Rhamnolipid fermentation and separation method

Abstract

The invention discloses a rhamnolipid fermentation and separation method. The rhamnolipid fermentation method comprises the following steps: carrying out fermentation culture on rhamnolipid-producing microorganisms in a nitrate-containing fermentation culture medium to obtain a strain liquid, and introducing nitric oxide-containing mixed gas in the fermentation culture process. The invention discloses a method for obtaining rhamnolipid by separating strain liquid. The method comprises the following steps: centrifuging; performing concentration; extraction: adding a lipophilic compound into the concentrated clear liquid for extraction, standing to layer the concentrated clear liquid, and collecting an upper lipophilic compound phase rich in rhamnolipid; evaporating to obtain concentrated rhamnolipid; and washing and drying the concentrated rhamnolipid to obtain a rhamnolipid pure product. According to the method, intracellular products can be promoted to be secreted outside cells, so that the yield of rhamnolipid is increased; meanwhile, through a purification method, the yield is effectively improved under the condition that the purity is ensured.

Description

Technical Field

[0001] The invention relates to the field of rhamnolipid production, and in particular to a rhamnolipid fermentation and separation method. Background Art

[0002] Rhamnolipid is a biosurfactant widely used in environmental remediation, cosmetics, food additives and pharmaceutical industries. It can be produced by microbial fermentation. Specifically, fermentation broth containing rhamnolipid is produced by microbial fermentation, and then pure rhamnolipid is separated and extracted from the fermentation broth.

[0003] At present, the types of microorganisms that can ferment and produce rhamnolipids include Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putica, etc., but the existing types of microorganisms all have the problem of low yield of rhamnolipids produced.

[0004] At the same time, in addition to the target product rhamnolipid, the fermentation broth also contains impurities such as microbial cells, unconsumed nutrients, metabolic byproducts, etc. Traditional separation and purification methods include centrifugation, filtration, extraction, chromatography, etc., but the pure rhamnolipid obtained by these methods has the problem of low purity or low yield, or has the problem of high cost. Summary of the invention

[0005] Therefore, the first technical problem to be solved by the present invention is that the yield of rhamnolipid produced by fermentation of existing strains is low, so as to provide a rhamnolipid fermentation method for increasing the yield of rhamnolipid.

[0006] A rhamnolipid fermentation method comprises fermenting rhamnolipid-producing microorganisms in a fermentation medium containing nitrate to obtain a bacterial liquid, and introducing a mixed gas containing nitric oxide during the fermentation process.

[0007] The initial introduction amount of the mixed gas is above 0.2 vvm, the volume percentage of nitric oxide in the mixed gas is 0.01-0.025%, and the dissolved oxygen is above 15% after the mixed gas is introduced.

[0008] Dissolved oxygen refers to the amount of oxygen dissolved in the fermentation medium, and the unit is percentage saturation (%). A dissolved oxygen saturation above 15% means that the oxygen content in the liquid is more than 15% of the maximum dissolved oxygen capacity at the corresponding temperature and pressure.

[0009] The nitrate is sodium nitrate.

[0010] During the fermentation culture, the fermentation temperature is 25-40° C., and the initial pH of the fermentation is 6.8-7.4.

[0011] The fermentation medium comprises, by mass percentage, 3-5% glycerol, 0.8-2.2% yeast powder, 2.0-4.6% peptone, 0.8-3% NaNO3, 0.1-0.5% KH2PO4, 0.1-0.8% Na2HPO4·12H2O, 0.1-0.8% MgSO4·7H2O, 0.8-1.2% CaCl2·2H2O, 0.1%-0.7% FeSO4, 0.5%-1.2% CoCl2, and the balance is water;

[0012] And / or, the inoculation amount of the microorganism is 3-8% of the mass of the fermentation medium.

[0013] The types of rhamnolipid-producing microorganisms include one or more of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas putida.

[0014] The concentration of rhamnolipid in the culture liquid is above 80g / L.

[0015] The second technical problem to be solved by the present invention is that the separation and purification methods in the prior art have the problems of low yield, low purity or high cost, so as to provide a method for separating and obtaining rhamnolipids by improving the purity and yield of rhamnolipids at a low cost.

[0016] Among them, the method of using bacterial liquid to separate and obtain rhamnolipids includes:

[0017] Centrifugation: Centrifuge the bacterial liquid to obtain a precipitate and a clear liquid;

[0018] Concentration: concentrating the clear liquid to obtain a concentrated clear liquid;

[0019] Extraction: Add lipophilic compounds to the concentrated clear solution for extraction, let it stand to separate into layers, and collect the upper lipophilic compound phase rich in rhamnolipid;

[0020] Evaporation: evaporating the lipophilic compound phase rich in rhamnolipid to remove the lipophilic compound and obtain concentrated rhamnolipid;

[0021] Purified rhamnolipids are obtained by washing and drying the concentrated rhamnolipids.

[0022] The centrifugal speed is 8000-10000rpm, and the centrifugal time is 8-15min;

[0023] And / or, the concentration method is reduced pressure concentration, and the volume of the concentrated clear liquid is 1 / 3-1 / 2 of the volume of the clear liquid;

[0024] And / or, the standing time is 2-4h;

[0025] and / or, the volume of the lipophilic compound added is 1 / 5-1 / 3 of the volume of the concentrated clear solution;

[0026] And / or, the evaporation is evaporation under reduced pressure.

[0027] The reduced pressure concentration is reduced pressure rotary evaporation, and the conditions are: rotation speed 150-200rpm, temperature 40-60°C, pressure 50-80pa;

[0028] And / or, the lipophilic compound includes at least one of n-butanol, isobutanol, and ethyl acetate;

[0029] And / or, the reduced pressure evaporation is reduced pressure rotary evaporation, and the conditions are: rotation speed 250-400 rpm, temperature 60-80° C., and pressure 10-40 Pa.

[0030] The technical solution of the present invention has the following advantages:

[0031] 1. The present invention provides a rhamnolipid fermentation method, which effectively achieves the purpose of significantly increasing the yield of rhamnolipid by cooperating with nitrate and introduced nitric oxide; the implementation mechanism may be: nitrate, as a nitrogen source, promotes the synthesis of rhamnolipid; nitric oxide is not only a nitrogen source, but also an important signal molecule, which may affect the physiological state of bacteria, for example: nitric oxide can induce biofilm dispersion by reducing the level of secondary messenger di-(3′-5′)-cyclic dimer guanosine monophosphate (c-di-GMP), thereby producing enzymes that decompose the biofilm matrix and promote dispersion, and promote the secretion of intracellular products to the extracellular, thereby increasing the yield of rhamnolipid.

[0032] 2. The method for separating rhamnolipid from bacterial liquid provided by the present invention, in addition to the conventional centrifugation step, also includes the steps of concentration, extraction, and evaporation, wherein the extraction step uses lipophilic compounds as extractants, effectively improving the extraction efficiency and purity of rhamnolipid at a lower cost; its implementation mechanism may be: lipophilic compounds have a higher partition coefficient in fat-soluble organic phase and aqueous phase, and they usually have a strong solubility in fat, oil, lipid and non-polar solvents, and rhamnolipid is a fat-soluble organic matter that can be dissolved in lipophilic compounds, and the density of lipophilic compounds is less than that of water, and can be separated from water. Therefore, using lipophilic compounds as extractants can effectively separate rhamnolipid from bacterial liquid, achieve the purpose of separation and purification, and effectively improve the yield while ensuring purity. And after testing, the purity of rhamnolipid obtained by the method of the present invention can reach 93.2%, and the yield can reach 98.6%, with significant effects.

[0033] 3. In the method of the present invention, the purity of the obtained pure rhamnolipid is relatively high, which meets the demand for high-purity products; at the same time, the lipophilic compounds can be recovered and reused by evaporation, and the recovery of the lipophilic compounds reduces the production cost, conforms to the principle of green chemistry, and is beneficial to environmental protection; and the overall production process of the pure rhamnolipid is easy to operate and can achieve large-scale production. DETAILED DESCRIPTION

[0034] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0035] If the specific experimental steps or conditions are not specified in the embodiments, the conventional experimental steps or conditions described in the literature in the art can be used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional reagent products that can be obtained commercially. The seed culture medium used in the present invention is selected from a conventional culture medium, for example: LB culture medium.

[0036] Pseudomonas aeruginosa was purchased from China Industrial Microorganism Culture Collection Administration Center with the accession number CICC 10351; Pseudomonas fluorescens was purchased from China Industrial Microorganism Culture Collection Administration Center with the accession number CICC 23246; Pseudomonas putica was purchased from China Industrial Microorganism Culture Collection Administration Center with the accession number CICC 21906.

[0037] Example 1

[0038] A method for preparing a pure rhamnolipid, including a method for obtaining a bacterial liquid containing rhamnolipid by fermenting rhamnolipid, and a method for obtaining rhamnolipid by separating the bacterial liquid. The specific preparation process is as follows:

[0039] 1. Prepare a fermentation medium, the composition of which is as follows: the mass percentage formula is 3% glycerol, 2.2% yeast powder, 4.6% peptone, 0.8% NaNO3, 0.1% KH2PO4, 0.1% Na2HPO4·12H2O, 0.1% MgSO4·7H2O, 0.8% CaCl2·2H2O, 0.1% FeSO4, 0.5% CoCl2, and the balance is water.

[0040] 2. Sterilize at 121°C for 15 min.

[0041] 3. Inoculate Pseudomonas aeruginosa into 200 ml of seed culture medium, culture at 33°C and 180 rpm for 28 h to obtain a primary seed solution.

[0042] 4. Inoculate the primary seed solution into 2000 ml of seed culture medium at an inoculation rate of 5% (mass percentage), and culture at 32°C and 220 rpm for 35 h to obtain the secondary seed solution.

[0043] 5. Inoculate the secondary seed liquid at a rate of 10% (volume ratio) into a 50L fermentation tank containing 20L fermentation medium for fermentation.

[0044] 6. When the microorganisms begin to ferment, a gas containing 0.01% by volume of nitric oxide is introduced. The nitric oxide gas forms a mixture with air and enters the fermentation tank through the air distributor at the bottom of the fermentation tank and dissolves into the fermentation liquid.

[0045] 7. The fermentation temperature is 32°C, the initial pH of the fermentation is 7.1, the initial rotation speed is 150rpm, the initial ventilation is 0.2vvm, and the rotation speed and ventilation are increased according to the dissolved oxygen to control the dissolved oxygen above 15%.

[0046] 8. Detect the concentration of rhamnolipid in the fermentation broth every hour; Method for determining the concentration of rhamnolipid: First, prepare a rhamnolipid standard curve, dilute the rhamnolipid standard to 0.1, 0.2, 0.5, and 1 g / L, and measure its absorbance at a wavelength of 620 nm, respectively, with the concentration as the horizontal axis and the absorbance value as the vertical axis, fit the standard curve, R2>0.99, and then measure the sample: centrifuge the fermentation broth at 10°C and 4000 rpm for 20 minutes to precipitate the bacteria, take the supernatant after centrifugation and dilute it a certain multiple; then take 1 mL of the dilution and add it to a test tube, put the test tube into ice water, and slowly drip 4 mL along the wall of the test tube 0.2% anthrone reagent was added and continuously shaken to mix evenly; then the test tube was immersed in a boiling water bath and heated for 15 minutes, taken out and immediately cooled with cold water to terminate the reaction, and placed at room temperature for 20 minutes; finally, the absorbance of the series of rhamnose solutions was measured at a wavelength of 620nm using an ultraviolet spectrophotometer; the uninoculated fermentation medium was used as a blank control, and the absorbance of the diluted supernatant was measured; finally, the measured absorbance was substituted into the rhamnolipid standard curve equation to obtain the rhamnolipid content.

[0047] 9. When the rhamnolipid concentration is greater than 80g / L for two consecutive hours, the tank can be stopped.

[0048] 10. Centrifuge the fermentation broth to separate the precipitate and supernatant at 10,000 rpm for 15 minutes.

[0049] 11. The supernatant after centrifugation was evaporated by vacuum rotary evaporation. The vacuum rotary evaporation conditions were: speed 200 rpm, temperature 60°C, pressure 50 Pa.

[0050] 12. Concentrate to 1 / 2 of the original volume.

[0051] 13. Add 1 / 5 volume of the lipophilic compound n-butanol to the concentrated supernatant and let it stand for 2 hours to allow it to separate naturally.

[0052] 14. Use a biological pump to extract the upper lipophilic compound phase rich in rhamnolipids.

[0053] 15. The lipophilic compound phase rich in rhamnolipid is subjected to reduced pressure evaporation to remove the lipophilic compounds and obtain concentrated rhamnolipid; the reduced pressure rotary evaporation conditions are: rotation speed 300 rpm, temperature 70°C, and pressure 30 Pa.

[0054] 16. Add 3 times the volume of sterile water to the concentrated rhamnolipid for washing and dry at 65°C to constant weight to obtain pure rhamnolipid.

[0055] Example 2

[0056] The difference from Example 1 is that in step 1, a fermentation medium is prepared, and its composition is: the mass percentage formula is 4% glycerol, 1.2% yeast powder, 3.2% peptone, 2.2% NaNO3, 0.4% KH2PO4, 0.5% Na2HPO4·12H2O, 0.5% MgSO4·7H2O, 1.1% CaCl2·2H2O, 0.4% FeSO4, 0.9% CoCl2, and the balance is water.

[0057] In step 6, when the microorganisms start to ferment, a gas containing 0.02% by volume of nitric oxide is introduced. The nitric oxide gas forms a mixture with air and enters the fermenter through the air distributor at the bottom of the fermenter and is dissolved in the fermentation liquid.

[0058] In step 7, the fermentation temperature is 32° C., the initial pH of the fermentation is 6.8, the initial rotation speed is 150 rpm, the initial ventilation is 0.2 vvm, and the rotation speed and ventilation are increased according to the dissolved oxygen to control the dissolved oxygen to be above 15%.

[0059] The other steps and parameter conditions are the same as those in Example 1.

[0060] Example 3

[0061] The difference from Example 1 is that in step 1, a fermentation medium is prepared, and its composition is: the mass percentage formula is 5% glycerol, 0.8% yeast powder, 2.0% peptone, 3% NaNO3, 0.5% KH2PO4, 0.8% Na2HPO4·12H2O, 0.8% MgSO4·7H2O, 1.2% CaCl2·2H2O, 0.7% FeSO4, 1.2% CoCl2, and the balance is water.

[0062] In step 7, the fermentation temperature is 32° C., the initial pH of the fermentation is 7.4, the initial rotation speed is 150 rpm, the initial ventilation is 0.2 vvm, and the rotation speed and ventilation are increased according to the dissolved oxygen to control the dissolved oxygen to be above 15%.

[0063] The other steps and parameter conditions are the same as those in Example 1.

[0064] Example 4

[0065] The difference from Example 1 is that in step 13, 1 / 3 of the volume of the lipophilic compound n-butanol is added to the concentrated supernatant, and the mixture is allowed to stand for 4 hours to allow natural stratification.

[0066] The other steps and parameter conditions are the same as those in Example 1.

[0067] Example 5

[0068] The difference from Example 1 is that in step 13, the lipophilic compound in the concentrated supernatant is 1 / 3 of the volume of isobutanol, and the supernatant is allowed to stand for 4 hours to allow natural stratification.

[0069] The other steps and parameter conditions are the same as those in Example 1.

[0070] Example 6

[0071] The difference from Example 1 is that in step 13, in the concentrated supernatant, the lipophilic compound is ethyl acetate, which accounts for 1 / 3 of the volume, and the supernatant is left to stand for 4 hours to allow natural stratification.

[0072] The other steps and parameter conditions are the same as those in Example 1.

[0073] Example 7

[0074] The difference from Example 1 is that in step 3, Pseudomonas fluorescens is inoculated into 200 ml of seed culture medium, and cultured at 33° C. and 180 rpm for 28 hours to obtain a primary seed solution.

[0075] The other steps and parameter conditions are the same as those in Example 1.

[0076] Example 8

[0077] The difference from Example 1 is that in step 3, Pseudomonas putica is inoculated into 200 ml of seed culture medium, and cultured at 33° C. and 180 rpm for 28 hours to obtain a primary seed solution.

[0078] The other steps and parameter conditions are the same as those in Example 1.

[0079] Example 9

[0080] The difference from Example 1 is that in step 13, 1 / 3 of the volume of water is added to the concentrated supernatant and the mixture is allowed to stand for 4 hours.

[0081] The other steps and parameter conditions are the same as those in Example 1.

[0082] Example 10

[0083] The difference from Example 1 is that in step 13, 1 / 3 of the volume of petroleum ether is added to the concentrated supernatant and the mixture is allowed to stand for 4 hours.

[0084] The other steps and parameter conditions are the same as those in Example 1.

[0085] Embodiment 11

[0086] A method for preparing a pure rhamnolipid, including a method for obtaining a bacterial liquid containing rhamnolipid by fermenting rhamnolipid, and a method for obtaining rhamnolipid by separating the bacterial liquid. The specific preparation process is as follows:

[0087] 1. Prepare a fermentation medium, the composition of which is as follows: 3% glycerol, 2.2% yeast powder, 4.6% peptone, 0.8% NaNO3, 0.1% KH2PO4, 0.1% Na2HPO4·12H2O, 0.1% MgSO4·7H2O, 0.8% CaCl2·2H2O, 0.1% FeSO4, 0.5% CoCl2, and the balance is water;

[0088] 2. Sterilize at 121°C for 15 min;

[0089] 3. Inoculate Pseudomonas aeruginosa into 200 ml of seed culture medium and culture at 33°C and 180 rpm for 28 h to obtain a primary seed solution;

[0090] 4. Inoculate the first-level seed solution into 2000 ml of seed culture medium at an inoculum volume of 5% (mass percentage), and culture at 32°C and 220 rpm for 35 h to obtain the second-level seed solution;

[0091] 5. Inoculate the secondary seed solution at a volume ratio of 10% into a 50L fermentation tank containing 20L fermentation medium for fermentation;

[0092] 6. When the microorganisms begin to ferment, a gas containing 0.01% by volume of nitric oxide is introduced. The nitric oxide gas forms a mixture with air and enters the fermenter through the air distributor at the bottom of the fermenter and dissolves into the fermentation liquid;

[0093] 7. The fermentation temperature is 25°C, the initial pH of the fermentation is 7.1, the initial speed is 150rpm, the initial ventilation is 0.2vvm, and the speed and ventilation are increased according to the dissolved oxygen to control the dissolved oxygen above 15%;

[0094] 8. Detect the concentration of rhamnolipid in the fermentation broth every hour;

[0095] 9. When the rhamnolipid concentration is greater than 80g / L for two consecutive hours, the tank can be stopped;

[0096] 10. Centrifuge the fermentation broth to separate the precipitate and supernatant at 9000 rpm for 8 min;

[0097] 11. The supernatant after centrifugation was evaporated by vacuum rotary evaporation. The vacuum rotary evaporation conditions were: speed 150 rpm, temperature 50 ° C, pressure 60 Pa;

[0098] 12. Concentrate to 1 / 3 of the original volume;

[0099] 13. Add 1 / 5 volume of lipophilic compound n-butanol to the concentrated supernatant and let it stand for 2 hours to allow it to separate naturally;

[0100] 14. Use a biological pump to extract the upper lipophilic compound phase rich in rhamnolipids;

[0101] 15. The lipophilic compound phase rich in rhamnolipid is subjected to reduced pressure evaporation to remove the lipophilic compound and obtain concentrated rhamnolipid; the reduced pressure rotary evaporation conditions are: speed 250 rpm, temperature 80°C, pressure 10 Pa;

[0102] 16. Add 3 times the volume of sterile water to the concentrated rhamnolipid for washing and dry at 65°C to constant weight to obtain pure rhamnolipid.

[0103] Example 12

[0104] A method for preparing a pure rhamnolipid, including a method for obtaining a bacterial liquid containing rhamnolipid by fermenting rhamnolipid, and a method for obtaining rhamnolipid by separating the bacterial liquid. The specific preparation process is as follows:

[0105] 1. Prepare a fermentation medium, the composition of which is as follows: 3% glycerol, 2.2% yeast powder, 4.6% peptone, 0.8% NaNO3, 0.1% KH2PO4, 0.1% Na2HPO4·12H2O, 0.1% MgSO4·7H2O, 0.8% CaCl2·2H2O, 0.1% FeSO4, 0.5% CoCl2, and the balance is water;

[0106] 2. Sterilize at 121°C for 15 min;

[0107] 3. Inoculate Pseudomonas aeruginosa into 200 ml of seed culture medium and culture at 33°C and 180 rpm for 28 h to obtain a primary seed solution;

[0108] 4. Inoculate the first-level seed solution into 2000 ml of seed culture medium at an inoculum volume of 5% (mass percentage), and culture at 32°C and 220 rpm for 35 h to obtain the second-level seed solution;

[0109] 5. Inoculate the secondary seed solution at a volume ratio of 10% into a 50L fermentation tank containing 20L fermentation medium for fermentation;

[0110] 6. When the microorganisms begin to ferment, a gas containing 0.01% by volume of nitric oxide is introduced. The nitric oxide gas forms a mixture with air and enters the fermenter through the air distributor at the bottom of the fermenter and dissolves into the fermentation liquid;

[0111] 7. The fermentation temperature is 40°C, the initial pH of the fermentation is 7.1, the initial speed is 150rpm, the initial ventilation is 0.2vvm, and the speed and ventilation are increased according to the dissolved oxygen to control the dissolved oxygen above 15%;

[0112] 8. Detect the concentration of rhamnolipid in the fermentation broth every hour;

[0113] 9. When the rhamnolipid concentration is greater than 80g / L for two consecutive hours, the tank can be stopped;

[0114] 10. Centrifuge the fermentation broth to separate the precipitate and supernatant at 8000 rpm for 12 min;

[0115] 11. The supernatant after centrifugation was evaporated by vacuum rotary evaporation. The vacuum rotary evaporation conditions were: speed 180 rpm, temperature 40 ° C, pressure 80 Pa;

[0116] 12. Concentrate to 1 / 3 of the original volume;

[0117] 13. Add 1 / 5 volume of lipophilic compound n-butanol to the concentrated supernatant and let it stand for 2 hours to allow it to separate naturally;

[0118] 14. Use a biological pump to extract the upper lipophilic compound phase rich in rhamnolipids;

[0119] 15. The lipophilic compound phase rich in rhamnolipid is subjected to reduced pressure evaporation to remove the lipophilic compound and obtain concentrated rhamnolipid; the reduced pressure rotary evaporation conditions are: speed 400 rpm, temperature 60°C, pressure 40 Pa;

[0120] 16. Add 3 times the volume of sterile water to the concentrated rhamnolipid for washing and dry at 65°C to constant weight to obtain pure rhamnolipid.

[0121] Comparative Example 1

[0122] The difference between this comparative example and Example 1 is that in step 1, NaNO3 is replaced by an equal amount of water.

[0123] Comparative Example 2

[0124] The difference between this comparative example and Example 1 is that air without nitric oxide is introduced in step 6.

[0125] Comparative Example 3

[0126] The difference between this comparative example and Example 1 is that in step 1, NaNO3 is replaced with an equal amount of water; and in step 6, air without nitric oxide is introduced.

[0127] The pure rhamnolipids of the above-mentioned examples and comparative examples were tested using the method for determining the concentration of rhamnolipids in step 8 to obtain the purity of the pure rhamnolipids, and the yield of the pure rhamnolipids was calculated.

[0128] Yield = (total weight of pure rhamnolipid * purity of pure rhamnolipid) / (concentration of rhamnolipid in fermentation broth * volume of fermentation broth) * 100%;

[0129] The results of various embodiments and comparative tests are shown in Table 1 below.

[0130] Table 1

[0131]

[0132]

[0133] Obviously, the above embodiments are only examples for clear explanation, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A rhamnolipid fermentation method, characterized in that: The method comprises the following steps: fermenting rhamnolipid-producing microorganisms in a fermentation medium containing nitrate to obtain bacterial liquid, and a mixed gas containing nitric oxide is introduced during the fermentation process.

2. The rhamnolipid fermentation method according to claim 1, characterized in that: The initial introduction amount of the mixed gas is above 0.2 vvm, the volume percentage of nitric oxide in the mixed gas is 0.01-0.025%, and the dissolved oxygen is above 15% after the mixed gas is introduced.

3. The rhamnolipid fermentation method according to claim 1 or 2, characterized in that: The nitrate is sodium nitrate.

4. The rhamnolipid fermentation method according to any one of claims 1 to 3, characterized in that: During the fermentation culture, the fermentation temperature is 25-40° C., and the initial pH of the fermentation is 6.8-7.

4.

5. The rhamnolipid fermentation method according to any one of claims 1 to 4, characterized in that: The fermentation medium comprises, by mass percentage, 3-5% glycerol, 0.8-2.2% yeast powder, 2.0-4.6% peptone, 0.8-3% NaNO3, 0.1-0.5% KH2PO4, 0.1-0.8% Na2HPO4·12H2O, 0.1-0.8% MgSO4·7H2O, 0.8-1.2% CaCl2·2H2O, 0.1%-0.7% FeSO4, 0.5%-1.2% CoCl2, and the balance is water; And / or, the inoculation amount of the microorganism is 3-8% of the mass of the fermentation medium.

6. The rhamnolipid fermentation method according to any one of claims 1 to 5, characterized in that: The types of rhamnolipid-producing microorganisms include one or more of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas putida.

7. The rhamnolipid fermentation method according to any one of claims 1 to 6, characterized in that: The concentration of rhamnolipid in the culture liquid is above 80g / L.

8. A method for separating rhamnolipids from bacterial culture liquid, characterized in that: include: Centrifugation: Centrifuge the bacterial liquid to obtain a precipitate and a clear liquid; Concentration: concentrating the clear liquid to obtain a concentrated clear liquid; Extraction: Add lipophilic compounds to the concentrated clear solution for extraction, let it stand to separate into layers, and collect the upper lipophilic compound phase rich in rhamnolipid; Evaporation: evaporating the lipophilic compound phase rich in rhamnolipid to remove the lipophilic compound and obtain concentrated rhamnolipid; Purified rhamnolipids are obtained by washing and drying the concentrated rhamnolipids.

9. The method according to claim 8, characterized in that The centrifugal speed is 8000-10000rpm, and the centrifugal time is 8-15min; And / or, the concentration method is reduced pressure concentration, and the volume of the concentrated clear liquid is 1 / 3-1 / 2 of the volume of the clear liquid; And / or, the standing time is 2-4h; and / or, the volume of the lipophilic compound added is 1 / 5-1 / 3 of the volume of the concentrated clear solution; And / or, the evaporation is evaporation under reduced pressure.

10. The method according to claim 9, characterized in that The reduced pressure concentration is reduced pressure rotary evaporation, and the conditions are: rotation speed 150-200rpm, temperature 40-60°C, pressure 50-80pa; And / or, the lipophilic compound includes at least one of n-butanol, isobutanol, and ethyl acetate; And / or, the reduced pressure evaporation is reduced pressure rotary evaporation, and the conditions are: rotation speed 250-400 rpm, temperature 60-80° C., and pressure 10-40 Pa.

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