A double membrane separation process for extracting oil phase from a sclareol fermentation broth

Abstract

This invention relates to a dual-membrane separation process for extracting the oil phase from perillaldehyde fermentation broth. In the microbial synthesis of perillaldehyde fermentation broth, dodecane (dodecane:water = 1:9) is typically used for in-situ extraction to reduce the inhibitory effect of the product perillaldehyde on microorganisms. However, because the fermentation broth contains various amphoteric substances such as proteins and polysaccharides, forming an oil-in-water emulsion, the in-situ extraction effect is severely affected, cell activity decreases, and microbial fermentation is hindered. Therefore, timely oil-water separation of the emulsion is necessary. However, traditional demulsification techniques are time-consuming, costly, and cannot be well coupled with the extraction process. This invention, based on ceramic membrane oil-water separation, proposes the use of a coupled hydrophilic-oleophobic membrane and a hydrophobic-oleophobic membrane, assembled into a dual-membrane separator. This allows for the simultaneous permeation and separation of the oil and aqueous phases from the extracted emulsion, achieving continuous extraction and demulsification of the perillaldehyde fermentation broth, ensuring the continuous and efficient progress of biological fermentation.

Description

Technical Field

[0001] This invention relates to a separation process for separating the oil phase containing the product from the fermentation broth of microbial synthesis of perillaldehyde, which belongs to the field of biological separation. Background Technology

[0002] Perillyl alcohol is a diterpenoid compound, an essential chemical for plant growth, development, and general metabolism, and has applications in pharmaceuticals, cosmetics, health products, fragrances, and pesticides. Currently, the main method for obtaining perillyl alcohol is extraction from the plant *Perillium sibiricum*. However, the yield is severely limited by environmental, climatic, and soil properties, and the plant-based method involves complex components and is difficult to separate and purify. Therefore, researchers have proposed a microbial method for preparing perillyl alcohol. By engineering *Saccharomyces cerevisiae*, they have achieved microbial synthesis of perillyl alcohol and proposed in-situ extraction at a dodecane:water ratio of 1:9 to transfer the perillyl alcohol product as quickly as possible, ensuring continuous and efficient fermentation. However, due to the large amount of proteins, polysaccharides, and other surfactants in the fermentation broth, the in-situ extraction process forms a relatively stable, poorly dispersed oil-in-water emulsion, resulting in close contact between the perillyl alcohol and the microorganisms, thus limiting the efficiency of bio-fermentation. Increasing the oil-water ratio (e.g., 2:8) leads to a larger volume of the extracted oil phase, further increasing the load on subsequent separation processes.

[0003] To address the impact of emulsion formation on fermentation efficiency during in-situ extraction, it is necessary to separate the product-containing oil phase from the formed oil-in-water emulsion, which requires demulsification technology. Currently, commercially available demulsification technology is chemical demulsification, which involves adding chemical reagents to disrupt the stability of emulsion droplets. However, this method is unsuitable for bio-fermentation broth systems because chemical demulsifiers can affect cell activity and product quality. Another emerging method is electro-demulsification, which uses a high-voltage electric field to disrupt emulsion stability. However, this method is not suitable for industrial scale-up due to its high technical risks, high energy consumption, and potential impact on cell activity.

[0004] Therefore, it can be seen that the existing technology for separating perillaldehyde fermentation broth has problems such as high separation difficulty and complex procedures. Summary of the Invention

[0005] This invention combines in-situ extraction and membrane separation, proposing a dual-membrane separation process. It employs both hydrophilic-oleophobic and oleophilic-hydrophobic membranes to continuously separate the product-containing oil phase from the emulsion after in-situ extraction of the bio-fermentation broth. The technical concept of this invention is as follows: Addressing the characteristics of the oil-in-water emulsion obtained from in-situ extraction of perillaldehyde fermentation broth under low oil-to-water ratio (dodecane:water = 1:9), a hydrophilic-oleophobic ceramic membrane is used to break the emulsion and separate the aqueous phase, obtaining a concentrated oil phase containing fermentation products. Simultaneously, the concentrated oil phase can be separated from the microorganisms and water using an oleophilic-hydrophobic membrane, allowing the perillaldehyde-containing oil phase to pass through. The resulting perillaldehyde-containing oil phase is then distilled based on the boiling point difference between dodecane and perillaldehyde, and the separated dodecane can be recovered and reused, thus achieving a continuous fermentation-separation process.

[0006] A dual-membrane separation process for extracting the oil phase from perillaldehyde fermentation broth includes the following steps:

[0007] Step 1: The perilla alcohol fermentation broth is extracted in situ using an organic solvent, and then emulsified by high-speed stirring to form an emulsion;

[0008] Step 2: The emulsion is separated using a hydrophilic and oleophobic membrane, allowing the aqueous phase to pass through while the oil phase is retained;

[0009] Step 3: The obtained oil phase is separated using an oleophilic-hydrophobic membrane, allowing the oil phase to pass through while water and suspended solids are retained;

[0010] Step 4: After the solvent is removed from the oil phase obtained in Step 3, perillyl alcohol is obtained, and the separated solvent is returned to the fermentation process.

[0011] The surface water droplet contact angle of the hydrophilic and oleophobic film is 15-50°, and the underwater oil contact angle is 110-170°.

[0012] The surface water droplet contact angle of the oleophilic-hydrophobic film is 100-165°, and the oil-water contact angle is 120-170°.

[0013] The average pore size of the hydrophilic and oleophobic membrane is 8-14 nm, and the separation layer is made of zirconium oxide.

[0014] The average pore size of the oleophilic-hydrophobic membrane is 80-130 nm, and the material of the separation layer is alumina with hydrophobic surface modification.

[0015] During the filtration process using the hydrophilic-hydrophobic membrane and / or the oleophilic-hydrophobic membrane, the operating pressure is 0.5–0.7 bar and the temperature range is 20–25°C.

[0016] Beneficial effects

[0017] 1. After in-situ extraction, perillyl alcohol is enriched in the dodecane oil phase. The oil phase containing perillyl alcohol cannot be obtained by using only an oleophilic-hydrophobic membrane. However, a dodecane solution of perillyl alcohol can be obtained by using the permeate side of the oleophilic-hydrophobic membrane in the double membrane method. The permeate rate of perillyl alcohol exceeds 90%.

[0018] 2. The dual-membrane method achieved stable and continuous separation of oil and water phases in an in-situ extraction system of bio-fermentation broth. During a 500-minute test, the stable flux of the hydrophilic-oleophobic membrane was 21 L / m³. -2 h -1 The stable flux of the oleophilic-hydrophobic membrane is 4.5 L / m³. - 2 h -1 The hydrophilic-oleophobic film has an oil retention rate of over 99%, and the oleophilic-hydrophobic film has a water retention rate of over 99%.

[0019] 3. Compared with existing commercial demulsification technologies, the dual-membrane separation process used in this invention saves energy and costs, is environmentally friendly, and is also more conducive to continuous automation. Membrane demulsification technology is based on the "pore size sieving effect" and "surface polymerization effect" of the membrane, achieving oil-water separation of emulsions through selective permeation of oil or water under pressure differential.

[0020] 4. The ceramic membrane used in this invention has good chemical and mechanical properties, is resistant to high temperature, acid and alkali, and has a long service life. Attached Figure Description

[0021] Figure 1 This is a flow chart of a dual-membrane separation process.

[0022] Figure 2 These are schematic diagrams of the device and photos showing the processing effects.

[0023] Figure 3 These are micrographs of oil-in-water emulsions.

[0024] Figure 4 The change in flux of a single hydrophobic membrane over time under different dodecane:water ratios.

[0025] Figure 5 The change in flux of the hydrophilic and hydrophobic membranes over time in a 1:9 dual-membrane separation process.

[0026] Figure 6 The separation effects of the hydrophilic and hydrophobic membranes in the two-membrane separation process are as follows: (a) Changes in TOC content in the permeate aqueous phase and water content in the permeate oil phase over time; (b) Changes in permethrin content in the permeate oil phase over time. Detailed Implementation

[0027] This invention relates to a dual-membrane separation process for extracting the oil phase from perillaldehyde fermentation broth. In the microbial synthesis of perillaldehyde fermentation broth, dodecane (dodecane:water = 1:9) is typically used for in-situ extraction to reduce the inhibitory effect of the product perillaldehyde on microorganisms. However, because the fermentation broth contains various amphoteric substances such as proteins and polysaccharides, forming an oil-in-water emulsion, the in-situ extraction effect is severely affected, cell activity decreases, and microbial fermentation is hindered. Therefore, timely oil-water separation of the emulsion is necessary. However, traditional demulsification techniques are time-consuming, costly, and cannot be well coupled with the extraction process. This invention, based on ceramic membrane oil-water separation, proposes the use of a coupled hydrophilic-oleophobic membrane and a hydrophobic-oleophobic membrane, assembled into a dual-membrane separator. This allows for the simultaneous permeation and separation of the oil and aqueous phases from the extracted emulsion, achieving continuous extraction and demulsification of the perillaldehyde fermentation broth, ensuring the continuous and efficient progress of biological fermentation.

[0028] The content determination of salinomyol by ultraviolet spectrophotometry involves preparing a standard solution of salinomyol dodecane and performing a full-wavelength scan using an ultraviolet spectrophotometer. This identifies a specific absorption peak of salinomyol within a certain wavelength range (200–300 nm), allowing for quantitative analysis based on the wavelength of the maximum absorption peak in the ultraviolet spectrum. Within the concentration range of 0.1–10 g / L, a good linear relationship exists between the concentration of salinomyol and the ultraviolet absorbance at the maximum absorption peak; therefore, ultraviolet spectrophotometry is used to determine the content of salinomyol.

[0029] Example 1

[0030] The process route used in this patent is as follows: Figure 1 As shown, the fermentation broth of perilla alcohol was extracted in situ (dodecane:water = 1:9) to form an oil-in-water emulsion, as follows: Figure 2 As shown, the emulsion is sequentially treated with a hydrophilic-oleophobic membrane and an oleophilic-hydrophobic membrane. The hydrophilic-oleophobic membrane is made of zirconium oxide with an average pore size of 10 nm. The oleophilic-hydrophobic membrane is made of alumina modified with hexadecyltrimethoxysilane hydrophobically with an average pore size of 100 nm. The surface water droplet contact angle of the hydrophilic-oleophobic membrane is 28.2°, and the underwater oil contact angle is 137.6°. The surface water droplet contact angle of the oleophilic-hydrophobic membrane is 141.3°, and the oil-water contact angle is 154.6°. The operating temperature is 25°C, and the operating pressure is 0.5 bar. The aqueous phase free of bacteria is obtained from the permeate side of the hydrophilic-oleophobic membrane and can be recycled. The concentrate from the retrieval side of the hydrophilic-oleophobic membrane is then filtered through the oleophilic-hydrophobic membrane, and the oil phase containing perillaldehyde is obtained from the permeate side of the oleophilic-hydrophobic membrane. The perillaldehyde content in the permeate oil phase is determined by ultraviolet spectrophotometry. The permeation flux of the water-permeable membrane and the oil-permeable membrane was tested and calculated. In addition, the total organic carbon (TOC) of the permeate aqueous phase was measured using a total organic carbon analyzer to indirectly evaluate the oil retention capacity of the hydrophilic-oleophobic membrane, and the water content in the permeate aqueous phase was measured using a Karl Fischer moisture analyzer to evaluate the water retention capacity of the hydrophilic-oleophobic membrane.

[0031] Comparative experiment:

[0032] Ideally, the process involves separating the oil phase containing the product from the emulsion formed by in-situ extraction of perillaldehyde fermentation broth using a uniphilic hydrophobic membrane. Based on this, the separation performance using only a uniphilic hydrophobic membrane was investigated under different dodecane:water ratios for in-situ extraction. The results are as follows: Figure 4 As shown in the figure, 20%, 30%, 40%, and 50% represent the volume fraction of dodecane in the emulsion. When the fermentation broth was extracted at a dodecane:water ratio of 1:9, the oleophilic-hydrophobic membrane used in Example 1 alone could not separate the perillaldehyde-containing oil phase from the formed emulsion. However, when the dodecane ratio was increased to 1:4, the perillaldehyde-containing oil phase could be separated from the formed emulsion using only the oleophilic-hydrophobic membrane, but the flux was only 3.6 L·m· -2 ·h -1 This phenomenon is mainly due to two factors: First, when the ratio of dodecane to water is 1:9, the water phase content is much higher than the oil phase, resulting in a very small chance for the oil phase to come into contact with the hydrophobic membrane surface; second, because it is in an emulsified state, it is difficult for the oil-in-water emulsion to break down on the hydrophobic membrane surface. Therefore, in actual separation processes, using oleophilic-hydrophobic membranes can lead to difficulties in the separation process.

[0033] In-situ extraction was performed using dodecane:water at a ratio of 1:9. The changes in permeation flux of the hydrophilic-oleophobic membrane and the oleophilic-hydrophobic membrane over time in the dual-membrane separation process are shown below. Figure 5 As shown, the initial flux of the hydrophilic-oleophobic film is approximately 70 L·m. -2 ·h -1 The obtained water was clear and transparent, indicating that the emulsified oil droplets were completely removed. Within the first 24 minutes, the hydrophilic membrane flux showed a sharp downward trend, while the hydrophobic-lipophilic membrane flux remained at zero. After 24 minutes, the hydrophobic membrane flux began to gradually increase and stabilize, while the downward trend of the hydrophilic membrane flux slowed and gradually stabilized. After 500 minutes of continuous operation, the hydrophobic-lipophilic membrane flux stabilized at 21 L·m⁻². -2 ·h -1 The flux of the oleophilic-hydrophobic film remained stable at around 4.5 L·m. -2 ·h -1The flux of the hydrophilic-oleophobic membrane was significantly higher than that of the oleophilic-hydrophobic membrane throughout the process. This was mainly due to two factors: firstly, the aqueous phase accounted for a larger proportion of the emulsion; secondly, the oil-in-water emulsion was prone to demulsification on the hydrophilic membrane surface, and the resulting aqueous phase easily formed a water film on the hydrophilic membrane surface. However, the high proportion of the aqueous phase on the hydrophobic membrane surface severely hindered the contact of the dispersed oil generated by the demulsification of the hydrophilic membrane with the hydrophobic membrane surface, resulting in lower flux. Compared with a single oleophilic-hydrophobic membrane, the dual-membrane separation process enabled the separation of the product-containing oil phase from the emulsion during in-situ extraction at a dodecane:water ratio of 1:9. This process can be combined with the in-situ extraction step of bio-fermentation to achieve continuous and efficient bio-fermentation.

[0034] Further analysis of the components in the permeate was conducted, and the results are as follows: Figure 6 As shown, the average TOC content in the aqueous phase permeated through the membrane was 53.2 mg / L, and the average water content in the oil phase permeated was 140.8 ppm. The hydrophilic-oleophobic membrane exhibited excellent oil retention performance, while the oleophilic-hydrophobic membrane demonstrated excellent water retention performance. The permeate oil phase obtained through the oleophilic-hydrophobic membrane was tested, and the average oleophilic-hydrophobic content was 19.9 g / L. It can be observed that the content in the oil phase after the dual-membrane separation process was not significantly different from that on the feed side, differing by only 0.1 g / L, indicating that the oleophilic-hydrophobic membrane essentially did not retain oleophilic-hydrophobic content.

Claims

1. A dual-membrane separation process for extracting the oil phase from perilla frutescens fermentation broth, characterized in that, Includes the following steps: Step 1: The fermentation broth of perilla alcohol is extracted in situ using dodecane, with a dodecane:water ratio of 1:9 (volume ratio). After high-speed stirring and emulsification, an emulsion is formed. Step 2: The emulsion is separated using a hydrophilic-oleophobic membrane, allowing the aqueous phase to pass through while the oil phase is retained; the surface water droplet contact angle of the hydrophilic-oleophobic membrane is 15-50°, and the underwater oil contact angle is 110-170°; the average pore size of the hydrophilic-oleophobic membrane is 8-14nm, and the separation layer is made of zirconium oxide. Step 3: The oil phase obtained in Step 2 is separated using an oleophilic-hydrophobic membrane, allowing the oil phase to pass through while water and suspended solids are retained. The surface water droplet contact angle of the oleophilic-hydrophobic membrane is 100-165°, and the oil-water contact angle is 120-170°. The average pore size of the oleophilic-hydrophobic membrane is 80-130 nm, and the separation layer is made of hydrophobic surface-modified alumina. Step 4: After removing dodecane from the oil phase obtained in Step 3, succinyl alcohol is obtained, and the separated dodecane is returned to the fermentation process. The operating pressure during filtration using the hydrophilic-oleophobic membrane and / or the oleophilic-hydrophobic membrane is 0.5~0.7 bar, and the temperature range is 20~25℃.

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