Continuous fermentation method and system

Abstract

A continuous fermentation method and system. The continuous fermentation method comprises: inoculating a microbial strain into a continuous fermentation system and carrying out fermentation culture in an aerobic environment; and collecting a fermentation broth and recovering and / or purifying the fermentation broth to obtain a fermentation product, wherein during fermentation culture, on the basis of the microbial concentration in the fermentation tank, the feed rate of the medium fed to the fermentation tank and the discharge rate of the fermentation broth discharged out of the fermentation tank are controlled. By detecting the microbial concentration, the discharge rate is changed in a self-adaptive mode to thus maintain the microbial concentration stable, and serious foaming of a fermentation broth is avoided, so that the continuous fermentation process can be efficiently and stably operated for a long time.

Description

Continuous fermentation method and continuous fermentation system Technical Field

[0001] The present invention relates to the technical field of biological fermentation, in particular to a continuous fermentation method and a continuous fermentation system capable of reducing bubbles during fermentation production. Background Art

[0002] Bubbles often form during microbial fermentation, and rhamnolipids pose a particularly serious foaming problem during fermentation. Rhamnolipids are glycolipid biosurfactants primarily produced by Pseudomonas aeruginosa and related strains. Their most prominent characteristic is their surface activity, which significantly reduces the surface tension of water and alters the wettability of solid surfaces. They possess a variety of functions, including emulsification, demulsification, foaming, washing, dispersion and flocculation, antistatic properties, and lubrication.

[0003] Because rhamnolipids are strong surfactants, conventional fermentation processes often require the addition of large amounts of defoamers to control the liquid level. This is particularly true in the later stages of rhamnolipid fermentation, where high rhamnolipid content leads to severe foaming in the fermentation broth, necessitating a dramatic increase in defoamer dosage. The use of defoamers can negatively impact rhamnolipid product quality and increase fermentation costs.

[0004] Therefore, there is an urgent need for a fermentation method that can reduce the generation of bubbles during fermentation production.

[0005] Summary of the Invention

[0006] The present invention aims to solve the problem of serious foaming of fermentation liquid in the prior art and provides a continuous fermentation method and a continuous fermentation system.

[0007] In one aspect, the present invention provides a continuous fermentation method, comprising:

[0008] Inoculate the strain into a continuous fermentation system and perform fermentation culture under an aerobic environment;

[0009] collecting the fermentation broth obtained from the fermentation culture, and recovering and / or purifying it to obtain a fermentation product;

[0010] Wherein, during the fermentation culture, the flow rate of the culture medium added to the fermentation tank and the discharge rate of the fermentation liquid out of the fermentation tank are controlled according to the bacterial cell concentration in the fermentation tank, including:

[0011] detecting the bacterial cell concentration in the fermentation tank;

[0012] When the bacterial cell concentration is greater than or equal to the first threshold, the culture medium is added and Qn1=K1-Kg×OD is calculated. 600 ×Kn, where Qn1 is the first rate, K1 is the first constant, Kg is the bacterial growth rate, OD600 is the absorbance value, reflecting the bacterial concentration, Kn is the culture medium conversion coefficient, and the discharge rate is controlled to be the first rate;

[0013] When the bacterial cell concentration is less than the first threshold, the culture medium is added and Qn2=K1+Kg×OD is calculated. 600 ×Kn, where Qn2 is the second rate, and the discharge rate is controlled to be the second rate.

[0014] In some embodiments, during the fermentation culture, the carbon source flow acceleration rate is further controlled according to the desired concentration of the fermentation product in the fermentor, comprising:

[0015] According to the desired concentration of the fermentation product in the fermentation tank, the carbon source flow acceleration rate is controlled as follows: Qc=Qm×Cm1 / Km, wherein Qc is the carbon source flow acceleration rate, Cm1 is the desired concentration of the fermentation product in the fermentation tank, Km is the carbon source conversion rate, Qm is the discharge flow rate of the fermentation liquid out of the fermentation tank, Qm=Vk×S×ρ, Vk is the bubble rise rate, ρ is the bubble density, and S is the diameter of the fermentation tank.

[0016] In some embodiments, during the fermentation culture, controlling the ventilation volume and the flow rate of the carbon source according to the foaming rate in the fermentor comprises:

[0017] At each sampling moment, the real-time concentration and ventilation volume of the fermentation product in the fermentation tank are detected, and the foam production rate is calculated based on the real-time concentration and the ventilation volume. If the foam production rate calculated at the current sampling moment is greater than or equal to the foam production rate calculated at the previous sampling moment, the ventilation volume is reduced; if the foam production rate calculated at the current sampling moment is less than the foam production rate calculated at the previous sampling moment, the ventilation volume is increased; wherein the foam production rate is: Vg=Km×exp(L / K2)×Cm×exp(T / K3), wherein Vg is the foam production rate, Km is the proportional coefficient, L is the ventilation volume, T is the stirring speed, Cm is the real-time concentration of the fermentation product, K2 is the second constant, K3 is the third constant, and exp is the natural exponent.

[0018] In some embodiments, the fermentation product is a rhamnolipid; the strain is a strain capable of producing rhamnolipid by fermentation, preferably Pseudomonas putida. It is understood that when the desired fermentation product is a substance other than rhamnolipid, the strain can be replaced with a strain capable of producing the desired fermentation product based on knowledge in the art.

[0019] In some embodiments, the first threshold is 65-75, preferably 70.

[0020] In some embodiments, the desired concentration of the fermentation product in the fermentor is 10-20 g / L.

[0021] [Corrected on 24.05.2024 according to Rule 26] In some embodiments, the carbon source added to the fermentation culture is selected from at least one of soybean oil, glycerol, glucose, palm oil, and waste cooking oil.

[0022] In some embodiments, the culture medium is a nitrogen source culture medium. It is understood that in the fermentation culture, in order to ensure the normal growth of the bacteria, a carbon source can be added according to conventional operations in the art. Preferably, the nitrogen source culture medium comprises: 2g / L NaNO3, 15.1g / L KH2PO4, 15.1g / L Na2HPO4, 0.08g / L FeCl3·6H2O, 0.75g / L ZnSO4·7H2O, 0.08g / L CoCl2·6H2O, 0.075g / L CuSO4·5H2O, 0.75g / L MnSO4·H2O, 0.15g / L H3BO3, and 0.05g / L Na2MoO4·2H2O.

[0023] In some embodiments, the culture medium may also be a recovery culture medium, which is the liquid obtained after removing the extractant, bacteria and / or fermentation products from the fermentation broth during the fermentation process, so as to reduce production costs.

[0024] In some preferred embodiments, during the fermentation culture, controlling the flow rate of the culture medium added to the fermenter and the discharge rate of the fermentation liquid out of the fermenter according to the bacterial cell concentration in the fermenter comprises:

[0025] detecting the bacterial cell concentration in the fermentation tank;

[0026] When the bacterial cell concentration is greater than or equal to the first threshold, the recovery medium is added and Qn1=K1-Kg×OD is calculated. 600 ×Kn, where Qn1 is the first rate, K1 is the first constant, Kg is the bacterial growth rate, OD 600 is the absorbance value, reflecting the bacterial concentration, Kn is the culture medium conversion coefficient, and the discharge rate is controlled to be the first rate;

[0027] When the bacterial cell concentration is less than the first threshold, the nitrogen source medium is added, and Qn2=K1+Kg×OD is calculated. 600 ×Kn, where Qn2 is the second rate, and the discharge rate is controlled to be the second rate.

[0028] In the fermentation method provided by the present invention, the continuous fermentation system can be a fermentation system commonly used in the art that can achieve continuous fermentation. Preferably, the continuous fermentation system provided below can be used to further reduce the generation of bubbles and improve fermentation efficiency.

[0029] On the other hand, the present invention provides a continuous fermentation system, which can be applied to the continuous fermentation method described in the present application. The continuous fermentation system provided by the present invention includes a carbon source feeding tank, a nitrogen source feeding tank, a culture medium buffer tank, a fermentor, an overflow tank, a ceramic membrane, a first extraction centrifugal device, a second extraction centrifugal device and a gas tower, wherein the discharge port of the carbon source feeding tank and the discharge port of the nitrogen source feeding tank are respectively controllably connected to the feed port of the fermentor, the overflow port of the fermentor is controllably connected to the feed port of the overflow tank, the discharge port of the overflow tank is respectively controllably connected to the feed port of the ceramic membrane and the feed port of the first extraction centrifugal device, the discharge port of the ceramic membrane is controllably connected to the feed port of the second extraction centrifugal device, the recovery port of the second extraction centrifugal device is connected to the feed port of the gas tower, the discharge port of the gas tower is controllably connected to the feed port of the culture medium buffer tank, and the discharge port of the culture medium buffer tank is controllably connected to the feed port of the fermentor.

[0030] In some embodiments, the continuous fermentation system further includes a mixing tank and a distillation tower, the discharge port of the first extraction centrifuge device and the discharge port of the second extraction centrifuge device are controllably connected to the feed port of the mixing tank, and the discharge port of the mixing tank is connected to the feed port of the distillation tower.

[0031] In some embodiments, the second extraction centrifugal device is a centrifugal extraction column.

[0032] In some embodiments, the extractant in the first extraction centrifugal device and the second extraction centrifugal device is selected from at least one of chloroform, dichloromethane, n-hexane, n-heptane, n-decane, ethyl acetate and acetonitrile.

[0033] In some embodiments, the fermentor is operated at a temperature of 30-37°C.

[0034] In some embodiments, the pH of the fermentation broth is 6.8-7.1.

[0035] The continuous fermentation method provided by the present invention controls the flow rate of the recovery culture medium and the nitrogen source culture medium added to the fermentation tank and the discharge rate of the fermentation liquid out of the fermentation tank according to the bacterial concentration in the fermentation tank during fermentation culture, so as to adaptively change the discharge rate of the fermentation liquid out of the fermentation tank, thereby maintaining the stability of the bacterial concentration and avoiding severe foaming of the fermentation liquid, so that the continuous fermentation process can operate efficiently and stably for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 is a flowchart of a continuous fermentation method according to an embodiment of the present invention;

[0037] FIG2 is a schematic diagram of a continuous fermentation system according to an embodiment of the present invention;

[0038] FIG3 is a production rate diagram of a continuous fermentation system in one embodiment of the present invention.

[0039] Marking instructions: 10-carbon source feeding tank; 11-nitrogen source feeding tank; 12-culture medium buffer tank; 13-fermentation tank; 14-overflow tank; 15-ceramic membrane; 16-first extraction centrifugal device; 17-second extraction centrifugal device; 18-mixing tank; 19-gas tower; 20-distillation tower; 21-first pump; 22-second pump. DETAILED DESCRIPTION

[0040] As shown in FIG1 , it is a workflow diagram of a continuous fermentation method of rhamnolipid in one embodiment of the present invention, comprising:

[0041] Step S101, inoculating a strain into a continuous fermentation system and performing fermentation culture under an aerobic environment;

[0042] Step S102, collecting the fermentation broth obtained by fermentation culture, and recovering and / or purifying it to obtain a fermentation product;

[0043] During the fermentation culture, the flow rate of the culture medium added to the fermentation tank and the discharge rate of the fermentation liquid out of the fermentation tank are controlled according to the concentration of the bacteria in the fermentation tank, including:

[0044] detecting the bacterial cell concentration in the fermentation tank;

[0045] When the bacterial concentration is greater than or equal to the first threshold, feed the culture medium and calculate Qn1=K1-Kg×OD 600 ×Kn, where Qn1 is the first rate, K1 is the first constant, Kg is the bacterial growth rate, OD 600 is the bacterial concentration, Kn is the culture medium conversion coefficient, and the discharge rate is controlled to be the first rate;

[0046] When the bacterial concentration is less than the first threshold, feed the culture medium and calculate Qn2 = K1 + Kg × OD 600 ×Kn, where Qn2 is the second rate, and the discharge rate is controlled to be the second rate.

[0047] Specifically, step S101 is performed to carry out fermentation culture, and step S102 is performed to collect the fermentation liquid, and recover and / or purify it to obtain a fermentation product.

[0048] During the fermentation culture, the flow rate of the culture medium added to the fermentation tank and the discharge rate of the fermentation liquid out of the fermentation tank are controlled according to the bacterial cell concentration in the fermentation tank.

[0049] Specifically, a mathematical model of bacterial concentration is introduced:

[0050] Online detection of bacterial concentration OD 600 , when OD 600 When the first threshold is reached, feed the culture medium, Qn1=K1-Kg×OD 600 ×Kn, where the first threshold is preferably 70, Qn1 is the first rate, Kg is the bacterial specific growth rate, which is related to bacterial activity and can be determined experimentally; preferably, Kg is 0.09-0.12. Kn is the culture medium conversion coefficient, K1 is the first constant obtained through experiments, and in the case of continuous fermentation of rhamnolipids, the first constant K is preferably 80. When OD 600 < the first threshold, feed culture medium, Qn2=K1+Kg×OD 600 × Kn, where Qn2 is the second rate. Control the discharge rate Qs = the first rate Qn1 or the second rate Qn2.

[0051] The discharge rate is the rate at which the fermentation liquid is discharged from the fermentation tank. Preferably, as shown in FIG2 , the discharge rate is the rate at which the fermentation liquid is discharged from the fermentation tank 13. By adjusting the discharge rate, the bacterial cell concentration is adjusted so that the bacterial cell concentration remains stable.

[0052] The continuous fermentation method for rhamnolipids of this embodiment utilizes extraction to separate rhamnolipids in situ during the fermentation process, thereby eliminating the impact of rhamnolipids on the fermentation system and improving the production efficiency of rhamnolipids. Furthermore, the continuous fermentation method for rhamnolipids of this embodiment controls the flow rate of the culture medium added to the fermenter and the discharge rate of the fermentation broth from the fermenter according to the bacterial cell concentration in the fermenter during fermentation. By detecting the bacterial cell concentration, the discharge rate is adaptively changed to maintain a stable bacterial cell concentration and avoid severe foaming in the fermentation broth, thereby enabling the continuous fermentation process to operate efficiently and stably for a long period of time.

[0053] In another embodiment, the workflow of the continuous fermentation method of rhamnolipid includes:

[0054] Step S101, inoculating a strain into a continuous fermentation system and performing fermentation culture under an aerobic environment;

[0055] Step S102, collecting the fermentation broth obtained by fermentation culture, and recovering and / or purifying it to obtain a fermentation product;

[0056] During the fermentation culture, the flow rate of the culture medium added to the fermentation tank and the discharge rate of the fermentation liquid out of the fermentation tank are controlled according to the concentration of the bacteria in the fermentation tank, including:

[0057] detecting the bacterial cell concentration in the fermentation tank;

[0058] When the bacterial concentration is greater than or equal to the first threshold, the recovery medium is added and Qn1 = K1-Kg×OD is calculated. 600 ×Kn, where Qn1 is the first rate, K1 is the first constant, Kg is the bacterial growth rate, OD 600 is the bacterial concentration, Kn is the culture medium conversion coefficient, and the discharge rate is controlled to be the first rate;

[0059] When the bacterial concentration is less than the first threshold, nitrogen source culture medium is added and Qn2 = K1 + Kg × OD is calculated. 600 ×Kn, where Qn2 is the second rate, and the discharge rate is controlled to be the second rate.

[0060] Specifically, step S101 is performed to carry out fermentation culture, and step S102 is performed to collect the fermentation liquid, and recover and / or purify it to obtain a fermentation product.

[0061] During the fermentation culture, the flow rate of the culture medium added to the fermentation tank and the discharge rate of the fermentation liquid out of the fermentation tank are controlled according to the bacterial cell concentration in the fermentation tank.

[0062] Specifically, a mathematical model of bacterial concentration is introduced:

[0063] Online detection of bacterial concentration OD 600 , when OD 600 When the value is greater than or equal to the first threshold, the culture medium is recovered by feeding, Qn1=K1-Kg×OD 600 ×Kn, where the first threshold is preferably 70, Qn1 is the first rate, Kg is the bacterial specific growth rate, which is related to bacterial activity and can be determined experimentally; preferably, Kg is 0.09-0.12. Kn is the culture medium conversion coefficient, K1 is the first constant obtained through experiments, and in the case of continuous fermentation of rhamnolipids, the first constant K is preferably 80. When OD 600 < the first threshold, feed nitrogen source culture medium, Qn2=K1+Kg×OD 600 × Kn, where Qn2 is the second rate. Control the discharge rate Qs = the first rate Qn1 or the second rate Qn2.

[0064] The discharge rate is the rate at which the fermentation liquid is discharged from the fermentation tank. Preferably, as shown in FIG2 , the discharge rate is the rate at which the fermentation liquid is discharged from the fermentation tank 13. By adjusting the discharge rate, the bacterial cell concentration is adjusted so that the bacterial cell concentration remains stable.

[0065] As shown in Figure 2, when OD 600 ≥ the first threshold, open valve V-521, close valve V-519, connect the fermentation tank 13 and the medium buffer tank 12, and feed the recovered medium; when OD 600When the temperature is less than the first threshold, valve V-519 is opened and valve V-521 is closed to connect the fermentation tank 13 with the nitrogen source feeding tank 11, and nitrogen source medium is fed. The medium buffer tank 12 contains the recovered medium, and the nitrogen source feeding tank 11 contains the nitrogen source medium.

[0066] The recovered culture medium is the liquid obtained after removing the extractant, bacteria and / or fermentation products from the fermentation liquid during the fermentation process.

[0067] The nitrogen source culture medium contains: 2g / L NaNO3, 15.1g / L KH2PO4, 15.1g / LNa2HPO4, 0.08g / L FeCl3·6H2O, 0.75g / L ZnSO4·7H2O, 0.08g / L CoCl2·6H2O, 0.075g / L CuSO4·5H2O, 0.75g / L MnSO4·H2O, 0.15g / L H3BO3, and 0.05g / L Na2MoO4·2H2O.

[0068] The rhamnolipid continuous fermentation method of this embodiment improves the production efficiency of rhamnolipid, avoids severe foaming of the fermentation liquid, and enables the continuous fermentation process to operate efficiently and stably for a long time, while also achieving the recycling of the fermentation liquid and reducing production costs.

[0069] In one embodiment, during the fermentation culture, the carbon source flow rate is controlled according to the desired concentration of the fermentation product (e.g., rhamnolipid) in the fermentor, comprising:

[0070] According to the desired concentration of the fermentation product in the fermentation tank, the carbon source flow acceleration rate is controlled as follows: Qc=Qm×Cm1 / Km, wherein Qc is the carbon source flow acceleration rate, Cm1 is the desired concentration of the fermentation product in the fermentation tank, Km is the carbon source conversion rate, Qm is the discharge flow rate of the fermentation liquid out of the fermentation tank, Qm=Vk×S×ρ, Vk is the bubble rise rate, ρ is the bubble density, and S is the diameter of the fermentation tank.

[0071] Specifically, the desired rhamnolipid concentration is pre-set, and the carbon source flow acceleration rate Qc is calculated and controlled based on the desired rhamnolipid concentration. Here, Qc = Qm × Cm1 / Km, where Qc is the carbon source flow acceleration rate, Cm1 is the desired rhamnolipid concentration in the fermenter, Km is the carbon source conversion rate, and in the case of continuous fermentation of rhamnolipid, the carbon source conversion rate is the conversion rate of the carbon source to rhamnolipid, preferably 0.51-0.55, Qm is the flow rate entering the overflow tank 14, and Qm = Vk × S × ρ, where Vk is the bubble rise rate, ρ is the bubble density, and S is the diameter of the fermenter 13.

[0072] In this embodiment, the carbon source flow rate is controlled to maintain a stable rhamnolipid concentration, thereby ensuring stable operation of continuous fermentation.

[0073] In one embodiment, during fermentation culture, the ventilation volume and the flow rate of the carbon source are controlled according to the foaming rate in the fermenter, including:

[0074] At each sampling moment, the real-time concentration and ventilation volume of the fermentation product (e.g., rhamnolipid) in the fermentation tank are detected, and the foam production rate is calculated based on the real-time concentration and ventilation volume. If the foam production rate calculated at the current sampling moment is greater than or equal to the foam production rate calculated at the previous sampling moment, the ventilation volume is reduced; if the foam production rate calculated at the current sampling moment is less than the foam production rate calculated at the previous sampling moment, the ventilation volume is increased; wherein the foam production rate is: Vg=Km×exp(L / K2)×Cm×exp(T / K3), wherein Vg is the foam production rate, Km is the proportional coefficient, L is the ventilation volume, T is the stirring speed, Cm is the real-time concentration of the fermentation product, K2 is the second constant, K3 is the third constant, and exp is the natural exponent.

[0075] Specifically, a bubble production rate model is introduced: Vg = Km × exp(L / K2) × Cm × exp(T / K3), where Km is the proportional coefficient, Vg is the bubble production rate (or bubble production rate), i.e., the height of bubbles rising per unit time, L is the ventilation volume, T is the stirring speed, Cm is the rhamnolipid concentration, K2 is the second constant, preferably 25, and K3 is the third constant, preferably 250. By fine-tuning the ventilation volume L, the bubble production rate Vg is kept constant.

[0076] The ventilation volume is the amount of air blown into the fermentation tank 13 , and the carbon source flow acceleration rate is the output flow rate of the carbon source feeding tank 10 .

[0077] In this embodiment, the ventilation volume is adjusted according to the foam production rate to keep the foam production rate constant, so that the continuous fermentation can run stably.

[0078] In some embodiments, a carbon source and a nitrogen source are continuously added to the fermentation tank 13, and the flow acceleration rate and the discharge rate are controlled according to the bacterial concentration of the fermentation tank 13, the carbon source flow acceleration rate is controlled according to the desired rhamnolipid concentration, and the ventilation volume and the carbon source flow acceleration rate are controlled according to the foaming rate in the fermentation tank 13.

[0079] [Corrected on 24.05.2024 according to Rule 26] In one embodiment, the carbon source used for fermentation culture is selected from at least one of soybean oil, glycerol, glucose, palm oil, and waste cooking oil.

[0080] In one embodiment, the pH of the fermentation broth is 6.8-7.1.

[0081] The continuous fermentation method provided by the present invention is applicable to fermentation and culture of any strain under an aerobic environment to obtain a fermentation product of the strain. In particular, the continuous fermentation method provided by the present invention is particularly suitable for situations where bubbles are easily generated during the fermentation process, thereby reducing foaming, improving fermentation efficiency and product yield (when the fermentation product is rhamnolipid, the yield is >99%). In one embodiment, the fermentation product is rhamnolipid, and accordingly, the strain is a strain capable of producing rhamnolipid by fermentation and culture, preferably Pseudomonas putida.

[0082] In one embodiment, the first threshold is 65-75, preferably 70.

[0083] The first threshold is used to limit the bacterial cell concentration in the fermentation tank, so that the bacterial cell concentration remains within the first threshold range. Therefore, if the first threshold is too high, the bacterial cell concentration is too high, which will affect the carbon source conversion rate; while if the first threshold is too low, the bacterial cell concentration is too low, which will reduce the yield.

[0084] In this embodiment, the first threshold is limited to a suitable range, so that the bacterial concentration is kept within a suitable range, which avoids affecting the carbon source conversion rate on the one hand and reducing the yield on the other hand.

[0085] In one embodiment, the desired concentration of the fermentation product in the fermenter is 10-20 g / L.

[0086] In this embodiment, the carbon source flow acceleration rate is set according to the desired concentration of the fermentation product in the fermentation tank, so that the concentration of the fermentation product in the fermentation tank is maintained at the desired concentration of the fermentation product in the fermentation tank, avoiding too low a concentration that reduces the yield, and avoiding too high a concentration that leads to too high a foaming rate and uncontrollable liquid level.

[0087] In this embodiment, the concentration of the desired fermentation product in the fermenter is limited to a suitable range, thereby controlling the carbon source flow rate to maintain the concentration of the fermentation product in the fermenter stable within a suitable range, thereby avoiding a decrease in yield and an inability to control the liquid level.

[0088] As shown in FIG2 , a continuous fermentation system in one embodiment of the present invention includes a carbon source feeding tank 10, a nitrogen source feeding tank 11, a culture medium buffer tank 12, a fermentation tank 13, an overflow tank 14, a ceramic membrane 15, a first extraction centrifugal device 16, a second extraction centrifugal device 17, and a gas tower 19, wherein the discharge port of the carbon source feeding tank 10 and the discharge port of the nitrogen source feeding tank 11 are controllably connected to the feed port of the fermentation tank 13, and the overflow port of the fermentation tank 13 is connected to the overflow port of the overflow tank 14. The feed port is controllably connected, the discharge port of the overflow tank 14 is controllably connected with the feed port of the ceramic membrane 15 and the feed port of the first extraction centrifugal device 16 respectively, the discharge port of the ceramic membrane 15 is controllably connected with the feed port of the second extraction centrifugal device 17, the recovery port of the second extraction centrifugal device 17 is connected with the feed port of the gas tower 19, the discharge port of the gas tower 19 is controllably connected with the feed port of the culture medium buffer tank 12, and the discharge port of the culture medium buffer tank 12 is controllably connected with the feed port of the fermentation tank 13.

[0089] Specifically, the discharge port of the carbon source feeding tank 10 is connected to the feed port of the fermentation tank 13 via valve V-515, and the discharge port of the nitrogen source feeding tank 11 is controllably connected to the feed port of the fermentation tank 13 via valve V-519. Clean air is also introduced into the fermentation tank 13. The overflow port of the fermentation tank 13 is controllably connected to the feed port of the overflow tank 14 via overflow valve V-516. The discharge port of the overflow tank 14 is controllably connected to the feed port of the ceramic membrane 15 and to the feed port of the first extraction centrifuge 16 via the first pump 21 and the second pump 22, respectively. The discharge port of the ceramic membrane 15 is controllably connected to the feed port of the second extraction centrifuge 17 via valve V-520. Simultaneously, the extractant enters the first extraction centrifuge 16 via valve V-525, and the extractant enters the second extraction centrifuge 17 via valve V-524. The recovery port of second extraction centrifuge 17 is connected to gas tower 19 via valve V-522. Gas tower 19 is controllably connected to the feed port of culture medium buffer tank 12 via valve V-518. The discharge port of culture medium buffer tank 12 is controllably connected to the feed port of fermentation tank 13 via valve V-521. Clean air is simultaneously introduced into gas tower 19, which then discharges organic waste gas.

[0090] In some embodiments, the recovery port of the ceramic membrane 15 is controllably connected to the recovery port of the fermentation tank 13 via valve V-523.

[0091] The continuous fermentation system of the present embodiment includes a carbon source feed tank, a nitrogen source feed tank, a culture medium buffer tank, a fermentation tank, an overflow tank, a ceramic membrane, a first extraction centrifuge, and a second extraction centrifuge. In the entire system, no defoaming agent is introduced, which improves product quality and reduces fermentation costs. The continuous fermentation system provided in this embodiment is particularly suitable for use in fermentation culture in conjunction with the continuous fermentation method provided by the present invention, and can further reduce the bubbles generated during the fermentation process. Therefore, the continuous fermentation system provided in this embodiment is also particularly suitable for the production of rhamnolipids. At the same time, the present embodiment is provided with an overflow tank. When the foam in the fermentation tank reaches a certain liquid level, the fermented liquid overflowing from the overflow port of the fermentation tank will flow into the overflow tank and stand still in the overflow tank. Due to the lack of ventilation and stirring, the fermented liquid stands still in the overflow tank, and the foam will gradually disappear, becoming a fermented liquid without foam, thereby further reducing the generation of bubbles.

[0092] In one embodiment, the continuous fermentation system further includes a mixing tank 18 and a distillation tower 20, the discharge port of the first extraction centrifuge 16 and the discharge port of the second extraction centrifuge 17 are controllably connected to the feed port of the mixing tank 18, and the discharge port of the mixing tank 18 is connected to the feed port of the distillation tower 20.

[0093] Specifically, the discharge port of the first extraction centrifuge 16 is controllably connected to the feed port of the mixing tank 18 via valve V-517. The first extraction centrifuge 16 discharges the bacterial waste liquid via valve V-514. The discharge port of the second extraction centrifuge 17 is connected to the feed port of the mixing tank 18, which in turn is connected to the feed port of the distillation column 20. Water is also injected into the water inlet of the mixing tank 18. The distillation column 20 discharges the extractant and crude product.

[0094] The first extraction centrifuge 16 is used to remove aged bacterial cells. A portion of the fermentation liquid flowing out of the overflow tank 14 enters the first extraction centrifuge 16, where it is separated into a fermentation product. The waste liquid containing aged bacterial cells is then discharged. The fermentation product separated by the first extraction centrifuge 16 passes through the mixing tank 18 and enters the distillation tower 20.

[0095] The second extraction centrifuge 17 is used to recover the fermentation broth to obtain a recovery culture medium. Another portion of the fermentation broth flowing out of the overflow tank 14 enters the ceramic membrane 15 to recover the bacterial cells. After the fermentation broth containing the fermentation product is separated from the fermentation product by the second extraction centrifuge 17, the remaining fermentation broth passes through the gas tower 19 to remove residual extractant and then enters the culture medium buffer tank 12 (as a recovery culture medium) for standby use. The fermentation product separated by the second extraction centrifuge 17 also enters the distillation tower 20 via the mixing tank 18. The distillation tower 20 distills the fermentation product separated from the first extraction centrifuge 16 and the fermentation product separated from the second extraction centrifuge 17, and then discharges the extractant and crude product.

[0096] This embodiment adds a mixing tank to achieve discharging after mixing.

[0097] In one embodiment, the fermentation tank is operated at a temperature of 30-37°C throughout the entire process.

[0098] In one embodiment, the second extraction centrifugal device 17 is a centrifugal extraction column.

[0099] In one embodiment, the extracting agent in the first extraction centrifugal device 16 and the second extraction centrifugal device 17 is one or more of chloroform, dichloromethane, n-hexane, n-heptane, n-decane, ethyl acetate and acetonitrile.

[0100] [Corrected on 24.05.2024 according to Rule 26] In one embodiment, the carbon source in the carbon source feeding tank is selected from at least one of soybean oil, glycerol, glucose, palm oil, and waste cooking oil.

[0101] In one embodiment, the nitrogen source culture medium in the nitrogen source feeding tank is 2g / L NaNO3, 15.1g / L KH2PO4, 15.1g / L Na2HPO4, 0.08g / L FeCl3·6H2O, 0.75g / L ZnSO4·7H2O, 0.08g / L CoCl2·6H2O, 0.075g / L CuSO4·5H2O, 0.75g / L MnSO4·H2O, 0.15g / L H3BO3, and 0.05g / L Na2MoO4·2H2O.

[0102] As a preferred embodiment of the present invention, a workflow of a rhamnolipid continuous fermentation method includes:

[0103] The aerobic fermentation stage includes: inoculating Almonas putida into the culture medium in the fermentation tank 13, and fermenting and culturing the culture medium under an aerobic environment until the cell concentration OD 600 is 65;

[0104] The initial accumulation stage includes: continuously adding carbon source to the fermentation tank 13 through the carbon source feeding tank 10, and adding nitrogen source culture medium to the fermentation tank 13 through the nitrogen source feeding tank 11. When the foam level of the fermentation liquid reaches 80%, the tank pressure of the fermentation tank 13 is continuously increased, and the ventilation volume of the fermentation tank 13 is reduced to maintain the foam level of the fermentation liquid at 80%. When the rhamnolipid content in the fermentation tank 13 is higher than 10 g / L, the overflow valve V-516 is opened to enter the steady-state continuous fermentation stage;

[0105] The steady-state continuous fermentation stage includes the following steps: continuously adding a carbon source and a nitrogen source to the fermentation tank 13. A portion of the fermentation broth flowing out of the overflow tank 14 enters the first extraction centrifuge 16 to separate rhamnolipids, and the waste liquid containing aged bacterial cells is discharged. The rhamnolipids separated from the first extraction centrifuge 16 are passed through a mixing tank 18 and then into a distillation tower 20. Another portion of the fermentation broth flowing out of the overflow tank 14 enters a ceramic membrane 15 to recover bacterial cells. The rhamnolipid-containing fermentation broth passes through a second extraction centrifuge 17 to separate rhamnolipids. The rhamnolipid-removed fermentation broth then passes through a gas column 19 to remove residual extractant before entering the culture medium buffer tank 12 for standby use. The rhamnolipids separated from the second extraction centrifuge 17 are also passed through a mixing tank 18 and then into a distillation tower 20. The distillation tower 20 rectifies the rhamnolipids separated from the first extraction centrifuge 16 and the second extraction centrifuge 17, and then discharges the extractant and crude product.

[0106] The post-fermentation treatment stage includes: when the rhamnolipid concentration continues to rise and the rhamnolipid concentration is greater than 20 g / L, the addition of the carbon source and nitrogen source to the fermentation tank 13 is stopped, and the fermentation liquid in the overflow tank 14 is extracted and separated by the second extraction centrifuge 17 to obtain rhamnolipid, and the remaining fermentation liquid is used as fermentation waste liquid.

[0107] Specifically, in the aerobic fermentation stage: Alphamonas putida is inoculated into the culture medium in the fermentation tank 13, and cultured under aerobic conditions until OD 600 is 65;

[0108] During the initial rhamnolipid accumulation phase, carbon and nitrogen sources are continuously added to the fermenter. When the foam level in the fermentation liquid reaches 80%, the tank pressure is continuously increased and the ventilation rate is reduced to maintain the foam level at 80%. When the rhamnolipid content exceeds 10g / L, the overflow valve is opened to enter steady-state continuous fermentation.

[0109] This is followed by a steady-state continuous fermentation phase, where fresh cells are continuously produced using a continuous nitrogen flow. When the fermentation broth level exceeds 80%, the foamy broth flows through an overflow line into an overflow tank, where it naturally defoams. A portion of the fermentation broth enters the first extraction centrifuge to recover rhamnolipids and remove aged cells. A portion of the fermentation broth enters a ceramic membrane to recover cells. The rhamnolipid-containing broth passes through a second extraction centrifuge to remove the rhamnolipids, then passes through a gas tower to remove residual extractant before entering the fermentation feed tank for standby use.

[0110] Finally, the post-fermentation treatment phase begins: when the rhamnolipid concentration continues to rise uncontrollably and reaches >20 g / L, feeding is stopped. After the fermentation broth in the tank is extracted and separated from the rhamnolipid by the second extraction centrifuge 17, the remaining fermentation broth is treated as fermentation waste.

[0111] Example 1: Different OD 600 Impact on average productivity

[0112] A rhamnolipid continuous fermentation method comprises the following steps:

[0113] Aerobic fermentation stage: inoculate Alphamonas putida into the culture medium in the fermentation tank 13 and culture under aerobic conditions until OD 600 is 65;

[0114] Initial Rhamnolipid Accumulation Stage: Continuously add carbon and nitrogen sources to the fermenter. When the foam level in the fermentation liquid reaches 80%, continuously increase the fermenter pressure and reduce the ventilation rate to maintain the foam level at 80%. When the rhamnolipid content exceeds 10g / L, open the overflow valve and enter steady-state continuous fermentation.

[0115] During the steady-state continuous fermentation phase, fresh cells are continuously produced using a continuous flow nitrogen source. When the fermentation broth level exceeds 80%, the foamy fermentation broth flows through the overflow line into the overflow tank, where it naturally defoams. A portion of the fermentation broth enters the first extraction centrifuge to recover rhamnolipids and remove aged cells. A portion of the fermentation broth enters the ceramic membrane to recover cells. The rhamnolipid-containing fermentation broth passes through the second extraction centrifuge to remove rhamnolipids, then passes through a gas tower to remove residual extractant before entering the fermentation feed tank for standby use.

[0116] Post-fermentation processing: The continued rise in rhamnolipid concentration leads to severe bubbles, which cannot be eliminated in time in the overflow tank. When the rhamnolipid concentration exceeds 20 g / L, the second extraction centrifuge 17 is no longer able to recover the excess rhamnolipid, disrupting the equilibrium of the continuous fermentation process. Feeding is stopped at this point. After the fermentation broth in the tank passes through the second centrifugal extraction device to separate the rhamnolipid, the remaining fermentation broth is considered fermentation waste.

[0117] The flow acceleration rate and discharge rate are controlled by the bacterial concentration mathematical model, and the bacterial concentration OD is maintained. 600 =65,OD 600 =70,OD 600 =75; the ventilation volume and carbon source flow acceleration rate were continuously controlled by a bubble production rate mathematical model to maintain the rhamnolipid concentration in the fermenter at 10-20 g / L.

[0118] Online OD detection 600 , when OD 600 When the pH value is ≥70, the culture medium is recovered by feeding, Qm=80-Kg×OD 600 ×Kn, Kg is the bacterial growth rate, which is related to bacterial activity, Kg is 0.09-0.12, Kn is the culture medium conversion coefficient, when OD 600 When the OD value is less than 70, nitrogen source culture medium is fed, Qn=80+Kg×OD600 × Kn. The rate of entry into the first extraction centrifuge is Qs = Qn or Qm. Using the bubble production rate model, Vg = Km × exp(L / 25) × Cm × exp(T / 250), where Km is the proportionality coefficient, Vg is the bubble production rate (i.e., the bubble rise height per unit time), L is the aeration volume, T is the agitation speed, and Cm is the rhamnolipid concentration. Qm = Vk × S × ρ, where S is the fermenter diameter and Qm is the flow rate into the overflow tank. Qc = Qm × Cm1 / Km, where Cm1 is the desired fermentation product concentration in the fermenter and Km is the carbon source conversion rate, which should be between 0.51 and 0.55. By fine-tuning L the aeration volume, Vg is kept constant, and by controlling Qc the carbon source flow rate, the rhamnolipid concentration is maintained at the desired fermentation product concentration in the fermenter. By maintaining a constant bubble production rate and rhamnolipid concentration, continuous fermentation is stable.

[0119] The fermentation tank operates at a temperature of 30-37°C throughout the entire process, the carbon source is soybean oil, and the pH of the fermentation liquid is 6.8-7.1.

[0120] The second extraction centrifugal device 17 is a three-stage centrifugal extraction, and the rhamnolipid yield is greater than 99%.

[0121] The nitrogen source culture medium in the nitrogen source feeding tank includes: 2g / L NaNO3, 15.1g / L KH2PO4, 15.1g / L Na2HPO4, 0.08g / L FeCl3·6H2O, 0.75g / L ZnSO4·7H2O, 0.08g / L CoCl2·6H2O, 0.075g / L CuSO4·5H2O, 0.75g / L MnSO4·H2O, 0.15g / L H3BO3, and 0.05g / L Na2MoO4·2H2O.

[0122] As shown in Table 1, the fermentation was continued for 108 h, and the OD 600 When the concentration was 65, the conversion rate was 52.2% and the average rhamnolipid production rate was 0.62 g / L·h. The conversion rate and average production rate were optimal.

[0123] Table 1 Different OD 600 Impact on average productivity

[0124] Example 2: Effect of different temperatures on average productivity

[0125] The content of this embodiment is consistent with that of embodiment 1, except that the fermentation temperatures for rhamnolipid concentrations are 30°C, 35°C, and 37°C, respectively.

[0126] The results are shown in Table 2. Higher temperatures significantly impacted bacterial activity, while higher temperatures also decreased carbon source conversion. At 35°C, rhamnolipid conversion was 53.5%, and the rhamnolipid production rate was 0.62 g / L·h. Considering both conversion efficiency and cost, 35°C was the optimal temperature.

[0127] Table 2 Effect of different temperatures on average production rate

[0128] Example 3: Continuous fermentation of rhamnolipids

[0129] The content of this embodiment is consistent with that of embodiment 1, except that the fermentation temperature is 35°C and the OD 600 Select 65 and extend the fermentation period to 216h.

[0130] The results are shown in Figure 3. The highest rhamnolipid yield was 0.738 g / L·h, the lowest rhamnolipid yield was 0.622 g / L·h, and the average rhamnolipid yield during the entire fermentation cycle was 0.640 g / L·h. The carbon source yield was 53.9%, indicating good economic benefits.

[0131] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A continuous fermentation method, characterized in that: include: Inoculate the strain into a continuous fermentation system and perform fermentation culture under an aerobic environment; collecting the fermentation broth obtained from the fermentation culture, and obtaining a fermentation product through recovery and / or purification; Wherein, in the fermentation culture, according to the bacterial cell concentration in the fermentation tank, the flow rate of the culture medium added to the fermentation tank and the discharge rate of the fermentation liquid discharged from the fermentation tank are controlled, including: Detecting the bacterial cell concentration in the fermentation tank; When the bacterial cell concentration is greater than or equal to the first threshold, the culture medium is added, and Qn1=K1-Kg×OD is calculated. 600 ×Kn, where Qn1 is the first rate, K1 is the first constant, Kg is the bacterial specific growth rate, OD 600 is the bacterial concentration, Kn is the culture medium conversion coefficient, and the discharge rate is controlled to be the first rate; When the bacterial cell concentration is less than the first threshold, the culture medium is added, and Qn2=K1+Kg×OD is calculated. 600 ×Kn, wherein Qn2 is the second rate, and the discharge rate is controlled to be the second rate.

2. The continuous fermentation method according to claim 1, characterized in that: In the fermentation culture, the carbon source flow acceleration rate is controlled according to the desired concentration of the fermentation product in the fermentation tank, including: According to the desired concentration of the fermentation product in the fermenter, the carbon source flow rate is controlled as follows: Qc=Qm×Cm1 / Km, wherein Qc is the carbon source flow acceleration rate, Cm1 is the desired concentration of the fermentation product in the fermenter, Km is the carbon source conversion rate, Qm is the discharge flow rate of the fermentation liquid out of the fermenter, Qm=Vk×S×ρ, Vk is the bubble rise rate, ρ is the bubble density, and S is the diameter of the fermenter.

3. The continuous fermentation method according to claim 1, characterized in that: During the fermentation culture, the ventilation volume and the flow rate of the carbon source are controlled according to the foaming rate in the fermenter, including: At each sampling moment, the real-time concentration and ventilation volume of the fermentation product in the fermenter are detected, and the foam production rate is calculated according to the real-time concentration and the ventilation volume. If the foam production rate calculated at the current sampling moment is greater than or equal to the foam production rate calculated at the previous sampling moment, the ventilation volume is reduced; if the foam production rate calculated at the current sampling moment is less than the foam production rate calculated at the previous sampling moment, the ventilation volume is increased; wherein the foam production rate is: Vg=Km×exp(L / K2)×Cm×exp(T / K3), wherein Vg is the foam production rate, Km is the proportional coefficient, L is the ventilation volume, T is the stirring speed, Cm is the real-time concentration of the fermentation product, K2 is the second constant, K3 is the third constant, and exp is the natural exponent.

4. The continuous fermentation method according to any one of claims 1 to 3, characterized in that: The fermentation product is rhamnolipid; the strain is a strain capable of producing rhamnolipid through fermentation culture, preferably Pseudomonas putida.

5. The continuous fermentation method according to claim 4, characterized in that: The first threshold is 65-75, preferably 70; The desired concentration of the fermentation product in the fermenter is 10-20 g / L.

6. [Corrected 24.05.2024 according to Rule 26] The continuous fermentation method according to any one of claims 1 to 3, characterized in that: In the fermentation culture, the added carbon source is selected from at least one of soybean oil, glycerol, glucose, palm oil, and kitchen waste oil; The culture medium is a nitrogen source culture medium, which contains: 2g / L NaNO3, 15.1g / L KH2PO4, 15.1g / L Na2HPO4, 0.08g / L FeCl3·6H2O, 0.75g / L ZnSO4·7H2O, 0.08g / L CoCl2·6H2O, 0.075g / L CuSO4·5H2O, 0.75g / L MnSO4·H2O, 0.15g / L H3BO3, and 0.05g / L Na2MoO4·2H2O.

7. A continuous fermentation system, characterized in that: The continuous fermentation system is applied to the continuous fermentation method according to any one of claims 1 to 5, comprising a carbon source feeding tank (10), a nitrogen source feeding tank (11), a culture medium buffer tank (12), a fermentation tank (13), an overflow tank (14), a ceramic membrane (15), a first extraction centrifugal device (16), a second extraction centrifugal device (17) and a gas tower (19), wherein the discharge port of the carbon source feeding tank (10) and the discharge port of the nitrogen source feeding tank (11) are controllably connected to the feed port of the fermentation tank (13), and the overflow port of the fermentation tank (13) is controllably connected to the overflow port of the overflow tank (14). The feed port is controllably connected, the discharge port of the overflow tank (14) is controllably connected with the feed port of the ceramic membrane (15) and the feed port of the first extraction centrifugal device (16), respectively, the discharge port of the ceramic membrane (15) is controllably connected with the feed port of the second extraction centrifugal device (17), the recovery port of the second extraction centrifugal device (17) is connected with the feed port of the gas tower (19), the discharge port of the gas tower (19) is controllably connected with the feed port of the culture medium buffer tank (12), and the discharge port of the culture medium buffer tank (12) is controllably connected with the feed port of the fermentation tank (13).

8. The continuous fermentation system according to claim 7, characterized in that: The continuous fermentation system further comprises a mixing tank (18) and a distillation tower (20); the discharge port of the first extraction centrifugal device (16) and the discharge port of the second extraction centrifugal device (17) are controllably connected to the feed port of the mixing tank (18); and the discharge port of the mixing tank (18) is connected to the feed port of the distillation tower (20).

9. The continuous fermentation system according to claim 7 or 8, characterized in that: The second extraction centrifugal device (17) is a centrifugal extraction tower; The extracting agent in the first extraction centrifugal device (16) and the second extraction centrifugal device (17) is selected from at least one of chloroform, dichloromethane, n-hexane, n-heptane, n-decane, ethyl acetate and acetonitrile.

10. The continuous fermentation system according to claim 7 or 8, characterized in that: The operating temperature of the fermentation tank (13) is 30-37°C; The pH of the fermentation broth is 6.8-7.1.

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