Method and device for producing rhamnolipid through fermentation

Abstract

The invention discloses a method and a device for producing rhamnolipid through fermentation. During fermentation, pseudomonas aeruginosa fermentation liquor circularly passes through an ultra-micro nano bubble generating device and is mixed with nano bubbles generated in the ultra-micro nano bubble generating device, bubbles generated in the fermentation process can be effectively reduced, the yield of rhamnolipid produced and fermented by matching with the strain is 65g / L, the yield of dirhamnolipid is 53.97 g / L, and the yield of dirhamnolipid is 53.97 g / L. The rhamnolipid accounts for 83.03% of the total amount of rhamnolipid, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method and a device for producing rhamnolipid by fermentation. Background Art

[0002] Rhamnolipids are a type of surfactant produced by microorganisms such as Pseudomonas or Burkholderia. Compared with general chemical surfactants, they are non-toxic, antibacterial, diverse in structure, and have strong emulsifying properties. As a type of glycolipid biosurfactant, rhamnolipids are excellent surfactants for water and can significantly reduce the surface tension of aqueous solutions. At the same time, they have the advantages of low toxicity, biodegradability, and low environmental toxicity and side effects. They can be widely used in oil production and pollution environment remediation to improve efficiency. At the same time, they can also be used as additives for cosmetics, medicines, detergents, foods, etc. to improve product performance. They are considered to be excellent substitutes for traditional chemical synthetic surfactants.

[0003] As a surfactant, rhamnolipids produce very serious foam due to ventilation and stirring during the fermentation process. The ensuing overflow phenomenon causes loss of fermentation liquid, filter blockage, and bacterial contamination, which will directly lead to fermentation failure in severe cases. Therefore, researchers have developed foam reflux fermentation processes, semi-switching fermentation processes, etc., and designed a defoaming process based on a stop valve. Although these processes have achieved some results, they have not yet solved the fundamental problem of high ventilation volume in the fermentation process. Based on this, the development of a new bioreactor with low ventilation volume is crucial to completely solve the overflow problem in the rhamnolipid fermentation process.

[0004] Nanobubbles have the characteristics of large specific surface area, long existence time, high gas-liquid mass transfer rate, large amount of free radicals, high interfacial potential, good flotation effect, high gas solubility rate, etc. These characteristics are conducive to promoting mass transfer, chemical reaction and biological effect, etc., and are an effective means to solve the problem of high overflow fermentation. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for producing rhamnolipids by fermentation in view of the deficiencies in the prior art. The yield of rhamnolipids produced by nanobubble fermentation is high, and the proportion of di-rhamnolipids reaches more than 80%. In addition, the bubble problem in the production of rhamnolipids by microorganisms is effectively suppressed during the fermentation process. Therefore, further research on the direction of nanobubble production of rhamnolipids has broad application prospects.

[0006] In order to solve the above technical problems, the present invention discloses a method for producing rhamnolipid by fermentation. During fermentation, Pseudomonas aeruginosa fermentation liquid is circulated through an ultramicro-nano bubble generating device and mixed with nanobubbles generated in the ultramicro-nano bubble generating device.

[0007] Among them, the Pseudomonas aeruginosa is classified and named Pseudomonas aeruginosa, the strain name is SLTZ-11, and it has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the preservation time is July 22, 2024, and the preservation number is CGMCC NO.31396.

[0008] Preferably, during the fermentation process, the fermentation temperature is 30-37° C., the aeration intensity is 0.2-2.0 vvm, and the pH is 6.5-7.5 for 72-120 hours.

[0009] During fermentation, the fermentation medium includes a carbon source, a nitrogen source, a phosphate, magnesium ions and trace elements, wherein the total concentration of the carbon source is 20-150 g / L, the total concentration of the nitrogen source is 4-10 g / L, the concentration of the phosphate is 4-8 g / L, the total concentration of the magnesium ions is 0.2-1.0 g / L, and the total concentration of the trace elements is 2-6 ml / L. The carbon source is any one of rapeseed oil, corn oil and peanut oil; the nitrogen source is any one of sodium nitrate, yeast powder, NH4Cl and (NH4)2SO4; the phosphate is any one of K2HPO4, KH2PO4 and Na2PO4 or a combination of several thereof; the magnesium ions are magnesium sulfate and magnesium chloride; and the trace elements are any one of FeCl3, ZnSO4, CuSO4, MnSO4 and H3BO3 or a combination of several thereof.

[0010] Preferably, the carbon source is one of corn oil and peanut oil; the total concentration of the carbon source is 20-90 g / L; the nitrogen source is sodium nitrate, and the total concentration of the nitrogen source is 6-8 g / L; the phosphate is a combination of Na2PO4 and KH2PO4, and the total concentration of the phosphate is 5.5-6.5 g / L; the magnesium ion is magnesium sulfate, and the total concentration of the magnesium ion is 0.2-0.3 g / L; the trace element is any one or a combination of FeCl3, ZnSO4, CuSO4, MnSO4, H3BO3, and the total concentration of the trace elements is 3.5-4.5 ml / L.

[0011] The Pseudomonas aeruginosa fermentation liquid is pumped out from the bottom of the stirred reactor into the ultramicro-nano bubble generating device, and then pumped out from the ultramicro-nano bubble generating device and returned to the stirred reactor to achieve mixing of the Pseudomonas aeruginosa fermentation liquid and the nanobubbles generated in the ultramicro-nano bubble generating device.

[0012] A rhamnolipid prepared by the above method is also within the protection scope of the present application. The yield of the rhamnolipid prepared by the present invention reaches 65 g / L, and the proportion of disrhamnolipid in its rhamnolipid components reaches 83.03%.

[0013] Furthermore, the present invention proposes an immobilized reactor for fermenting and producing rhamnolipid, comprising a stirred reactor and an ultramicro-nano bubble generating device connected to each other, wherein the ultramicro-nano bubble generating device is used to generate nanobubbles, and a constant flow pump is arranged between the stirred reactor and the ultramicro-nano bubble generating device.

[0014] The ultramicro-nano bubble generating device comprises an ultramicro-nano bubble generating unit and a ventilation unit, wherein the ultramicro-nano bubble generating unit is provided with an automatic temperature control system and a perforated gas distributor, and the perforated gas distributor is arranged at the bottom of the ultramicro-nano bubble generating unit; the ventilation unit is used to provide gas to the ultramicro-nano bubble generating unit, and comprises an air flow meter and an air filter connected in sequence; the stirred reactor comprises a stirred reactor, a temperature probe, an exhaust device and a stirring device, wherein the stirring device and the temperature probe are arranged in the stirred reactor, and the exhaust device comprises an exhaust pipe.

[0015] The present invention also proposes a Pseudomonas aeruginosa, which is classified and named Pseudomonas aeruginosa, and the strain is named SLTZ-11. It has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on July 22, 2024, and the deposit number is CGMCC NO.31396. The strain was screened by the inventor from oil field soil in May 2023. The full length of the 16S rDNA sequence of Pseudomonas aeruginosa SLTZ-11 is 1327bp. By comparing the blastN sequence comparison tool in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), the strain of the present invention is identified as Pseudomonas aeruginosa, named SLTZ-11.

[0016] The present invention also proposes the use of the Pseudomonas aeruginosa in fermentation production of rhamnolipid.

[0017] Beneficial effects: The present application can efficiently produce disrhamnolipids through the nanobubble method, reduce the foam generated during the fermentation process, and with the strain of the present invention, the rhamnolipid yield of the fermented rhamnolipids is 65g / L, and the disrhamnolipid yield is 53.97g / L, accounting for 83.03% of the total rhamnolipids, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of the structure of an immobilized reactor device for fermentation production of rhamnolipids;

[0019] Figure 2 The bar graph is a graph showing the rhamnolipid production of different strains in Example 1;

[0020] Figure 3 is the fermentation kinetics curve in Example 2;

[0021] Figure 4 , wherein a is a state diagram of the fermentation process of rhamnolipid in a common fermentation tank; b is a state diagram of the fermentation process of rhamnolipid in a nanobubble-immobilized reactor;

[0022] Figure 5 This is the liquid chromatogram of the rhamnolipid standard in Example 3;

[0023] Figure 6 This is a liquid chromatogram of the rhamnolipid fermentation broth in Example 3. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0025] like Figure 1 As shown, this embodiment proposes a fixed reactor for fermenting and producing rhamnolipids, which is composed of a stirring reactor and an ultra-micro-nano bubble generating device. The two parts are connected by a constant flow pump 9 for material circulation.

[0026] The stirred reactor part is composed of a stirred reactor 1, an exhaust device and a stirring device 4; the stirring device 4 is placed in the stirred reactor 1 and driven by a motor 5; the exhaust device is composed of an exhaust pipe 10, an alkaline solution 11 and interconnected pipes, and the exhaust gas is discharged into the alkaline solution 11 through the exhaust pipe 10 to avoid bacterial contamination; the alkaline solution is a sodium hydroxide solution. The temperature probe 6 is connected to the stirred reactor 1 and contacts the fermentation liquid in the reactor.

[0027] The ultra-micro-nano bubble generating device part is composed of an ultra-micro-nano bubble generating device 2, a ventilation device and interconnected pipes. The ultra-micro-nano bubble generating device 2 cuts the specified gas to obtain nanobubbles and mixes them with the fermentation material; the cutting carrier is a nano-ceramic membrane. The ultra-micro-nano bubble generating device 2 is also equipped with an automatic temperature control system 12 and a perforated gas distributor 3; the automatic temperature control system 12 controls the temperature of the nanobubble-fermentation mixed liquid in the ultra-micro-nano bubble generating device 2 to be consistent with that in the stirring reactor 1; the ventilation device is composed of an air flow meter 7, an air filter 8, a constant flow pump 9 and interconnected pipes, and the air entering the ultra-micro-nano bubble generating device 2 is uniformly distributed by the bottom perforated gas distributor 3 in the ultra-micro-nano bubble generating device 2 after the flow rate is adjusted by the air flow meter 7, and filtered by the air filter 8 and then passed into the ultra-micro-nano bubble generating device 2.

[0028] The specific production process is as follows: Pseudomonas aeruginosa SLTZ-11 is inoculated in a stirred reactor 1 pre-filled with a fermentation medium at an inoculation rate of 4% (v / v), a constant flow pump 9 is turned on to realize material circulation in the stirred reactor 1 and the ultramicro-nano bubble generating device 2 (the fermentation liquid is extracted from the bottom of the stirred reactor and enters the ultramicro-nano bubble generating device 2, and then extracted from the ultramicro-nano bubble generating device 2 and returned to the stirred reactor), the automatic temperature control system 12 is turned on to realize a constant material temperature in the ultramicro-nano bubble generating device 2, and the tail gas is discharged from the exhaust device. The fermentation temperature in the stirred reactor is 37° C., the initial pH value is 7.0, the ventilation volume is 1.0 vvm, the stirring speed is 300 rpm / min, and the culture is carried out for 72-120 hours to realize the continuous supply of nanobubbles and fermentation of material circulation of Pseudomonas aeruginosa SLTZ-11.

[0029] Example 1 Screening of rhamnolipid-producing strains.

[0030] Weigh 2g of oil-contaminated soil (oilfield soil) and add it to a centrifuge tube containing 48ml of sterile saline and stir evenly. Pipette 1ml of the above bacterial suspension into a test tube containing 9ml of sterile saline and dilute it to 10 -1 times, and dilute to 10 -5 Take 100 μL and dilute it to 10 -5 The bacterial suspension was spread on CTAB-methylene blue plates and cultured at 37°C for 72-96 hours. The strains producing dark blue halos were selected and cultured on LB solid medium at 37°C for 16-24 hours to isolate single colonies.

[0031] CTAB-methylene blue medium is used to screen rhamnolipid-producing strains. The formula is: 0.7g / L KH2PO4, 0.9g / L Na2HPO4, 2g / L NaNO3, 0.4g / L MgSO4, 20g / L glycerol, 0.2g / L CTAB, 0.005g / L methylene blue, 15g / L agar powder, 1mL / L trace element stock solution, sterilized at 121℃ for 20min.

[0032] Five selected single colonies producing rhamnolipid were inoculated into a fresh 30 ml glass sample bottle pre-filled with 5 ml LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain seed solution. The seed solution was inoculated at a rate of 2% (v / v) into a 30 ml glass sample bottle pre-filled with 5 ml sterilized inorganic salt medium, and then the fermentation process was controlled at 37°C for 72-120 hours to obtain fermentation broth of different strains.

[0033] Inorganic salt medium, used for rhamnolipid fermentation, formula: 2g / L NaNO3, 3g / L KH2PO4, 0.3g / LNa2HPO4, 0.25g / L MgSO4, 1mL / L trace element mother liquor, sterilized at 121℃ for 20min. After the fermentation, the rhamnolipid yield was determined using the anthrone sulfuric acid method. Take 1ml of fermentation broth in a centrifuge tube, centrifuge at 12000rpm for 10min, take 1.25ml of fermentation supernatant (diluted 50 times) in a centrifuge tube, slowly add 2.5mL 0.2% anthrone sulfate solution under ice bath conditions, mix well and then boil in water for 15min, cool naturally to room temperature, and use an ELISA reader to measure its absorbance at 625nm.

[0034] According to the molecular weight relationship between rhamnolipid and rhamnose, the conversion formula between rhamnolipid and L-rhamnose concentration is: rhamnolipid concentration (g / L) = L-rhamnose concentration (g / L) × 3.4.

[0035] Among them, the preparation of anthrone sulfate solution is to weigh 0.2g of anthrone and dissolve it in 100mL of concentrated sulfuric acid. Prepare it before use and avoid light.

[0036] The experimental results are as follows Figure 2 As shown, among the five strains screened, SLTZ-11 had the highest rhamnolipid production of 9.10 g / L. The species information of the rhamnolipid-producing bacteria was identified by 16s rDNA gene, and the SLTZ-11 strain was determined to be Pseudomonas aeruginosa.

[0037] Example 2 Rhamnolipid was produced by fermentation in a 5 L fermenter.

[0038] Rhamnolipids were produced by fermentation using rapeseed oil as carbon source and Pseudomonas aeruginosa SLTZ-11 obtained in our laboratory.

[0039] Seed culture medium (LB liquid medium): peptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, sterilized at 121°C for 20 min.

[0040] Fermentation medium: 80 g / L rapeseed oil; 8 g / L sodium nitrate; 3.6 g / L Na2HPO4; 2.4 g / L KH2PO4; 0.5 g / LMgSO4; 4 ml / L trace element mother solution.

[0041] A single colony of Pseudomonas aeruginosa SLTZ-11 was selected and inoculated into a fresh 500 ml triangular flask pre-filled with 100 ml of LB liquid medium, and the flask was placed at 37° C. and 200 rpm for 16 hours to obtain a seed solution. The seed solution was cultured in a 5 L stirred reactor pre-filled with 3.0 L of sterilized fermentation medium at an inoculum amount of 4% (v / v), and then the fermentation process was controlled at 37° C., pH 7.0, ventilation volume 0.5 vvm (the aeration device was connected to a nano bubble device for ventilation), stirring speed 300 rpm, and fermented for 72-120 hours to obtain a Pseudomonas aeruginosa SLTZ-11 fermentation liquid.

[0042] After the fermentation, the rhamnolipid production was determined using the anthrone sulfuric acid method. 1 ml of the fermentation liquid was placed in a centrifuge tube and centrifuged at 12,000 rpm for 10 min. 1.25 ml of the fermentation supernatant (diluted 300 times) was placed in a centrifuge tube and 2.5 mL of 0.2% anthrone sulfate solution was slowly added under ice bath conditions. The mixture was thoroughly mixed and then placed in a boiling water bath for 15 min. After cooling naturally to room temperature, the absorbance at 625 nm was determined using an ELISA reader.

[0043] The method for measuring rhamnolipid concentration is the same as the method for measuring rhamnolipid concentration in Example 1.

[0044] The experimental results are as follows Figure 3 As shown, this example illustrates that the final yield of rhamnolipid in the fermentation broth was 65 g / L at 72 h, and there was no significant change in the yield after 72 h.

[0045] like Figure 4 As shown in a, it is a schematic diagram of using a common fermentation tank to produce rhamnolipids. The amount of foam is large and overflows from the exhaust device. Figure 4 As shown in b, the nanobubble-immobilized reactor of the present invention is used to effectively reduce the amount of foam during the fermentation of rhamnolipid.

[0046] Example 3 Determination of rhamnolipid components in the fermentation broth of a 5 L fermenter.

[0047] After the fermentation, 1 ml of the fermentation liquid was centrifuged at 12000 rpm for 10 min in a centrifuge, and then 0.1 ml of the supernatant was mixed with 5.9 ml of ultrapure water, and the mixture was shaken for 1 min. Then, a high performance liquid chromatography-evaporative light scattering detector was used to detect the proportion of mono- and di-rhamnolipids in the fermentation liquid components.

[0048] The rhamnolipid component determination method is HPLC, the chromatographic column adopts C18 chromatographic column, the concentration of the standard product (95% purity rhamnolipid) is 1g / L; the detector is an evaporative light scattering detector, the column oven temperature is set to 25°C, the mobile phase A is acetonitrile, the mobile phase B is pure water, the separation temperature is 60°C, the oil-free air pump provides the carrier gas, and the flow rate is 1.5L / min. Gradient elution conditions: 0-40min, 30%-90% B. Figure 5 This is the liquid chromatogram of the rhamnolipid standard in Example 3.

[0049] This example shows that among the rhamnolipid components of Pseudomonas aeruginosa SLTZ-11 strain, di-rhamnolipid and mono-rhamnolipid are the main products. When rhamnolipid is produced by nanobubble-immobilization reaction, di-rhamnolipid accounts for 83.03% ( Figure 6 Table 1 shows the rhamnolipid components produced by Pseudomonas aeruginosa SLTZ-11 strain analyzed by HPLC-ELSD.

[0050] Table 1

[0051] Components Rhamnolipid Monorhamnolipid Standards 47.51％ 54.29％ Rapeseed oil fermentation 83.03％ 16.97％

[0052] The present invention provides a method and idea for high-yield rhamnolipid based on nanobubbles. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for producing rhamnolipid by fermentation, characterized in that: During fermentation, the Pseudomonas aeruginosa fermentation liquid is circulated through the ultrafine nano bubble generating device to be mixed with the nano bubbles generated in the ultrafine nano bubble generating device.

2. The method according to claim 1, characterized in that: The Pseudomonas aeruginosa is classified and named Pseudomonas aeruginosa The strain is named SLTZ-11 and has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is July 22, 2024, and the deposit number is CGMCC NO.31396.

3. The method according to claim 1, characterized in that During the initial fermentation, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 4% (v / v). During the fermentation process, the fermentation temperature is 30-37°C, the ventilation intensity is 0.2-2.0 vvm, and the pH is 6.5-7.5 for 72-120 hours.

4. The method according to claim 1, characterized in that: During fermentation, the fermentation medium includes a carbon source, a nitrogen source, a phosphate, magnesium ions and trace elements, wherein the total concentration of the carbon source is 20-150 g / L, the total concentration of the nitrogen source is 4-10 g / L, the concentration of the phosphate is 4-8 g / L, the total concentration of the magnesium ions is 0.2-1.0 g / L, and the total concentration of the trace elements is 2-6 ml / L. The carbon source is any one of rapeseed oil, corn oil and peanut oil; the nitrogen source is any one of sodium nitrate, yeast powder, NH4Cl and (NH4)2SO4; the phosphate is any one of K2HPO4, KH2PO4 and Na2PO4 or a combination of several thereof; the magnesium ions are magnesium sulfate and magnesium chloride; and the trace elements are any one of FeCl3, ZnSO4, CuSO4, MnSO4 and H3BO3 or a combination of several thereof.

5. The method according to claim 1, characterized in that The Pseudomonas aeruginosa fermentation liquid is pumped out from the bottom of the stirring reactor into the ultramicro-nano bubble generating device, and then pumped out from the ultramicro-nano bubble generating device and returned to the stirring reactor to achieve mixing of the Pseudomonas aeruginosa fermentation liquid and the nanobubbles generated in the ultramicro-nano bubble generating device.

6. A rhamnolipid, characterized in that It is prepared by the method according to claims 1-5.

7. An immobilized reactor for fermentation production of rhamnolipids, characterized in that: The invention comprises a stirring reactor and an ultra-micro-nano bubble generating device which are connected to each other. The ultra-micro-nano bubble generating device is used to generate nano bubbles. A constant flow pump is arranged between the stirring reactor and the ultra-micro-nano bubble generating device.

8. The device according to claim 7, characterized in that The ultramicro-nano bubble generating device comprises an ultramicro-nano bubble generating unit and a ventilation unit, wherein the ultramicro-nano bubble generating unit is provided with an automatic temperature control system and a perforated gas distributor, and the perforated gas distributor is arranged at the bottom of the ultramicro-nano bubble generating unit; the ventilation unit is used to provide gas to the ultramicro-nano bubble generating unit, and comprises an air flow meter and an air filter connected in sequence; the stirred reactor comprises a stirred reactor, a temperature probe, an exhaust device and a stirring device, wherein the stirring device and the temperature probe are arranged in the stirred reactor, and the exhaust device comprises an exhaust pipe.

9. Use of the Pseudomonas aeruginosa according to claim 1 in fermentation production of rhamnolipid.

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