Preparation method and device of rhamnolipid and polyhydroxyalkanoate

Abstract

The invention provides a preparation method and device of rhamnolipid and polyhydroxyalkanoate. The preparation method comprises the following steps: inoculating strains for co-producing rhamnolipid and polyhydroxyalkanoate into a culture medium, and carrying out fermentation reaction on the strains to obtain fermentation liquor; continuously outputting the fermentation liquor from the fermentation system, and defoaming the output fermentation liquor; sequentially separating the fermentation liquor subjected to the defoaming treatment to obtain the polyhydroxyalkanoate and the rhamnolipid; and returning the fermentation liquor from which the polyhydroxyalkanoate and the rhamnolipid are separated to a fermentation system, and continuing the fermentation reaction. According to the method, the production efficiency of continuously producing rhamnolipid and polyhydroxyalkanoate can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field related to rhamnolipid production, and in particular to a method and device for preparing rhamnolipid and polyhydroxy fatty acid. Background Art

[0002] Rhamnolipids are glycolipid biosurfactants, mainly produced by Pseudomonas aeruginosa and related strains. Their most prominent characteristic is high surface activity, such as significantly reducing the surface tension of water and changing the wettability of solid surfaces. They have multiple functions such as emulsification, demulsification, foaming, washing, dispersion and flocculation, antistatic and lubrication. Rhamnolipids are strong surfactants. In the traditional fermentation process of preparing rhamnolipids, a large amount of defoamers must be introduced to control foam and liquid level, which affects production efficiency.

[0003] In addition, during the production of rhamnolipids, a certain amount of polyhydroxy fatty acids (PHA) will be produced in the bacterial cells. As the fermentation process progresses and the bacterial cells age, the PHA in the cells will be released into the fermentation broth, causing the viscosity of the fermentation broth to increase rapidly, resulting in a decrease in the dissolved oxygen in the fermentation broth, reducing the fermentation activity of the bacteria and reducing production efficiency. This situation is particularly prone to occur in the later stages of rhamnolipid fermentation.

[0004] Polyhydroxy fatty acids (PHA) are a type of biopolymer synthesized by various microorganisms as carbon sources and energy storage when carbon and nitrogen nutrition is unbalanced. Its accumulation in bacterial cells is a common phenomenon in nature. It has been found that more than 90 genera of bacteria synthesize more than 150 PHAs with different structures. PHA is composed of hydroxy fatty acid monomers with 3 to 14 carbon atoms. PHA has good biodegradability and biocompatibility and can be used as various medical materials or drug carriers.

[0005] Therefore, a method that can effectively control the foam and viscosity of the fermentation broth during the fermentation production of rhamnolipids and co-produce PHA is a research focus in this field. Summary of the invention

[0006] The present invention provides a method and device for preparing rhamnolipid and polyhydroxy fatty acid. The method can continuously co-produce rhamnolipid and polyhydroxy fatty acid, improves the production efficiency of rhamnolipid and polyhydroxy fatty acid, and solves the problem of difficult control of foam and fermentation liquid viscosity in the production process of rhamnolipid in the prior art.

[0007] The invention provides a preparation device for rhamnolipid and polyhydroxy fatty acid, which can be used for continuous co-production of rhamnolipid and polyhydroxy fatty acid, thereby improving the production efficiency of rhamnolipid and polyhydroxy fatty acid.

[0008] The invention provides a method for preparing rhamnolipid and polyhydroxy fatty acid, comprising: inoculating a strain for co-producing rhamnolipid and polyhydroxy fatty acid into a culture medium, and subjecting the strain to a fermentation reaction to obtain a fermentation liquid; continuously outputting the fermentation liquid from a fermentation system, and performing a defoaming treatment on the outputted fermentation liquid; and sequentially separating the polyhydroxy fatty acid and the rhamnolipid from the fermentation liquid after the defoaming treatment.

[0009] Optionally, after the polyhydroxy fatty acids and the rhamnolipid are sequentially separated from the fermentation broth after the defoaming treatment, the method further comprises: returning the fermentation broth from which the polyhydroxy fatty acids and the rhamnolipid are separated to the fermentation system, continuing the fermentation reaction to produce the fermentation broth, until the concentration of rhamnolipid in the fermentation broth is greater than 20 g / L and the viscosity of the fermentation broth is greater than 15 cp, and then stopping the fermentation reaction.

[0010] Optionally, the process of inoculating a strain of bacteria that co-produces rhamnolipids and polyhydroxyalkanoates into a culture medium and subjecting the strain to a fermentation reaction to obtain a fermentation broth includes the following stages in sequence: a seed growth stage, a rhamnolipid initial accumulation stage, a polyhydroxyalkanoate initial accumulation stage, and a steady-state continuous fermentation stage; or; a seed growth stage, a polyhydroxyalkanoate initial accumulation stage, a rhamnolipid initial accumulation stage, and a steady-state continuous fermentation stage; wherein the feed amount in the initial rhamnolipid accumulation stage is less than the feed amount in the initial polyhydroxyalkanoate accumulation stage, and the feed amount in the initial polyhydroxyalkanoate accumulation stage is the same as the feed amount in the steady-state continuous fermentation stage.

[0011] Optionally, the seed growth stage includes: inoculating the culture medium with a strain that co-produces rhamnolipids and polyhydroxy fatty acids, culturing the strain in an aerobic environment until the OD600 of the culture medium is greater than or equal to 65, and obtaining a seed culture solution; the initial accumulation stage of rhamnolipid includes: continuously adding a first carbon source and a first nitrogen source to the fermentation system so that the concentration of the carbon source in the fermentation system is 3 to 5 g / L, and the first carbon source and the first nitrogen source added continuously satisfy a mass ratio of carbon element to nitrogen element of 1.8 to 2.2, performing a first fermentation reaction, and obtaining a first fermentation liquid; after the concentration of the rhamnolipid in the first fermentation liquid is greater than 15 g / L, stopping the addition of the first carbon source and the first nitrogen source; the initial accumulation stage of polyhydroxy fatty acids includes: continuously adding a second carbon source and a second nitrogen source to the fermentation system so that the concentration of the carbon source in the fermentation system is 10 to 15 g / L , and the second carbon source and the second nitrogen source added continuously satisfy the mass ratio of carbon element to nitrogen element of 9.8-10.2, a second fermentation reaction is carried out to obtain a second fermentation liquid; when the concentration of the polyhydroxy fatty acid in the second fermentation liquid is greater than 10g / L, the addition of the second carbon source and the second nitrogen source is stopped; the steady-state continuous fermentation stage includes: continuously adding the second carbon source and the second nitrogen source to the fermentation system so that the sum of the concentrations of the first carbon source and the second carbon source in the fermentation system is 10-15g / L, and the second carbon source and the second nitrogen source added continuously satisfy the mass ratio of carbon element to nitrogen element of 9.8-10.2, a third fermentation reaction is carried out to continuously obtain a fermentation liquid, and the fermentation liquid is continuously output from the fermentation system; until the concentration of rhamnolipid in the fermentation liquid is greater than 20g / L, and the viscosity of the fermentation liquid is greater than 15cp, the third fermentation reaction is stopped.

[0012] Optionally, in the steady-state continuous fermentation stage, after the fermentation broth is continuously output from the fermentation system, the second carbon source and the second nitrogen source are continuously added to the third fermentation system, and the steady-state continuous fermentation stage is continued to continuously obtain the fermentation broth; and / or, in the steady-state continuous fermentation stage, the OD600 of the fermentation broth is 65-75, the concentration of the polyhydroxy fatty acid in the fermentation broth is 5-10 g / L, and the concentration of the rhamnolipid is 20-25 g / L.

[0013] Optionally, the process of sequentially separating the polyhydroxy fatty acids and the rhamnolipids from the fermentation broth after the defoaming treatment comprises: after lysing the strain in the fermentation broth after the defoaming treatment, adding a polar solvent to precipitate the polyhydroxy fatty acids, and separating the polyhydroxy fatty acids; after removing the polar solvent in the fermentation broth after the polyhydroxy fatty acids are separated, passing it into an adsorption column to adsorb the rhamnolipid, and separating the rhamnolipid.

[0014] Optionally, the process of continuously outputting the fermentation liquid from the fermentation system includes: when the liquid level of the fermentation liquid reaches 75% to 85% of the maximum liquid level, continuously outputting the fermentation liquid from the fermentation system.

[0015] Optionally, the temperature of the fermentation reaction is 30-37° C., the dissolved oxygen of the fermentation reaction liquid is greater than 60%, and the pH is 6.8-7.1.

[0016] Optionally, the first carbon source includes one or more of glucose, glycerol, and soybean oil; and / or, the second carbon source includes one or more of glucose, glycerol, and soybean oil; and / or, the first nitrogen source includes an aqueous sodium nitrate solution with a sodium nitrate concentration of 1.5 to 2.5 g / L; and / or, the second nitrogen source includes an aqueous sodium nitrate solution with a sodium nitrate concentration of 1.5 to 2.5 g / L.

[0017] The invention provides a preparation device for rhamnolipid and polyhydroxy fatty acid, comprising: a carbon source feeding tank, a nitrogen source feeding tank, a culture medium buffer tank, a fermentation tank, an overflow tank, a cracking tank, a sedimentation tank, a centrifuge, a distillation tower and an adsorption column; the discharge port of the carbon source feeding tank, the discharge port of the nitrogen source feeding tank and the discharge port of the culture medium buffer tank are all connected with the feed port of the fermentation tank; the overflow port of the fermentation tank is connected with the feed port of the overflow tank, the discharge port of the overflow tank is connected with the feed port of the cracking tank, the discharge port of the cracking tank is connected with the feed port of the sedimentation tank, the discharge port of the sedimentation tank is connected with the feed port of the centrifuge, the discharge port of the centrifuge is connected with the feed port of the distillation tower, the discharge port of the distillation tower is connected with the feed port of the adsorption column, and the liquid discharge port of the adsorption column is connected with the feed port of the fermentation tank.

[0018] The present invention provides a method and device for preparing rhamnolipid and polyhydroxy fatty acid. The method can continuously co-produce rhamnolipid and polyhydroxy fatty acid, avoids the problem that a large amount of PHA is accumulated in the production process of rhamnolipid, resulting in an increase in the viscosity of the fermentation liquid and a continuous decrease in the fermentation liquid solution, and improves the production efficiency of rhamnolipid and polyhydroxy fatty acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram of the preparation device of rhamnolipid in Comparative Example 1;

[0020] Figure 2 This is a diagram of the preparation device of rhamnolipid and polyhydroxy fatty acid of Comparative Example 2;

[0021] Figure 3 This is a diagram of the continuous preparation device of rhamnolipid and polyhydroxy fatty acid of Example 1;

[0022] Figure 4 This is a graph showing the relationship between time and production rate during the preparation of rhamnolipid in Comparative Example 1;

[0023] Figure 5 This is a graph showing the relationship between time and production rate during the preparation of rhamnolipid and polyhydroxy fatty acid in Comparative Example 2;

[0024] Figure 6 This is a graph showing the relationship between time and production rate during the continuous preparation of rhamnolipid and polyhydroxy fatty acid in Example 1.

[0025] Reference numerals:

[0026] 10-glucose feeding tank; 11-glycerol feeding tank; 12-soybean oil feeding tank; 13-nitrogen source feeding tank; 14-culture medium buffer tank; 15-fermentation tank; 16-overflow tank; 17-continuous cracking tank; 18-ethanol sedimentation tank; 19-disc centrifuge; 20-continuous distillation tower; 21-adsorption column. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific implementation methods listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0028] In the existing process of producing rhamnolipids, a large amount of foam is generated, which affects the production efficiency. Polyhydroxy fatty acids (PHA) are also produced as by-products, which causes the viscosity of the fermentation liquid to increase rapidly, resulting in a decrease in the dissolved oxygen in the fermentation liquid, reducing the fermentation activity of the bacteria and reducing the production efficiency.

[0029] Based on the problems existing in the prior art, an embodiment of the present invention provides a method for preparing rhamnolipid and polyhydroxy fatty acid (PHA), which comprises: inoculating a strain of bacteria for co-producing rhamnolipid and polyhydroxy fatty acid into a culture medium, and causing the strain to undergo a fermentation reaction to obtain a fermentation liquid; continuously outputting the fermentation liquid from a fermentation system, and performing a defoaming treatment on the outputted fermentation liquid; and sequentially separating polyhydroxy fatty acid and rhamnolipid from the fermentation liquid after the defoaming treatment.

[0030] According to the inventor's research and analysis: continuously outputting the fermentation liquid from the fermentation system helps to continuously vacate the liquid level, helps to continuously produce fermentation liquid, and improves production efficiency. The continuously output fermentation liquid is subjected to defoaming treatment to avoid large amounts of foam accumulating in the fermentation system and occupying the effective liquid level, so that the actual production liquid level is increased, thereby improving production efficiency. In addition, polyhydroxy fatty acids and rhamnolipids are separated from the fermentation liquid after defoaming treatment. On the one hand, the products (polyhydroxy fatty acids and rhamnolipids) can be obtained, and on the other hand, the accumulation of polyhydroxy fatty acids in the fermentation system can be avoided, solving the problem of increased viscosity of the fermentation system caused by the accumulation of polyhydroxy fatty acids, thereby avoiding the problems of decreased dissolved oxygen in the fermentation liquid and decreased fermentation activity of the bacteria caused by increased viscosity, and helping to improve production efficiency.

[0031] Therefore, the embodiments of the present invention help to improve the production efficiency of rhamnolipids and polyhydroxy fatty acids, achieve the co-production of rhamnolipids and polyhydroxy fatty acids, and reduce production costs.

[0032] In some embodiments, the bacterial species for co-producing rhamnolipids and polyhydroxyalkanoates includes Alphamonas aeruginosa.

[0033] The temperature of the above fermentation reaction (the whole operation temperature of the fermentation tank) can be 30-37°C, for example, 30°C, 32°C, 34°C, 36°C, 37°C or a range composed of any two thereof. The dissolved oxygen of the fermentation reaction liquid can be greater than 60% and the pH is 6.8-7.1, which is helpful to improve the activity of the strain and thus improve the production efficiency of rhamnolipids and polyhydroxy fatty acids. The dissolved oxygen content of the fermentation reaction liquid can be maintained by controlling ventilation (introducing clean air) and stirring; in addition, the pH can be adjusted to meet the above range by adding one or more of phosphoric acid, sulfuric acid, and hydrochloric acid to the fermentation reaction liquid. The above dissolved oxygen refers to the percentage saturation of dissolved oxygen in the liquid, the unit is %, and can be measured by a dissolved oxygen electrode (dissolved oxygen electrode).

[0034] The above culture medium can be formulated according to the specific requirements of the bacteria for co-producing rhamnolipid and polyhydroxy fatty acid.

[0035] The process of inoculating the culture medium with the strain that co-produces rhamnolipid and polyhydroxy fatty acid and allowing the strain to ferment to obtain the fermentation liquid may include a seed growth stage, an initial accumulation stage of rhamnolipid, an initial accumulation stage of polyhydroxy fatty acid, and a steady-state continuous fermentation stage; or a seed growth stage, an initial accumulation stage of polyhydroxy fatty acid, an initial accumulation stage of rhamnolipid, and a steady-state continuous fermentation stage.

[0036] Specifically, the seed growth stage includes: inoculating the culture medium with the bacteria that co-produce rhamnolipids and polyhydroxy fatty acids, and culturing the bacteria in an aerobic environment until the OD of the culture medium reaches 600Greater than or equal to 65, to obtain seed culture solution.

[0037] The initial accumulation stage of rhamnolipid includes: continuously adding the first carbon source and the first nitrogen source to the first fermentation system, so that the concentration of the carbon source in the first fermentation system is 3-5g / L, for example, 3g / L, 4g / L, 5g / L or any two of them, and the first carbon source and the first nitrogen source added continuously satisfy the mass ratio of carbon element to nitrogen element of 1.8-2.2, for example, 1.8, 2, 2.2 or any two of them, and the first fermentation reaction is carried out to obtain the first fermentation liquid; after the concentration of rhamnolipid in the first fermentation liquid is greater than 15g / L, stop adding the first carbon source and the first nitrogen source. It can be understood that the carbon source in the first fermentation system includes all carbon sources in the first fermentation system, such as the first carbon source.

[0038] The fermentation time of the initial accumulation stage of rhamnolipid can be 34 to 38 hours, for example, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours or a range consisting of any two of them. Continuous fermentation for 34 to 38 hours is conducive to sufficient accumulation of rhamnolipid and improve its production efficiency.

[0039] The initial accumulation stage of polyhydroxy fatty acids includes: continuously adding a second carbon source and a second nitrogen source to the second fermentation system, so that the concentration of the carbon source in the second fermentation system is 10-15 g / L, for example, 10 g / L, 12 g / L, 15 g / L or any two of them, and the continuously added second carbon source and second nitrogen source satisfy the mass ratio of carbon element to nitrogen element of 9.8-10.2, for example, 9.8, 10, 10.2 or any two of them, and the second fermentation reaction is carried out to obtain a second fermentation liquid; when the concentration of polyhydroxy fatty acids in the second fermentation liquid is greater than 10 g / L, stop adding the second carbon source and the second nitrogen source. It can be understood that the carbon source in the second fermentation system includes all carbon sources in the second fermentation system, such as the second carbon source.

[0040] The steady-state continuous fermentation stage includes: continuously adding a second carbon source and a second nitrogen source to the third fermentation system, so that the sum of the concentrations of the first carbon source and the second carbon source in the third fermentation system is 10 to 15 g / L, for example, 10 g / L, 12 g / L, 15 g / L or a range consisting of any two thereof, and the continuously added second carbon source and the second nitrogen source satisfy a mass ratio of carbon element to nitrogen element of 9.8 to 10.2, for example, 9.8, 10, 10.2 or a range consisting of any two thereof, performing a third fermentation reaction, continuously obtaining a fermentation liquid, and continuously outputting the fermentation liquid from the fermentation system; until the concentration of rhamnolipid in the fermentation liquid is greater than 20 g / L and the viscosity of the fermentation liquid is greater than 15 cp, stopping the third fermentation reaction.

[0041] In the above-mentioned steady-state continuous fermentation stage, after the fermentation liquid is continuously output from the fermentation system, the second carbon source and the second nitrogen source are continuously added to the third fermentation system, and the steady-state continuous fermentation stage is continuously carried out to continuously obtain the fermentation liquid. In the above-mentioned steady-state continuous fermentation stage that is carried out continuously, the strain continuously produces fresh bacteria by continuously adding nitrogen source and carbon source (that is, the above-mentioned second carbon source and second nitrogen source), continuously carries out fermentation reaction, continuously produces fermentation liquid, and at the same time, the fermentation liquid is continuously output from the fermentation system, thereby realizing the continuous production of rhamnolipids and polyhydroxy fatty acids, and improving production efficiency.

[0042] In some embodiments, during the above-mentioned steady-state continuous fermentation stage, the OD 600 The range of 65 to 75, such as 65, 70, 75 or any two thereof, is helpful to improve the production efficiency of rhamnolipid and polyhydroxy fatty acid.

[0043] In some embodiments, in the above-mentioned steady-state continuous fermentation stage, the concentration of polyhydroxy fatty acids in the fermentation broth is 5-10 g / L, and the concentration of rhamnolipid is 20-25 g / L. Controlling the concentrations of polyhydroxy fatty acids and rhamnolipid in the fermentation broth within the above-mentioned ranges helps to inhibit the increase in viscosity of the fermentation system and avoid the influence of polyhydroxy fatty acids and rhamnolipid on the fermentation system, thereby improving the production efficiency of rhamnolipid and polyhydroxy fatty acids.

[0044] In a specific implementation, the flow acceleration of the carbon source and the nitrogen source (the second carbon source and the second nitrogen source) can be controlled in the steady-state continuous fermentation stage to maintain the bacterial concentration (OD 600 ), the concentration of polyhydroxy fatty acids and the concentration of rhamnolipids satisfy the above ranges.

[0045] The first carbon source may include one or more of glucose, glycerol, and soybean oil, preferably soybean oil.

[0046] The second carbon source may include one or more of glucose, glycerol, and soybean oil, preferably glucose and glycerol.

[0047] It is understandable that the first carbon source and the second carbon source may be the same or different.

[0048] The first nitrogen source may include a sodium nitrate (NaNO3) aqueous solution having a sodium nitrate concentration of 1.5 to 2.5 g / L. Exemplarily, the sodium nitrate concentration in the sodium nitrate (NaNO3) aqueous solution may be 1.5 g / L, 2 g / L, 2.5 g / L, or a range consisting of any two of them. In addition, the first nitrogen source may also include KH2PO4, Na2HPO4, and a weighing element. In the first nitrogen source, the concentration of KH2PO4 may be 14 to 16 g / L, such as 14 g / L, 15.1 g / L, 16 g / L, or a range consisting of any two of them, the concentration of Na2HPO4 may be 14 to 16 g / L, such as 14 g / L, 15.1 g / L, 16 g / L, or a range consisting of any two of them, and the concentration of the weighing element may be 1.8 to 2.2 g / L, such as 1.8 g / L, 2 g / L, 2.2 g / L, or a range consisting of any two of them. The above-mentioned measuring elements may include one or more of FeCl3·6H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, MnSO4·H2O, H3BO3, and Na2MoO4·2H2O, and in the first nitrogen source, the concentration of FeCl3·6H2O may be 0.05-0.1 g / L, for example, 0.05 g / L, 0.08 g / L, 0.1 g / L, or a range consisting of any two thereof; the concentration of ZnSO4·7H2O may be 0.5-1 g / L, for example, 0.5 g / L, 0.6 g / L, 0.75 g / L, 1 g / L, or a range consisting of any two thereof; the concentration of CoCl2·6H2O may be 0.05-0.1 g / L, for example, 0.05 g / L, 0.06 g / L, 0.08 g / L, The concentration of MnSO4·H2O can be 0.5~1g / L, for example, 0.5g / L, 0.6g / L, 0.75g / L, 1g / L, or a range consisting of any two thereof; the concentration of H3BO3 can be 0.1~0.2g / L, for example, 0.1g / L, 0.15g / L, 0.2g / L, or a range consisting of any two thereof; the concentration of Na2MoO4·2H2O can be 0.03~0.1g / L, for example, 0.03g / L, 0.05g / L, 0.1g / L, or a range consisting of any two thereof.

[0049] The second nitrogen source may include a sodium nitrate (NaNO3) aqueous solution having a sodium nitrate concentration of 1.5 to 2.5 g / L. Exemplarily, the sodium nitrate concentration in the sodium nitrate (NaNO3) aqueous solution may be 1.5 g / L, 2 g / L, 2.5 g / L, or a range consisting of any two of them. In addition, the second nitrogen source may also include KH2PO4, Na2HPO4, and a weighing element. In the second nitrogen source, the concentration of KH2PO4 may be 14 to 16 g / L, such as 14 g / L, 15.1 g / L, 16 g / L, or a range consisting of any two of them, the concentration of Na2HPO4 may be 14 to 16 g / L, such as 14 g / L, 15.1 g / L, 16 g / L, or a range consisting of any two of them, and the concentration of the weighing element may be 1.8 to 2.2 g / L, such as 1.8 g / L, 2 g / L, 2.2 g / L, or a range consisting of any two of them. The above-mentioned measuring elements may include one or more of FeCl3·6H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, MnSO4·H2O, H3BO3, and Na2MoO4·2H2O, and in the second nitrogen source, the concentration of FeCl3·6H2O may be 14-16 g / L, for example, 14 g / L, 15.1 g / L, 16 g / L, or a range consisting of any two thereof, the concentration of ZnSO4·7H2O may be 0.5-1 g / L, for example, 0.5 g / L, 0.6 g / L, 0.75 g / L, 1 g / L, or a range consisting of any two thereof, and the concentration of CoCl2·6H2O may be 0.05-0.1 g / L, for example, 0.05 g / L, 0.06 g / L, 0.08 g / L, 0.1 g / L / L or a range consisting of any two thereof, the concentration of CuSO4··5H2O can be 0.05~0.1g / L, for example 0.05g / L, 0.06g / L, 0.075g / L, 0.1g / L or a range consisting of any two thereof, the concentration of MnSO4·H2O can be 0.5~1g / L, for example 0.5g / L, 0.6g / L, 0.75g / L, 1g / L or a range consisting of any two thereof, the concentration of H3BO3 can be 0.1~0.2g / L, for example 0.1g / L, 0.15g / L, 0.2g / L or a range consisting of any two thereof, and the concentration of Na2MoO4·2H2O can be 0.03~0.1g / L, for example 0.03g / L, 0.05g / L, 0.1g / L or a range consisting of any two thereof.

[0050] It is understandable that the first nitrogen source and the second nitrogen source may be the same or different.

[0051] The above-mentioned process of obtaining the fermentation broth can be carried out in a fermenter. Specifically, the culture medium can be added to the fermenter through a culture medium buffer tank, the carbon source (the first carbon source and / or the second carbon source) can be added to the fermenter through a carbon source feeding tank, and the nitrogen source (the first nitrogen source and / or the second nitrogen source) can be added to the fermenter through a nitrogen source feeding tank. By finely controlling the flow rates of the carbon source and the nitrogen source, the rhamnolipid and PHA in the fermentation broth can reach a suitable production amount and ratio, which is beneficial to improving the production stability of rhamnolipid, reducing the production cost of rhamnolipid, and co-producing PHA, which has good economic benefits.

[0052] The process of continuously outputting the fermentation liquid from the fermentation system includes: when the liquid level of the fermentation liquid reaches 75% to 85% of the maximum liquid level, such as 75%, 80%, 85% or a range composed of any two thereof, continuously outputting the fermentation liquid from the fermentation system.

[0053] Next, the output fermentation liquid is subjected to defoaming treatment, so that the output fermentation liquid is defoamed outside the fermentation system, thereby avoiding the introduction of defoaming agents into the fermentation system, which is helpful to improve the quality of rhamnolipids and polyhydroxy fatty acids.

[0054] The defoaming treatment may include a static defoaming treatment. In specific implementation, the output fermentation liquid (containing a large amount of foam and bacteria) can be allowed to enter the overflow tank through the overflow pipeline, and the fermentation liquid can be allowed to stand in the overflow tank to defoam naturally.

[0055] The process of sequentially separating polyhydroxy fatty acids and rhamnolipids from the fermentation broth after defoaming treatment comprises: after the bacterial strain in the fermentation broth after defoaming treatment is lysed, a polar solvent is added to precipitate the polyhydroxy fatty acids, and the polyhydroxy fatty acids are separated; after the polar solvent in the fermentation broth after the polyhydroxy fatty acids are separated is removed, the broth is passed into an adsorption column to adsorb rhamnolipid, and the rhamnolipid is separated.

[0056] In the specific implementation, in the process of lysing the strains in the fermentation broth after defoaming treatment, a lysing enzyme can be used to lyse the strains in the fermentation broth after defoaming treatment, so that the strain cells are lysed, thereby releasing the polyhydroxy fatty acids inside the strain cells. For example, the fermentation broth after defoaming treatment can be allowed to enter a lysis tank (continuous lysis tank), and a fully mixed flow continuous reactor can be used to fix the lysing enzyme using a metal organic framework composite carrier material (MOFs composite carrier material). The above-mentioned MOFs composite carrier material may include iron oxide magnetic nanoparticles-metal organic framework composite materials (Fe3O4-MIL-Fe). The above-mentioned lysing enzyme can use conventional lysing enzymes in the art (for example, lysing enzyme LysWH8). The concentration of the above-mentioned lysing enzyme in the fermentation broth can be 180 to 220ug / L, for example, 180ug / L, 200ug / L, 220ug / L or a range consisting of any two of them.

[0057] The polar solvent may include ethanol, that is, the polyhydroxy fatty acid is precipitated by using an ethanol precipitation method, thereby separating and obtaining the polyhydroxy fatty acid.

[0058] In specific implementation, the fermentation liquid after cleavage and the polar solvent can be respectively placed in a sedimentation tank (e.g., an ethanol sedimentation tank) and then mixed, and then cooled to 4-8°C to allow PHA to precipitate, and then the mixed liquid can be placed in a centrifuge (e.g., a disc centrifuge) to separate (recover) PHA. A fully mixed flow continuous reactor can be used in the above-mentioned sedimentation tank. For example, the fermentation liquid after cleavage (containing free PHA) and the polar solvent (e.g., ethanol) are mixed in equal volumes, then cooled to 4-8°C to allow PHA to precipitate, and then separated (recovered) PHA by centrifugation (e.g., using a disc centrifuge). In addition, in the settling tank, the ratio of the feed rate of the polar solvent (such as ethanol) to the feed rate (distillation rate) of the fermentation broth after the PHA is settled in the process of removing the polar solvent can be 0.3 to 0.5 (that is, the feed rate of the polar solvent (such as ethanol) is 0.3 to 0.5 times the distillation rate of the fermentation broth after the PHA is settled). The specific ratio can be adjusted according to the concentration of PHA in the fermentation broth after the PHA is settled.

[0059] The process of removing the polar solvent from the fermentation broth after the polyhydroxyalkanoic acid (PHA) is separated may include: allowing the fermentation broth after the polyhydroxyalkanoic acid (PHA) is separated to enter a distillation tower to remove the polar solvent therein.

[0060] The fermentation liquid after the polar solvent is removed is passed into an adsorption column to adsorb the rhamnolipid, thereby separating the rhamnolipid.

[0061] The above-mentioned adsorption column can be filled with a macroporous adsorption resin (such as the macroporous adsorption resin produced by Dandong Mingzhu), and specifically, ethanol can be used to regenerate the adsorption column.

[0062] After polyhydroxy fatty acids and rhamnolipids are separated in sequence from the fermentation liquid after defoaming treatment, the method further includes: returning the fermentation liquid after separating polyhydroxy fatty acids and rhamnolipids to the fermentation system, continuing the fermentation reaction to produce fermentation liquid, until the concentration of rhamnolipids in the fermentation liquid is greater than 20g / L, and the viscosity of the fermentation liquid is greater than 15cp, and the fermentation reaction is stopped. The fermentation liquid after separating polyhydroxy fatty acids and rhamnolipids does not contain polyhydroxy fatty acids and rhamnolipids, and this part of the fermentation liquid (column filtrate) is returned to the fermentation system as a growth raw material for the strain, and the fermentation reaction is continued, which is beneficial to saving costs, realizing the continuous co-production of polyhydroxy fatty acids and rhamnolipids, and helping to improve production efficiency.

[0063] Generally, when the concentration of rhamnolipid in the fermentation broth is greater than 20 g / L and the viscosity of the fermentation broth is greater than 15 cp, the concentration of rhamnolipid in the fermentation broth continues to rise and cannot be controlled. At this time, the addition of carbon source and nitrogen source to the fermentation system can be stopped (feeding can be stopped) to end the fermentation reaction; then, after recovering PHA and rhamnolipid in the fermentation broth in the fermenter, the remaining feed liquid can be treated as fermentation waste liquid.

[0064] The embodiment of the present invention also provides a preparation device for rhamnolipid and polyhydroxy fatty acid, comprising: a carbon source feeding tank, a nitrogen source feeding tank, a culture medium buffer tank, a fermentation tank, an overflow tank, a cracking tank, a sedimentation tank, a centrifuge, a distillation tower, and an adsorption column; the discharge port of the carbon source feeding tank, the discharge port of the nitrogen source feeding tank, and the discharge port of the culture medium buffer tank are all connected to the feed port of the fermentation tank; the overflow port of the fermentation tank is connected to the feed port of the overflow tank; the discharge port of the overflow tank is connected to the feed port of the cracking tank; the discharge port of the cracking tank is connected to the feed port of the sedimentation tank; the discharge port of the sedimentation tank is connected to the feed port of the centrifuge; the discharge port of the centrifuge is connected to the feed port of the distillation tower; the discharge port of the distillation tower is connected to the feed port of the adsorption column; the liquid discharge port of the adsorption column is connected to the feed port of the fermentation tank.

[0065] The above-mentioned rhamnolipid and polyhydroxy fatty acid preparation device (continuous fermentation system) can be used for the preparation of rhamnolipid and polyhydroxy fatty acid, which helps to achieve the continuous production of rhamnolipid and polyhydroxy fatty acid and improve the production efficiency of rhamnolipid and polyhydroxy fatty acid.

[0066] like Figure 3 As shown, the carbon source feeding tank may also include a glucose feeding tank (10), a glycerol feeding tank (11), and a soybean oil feeding tank (12);

[0067] It can be understood that the above-mentioned discharge ports and feed ports are connected in a controllable manner.

[0068] Specifically, the discharge port of the fermentation tank and the feed port of the overflow tank can be connected through an overflow pipeline, and other discharge ports and feed ports are connected through corresponding pipelines.

[0069] The present invention is further described below by specific examples and comparative examples. Unless otherwise specified, the reagents, materials and instruments used below are all conventional reagents, conventional materials and conventional instruments, which can be commercially available, and the reagents and materials involved can also be synthesized by conventional synthesis methods.

[0070] In each embodiment and comparative example, the nitrogen source is a sodium nitrate aqueous solution with a sodium nitrate concentration of 2 g / L; the sodium nitrate aqueous solution also includes KH2PO4, Na2HPO4, and a weighing element; in the above sodium nitrate aqueous solution, the concentration of KH2PO4 is 15.1 g / L, the concentration of Na2HPO4 is 15.1 g / L, the concentration of the weighing element is 2 g / L, and the above weighing element includes FeCl3·6H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, MnSO4·H2 O, H3BO3, Na2MoO4·2H2O, and in the above sodium nitrate aqueous solution, the concentration of FeCl3·6H2O is 0.08g / L, the concentration of ZnSO4·7H2O is 0.75g / L, the concentration of CoCl2·6H2O is 0.08g / L, the concentration of CuSO4·5H2O is 0.075g / L, the concentration of MnSO4·H2O is 0.75g / L, the concentration of H3BO3 can be 0.15g / L, and the concentration of Na2MoO4·2H2O is 0.05g / L;

[0071] The whole operation temperature of the fermentation tank is 30-37°C, and the ventilation and stirring are controlled to maintain the dissolved oxygen>60%; and the pH of the fermentation liquid is 6.8-7.1, which is regulated by one or more of phosphoric acid, sulfuric acid or hydrochloric acid;

[0072] The continuous cracking tank adopts a fully mixed flow continuous reactor, and uses a metal organic framework composite carrier material (MOFs composite carrier material) to fix the lyase; the MOFs composite carrier material includes iron oxide magnetic nanoparticles-metal organic framework composite material (Fe3O4-MIL-Fe); the lyase uses lyase LysWH8; the concentration of the lyase in the fermentation broth can be 200ug / L;

[0073] Example 1

[0074] This embodiment provides a continuous preparation device of rhamnolipid and polyhydroxy fatty acid, such as Figure 3As shown, it comprises: a glucose feeding tank (10), a glycerol feeding tank (11), a soybean oil feeding tank (12), a nitrogen source feeding tank (13), a culture medium buffer tank (14), a fermentation tank (15), an overflow tank (16), a continuous cracking tank (17), an ethanol sedimentation tank (18), a disc centrifuge (19), a continuous distillation tower (20), and an adsorption column (21); wherein the discharge ports of the glucose feeding tank (10), the glycerol feeding tank (11), the soybean oil feeding tank (12), the nitrogen source feeding tank (13), and the culture medium buffer tank (14) are all connected to the fermentation tank (15). The feed port of the fermentation tank (15) is controllably connected, the overflow port of the fermentation tank (15) is connected to the feed port of the overflow tank (16), the discharge port of the overflow tank (16) is controllably connected to the feed port of the continuous cracking tank (17), the discharge port of the continuous cracking tank (17) is controllably connected to the feed port of the ethanol settling tank (18), the discharge port of the ethanol settling tank (18) is controllably connected to the feed port of the disc centrifuge (19), the discharge port of the disc centrifuge (19) is controllably connected to the feed port of the continuous distillation tower (20), and the discharge port of the continuous distillation tower (20) is controllably connected to the feed port of the adsorption column (21).

[0075] This embodiment also provides a method for preparing rhamnolipid and polyhydroxy fatty acid, using Figure 3 The device shown is carried out, comprising:

[0076] Aeruginosa is inoculated into the culture medium in the fermentation tank (15), and clean air is introduced to culture the culture medium in an aerobic environment until the OD value of the culture medium reaches 0. 600 is 65, and the seed culture solution is obtained;

[0077] Then, glucose, glycerol and a nitrogen source are continuously added to the fermentation tank (15) to make the sum of the concentrations of glucose and glycerol in the fermentation tank 10-15 g / L, and the mass ratio of the continuously added glucose, glycerol and nitrogen source to the carbon element to the nitrogen element is 10, and a second fermentation reaction (PHA initial accumulation stage) is carried out to obtain a second fermentation liquid, until the concentration of the polyhydroxy fatty acid (PHA) is greater than 10 g / L, and the addition of glucose, glycerol and nitrogen source is stopped;

[0078] Then, soybean oil and a nitrogen source are continuously added to the fermentation tank (15) to make the concentration of soybean oil in the fermentation tank 3-5 g / L, and the mass ratio of carbon element to nitrogen element of the continuously added soybean oil and nitrogen source is 2, and the first fermentation reaction (initial accumulation stage of rhamnolipid) is carried out, and the fermentation is continued for 36 hours to obtain a first fermentation liquid; after the concentration of rhamnolipid in the first fermentation liquid is greater than 15 g / L, the addition of soybean oil is stopped;

[0079] Then, glucose, glycerol and a nitrogen source are continuously added to the fermentation tank (15) to make the total concentration of glucose, glycerol and soybean oil in the fermentation tank (15) be 10-15 g / L, and the glucose, glycerol and nitrogen source added continuously are made to satisfy the mass ratio of carbon element to nitrogen element of 10 to carry out the third fermentation reaction. At this time, the fermentation reaction in the fermentation tank (15) enters the steady-state continuous fermentation stage, during which the flow rate of glucose, soybean oil, glycerol and nitrogen source is controlled to maintain the bacterial concentration in the fermentation tank (15) to satisfy the OD 600 The concentration of rhamnolipid is 20-25 g / L, the concentration of PHA is 5-10 g / L, and the fermentation liquid is obtained;

[0080] After the liquid level of the fermented liquid is higher than 80% of the highest liquid level of the fermentation tank (15), the fermented liquid is continuously discharged from the fermentation tank (15) and enters the overflow tank (16) through the overflow pipeline. After the fermented liquid is naturally defoamed in the overflow tank (16), it enters the continuous lysis tank (17) to break the cells and release the PHA in the cells. The feed liquid in the continuous lysis tank (17) enters the ethanol sedimentation tank (18) and is mixed with an equal volume of ethanol and then cooled to 4-8°C to allow the PHA to settle. Next, the feed liquid in the ethanol sedimentation tank (18) enters the disc centrifuge (19) and is separated by centrifugation to obtain PHA (crude PHA).

[0081] The feed liquid in the disc centrifuge (19) enters the continuous distillation tower (20) to remove ethanol, then enters the adsorption column (21) to remove rhamnolipid, and then returns the feed liquid to the fermentation tank to continue the fermentation reaction; wherein the feed rate of ethanol in the ethanol settling tank (18) is 0.3 to 0.5 times the distillation rate of the feed liquid in the continuous distillation tower (20);

[0082] After the fermentation time reaches 720 hours, the feeding is stopped, and the fermentation liquid in the fermenter is continuously passed through a disc centrifuge (19) and an adsorption column (21) to recover rhamnolipid and PHA, and then treated as fermentation waste liquid.

[0083] like Figure 6 As shown, the fermentation process of producing rhamnolipids and PHA in Example 1 lasts for about 720 hours, the average yield (production rate) of rhamnolipids is 0.42 g / L / h, and the average yield (production rate) of PHA is 0.17 g / L / h. Due to the increase in the concentration of rhamnolipids (R fat) in the fermentation broth. Under the action of high content of R fat, the foam in the overflow pipe cannot be controlled, resulting in premature cessation of fermentation. In this embodiment, the production rates of rhamnolipids and PHA are still relatively high after the fermentation lasts for 720 hours (30 days). In order to avoid phage infection of the fermentation system, the fermentation is terminated in advance.

[0084] Example 2

[0085] This embodiment provides a method for preparing rhamnolipid and polyhydroxy fatty acid, using Figure 3 The device shown is carried out, comprising:

[0086] Inoculate Almonas aeruginosa into the culture medium in the fermentation tank (15), introduce clean air, and culture under an aerobic environment until the OD600 of the culture medium reaches 65, thereby obtaining a seed culture solution;

[0087] Then, soybean oil and a nitrogen source are continuously added to the fermentation tank (15) to make the concentration of soybean oil in the fermentation tank 3-5 g / L, and the mass ratio of carbon element to nitrogen element of the continuously added soybean oil and nitrogen source is 2, and the first fermentation reaction (initial accumulation stage of rhamnolipid) is carried out, and the fermentation is continued for 36 hours to obtain a first fermentation liquid; after the concentration of rhamnolipid in the first fermentation liquid is greater than 15 g / L, the addition of soybean oil is stopped;

[0088] Then, glucose, glycerol and a nitrogen source are continuously added to the fermentation tank (15) to make the sum of the concentrations of glucose and glycerol in the fermentation tank 10-15 g / L, and the glucose, glycerol and nitrogen source added continuously satisfy the mass ratio of carbon element to nitrogen element of 10, and a second fermentation reaction (PHA initial accumulation stage) is carried out to obtain a second fermentation liquid, until the concentration of polyhydroxy fatty acid (PHA) is greater than 10 g / L, at which time the fermentation reaction in the fermentation tank (15) enters a steady-state continuous fermentation stage, during which the flow rate of glucose, soybean oil, glycerol and nitrogen source is controlled to maintain the bacterial concentration in the fermentation tank (15) to satisfy OD600 of 65-75, rhamnolipid concentration of 20-25 g / L, and PHA concentration of 5-10 g / L, to obtain a fermentation liquid;

[0089] After the liquid level of the fermented liquid is higher than 80% of the highest liquid level of the fermentation tank (15), the fermented liquid is continuously discharged from the fermentation tank (15) and enters the overflow tank (16) through the overflow pipeline. After the fermented liquid is naturally defoamed in the overflow tank (16), it enters the continuous lysis tank (17) to break the cells and release the PHA in the cells. The feed liquid in the continuous lysis tank (17) enters the ethanol sedimentation tank (18) and is mixed with an equal volume of ethanol and then cooled to 4-8°C to allow the PHA to settle. Next, the feed liquid in the ethanol sedimentation tank (18) enters the disc centrifuge (19) and is separated by centrifugation to obtain PHA (crude PHA).

[0090] The feed liquid in the disc centrifuge (19) enters the continuous distillation tower (20) to remove ethanol, then enters the adsorption column (21) to remove rhamnolipid, and then returns the feed liquid to the fermentation tank to continue the fermentation reaction; wherein the feed rate of ethanol in the ethanol settling tank (18) is 0.3 to 0.5 times the distillation rate of the feed liquid in the continuous distillation tower (20);

[0091] After the fermentation time reaches 720 hours, the feeding is stopped, and the fermentation liquid in the fermenter is continuously passed through a disc centrifuge (19) and an adsorption column (21) to recover rhamnolipid and PHA, and then treated as fermentation waste liquid.

[0092] Example 2 The fermentation process for producing rhamnolipids and PHA lasts for about 720 hours, and the average yield (production rate) of rhamnolipids is 0.41 g / L / h, and the average yield (production rate) of PHA is 0.17 g / L / h. Due to the increase in the concentration of rhamnolipids (R fat) in the fermentation broth. Under the action of high-content R fat, the foam in the overflow pipe cannot be controlled, resulting in premature cessation of fermentation. In this embodiment, the production rates of rhamnolipids and PHA are still relatively high during the fermentation for 720 hours (30 days). In order to avoid phage infection of the fermentation system, the fermentation is terminated in advance.

[0093] Comparative Example 1

[0094] This comparative example provides a preparation device for rhamnolipid, such as Figure 1 As shown, it comprises: a glucose feeding tank (10), a glycerol feeding tank (11), a soybean oil feeding tank (12), a nitrogen source feeding tank (13), a culture medium buffer tank (14), a fermentation tank (15), an overflow tank (16), a disc centrifuge (19), and an adsorption column (21); wherein the discharge ports of the glucose feeding tank (10), the glycerol feeding tank (11), the soybean oil feeding tank (12), the nitrogen source feeding tank (13), and the culture medium buffer tank (14) are all controllably connected to the feed port of the fermentation tank (15), the overflow port of the fermentation tank (15) is connected to the feed port of the overflow tank (16), the discharge port of the overflow tank (16) is controllably connected to the feed port of the disc centrifuge (19), and the discharge port of the disc centrifuge (19) is controllably connected to the feed port of the adsorption column (21) (which is an ethanol regeneration adsorption column).

[0095] This comparative example also provides a method for preparing rhamnolipid, using Figure 1 The device shown is carried out, comprising:

[0096] Aeruginosa is inoculated into the culture medium in the fermentation tank (15), and clean air is introduced to culture the culture medium in an aerobic environment until the OD value of the culture medium reaches 0. 600 is 65, and the seed culture solution is obtained;

[0097] Then, soybean oil and a nitrogen source are continuously added to the fermentation tank (15) to make the concentration of soybean oil in the fermentation tank 3-5 g / L, and the mass ratio of carbon element to nitrogen element of the continuously added soybean oil and nitrogen source is 2, and a first fermentation reaction is carried out, and the fermentation is continued for 36 hours to obtain a first fermentation liquid; after the concentration of rhamnolipid in the first fermentation liquid is greater than 15 g / L, the addition of soybean oil is stopped; then, glucose and a nitrogen source are continuously added to the fermentation tank (15) to make the concentration of glucose and soybean oil in the fermentation tank (15) 10-15 g / L, and the mass ratio of carbon element to nitrogen element of the continuously added glucose and nitrogen source is 9.8-10.2, and a third fermentation reaction is carried out. At this time, the fermentation reaction in the fermentation tank (15) enters a steady-state continuous fermentation stage, during which the flow rate of glucose, soybean oil and nitrogen source is controlled to maintain the bacterial concentration in the fermentation tank (15) to meet OD 600 The concentration of rhamnolipid is 20-25 g / L, and the fermentation liquid is obtained;

[0098] After the liquid level of the fermented liquid is higher than 80% of the highest liquid level of the fermentation tank (15), the fermented liquid is continuously discharged from the fermentation tank (15) and enters the overflow tank (16) through the overflow pipeline. After the fermented liquid is naturally defoamed in the overflow tank (16), it continuously enters the disc centrifuge (19) to separate the bacteria and the adsorption column (21) to recover the rhamnolipid, and then the fermented liquid is returned to the fermentation tank (15) to continue the fermentation reaction;

[0099] When the viscosity of the fermentation liquid reaches >15cp, the feeding is stopped, and the fermentation liquid in the fermentation tank (15) is continuously passed through a disc centrifuge (19) to separate the bacteria and an adsorption column (21) to recover rhamnolipid, and then treated as fermentation waste liquid.

[0100] like Figure 4 As shown, the fermentation process of producing rhamnolipid in Comparative Example 1 lasts for about 170 hours, and the average yield (production rate) of rhamnolipid is 0.32 g / L / h. Due to the shearing action of the disc centrifuge (19), part of the bacterial cells will be broken, so that the PHA in the bacterial cells will be released, causing the viscosity of the fermentation liquid to slowly increase. When the fermentation time is 172 hours, the viscosity of the fermentation liquid reaches 18.5 cp (greater than 15 cp). The high viscosity of the fermentation liquid causes the disc centrifuge (19) to be unable to completely separate the bacterial cells from the fermentation liquid, causing the fermentation liquid containing bacteria to enter the adsorption column, causing the efficiency of the adsorption column to adsorb rhamnolipid to decrease, and then causing the concentration of rhamnolipid (R lipid) in the fermentation liquid returned to the fermentation tank (15) to increase. Under the action of high viscosity and high content of rhamnolipid (R lipid), the overflow pipe foam cannot be controlled, resulting in premature cessation of fermentation.

[0101] Comparative Example 2

[0102] This comparative example provides a preparation device for rhamnolipid and polyhydroxy fatty acid, such as Figure 2 As shown, it comprises: a glucose feed tank (10), a glycerol feed tank (11), a soybean oil feed tank (12), a nitrogen source feed tank (13), a culture medium buffer tank (14), a fermentation tank (15), an overflow tank (16), a continuous cracking tank (17), an ethanol sedimentation tank (18), a disc centrifuge (19), and a continuous distillation tower (20); wherein the output of the glucose feed tank (10), the glycerol feed tank (11), the soybean oil feed tank (12), the nitrogen source feed tank (13), and the culture medium buffer tank (14) The feed ports are controllably connected to the feed port of the fermentation tank (15), the overflow port of the fermentation tank (15) is connected to the feed port of the overflow tank (16), the discharge port of the overflow tank (16) is controllably connected to the feed port of the continuous cracking tank (17), the discharge port of the continuous cracking tank (17) is controllably connected to the feed port of the ethanol settling tank (18), the discharge port of the ethanol settling tank (18) is controllably connected to the feed port of the disc centrifuge (19), and the discharge port of the disc centrifuge (19) is controllably connected to the feed port of the continuous distillation tower (20).

[0103] This comparative example also provides a method for preparing rhamnolipid and polyhydroxy fatty acid, using Figure 2 The device shown is carried out, comprising:

[0104] Aeruginosa is inoculated into the culture medium in the fermentation tank (15), and clean air is introduced to culture the culture medium in an aerobic environment until the OD value of the culture medium reaches 0. 600 is 65, and the seed culture solution is obtained;

[0105] Then, glucose, glycerol and a nitrogen source are continuously added to the fermentation tank (15) to make the sum of the concentrations of glucose and glycerol in the fermentation tank 10-15 g / L, and the mass ratio of the continuously added glucose, glycerol and nitrogen source to the carbon element to the nitrogen element is 10, and a second fermentation reaction (PHA initial accumulation stage) is carried out to obtain a second fermentation liquid, until the concentration of the polyhydroxy fatty acid (PHA) is greater than 10 g / L, and the addition of glucose, glycerol and nitrogen source is stopped;

[0106] Then, soybean oil and a nitrogen source are continuously added to the fermentation tank (15) to make the concentration of soybean oil in the fermentation tank 3-5 g / L, and the mass ratio of carbon element to nitrogen element of the continuously added soybean oil and nitrogen source is 2, and the first fermentation reaction (initial accumulation stage of rhamnolipid) is carried out, and the fermentation is continued for 36 hours to obtain a first fermentation liquid; after the concentration of rhamnolipid in the first fermentation liquid is greater than 15 g / L, the addition of soybean oil is stopped;

[0107] Then, glucose, glycerol and a nitrogen source are continuously added to the fermentation tank (15) to make the total concentration of glucose, glycerol and soybean oil in the fermentation tank (15) be 10-15 g / L, and the glucose, glycerol and nitrogen source added continuously are made to satisfy the mass ratio of carbon element to nitrogen element of 10 to carry out the third fermentation reaction. At this time, the fermentation reaction in the fermentation tank (15) enters the steady-state continuous fermentation stage, during which the flow rate of glucose, soybean oil, glycerol and nitrogen source is controlled to maintain the bacterial concentration in the fermentation tank (15) to satisfy the OD 600 The concentration of rhamnolipid is 20-25 g / L, the concentration of PHA is 5-10 g / L, and the fermentation liquid is obtained;

[0108] After the liquid level of the fermented liquid is higher than 80% of the highest liquid level of the fermentation tank (15), the fermented liquid is continuously discharged from the fermentation tank (15) and enters the overflow tank (16) through the overflow pipeline. After the fermented liquid is naturally defoamed in the overflow tank (16), it enters the continuous lysis tank (17) to break the cells and release the PHA in the cells. The feed liquid in the continuous lysis tank (17) enters the ethanol sedimentation tank (18) and is mixed with an equal volume of ethanol and then cooled to 4-8°C to allow the PHA to settle. Next, the feed liquid in the ethanol sedimentation tank (18) enters the disc centrifuge (19) and is separated by centrifugation to obtain PHA (crude PHA).

[0109] After the feed liquid in the disc centrifuge (19) enters the continuous distillation tower (20) to remove ethanol, the feed liquid from which ethanol has been removed is returned to the fermentation tank to continue the fermentation reaction; wherein the feed rate of ethanol in the ethanol settling tank (18) is 0.3 to 0.5 times the distillation rate of the feed liquid in the continuous distillation tower (20);

[0110] When the concentration of rhamnolipid in the fermentation broth continues to rise uncontrollably and the concentration of rhamnolipid is >130 g / L, the feeding is stopped, and the fermentation broth in the fermenter is continuously passed through a disc centrifuge (19) and an adsorption column to recover rhamnolipid and PHA, and then treated as fermentation waste liquid.

[0111] like Figure 5 As shown, the fermentation process of producing rhamnolipid and PHA in Comparative Example 2 lasted for about 360 hours, the average yield (production rate) of rhamnolipid was 0.37 g / L / h, and the average yield (production rate) of PHA was 0.15 g / L / h. Due to the increase in the concentration of rhamnolipid (R lipid) in the fermentation broth, the foam in the overflow pipe could not be controlled under the action of high content of R lipid, resulting in premature cessation of fermentation.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing rhamnolipid and polyhydroxy fatty acid, characterized in that: include: Inoculating a bacterial strain that co-produces rhamnolipid and polyhydroxy fatty acid into a culture medium, and allowing the bacterial strain to undergo a fermentation reaction to obtain a fermentation liquid; The fermentation liquid is continuously output from the fermentation system, and the output fermentation liquid is subjected to a defoaming treatment; The polyhydroxy fatty acid and the rhamnolipid are sequentially separated from the fermentation liquid after the defoaming treatment.

2. The preparation method according to claim 1, characterized in that: After sequentially separating the polyhydroxy fatty acid and the rhamnolipid from the fermentation liquid after the defoaming treatment, the method further comprises: The fermentation liquid after separating the polyhydroxy fatty acids and the rhamnolipid is returned to the fermentation system, and the fermentation reaction is continued to produce the fermentation liquid until the concentration of rhamnolipid in the fermentation liquid is greater than 20 g / L and the viscosity of the fermentation liquid is greater than 15 cp, and the fermentation reaction is stopped.

3. The preparation method according to claim 1, characterized in that: The process of inoculating the culture medium with the bacteria for co-producing rhamnolipid and polyhydroxy fatty acid and allowing the bacteria to undergo fermentation reaction to obtain the fermentation liquid includes the following steps in sequence: Seed growth stage, rhamnolipid initial accumulation stage, polyhydroxyalkanoic acid initial accumulation stage, steady-state continuous fermentation stage; or; Seed growth stage, initial accumulation stage of polyhydroxyalkanoic acid, initial accumulation stage of rhamnolipid, steady-state continuous fermentation stage; The feed amount in the initial accumulation stage of rhamnolipid is less than that in the initial accumulation stage of polyhydroxyalkanoic acid, and the feed amount in the initial accumulation stage of polyhydroxyalkanoic acid is the same as that in the steady-state continuous fermentation stage.

4. The preparation method according to claim 3, characterized in that: The seed growth stage includes: inoculating the culture medium with a strain of bacteria that co-produces rhamnolipid and polyhydroxy fatty acid, and culturing the strain in an aerobic environment until the OD of the culture medium reaches 600 Greater than or equal to 65, obtaining seed culture solution; The initial accumulation stage of rhamnolipid comprises: continuously adding a first carbon source and a first nitrogen source to the fermentation system, so that the concentration of the carbon source in the fermentation system is 3-5 g / L, and the first carbon source and the first nitrogen source continuously added satisfy a mass ratio of carbon element to nitrogen element of 1.8-2.2, performing a first fermentation reaction, and obtaining a first fermentation liquid; after the concentration of the rhamnolipid in the first fermentation liquid is greater than 15 g / L, stopping adding the first carbon source and the first nitrogen source; The initial accumulation stage of polyhydroxy fatty acids comprises: continuously adding a second carbon source and a second nitrogen source to the fermentation system, so that the concentration of the carbon source in the fermentation system is 10-15 g / L, and the mass ratio of the second carbon source and the second nitrogen source added continuously satisfies the carbon element and the nitrogen element of 9.8-10.2, performing a second fermentation reaction, and obtaining a second fermentation liquid; when the concentration of the polyhydroxy fatty acid in the second fermentation liquid is greater than 10 g / L, stopping adding the second carbon source and the second nitrogen source; The steady-state continuous fermentation stage includes: continuously adding the second carbon source and the second nitrogen source to the fermentation system so that the sum of the concentrations of the first carbon source and the second carbon source in the fermentation system is 10-15 g / L, and the continuously added second carbon source and the second nitrogen source satisfy a mass ratio of carbon element to nitrogen element of 9.8-10.2, performing a third fermentation reaction, continuously obtaining a fermentation liquid, and continuously outputting the fermentation liquid from the fermentation system; until the concentration of rhamnolipid in the fermentation liquid is greater than 20 g / L and the viscosity of the fermentation liquid is greater than 15 cp, stopping the third fermentation reaction.

5. The preparation method according to claim 4, characterized in that: In the steady-state continuous fermentation stage, after the fermentation liquid is continuously output from the fermentation system, the second carbon source and the second nitrogen source are continuously added to the third fermentation system, and the steady-state continuous fermentation stage is continuously performed to continuously obtain the fermentation liquid; And / or, in the steady-state continuous fermentation stage, the OD of the fermentation broth is 600 is 65-75, the concentration of the polyhydroxy fatty acid in the fermentation broth is 5-10 g / L, and the concentration of the rhamnolipid is 20-25 g / L.

6. The preparation method according to claim 1, characterized in that: The process of sequentially separating the polyhydroxy fatty acid and the rhamnolipid from the fermentation broth after the defoaming treatment comprises: After the strain in the fermentation broth after the defoaming treatment is lysed, a polar solvent is added to precipitate the polyhydroxy fatty acid, and the polyhydroxy fatty acid is separated; after the polar solvent in the fermentation broth after the polyhydroxy fatty acid is separated is removed, the broth is passed into an adsorption column to adsorb the rhamnolipid, and the rhamnolipid is separated.

7. The preparation method according to claim 1, characterized in that: The process of continuously outputting the fermentation liquid from the fermentation system includes: when the liquid level of the fermentation liquid reaches 75% to 85% of the maximum liquid level, continuously outputting the fermentation liquid from the fermentation system.

8. The preparation method according to any one of claims 1 to 6, characterized in that: The temperature of the fermentation reaction is 30-37° C., the dissolved oxygen of the fermentation reaction liquid is greater than 60%, and the pH is 6.8-7.

1.

9. The preparation method according to claim 4, characterized in that: The first carbon source includes one or more of glucose, glycerol, and soybean oil; And / or, the second carbon source includes one or more of glucose, glycerol, and soybean oil; And / or, the first nitrogen source comprises a sodium nitrate aqueous solution having a sodium nitrate concentration of 1.5 to 2.5 g / L; And / or, the second nitrogen source comprises a sodium nitrate aqueous solution having a sodium nitrate concentration of 1.5 to 2.5 g / L.

10. A device for preparing rhamnolipid and polyhydroxy fatty acid, characterized in that: include: Carbon source feed tank, nitrogen source feed tank, culture medium buffer tank, fermentation tank, overflow tank, cracking tank, sedimentation tank, centrifuge, distillation tower, adsorption column; The discharge port of the carbon source feeding tank, the discharge port of the nitrogen source feeding tank, and the discharge port of the culture medium buffer tank are all connected to the feed port of the fermentation tank; The overflow port of the fermentation tank is connected with the feed port of the overflow tank, the discharge port of the overflow tank is connected with the feed port of the cracking tank, the discharge port of the cracking tank is connected with the feed port of the sedimentation tank, the discharge port of the sedimentation tank is connected with the feed port of the centrifuge, the discharge port of the centrifuge is connected with the feed port of the distillation tower, the discharge port of the distillation tower is connected with the feed port of the adsorption column, and the liquid discharge port of the adsorption column is connected with the feed port of the fermentation tank.

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