Process for extracting rhamnolipid from microbial fermentation liquor
By adjusting pH and adding specific compounds, rhamnolipid is extracted from microbial fermentation broth, solving the problems of high costs and pollution risks in the existing technology, and achieving a cost-effective, green and environmentally friendly extraction process, suitable for large-scale production .
Patent Information
- Application Number
- CN202411231096.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-04
- Publication Date
- 2024-12-10
AI Technical Summary
In the prior art, rhamnolipid extraction cost is high, the pollution risk is high, and the operation is cumbersome, making it difficult to achieve a cost-effective, green and environmentally friendly extraction process.
After filtration, adjust the pH, add ferric chloride and polymeric ferric chloride, then add ethanol, distillation and pH adjustment. Through these steps, rhamnolipid is extracted from the microbial fermentation broth, avoiding the toxic organic solvents. use.
It realizes the use of non-toxic and harmful organic solvents, and the recycling of cheap and easy-to-get reagents and ethanol, which significantly reduces production costs, is simple and efficient in the process, and is suitable for large-scale production.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of fermentation engineering, and in particular to a process for extracting rhamnolipid from microbial fermentation liquid. Background Art
[0002] With the enhancement of social environmental awareness and the advancement of ecological civilization construction, biosurfactants have received more and more attention due to their many advantages such as high activity, low toxicity, and environmental friendliness, and their demand has also been increasing.
[0003] Rhamnolipid is a glycolipid biosurfactant, which has been studied for a long time and has mature application technology. Rhamnolipid can be used in many fields such as oil extraction, environmental protection, agriculture, cosmetics, etc. However, the relatively high production cost is the bottleneck of rhamnolipid application.
[0004] In the fermentation production of rhamnolipids, the extraction cost of rhamnolipids accounts for 50% or even higher of the production cost. After the fermentation of rhamnolipids is completed, the amount of fermentation liquid in the tank is large; in addition to microbial cells, there are other components in the fermentation liquid, and it is difficult to extract rhamnolipids from it. The chromatographic separation and extraction method of rhamnolipids has high requirements for instruments and equipment, and the rhamnolipid extraction method is highly dependent on toxic organic solvents. Therefore, the development of an economical, efficient, green, and easy-to-operate rhamnolipid extraction process is of great significance to the fermentation production and application of rhamnolipids. Summary of the invention
[0005] The purpose of the present invention is to provide a process for extracting rhamnolipid from microbial fermentation broth, which is conducive to solving the current problems of high cost, pollution risk, cumbersome operation, etc. of rhamnolipid extraction. Compared with the prior art, the rhamnolipid extraction process of the present invention is economical and efficient, green and environmentally friendly, and easy to operate, and can be applied to the fermentation production of rhamnolipid.
[0006] To achieve the above object, the present invention adopts the following technical solution: A process for extracting rhamnolipid from microbial fermentation broth, comprising the following steps: (1) filtering the rhamnolipid fermentation broth and collecting the filtrate A; (2) adjusting the pH of A to 2.5, adding ferric chloride and polyferric chloride, stirring, standing, centrifuging, discarding the supernatant, and obtaining a precipitate B; (3) adding ethanol to B, stirring and mixing, centrifuging and collecting the supernatant to obtain liquid C; (4) distilling C, recovering ethanol, and obtaining a distillation residue D; (5) cooling D, adjusting its pH to ≤2.0, standing, and centrifuging to collect the precipitate, thereby obtaining a rhamnolipid extract.
[0007] The filtration treatment in step (1) refers to removing microbial cells and other insoluble substances in the rhamnolipid fermentation broth using a filter, a filter membrane, or a filter material.
[0008] In step (2), the pH of the filtrate is first adjusted to 2.5 with a 2-6 mol / L hydrochloric acid aqueous solution, and then polyferric chloride and ferric chloride with a final concentration of 1-3 g / L are added respectively; the standing temperature is 20°C-40°C and the time is 1-3 h; and the centrifugation is performed at 6000g-10000g for 10-20 min.
[0009] In step (3), 4-10 mL of 90%-100% ethanol reagent is added to every 1 g of precipitate B, the mixture is stirred and evenly mixed, and then centrifuged at 6000g-10000g for 10-20 min to collect the supernatant.
[0010] The distillation treatment described in step (4) refers to atmospheric distillation.
[0011] In step (5), after the distillation residue D is cooled to 20°C to 40°C, its pH is adjusted to less than 2.0 with a 2-6 mol / L hydrochloric acid aqueous solution, allowed to stand for 1-3 h, and then centrifuged at 6000g to 10000g for 10-20 min to collect the precipitate.
[0012] The extraction process is applied in the fermentation production of rhamnolipid.
[0013] Compared with the prior art, the rhamnolipid extraction process is green, environmentally friendly, economical and efficient, and easy to operate.
[0014] The beneficial effects of the present invention are: The rhamnolipid extraction process of the present invention does not use toxic and harmful organic solvents, and is more environmentally friendly. Reagents such as ferric chloride and polyferric chloride ethanol are cheap and easy to obtain, and the latter stage of the extraction process adopts atmospheric distillation to recover ethanol, which embodies the economy of the extraction process. If large-scale equipment such as ceramic filters and plate-and-frame filters are used in conjunction with the extraction process of the present invention, rhamnolipid products can also be extracted from large-scale rhamnolipid fermentation broth. Compared with the prior art, the rhamnolipid extraction process of the present invention is economical and efficient, environmentally friendly, and easy to operate, and can be applied to the fermentation production field of biosurfactants such as rhamnolipids. DETAILED DESCRIPTION
[0015] The present invention is described in detail in conjunction with the following specific examples so that those skilled in the art can more fully understand the present invention, but the present invention is not limited in any way. In the following examples, unless otherwise specified, the materials and reagents used can be purchased from biochemical reagent material companies.
[0016] Example 1 A process for extracting rhamnolipid from microbial fermentation broth, comprising the following steps: (1) filtering the rhamnolipid fermentation broth and collecting the filtrate A; (2) adjusting the pH of A to 2.5, adding ferric chloride and polyferric chloride, stirring, standing, centrifuging, discarding the supernatant, and obtaining a precipitate B; (3) adding ethanol to B, stirring and mixing, centrifuging and collecting the supernatant to obtain liquid C; (4) distilling C, recovering ethanol, and obtaining a distillation residue D; (5) cooling D, adjusting its pH to ≤2.0, standing, and centrifuging to collect the precipitate, thereby obtaining a rhamnolipid extract.
[0017] In this example, rhamnolipid-producing bacteria Pseudomonas aeruginosa ( Pseudomonas aeruginosa )LP20 (Liet al. High mono-rhamnolipids production by a novel isolate Pseudomonas aeruginosa LP20 from oily sludge: characterization, optimization, and potential application. Letters in Applied Microbiology, 2024, 77(2):ovae016.) was used as the fermentation strain. LB medium was prepared with the following formula: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH 7.0. The slant culture of strain LP20 was inoculated into 80 mL of LB medium and cultured at 35°C and 160 rpm for 12 hours to obtain the seed solution of strain LP20.
[0018] Prepare 1.5L of fermentation medium with glycerol as carbon source, the formula is as follows: 30.0 g / L glycerol, 3.5 g / L NaNO3, 4.5 g / L KH2PO4, 5.5 g / L K2HPO4∙3H2O, 1.0 g / L MgSO4∙7H2O, pH 6.8. After packaging, sterilize at 121°C for 20 min. After the culture medium is cooled to room temperature, inoculate the Pseudomonas aeruginosa LP20 seed liquid into the fermentation medium with glycerol as carbon source at a 5% inoculation amount (v / v), and ferment and culture at 35°C and 160 rpm for 5 days to obtain a total of 1.5 L of rhamnolipid fermentation liquid.
[0019] The rhamnolipid fermentation broth was filtered with a 0.22 μm filter membrane to remove microbial cells and other insolubles to obtain a filtrate containing rhamnolipids. The pH of the filtrate was adjusted to 2.5 with a 4 mol / L hydrochloric acid aqueous solution, and then ferric chloride and 2 g / L polyferric chloride were added at a final concentration of 8 g / L, stirred and mixed, and allowed to stand at room temperature of 30°C for 1.5 h, and then centrifuged at 7500g for 10 min, and the supernatant was discarded to obtain a precipitate.
[0020] For every 1 g of precipitate, add 6 mL of 95% ethanol reagent, stir and mix, centrifuge at 7500g for 10 min, and collect the supernatant.
[0021] The supernatant was distilled at atmospheric pressure to recover ethanol and obtain a distillation residue containing rhamnolipid. After the distillation residue was cooled to 30°C, its pH was adjusted to 2.0 with a 4 mol / L hydrochloric acid aqueous solution, allowed to stand at 4°C for 1.5 h, and then centrifuged at 7500 g for 10 min to collect the precipitate, i.e., the rhamnolipid extract.
[0022] The precipitate was transferred to a glass dish and then placed in an oven at 50°C. After drying, it was weighed to obtain 5.13 g of rhamnolipid extract.
[0023] Example 2 A process for extracting rhamnolipid from microbial fermentation broth, comprising the following steps: (1) filtering the rhamnolipid fermentation broth and collecting the filtrate A; (2) adjusting the pH of A to 2.5, adding ferric chloride and polyferric chloride, stirring, standing, centrifuging, discarding the supernatant, and obtaining a precipitate B; (3) adding ethanol to B, stirring and mixing, centrifuging and collecting the supernatant to obtain liquid C; (4) distilling C, recovering ethanol, and obtaining a distillation residue D; (5) cooling D, adjusting its pH to ≤2.0, standing, and centrifuging to collect the precipitate, thereby obtaining a rhamnolipid extract.
[0024] In this embodiment, rhamnolipid producing bacteria P. aeruginosa LP20 is a fermentation strain, and the seed solution of strain LP20 is prepared using LB medium. The fermentation medium of this embodiment uses glucose as the carbon source.
[0025] Prepare 1.5L of fermentation medium with glucose as carbon source, the formula is as follows: glucose 30.0 g / L, NaNO3 3.5 g / L, KH2PO4 4.5 g / L, K2HPO4∙3H2O 5.5 g / L, MgSO4∙7H2O 1.0 g / L, pH 6.8. After packaging, sterilize at 115°C for 30 min. After the culture medium cools to room temperature, inoculate the strain LP20 seed liquid into the fermentation medium with glucose as carbon source at a 5% inoculation amount (v / v), and ferment and culture at 35°C and 160 rpm for 5 days to obtain a total of 1.5 L of rhamnolipid fermentation liquid.
[0026] The rhamnolipid fermentation broth was filtered with a 0.22 μm filter membrane to remove microbial cells and other insolubles to obtain a filtrate containing rhamnolipids. The pH of the filtrate was adjusted to 2.5 with a 4 mol / L hydrochloric acid aqueous solution, and then ferric chloride and 2 g / L polyferric chloride were added at a final concentration of 8 g / L, stirred and mixed, and allowed to stand at room temperature of 30°C for 1.5 h, and then centrifuged at 7500g for 10 min, and the supernatant was discarded to obtain a precipitate.
[0027] For every 1 g of precipitate, add 6 mL of 95% ethanol reagent, stir and mix, centrifuge at 7500g for 10 min, and collect the supernatant.
[0028] The supernatant was distilled at atmospheric pressure to recover ethanol and obtain a distillation residue containing rhamnolipid. After the distillation residue was cooled to 30°C, its pH was adjusted to 2.0 with a 4 mol / L hydrochloric acid aqueous solution, allowed to stand at 4°C for 1.5 h, and then centrifuged at 7500 g for 10 min to collect the precipitate, i.e., the rhamnolipid extract.
[0029] The precipitate was transferred to a glass dish and then placed in an oven at 50°C. After drying, it was weighed to obtain 4.06 g of rhamnolipid extract.
[0030] Example 3 A process for extracting rhamnolipid from microbial fermentation broth, comprising the following steps: (1) filtering the rhamnolipid fermentation broth and collecting the filtrate A; (2) adjusting the pH of A to 2.5, adding ferric chloride and polyferric chloride, stirring, standing, centrifuging, discarding the supernatant, and obtaining a precipitate B; (3) adding ethanol to B, stirring and mixing, centrifuging and collecting the supernatant to obtain liquid C; (4) distilling C, recovering ethanol, and obtaining a distillation residue D; (5) cooling D, adjusting its pH to ≤2.0, standing, and centrifuging to collect the precipitate, thereby obtaining a rhamnolipid extract.
[0031] In this embodiment, rhamnolipid producing bacteria P. aeruginosaLP20 is a fermentation strain, and the seed liquid of strain LP20 is prepared using LB medium. The fermentation medium of this embodiment uses soybean oil as a carbon source.
[0032] Prepare 1.5L of fermentation medium with soybean oil as carbon source, the formula is as follows: soybean oil 30.0 g / L, NaNO3 3.5 g / L, KH2PO4 4.5 g / L, K2HPO4∙3H2O 5.5 g / L, MgSO4∙7H2O 1.0 g / L, pH 6.8. The sterilized medium was cooled to room temperature, and the strain LP20 seed liquid was inoculated into the fermentation medium with soybean oil as carbon source at a 5% inoculum (v / v), and fermented at 35°C and 160 rpm for 5 days to obtain a total of 1.5L of rhamnolipid fermentation liquid.
[0033] The rhamnolipid fermentation broth was filtered through a No. 6 vertical fused glass filter to remove microbial cells and other insolubles to obtain a filtrate containing rhamnolipids. The rhamnolipid fermentation broth was filtered through a 0.22 μm filter membrane to remove microbial cells and other insolubles to obtain a filtrate containing rhamnolipids. The pH of the filtrate was adjusted to 2.5 with a 4 mol / L hydrochloric acid aqueous solution, and then ferric chloride and 2 g / L polyferric chloride were added at a final concentration of 8 g / L, stirred and mixed, and allowed to stand at room temperature of 30°C for 1.5 h, and then centrifuged at 7500g for 10 min, and the supernatant was discarded to obtain a precipitate.
[0034] For every 1 g of precipitate, add 6 mL of 95% ethanol reagent, stir and mix, centrifuge at 7500g for 10 min, and collect the supernatant.
[0035] The supernatant was distilled at atmospheric pressure to recover ethanol and obtain a distillation residue containing rhamnolipid. After the distillation residue was cooled to 30°C, its pH was adjusted to 2.0 with a 4 mol / L hydrochloric acid aqueous solution, allowed to stand at 4°C for 1.5 h, and then centrifuged at 7500 g for 10 min to collect the precipitate, i.e., the rhamnolipid extract.
[0036] The precipitate was transferred to a glass dish and then placed in an oven at 50°C. After drying, it was weighed to obtain 4.98 g of rhamnolipid extract.
[0037] Example 4 A process for extracting rhamnolipid from microbial fermentation broth, comprising the following steps: (1) filtering the rhamnolipid fermentation broth and collecting the filtrate A; (2) adjusting the pH of A to 2.5, adding ferric chloride and polyferric chloride, stirring, standing, centrifuging, discarding the supernatant, and obtaining a precipitate B; (3) adding ethanol to B, stirring and mixing, centrifuging and collecting the supernatant to obtain liquid C; (4) distilling C, recovering ethanol, and obtaining a distillation residue D; (5) cooling D, adjusting its pH to ≤2.0, standing, and centrifuging to collect the precipitate, thereby obtaining a rhamnolipid extract.
[0038] In this embodiment, the genetically engineered bacterium Pseudomonas stutzeri ( Pseudomonas stutzer )Rhl(Zhao et al. Heterologous production of Pseudomonas aeruginosa rhamnolipid under anaerobic conditions for microbial enhanced oil recovery. Journal of Applied Microbiology 2015, 118: 379-389) was used as the fermentation strain, and the seed solution of the strain Rhl was prepared using LB medium. The fermentation medium of this example used glycerol as the carbon source.
[0039] Prepare 1.5 L of fermentation medium with glycerol as carbon source, the formula is as follows: 30.0 g / L glycerol, 3.5 g / L NaNO3, 4.5 g / L KH2PO4, 5.5 g / L K2HPO4∙3H2O, 1.0 g / L MgSO4∙7H2O, pH 6.8. The sterilized medium was cooled to room temperature, and the strain Rhl seed liquid was inoculated into the fermentation medium with glycerol as carbon source at a 5% inoculation amount (v / v). Fermentation was cultured at 35°C and 160 rpm for 5 days to obtain a total of 1.5 L rhamnolipid fermentation liquid.
[0040] The rhamnolipid fermentation broth was filtered with a 0.22 μm filter membrane to remove microbial cells and other insolubles to obtain a filtrate containing rhamnolipids. The pH of the filtrate was adjusted to 2.5 with a 4 mol / L hydrochloric acid aqueous solution, and then ferric chloride and 2 g / L polyferric chloride were added at a final concentration of 8 g / L, stirred and mixed, and allowed to stand at room temperature of 30°C for 1.5 h, and then centrifuged at 7500g for 10 min, and the supernatant was discarded to obtain a precipitate.
[0041] For every 1 g of precipitate, add 6 mL of 95% ethanol reagent, stir and mix, centrifuge at 7500g for 10 min, and collect the supernatant.
[0042] The supernatant was distilled at atmospheric pressure to recover ethanol and obtain a distillation residue containing rhamnolipid. After the distillation residue was cooled to 30°C, its pH was adjusted to 2.0 with a 4 mol / L hydrochloric acid aqueous solution, allowed to stand at 4°C for 1.5 h, and then centrifuged at 7500 g for 10 min to collect the precipitate, i.e., the rhamnolipid extract.
[0043] The precipitate was transferred to a glass dish, then placed in an oven at 50°C, dried, and weighed to obtain 0.72 g of rhamnolipid extract.
[0044] Example 5 In this example, rhamnolipid-producing bacteria P. aeruginosa LP20 is a fermentation strain, and the seed solution of strain LP20 is prepared using LB medium. The fermentation medium of this embodiment uses glycerol as a carbon source.
[0045] Prepare 2.0 L of fermentation medium with glycerol as carbon source, the formula is as follows: 30.0 g / L glycerol, 3.5 g / L NaNO3, 4.5 g / L KH2PO4, 5.5 g / L K2HPO4∙3H2O, 1.0 g / L MgSO4∙7H2O, pH 6.8. The sterilized medium was cooled to room temperature, and the strain LP20 seed liquid was inoculated into the fermentation medium with glycerol as carbon source at a 5% inoculation amount (v / v). Fermentation was cultured at 35°C and 160 rpm for 5 days to obtain a total of 2.0 L rhamnolipid fermentation liquid.
[0046] The fermentation broth was divided into two equal parts, each of 1 L. The two rhamnolipid fermentation broths were filtered using a 0.22 μm filter membrane to remove microbial cells and other insoluble matter to obtain two filtrates containing rhamnolipid.
[0047] One portion of the filtrate is subjected to the extraction process of the present invention, and the pH of the filtrate is adjusted to 2.5 with a 4 mol / L hydrochloric acid aqueous solution, and then ferric chloride and 2 g / L polyferric chloride are added at a final concentration of 8 g / L, stirred and mixed, and allowed to stand at room temperature of 30°C for 1.5 h, and then centrifuged at 7500g for 10 min, and the supernatant is discarded to obtain a precipitate. 6 mL of 95% ethanol reagent is added to each 1 g of precipitate, and after stirring and mixing, the supernatant is collected at 7500g for 10 min. The supernatant is subjected to atmospheric distillation to recover ethanol and obtain a distillation residue concentrated in rhamnolipid. After the distillation residue is cooled to 30°C, its pH is adjusted to 2.0 with a 4 mol / L hydrochloric acid aqueous solution, allowed to stand at 4°C for 1.5 h, and then centrifuged at 7500g for 10 min, and the precipitate is collected, i.e., the rhamnolipid extract. The precipitate was transferred to a glass dish and then placed in an oven at 50°C. After drying, it was weighed to obtain 3.53 g of rhamnolipid extract.
[0048] Another portion of the filtrate was extracted by acid precipitation. The pH of the filtrate was adjusted to 2.0 with 4 mol / L hydrochloric acid aqueous solution, and the filtrate was allowed to stand at 4°C for 6 hours, and then centrifuged at 7500 g for 10 minutes to collect the precipitate, i.e., the rhamnolipid extract. The precipitate was transferred to a glass dish, then placed in a 50°C oven, dried, and weighed to obtain 1.26 g of rhamnolipid extract.
[0049] The comparative example results show that the rhamnolipid extraction process of the present invention has a higher extraction efficiency.
[0050] The rhamnolipid extraction process of the present invention does not use toxic or harmful organic solvents, and is a green and environmentally friendly process. The reagents used are all cheap and readily available, and the ethanol reagent therein can also be recycled and reused. The embodiments show that the rhamnolipid extraction process of the present invention can extract rhamnolipid from rhamnolipid fermentation broths of different carbon source substrates, and has a wide range of applications. The rhamnolipid extraction process of the present invention has the advantages of being economical and efficient, green and environmentally friendly, and easy to operate, and can be applied to the fermentation production field of rhamnolipid.
Claims
1. A process for extracting rhamnolipid from microbial fermentation broth, comprising the following steps: (1) The rhamnolipid fermentation broth is filtered to collect filtrate A; (2) After adjusting the pH of A to 2.5, ferric chloride and polyferric chloride are added, stirred, allowed to stand, centrifuged, and the supernatant is discarded to obtain a precipitate B; (3) Ethanol is added to B, stirred and mixed, and the supernatant is collected by centrifugation to obtain liquid C; (4) C is treated by distillation, ethanol is recovered, and a distillation residue D is obtained; (5) After cooling D, the pH value is adjusted to ≤ 2.0, allowed to stand, and the precipitate is collected by centrifugation to obtain a rhamnolipid extract.
2. The process for extracting rhamnolipid according to claim 1, characterized in that: The filtration treatment in step (1) refers to removing microbial cells and other insoluble substances in the rhamnolipid fermentation broth using a filter, a filter membrane, or a filter material.
3. The process for extracting rhamnolipid according to claim 1, characterized in that: In step (2), the pH of the filtrate is first adjusted to 2.5 with a 2-6 mol / L hydrochloric acid aqueous solution, and then polyferric chloride and ferric chloride are respectively added at a final concentration of 1-3 g / L and 5-15 g / L; The standing temperature is 20°C to 40°C and the time is 1 to 3 hours; the centrifugation is performed at 6000g to 10000g for 10 to 20 minutes.
4. The process for extracting rhamnolipid according to claim 1, characterized in that: In step (3), 4-10 mL of 90%-100% ethanol reagent is added to every 1 g of precipitate B, the mixture is stirred and evenly mixed, and then centrifuged at 6000g-10000g for 10-20 min to collect the supernatant.
5. The process for extracting rhamnolipid according to claim 1, characterized in that: The distillation treatment in step (4) refers to atmospheric distillation.
6. The process for extracting rhamnolipid according to claim 1, characterized in that: In step (5), after the distillation residue D is cooled to 20°C to 40°C, its pH is adjusted to less than 2.0 with a 2-6 mol / L hydrochloric acid aqueous solution, allowed to stand for 1-3 h, and then centrifuged at 6000g to 10000g for 10-20 min to collect the precipitate.
7. The application of the process for extracting rhamnolipids according to claim 1, characterized in that: The extraction process is applied in the fermentation production of rhamnolipid.