Separation process for high-viscosity rhamnolipid fermentation liquor obtained under abnormal working condition

Abstract

Disclosed in the present application is a separation process for a high-viscosity rhamnolipid fermentation liquor obtained under an abnormal working condition, which comprises the following steps: 1) adding a soluble inorganic alkali to a high-viscosity rhamnolipid fermentation liquor to adjust the pH value to 10 or above, and performing standing treatment; 2) adding a normal primary alcohol to the rhamnolipid fermentation liquor after the standing treatment and stirring same; (3) performing centrifugal separation on the stirred fermentation liquor, and collecting a supernatant; 4) filtering the supernatant by using an organic microfiltration membrane, and collecting a filtrate; and 5) carrying out vacuum rectification on the filtrate, so as to obtain a rhamnolipid aqueous solution from a column bottom. The separation process provided by the present application involves a simple route; separation devices used therein are all conventional devices; and the separation of rhamnolipid and high-viscosity PHA can be achieved well.

Description

A separation process for high-viscosity fermentation broth under abnormal rhamnolipid working conditions Technical Field

[0001] The present application relates to a separation process, for example, a separation process for a highly viscous fermentation broth under abnormal rhamnolipid working conditions. Background Art

[0002] Rhamnolipids, produced by Pseudomonas aeruginosa under certain conditions, are extracellular metabolites. They possess excellent surface and interfacial activity and are widely used in the petrochemical, environmental, pharmaceutical, food, and agricultural sectors. They are non-toxic and biodegradable, making them a biosurfactant with the longest research history, the best results, and the most mature application technology.

[0003] Polyhydroxyalkanoate (PHA) is a byproduct produced during the metabolism of Pseudomonas aeruginosa. It is an intracellular metabolite and a high molecular weight polymer. During the fermentation process of Pseudomonas aeruginosa, rhamnolipids and PHA are produced simultaneously, but one is produced extracellularly and the other is produced intracellularly. In the normal fermentation metabolism process, rhamnolipids are mainly produced, and the output of PHA is small, and all of it is intracellular and does not diffuse into the fermentation broth in large quantities. Therefore, the viscosity of the fermentation broth is normal and generally remains below 5 cP. At the end of fermentation, it can be sterilized and further separated and purified by conventional solid-liquid separation and other methods. Since the fermentation process is a microbial metabolic process, process abnormalities will lead to abnormal microbial growth and metabolic processes, causing the death of microorganisms. After the death of the bacteria, the viscosity of the fermentation broth increases rapidly, and the product cannot be separated by conventional solid-liquid separation methods, resulting in huge production losses.

[0004] In the case of abnormal viscosity of the fermentation broth due to process operation, if the fermentation broth can be effectively separated and rhamnolipids can be recovered, product losses caused by uncontrollable factors in the production stage can be avoided, which is of great significance for the industrial production of rhamnolipids.

[0005] Summary of the Invention

[0006] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0007] Through continuous research, the inventors have discovered that there are many uncontrollable factors in the fermentation process. These factors may result in the death and rupture of a large number of bacterial cells, and the transfer of PHA from the intracellular to the extracellular space, causing the viscosity of the fermentation broth to instantly increase to tens or even thousands of centipoises (cP). The highly viscous fermentation broth cannot be separated into solids and liquids, thus affecting normal production and causing rhamnolipid loss.

[0008] There is currently no literature report on how to recover rhamnolipids with high yield in fermentation broths with abnormal viscosity. In order to solve the above technical problems, this application proposes a separation process for high-viscosity fermentation broths under abnormal rhamnolipid working conditions.

[0009] To achieve the above objectives, the technical solutions adopted in this application are as follows:

[0010] A process for separating high-viscosity fermentation broth under abnormal rhamnolipid working conditions comprises the following steps:

[0011] 1) adding a soluble inorganic base to the high-viscosity rhamnolipid fermentation broth, adjusting the pH to above 10, and allowing to stand;

[0012] 2) adding n-primary alcohol to the rhamnolipid fermentation broth after standing and stirring;

[0013] 3) centrifuging the fermentation broth and collecting the supernatant;

[0014] 4) filtering the supernatant using an organic microfiltration membrane and collecting the filtrate;

[0015] 5) The filtrate is subjected to vacuum distillation to obtain a rhamnolipid aqueous solution in the bottom of the tower.

[0016] As an optional embodiment, the viscosity of the rhamnolipid fermentation broth is 50-3000 cp, the rhamnolipid content is 30-90 g / L, and the pH is 6.5-8.0.

[0017] Through ongoing research, the applicant has discovered that high levels of PHA can be detected in rhamnolipid fermentation broths with abnormal viscosity using gel permeation chromatography (GPC), while PHA cannot be detected in normal fermentation broths. This indicates that PHA is transferred from the intracellular to the extracellular space, which is the main cause of the increased viscosity of rhamnolipid fermentation broth. Furthermore, for rhamnolipid fermentation broths with a viscosity of 50-3000 cp, the PHA content detected by GPC is 1-50 g / L. Due to the limitations of the detection method, the rhamnolipid content and PHA content in this application both represent the extracellular content of the fermentation broth.

[0018] As an optional embodiment, the soluble inorganic base is selected from one or more of NaOH, KOH, and ammonia water.

[0019] As an optional embodiment, the standing treatment time in step 1) is 5-12 hours.

[0020] As an optional embodiment, the normal primary alcohol is selected from small molecule alcohols with a carbon chain length of 1-4, and can be one or more of methanol, ethanol, propanol, and butanol;

[0021] In one embodiment, the amount of the normal primary alcohol added is 0.5-1.5 times the mass of the rhamnolipid fermentation broth.

[0022] As an optional embodiment, the stirring condition in step 2) is stirring at 150-250 rpm for 1-3 hours.

[0023] As an optional embodiment, the centrifugation conditions in step 3) are: temperature 5-15°C, separation factor 5000-8000g, and residence time 3-10 min.

[0024] As an optional embodiment, the organic microfiltration membrane is selected from one or more of a hollow fiber membrane, a spiral membrane, and a tubular membrane;

[0025] In one embodiment, the material of the organic microfiltration membrane is selected from one or more of polyethersulfone, polyacrylonitrile, and polyvinylidene fluoride;

[0026] In one embodiment, the pore size of the organic microfiltration membrane is in the range of 0.11-0.45 μm.

[0027] As an optional implementation scheme, the vacuum distillation conditions in step 5) are 50-200 hPa in vacuum and 60-80° C. in the bottom of the tower; the distillation is stopped after the concentration reaches 1 / 10-1 / 5 of the original fermentation liquid mass.

[0028] The rhamnolipid aqueous solution prepared by the method of the present application has a content of 10-30% and a rhamnolipid yield of more than 90%.

[0029] The separation process provided by the application is simple, and the separation equipment used is conventional equipment, which can well realize the separation of rhamnolipid and PHA, and the rhamnolipid yield is improved. The viscosity increase in the high-viscosity fermentation broth is mainly caused by PHA. After adding alkali solution to increase the pH, the thalline is completely broken, rhamnolipid and PHA are all released, and the solubility of rhamnolipid is significantly increased. After that, ethanol is added to cause the denaturation precipitation of thalline protein, and the particle size increases after PHA contacts ethanol and precipitates in the form of large-particle colloids, and most of PHA and thalline can be separated by centrifugation. After that, an organic membrane with good hydrophilicity is selected to filter the centrifugal supernatant, and the residual thalline and PHA can be thoroughly removed. Finally, rhamnolipid is basically all recovered to the aqueous phase, which can be further refined according to subsequent needs.

[0030] Still other aspects will become apparent upon reading and understanding the detailed description. DETAILED DESCRIPTION

[0031] The present application is further described below through specific examples. The examples described in this application are only used to illustrate the present application and do not limit the scope of the present application.

[0032] In the following examples and comparative examples of the present application, the separation equipment used are as follows: Zhongke Ruiyang rolled membrane SS-MF0452319-AF (0.45 μm, polyvinylidene fluoride), SS-ME0222319-AF (0.22 μm, polyethersulfone), Zhongke Ruiyang tubular membrane TGMN0205410 (0.2 μm, polyacrylonitrile), Asahi Kasei hollow fiber membrane UNA-620A (0.1 μm, polyvinylidene fluoride), Hitachi desktop centrifuge CP-NX, Japan Rika rotary evaporator N-1100D-W, 3L three-necked flask, Shanghai Jinghong water bath.

[0033] The reagents used, their sources, and purities are as follows: anhydrous ethanol, analytical grade, purchased from Kermiou; NaOH, analytical grade, purchased from Sinopharm; concentrated sulfuric acid, 98% analytical grade, purchased from Sinopharm; and rhamnose standard, analytical grade, purchased from Sinopharm.

[0034] High viscosity rhamnolipid fermentation broth A: viscosity 89cp, rhamnolipid content 61g / L, PHA content 2.3g / L, pH=7.2;

[0035] High viscosity rhamnolipid fermentation broth B: viscosity 950cp, rhamnolipid content 35g / L, PHA content 10.5g / L, pH = 6.8;

[0036] High viscosity rhamnolipid fermentation broth C: viscosity 2800 cp, rhamnolipid content 82 g / L, PHA content 45.2 g / L, pH = 7.8;

[0037] High viscosity rhamnolipid fermentation broth D: viscosity 54 cp, rhamnolipid content 43 g / L, PHA content 1.3 g / L, pH = 6.5.

[0038] GPC detection uses gel chromatography for quantitative analysis. The specific operation is as follows:

[0039] 1) Prepare 1 g / L PHA standard solution: Weigh 0.1 g PHA into a 100 mL volumetric flask and dissolve in THF to prepare a 1 g / L PHA standard solution.

[0040] 2) Take 1 g of fermentation broth, dilute to 10 mL volumetric flask, and dissolve in THF solvent to prepare a 100 g / L test solution.

[0041] 3) Take 1 mL of the standard solution and the test solution, filter them separately through a 0.2 μm filter membrane, and then determine the content by gel chromatography. The chromatographic conditions are as follows:

[0042] Column temperature: 40°C, flow rate: 1 mL / min, running time: 40 min, mobile phase: A: water, B: acetonitrile, running program as shown in Table 1.

[0043] Table 1

[0044] Based on the comparison of the peak areas of the standard sample and the sample to be tested at the same time, the PHA content in the sample to be tested (in g / L) can be calculated. The calculation formula is as follows: PHA content in the sample to be tested = peak area of ​​the sample to be tested / peak area of ​​the standard * 10

[0045] The method for detecting rhamnolipid content is the sulfuric acid-anthrone method. The specific operation method is as follows:

[0046] 1) Prepare anthrone solution: dissolve 0.2 g of anthrone in 100 mL of 80% sulfuric acid. Protect from light and prepare immediately before use. Do not store.

[0047] 2) Place 0.5 mL of the sample in a 10-15 mL graduated stoppered test tube and cool thoroughly in an ice-water bath. While still in the ice-water bath, add 2 mL of the anthrone solution and mix rapidly (minimize the reaction). Then, place in a boiling water bath for 10 minutes. Remove from the test tube and cool to room temperature in an ice-water bath. Measure the absorbance at 620 nm.

[0048] 3) Rhamnose standard solution: Weigh 0.1 g of rhamnose and dilute to a 250 mL volumetric flask to make a 400 mg / L rhamnose stock solution. Then dilute the solution proportionally to obtain standards of different concentrations, as shown in Table 2 below:

[0049] Table 2. Rhamnose standard curve with different concentrations of samples

[0050] The absorbance was determined by the anthrone method and a standard curve was drawn.

[0051] y=ax+b

[0052] Where y is the rhamnose content and x is the absorbance.

[0053] The calculation formula for the final rhamnolipid content is: Rhamnolipid 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] 200 g of high-viscosity rhamnolipid fermentation broth A was prepared, and NaOH was added to adjust the pH to 10.2. The mixture was allowed to stand at 25°C for 5 hours, followed by the addition of 100 g of ethanol and stirring at 25°C for 2 hours at 150 rpm. The mixture was then centrifuged at 5°C for solid-liquid separation, with a separation factor of 5000 g and a residence time of 3 minutes. Approximately 265 g of the supernatant was collected. GPC analysis revealed a rhamnolipid content of 45 g / L and a PHA content of 0.35 g / L. The supernatant was then filtered using an Asahi Kasei hollow fiber membrane UNA-620A, and approximately 230 g of the filtrate was collected. GPC analysis revealed a rhamnolipid content of 49.56 g / L, with no PHA detected. The filtrate was concentrated to 40 g at 75°C under a vacuum of 50 hPa. The rhamnolipid content was approximately 28.5%, resulting in a yield of 93.44%.

[0057] Example 2

[0058] 300 g of high-viscosity rhamnolipid fermentation broth B was taken, KOH was added to adjust the pH to 11, and the mixture was allowed to stand at 20°C for 12 hours. Then, 300 g of methanol was added and stirred at 30°C for 1 hour at 200 rpm. The solid and liquid were then centrifuged at 5°C with a separation factor of 8000 g and a residence time of 5 minutes. Approximately 510 g of the supernatant was collected. GPC analysis showed that the rhamnolipid content in the supernatant was 20.09 g / L and the PHA content was 1.23 g / L. The supernatant was then filtered using a Zhongke Ruiyang spiral membrane SS-ME0222319-AF, and approximately 490 g of the filtrate was collected. GPC analysis showed that the rhamnolipid content in the filtrate was 20.42 g / L, and no PHA was detected. The filtrate was concentrated to 55 g at 60°C under a vacuum of 150 hPa. The rhamnolipid content was approximately 18.2%, with a yield of 95.3%.

[0059] Example 3

[0060] Take 200g of high-viscosity rhamnolipid fermentation broth C, add ammonia water to adjust the pH to 10.5, let it stand at 30°C for 8 hours, then add 300g of propanol, stir at 20°C for 3 hours, stir at 250rpm, then centrifuge at 15°C to separate the solid and liquid, with a separation factor of 7000g and a residence time of 10 minutes. Collect about 470g of supernatant. GPC analysis of the supernatant showed a rhamnolipid content of 32.9g / L and a PHA content of 1.09g / L. The supernatant was then filtered using a Zhongke Ruiyang spiral membrane SS-MF0452319-AF, collecting about 410g of filtrate. GPC analysis of the filtrate showed a rhamnolipid content of 39.12g / L, and no PHA was detected. The filtrate was concentrated to 55g at 80°C under a vacuum of 200hPa. The rhamnolipid content was detected to be approximately 29.2%, with a yield of 97.8%.

[0061] Example 4

[0062] Take 200g of high-viscosity rhamnolipid fermentation broth D, add ammonia water to adjust the pH to 10.5, let it stand at 30°C for 8 hours, then add 100g of butanol, stir at 20°C for 3 hours at a stirring speed of 250rpm, then centrifuge at 15°C to separate the solid and liquid, with a separation factor of 8000g and a residence time of 10 minutes. Collect about 270g of supernatant. GPC analysis of the supernatant showed a rhamnolipid content of 65.9g / L and a PHA content of 0.08g / L. The supernatant was then filtered using a Zhongke Ruiyang tubular membrane TGMN0205410, collecting about 205g of filtrate. GPC analysis of the filtrate showed a rhamnolipid content of 87.6g / L, and no PHA was detected. The filtrate was concentrated to 65g at 80°C under a vacuum of 200hPa. The rhamnolipid content was detected to be approximately 13.2%, with a yield of 99.8%.

[0063] Comparative Example 1

[0064] 200 g of high-viscosity rhamnolipid fermentation broth A was adjusted to pH 4 by adding HCl. The mixture was allowed to stand at 25°C for 5 hours, followed by the addition of 100 g of ethanol and stirring at 25°C for 2 hours at 150 rpm. The solid-liquid separation was then performed by centrifugation at 5°C with a separation factor of 5000 g and a residence time of 3 minutes. Approximately 187 g of the supernatant was collected. GPC analysis revealed a rhamnolipid content of 17.2 g / L and a PHA content of 2.78 g / L. The supernatant was then filtered using an Asahi Kasei hollow fiber membrane UNA-620A. Approximately 150 g of the filtrate was collected. GPC analysis revealed a rhamnolipid content of 38 g / L, with no PHA detected. The filtrate was concentrated to 42 g at 75°C under a vacuum of 50 hPa. The rhamnolipid content was approximately 13.6%, resulting in a yield of 46.8%.

[0065] Comparative Example 2

[0066] To 200 g of high-viscosity rhamnolipid fermentation broth A, 100 g of ethanol was added directly. The mixture was stirred at 25°C for 2 hours at 150 rpm. The solid-liquid separation was then performed by centrifugation at 5°C with a separation factor of 5000 g and a residence time of 3 minutes. Approximately 206 g of the supernatant was collected. GPC analysis revealed a rhamnolipid content of 27.6 g / L and a PHA content of 2.98 g / L. The supernatant was then filtered using an Asahi Kasei hollow fiber membrane UNA-620A. Approximately 198 g of the filtrate was collected. GPC analysis revealed a rhamnolipid content of 48 g / L, with no PHA detected. The filtrate was concentrated to 45 g at 75°C under a vacuum of 50 hPa. The rhamnolipid content was approximately 21.4%, resulting in a yield of 78.9%.

[0067] Comparative Example 3

[0068] To 200 g of high-viscosity rhamnolipid fermentation broth A, 100 g of ethanol was added, and NaOH was added to adjust the pH to 10.2. After stirring, the mixture was centrifuged at 5°C to separate the solid and liquid, with a separation factor of 5000 g and a residence time of 3 minutes. Approximately 223 g of the supernatant was collected. GPC analysis revealed a rhamnolipid content of 43 g / L and a PHA content of 3.54 g / L. The supernatant was then filtered using an Asahi Kasei hollow fiber membrane UNA-620A, and approximately 205 g of the filtrate was collected. GPC analysis revealed a rhamnolipid content of 44 g / L, with no PHA detected. The filtrate was concentrated to 40 g at 75°C under a vacuum of 50 hPa. The rhamnolipid content was approximately 22.6%, resulting in a yield of 74.1%.

[0069] Comparative Example 4

[0070] 200 g of high-viscosity rhamnolipid fermentation broth A was taken, and NaOH was added to adjust the pH to 10.2. The mixture was allowed to stand at 25°C for 5 hours, followed by the addition of 80 g of ethanol and stirring at 25°C for 2 hours at 150 rpm. The solid-liquid separation was then performed by centrifugation at 5°C with a separation factor of 5000 g and a residence time of 3 minutes. Approximately 196 g of the supernatant was collected. GPC analysis revealed a rhamnolipid content of 54.5 g / L and a PHA content of 3.1 g / L. The supernatant was then filtered using an Asahi Kasei hollow fiber membrane UNA-620A, and approximately 209 g of the filtrate was collected. GPC analysis revealed a rhamnolipid content of 43.6 g / L, with no PHA detected. The filtrate was concentrated to 40 g at 75°C under a vacuum of 50 hPa. The rhamnolipid content was approximately 22.5%, resulting in a yield of 73.8%.

[0071] Comparative Example 5

[0072] 200 g of high-viscosity rhamnolipid fermentation broth A was diluted with 400 g of water to reduce the viscosity to 32 cp. The broth was sterilized by centrifugation at a separation factor of 8000 g and a residence time of 10 minutes to obtain 412 g of supernatant. GPC analysis of the supernatant revealed a rhamnolipid content of 21.3 g / L and a PHA content of 1.1 g / L, for a rhamnolipid yield of 71.9%.

[0073] The above is only an optional implementation method of the present application. It should be pointed out that ordinary technicians in this field can make several improvements and supplements without departing from the method of the present application. These improvements and supplements should also be regarded as the scope of protection of the present application.

Claims

1. A process for separating high-viscosity fermentation broth under abnormal rhamnolipid working conditions, comprising the following steps: 1) adding a soluble inorganic base to the high-viscosity rhamnolipid fermentation broth, adjusting the pH to above 10, and allowing to stand for treatment; 2) adding n-primary alcohol to the rhamnolipid fermentation broth after standing and stirring; 3) centrifuging the fermentation broth and collecting the supernatant; 4) filtering the supernatant with an organic microfiltration membrane and collecting the filtrate; 5) The filtrate is subjected to vacuum distillation to obtain a rhamnolipid aqueous solution in the bottom of the tower.

2. The separation process of high-viscosity fermentation broth under abnormal rhamnolipid working conditions according to claim 1, wherein: The rhamnolipid fermentation liquid has a viscosity of 50-3000 cp, a rhamnolipid content of 30-90 g / L, and a pH of 6.5-8.

0.

3. The separation process of high-viscosity fermentation broth under abnormal rhamnolipid working conditions according to claim 2, wherein: The soluble inorganic base is selected from one or more of NaOH, KOH and ammonia water.

4. The separation process of high-viscosity fermentation broth under abnormal rhamnolipid working conditions according to claim 2 or 3, characterized in that: The standing treatment time in step 1) is 5-12 hours.

5. The separation process of high-viscosity fermentation broth under abnormal rhamnolipid working conditions according to any one of claims 1 to 3, wherein: The normal primary alcohol is selected from small molecule alcohols with a carbon chain length of 1 to 4, and can be selected from one or more of methanol, ethanol, propanol, and butanol; Optionally, the added amount of the normal primary alcohol is 0.5-1.5 times the mass of the rhamnolipid fermentation broth.

6. The separation process of high-viscosity fermentation broth under abnormal rhamnolipid working conditions according to any one of claims 1 to 3, wherein: The stirring condition in step 2) is 150-250 rpm for 1-3 hours.

7. The separation process of high-viscosity fermentation broth under abnormal rhamnolipid working conditions according to any one of claims 1 to 3, wherein: The centrifugal conditions in step 3) are: temperature 5-15°C, separation factor 5000-8000g, and residence time 3-10min.

8. The separation process of high-viscosity fermentation broth under abnormal rhamnolipid working conditions according to any one of claims 1 to 3, wherein: The organic microfiltration membrane is selected from one or more of a hollow fiber membrane, a spiral membrane, and a tubular membrane; Optionally, the material of the organic microfiltration membrane is selected from one or more of polyethersulfone, polyacrylonitrile, and polyvinylidene fluoride; Optionally, the pore size of the organic microfiltration membrane ranges from 0.11 to 0.45 μm.

9. The separation process of high-viscosity fermentation broth under abnormal rhamnolipid working conditions according to any one of claims 1 to 3, wherein: Step 5) The vacuum distillation conditions are as follows: vacuum degree 50-200 hpa, tower bottom temperature 60-80° C.; and the distillation is stopped after the concentration reaches 1 / 10-1 / 5 of the original fermentation liquid mass.

Images