Microbacter sp. Strain for producing lipopeptide surfactant and application of microbacter sp. Strain

Abstract

The invention relates to a bacillus sp. Strain for producing a lipopeptide surfactant and application thereof, the bacillus sp. Strain is named as R3-1, and is preserved in the China General Microbiological Culture Collection Center on May 16, 2024, and the preservation number is CGMCC No.30660. The invention further discloses a preparation method of the bacillus sp. Strain for producing the lipopeptide surfactant and application of the bacillus sp. Strain for producing the lipopeptide surfactant. The lipopeptide surfactant produced by culturing and fermenting the cyst bacillus strain R3-1 comprises a surfactin and a progen. Compared with the prior art, the bacillus sp. Strain disclosed by the invention can simultaneously generate two lipopeptide surfactants, is strong in acid and alkali resistance, salt resistance and temperature adaptability, has a relatively good emulsifying effect on various hydrocarbon substances, and also has relatively good emulsifying capacity at relatively low concentration. Therefore, the method has great application potential in the aspects of oil exploitation and environmental pollution treatment caused by oil sludge.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a cystic bacillus strain producing lipopeptide surfactants and application thereof. Background Art

[0002] Biosurfactants are a class of amphiphilic natural compounds containing hydrophilic and hydrophobic groups produced by microbial metabolism. They can be arranged in a directional manner at the interface between the two phases to form a monolayer, replacing the original water or oil molecules on the interface, thereby effectively reducing the intermolecular forces of the solvent molecules and thus reducing the surface / interfacial tension. According to the different molecular structures, biosurfactants can be divided into glycolipids, lipopeptides, phospholipids, fatty acids, polymer surfactants and particulate surfactants. Compared with chemical synthetic surfactants, biosurfactants have broad application prospects in many fields such as petrochemicals, agriculture, environment, daily chemicals, and food due to their low toxicity, biodegradability, stability and excellent surface activity.

[0003] Lipopeptide biosurfactants are the most effective and most studied biosurfactants. According to reports, strains producing lipopeptide surfactants include Bacillus subtilis, Pseudomonas, Bacillus licheniformis, Acinetobacter, Bacillus amyloliquefaciens, etc. Different types of microorganisms produce different types of lipopeptides. At present, the reported lipopeptide surfactants include 34 families with more than 300 members. Among them, there are 28 families of cyclic lipopeptides with more than 260 members; there are 6 families of linear lipopeptides with more than 40 members. Cyclic lipopeptides include: fatty acid cyclic lipopeptides (such as surfactin, β-hydroxy fatty acid cyclic lipopeptides, lichenin, etc.), fatty acid cyclic lipopeptides (such as cyclic spores, echinocandins, etc.), fatty acid cyclic lipopeptides (such as fengyuan, phospholipids, kurstak, etc.). Most strains producing lipopeptide surfactants usually produce one lipopeptide compound, and there are limited reports of producing lipopeptide compounds with different structures at the same time. Structure determines performance. The use scenarios of lipopeptide surfactants with single performance are limited, and their acid, alkali, salt and temperature resistance are narrow. At the same time, although lipopeptide surfactants have strong surface activity, their emulsification properties are generally weak, and their fermentation costs are high and their yields are low, which further limits their application. Summary of the invention

[0004] The purpose of the present invention is to provide a lipopeptide surfactant-producing Bacillus strain and its application to solve the problems of the existing strains in the prior art, such as single type of surfactant production, weak emulsification performance, poor acid and alkali resistance, salt resistance and temperature adaptability, and limited application sites.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] One of the technical solutions of the present invention is to provide a Bacillus cystis strain that produces lipopeptide surfactants. The Bacillus cystis strain is named R3-1 and was deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on May 16, 2024, with a deposit number of CGMCC No. 30660.

[0007] In some specific embodiments, the lipopeptide surfactants produced by the Bacillus cystis strain R3-1 include surfactin and fengyrin.

[0008] A second technical solution of the present invention is to provide a use of the Bacillus cystis strain as described in one of the above technical solutions in emulsifying hydrocarbon compounds.

[0009] In some specific embodiments, the hydrocarbon compound includes n-hexane, n-hexadecane, benzene, toluene, kerosene, diesel, liquid paraffin, and crude oil.

[0010] In some specific embodiments, the fermentation broth obtained by fermenting the Bacillus cystis strain or the surfactant sample obtained by purifying the fermentation broth is used to emulsify hydrocarbon compounds.

[0011] In some embodiments, the method of fermenting the Bacillus cystis strain to obtain a fermentation broth comprises the following steps:

[0012] S1, culturing the seed solution of the Bacillus cystis strain R3-1 as described in claim 1 or 2;

[0013] S2. Inoculate the seed solution from step S1 into the fermentation medium for shaking fermentation, and collect the fermentation supernatant by centrifugation.

[0014] In some specific embodiments, in step S1, the seed solution culture process is as follows: inoculate the Bacillus cystis strain R3-1 into LB medium, and culture at 37° C. and 130 rpm on a shaker for (18 to 24) hours to form a seed culture solution;

[0015] In some specific embodiments, in step S2, the fermentation process is as follows: the seed liquid is inoculated into the fermentation medium at a volume ratio of 5%, and cultured at 37° C. and 130 rpm on a shaker for (70-72) h;

[0016] The fermentation medium includes the following raw material components with final concentrations: sucrose 15.0 g / L, ammonium sulfate 3.0 g / L, yeast powder 4.0 g / L, sodium chloride 1.1 g / L, potassium chloride 1.1 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 2.0 g / L, and pH is 7.0-7.2.

[0017] In some embodiments, the method for purifying the surfactant sample obtained from the fermentation broth comprises the following steps:

[0018] S3, adjusting the pH of the fermentation supernatant obtained in step S2 to alkaline, collecting the supernatant by centrifugation, adjusting the pH of the supernatant to acidic, letting it stand, and collecting the precipitate by centrifugation;

[0019] S4. Wash the precipitate obtained in step S3 until it is neutral, extract and collect the organic phase, and rotary evaporate and dry to obtain a surfactant sample, which contains surfactin and fengin.

[0020] In some embodiments, in step S3, the pH of the fermentation supernatant is adjusted to an alkaline pH of 12.0;

[0021] Adjust the pH of the supernatant to an acidic pH of ≤2.0;

[0022] The temperature of the static state is 4°C and the static state time is at least (10-12) hours;

[0023] In some specific embodiments, in step S4, the organic solvent used for extraction is ethyl acetate; and the temperature of rotary evaporation is 55°C to 65°C.

[0024] In some embodiments, the environmental conditions of the Bacillus cystis strain when emulsifying hydrocarbon compounds are:

[0025] The temperature is 20°C to 60°C, the pH is 5.0 to 9.0, and the NaCl concentration is 10g / L to 100g / L.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention provides a Bacillus cystis strain that simultaneously produces two lipopeptide surfactants, surfactin and fengyrin, thereby overcoming the problems of the prior art, such as the single type of surfactant produced by the strain and the large limitation of usage scenarios.

[0028] (2) Compared with the prior art, the surfactant produced by the Bacillus cystis strain provided by the present invention has strong acid and alkali resistance, salt resistance and temperature adaptability, and has a strong emulsification effect on liquid paraffin under the conditions of pH value of 5.0-9.0, temperature of 20°C-60°C and NaCl concentration of 10g / L-100g / L. 24 The index range is 32% to 67%;

[0029] It has good emulsification effect on various hydrocarbon substances. After mixing with hydrocarbons of different carbon chain lengths at a volume ratio of 1:1, it has strong emulsification ability. 24 The index range is 62.90% to 72.36%;

[0030] It also has a good emulsification effect at a lower concentration. It can still emulsify n-hexadecane and toluene at a low concentration (0.05g / L). The emulsification index E 24 All above 40%.

[0031] Therefore, it has great application potential in oil extraction, environmental pollution control caused by oil sludge, food, medical treatment, etc.

[0032] (3) The method for preparing lipopeptide surfactants provided by the present invention is simple, has low fermentation cost, is economical and environmentally friendly, and the two prepared lipopeptide surfactants have outstanding emulsifying properties and can replace chemical surfactants in scenarios with high environmental protection requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the phylogenetic tree of the Bacillus cystis strain R3-1 of the present invention.

[0034] Figure 2 This is the electrospray ionization mass spectrum of the lipopeptide surfactant prepared by the Bacillus cystis strain R3-1 of the present invention.

[0035] Figure 3 The fermentation liquid containing lipopeptide surfactant prepared by the cystic Bacillus strain R3-1 of the present invention is used to emulsify different hydrocarbon substances. 24 index.

[0036] Figure 4 The E of different concentrations of lipopeptide surfactant prepared by the Bacillus cystis strain R3-1 of the present invention to emulsify hydrocarbon substances 24 index.

[0037] Figure 5 The lipopeptide surfactant prepared for the Bacillus cystis strain R3-1 has temperature resistance, pH resistance and NaCl resistance. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0039] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0040] Embodiment 1:

[0041] This embodiment provides a Bacillus cystis strain producing two lipopeptide surfactants and a screening method thereof.

[0042] The screening method is as follows:

[0043] (1) Sample enrichment culture: The oil well output fluid obtained from Daqing Oilfield was inoculated into 100 mL of enrichment medium (LB medium) at a volume ratio of 5%, and cultured at 37°C and 130 rpm for 5 days; then, the inoculation volume was transferred to fresh LB medium at a volume ratio of 5%, and cultured at 37°C and 130 rpm. The transfer was repeated every 5 days for 3 times to obtain enriched culture fluid. The enriched culture fluid was inoculated into an inorganic salt medium containing 50 g / L crude oil at a volume ratio of 5%, and cultured at 37°C and 130 rpm for 7 days until the crude oil was completely emulsified;

[0044] The inorganic salt medium containing 50g / L crude oil includes: 50g / L crude oil, 3.0g / L ammonium sulfate, 4.0g / L yeast powder, 1.1g / L sodium chloride, 1.1g / L potassium chloride, 0.5g / L magnesium sulfate, 2.0g / L potassium dihydrogen phosphate. The pH is adjusted to 7.0-7.2, the temperature during sterilization is 121℃, and the sterilization time is 30min.

[0045] (2) Strain isolation and screening: The culture obtained in (1) was diluted to 10 -8 , take 10 -5 Up to 10 -8 0.1 mL of each dilution solution was applied to a culture dish containing LB solid medium prepared in advance, and the dish was placed in a 37°C incubator for constant temperature culture. After culturing for 24 h, a single colony was picked and its emulsification index (E) was detected using liquid paraffin (v:v = 1:1). 24 Index), the single bacteria with emulsification effect (E 24 >20%), repeatedly streaking and purifying, obtaining pure strains for identification and preservation.

[0046] (3) Fermentation production: The single colony prepared in step (2) was cultured as a seed solution in LB medium, and then inoculated into an inorganic salt medium containing 15.0 g / L sucrose at a volume ratio of 5% for fermentation culture at 37°C with a shaker speed of 130 rpm for 72 h to obtain a fermentation liquid.

[0047] The inorganic salt medium containing 15.0 g / L sucrose includes the following raw material components at final concentrations: sucrose 15.0 g / L, ammonium sulfate 3.0 g / L, yeast powder 4.0 g / L, sodium chloride 1.1 g / L, potassium chloride 1.1 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 2.0 g / L. The pH is adjusted to 7.0-7.2, the temperature during sterilization is 121°C, and the sterilization time is 30 min.

[0048] Verification of the emulsification performance of fermentation products: Take 20 mL of fermentation broth, centrifuge at 5000 rpm for 10 min to remove the bacteria, and use liquid paraffin as the emulsification object to measure its E 24 index.

[0049] Where E 24 The index determination method is as follows: the fermentation liquid is centrifuged to remove the bacteria, the fermentation supernatant is mixed with liquid paraffin at a volume ratio of 1:1, and vortexed on a vortex oscillator for 3 minutes to fully mix to form an emulsion of oil and water. After 24 hours, the height of the emulsion layer is read and the emulsification index (E) is calculated. 24 ) = emulsion layer height / total liquid height × 100%.

[0050] (4) Identification of strain taxonomy: Gene amplification was performed using the bacterial 16S rRNA universal primers 27F: 5'-AGAGTTTGATC MTGGCTCAG-3' (SEQ ID NO. 1) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2). The obtained sequences were aligned in Genbank, and a phylogenetic tree was constructed based on the 16S rRNA gene sequence using MEGA 7.0 ( Figure 1 ), the results showed that the strain obtained by screening, named R3-1, had a similarity of 99.93% with the currently reported Cytobacillus sp. strain B1013 / 17.

[0051] The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 16, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is Cytobacillus sp. and its deposit number is CGMCC No. 30660.

[0052] Embodiment 2:

[0053] This example provides a method for preparing two lipopeptide surfactants using the Bacillus cystis strain R3-1 and product identification.

[0054] (1) The Bacillus cystis strain R3-1 was inoculated into LB medium and cultured at 37° C. and 130 rpm for 24 h to form a seed culture solution.

[0055] (2) inoculating the seed liquid into the fermentation medium at a volume ratio of 5%, culturing at 37° C. and 130 rpm on a shaker for 72 h, and collecting the fermentation supernatant by centrifugation as a crude surfactant;

[0056] The fermentation medium includes the following raw material components with final concentrations: sucrose 15.0 g / L, ammonium sulfate 3.0 g / L, yeast powder 4.0 g / L, sodium chloride 1.1 g / L, potassium chloride 1.1 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 2.0 g / L, and pH is 7.0-7.2.

[0057] (3) Add 1.0 mol / L NaOH to the fermentation supernatant to adjust the pH to 12.0, centrifuge at 5000 rpm for 10 min, and discard the precipitate. Add 6.0 mol / L hydrochloric acid to the supernatant to adjust the pH to 2.0, let stand at 4°C for 12 h, and centrifuge again at 5000 rpm for 10 min to obtain a precipitate as a surfactant mixture.

[0058] (4) The surfactant mixture was washed with water until neutral, then extracted three times with an equal volume of ethyl acetate, and the organic phases were combined and rotary evaporated at 55°C to 65°C until it became a paste. The precipitate was then dried using a low-temperature vacuum freeze dryer to obtain a surfactant sample.

[0059] (5) The substances produced by the Bacillus cystis strain R3-1 were characterized by electrospray ionization mass spectrometry (ESI-MS). About 3.0 to 5.0 mg of the product obtained in step (4) was weighed and dissolved in a methanol solution containing 0.10% formic acid. The mass-to-charge ratio characteristics of the product were determined by electrospray ionization mass spectrometry.

[0060] like Figure 2 , calculated by ESI-MS positive and negative ion mass spectrometry, the substances produced by the Bacillus cystis strain R3-1 contain two lipopeptide surfactant substances with different structures, one is a homologue with molecular weights of 1007.6, 1021.7 and 1035.7, and the other is a homologue with molecular weights of 1462.8, 1476.8, 1490.8 and 1504.8. As far as we know, these two biosurfactants match the lipopeptide biosurfactants, corresponding to surfactin and fengycin, respectively.

[0061] (6) HPLC determination of amino acid composition

[0062] The amino acid composition of the product was analyzed by high performance liquid chromatography. The main amino acids in the sample were Asp, Glu, Gly, Thr, Ala, Pro, Tyr, Val, Ile, Leu and Lys. It is reported that the fengyuansu family contains different members, but they all have the same peptide ring, which is composed of 2 Tyr and Glu and Gln, Thr, Pro, Ile and Ala or Val. The peptide ring part of the surfactin family is usually composed of Glu, Asp, Val and 4 Leu. According to the structure of the reported biosurfactants and their molecular weight fingerprint analysis, all the amino acids in the peptide ring of surfactin and fengyuansu were detected in the sample, proving that these two lipopeptide surfactants belong to the surfactin family and the fengyuansu family, respectively.

[0063] (7) Fourier transform infrared spectroscopy (FTIR) was used to characterize the functional groups of substances produced by the metabolism of Bacillus cystis strain R3-1:

[0064] 2.0 mg of the sample purified in step (4) was measured on a Fourier transform infrared spectrometer at a frequency of 4,000 to 400 cm -1 Carry out at 25℃.

[0065] The infrared spectrum obtained by scanning is at 3418.9cm -1 There is a strong absorption band, indicating that the substance has CH and NH hydrocarbons containing amino groups; at 2973.6cm -1 There is an absorption peak at 2928.6, which is the stretching vibration of the CH bond on the aliphatic carbon chain, indicating that the CH 2 and CH 3 The group has symmetrical stretching (γC-H). -1 There is a strong absorption peak, indicating the presence of -CO-NH amide bond (peptide bond). 1404.1cm -1 It is the CH bending vibration absorption band on the carbon chain, at 1048.2 cm -1 There is a strong absorption peak of -OCO stretching vibration absorption band. Therefore, this substance is a lipopeptide surfactant, which was confirmed by Fourier transform infrared analysis.

[0066] Embodiment 3:

[0067] This example provides a test for the emulsifying properties of the metabolites of Bacillus cystis strain R3-1:

[0068] (1) Emulsification ability of fermentation broth for different hydrocarbon substances:

[0069] The fermentation supernatant obtained by fermentation in step (2) of Example 2 was mixed with 8 hydrocarbon substances with different carbon chain lengths (n-hexane, n-hexadecane, benzene, toluene, kerosene, diesel, liquid paraffin, and crude oil) at a volume ratio of 1:1 for 3 minutes and left at room temperature for 24 hours to measure E 24 Index. 24 Index such as Figure 3 shown.

[0070] pass Figure 3 It can be seen that the substances produced by the metabolism of the Bacillus cystis strain R3-1 have a good emulsification effect on 8 hydrocarbons with different carbon chain lengths. 24 Among them, n-hexadecane has the best emulsification effect, and the emulsification index E 24 Reaching 72.36%, followed by crude oil, whose E 24 The index is 67.36%.

[0071] (2) Emulsification properties of lipopeptide biosurfactants at different concentrations:

[0072] The lipopeptide surfactant purified by ethyl acetate in step (4) of Example 2 was prepared into different concentrations (0.14 g / L, 0.10 g / L and 0.05 g / L), and toluene, n-hexadecane and liquid paraffin were used as emulsification objects, respectively, and mixed for 3 min at a volume ratio (v:v=1:1), and placed at room temperature for 24 h to measure E 24 Index. 24 Index such as Figure 4 shown.

[0073] pass Figure 4 It can be seen that at concentrations of 0.14 g / L and 0.10 g / L, the lipopeptide surfactant has a good emulsification effect on the three substances. The higher the concentration, the better the emulsification effect. When the concentration is 0.05 g / L, it still has emulsification ability for toluene and n-hexadecane, but has a weak effect on the E of liquid paraffin. 24 The index is zero.

[0074] Embodiment 4:

[0075] This example provides an environmental tolerance test for the metabolites of Bacillus cystis strain R3-1. 24 Index such as Figure 5 As shown:

[0076] (1) Optimum temperature of lipopeptide surfactants

[0077] The fermentation supernatant obtained in step (2) of Example 2 was divided into different test tubes, each containing 10 mL of liquid. They were kept at 20°C, 30°C, 40°C, 50°C and 60°C for 20 min. The emulsification index (E) in Example 1 was calculated. 24 ) assay method was used to determine the emulsification index, and the emulsification activity of the surfactant produced by the Bacillus cystis strain R3-1 at different temperatures was determined.

[0078] The results show that the emulsification activity of the surfactant is above 45% in the range of 20-60℃. The optimum temperature for the highest emulsification capacity is 20℃, and the emulsification index E 24 The value is 67.1%.

[0079] (2) Optimal pH of lipopeptide surfactants

[0080] The fermentation supernatant obtained by fermentation in step (2) of Example 2 was divided into different test tubes, each test tube containing 10 mL of liquid. The pH of the fermentation supernatant was adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0 with 1.0 mol / L NaOH or 6.0 mol / L HCl and maintained for 20 min. The emulsification index (E) in Example 1 was used to determine the pH of the fermentation supernatant. 24 ) method for determining the emulsification index.

[0081] The results showed that the emulsification index of the surfactant was within the range of pH 5 to 9. 24 The optimum pH value for the highest emulsification capacity is 7.0, and the emulsification index E 24 The value is 67.0%.

[0082] (3) Optimal NaCl concentration of lipopeptide surfactants

[0083] The fermentation supernatant obtained by fermentation in step (2) of Example 2 was divided into different test tubes, and 10 g / L, 12 g / L, 16 g / L, 20 g / L, 40 g / L, 60 g / L, 80 g / L and 100 g / L of reagent grade NaCl powder were added thereto respectively, and after dissolution, the mixture was left to stand for 20 min. 24 ) method for determining the emulsification index.

[0084] The results show that the emulsification ability is good when the NaCl addition amount is 10-100g / L. 24 Greater than 30%. The optimum NaCl concentration for the highest emulsification capacity is 10 g / L, and the emulsification index E 24 The value was 67.4%, indicating that the surfactant produced by the Bacillus cystis strain R3-1 had a certain salt tolerance in a wide range of NaCl.

[0085] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A cystic bacillus strain producing a lipopeptide surfactant, characterized in that: The Bacillus cystis strain was named R3-1 and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 16, 2024, with the deposit number CGMCC No.30660.

2. The cystic bacillus strain producing lipopeptide surfactant according to claim 1, characterized in that: The lipopeptide surfactants produced by the Bacillus cystis strain R3-1 include surfactin and fengyrin.

3. Use of the Bacillus cystis strain as claimed in claim 1 or 2 in emulsifying hydrocarbon compounds.

4. The use according to claim 3, characterized in that: The hydrocarbon compounds include n-hexane, n-hexadecane, benzene, toluene, kerosene, diesel, liquid paraffin, and crude oil.

5. The use according to claim 3, characterized in that: The fermentation broth obtained by fermenting the Bacillus cystis strain or the surfactant sample obtained by purifying the fermentation broth is used to emulsify hydrocarbon compounds.

6. The use according to claim 5, characterized in that: The method for fermenting the cystic bacillus strain to obtain a fermentation liquid comprises the following steps: S1, culturing the seed solution of the Bacillus cystis strain R3-1 as described in claim 1 or 2; S2. Inoculate the seed solution from step S1 into the fermentation medium for shaking fermentation, and collect the fermentation supernatant by centrifugation.

7. The use according to claim 6, characterized in that: In step S1, the seed solution culture process is as follows: the Bacillus cystis strain R3-1 is inoculated into LB medium, and cultured at 37° C. and 130 rpm on a shaker for (18-24) hours to form a seed culture solution; In step S2, the fermentation process is as follows: the seed liquid is inoculated into the fermentation medium at a volume ratio of 5%, and cultured at 37° C. and 130 rpm on a shaker for (70-72) h; The fermentation medium includes the following raw material components with final concentrations: sucrose 15.0 g / L, ammonium sulfate 3.0 g / L, yeast powder 4.0 g / L, sodium chloride 1.1 g / L, potassium chloride 1.1 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 2.0 g / L, and pH is 7.0-7.

2.

8. The use according to claim 6, characterized in that: The method for purifying the surfactant sample obtained by fermentation broth comprises the following steps: S3, adjusting the pH of the fermentation supernatant obtained in step S2 to alkaline, collecting the supernatant by centrifugation, adjusting the pH of the supernatant to acidic, letting it stand, and collecting the precipitate by centrifugation; S4. Wash the precipitate obtained in step S3 until it is neutral, extract and collect the organic phase, and rotary evaporate and dry to obtain a surfactant sample, which contains surfactin and fengin.

9. The use according to claim 8, characterized in that: In step S3, the pH of the fermentation supernatant is adjusted to an alkaline pH value of 12.0; Adjust the pH of the supernatant to an acidic pH of ≤2.0; The temperature of the static state is 4°C and the static state time is at least (10-12) hours; In step S4, the organic solvent for extraction is ethyl acetate; the temperature of rotary evaporation is 55°C to 65°C.

10. The use according to claim 3, characterized in that: The environmental conditions of the cystic bacillus strain when emulsifying hydrocarbon compounds are: The temperature is 20°C to 60°C, the pH is 5.0 to 9.0, and the NaCl concentration is 10g / L to 100g / L.

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