CN117126904B - Method for circularly and continuously fermenting rhamnolipid - Google Patents

Method for circularly and continuously fermenting rhamnolipid Download PDF

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CN117126904B
CN117126904B CN202311400338.1A CN202311400338A CN117126904B CN 117126904 B CN117126904 B CN 117126904B CN 202311400338 A CN202311400338 A CN 202311400338A CN 117126904 B CN117126904 B CN 117126904B
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刘开开
张雅萍
杨付伟
姜西娟
石森
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Wanhua Chemical Group Co Ltd
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Abstract

The invention discloses a method for circularly and continuously fermenting rhamnolipid. The invention belongs to the technical field of biology, and aims to improve the synthesis yield and substrate conversion rate of fermented rhamnolipid. The method for circularly and continuously fermenting rhamnolipid disclosed by the invention comprises the steps of fermenting and culturing rhamnolipid-producing microorganisms in a fermentation culture medium, controlling the carbon-nitrogen ratio in the fermentation process, taking out part of fermentation liquor under a certain condition and the like. By adopting the method, the synthesis yield of the rhamnolipid reaches more than 86 g/L, the substrate conversion rate reaches 68%, and the uninterrupted production of the rhamnolipid can be realized.

Description

Method for circularly and continuously fermenting rhamnolipid
Technical Field
The invention belongs to the technical field of biology, and relates to a method for circularly and continuously fermenting rhamnolipid.
Background
Rhamnolipid is a high-efficiency anionic biosurfactant which is most widely used at present, has multiple functions of emulsification, foaming, antibiosis, antivirus, mycoplasma resistance and the like, and is mainly applied to the fields of fine chemical industry, petroleum exploitation, environmental protection, soil restoration and the like at present.
The rhamnolipid is prepared mainly by a microbial fermentation method, and the method has the following advantages: a. the target molecule with complex structure can be generated by utilizing the metabolic process of the microorganism, other methods are difficult to synthesize, and in addition, the biotransformation way of the microorganism can be modified or changed by a genetic engineering means, so that the complex molecular structure is modified, and the performance is more excellent. b. The source of raw materials is wide, the product can be completely degraded, the fermentation liquid is nontoxic to human body, and the harm to the environment is small. c. The fermentation production process is simple, low in cost, safe and economic, and has good industrial value. Of course, this method also has drawbacks such as a low concentration of the product in the fermentation broth, secretion of the product outside the cell, making isolation and purification of the product cumbersome.
Compared with the traditional chemical surfactant product, the rhamnolipid product belongs to an environment-friendly product and has the advantages of good product stability, wide application range, environmental protection, biodegradability, no secondary pollution and the like.
Rhamnolipids are a secondary metabolite of heterogeneous chemical structure formed by the linkage of one or two rhamnose molecules and one or two 3-hydroxy fatty acids of different chain lengths through beta-glycosidic bonds, wherein rhamnose constitutes its hydrophilic group and fatty acid constitutes its hydrophobic group. Common rhamnolipids are mixtures and, apart from the specific experimental use, the general products contain various homologs. There are statistics showing that up to 28 rhamnolipid structures have been reported, and that differences between the various homologs are mainly due to differences in the modification of the glycosyl moiety and the aglycone moiety. Can be largely divided into mono-rhamnolipids and di-rhamnolipids. The specific structure of the rhamnolipid product is influenced by relevant parameters such as strain, carbon source type, carbon nitrogen ratio and fermentation temperature. In the large-scale industrial fermentation process, four common rhamnolipid structures in the product are respectively a disaccharide double-lipid structure RL1, a monosaccharide double-lipid structure RL2, a disaccharide single-lipid structure RL3 and a monosaccharide single-lipid structure RL4.
The yield of fermented rhamnolipid is not only influenced by the nitrogen source species, but also significantly influenced by the C/N ratio, and the nutritional conditions can significantly influence the expression of the relevant genes and thus the biosynthesis of rhamnolipid. Many research results show that hydrophobic carbon sources are advantageous for obtaining high yields of rhamnolipids compared to hydrophilic carbon sources. If the fermentation process is unreasonable in design, the yield of rhamnolipid is low, and the problems that foam in fermentation liquid is increased due to the generation of rhamnolipid in the fermentation process, ventilation amount in the fermentation process is small, oxygen supply amount of strains is insufficient, and the metabolism speed of the strains in the fermentation process is influenced exist.
Disclosure of Invention
The invention aims to solve the technical problems of improving the synthesis yield and substrate conversion rate of fermented rhamnolipid.
To this end, the invention provides a method for cyclically and continuously fermenting rhamnolipid, comprising the following steps:
(1) Fermenting and culturing rhamnolipid-producing microorganisms in a fermentation medium;
(2) Feeding a carbon source and a nitrogen source during fermentation of 24-28 h; after the carbon source and the nitrogen source are fed in, controlling the carbon-nitrogen ratio to be 200-400:1 in the process of fermenting 48-52 h; then, controlling the carbon-nitrogen ratio to be 80-100:1; the carbon source flow rate is fixed to be 0.02-0.04% of the initial fermentation volume fed per minute, and the nitrogen source flow rate is regulated according to the carbon nitrogen ratio;
(3) When the volume of the fermentation liquor reaches 150% -200% of the initial fermentation volume and the synthetic yield of the rhamnolipid is more than 80 g/L, taking out 1/3-2/3 volume of the fermentation liquor, and continuing fermentation in the fermentation tank; performing solid-liquid separation on the taken fermentation liquor to obtain a fermentation liquor supernatant and a thallus precipitate; using the fermentation broth supernatant to extract rhamnolipids; the bacterial precipitate is processed by breaking bacteria to prepare a bacterial feed supplement;
(4) After the fermentation liquor is taken out from the step (3) and 4-6 and h are fed with the thallus feed, the flow rate is 0.01-0.03% of the initial fermentation volume fed per minute;
(5) Continuing fermentation culture, and repeating the steps (3) - (4) at least once;
the method of cyclically fermenting rhamnolipids may continue for at least 1248 h.
In some embodiments, in the above methods, the composition of the fermentation medium is: glycerol 2-3%, naNO 3 0.5-2%、KH 2 PO 4 0.05-0.1%、Na 2 HPO 4 ·12H 2 O 0.05-0.1%、MgSO 4 ·7H 2 O 0.01-0.1%、CaCl 2 ·2H 2 O 0.5-0.8%、FeSO 4 0.01%-0.1%、CoCl 2 0.01% -0.1% and the balance of water.
In some embodiments, the initial pH of the fermentation culture is in the range of 6.8-7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or a range or number between any two of these values.
In some embodiments, in the method of any one of the above, the initial OD of the fermentation culture 600 From 0.8 to 4, such as from 2 to 3, for example, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, or any range or value therebetween.
In some embodiments, in any of the above methods, the fermentation temperature of the fermentation culture is 25-40 ℃, e.g., 35-37 ℃; the initial stirring speed is 150-200 rpm, the initial ventilation speed is 0.2-0.6 vvm, the rotation speed and ventilation quantity are gradually increased along with the decrease of dissolved oxygen in the fermentation process, and the dissolved oxygen is ensured to be more than 20%.
In some embodiments, in any of the above described methods, the carbon source fed in step (2) is an aqueous solution containing 10-30 g/100ml glycerol and 10-30 g/100ml propylene-1, 2-diol, preferably wherein the mass ratio of glycerol to propylene-1, 2-diol is 1:1.
In some embodiments, in any of the above methods, the nitrogen source fed in step (2) is sterile filtered NaNO containing 1-5 g/100ml 3 And 0.1-1 g/100ml of aqueous glutamic acid.
In some embodiments, the method of any one of the above, the extracting rhamnolipid comprises the steps of: adjusting the pH of the broth supernatant to a pH of 1.5-2.4 (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or any range or value therebetween), wherein the rhamnolipid has very low solubility under acidic conditions and precipitates with impurities in a precipitation manner, and standing to obtain a precipitate; adding ethyl acetate with the volume of 1/5-1/10 into the precipitate for extraction to obtain a lower layer extract; distilling ethyl acetate from the lower extract at 80-85deg.C to obtain red paste containing rhamnolipid and impurities; adding ethyl acetate 1/5-1/10 volume into the paste, extracting for 2-3 times to obtain lower layer extract, and distilling ethyl acetate at 80-85deg.C to obtain rhamnolipid pure product with purity of above 92%.
In some embodiments, the method of any one of the above, the method further comprises the step of: adding lysozyme into the bacterial precipitate for enzymolysis, then carrying out low-temperature (2-8 ℃) high-pressure bacteria breaking, adding water into the homogenized liquid after bacteria breaking to prepare 20-50 g/100ml bacterial feed, and sterilizing (for example, sterilizing at 121 ℃ for 30 minutes).
In some embodiments, in any of the methods described above, a seed culture step is further performed prior to step (1);
the composition of the seed culture medium during seed culture is as follows: glucose 1-3%, naNO 3 0.5-3%, yeast extract 0.5-2%, tryptone 1-3%, and water in balance, and pH 7-7.4.
In some embodiments, in the above methods, the seed culture comprises a two-stage seed culture step;
the first seed culture condition is 25-40deg.C, 150-220 rpm culture 26-30 h; for example, at 33℃and 180 rpm, 28. 28 h;
culturing the second seed at 25-40deg.C and 200-250 rpm for 30-38 h; for example, 35 h is cultured at 32℃and 220 rpm.
In some embodiments, in any of the above methods, the rhamnolipid producing microorganism is a pseudomonas microorganism, preferably pseudomonas aeruginosa @Pseudomonas aeruginosa) Pseudomonas fluorescens @Pseudomonas fluorescens) Pseudomonas putida @Pseudomonas putica) One or more of the following.
The invention has the following advantages:
1. the microelements cobalt and ferrous ions in the fermentation medium can promote the synthesis of rhamnolipid;
2. the carbon source fed in the invention is glycerol and propylene-1, 2-diol, the glycerol and propylene-1, 2-diol are 1:1 combined to be precursor substances for synthesizing rhamnolipid, and the carbon source combination enables the rhamnolipid to be synthesized faster;
3. the nitrogen source fed by the invention is NaNO 3 And glutamic acid, the nitrate nitrogen can promote the production of rhamnolipid more than ammonia nitrogen, and the glutamic acid is beneficial to the synthesis of rhamnolipid by thalli;
4. according to different carbon-nitrogen ratio requirements of the thalli in different growth periods, the carbon-nitrogen ratio is controlled to be 200-400:1 in the early fermentation period, so that the thalli can grow rapidly; the carbon-nitrogen ratio is controlled to be 80-100:1 in the later period, which is more beneficial for the thalli to synthesize rhamnolipid;
5. treatment of fermentation broth supernatant: the extraction of ethyl acetate is carried out for three times, so that the yield and purity are greatly improved;
6. according to the invention, bacterial precipitation of fermentation liquor is recycled, and the low-temperature and high-pressure bacteria are broken through enzymolysis, so that the rhamnolipid which is not secreted in the bacterial body is released, the yield of rhamnolipid is improved, the separated bacterial body can be continuously used as a nutritional ingredient, the fermentation period is greatly shortened, the generation of waste is reduced, and the method has the competitive cost advantage;
7. by adopting the process, the synthesis yield of the rhamnolipid is more than 86 g/L, the substrate conversion rate reaches 68%, the production speed can be continuously kept, the uninterrupted production of the rhamnolipid can be realized, the rhamnolipid can be supplemented for at least 9 times, and the fermentation is performed for 1248 and h, so that the accumulation of harmful metabolites is effectively relieved, and the supplemented thallus lysate and the supplemented material exactly fit for the thallus growth;
8. the invention does not need frequent culture medium sterilization, does not need complicated process regulation and control, and has less foam in the fermentation process.
In the traditional fermentation method, the rhamnolipid is produced in a single batch by batch manner, a culture medium is prepared at the beginning of fermentation, defoaming regulation and control are performed in the fermentation process, and rhamnolipid is extracted from bacterial liquid at the later stage. According to the method for circularly and continuously fermenting rhamnolipid, provided by the invention, the culture medium composition is optimized to promote the quick growth of thalli, the process regulation strategy is improved to reduce foam generation, when the fermentation volume reaches a proper range and the rhamnolipid synthesis rate is maximum, discharging is carried out according to a proportion, separating supernatant of a discharged fermentation liquid is used for extracting rhamnolipid, and after the precipitation thalli are treated, feeding is carried out for continuous recycling, so that the primary metabolite rhamnolipid which is accumulated in the thalli is returned to the fermentation liquid again.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Unless otherwise indicated, all technical means used in the examples are routine in the art or according to the experimental methods suggested by the manufacturers of the kits and instruments. Reagents and biological materials used in the examples were obtained commercially unless otherwise specified.
Concentration detection of rhamnolipid: the rhamnolipid content was determined using the anthrone-sulfuric acid method, wherein the rhamnolipid content was calculated as the measured rhamnose content multiplied by a factor of 3.4.
Calculation method of substrate conversion (%):
multiplying the concentration (g/L) of rhamnolipid in the fermentation broth by the total volume (L) of the fermentation broth to obtain the total amount (g) of rhamnolipid in the fermentation broth, dividing the total amount (g) of rhamnolipid in the fermentation broth by the mass (g) of all carbon sources in the basic medium and the fed-in feed medium fed in at the current time to obtain the substrate conversion rate (%).
Seed culture medium: glucose 2.5%, naNO 3 0.5%, yeast extract 1%, tryptone 2%, the balance being water, pH 7.2, and sterilizing at 121 ℃ for 20 minutes.
Fermentation medium: glycerol 2%, naNO 3 0.5%、KH 2 PO 4 0.05%、Na 2 HPO 4 ·12H 2 O 0.05%、MgSO 4 ·7H 2 O 0.01%、CaCl 2 ·2H 2 O 0.5%、FeSO 4 0.01%、CoCl 2 0.01% of water, the balance being water, in the absence of FeSO 4 And CoCl 2 Sterilizing the culture medium at 121deg.C for 20 min, cooling, sterilizing with 0.25 μm filter membrane, and filtering to obtain FeSO 4 And CoCl 2 Adding cultureIn the base.
The "%" in the seed medium and the fermentation medium means "g/100ml".
Example 1: circulating continuous fermentation rhamnolipid
1. The pseudomonas aeruginosa is treatedPseudomonas aeruginosa) (number from China center for Industrial microbiological culture Collection center: CICC 10351) was inoculated into 200 ml seed medium, and cultured at 33℃and 180 rpm for 28h to obtain a first seed liquid.
2. The primary seed solution was inoculated into a 2000 ml seed medium at a 10% (by volume) inoculum size, and cultured at 32℃and 220 rpm for 35 h to obtain a secondary seed solution.
3. Inoculating the secondary seed solution into 50L fermentation tank containing 20L fermentation medium according to 10% (volume ratio) of inoculating amount, adjusting initial pH to 7.1 with hydrochloric acid or sodium hydroxide as required, and initial OD 600 2.1, fermenting and culturing at 35 ℃, wherein the initial rotating speed is 200 rpm, the ventilation is 0.2 vvm, and the rotating speed and ventilation are gradually increased along with the decrease of dissolved oxygen, so that the dissolved oxygen is ensured to be more than 20%.
4. Component (1) (carbon source) and component (2) (nitrogen source) were fed at 24 h (i.e., just after the start of the stationary phase), wherein the carbon source was an aqueous solution containing 30 g/100ml of glycerin and 30 g/100ml of propylene-1, 2-diol, and the nitrogen source was a sterile-filtered NaNO containing 5 g/100ml 3 And 1 g/100ml of aqueous glutamic acid; the carbon-nitrogen ratio is controlled to be 200:1 in the process of fermenting 24 h-52 h, so that the quick growth of thalli is facilitated; after 52 and h are fermented, the carbon-nitrogen ratio is controlled to be 80:1, so that the synthesis of rhamnolipid by thalli is facilitated, the carbon-nitrogen ratio is regulated and controlled by controlling the flow acceleration of a carbon source and a nitrogen source in the fermentation process, the flow rate of the carbon source is fixed to be 4 ml/min, and the flow rate of the nitrogen source is regulated according to the carbon-nitrogen ratio.
5. At fermentation 168 h, the rhamnolipid synthesis yield (i.e., rhamnolipid concentration in the broth) was 86.7 g/L at a broth volume of 40L (up to 200% of the initial fermentation broth volume), 16L volumes of broth was withdrawn and fermentation in the fermentor continued.
The obtained fermentation broth was centrifuged at 10℃and 10000 rpm for 20 minutes to obtain a fermentation broth supernatant and a bacterial cell pellet, which were subjected to the following treatments, respectively:
regulating the pH of the supernatant of the fermentation broth to 2.2 by hydrochloric acid, and standing to obtain a precipitate; adding 1/5 volume of ethyl acetate into the precipitate for extraction to obtain a lower layer extract; then the lower layer extract is distilled out of ethyl acetate at 85 ℃ to obtain red paste containing rhamnolipid and impurities; adding 1/10 volume of ethyl acetate into the paste for first extraction to obtain a first lower layer extract, adding 1/10 volume of ethyl acetate into the first lower layer extract for second extraction to obtain a second lower layer extract, adding 1/10 volume of ethyl acetate into the second lower layer extract for third extraction to obtain a third lower layer extract, extracting with ethyl acetate for three times, and distilling the finally obtained lower layer extract (namely, the third lower layer extract) at 85 ℃ for 20 h to obtain a rhamnolipid pure product with the purity reaching 92.7 percent.
Adding lysozyme with the concentration of 1 mg/ml in the bacterial precipitate, performing enzymolysis treatment at 37 ℃ for 2 h (destroying cell walls and helping subsequent bacterial breaking), and then performing bacterial breaking for 2-3 times at 2-8 ℃ by a high-pressure homogenizer; the homogenized solution after the sterilization was prepared into a 20 g/100ml solution with water, and sterilized at 121℃for 30 minutes as the component (3).
6. After part of the broth 4 h was withdrawn in step 5, the addition of component (3) was started with a flow acceleration of 4 ml/min.
7. Continuing fermentation culture, repeating the steps 5-6 at least once, and realizing uninterrupted production of rhamnolipid.
In this example, all of the broth in the fermenter was discharged during fermentation 1248 h (9 partial extractions and 9 component (3) recycle feeds, when the final broth volume reached 80% of the total fermenter volume).
In this example, the extraction time point of the fermentation broth in step 5, the volume of the extracted fermentation broth, the synthetic yield of rhamnolipid calculated by taking the extracted fermentation broth as a sample, and the purity of rhamnolipid obtained by extracting the extracted fermentation broth with ethyl acetate are shown in table 1.
The synthetic yield of the rhamnolipid reaches more than 86 g/L, the substrate conversion rate reaches 68% when the rhamnolipid is taken out for the first time, no foam is generated in the fermentation process, the production speed can be continuously maintained, and the continuous production of the rhamnolipid can be realized.
TABLE 1
Example 2: circulating continuous fermentation rhamnolipid
1. The pseudomonas aeruginosa is treatedPseudomonas aeruginosa) CICC 10351 is inoculated into 200 ml seed culture medium, and cultured at 33 ℃ and 180 rpm for 28h to obtain primary seed liquid.
2. The primary seed solution was inoculated into a 2000 ml seed medium at a 10% (by volume) inoculum size, and cultured at 32℃and 220 rpm for 35 h to obtain a secondary seed solution.
3. Inoculating the secondary seed solution into 50L fermentation tank containing 20L fermentation medium according to 10% (volume ratio) of inoculating amount, adjusting initial pH to 7.1 with hydrochloric acid or sodium hydroxide as required, and initial OD 600 2.3, fermenting and culturing at 35 ℃, wherein the initial rotating speed is 170 rpm, the ventilation is 0.6 vvm, and the rotating speed and ventilation are gradually increased along with the decrease of dissolved oxygen, so that the dissolved oxygen is ensured to be more than 20%.
4. Component (1) (carbon source) and component (2) (nitrogen source) were fed at the time of fermentation 26 h (i.e., just at the beginning of the stationary phase), wherein the carbon source and nitrogen source were the same as in example 1; the carbon-nitrogen ratio is controlled to be 300:1 in the process of fermenting 26 h-52 h, so that the quick growth of thalli is facilitated; after 52 and h are fermented, the carbon-nitrogen ratio is controlled to be 90:1, so that the synthesis of rhamnolipid by thalli is facilitated, the carbon-nitrogen ratio is regulated and controlled by controlling the flow acceleration of a carbon source and a nitrogen source in the fermentation process, the flow rate of the carbon source is fixed to be 6 ml/min, and the flow rate of the nitrogen source is regulated according to the carbon-nitrogen ratio.
5. When 168 was fermented h, the yield of rhamnolipid synthesis was 86.7 g/L when the volume of the fermentation broth was 40L (up to 200% of the volume of the initial fermentation medium), and the fermentation broth of 14L volumes was taken out, and fermentation in the fermenter was continued.
The obtained fermentation broth was centrifuged at 10℃and 10000 rpm for 20 minutes to obtain a fermentation broth supernatant and a bacterial cell pellet, which were subjected to the following treatments, respectively:
regulating the pH of the supernatant of the fermentation broth to 2.2 by hydrochloric acid, and standing to obtain a precipitate; adding 1/5 volume of ethyl acetate into the precipitate to extract the precipitate to obtain a lower layer extract; then the lower layer extract is distilled out of ethyl acetate at 85 ℃ to obtain red paste containing rhamnolipid and impurities; adding 1/10 volume of ethyl acetate into the paste for first extraction to obtain a first lower layer extract, adding 1/10 volume of ethyl acetate into the first lower layer extract for second extraction to obtain a second lower layer extract, adding 1/10 volume of ethyl acetate into the second lower layer extract for third extraction to obtain a third lower layer extract, extracting with ethyl acetate for three times, and distilling the finally obtained lower layer extract (namely, the third lower layer extract) at 85 ℃ for 20 h to obtain a rhamnolipid pure product with the purity reaching 92.7 percent.
Adding lysozyme with the concentration of 1 mg/ml in the bacterial precipitate, performing enzymolysis treatment at 37 ℃ for 2 h (destroying cell walls and helping subsequent bacterial breaking), and then performing bacterial breaking for 2-3 times at 2-8 ℃ by a high-pressure homogenizer; the homogenized solution after the sterilization was prepared into a 20 g/100ml solution with water, and sterilized at 121℃for 30 minutes as the component (3).
6. After part of the broth 4 h was withdrawn in step 5, the addition of component (3) was started with a flow acceleration of 4 ml/min.
7. Continuing fermentation culture, repeating the steps 5-6 at least once, and realizing uninterrupted production of rhamnolipid.
In this example, all of the broth in the fermenter was discharged during fermentation 1248 h (9 partial extractions and 9 component (3) recycle feeds, when the final broth volume reached 80% of the total fermenter volume).
In this example, the extraction time point of the fermentation broth in step 5, the volume of the extracted fermentation broth, the synthetic yield of rhamnolipid calculated by taking the extracted fermentation broth as a sample, and the purity of rhamnolipid obtained by extracting the extracted fermentation broth with ethyl acetate are shown in table 2.
The synthetic yield of the rhamnolipid reaches more than 86 g/L, the substrate conversion rate reaches 68% when the rhamnolipid is taken out for the first time, no foam is generated in the fermentation process, the production speed can be continuously maintained, and the continuous production of the rhamnolipid can be realized.
TABLE 2
Example 3: circulating continuous fermentation rhamnolipid
1. The pseudomonas aeruginosa is treatedPseudomonas aeruginosa) CICC 10351 is inoculated into 200 ml seed culture medium, and cultured at 33 ℃ and 180 rpm for 28h to obtain primary seed liquid.
2. The primary seed solution was inoculated into a 2000 ml seed medium at a 10% (by volume) inoculum size, and cultured at 32℃and 220 rpm for 35 h to obtain a secondary seed solution.
3. Inoculating the secondary seed solution into 50L fermentation tank containing 20L fermentation medium according to 10% (volume ratio) of inoculating amount, adjusting initial pH to 7.1 with hydrochloric acid or sodium hydroxide as required, and initial OD 600 2.2, fermenting and culturing at 35 ℃, wherein the initial rotating speed is 150 rpm, the ventilation is 0.4 vvm, and the rotating speed and ventilation are gradually increased along with the decrease of dissolved oxygen, so that the dissolved oxygen is ensured to be more than 20%.
4. At 28h of fermentation (i.e., just after starting to enter the stationary phase), component (1) (carbon source) and component (2) (nitrogen source) were fed, wherein the carbon source and nitrogen source were the same as in example 1; the carbon-nitrogen ratio is controlled to be 400:1 in the process of fermenting 28 h-52 h, so that the quick growth of thalli is facilitated; after 52 and h are fermented, the carbon-nitrogen ratio is controlled to be 100:1, so that the synthesis of rhamnolipid by thalli is facilitated, the carbon-nitrogen ratio is regulated and controlled by controlling the flow acceleration of a carbon source and a nitrogen source in the fermentation process, the flow rate of the carbon source is fixed to be 8 ml/min, and the flow rate of the nitrogen source is regulated according to the carbon-nitrogen ratio.
5. When 168 h was fermented, the yield of rhamnolipid synthesis was 86.5 g/L when the volume of the fermentation broth was 40L (up to 200% of the volume of the initial fermentation medium), 18L volumes of fermentation broth was withdrawn, and fermentation in the fermenter continued.
The obtained fermentation broth was centrifuged at 10℃and 10000 rpm for 20 minutes to obtain a fermentation broth supernatant and a bacterial cell pellet, which were subjected to the following treatments, respectively:
regulating the pH of the supernatant of the fermentation broth to 2.2 by hydrochloric acid, and standing to obtain a precipitate; adding 1/5 volume of ethyl acetate into the precipitate to extract the precipitate to obtain a lower layer extract; then the lower layer extract is distilled out of ethyl acetate at 85 ℃ to obtain red paste containing rhamnolipid and impurities; adding 1/10 volume of ethyl acetate into the paste for first extraction to obtain a first lower layer extract, adding 1/10 volume of ethyl acetate into the first lower layer extract for second extraction to obtain a second lower layer extract, adding 1/10 volume of ethyl acetate into the second lower layer extract for third extraction to obtain a third lower layer extract, extracting with ethyl acetate for three times, and distilling the finally obtained lower layer extract (namely, the third lower layer extract) at 85 ℃ for 20 h to obtain a rhamnolipid pure product with the purity reaching 92.8%.
Adding lysozyme with the concentration of 1 mg/ml in the bacterial precipitate, performing enzymolysis treatment at 37 ℃ for 2 h (destroying cell walls and helping subsequent bacterial breaking), and then performing bacterial breaking for 2-3 times at 2-8 ℃ by a high-pressure homogenizer; the homogenized solution after the sterilization was prepared into a 20 g/100ml solution with water, and sterilized at 121℃for 30 minutes as the component (3).
6. After part of the broth 4 h was withdrawn in step 5, the addition of component (3) was started with a flow acceleration of 4 ml/min.
7. Continuing fermentation culture, repeating the steps 5-6 at least once, and realizing uninterrupted production of rhamnolipid.
In this example, all of the broth in the fermenter was discharged during fermentation 1248 h (9 partial extractions and 9 component (3) recycle feeds, when the final broth volume reached 80% of the total fermenter volume).
In this example, the extraction time point of the fermentation broth in step 5, the volume of the fermentation broth extracted, the synthetic yield of rhamnolipid calculated by taking the fermentation broth extracted as a sample, and the purity of rhamnolipid obtained by extracting the fermentation broth extracted with ethyl acetate are shown in table 3.
The synthetic yield of the rhamnolipid reaches more than 86 g/L, the substrate conversion rate reaches 68% when the rhamnolipid is taken out for the first time, no foam is generated in the fermentation process, the production speed can be continuously maintained, and the continuous production of the rhamnolipid can be realized.
TABLE 3 Table 3
Comparative example 1: single batch fermentation rhamnolipid
Fermentation broth of fermentation 168 h was obtained as in steps 1-4 of example 1, followed by the following steps:
5. taking out the fermentation liquor completely, and centrifuging at 10 ℃ and 10000 rpm for 20 minutes to obtain a fermentation liquor supernatant; and extracting by ethyl acetate in the step 5 of the embodiment 1 to obtain the rhamnolipid pure product.
The synthetic yield of rhamnolipid by single batch fermentation in this example was detected to be 87.2 g/L, the substrate conversion was 68.4%, and the rhamnolipid purity was 92.8%.
Comparative example 2: the carbon source is replaced by edible oil
Example 1 was repeated except that component (1) (carbon source) was replaced with edible oil; and when the volume of the fermentation liquid is 40L (reaching 200% of the volume of the initial fermentation medium) during the fermentation of 168 h, 1/3-2/3 of the volume of the fermentation liquid is discharged, and the fermentation in the fermentation tank is continued.
In this example, the extraction time point of the fermentation liquid, the volume of the extracted fermentation liquid, the synthetic yield of rhamnolipid calculated by taking the extracted fermentation liquid as a sample, and the purity of rhamnolipid obtained by extracting the extracted fermentation liquid with ethyl acetate are shown in table 4.
The synthetic yield of rhamnolipid is about 48 g/L, the substrate conversion rate is 55% when the rhamnolipid is taken out for the first time, a large amount of foam is generated in the fermentation process, and the foam is required to be continuously defoamed by adding a defoaming agent after adding edible oil.
TABLE 4 Table 4
Comparative example 3: the nitrogen source is replaced by ammonium sulfate
Example 1 was repeated except that component (2) (nitrogen source) was replaced with an aqueous solution containing 30 g/100ml of ammonium sulfate; and when the volume of the fermentation liquid is 40L (reaching 200% of the volume of the initial fermentation medium) during the fermentation of 168 h, 1/3-2/3 of the volume of the fermentation liquid is discharged, and the fermentation in the fermentation tank is continued.
In this example, the extraction time point of the fermentation liquid, the volume of the extracted fermentation liquid, the synthetic yield of rhamnolipid calculated by taking the extracted fermentation liquid as a sample, and the purity of rhamnolipid obtained by extracting the extracted fermentation liquid with ethyl acetate are shown in table 5.
The synthetic yield of rhamnolipid is about 52 g/L, the substrate conversion rate is 58% when the rhamnolipid is taken out for the first time, a large amount of foam is generated in the fermentation process, and a defoaming agent is added for defoaming after the foam is generated.
TABLE 5
Comparative example 4: the fermentation culture medium is free of ferrous sulfate and cobalt chloride
Example 1 was repeated except that no ferrous sulfate and no cobalt chloride were added to the fermentation medium; and when the volume of the fermentation liquid is 40L (reaching 200% of the volume of the initial fermentation medium) during the fermentation of 168 h, 1/3-2/3 of the volume of the fermentation liquid is discharged, and the fermentation in the fermentation tank is continued.
In this example, the extraction time point of the fermentation liquid, the volume of the extracted fermentation liquid, the synthetic yield of rhamnolipid calculated by taking the extracted fermentation liquid as a sample, and the purity of rhamnolipid obtained by extracting the extracted fermentation liquid with ethyl acetate are shown in table 6.
The synthetic yield of rhamnolipid is about 46 g/L, the substrate conversion rate is 48% when the rhamnolipid is taken out for the first time, a large amount of foam is generated in the fermentation process, and a defoaming agent is added for defoaming after the foam is generated.
TABLE 6
Comparative example 5: the carbon-nitrogen ratio is fixed to be 150:1
Example 1 was repeated except that the carbon-nitrogen ratio was controlled to 150:1 during the whole fermentation, the carbon source flow rate was fixed to 6 ml/min, and the nitrogen source flow rate was adjusted according to the carbon-nitrogen ratio; and when the volume of the fermentation liquid is 40L (reaching 200% of the volume of the initial fermentation medium) during the fermentation of 168 h, 1/3-2/3 of the volume of the fermentation liquid is discharged, and the fermentation in the fermentation tank is continued.
In this example, the extraction time point of the fermentation liquid, the volume of the extracted fermentation liquid, the synthetic yield of rhamnolipid calculated by taking the extracted fermentation liquid as a sample, and the purity of rhamnolipid obtained by extracting the extracted fermentation liquid with ethyl acetate are shown in table 7.
The synthesis yield of rhamnolipid reaches about 51 g/L, the substrate conversion rate is 55% when the rhamnolipid is taken out for the first time, a large amount of foam is generated in the fermentation process, and a defoaming agent is added for defoaming after the foam is generated.
TABLE 7
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Claims (6)

1. A method for cyclically and continuously fermenting rhamnolipid, comprising the following steps:
(1) Fermenting and culturing rhamnolipid-producing microorganisms in a fermentation medium;
(2) Feeding a carbon source and a nitrogen source during fermentation of 24-28 h; after the carbon source and the nitrogen source are fed in, controlling the carbon-nitrogen ratio to be 200-400:1 in the process of fermenting 48-52 h; then, controlling the carbon-nitrogen ratio to be 80-100:1; the carbon source flow rate is fixed to be 0.02-0.04% of the initial fermentation volume fed per minute, and the nitrogen source flow rate is regulated according to the carbon nitrogen ratio;
(3) When the volume of the fermentation liquor reaches 150% -200% of the initial fermentation volume and the synthetic yield of the rhamnolipid is more than 80 g/L, taking out 1/3-2/3 volume of the fermentation liquor, and continuing fermentation in the fermentation tank; performing solid-liquid separation on the taken fermentation liquor to obtain a fermentation liquor supernatant and a thallus precipitate; using the fermentation broth supernatant to extract rhamnolipids; the bacterial precipitate is processed by breaking bacteria to prepare a bacterial feed supplement;
(4) After the fermentation liquor is taken out from the step (3) and 4-6 and h are fed with the thallus feed, the flow rate is 0.01-0.03% of the initial fermentation volume fed per minute;
(5) Continuing fermentation culture, and repeating the steps (3) - (4) at least once;
the composition of the fermentation medium is as follows: glycerol 2-3%, naNO 3 0.5-2%、KH 2 PO 4 0.05-0.1%、Na 2 HPO 4 ·12H 2 O 0.05-0.1%、MgSO 4 ·7H 2 O 0.01-0.1%、CaCl 2 ·2H 2 O 0.5-0.8%、FeSO 4 0.01%-0.1%、CoCl 2 0.01% -0.1% and the balance of water;
the carbon source fed in the step (2) is an aqueous solution containing 10-30 g/100ml of glycerin and 10-30 g/100ml of propylene-1, 2-diol;
the nitrogen source fed in the step (2) is NaNO with the concentration of 1-5 g/100ml 3 And 0.1-1 g/100ml of aqueous glutamic acid;
the microorganism producing rhamnolipid is pseudomonas aeruginosaPseudomonas aeruginosa)。
2. The method according to claim 1, characterized in that: the initial pH of the fermentation culture is 6.8-7.4;
initial OD of the fermentation culture 600 0.8-4; and/or
The fermentation temperature of the fermentation culture is 25-40 ℃, and dissolved oxygen is ensured to be more than 20% through stirring and ventilation.
3. The method according to claim 1 or 2, characterized in that: the extraction of the rhamnolipid comprises the following steps: regulating the pH value of the supernatant of the fermentation liquor to 1.5-2.4, and standing to obtain a precipitate; adding ethyl acetate with the volume of 1/5-1/10 into the precipitate for extraction to obtain a lower layer extract; distilling the lower layer extract at 80-85deg.C to obtain red paste; adding ethyl acetate 1/5-1/10 volume into the paste, extracting for 2-3 times to obtain lower extract, and distilling ethyl acetate at 80-85deg.C to obtain rhamnolipid pure product.
4. The method according to claim 1 or 2, characterized in that: the bacteria breaking treatment comprises the following steps: adding lysozyme into the bacterial precipitate for enzymolysis, then carrying out low-temperature Gao Yapo bacteria, adding water into the homogenized liquid after bacterial breaking to prepare 20-50 g/100ml bacterial feed, and sterilizing.
5. The method according to claim 1 or 2, characterized in that: a seed culture step is also carried out before the step (1);
the composition of the seed culture medium during seed culture is as follows: glucose 1-3%, naNO 3 0.5-3%, yeast extract 0.5-2%, tryptone 1-3%, and water in balance.
6. The method according to claim 5, wherein: the seed culture comprises two stages of seed culture steps;
the first seed culture condition is 25-40deg.C, 150-220 rpm culture 26-30 h;
the secondary seed culture condition is that the temperature is 25-40 ℃ and the rpm is 200-250, and the culture is 30-38 h.
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