A rhamnolipid fermentation process

Abstract

The present invention provides a rhamnolipid fermentation process. During the fermentation process of this process, perfluoropolyether emulsion is added to increase the yield of rhamnolipid. At the same time, an aeration process combining sterile air and sterile oxygen-rich gas is adopted to reduce the amount of antifoaming agent added. The present invention provides a new development strategy for increasing the yield of fermentation products and reducing the dosage of antifoaming agent during the microbial fermentation process.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation, and specifically relates to a rhamnolipid fermentation process. Background Art

[0002] Rhamnolipid is a biosurfactant that has been relatively deeply studied and widely applied at present. It is an anionic surfactant of glycolipid produced by Pseudomonas, and has the same emulsifying, wetting, foaming and other properties as chemical surfactants. At the same time, it has the characteristics of being healthy, non-toxic, green, environmentally friendly and biodegradable, and has unique application prospects in many industrial fields such as medicine, food, cosmetics, and environmental protection.

[0003] Rhamnolipid fermentation is an aerobic fermentation. The whole fermentation process requires continuous ventilation and stirring, so a large amount of foam will be generated. Moreover, the excellent foaming property of rhamnolipid itself further exacerbates the formation of fermentation foam. In order to prevent foam overflow and avoid the occurrence of fermentation broth leakage and bacterial contamination, conventional fermentation usually uses antifoaming agents for defoaming. However, on the one hand, the antifoaming agents are expensive, increasing the production cost of the product. On the other hand, the use of excessive antifoaming agents is also not conducive to the separation and purification of downstream rhamnolipid.

[0004] In CN201010136140.3, Meng Qin et al. used spraying ethanol to control foam, but ethanol itself has a bactericidal effect, and locally excessive ethanol has a certain impact on the growth of bacteria, thereby affecting the yield of rhamnolipid. In CN202010017299.7, Wang Jinghui et al. also tried to control fermentation foam by adjusting different pH values and tank pressures in stages, but it is not applicable to pH- and tank pressure-sensitive strains.

[0005] Therefore, a process method that can reduce foam and does not affect the yield of the target product is needed in the rhamnolipid fermentation process. Summary of the Invention

[0006] To solve the above technical problems, the purpose of the present invention is to provide a fermentation process that can reduce the addition amount of antifoaming agent during the rhamnolipid fermentation process and does not affect the yield of rhamnolipid.

[0007] The inventor of the present invention surprisingly found that the above technical problems can be solved by adding perfluoropolyether. Perfluoropolyether (PFPE, English name Perfluom Polyethers) is a polymer formed by the photooxidation of hexafluoropropylene (HFP) under the action of ultraviolet light. Due to its special structure, it can dissolve a large amount of oxygen and can be used to improve the dissolved oxygen level in the fermentation broth. At the same time, as a non-ionic surfactant itself, it has good defoaming and foam inhibition functions.

[0008] The specific technical solution adopted by the present invention is as follows:

[0009] A rhamnolipid fermentation process method, in which a perfluoropolyether emulsion is added during the fermentation process.

[0010] Introducing perfluoropolyether in the fermentation, its special structure endows it with good oxygen-carrying capacity, thus improving the dissolved oxygen level in the fermentation broth, which is beneficial to the production of rhamnolipid. At the same time, it has the properties of polyether itself, which can reduce the surface tension of the foaming liquid surface, thus playing the role of defoaming and foam inhibition, and further reducing the dosage of defoamer.

[0011] In the present invention, the structure of the perfluoropolyether is [CF(CF 3 )CF 2 O] x (CF 2 O) y , where x is an integer from 10 to 20, and y is an integer from 40 to 60; preferably, the molecular weight of the perfluoropolyether is 5000 - 6000.

[0012] In the present invention, the preparation method of the perfluoropolyether emulsion is to prepare the perfluoropolyether, polyethylene glycol and water in a ratio of 1:(0.2 - 0.5):(0.2 - 0.5); preferably, the polyethylene glycol is one or more of polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, and preferably polyethylene glycol 600.

[0013] In the present invention, continuous stirring is carried out at 40 - 70 °C for 1 - 3 h during the preparation.

[0014] In the present invention, the perfluoropolyether emulsion is added in batches; preferably, it is added at 0 - 144 h, preferably at 0 h, 48 h, 96 h, and 144 h respectively.

[0015] In the present invention, the volume ratio of each addition of the perfluoropolyether emulsion is 0.1% - 3% of the volume of the fermentation broth.

[0016] In the present invention, a ventilation process combining sterile air and sterile oxygen-rich gas is adopted during the fermentation process; preferably, the ventilation process is staged ventilation; preferably, the first stage is from 0 h to 24 h of fermentation, and sterile air is used for ventilation with a ventilation volume of 0.5 - 1.5 vvm, and the second stage is from 24 h to 192 h of fermentation, and sterile oxygen-rich gas is used for ventilation with a ventilation volume of 0.25 - 0.5 vvm.

[0017] In the present invention, the strain used for fermentation is Pseudomonas aeruginosa CCICC 21100.

[0018] Another object of the present invention is to provide a rhamnolipid.

[0019] A rhamnolipid is prepared by the above process method, and a perfluoropolyether emulsion is added during the fermentation process of the rhamnolipid.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) At different stages of fermentation, a perfluoropolyether emulsion is added. On the one hand, the perfluoropolyether emulsion itself has a high oxygen solubility, which can improve the oxygen concentration in the fermentation broth, enhance oxygen transfer, promote the growth of production strains, and increase the yield of rhamnolipid. On the other hand, the polyether structure of the perfluoropolyether emulsion itself has good defoaming and foam suppression functions, further reducing the dosage of the defoamer and not affecting the yield of the target product.

[0022] (2) The traditional sterile air ventilation process is optimized into a ventilation process combining sterile air and sterile oxygen-rich gas, reducing the dosage of the defoamer during the whole fermentation process. Specific Embodiments

[0023] Biological experimental methods not specifically described in the following examples are all referred to conventional methods or carried out according to the kits and product instructions.

[0024] The main reagent raw materials in the following examples: perfluoropolyether, with the structure: [CF(CF 3 )CF 2 O] x (CF 2 O) y , purchased from Wuhan Kemike Biopharmaceutical Technology Co., Ltd.; polyethylene glycol is of analytical grade, purchased from Shanghai Bioengineering Co., Ltd.; soybean oil is of food grade, purchased from Yihai Kerry Food Co., Ltd.; sodium nitrate is of industrial grade, purchased from Shijiazhuang Fengshan Chemical Industry.

[0025] The strain used for fermentation is Pseudomonas aeruginosa CCICC 21100.

[0026] Culture Medium:

[0027] Seed culture medium: yeast powder 15 g / L, peptone 5 g / L, sodium chloride 5 g / L.

[0028] Basic culture medium: soybean oil 40 g / L, sodium nitrate 7.5 g / L, peptone 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, disodium hydrogen phosphate 2.5 g / L, magnesium sulfate 0.5 g / L, calcium chloride 0.5 g / L.

[0029] Fed-batch nitrogen source medium: sodium nitrate 25 g / L, yeast powder 12 g / L, dipotassium hydrogen phosphate 7.5 g / L, disodium hydrogen phosphate 7.5 g / L, magnesium sulfate 1.5 g / L, calcium chloride 1.5 g / L.

[0030] Main equipment and instruments: Shanghai Bailun 50 L fermenter, Molecular Device microplate reader Spectra MAX 190,

[0031] Determination of rhamnolipid content

[0032] 1) Preparation of anthrone solution: Prepare 100 mL of concentrated sulfuric acid with a volume fraction of 80%, add 0.2 g of anthrone, shake well until completely mixed, wrap the bottle with tin foil to avoid light, and prepare it freshly for immediate use;

[0033] 2) Preparation of rhamnose standard solution: Prepare rhamnose solutions with concentrations of 20, 40, 60, 80, and 100 mg / L respectively for standby;

[0034] 3) Place the test tube rack with 10 mL glass test tubes in an ice bath. Add 0.5 mL of each rhamnose gradient solution and sample in sequence, then add 2 mL of sulfuric acid anthrone solution. After quickly shaking well, place the test tubes in a pre-prepared boiling water bath at 100 °C for 10 min. After the reaction is completed, place the test tubes in an ice bath to cool to room temperature, and measure the absorbance value at 620 nm. Calculate the rhamnose concentration in the sample according to the rhamnose standard curve, and then convert it to the rhamnolipid content in the sample according to the 3.4 coefficient.

[0035] Preparation of perfluoropolyether emulsion:

[0036] Perfluoropolyether A emulsion: The structure of perfluoropolyether is [CF(CF 3 )CF 2 O] 10 (CF 2 O) 50 , its molecular weight is 4960, and polyethylene glycol 200. Weigh 60 g of perfluoropolyether, 30 g of polyethylene glycol 200, and 12 g of pure water according to the mass ratio of 1:0.5:0.2 to form a suspension. Heat the suspension to 40 °C and stir continuously for 3 h to prepare perfluoropolyether emulsion A.

[0037] Perfluoropolyether B emulsion: The structure of perfluoropolyether is [CF(CF 3 )CF 2 O] 20 (CF 2 O) 40, with a molecular weight of 5960, polyethylene glycol 400. Weigh 60 g of perfluoropolyether, 15 g of polyethylene glycol 400, and 30 g of pure water according to the mass ratio of 1:0.25:0.5 to prepare a suspension. Heat the suspension to 70 °C and continuously stir for 1 h to prepare perfluoropolyether emulsion B.

[0038] Perfluoropolyether C emulsion: The perfluoropolyether structure is [CF(CF 3 )CF 2 O] 16 (CF 2 O) 50 , with a molecular weight of 5956, polyethylene glycol 600. Weigh 60 g of perfluoropolyether, 15 g of polyethylene glycol 600, and 15 g of pure water according to the mass ratio of 1:0.25:0.25 to prepare a suspension. Heat the suspension to 60 °C and continuously stir for 1.5 h to prepare perfluoropolyether emulsion C.

[0039] Perfluoropolyether D emulsion: The perfluoropolyether structure is [CF(CF 3 )CF 2 O] 15 (CF 2 O) 60 , with a molecular weight of 6450, polyethylene glycol 1000. Weigh 60 g of perfluoropolyether, 12 g of polyethylene glycol 1000, and 24 g of pure water according to the mass ratio of 1:0.2:0.4 to prepare a suspension. Heat the suspension to 50 °C and continuously stir for 2 h to prepare perfluoropolyether emulsion D.

[0040] Example 1

[0041] Pick 1 loop of Pseudomonas aeruginosa and inoculate it into the seed medium. After shaking culture at 30 °C and 200 rpm for 24 h, inoculate it into a 50 L fermenter containing 25 L of fermentation medium at an inoculation amount of 10% (v / v). Add 0.1% (v / v) of perfluoropolyether A emulsion at 0 h, 48 h, 96 h, and 144 h respectively. Control the fermentation temperature at 30 °C, pH = 7.5, the rotation speed at 500 r / min, introduce sterile air, and the ventilation volume is 1.5 vvm. Stop supplying sterile air at 24 h of fermentation, introduce sterile oxygen-enriched gas with a volume ratio of oxygen to nitrogen of 1:1, and the ventilation volume is 0.5 vvm. At the same time, start feeding. The feeding speed of the carbon source is 25 g / h, and the feeding speed of the nitrogen source is 60 g / h. Ferment for 192 h, end the fermentation, centrifuge at 8000 rpm at room temperature for 10 min, take the supernatant, and determine the rhamnolipid content by the anthrone method. The content of rhamnolipid is the content of rhamnose multiplied by the coefficient 3.4. The results are shown in Table 1.

[0042] Example 2

[0043] Pick 1 loop of Pseudomonas aeruginosa and inoculate it into the seed medium. After shaking culture at 30 °C and 200 rpm for 24 h, inoculate it into a 50 L fermenter containing 25 L of fermentation medium at an inoculation amount of 10% (v / v). Add 0.2% (v / v) of perfluoropolyether B emulsion at 0 h, 48 h, 96 h, and 144 h respectively. Control the fermentation temperature at 30 °C, pH = 7.5, rotation speed at 500 r / min, introduce sterile air, and the ventilation volume is 1 vvm. Ferment for 24 h, then stop supplying sterile air and introduce sterile oxygen-enriched gas with a volume ratio of oxygen to nitrogen of 1:1, and the ventilation volume is 0.5 vvm. At the same time, start feeding. The feeding rate of the carbon source is 25 g / h, and the feeding rate of the nitrogen source is 60 g / h. Ferment for 192 h to end the fermentation. Centrifuge at 8000 rpm at room temperature for 10 min, take the supernatant, and determine the rhamnolipid content by the anthrone method. The content of rhamnolipid is the content of rhamnose multiplied by the coefficient 3.4. The results are shown in Table 1.

[0044] Example 3

[0045] Pick 1 loop of Pseudomonas aeruginosa and inoculate it into the seed medium. After shaking culture at 30 °C and 200 rpm for 24 h, inoculate it into a 50 L fermenter containing 25 L of fermentation medium at an inoculation amount of 10% (v / v). Add 0.5% (v / v) of perfluoropolyether C emulsion at 0 h, 48 h, 96 h, and 144 h respectively. Control the fermentation temperature at 30 °C, pH = 7.5, rotation speed at 500 r / min, introduce sterile air, and the ventilation volume is 1.5 vvm. Ferment for 24 h, then stop supplying sterile air and introduce sterile oxygen-enriched gas with a volume ratio of oxygen to nitrogen of 1:1, and the ventilation volume is 0.25 vvm. At the same time, start feeding. The feeding rate of the carbon source is 25 g / h, and the feeding rate of the nitrogen source is 60 g / h. Ferment for 192 h to end the fermentation. Centrifuge at 8000 rpm at room temperature for 10 min, take the supernatant, and determine the rhamnolipid content by the anthrone method. The content of rhamnolipid is the content of rhamnose multiplied by the coefficient 3.4. The results are shown in Table 1.

[0046] Example 4

[0047] Pick 1 loop of Pseudomonas aeruginosa and inoculate it into the seed medium. After shaking culture at 30°C and 200 rpm for 24 h, inoculate it into a 50 L fermenter containing 25 L of fermentation medium at an inoculation amount of 10% (v / v). Add 1% (v / v) of perfluoropolyether C emulsion at 0 h, 48 h, 96 h, and 144 h respectively. Control the fermentation temperature at 30°C, pH = 7.5, the rotation speed at 500 r / min, introduce sterile air, and the ventilation rate at 0.5 vvm. After fermentation for 24 h, stop supplying sterile air and introduce sterile oxygen-enriched gas with a volume ratio of oxygen to nitrogen of 1:1, and the ventilation rate is 0.25 vvm. At the same time, start feeding. The feeding rate of the carbon source is 25 g / h, and the feeding rate of the nitrogen source is 60 g / h. Ferment for 192 h, end the fermentation, centrifuge at 8000 rpm at room temperature for 10 min, take the supernatant, and determine the rhamnolipid content by the anthrone method. The content of rhamnolipid is the content of rhamnose multiplied by the coefficient 3.4. The results are shown in Table 1.

[0048] Example 5

[0049] Pick 1 loop of Pseudomonas aeruginosa and inoculate it into the seed medium. After shaking culture at 30°C and 200 rpm for 24 h, inoculate it into a 50 L fermenter containing 25 L of fermentation medium at an inoculation amount of 10% (v / v). Add 0.3% (v / v) of perfluoropolyether D emulsion at 1 h, 40 h, 90 h, and 140 h respectively. Control the fermentation temperature at 30°C, pH = 7.5, the rotation speed at 500 r / min, introduce sterile air, and the ventilation rate at 0.5 vvm. After fermentation for 24 h, stop supplying sterile air and introduce sterile oxygen-enriched gas with a volume ratio of oxygen to nitrogen of 1:1, and the ventilation rate is 0.5 vvm. At the same time, start feeding. The feeding rate of the carbon source is 25 g / h, and the feeding rate of the nitrogen source is 60 g / h. Ferment for 192 h, end the fermentation, centrifuge at 8000 rpm at room temperature for 10 min, take the supernatant, and determine the rhamnolipid content by the anthrone method. The content of rhamnolipid is the content of rhamnose multiplied by the coefficient 3.4. The results are shown in Table 1.

[0050] Comparative Example 1

[0051] Compared with Example 3, this comparative example does not use perfluoropolyether emulsion.

[0052] Pick one loop of Pseudomonas aeruginosa and inoculate it into the seed medium. After culturing with shaking at 30 °C and 200 rpm for 24 h, inoculate it into a 50 L fermenter containing 25 L of fermentation medium at an inoculation amount of 10% (v / v). Control the fermentation temperature at 30 °C, pH = 7.5, rotation speed at 500 r / min, introduce sterile air, and the aeration rate is 1.5 vvm. Ferment for 24 h, then stop supplying sterile air and introduce sterile oxygen-enriched gas with a volume ratio of oxygen to nitrogen of 1:1, and the aeration rate is 0.25 vvm. At the same time, start feeding. The feeding rate of the carbon source is 25 g / h, and the feeding rate of the nitrogen source is 60 g / h. Ferment for 192 h to end the fermentation. Centrifuge at 8000 rpm at room temperature for 10 min, take the supernatant, and determine the rhamnolipid content by the anthrone method. The content of rhamnolipid is the content of rhamnose multiplied by the coefficient 3.4. The results are shown in Table 1.

[0053] Comparative Example 2

[0054] Compared with Example 3, this comparative example does not use perfluoropolyether emulsion but uses sodium percarbonate.

[0055] Pick one loop of Pseudomonas aeruginosa and inoculate it into the seed medium. After culturing with shaking at 30 °C and 200 rpm for 24 h, inoculate it into a 50 L fermenter containing 25 L of fermentation medium at an inoculation amount of 10% (v / v). Add 0.5% (m / v) of sodium percarbonate at 0 h, 48 h, 96 h, and 144 h respectively. Control the fermentation temperature at 30 °C, pH = 7.5, rotation speed at 500 r / min, introduce sterile air, and the aeration rate is 1.5 vvm. Ferment for 24 h, then stop supplying sterile air and introduce sterile oxygen-enriched gas with a volume ratio of oxygen to nitrogen of 1:1, and the aeration rate is 0.25 vvm. At the same time, start feeding. The feeding rate of the carbon source is 25 g / h, and the feeding rate of the nitrogen source is 60 g / h. Ferment for 192 h to end the fermentation. Centrifuge at 8000 rpm at room temperature for 10 min, take the supernatant, and determine the rhamnolipid content by the anthrone method. The content of rhamnolipid is the content of rhamnose multiplied by the coefficient 3.4. The results are shown in Table 1.

[0056] Table 1 Evaluation Results

[0057]

[0058]

[0059] The results show that compared with not adding perfluoropolyether emulsion, when different amounts of perfluoropolyether emulsion are added in batches for rhamnolipid fermentation, its yield can be improved to varying degrees, and the dosage of the defoamer is also significantly reduced.

Claims

1. A method for rhamnolipid fermentation process, characterized in that, perfluoropolyether emulsion is added during the fermentation process in the process method; the strain used for the fermentation is Pseudomonas aeruginosa CCICC 21100; the preparation method of the perfluoropolyether emulsion is to prepare perfluoropolyether, polyethylene glycol and water according to a ratio of 1:(0.2 - 0.5):(0.2 - 0.5).

2. The method according to claim 1, characterized in that, The structure of the perfluoropolyether is [CF(CF 3 )CF 2 O] x (CF 2 O) y , where x is an integer from 10 to 20 and y is an integer from 40 to 60.

3. The method according to claim 2, characterized in that, the molecular weight of the perfluoropolyether is 5000 - 6000.

4. The method according to claim 1 or 2, characterized in that, when preparing the perfluoropolyether emulsion, it is continuously stirred at 40 - 70 °C for 1 - 3 h.

5. The method according to claim 1 or 2, characterized in that, the polyethylene glycol is one or more of polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, and polyethylene glycol 1000.

6. The method according to claim 5, characterized in that, the polyethylene glycol is polyethylene glycol 600.

7. The method according to claim 1, characterized in that, the perfluoropolyether emulsion is added in batches; and / or, the volume ratio of the perfluoropolyether emulsion added each time is 0.1% - 3% of the volume of the fermentation broth.

8. The method according to claim 7, characterized in that, the perfluoropolyether emulsion is added at 0 - 144 h respectively.

9. The method according to claim 8, characterized in that, the perfluoropolyether emulsion is added at 0 h, 48 h, 96 h, and 144 h respectively.

10. The method according to claim 1, characterized in that, a ventilation process combining sterile air and sterile oxygen-rich gas is adopted during the fermentation process.

11. The method according to claim 10, characterized in that, the ventilation process is staged ventilation.

12. The method according to claim 11, characterized in that, in the first stage of the ventilation process, for fermentation from 0 h to 24 h, sterile air is used for ventilation, and the ventilation volume is 0.5 - 1.5 vvm; in the second stage of fermentation from 24 h to 192 h, sterile oxygen-rich gas is used for ventilation, and the ventilation volume is 0.25 - 0.5 vvm.

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