A new type of microbubble enhanced gas supply bioreactor

Abstract

The utility model relates to a microorganism fermentation equipment technical field, especially in kind of novel microbubble reinforced gas supply biological reactor. Including fermentation tank, liquid delivery pump, gas supply system, membrane module and circulation pipeline, wherein fermentation tank has liquid outlet and gas -liquid inlet, and liquid delivery pump and membrane module are connected in series through circulation pipeline, and the both ends of circulation pipeline are connected with the liquid outlet and gas -liquid inlet of fermentation tank respectively, and liquid delivery pump is used for delivering fermentation broth to membrane module, gas supply system is connected with membrane module through gas supply pipeline, and gas is delivered to membrane module, and high flow rate fermentation broth in membrane module is wrapped and carried gas to form microbubble, and microbubble is backflowed to fermentation tank through circulation pipeline with fermentation broth. The utility model improves gas mass transfer efficiency under low ventilation and low stirring speed, reduces energy consumption, avoids cell damage, reduces foam, is applicable to aerobic fermentation, anaerobic fermentation and gas as nutrient substance fermentation, can improve target product yield and yield, and reduces fermentation cost.

Description

Technical Field

[0001] This utility model relates to the field of microbial fermentation equipment technology, and in particular to a novel microbubble-enhanced gas supply bioreactor. Background Technology

[0002] Oxygen supply is a challenging task in industrial fermentation processes, closely related to microbial growth and metabolism. Aerobic microorganisms obtain energy by oxidizing the substrate in an aerobic environment to power their cell growth processes, including cell synthesis, product synthesis, cell repair, and substance transport, as well as non-growth processes such as cell movement. High-density fermentation processes require and consume even more oxygen, thus necessitating higher oxygen transfer rates.

[0003] Currently, strategies to increase oxygen levels include continuously increasing aeration and stirring rates. However, these conventional strategies result in higher energy consumption and mechanical energy requirements, which are not conducive to large-scale industrial production. Therefore, improving oxygen mass transfer efficiency under mild, low-energy conditions is a key area of ​​innovation in bioreactor device development.

[0004] Studies have shown that reducing bubble size can increase surface area and gas residence time, thereby improving oxygen mass transfer efficiency and achieving an oxygenation effect. Microbubbles are micrometer-sized bubbles. Compared to ordinary bubbles, their particle size is much smaller, thus exhibiting some unique advantages, such as longer residence time, higher mass transfer efficiency, slower rise speed, and higher zeta potential formed by surface charge. These characteristics have led to the widespread application of microbubbles in recent years in fields such as the degradation of pollutants in water, aquaculture, and agricultural soil improvement.

[0005] Simple aeration heads produce bubbles with diameters on the millimeter scale, which are insufficient to form microbubbles; specialized equipment is required to obtain microbubbles. Major microbubble generation technologies include pressurized dissolved gas release, air dispersion, electrolysis, and air flotation pump generation. These technologies have high energy consumption and are limited by gas solubility, thus restricting their gas production efficiency. Passing air through a perforated plate or porous medium into a liquid can generate a large number of bubbles with low energy consumption; this method is one of the commonly used aeration methods. To reduce the particle size of the obtained bubbles, smaller pore sizes can be used; however, when the membrane pore size is reduced to a certain extent, it is difficult to further reduce the bubble diameter by decreasing the membrane pore size.

[0006] Janus membrane is a special membrane material with different properties on both sides. Although Janus membrane has been used in many fields, there is currently no device or research to couple Janus membrane system with fermenter. Utility Model Content

[0007] To address the aforementioned problems, the purpose of this invention is to provide a novel microbubble-enhanced gas supply bioreactor that achieves efficient gas mass transfer under low aeration rates and low stirring speeds, thereby reducing fermentation energy consumption and increasing the yield of the target product.

[0008] To achieve the above objectives, this utility model provides the following technical solution:

[0009] This invention provides a novel microbubble-enhanced gas supply bioreactor, comprising a fermenter, a liquid transfer pump, a gas supply system, a membrane module, and a circulation pipeline. The fermenter has a liquid outlet and a gas-liquid inlet. The liquid transfer pump and the membrane module are connected in series via the circulation pipeline, with both ends of the pipeline connected to the liquid outlet and gas-liquid inlet of the fermenter, respectively. The liquid transfer pump is used to transport the fermentation broth in the fermenter to the membrane module. The gas supply system is connected to the membrane module via the gas supply pipeline and supplies gas to the membrane module. The high-velocity fermentation broth in the membrane module carries the gas to form microbubbles. The microbubbles flow back to the fermenter with the fermentation broth through the circulation pipeline, achieving efficient gas mass transfer.

[0010] The membrane assembly includes an outer tube and multiple hollow fiber membrane filaments disposed within the outer tube. One end of the outer tube has an inlet 1 that communicates with the inner cavity of the outer tube and is connected to the circulation pipeline. The other end of the outer tube has an inlet 2 that communicates with the inner cavity of each hollow fiber membrane filament and is connected to the gas supply pipeline. The hollow fiber membrane filaments have a liquid-proof and gas-permeable function. Gas permeating through the hollow fiber membrane filaments is entrained by the high-velocity liquid in the outer tube to form microbubbles. The other end of the outer tube has a gas-liquid outlet, which is connected to the gas-liquid inlet of the fermenter through the circulation pipeline.

[0011] The hollow fiber membrane is made of Janus hollow fiber membrane, with a hydrophobic layer on the inner side and a hydrophilic layer on the outer side.

[0012] The aspect ratio of the hollow fiber membrane filaments is from 100:1 to 1000:1.

[0013] The outer tube (114) is made of stainless steel or plastic.

[0014] The circulation pipeline between the liquid transfer pump and the membrane module is sequentially equipped with a control valve I, a pressure gauge I, and a flow meter. The control valve I is used to control the flow rate of the fermentation broth drawn from the fermenter to the membrane module, the pressure gauge I is used to monitor the pressure of the fermentation broth delivered to the membrane module, and the flow meter is used to monitor the flow rate of the fermentation broth delivered to the membrane module.

[0015] The gas supply pipeline is equipped with a pressure gauge II and a control valve II, wherein the control valve II is used to control the gas flow rate in the gas supply pipeline, and the pressure gauge II is used to control the gas pressure in the gas supply pipeline.

[0016] A gas flow meter is installed on the top of the fermenter.

[0017] The fermentation tank is equipped with a stirring device, which is used to stir the fermentation liquid inside the fermentation tank.

[0018] The advantages and beneficial effects of this invention are as follows: This invention provides a novel microbubble-enhanced gas supply bioreactor that can operate at relatively low aeration rates (<0.1 vvm) and stirring speeds (typically 0-50 rpm). This not only significantly improves the gas mass transfer rate during fermentation but also offers the following benefits:

[0019] 1. Reduce energy loss caused by stirring and high ventilation during production;

[0020] 2. Mild conditions prevent cells from being damaged by shear forces;

[0021] 3. Reduce foaming and increase the effective volume of the fermentation tank;

[0022] 4. For fermentation containing volatile substances, it greatly reduces the volatilization of volatile substances, increases yield and productivity, while reducing gas emissions and protecting the environment;

[0023] 5. This reactor can be used for gas supply in fermentation where gas is used as a nutrient, greatly improving gas utilization.

[0024] 6. For anaerobic fermentation, this reactor can be used to introduce protective gas, which can improve the utilization rate of protective gas, reduce the amount used, and also has a stirring effect. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is a schematic diagram of the structure of a novel microbubble-enhanced gas supply bioreactor according to this utility model; Figure 2 This is a schematic diagram of the membrane module in this utility model.

[0027] In the diagram: 1. Fermentation tank; 2. Liquid outlet; 3. Gas flow meter; 4. Liquid transfer pump; 5. Control valve I; 6. Pressure gauge I; 7. Flow meter; 8. Pressure gauge II; 9. Control valve II; 10. Gas supply system; 11. Membrane module; 111. Inlet 1; 112. Inlet 2; 113. Hollow fiber membrane filament; 114. Outer pipe; 115. Gas-liquid outlet; 12. Gas-liquid inlet; 13. Stirring device. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this utility model clearer, the present utility model will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Example 1

[0030] See Figure 1 As shown, this embodiment provides a novel microbubble-enhanced gas supply bioreactor, including a fermenter 1, a liquid transfer pump 4, a gas supply system 10, a membrane module 11, and a circulation pipeline. The fermenter 1 has a liquid outlet 2 and a gas-liquid inlet 12. The liquid transfer pump 4 and the membrane module 11 are connected in series through the circulation pipeline, with both ends of the circulation pipeline connected to the liquid outlet 2 and the gas-liquid inlet 12 of the fermenter 1, respectively. The liquid transfer pump 4 is used to transport the fermentation broth in the fermenter 1 to the membrane module 11. The gas supply system 10 is connected to the membrane module 11 through the gas supply pipeline. The gas supply system 10 supplies gas to the membrane module 11. The high-flow-rate fermentation broth in the membrane module 11 carries the gas to form microbubbles. The microbubbles flow back to the fermenter 1 through the circulation pipeline with the fermentation broth, realizing efficient gas mass transfer.

[0031] See Figure 1 As shown, the fermenter 1 is equipped with a stirring device 13, which is used to stir the fermentation liquid inside the fermenter 1. The gas-liquid inlet 12 is located at the bottom of the fermenter 1, and the top of the fermenter 1 is equipped with a gas flow meter 3.

[0032] Furthermore, a control valve I5, a pressure gauge I6, and a flow meter 7 are sequentially installed on the circulation pipeline between the liquid transfer pump 4 and the membrane module 11. The control valve I5 is used to control the flow rate of the fermentation broth drawn from the fermenter 1 to the membrane module 11, the pressure gauge I6 is used to monitor the pressure of the fermentation broth delivered to the membrane module 11, and the flow meter 7 is used to monitor the flow rate of the fermentation broth delivered to the membrane module 11.

[0033] Furthermore, the gas supply pipeline is equipped with a pressure gauge II8 and a control valve II9, wherein the control valve II9 is ​​used to control the gas flow rate in the gas supply pipeline, and the pressure gauge II8 is used to control the gas pressure in the gas supply pipeline.

[0034] See Figure 2 As shown, the membrane module 11 includes an outer tube 114 and a plurality of hollow fiber membrane filaments 113 disposed within the outer tube 114. One end of the outer tube 114 has an inlet 111 communicating with the inner cavity of the outer tube 114 and is connected to a circulation pipeline. One end of the outer tube 114 has an inlet 112 communicating with the inner cavity of each hollow fiber membrane filament 113 and is connected to a gas supply pipeline. The hollow fiber membrane filaments 113 have a liquid-proof and gas-permeable function. The gas permeating from the hollow fiber membrane filaments 113 is entrained by the high-velocity liquid in the outer tube 114 to form microbubbles. The other end of the outer tube 114 has a gas-liquid outlet 115, which is connected to the gas-liquid inlet 12 of the fermenter 1 through a circulation pipeline.

[0035] In this embodiment, the hollow fiber membrane 113 is made of Janus membrane, with a hydrophobic inner layer and a hydrophilic outer layer. The gap between the hollow fiber membrane 113 and the outer tube 114 at the second air inlet 112 is sealed with adhesive, and the other end of the hollow fiber membrane 113 is also sealed with adhesive. Therefore, gas entering the hollow fiber membrane 113 from the second air inlet 112 can only be discharged outside the membrane through the micropores of the membrane wall. Since the volumetric surface area of ​​the membrane increases as the inner diameter decreases, from the perspective of cost saving and efficiency improvement, the inner diameter of the hollow fiber membrane 113 should be as small as possible within a certain range. In one embodiment, a Janus membrane with an inner diameter of 0.2 mm, 0.5 mm, 1 mm, 2 mm, or 3 mm is selected, but it is not limited to these.

[0036] The aspect ratio of the hollow fiber membrane filaments 113 is typically within a suitable range, preferably 100:1 to 1000:1. In one embodiment, Janus membranes with aspect ratios of 50:1, 100:1, 500:1, 750:1, and 1000:1 are selected, but are not limited thereto.

[0037] The outer tube 114 is made of a material with certain pressure resistance and corrosion resistance, such as stainless steel or plastic. In one embodiment, a PE plastic pipe is selected as the outer tube 114, and in another embodiment, a 304 stainless steel pipe is selected as the outer tube 114, but it is not limited to these.

[0038] Liquid transfer pump 4 is a conventional infusion pump. Considering shear force, self-priming capability and cost-effectiveness, in one implementation, a diaphragm pump, rotary pump, screw pump or centrifugal pump may be selected as the liquid transfer pump, but it is not limited to this.

[0039] This embodiment provides a novel microbubble-enhanced gas supply bioreactor, the working principle of which is as follows:

[0040] The liquid transfer pump 4 draws fermentation broth from the fermenter 1. The fermentation broth enters the outer tube 114 of the membrane module 11 through inlet 111 and then flows back into the fermenter 1. Gas enters each hollow fiber membrane filament 113 through inlet 112. The gas permeates from the membrane wall of the hollow fiber membrane filament 113 to the outside and is entrained by the high-velocity liquid outside the membrane to form microbubbles that enter the fermenter 1. This cycle is repeated, thereby reducing the bubble size, enhancing its stability in the solution, and prolonging its existence time. Under lower aeration rates and stirring speeds, this improves the gas mass transfer efficiency of the fermentation system, increases the yield of the target substance, and significantly reduces fermentation costs.

[0041] During fermentation, it is necessary to select a membrane module 11 with an appropriate area, gas flow rate, and liquid flow rate according to the volume of the fermentation liquid. The liquid flow rate is adjusted by control valve I5, and the liquid pressure and flow rate are monitored in real time by pressure gauge I6 and flow meter 7. The gas flow rate is adjusted by control valve II9, and the gas pressure is monitored in real time by pressure gauge II8.

[0042] Janus membranes are a special type of membrane material composed of hydrophilic and hydrophobic materials. The different properties on each side give them many unique characteristics. Composed of hydrophilic and hydrophobic materials, Janus membranes combine the advantages of both. As a bubbler membrane material, the hydrophilic side provides a smaller bubble diameter to the liquid, while the hydrophobic side ensures the membrane pores remain unwetted, reducing gas permeation pressure. Therefore, this ensures a stable microbubble fabrication process over a long period.

[0043] The membrane area in membrane module 11 is related to the fermentation broth volume, and the membrane area per liter of fermentation broth should be ≥12 cm². 2 The preferred membrane area is 20-100 cm². 2 A more optimal membrane area is 30-60 cm². 2 .

[0044] In one implementation scheme, a 5L fermenter (containing 2.5-4.0L of fermentation broth) is used, with a surface area of ​​47.8cm². 2 (corresponding to 12-19 cm) 2 / L fermentation broth), 150 cm 2 (corresponding to 38-60 cm) 2 / L fermentation broth), 225 cm 2 (corresponding to 56-90 cm) 2 Fermentation is carried out using a membrane with a surface area of ​​508 cm² (containing 25-40 L of fermentation broth), but is not limited to this; in another embodiment, a 50 L fermenter (containing 25-40 L of fermentation broth) is used, with a membrane having a surface area of ​​508 cm². 2 (corresponding to 13-20 cm) 2 / L fermentation broth), 1230 cm 2 (corresponding to 31-49 cm) 2 / L fermentation broth) and 2458 cm 2 (corresponding to 62-98 cm) 2 Fermentation can be carried out using a membrane containing 1 / L of fermentation broth, but is not limited to this.

[0045] Gas flow rate is related to membrane area; the corresponding gas flow rate per square meter of membrane should be ≥0.15 m³ / s. 3 / h, with a preferred ventilation rate of 0.5-5 m³ / h. 3 / h, with an optimal ventilation rate of 1.2-2.4 m³ / h. 3 / h.

[0046] In one implementation scheme, a 5L fermenter with a surface area of ​​150 cm² is used. 2 The air permeability of the membrane was set at 0.15, 1, 2, 2.5, and 4 m³ per square meter, respectively. 3 / h, but not limited to this; in another embodiment, a 50L fermenter with an area of ​​508 cm² is used. 2 The air permeability of the membrane was set at 1, 1.2, and 4 m³ / m², respectively. 3 / h, but not limited to this.

[0047] Liquid flow rate is related to membrane area; the liquid flow rate per square meter of membrane should be ≥6 m³ / s. 3 / h, with a preferred liquid flow rate of 10-50 m³ / h. 3 / h, with an optimal liquid flow rate of 15-25 m³ / h. 3 / h.

[0048] In one implementation scheme, a 5L fermenter with a surface area of ​​150 cm² is used. 2 The liquid flow rate was controlled at 6, 12, 18, 24, and 30 m³ / m² of membrane. 3 / h, but not limited to this; in another embodiment, a 50L fermenter with an area of ​​508 cm² is used. 2 The liquid flow rate was controlled at 10, 20, and 40 m³ / m² of membrane, respectively. 3 / h, but not limited to this.

[0049] To improve dissolved oxygenation, the number of gas-liquid inlets 12 in fermenter 1 is related to the volume of fermentation liquid. There can be one or more gas-liquid inlets 12, and the volume of fermentation liquid corresponding to each gas-liquid inlet 12 should be ≤500L. Preferably, the volume of fermentation liquid is ≤300L, and more preferably, the volume of fermentation liquid is ≤200L.

[0050] In one embodiment, fermenters with volumes of 5L and 50L are used, with one gas-liquid inlet, but not limited to this; in another embodiment, a fermenter with a volume of 1000L and an area of ​​9500 cm² is used. 2 The membrane can be configured with 2, 3, 5, or 8 gas-liquid inlets, but is not limited to these.

[0051] To improve dissolved oxygenation, membrane modules 11 can be one or more connected in parallel. All membrane modules 11 can be supplied with liquid by one liquid transfer pump 4 or by multiple liquid transfer pumps 4. Preferably, a single membrane module 11 is supplied with liquid by a single liquid transfer pump 4. In one embodiment, one, two, or three liquid transfer pumps 4 are used to supply liquid to one membrane module 11, but this is not a limitation.

[0052] This novel microbubble-enhanced gas supply bioreactor can be used for aerobic fermentation, anaerobic fermentation, and gas-based nutrient fermentation. It can significantly improve the gas mass transfer rate under low aeration and stirring conditions.

[0053] Example 2

[0054] Volumetric oxygen mass transfer coefficient (K) L a) is a key parameter for measuring the rate at which oxygen transfers from the gas phase to the liquid phase. A higher value indicates a faster rate of gas-to-liquid transfer, which is more conducive to maintaining dissolved oxygen concentration during fermentation. Using the reactor provided by this invention, the volumetric oxygen mass transfer coefficient was measured under different conditions, as follows:

[0055] The volumetric oxygen transfer coefficient was determined using the dynamic ventilation method. Nitrogen gas was initially introduced into the culture medium until the dissolved oxygen concentration dropped to below 6% oxygen saturation. The nitrogen flow was then interrupted, and air was introduced into the solution. The increase in dissolved oxygen concentration was monitored over time until >90% oxygen saturation was achieved. Ka was then calculated using the formula... L a-fit:

[0056] (1)

[0057] In the formula, The saturated oxygen concentration is expressed in mg·L⁻¹. -1 ; These are oxygen concentrations at different time points, in mg·L⁻¹ -1 t represents time, in seconds.

[0058] The results are shown in Table 1:

[0059] Table 1 Oxygen mass transfer coefficients under different conditions 1 2.5 150 0.0023 0.09 1 1 0.032 0.119-0.38 2 2.5 150 0.0150 0.18 1 1 0.044 0.091-0.25 3 2.5 150 0.0300 0.27 1 1 0.049 0.085-0.18 4 2.5 150 0.0375 0.36 1 1 0.048 0.092-0.22 5 2.5 150 0.0600 0.45 1 1 0.046 0.092-0.24 6 2.5 29.8 0.0060 0.06 1 1 0.043 0.090-0.27 7 2.5 225 0.0450 0.45 1 1 0.045 0.088-0.26 8 40 508 0.051 2.04 1 1 0.033 0.122-0.30 9 40 508 0.204 0.51 1 1 0.038 0.101-0.29 10 40 508 0.061 1.12 1 1 0.041 0.095-0.27 11 700 9500 1.90 9.50 2 2 0.033 0.120-0.39 12 700 9500 1.90 9.50 3 3 0.036 0.106-0.38 13 700 9500 1.90 9.50 5 5 0.039 0.100-0.32 14 700 9500 1.90 9.50 8 8 0.035 0.109-0.39

[0060] When the liquid volume is constant, K L The α value is related to the membrane area, gas flow rate, liquid flow rate, and the number of gas and liquid inlets. The optimal condition is Experiment 3, where the liquid volume in the system is 2.5 L, and the membrane area is 150 cm². 2 (60 cm per liter of liquid) 2 (membrane), gas flow rate 0.03 m³ 3 / h (2 m per square meter of membrane) 3 / h), liquid flow rate 0.27 m 3 / h (18 m per square meter of membrane) 3 / h), number of gas-liquid inlets: 1, K L a is 0.049 s -1 The measured bubble diameter distribution range was (0.085-0.18 mm).

[0061] When the liquid volume (40 L) and membrane area (508 cm²) in the system are... 2 When the conditions are fixed, the optimal condition is Experiment 10, where the gas flow rate is 0.061 m³ / s. 3 / h (1.2 m per square meter of membrane) 3 / h), liquid flow rate 1.12 m 3 / h (20 m² of membrane per square meter) 3 / h), with 1 gas-liquid inlet. At this time, K L a is 0.041 s -1 The measured bubble diameter distribution range was (0.095-0.27 mm).

[0062] When the liquid volume of the stationary system is 700 L and the membrane area is 9500 cm², 2 (13.6 cm per liter of liquid) 2 / L), gas flow rate 1.9 m 3 / h (2 m per square meter of membrane) 3 / h), liquid flow rate 9.50 m 3 / h (10 m² of membrane per square meter) 3 When the flow rate is / h), the optimal condition is Experiment 13, where there are 5 gas-liquid inlets (each inlet supplies an average of 140 L of liquid gas). At this time, K L a is 0.039 s -1 The measured bubble diameter distribution range was (0.100-0.32 mm).

[0063] Comparative Example 1

[0064] Using the reactor provided by this utility model, K is carried out under conditions other than those described in this application. L The results of the determination are shown in Table 2:

[0065] Table 2 Oxygen mass transfer coefficients under different conditions 1 2.5 24.2 0.00363 0.0363 1 1 0.014 0.40-0.95 2 2.5 24.2 0.00363 0.0121 1 1 0.010 0.45-0.98 3 2.5 24.2 0.00029 0.0363 1 1 0.004 0.49-1.02 4 2.5 24.2 0.00029 0.0121 1 1 0.002 0.82-1.28 5 2.5 150 0.0375 0.360 1 1 0.048 0.09-0.22 6 2.5 150 0.0375 0.075 1 1 0.027 0.16-0.56 7 2.5 150 0.0018 0.360 1 1 0.018 0.23-0.69 8 2.5 150 0.0018 0.075 1 1 0.015 0.42-0.89

[0066] Compared to the better conditions in Example 2 (Experiment 5), K L The a-values ​​all decreased significantly (0.008-0.027 s). -1 The bubble size also shows a trend of increasing.

[0067] Comparative Example 2

[0068] Using a traditional fermentation system, K L The results of the determination are shown in Table 3:

[0069] Table 3 Oxygen mass transfer coefficients under different conditions in traditional fermenters 1 2.5 0.30 400 0.011 0.53-2.94 2 2.5 0.30 500 0.021 0.24-2.02 3 2.5 0.30 600 0.032 0.21-1.56 4 2.5 0.30 700 0.043 0.20-0.76 5 2.5 0.30 800 0.052 0.20-0.60 6 2.5 0.45 600 0.032 0.25-1.64 7 2.5 0.15 600 0.017 0.20-1.32

[0070] When the ventilation rate is maintained at 2 vvm, K L The a value increases with increasing stirring speed, reaching 0.052s at 800 rpm. -1 When the fixed speed is 600 rpm, the ventilation rate of 2VVM and 3VVM is optimal, at 0.032 s. -1 By comparing the two systems, for the same liquid volume, a surface area of ​​29.8 cm² was used. 2 The membrane exhibits mass transfer performance comparable to that of a traditional fermentation system at 700 rpm and 2 vvm, but with a 10-fold reduction in stirring speed and a more than 15-fold reduction in aeration rate. Therefore, the bioreactor provided by this invention can significantly reduce energy consumption and improve oxygen utilization.

[0071] Example 3

[0072] Applied to fermentation of Pichia pastoris FXY-17H strain

[0073] The 5L fermentation system constructed in this study was used to ferment the metabolically engineered Pichia pastoris 3-hydroxypropionic acid producing strain FXY-17H (ACS Sustainable Chemistry & Engineering 2023 11 (16), 6445-6453), specifically including:

[0074] (1) Fermentation strain selected: Pichia pastoris metabolic engineering modified strain FXY-17H.

[0075] (2) Grade 1 seed culture medium (L) -1 ): 20 g glucose, 20 g peptone, 10 g yeast powder;

[0076] (3) Level 2 seed culture medium (L) -1 ): 20 g glucose, 0.5 g magnesium sulfate, 14.4 g potassium dihydrogen phosphate, 2.5 g ammonium sulfate, 2 ml trace element solution (1000*), 1 ml vitamin solution (1000*).

[0077] (4) Fermentation medium (L) -1 Ingredients: 10 g methanol, 0.5 g magnesium sulfate, 14.4 g potassium dihydrogen phosphate, 2.5 g ammonium sulfate, 2 mL trace element solution (1000*), 1 mL vitamin solution (1000*).

[0078] (5) Trace element solution: FeSO4•7H2O (3.0 g / L), ZnSO4•7H2O (4.5 g / L), CaCl2•2H2O (4.5 g / L), MnCl2•4H2O (1 g / L), CoCl2•6H2O (0.3 g / L), CuSO4•5H2O (0.3 g / L), Na2MoO4•2H2O (0.4 g / L), H3BO3 (0.1 g / L), KI (0.1 g / L), Na2EDTA•2H2O (19 g / L);

[0079] (6) Vitamin solution The vitamin mixture contains: D-pantothenic acid hemicalcium salt (1.0 g / L), thiamine hydrochloride (1.0 g / L), pyridoxine hydrochloride (1.0 g / L), nicotinic acid (1.0 g / L), 4-aminobenzoic acid (0.2 g / L), biotin (0.05 g / L), m-inositol (25 g / L), which are added to the fermentation medium after filtration and sterilization.

[0080] (7) Sterilization of membrane components: Soak the membrane system and pipelines in 75% ethanol solution for 24 hours, and then rinse the system twice with sterile water, 3L each time.

[0081] (8) Seed preparation: Pick a single colony from the solid plate culture medium and transfer it to an Erlenmeyer flask containing 20 mL of YPD liquid culture medium. Set the shaker temperature to 30 °C and the rotation speed to 260 rpm for 24 h. Then, transfer it to an Erlenmeyer flask containing 50 mL of grade 2 seed culture medium with an inoculation OD of 0.5. After culturing for another 24 h, the OD reaches 9.0. Then, inoculate it into the bioreactor provided by this utility model with an inoculation OD of 1.0.

[0082] (9) Fermentation process: Batch feeding fermentation was adopted. A membrane with a surface area of ​​150 cm² was used. 2 The membrane module maintains an air permeability of 0.03-0.06 m³ / s. 3 The liquid flow rate is maintained at 0.3-0.4 m / h. 3 / h. Dissolved oxygen was maintained above 10%, pH 5.6, and temperature 30 degrees Celsius. When the methanol concentration fell below 1 g / L, methanol was added to maintain the methanol concentration at 1-5 g / L. At the same time, fed culture medium (10* fermentation medium, methanol-free) was added.

[0083] After 234 hours of fermentation, the final fermentation liquid volume was 4.0 L, the total amount of 3-hydroxypropionic acid was 349 g, and the yield reached 0.42 g / g methanol.

[0084] Comparative Example 3

[0085] Fermentation of strain FXY-17H was carried out using a conventional fermenter. The aeration rate was 0.3-0.48 m³ / s. 3 The stirring speed was 300-700 rpm, and the other conditions were the same as in Example 3. The results showed that after 234 hours of fermentation, 2.6 L of fermentation broth was finally obtained, with a total amount of 260 g of 3-hydroxypropionic acid and a yield of 0.24 g / g methanol.

[0086] A comparison of the results from the two fermentation systems revealed that the new fermentation system provided by this invention significantly improved the product yield and efficiency of Pichia pastoris strain FXY-17H.

[0087] Example 4

[0088] Fermentation of Clostridium carboxidivorans P7

[0089] The 5L fermentation system provided by this invention is used to ferment Clostridium carboxidivorans P7 strain, specifically including:

[0090] (1) Fermentation strain selected: Clostridium carboxidivorans P7.

[0091] (2) C. carboxidivorans P7 activation medium formulation (L -1 The following ingredients were added: 10 g peptone, 10 g fish peptone, 5 g yeast extract, 2 g glucose, 5 g NaCl, 1 g L-Arginine, 1 g 0.1% Sodium pyruvate, 1 mL resazurin solution, 0.3 g L-cysteine, 1 mL stock solution c, 10 mL stock solution a, and 200 µL stock solution b. Stock solution a consisted of 0.5 g heme + 10 mL 1N NaOH + 990 mL H2O; stock solution b consisted of 0.05 mL Vk1 solution + 20 mL 95% ethanol; and stock solution c consisted of 0.1 g NaHCO3 + 100 mL H2O.

[0092] (3) Fermentation medium formulation (L) -1The following ingredients were added: NaCl 2.4 g, NH4Cl 3 g, KCl 0.3 g, KH2PO4 0.3 g, MgSO4 0.6 g, CaCl2 0.12 g, L-cystein·HCl 0.2 g, L-cystein 0.2 g, yeast extract 0.5 g, morpholine ethanesulfonic acid 5 g, trace elements 2 mL, vitamins 1 mL, and 0.1% resazurite 1 mL. The trace element formula (L...) -1 Ingredients: 10 g triacetic acid, 5 g MnSO4, 4 g FeSO4·6H2O, 1 g CoCl2·6H2O, 1 g ZnSO4·7H2O, 0.1 g CuCl2·2H2O, 0.1 g NiCl2·6H2O, 0.1 g Na2WO4·2H2O, 0.1 g Na2MoO4·2H2O, 0.1 g Na2SeO4. Vitamin solvent formula (L... -1 ): VB6 0.1 g, nicotinic acid 0.05 g, thiamine (VB1) 0.05 g, VB2 0.05 g, folic acid 0.02 g. After filtration and sterilization, add to the fermentation medium.

[0093] (4) Preservation of the strain: Add 4 mL of 50% glycerol (glycerol + water) to a glycerol tube, seal with a rubber stopper, remove oxygen from the glycerol tube using nitrogen, and sterilize at 121 °C for 20 min; prepare a Na2S and L-cysteine ​​solution (concentrated solution), and filter to sterilize; use a syringe to add 3 mL of logarithmic phase bacterial culture and 0.5 mL of Na2S and L-cysteine ​​solution to the glycerol tube, so that the final concentration of Na2S and L-cysteine ​​in the glycerol tube is 0.1 g / L. After mixing evenly, freeze the strain at -80 °C.

[0094] (5) C. carboxidivorans P7 activation culture: Take out the bacterial strain stored at -80℃ and place it at room temperature for about 15 min. After it is completely thawed, inoculate it into activation medium and incubate at 37℃. After about 36 h, the bacteria in the first batch of activation medium enter the logarithmic phase, at which time the OD600 is about 0.8. Take 5 mL of bacterial solution and inoculate it into a new activation medium. Perform the second batch of activation culture.

[0095] (6) Fermentation of C. carboxidivorans P7: using a membrane with a surface area of ​​29.8 cm². 2The membrane module was prepared as follows: First, 300 ml of the second activated bacterial culture (OD approximately 0.8) was inoculated into the fermenter provided by this invention (containing 2.5 L of fermentation medium). The temperature was maintained at 37 ℃, the rotation speed at 300 rpm, and the pH at 5.5. Syngas (CO 50% / CO2 35% / H2 15%) was used as the carbon source. The gas flow rate was set to 0.05-0.1 L / min, the pump flow rate was 1-1.2 L / min, and the OD600 was measured every 24 h.

[0096] Results: After 144 hours of fermentation, the OD600 reached its maximum of 3.7. The utilization rates of CO and H2 reached 85.5% and 93.7%, respectively.

[0097] Comparative Example 4

[0098] Fermentation of C. carboxidivorans P7 strain was carried out using a conventional fermenter. The aeration rate was 0.1-0.2 L / min, the stirring speed was 500 rpm, and other conditions were the same as in Example 4. Results showed that after 144 h of fermentation, the OD600 reached a maximum of 3.1. CO and H2 utilization rates were 42.6% and 53.9%, respectively.

[0099] A comparison of the two fermentation systems revealed that the fermentation system provided by this invention has a significantly better fermentation effect on Clostridium carboxidivorans P7 strain than traditional fermenters, with a substantial increase in cell growth per unit time and syngas utilization rate.

[0100] Example 5

[0101] Thermophilic streptococcal anaerobic fermentation

[0102] The 5L fermentation system provided by this invention is used to ferment Streptococcus thermophilus, specifically including:

[0103] (1) Fermentation strain: Streptococcus thermophilus.

[0104] (2) Seed preparation: The slant culture was inoculated into 100 mL of liquid M17 + 1% lactose medium and cultured at 42°C and 200 rpm for 10 h. OD 600 Up to 2.

[0105] (3) Fermentation tank culture: 2.5L culture medium (formula: sucrose 25 g / L, yeast extract 15 g / L, casein hydrolysate 10 g / L, 0.1% Tween-80, 0.2% MgSO4·7H2O, dipotassium hydrogen phosphate 3 g / L, triammonium citrate 2 g / L, MnSO4 0.05 g / L, FeSO4 0.01 g / L, adjusted to 6.7 with NaOH before sterilization) was sterilized. After sterilization, high-purity nitrogen was introduced to replace oxygen, so that DO < 0.3ppm. The inoculum size was 3%, the culture temperature was 42°C, the pH was controlled at 5.8-6.3 (10% ammonia water was added automatically), the stirring speed was 0 rpm, and the high-purity nitrogen flow rate was 0.02vvm. When the residual sugar was < 8g / L, 40% lactose solution was added. After 8 hours of fermentation, the viable cell count reached 5×10 9 CFU / mL.

[0106] Comparative Example 5

[0107] A conventional 5L fermentation system was used to ferment *Streptococcus thermophilus*. The high-purity nitrogen gas flow rate in the fermenter was 0.05 vvm, the stirring speed was 300 rpm, and other conditions were the same as in Example 5. The results showed that after 8 hours of fermentation, the viable cell count reached 4 × 10⁻⁶. 9 CFU / mL.

[0108] A comparison of the two fermentation systems revealed that the fermentation system provided by this invention achieves the same effect as a traditional fermenter by using lower aeration and stirring during anaerobic fermentation.

[0109] The above description is merely an embodiment of this utility model and is not intended to limit the scope of protection of this utility model. Any modifications, equivalent substitutions, improvements, extensions, etc., made within the spirit and principles of this utility model are included within the scope of protection of this utility model.

Claims

1. A novel microbubble-enhanced gas supply bioreactor, characterized in that, The system includes a fermenter (1), a liquid transfer pump (4), a gas supply system (10), a membrane module (11), and a circulation pipeline. The fermenter (1) has a liquid outlet (2) and a gas-liquid inlet (12). The liquid transfer pump (4) and the membrane module (11) are connected in series through the circulation pipeline. The two ends of the circulation pipeline are connected to the liquid outlet (2) and the gas-liquid inlet (12) of the fermenter (1), respectively. The liquid transfer pump (4) is used to transport the fermentation liquid in the fermenter (1) to the membrane module (11). The gas supply system (10) is connected to the membrane module (11) through the gas supply pipeline. The gas supply system (10) supplies gas to the membrane module (11). The high-flow-rate fermentation liquid in the membrane module (11) carries the gas to form microbubbles. The microbubbles flow back to the fermenter (1) with the fermentation liquid through the circulation pipeline, thereby achieving efficient gas mass transfer.

2. The novel microbubble-enhanced gas supply bioreactor according to claim 1, characterized in that, The membrane module (11) includes an outer tube (114) and a plurality of hollow fiber membrane filaments (113) disposed in the outer tube (114). One end of the outer tube (114) is provided with an inlet (111) communicating with the inner cavity of the outer tube (114). The inlet (111) is connected to the circulation pipeline. One end face of the outer tube (114) is provided with an inlet (112) communicating with the inner cavity of each hollow fiber membrane filament (113). The inlet (112) is connected to the gas supply pipeline. The hollow fiber membrane filaments (113) have a liquid-proof and gas-permeable function. The gas permeating from the hollow fiber membrane filaments (113) is entrained by the high-velocity liquid in the outer tube (114) to form microbubbles. The other end of the outer tube (114) is provided with a gas-liquid outlet (115). The gas-liquid outlet (115) is connected to the gas-liquid inlet (12) of the fermenter (1) through the circulation pipeline.

3. The novel microbubble-enhanced gas supply bioreactor according to claim 2, characterized in that, The hollow fiber membrane filament (113) is made of Janus membrane, with a hydrophobic layer on the inner side and a hydrophilic layer on the outer side.

4. The novel microbubble-enhanced gas supply bioreactor according to claim 2, characterized in that, The aspect ratio of the hollow fiber membrane filament (113) is 100:1 to 1000:

1.

5. The novel microbubble-enhanced gas supply bioreactor according to claim 2, characterized in that, The outer tube (114) is made of stainless steel or plastic.

6. The novel microbubble-enhanced gas supply bioreactor according to claim 1, characterized in that, The circulation pipeline between the liquid transfer pump (4) and the membrane module (11) is provided with a control valve I (5), a pressure gauge I (6) and a flow meter (7) in sequence. The control valve I (5) is used to control the flow rate of the fermentation liquid drawn from the fermenter (1) to the membrane module (11), the pressure gauge I (6) is used to monitor the pressure of the fermentation liquid delivered to the membrane module (11), and the flow meter (7) is used to monitor the flow rate of the fermentation liquid delivered to the membrane module (11).

7. The novel microbubble-enhanced gas supply bioreactor according to claim 1, characterized in that, The gas supply pipeline is equipped with a pressure gauge II (8) and a control valve II (9), wherein the control valve II (9) is used to control the gas flow rate in the gas supply pipeline, and the pressure gauge II (8) is used to control the gas pressure in the gas supply pipeline.

8. The novel microbubble-enhanced gas supply bioreactor according to claim 1, characterized in that, The fermenter (1) is equipped with a gas flow meter (3) at the top.

9. The novel microbubble-enhanced gas supply bioreactor according to claim 1, characterized in that, The fermentation tank (1) is equipped with a stirring device (13), which is used to stir the fermentation liquid in the fermentation tank (1).

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