.0001), as carbon source content can improve the nutrient limitation of bacteria. Conversely, pH and temperature did not have a significant effect on surfactant yield, indicating that *Bacillus amyloliquefaciens* A3 exhibits strong adaptability to harsh environments within the pH and temperature limits. The final results show that at a temperature of 25℃, pH of 8.0, and glycerol concentration of 40 g / L, *Bacillus amyloliquefaciens* A3 exhibits the optimal theoretical biosurfactant yield of 1.3976 g / L. Validation under the same culture and nutrient conditions revealed an actual yield of 1.4114 g / L, with the theoretical and actual yields being very close, thus proving the accuracy of the model. The model formula is shown below:

[0076] Biosurfactant yield (g / L)

[0077] =+0.91+0.22B-0.042AB-0.11AC+0.026BC-0.029A2-0.013C2+0.039ABC+0.18A2C+0.067AB2

[0078] Example 4: Characterization of surfactants produced by Bacillus amyloliquefaciens A3.

[0079] (1) Extraction of surfactants produced by Bacillus amyloliquefaciens A3

[0080] The seed culture of Bacillus amyloliquefaciens A3 from Example 1 was inoculated into MSM medium (the carbon source (glucose) in the above MSM was replaced with glycerol, the amount of which was 40 g / L; the nitrogen source (ammonium sulfate) was replaced with urea, the amount of which was 2 g / L) and the surfactant was produced under the optimal conditions (temperature of 25°C, pH of 8.0, carbon source glycerol amount of 40 g / L, and rotation speed of 150 rpm). After culturing for 72 h, the bacterial culture was centrifuged at 10000 rpm for 10 min, and the pH of the obtained supernatant was adjusted to 2 with 6 mol / L hydrochloric acid. It was then refrigerated overnight at 4°C and centrifuged again at 10000 rpm for 10 min. The centrifuged product was placed in a refrigerated dryer for refrigeration. The brownish-yellow product obtained was the crude surfactant extracted.

[0081] (2) The products of Bacillus amyloliquefaciens A3 were characterized by thin-layer chromatography (TLC).

[0082] The crude surfactant obtained from extraction was dissolved in methanol, and 5 μL was spotted onto a silica gel plate for thin-layer chromatography. The silica gel plate was placed in a chromatography tank containing chloroform:methanol:acetic acid:water = 25:15:4:2 (v / v / v / v) chromatography buffer and chromatographically analyzed for 10–15 minutes. Afterward, the chromatography plate was removed. One treatment was subjected to color development with 0.5% ninhydrin solution, while the other treatment was acidified with hydrochloric acid and then color developed with 0.5% ninhydrin solution. Both treatments were placed in an oven at 105°C for 20 minutes for color development. The ratio shift (Rf) of the colored spots was then calculated.

[0083] The result after color development is as follows Figure 4 As shown, a total of 4 colored spots appeared, located at positions with Rf values ​​of 0.08, 0.34, 0.67, and 0.96, respectively. After consulting the literature, it was finally found that the spots at positions of 0.34 and 0.67 should be lipopeptide surfactants, while the spots at positions of 0.08 and 0.96 should be impurities.

[0084] (3) The products of Bacillus amyloliquefaciens A3 were characterized by Fourier transform infrared spectroscopy (FTIR).

[0085] The extracted surfactant powder was directly measured on a Fourier transform infrared spectrometer, with the measurement frequency set between 4000 and 400 cm⁻¹. -1 Within the range, each sample was scanned 32 times to obtain the average value.

[0086] The infrared spectrum obtained by scanning is as follows Figure 5 As shown, the surfactant produced by Bacillus amyloliquefaciens A3, judging from the absorption peaks of its functional groups, is chemically classified as a lipopeptide and does not contain the characteristic peaks of glycolipid surfactants.

[0087] Example 5: Stability and emulsifying activity of surfactants produced by Bacillus amyloliquefaciens A3.

[0088] (1) Stability of surfactants produced by Bacillus amyloliquefaciens A3

[0089] The seed culture from Example 1 was centrifuged at 10,000 rpm for 10 min to obtain the supernatant. The supernatant was then kept within the set temperature, pH, and salinity ranges for 2 h before measuring the surface tension at room temperature. Temperature range: 0℃, 20℃, 40℃, 60℃, 80℃, 100℃, 121℃; pH range: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; Salinity range: 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 10%, 12%, 14%, 16%, 18%, 20% (w / v).

[0090] Stability results are as follows Figure 6As shown, the surfactant produced by Bacillus amyloliquefaciens A3 exhibits good stability within a pH range of 4-12, a temperature range of 4℃-121℃, and a salinity range of 0%-20%, indicating that Bacillus amyloliquefaciens BA has good environmental adaptability and great potential for application in various fields.

[0091] (2) Emulsifying activity of surfactants produced by Bacillus amyloliquefaciens A3

[0092] The seed culture from Example 1 was centrifuged at 10,000 rpm for 10 min to obtain the supernatant. 2 mL of the supernatant and 2 mL of the hydrophobic matrix were placed in 15 mL test tubes and shaken vigorously for 2 min. The mixtures were then allowed to stand at room temperature for 24 h, and the emulsification index (E24) was measured. All tests were performed in triplicate. The hydrophobic matrices tested included: n-hexane, pentane, n-dodecane, n-hexadecane, diesel oil, liquid paraffin, soybean oil, toluene, and petroleum ether.

[0093]

[0094] Emulsification activity results as follows Figure 7 As shown, the supernatant of Bacillus amyloliquefaciens A3 exhibited the highest emulsification index for n-hexane at 51.20%, followed by petroleum ether (50.81%), liquid paraffin (50.00%), n-hexadecane (49.25%), diesel oil (46.21%), toluene (12.78%), soybean oil (12.70%), pentane (7.21%), and n-dodecane (4.76%). Specifically, Bacillus amyloliquefaciens A3 demonstrates excellent emulsification capabilities for hydrophobic matrices and shows promising performance in future applications involving the emulsification and solubilization of petroleum hydrocarbons.

[0095] Example 6: Application of Bacillus amyloliquefaciens BA in soil column leaching experiment.

[0096] Examples 2 and 3 show that *Bacillus amyloliquefaciens* A3 has a high ability to produce surfactants. Example 5 demonstrates that the surfactants produced by *Bacillus amyloliquefaciens* A3 have high stability and emulsifying activity. Therefore, in this example, the bacterial solution and crude surfactant produced by *Bacillus amyloliquefaciens* A3 are used to leach petroleum hydrocarbon-contaminated soil, and the soil remediation effect is observed. The specific steps are as follows:

[0097] (1) Preparation of contaminated soil:

[0098] Soil samples were taken from the top 0-10 cm layer of farmland soil in the suburbs of Beijing that was not contaminated by petroleum hydrocarbons. After removing impurities, the soil was air-dried and passed through a 2 mm sieve. 20 g of diesel oil and 20 g of engine oil were added to each kilogram of soil, mixed evenly, and aged for one week under dark and ventilated conditions.

[0099] (2) Soil column rinsing experiment:

[0100] Soil column washing experiment in Figure 8 The experiment was conducted in a reactor filled with 480g of a 3:1 (w / w) mixture of contaminated soil and quartz sand, totaling 20cm in depth. The soil was covered at both ends with cotton and 1cm high glass beads were added to evenly disperse the leaching solution. Two experimental groups were set up: one using deionized water and the other using Bacillus amyloliquefaciens A3 bacterial solution. Soil columns (200mL) were soaked in deionized water and a solution containing 2g of crude surfactant extract for 24h, respectively. The soil was then leached with either deionized water or Bacillus amyloliquefaciens A3 bacterial solution at a rate of 1mL / min for 48h. This soaking and leaching process was repeated 6 times. The soil columns were then removed, the soil was air-dried, and the quartz sand was removed. The content of n-alkanes in the soil and the activities of catalase (CAT), lipase (LPS), and dehydrogenase (DHA) were measured.

[0101] The final rinsing results are as follows Figure 9 visible, Figure 9 (A) shows that the removal rate of n-alkanes by the Bacillus amyloliquefaciens A3 treatment group was 45.44%, while the removal rate of n-alkanes by the deionized water treatment group was 38.75%. However, in terms of leached components, the amount of n-alkanes leached by the BA treatment group was 13.26% of the total pollution, while that by the deionized water treatment group was 3.46%. This indicates that the surfactant produced by Bacillus amyloliquefaciens A3 effectively activated the activity of hydrocarbons in the soil and reduced the tightness of the binding of hydrocarbons to soil particles. Figure 9 (C) shows that the solubilization in the A3 treatment group was higher than that in the deionized water treatment group in the range of n-alkanes C13-C18, and the removal rate of high-chain n-alkanes above C28 was 100%, which also demonstrates the effectiveness of Bacillus amyloliquefaciens A3 for leaching remediation.

[0102] Changes in enzyme activity during soil column leaching experiments, such as Figure 9 As shown in (DF), after rinsing treatment, compared with the deionized water experimental group, the activity of catalase (CAT) increased by 44.86%, the activity of lipase (LPS) increased by 261.11%, and the activity of dehydrogenase (DHA) decreased by 49.37%. This indicates that the addition of Bacillus amyloliquefaciens A3 has a stimulating effect on in-situ microorganisms, promoting the degradation and solubilization of hydrocarbons by regulating enzyme activity.

[0103] In summary, the *Bacillus amyloliquefaciens* A3 of this invention exhibits a high surfactant yield, producing 1.3976 g / L of lipopeptide surfactants under optimal conditions. These lipopeptide surfactants possess high stability and emulsifying ability. Application in the remediation of petroleum hydrocarbon-contaminated soil reveals that *Bacillus amyloliquefaciens* A3 and its produced surfactants can effectively promote the solubilization and emulsification of hydrocarbons tightly bound to the soil. Furthermore, it can influence enzyme activity through stimulation of in-situ microorganisms, thereby achieving the goals of solubilizing, degrading, and emulsifying hydrocarbons. Therefore, *Bacillus amyloliquefaciens* A3 can be used in the bioremediation of petroleum hydrocarbon-contaminated soil.

Claims

1. A method for preparing a lipopeptide surfactant, comprising the following steps: reacting Bacillus amyloliquefaciens (… Bacillus amyloliquefaciens Fermentation culture of strain A3 yielded a fermentation broth containing the lipopeptide surfactant; The Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The A3 strain has the accession number CGMCCNo.6676. The fermentation conditions were as follows: 25-35℃, pH 6.0-8.0, and 120-180 rpm for 72 h. The culture medium for fermentation contains a carbon source and a nitrogen source. The carbon source is selected from at least one of the following: glycerol, glucose, sodium citrate, sucrose, crude oil, and waste cooking oil. The nitrogen source is selected from at least one of the following: urea, ammonium sulfate, sodium nitrate, sodium nitrite, peptone, and ammonium nitrate. The method further includes: The specific method for separating the lipopeptide surfactant from the fermentation broth containing the lipopeptide surfactant is as follows: centrifuge the fermentation broth containing the lipopeptide surfactant, adjust the pH of the supernatant to 2-3 with hydrochloric acid, refrigerate it overnight at 4°C, centrifuge it again, freeze-dry the centrifuged product, and the brownish-yellow product obtained is the crude surfactant extracted.

2. The preparation method according to claim 1, characterized in that: The fermentation conditions were as follows: 25°C, pH 8.0, and 150 rpm for 72 h. The carbon source is glycerol; the nitrogen source is urea. The carbon source content in the culture medium is 40 g / L.

3. The preparation method according to claim 1, characterized in that: The culture medium also contains the following inorganic salts: dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, and calcium chloride.

4. The preparation method according to claim 3, characterized in that: The culture medium is composed of the following: glycerol 40 g / L, urea 2 g / L, dipotassium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 1.1 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.01 g / L, calcium chloride 0.01 g / L, pH 7.

2.

5. The preparation method according to claim 1, characterized in that: The centrifugation conditions are: centrifugation at 8000-10000 rpm for 10 min; The hydrochloric acid is 6 mol / L hydrochloric acid.

6. The lipopeptide surfactant or fermentation broth containing the lipopeptide surfactant prepared by the preparation method of any one of claims 1-5.

7. The application of the lipopeptide surfactant prepared by the preparation method according to any one of claims 1-5, or the fermentation broth containing said lipopeptide surfactant, is selected from at least one of the following aspects: 1) Applications in the emulsification, solubilization, and / or degradation of petroleum hydrocarbons; 2) Application in the bioremediation of petroleum hydrocarbon contaminated soil.

8. The Bacillus amyloliquefaciens in the preparation method of claim 1 ( Bacillus amyloliquefaciens A3 is used in at least one of the following applications: a) Application in the fermentation production of lipopeptide surfactants by bacterial strains; b) Applications in the emulsification, solubilization and / or degradation of petroleum hydrocarbons; c) Application in the bioremediation of petroleum hydrocarbon contaminated soil.

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