Separation process of high viscosity fermentation liquor of rhamnolipid under abnormal working condition
By adding soluble inorganic base and n-primary alcohol to high-viscosity fermentation broth, combined with centrifugation and organic microfiltration membrane filtration, rhamnolipin was successfully separated and recovered, solving the problem of separating rhamnolipin from high-viscosity fermentation broth and achieving high-yield recovery of rhamnolipin.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WANHUA CHEM GRP CO LTD
- Filing Date
- 2023-11-27
- Publication Date
- 2026-07-10
AI Technical Summary
During the fermentation process, uncontrollable factors can lead to high viscosity in the fermentation broth, making it impossible to effectively separate rhamnolipids and PHAs, resulting in product loss and affecting normal production.
The pH was adjusted to above 10 by adding a soluble inorganic base to the high-viscosity fermentation broth, and after standing, n-primary alcohol was added and stirred. Then, the broth was centrifuged, filtered through an organic microfiltration membrane, and distilled under reduced pressure to separate and recover rhamnolipin.
A high-yield separation of rhamnolipin was achieved, with a yield of over 90%, solving the problem of rhamnolipin recovery from high-viscosity fermentation broth.
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Figure BDA0004569898620000061
Abstract
Description
Technical Field
[0001] This invention relates to a separation process, and more particularly to a separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions. Background Technology
[0002] Rhamnolipids, produced by Pseudomonas aeruginosa under certain conditions, are extracellular metabolites. They possess excellent surface and interfacial activity and can be widely used in petrochemical, environmental, pharmaceutical, food, and agricultural fields. They are non-toxic, biodegradable, and represent one of the longest-researched, most effective, and most technologically mature biosurfactants.
[0003] Polyhydroxyalkanoates (PHAs) are byproducts of Pseudomonas aeruginosa metabolism. They are intracellular metabolites and high-molecular-weight polymers. During Pseudomonas aeruginosa fermentation, rhamnolipids and PHAs are produced simultaneously, one extracellularly and the other intracellularly. In normal fermentation metabolism, rhamnolipid production is dominant, with very little PHA produced, all of which remains intracellular and does not diffuse significantly into the fermentation broth. Therefore, the fermentation broth viscosity is normal, generally maintained below 5 cP. At the end of fermentation, conventional solid-liquid separation methods can be used for sterilization and further purification. However, through continuous research, the inventors have discovered that there are many uncontrollable factors during fermentation. The potential result is mass cell death and rupture, causing PHA to migrate from intracellular to extracellular space, leading to a sudden increase in fermentation broth viscosity to tens or even thousands of centipoises (cP). This high-viscosity fermentation broth cannot be separated into solid and liquid components, thus affecting normal production and causing rhamnolipid loss.
[0004] In cases where the viscosity of the fermentation broth is abnormal due to process operations, if the fermentation broth can be effectively separated and rhamnolipin recovered, product losses due to uncontrollable factors during the production stage can be avoided, which is of great significance for the industrial production of rhamnolipin. Summary of the Invention
[0005] There are currently no reports in the literature on how to recover rhamnolipin in high-yield fermentation broth with abnormal viscosity. To solve the above technical problems, this invention proposes a separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0007] A separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions includes the following steps:
[0008] 1) Add soluble inorganic base to the high-viscosity rhamnolipin fermentation broth, adjust the pH to above 10, and let it stand.
[0009] 2) Add n-primary alcohol to the rhamnolipin fermentation broth after it has been allowed to stand, and stir.
[0010] 3) Centrifuge the fermentation broth and collect the supernatant;
[0011] 4) Filter the supernatant using an organic microfiltration membrane and collect the filtrate;
[0012] 5) The filtrate was subjected to vacuum distillation to obtain an aqueous solution of rhamnolipin from the bottom of the column.
[0013] As a preferred embodiment, the rhamnolipin fermentation broth has a viscosity of 50-3000 cp, a rhamnolipin content of 30-90 g / L, and a pH of 6.5-8.0.
[0014] Through continuous research, the inventors discovered that rhamnolipin fermentation broth with abnormal viscosity can be detected with high levels of PHA by gel permeation chromatography (GPC), while it is undetectable in normal fermentation broth. This indicates that the translocation of PHA from intracellular to extracellular space is the main cause of the increased viscosity of rhamnolipin fermentation broth. Furthermore, for rhamnolipin fermentation broth with a viscosity of 50-3000 cp, the PHA content detected by GPC was 1-50 g / L. Due to limitations in detection methods, in this invention, both rhamnolipin content and PHA content refer to the extracellular content of the fermentation broth.
[0015] In a preferred embodiment, the soluble inorganic base is selected from one or more of NaOH, KOH, and ammonia water.
[0016] As a preferred embodiment, the settling time in step 1) is 5-12 hours.
[0017] In a preferred embodiment, the n-type primary alcohol is selected from small molecule alcohols with a chain length of 1-4 carbons, preferably one or more of methanol, ethanol, propanol, and butanol;
[0018] Preferably, the amount of the n-primary alcohol added is 0.5-1.5 times the mass of the rhamnolipin fermentation broth.
[0019] As a preferred embodiment, the stirring conditions in step 2) are: stirring at 150-250 rpm for 1-3 hours.
[0020] As a preferred embodiment, the centrifugation conditions in step 3) are: temperature 5-15℃, separation factor 5000-8000g, and residence time 3-10min.
[0021] In a preferred embodiment, the organic microfiltration membrane is selected from one or more of hollow fiber membranes, spiral wound membranes, and tubular membranes;
[0022] Preferably, the material of the organic microfiltration membrane is selected from one or more of polyethersulfone, polyacrylonitrile, and polyvinylidene fluoride;
[0023] Preferably, the pore size of the organic microfiltration membrane is in the range of 0.11-0.45 μm.
[0024] As a preferred embodiment, step 5) involves vacuum distillation under the following conditions: vacuum degree of 50-200 hPa and bottom temperature of 60-80℃; concentration is stopped after the volume of the original fermentation broth is reduced to 1 / 10-1 / 5.
[0025] The rhamnolipin aqueous solution prepared by the method of the present invention has a content of 10-30% and a rhamnolipin yield of over 90%.
[0026] The separation process provided by this invention is simple, using conventional equipment that effectively separates rhamnolipids and PHA, while increasing the rhamnolipid yield. The increased viscosity in the high-viscosity fermentation broth is mainly caused by PHA. Adding alkali to raise the pH completely ruptures the bacteria, releasing both rhamnolipids and PHA. The solubility of rhamnolipids increases significantly. Adding ethanol causes denaturation and precipitation of bacterial proteins, while PHA, upon contact with ethanol, increases in particle size and precipitates as large colloidal particles. Centrifugation then separates most of the PHA from the bacteria. Filtering the supernatant with a hydrophilic organic membrane thoroughly removes residual bacteria and PHA, ultimately recovering almost all rhamnolipids to the aqueous phase for further purification as needed. Detailed Implementation
[0027] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention.
[0028] The separation equipment used in the following embodiments and comparative examples of the present invention is as follows: Zhongke Ruiyang spiral wound membrane SS-MF0452319-AF (0.45μm, polyvinylidene fluoride), SS-ME0222319-AF (0.22μm, polyethersulfone), Zhongke Ruiyang tubular membrane TGMN0205410 (0.2μm, polyacrylonitrile), Asahi Kasei hollow fiber membrane UNA-620A (0.1μm, polyvinylidene fluoride), Hitachi benchtop centrifuge CP-NX, Nippon Rikka rotary evaporator N-1100D-W, 3L three-necked flask, and Shanghai Jinghong water bath.
[0029] The reagents used, their sources, and purities are as follows: anhydrous ethanol, analytical grade, purchased from Comio; NaOH, analytical grade, purchased from Sinopharm; concentrated sulfuric acid, 98% purity, analytical grade, purchased from Sinopharm; rhamnose standard, analytical grade, purchased from Sinopharm.
[0030] High-viscosity rhamnolipin fermentation broth A: viscosity 89cp, rhamnolipin content 61g / L, PHA content 2.3g / L, pH=7.2;
[0031] High-viscosity rhamnolipin fermentation broth B: viscosity 950cp, rhamnolipin content 35g / L, PHA content 10.5g / L, pH=6.8;
[0032] High-viscosity rhamnolipin fermentation broth C: viscosity 2800cp, rhamnolipin content 82g / L, PHA content 45.2g / L, pH=7.8;
[0033] High-viscosity rhamnolipin fermentation broth D: viscosity 54cp, rhamnolipin content 43g / L, PHA content 1.3g / L, pH=6.5.
[0034] GPC assays were performed using gel permeation chromatography for quantitative analysis. The specific procedure is as follows:
[0035] 1) Prepare a 1 g / L PHA standard solution: Weigh 0.1 g PHA and dilute to 100 mL in a volumetric flask. Dissolve the PHA in THF solvent to prepare a 1 g / L PHA standard solution.
[0036] 2) Take 1g of fermentation broth, dilute to 10mL in a volumetric flask, dissolve in THF solvent, and prepare a 100g / L test solution.
[0037] 3) Take 1 mL of standard solution and test solution, filter them separately through a 0.2 μm filter membrane, and then determine the content by gel chromatography under the following chromatographic conditions:
[0038] Column temperature: 40℃, flow rate: 1mL / min, running time: 40min, mobile phase: A: water, B: acetonitrile, running procedure as shown in Table 1.
[0039] Table 1
[0040] runtime min B mobile phase percentage 0.01 95 10 95 35 5
[0041] By comparing the peak areas of the standard sample and the test sample at the same time, the PHA content in the test sample (in g / L) can be calculated. The calculation formula is as follows:
[0042] PHA content in the sample = (peak area of sample / peak area of standard) * 10
[0043] The method for determining rhamnolipid content is the sulfuric acid-anthrone method, and the specific operation method is as follows:
[0044] 1) Preparation of anthrone solution: Dissolve 0.2g of anthrone in 100mL of 80% sulfuric acid, protect from light and use immediately. Do not store.
[0045] 2) Take 0.5 mL of the sample to be tested into a 10-15 mL stoppered graduated test tube, place it in an ice-water bath to cool it completely, add 2 mL of anthrone solution while keeping the ice-water bath, mix quickly (minimize the reaction), then boil in a water bath for 10 min, take it out and place it in an ice-water bath to cool to room temperature, and measure the absorbance at 620 nm.
[0046] 3) Rhamnose standard: Weigh 0.1g of rhamnose and dilute to a 250mL volumetric flask to prepare a 400mg / L rhamnose stock solution. Then dilute according to the ratio to obtain standard samples of different concentrations, as shown in Table 2 below:
[0047] Table 2. Samples of different concentrations used in rhamnose standard koji
[0048]
[0049] The absorbance was determined using the anthrone method, and a standard curve was plotted.
[0050] y = ax + b
[0051] Where y is the rhamnose content and x is the absorbance.
[0052] The final formula for calculating rhamnolipid content is:
[0053] Rhamnose content = rhamnose content in the reaction solution × dilution factor × 3.4.
[0054] The yield is calculated as follows: Rhamnolipid content in the concentrate * Mass of the concentrate / (Rhamnolipid content in the fermentation broth * Mass of the fermentation broth)
[0055] Example 1
[0056] 200g of high-viscosity rhamnolipin fermentation broth A was taken, and the pH was adjusted to 10.2 with NaOH. The mixture was allowed to stand at 25℃ for 5 hours, then 100g of ethanol was added, and the mixture was stirred at 25℃ for 2 hours at 150rpm. The solid and liquid phases were then separated by centrifugation at 5℃ with a separation factor of 5000g and a residence time of 3 minutes. Approximately 265g of supernatant was collected. GPC analysis showed that the rhamnolipin content in the supernatant was 45g / L, and the PHA content was 0.35g / L. The supernatant was then filtered through an Asahi Kasei hollow fiber membrane UNA-620A, and approximately 230g of filtrate was collected. GPC analysis showed that the rhamnolipin content in the filtrate was 49.56g / L, and PHA was not detected. The filtrate was concentrated to 40g under a vacuum of 50hpa at 75℃, and the rhamnolipin content was determined to be approximately 28.5%, with a yield of 93.44%.
[0057] Example 2
[0058] Take 300g of high-viscosity rhamnolipin fermentation broth B, add KOH to adjust the pH to 11, let it stand at 20℃ for 12 hours, then add 300g of methanol, stir at 30℃ for 1 hour at 200rpm, then centrifuge at 5℃ to separate the solid and liquid, separation factor 8000g, residence time 5 minutes, collect about 510g of supernatant. GPC analysis shows that the rhamnolipin content in the supernatant is 20.09g / L and the PHA content is 1.23g / L. Then filter the supernatant using a Zhongke Ruiyang spiral wound membrane SS-ME0222319-AF, collect about 490g of filtrate. GPC analysis shows that the rhamnolipin content in the filtrate is 20.42g / L, and PHA is not detected. Concentrate the filtrate to 55g under a vacuum of 150hpa at 60℃, the rhamnolipin content is about 18.2%, and the yield is 95.3%.
[0059] Example 3
[0060] 200g of high-viscosity rhamnolipin fermentation broth (C) was taken, and the pH was adjusted to 10.5 with ammonia. The mixture was allowed to stand at 30℃ for 8 hours, then 300g of propanol was added, and the mixture was stirred at 20℃ for 3 hours at 250 rpm. The solid and liquid phases were then separated by centrifugation at 15℃ with a separation factor of 7000g and a residence time of 10 minutes. Approximately 470g of the supernatant was collected. GPC analysis showed that the rhamnolipin content in the supernatant was 32.9g / L, and the PHA content was 1.09g / L. The supernatant was then filtered using a Zhongke Ruiyang spiral wound membrane SS-MF0452319-AF, and approximately 410g of the filtrate was collected. GPC analysis showed that the rhamnolipin content in the filtrate was 39.12g / L, and PHA was not detected. The filtrate was concentrated to 55g under a vacuum of 200 hPa at 80℃, and the rhamnolipin content was determined to be approximately 29.2%, with a yield of 97.8%.
[0061] Example 4
[0062] Take 200g of high-viscosity rhamnolipin fermentation broth D, add ammonia to adjust the pH to 10.5, let it stand at 30℃ for 8 hours, then add 100g of butanol, stir at 20℃ for 3 hours at 250rpm, then centrifuge at 15℃ to separate the solid and liquid, with a separation factor of 8000g and a residence time of 10 minutes, collecting approximately 270g of supernatant. GPC analysis showed that the rhamnolipin content in the supernatant was 65.9g / L and the PHA content was 0.08g / L. Then filter the supernatant using a Zhongke Ruiyang tubular membrane TGMN0205410, collecting approximately 205g of filtrate. GPC analysis showed that the rhamnolipin content in the filtrate was 87.6g / L, and PHA was not detected. The filtrate was concentrated to 65g under a vacuum of 200hpa at 80℃, and the rhamnolipin content was found to be approximately 13.2%, with a yield of 99.8%.
[0063] Comparative Example 1
[0064] 200g of high-viscosity rhamnolipin fermentation broth A was taken, and the pH was adjusted to 4 with HCl. The mixture was allowed to stand at 25°C for 5 hours, then 100g of ethanol was added, and the mixture was stirred at 25°C for 2 hours at 150 rpm. The solid and liquid phases were then separated by centrifugation at 5°C with a separation factor of 5000g and a residence time of 3 minutes. Approximately 187g of the supernatant was collected. GPC analysis showed that the rhamnolipin content in the supernatant was 17.2g / L, and the PHA content was 2.78g / L. The supernatant was then filtered through an Asahi Kasei hollow fiber membrane UNA-620A, and approximately 150g of the filtrate was collected. GPC analysis showed that the rhamnolipin content in the filtrate was 38g / L, and PHA was not detected. The filtrate was concentrated to 42g under a vacuum of 50 hPa at 75°C, and the rhamnolipin content was determined to be approximately 13.6%, with a yield of 46.8%.
[0065] Comparative Example 2
[0066] 200g of high-viscosity rhamnolipin fermentation broth A was taken and 100g of ethanol was added directly. The mixture was stirred at 25℃ for 2 hours at 150rpm. Then, the solid and liquid phases were separated by centrifugation at 5℃ with a separation factor of 5000g and a residence time of 3 minutes. Approximately 206g of supernatant was collected. GPC analysis showed that the rhamnolipin content in the supernatant was 27.6g / L and the PHA content was 2.98g / L. The supernatant was then filtered through an Asahi Kasei hollow fiber membrane UNA-620A, and approximately 198g of filtrate was collected. GPC analysis showed that the rhamnolipin content in the filtrate was 48g / L, and PHA was not detected. The filtrate was concentrated to 45g under a vacuum of 50hpa at 75℃, and the rhamnolipin content was found to be approximately 21.4%, with a yield of 78.9%.
[0067] Comparative Example 3
[0068] 200g of high-viscosity rhamnolipin fermentation broth A was taken, 100g of ethanol was added, and then NaOH was added to adjust the pH to 10.2. After stirring and mixing, the solid and liquid were separated by centrifugation at 5℃ with a separation factor of 5000g and a residence time of 3 minutes. Approximately 223g of supernatant was collected. GPC analysis showed that the rhamnolipin content in the supernatant was 43g / L and the PHA content was 3.54g / L. The supernatant was then filtered through an Asahi Kasei hollow fiber membrane UNA-620A, and approximately 205g of filtrate was collected. GPC analysis showed that the rhamnolipin content in the filtrate was 44g / L, and PHA was not detected. The filtrate was concentrated to 40g under a vacuum of 50hpa at 75℃, and the rhamnolipin content was found to be approximately 22.6%, with a yield of 74.1%.
[0069] Comparative Example 4
[0070] 200g of high-viscosity rhamnolipin fermentation broth A was taken, and the pH was adjusted to 10.2 with NaOH. The mixture was allowed to stand at 25℃ for 5 hours, then 80g of ethanol was added, and the mixture was stirred at 25℃ for 2 hours at 150rpm. The solid and liquid phases were then separated by centrifugation at 5℃ with a separation factor of 5000g and a residence time of 3 minutes. Approximately 196g of the supernatant was collected. GPC analysis showed that the rhamnolipin content in the supernatant was 54.5g / L, and the PHA content was 3.1g / L. The supernatant was then filtered through an Asahi Kasei hollow fiber membrane UNA-620A, and approximately 209g of the filtrate was collected. GPC analysis showed that the rhamnolipin content in the filtrate was 43.6g / L, and PHA was not detected. The filtrate was concentrated to 40g under a vacuum of 50hpa at 75℃, and the rhamnolipin content was determined to be approximately 22.5%, with a yield of 73.8%.
[0071] Comparative Example 5
[0072] Take 200g of high-viscosity rhamnolipin fermentation broth A, add 400g of water to dilute until the viscosity is reduced to 32cp, centrifuge to sterilize, with a separation factor of 8000g and a residence time of 10 minutes, and obtain 412g of centrifuged supernatant. GPC analysis showed that the rhamnolipin content in the supernatant was 21.3g / L, the PHA content was 1.1g / L, and the rhamnolipin yield was 71.9%.
[0073] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.
Claims
1. A separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions, characterized in that, Includes the following steps: 1) Add a soluble inorganic base to the high-viscosity rhamnolipin fermentation broth, adjust the pH to 10-11, and let it stand; the viscosity of the rhamnolipin fermentation broth is 50-3000 cp. 2) Add n-primary alcohol to the rhamnolipin fermentation broth after it has been allowed to stand, and stir. The n-primary alcohol is selected from small molecule alcohols with a chain length of 1-4 carbons. 3) Centrifuge the fermentation broth and collect the supernatant; 4) Filter the supernatant using an organic microfiltration membrane and collect the filtrate; 5) The filtrate was subjected to vacuum distillation to obtain an aqueous solution of rhamnolipin from the bottom of the column.
2. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to claim 1, characterized in that, The rhamnolipin fermentation broth contains 30-90 g / L of rhamnolipin and has a pH of 6.5-8.
0.
3. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to claim 2, characterized in that, The soluble inorganic base is selected from one or more of NaOH, KOH, and ammonia water.
4. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to claim 2 or 3, characterized in that, The settling time in step 1) is 5-12 hours.
5. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to any one of claims 1-3, characterized in that, The primary alcohol is selected from one or more of methanol, ethanol, propanol, and butanol.
6. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to claim 5, characterized in that, The amount of the n-type primary alcohol added is 0.5-1.5 times the mass of the rhamnolipin fermentation broth.
7. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to any one of claims 1-3, characterized in that, The stirring conditions in step 2) are: stirring at 150-250 rpm for 1-3 hours.
8. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to any one of claims 1-3, characterized in that, The centrifugation conditions in step 3) are: temperature 5-15℃, separation factor 5000-8000g, and residence time 3-10min.
9. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to any one of claims 1-3, characterized in that, The organic microfiltration membrane is selected from one or more of hollow fiber membranes, spiral wound membranes, and tubular membranes.
10. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to claim 9, characterized in that, The organic microfiltration membrane is made of one or more of polyethersulfone, polyacrylonitrile, and polyvinylidene fluoride.
11. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to claim 10, characterized in that, The organic microfiltration membrane has a pore size range of 0.11-0.45 μm.
12. The separation process for high-viscosity fermentation broth under abnormal rhamnolipin conditions according to any one of claims 1-3, characterized in that, Step 5) The vacuum distillation conditions are 50-200 hPa and 60-80℃; the distillation is stopped after the concentration is reduced to 1 / 10-1 / 5 of the original fermentation broth mass.