An oxygen micro-nano bubble enhanced aerobic fermentation bioreactor and its application

Abstract

The present invention discloses an oxygen micro-nano bubble enhanced aerobic fermentation bioreactor and its application, comprising an oxygen generator, a micro-nano bubble generator, a fermentation tank, a gas-liquid separation tank and a fermentation product collection tank connected in sequence. Oxygen and seed liquid form oxygen micro-nano bubbles through the micro-nano bubble generator and enter the fermentation tank, and the stirring paddle and the oxygen micro-nano bubbles are synergistically oxygenated to improve the oxygen mass transfer efficiency of the fermentation system. In addition, the circulation of oxygen micro-nano bubbles can reduce the bubble size, enhance its stability in the solution and prolong the existence time while maintaining the dissolved oxygen content of the fermentation liquid, thereby facilitating the growth and proliferation of immobilized cells, improving the self-renewal ability of microorganisms in the fermentation tank, and further enhancing the continuous fermentation degree of immobilized cells.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and in particular relates to an oxygen micro-nano bubble enhanced aerobic fermentation bioreactor. Background Art

[0002] In the process of aerobic submerged fermentation, the supply of oxygen is often one of the important limiting factors for the success of fermentation. Aerobic microorganisms can only grow in the presence of molecular oxygen, but since the solubility of oxygen in water is very small, the solubility in the fermentation liquid is also the same. Therefore, continuous ventilation and stirring are required to meet the oxygen demand of aerobic microorganisms.

[0003] Micro-nano bubbles are micro-nano bubbles. Compared with ordinary bubbles, they have a very small particle size, so they show some special advantages, such as long existence time, high mass transfer efficiency, slow rise speed, and high Zeta potential formed by surface charge. These characteristics have made micro-nano bubbles widely used in the fields of water pollutant degradation, aquaculture, agricultural soil improvement, etc. in recent years. Lu Liang et al. (Shanghai Agricultural Science and Technology, 2017, 06: 52+73) used bubble water generated by a nano-micro gas-liquid dispersed phase interface mixing generator to irrigate rice, which can improve the vitality of rice roots, increase the number of effective panicles, the number of grains per panicle, and the thousand-grain weight, thereby increasing yields, and can reduce the use of chemical fertilizers, which is helpful to control agricultural non-point source pollution and protect the ecological environment. Yuan Xiaoling et al. (Industrial Water Treatment, 2020, 40(07): 47-50) used micro-nano bubbles combined with ozonation technology to treat sludge. Compared with ordinary ozone bubbles, ozone micro-nano bubbles have a higher sludge reduction effect, and the sludge reduction rate can reach about 15%. CN114180670 A proposes an ozone micro-nano bubble wastewater treatment system and a wastewater treatment method, which reduces the size of micro-nano bubbles through primary and secondary gas dissolution processes, enhances the stability of micro-nano bubbles in water, and thus improves the wastewater treatment effect. Summary of the invention

[0004] Purpose of the invention: Existing literature reports have demonstrated the potential application value of micro-nano bubbles, but currently there is no application of micro-nano bubbles to aerobic microbial fermentation systems. Therefore, in view of the problems existing in the immobilized fermentation process of aerobic microorganisms, such as insufficient oxygen supply, low mass transfer efficiency, and increased stirring speed causing shear force to damage cells, the present invention proposes an oxygen micro-nano bubble enhanced aerobic fermentation bioreactor for improving the continuous fermentation performance of immobilized cells of aerobic microorganisms.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] An oxygen micro-nano bubble enhanced aerobic fermentation bioreactor, comprising an oxygen generator, a seed tank, a gas-liquid mixing pump, a micro-nano bubble generator, a fermentation tank, a batching tank, a gas-liquid separation tank and a fermentation product collection tank;

[0007] The gas-liquid mixing pump, micro-nano bubble generator, fermentation tank, gas-liquid separation tank and fermentation product collection tank are connected in sequence through pipelines; the batching tank is connected to the fermentation tank;

[0008] The oxygen generator and the seed tank are respectively connected to the first inlet and the second inlet of the gas-liquid mixing pump, and oxygen and seed liquid are sent to the gas-liquid mixing pump for mixing, and then sent to the micro-nano bubble generator for shearing to form oxygen micro-nano bubbles, and then sent to the fermentation tank for fermentation together with the raw materials sent from the batching tank. The fermentation product is separated by the gas-liquid separation tank and sent to the fermentation product collection tank.

[0009] Specifically, the micro-nano bubble generator includes a nano bubble pump, a safety valve, a pressure tank and a bubble atomizer; the feed port of the nano bubble pump is connected to the discharge port of the gas-liquid mixing pump; the nano bubble pump, the pressure tank and the bubble atomizer are connected in sequence; the discharge port of the bubble atomizer is connected to the feed port of the fermentation tank; the nano bubble pump and the pressure tank are also connected through a gas circulation pipe to return excess oxygen in the pressure tank to the nano bubble pump, and the safety valve is installed on the gas circulation pipe.

[0010] Furthermore, the discharge port of the bubble atomizer is connected to the feed port at the lower side of the fermentation tank, and a water pump is provided on the connecting pipeline; the gas outlet at the top of the fermentation tank is connected to the pressure tank through a gas circulation pipe to return excess oxygen in the fermentation tank to the pressure tank, and the gas circulation pipe is provided with an exhaust pump.

[0011] Specifically, the fermenter is a mechanical stirring fermenter, the feed port at the lower side thereof is connected to the micro-nano bubble generator, the feed port at the top is connected to the batching tank, and the discharge port at the bottom is connected to the gas-liquid separation tank;

[0012] A stirring paddle is provided in the fermentation tank, which is connected to a motor located above the top of the fermentation tank and drives the stirring paddle through the motor to stir the materials in the fermentation tank;

[0013] A cylindrical, hollow wire mesh holder is provided in the center of the reaction chamber in the fermentation tank for fixing the cotton fibers; a stirring paddle runs through the hollow part in the middle of the wire mesh holder;

[0014] The fermentation tank is also provided with a heat exchanger, through which the temperature of the fermentation liquid in the fermentation tank is adjusted.

[0015] Furthermore, the heat exchanger selects a built-in multi-group coil heat exchanger, which is fixed on the inner wall of the reaction chamber of the mechanically stirred fermentation tank. The heat exchanger is connected to the water tank outside the fermentation tank through a water pipe. The water pipe is provided with a water pump. The pre-cooled sterile water in the water tank is sent into the coil in the fermentation tank through the water pump to exchange heat with the fermentation liquid in the tank.

[0016] Furthermore, a first gas phase balance pipe is provided on the pipeline connecting the top feed port of the fermentation tank and the batching tank; a second gas phase balance pipe is provided on the pipeline connecting the bottom discharge port of the fermentation tank and the gas-liquid separation tank.

[0017] Furthermore, a gas filter is provided on the pipeline connecting the oxygen generator and the gas-liquid mixing pump to prevent the seed liquid from being contaminated by bacteria in the oxygen.

[0018] Specifically, the bottom feed port of the gas-liquid separation tank is connected to the feed port of the fermentation tank, the middle separation port is connected to the fermentation product collection tank, and the top gas outlet is connected to the water separator.

[0019] Specifically, the water separator has a water outlet, an air inlet, an air outlet, a circulating gas outlet and a circulating gas inlet, wherein the circulating gas inlet is connected to the top air outlet of the gas-liquid separation tank, the circulating gas outlet is connected to the air inlet in the water separator body, the water outlet is connected to the fermentation product collection tank, and the air outlet is connected to the atmosphere; a valve is provided on the water separator for controlling the circulation of the gas and the circulation of the fermentation liquid.

[0020] Furthermore, the present invention also claims to protect the use of the above-mentioned oxygen micro-nano bubble enhanced aerobic fermentation bioreactor in the fermentation production of L-lysine by immobilized Corynebacterium glutamicum.

[0021] Furthermore, the specific steps of the above reactor for immobilizing Corynebacterium glutamicum to ferment and produce L-lysine include:

[0022] (1) First, the materials in the seed tank, the batching tank, and the immobilized carrier in the fermentation tank are respectively sterilized by steam and then used for later use;

[0023] (2) Then, the filtered and sterilized oxygen and the activated seed liquid are simultaneously introduced into the micro-nano bubble generator, and the oxygen flow rate is controlled at 200-250 g / h to form oxygen micro-nano bubbles containing cells, which are further introduced into the fermentation tank at a flow rate of 6-8 L / min for immobilized cell fermentation; wherein the average size of the oxygen micro-nano bubbles is less than 220 nm and the existence time is more than 630 s; the stirring rate in the fermentation tank is controlled at less than 180 rpm;

[0024] (3) After the fermentation is completed, the fermentation liquid is circulated and separated using a gas-liquid separation tank and a water separator, and the product finally flows into a fermentation product collection tank.

[0025] Compared with the existing aerobic fermentation bioreactor, the formation of oxygen micro-nano bubbles in the present invention can reduce the stirring speed by 10%, increase the L-lysine production by more than 20%, and increase the L-lysine yield by more than 68%. Further recycling will extend the existence time by more than 6%.

[0026] Beneficial effects:

[0027] (1) The large bubbles formed by the traditional oxygenation method show a tendency to gradually rise and grow larger, then burst near the liquid surface and produce a large amount of foam in the fermentation liquid, which in turn leads to limited oxygen mass transfer, slow formation of fermentation products, and a significant extension of the fermentation cycle. At the same time, the foam rises to the top of the fermentation tank and seeps out from the shaft seal, which increases the risk of bacterial contamination. In contrast, micro-nano bubbles can persist in the fermentation liquid for a long time without bursting until they disappear, which not only improves the mass transfer efficiency, but also reduces the risk of bacterial contamination during the fermentation process, and avoids the effect of adding defoaming agents on the activity and metabolism of the strain.

[0028] (2) The coupling of the micro-nano bubble generator and the immobilized cell fermentation system can achieve the effect of efficient oxygen supply without increasing the stirring speed of the fermentation tank, thus avoiding damage to the cells caused by water shear force and mechanical force due to excessive stirring speed.

[0029] (3) The recycling and good physiological activity of oxygen micro-nano bubbles can accelerate cell metabolism and improve cell growth and proliferation. On the other hand, it can also help to efficiently enrich nutrients on the surface of immobilized cells in the fermentation tank, thereby improving the continuous fermentation performance of immobilized cells and promoting product conversion.

[0030] (4) During the primary and secondary gas dissolution process of the seed liquid, oxygen micron bubbles carry microbial cells into the nanobubble pump, which improves the initial cell activity of the seed liquid to a certain extent. At the same time, appropriate pressure can enhance the permeability of the cell membrane, improve the mass transfer performance, and enhance the cell metabolic flux in the direction of the target product. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0032] Figure 1 It is a schematic diagram of the overall structure of the oxygen micro-nano bubble enhanced aerobic fermentation bioreactor of the present invention.

[0033] Wherein, each reference numeral represents:

[0034] 1. Oxygen generator; 2. Seed tank; 3. Gas filter; 4. Gas-liquid mixing pump; 5. Micro-nano bubble generator; 6. Nano bubble pump; 7. Safety valve; 8. Pressure tank; 9. Bubble atomizer; 10. Water pump; 11. Air pump; 12. Motor; 13. Stirring paddle; 14. Heat exchanger; 15. First gas phase balance pipe; 16. Mixing tank; 17. Fermentation tank; 18. Water pump; 19. Water tank; 20. Second gas phase balance pipe; 21. Gas-liquid separation tank; 22. Fermentation product collection tank; 23. Valve; 24. Water distributor. DETAILED DESCRIPTION

[0035] The present invention can be better understood with reference to the following examples.

[0036] Embodiment 1:

[0037] like Figure 1 As shown, the oxygen micro-nano bubble enhanced aerobic fermentation bioreactor of the present invention includes an oxygen generator 1, a seed tank 2, a gas-liquid mixing pump 4, a micro-nano bubble generator 5, a fermentation tank 17, a batching tank 16, a gas-liquid separation tank 21 and a fermentation product collection tank 22.

[0038] Among them, the gas-liquid mixing pump 4, the micro-nano bubble generator 5, the fermentation tank 17, the gas-liquid separation tank 21 and the fermentation product collection tank 22 are connected in sequence through pipelines; the batching tank 16 is connected to the fermentation tank 17.

[0039] The oxygen generator 1 and the seed tank 2 are respectively connected to the first inlet and the second inlet of the gas-liquid mixing pump 4. The oxygen and the seed liquid are sent into the gas-liquid mixing pump 4 for mixing, and then sent to the micro-nano bubble generator 5 for shearing to form oxygen micro-nano bubbles, and then sent to the fermentation tank 17 for fermentation together with the raw materials sent from the batching tank 16. The fermentation product is separated by the gas-liquid separation tank 21 and sent to the fermentation product collection tank 22.

[0040] The micro-nano bubble generator 5 includes a nano bubble pump 6, a safety valve 7, a pressure tank 8 and a bubble atomizer 9; the feed port of the nano bubble pump 6 is connected to the discharge port of the gas-liquid mixing pump 4; the nano bubble pump 6, the pressure tank 8 and the bubble atomizer 9 are connected in sequence; the discharge port of the bubble atomizer 9 is connected to the feed port of the fermentation tank 17; the nano bubble pump 6 and the pressure tank 8 are also connected through a gas circulation pipe to return excess oxygen in the pressure tank 8 to the nano bubble pump 6, and the safety valve 7 is installed on the gas circulation pipe.

[0041] The discharge port of the bubble atomizer 9 is connected to the feed port at the lower side of the fermentation tank 17, and a water pump 10 is provided on the connecting pipeline; the gas outlet at the top of the fermentation tank 17 is connected to the pressure tank 8 through a gas circulation pipe to return the excess oxygen in the fermentation tank 17 to the pressure tank 8, and the gas circulation pipe is provided with an air pump 11.

[0042] The fermentation tank 17 is a mechanical stirring fermentation tank, the feed port at the lower side thereof is connected to the micro-nano bubble generator 5 , the feed port at the top is connected to the batching tank 16 , and the discharge port at the bottom is connected to the gas-liquid separation tank 21 .

[0043] A stirring paddle 13 is provided in the fermenter 17, which is connected to a motor 12 located above the top of the fermenter 17, and the stirring paddle 13 is driven by the motor 12 to stir the material in the fermenter 17. The stirring device can be a propeller stirrer, a vortex stirrer, a paddle stirrer, a shaft stirrer and a ribbon stirrer, and the stirring speed is controlled at 150-240 rpm to prevent the shear force formed by too high a stirring speed from causing irreversible damage to the cells, and too low a stirring speed will result in insufficient dissolved oxygen during the fermentation process.

[0044] A cylindrical, hollow wire mesh holder is provided in the center of the reaction chamber in the fermentation tank 17 for fixing the cotton fibers; a stirring paddle 13 passes through the hollow part in the middle of the wire mesh holder. The size of the cotton fibers is 60cm*120cm~80cm*160cm, and the addition amount is 30~50g / L.

[0045] The fermentation tank 17 is also provided with a heat exchanger 14, through which the temperature of the fermentation liquid in the fermentation tank 17 is adjusted. The heat exchanger can be a jacketed heat exchanger, a U-shaped tube plate heat exchanger, a floating head heat exchanger, a plate heat exchanger, etc. The refrigerant used in the heat exchanger is sterile water, and the water temperature is controlled at 4-10°C.

[0046] The heat exchanger 14 uses a built-in multi-group coil heat exchanger, which is fixed on the inner wall of the reaction chamber of the mechanical stirring fermentation tank. The heat exchanger 14 is connected to the water tank 19 outside the fermentation tank through a water pipe. A water pump 18 is provided on the water pipe. The pre-cooled sterile water in the water tank 19 is sent into the coil in the fermentation tank through the water pump 18 to exchange heat with the fermentation liquid in the tank.

[0047] A first gas phase balance pipe 15 is provided on the pipeline connecting the top feed port of the fermentation tank 17 and the batching tank 16; the batching tank 16 is used to hold the sterile fermentation medium, and the sterile fermentation medium is 4 to 10 m 3 / h rate into the reaction chamber through the feed port of the fermentation tank 17. To balance the pressure of the batching tank 16 and the fermentation tank 17, a gas phase balance pipe is provided on the connecting pipe between the two. A second gas phase balance pipe 20 is provided on the pipe connecting the bottom discharge port of the fermentation tank 17 and the gas-liquid separation tank 21 to maintain the pressure balance between the gas-liquid separation tank and the mechanical stirring fermentation tank.

[0048] A gas filter 3 is provided on the pipeline connecting the oxygen generator 1 and the gas-liquid mixing pump 4 to prevent the contamination of the seed liquid by bacteria in the oxygen. The oxygen flow rate is controlled to be 200-500 g / h, and the speed of the seed liquid entering the gas-liquid mixing pump after secondary activation is 3-10 m / s. 3 / h.

[0049] The mixed liquid of oxygen microbubbles and seed liquid flows out from the liquid outlet of the gas-liquid mixing pump 4 at a speed of 0.5 to 3 m 3 / h of liquid flow rate flows into the nano bubble pump 6, and secondary pressurization and gas dissolution are carried out in the pressure tank 8. During this process, the pressure of the pressure tank 8 is controlled within the range of 0.5-20MPa. In order to prevent the pressure in the tank from being too high, a safety valve 7 is provided at the air inlet on the top of the pressure tank. When the pressure in the tank is too high, the safety valve 7 is manually opened, and the oxygen in the pressure tank can be circulated to the nano bubble pump 6 through the connecting pipeline for secondary use.

[0050] After the micro-nano bubbles entrained with cells are formed, they are discharged from the outlet of the bubble atomizer 9 at a flow rate of 6 to 10 L / min, and enter the reaction chamber through the air inlet at the bottom of the shell of the mechanical stirring fermenter 17 through the connecting pipeline, so that the micro-nano bubbles enter the fermentation liquid from bottom to top.

[0051] The bottom feed port of the gas-liquid separation tank 21 is connected to the discharge port of the fermentation tank 17, the middle separation port is connected to the fermentation product collection tank 22, and the top gas outlet is connected to the water separator 24. The water separator 24 is used to realize the circulation separation of the fermentation product and achieve the diversion and pressure stabilization effect of the gas-liquid separation tank and the fermentation product collection tank.

[0052] The water separator 24 has a water outlet, an air inlet, an air outlet, a circulating gas outlet and a circulating gas inlet. The circulating gas inlet is connected to the top air outlet of the gas-liquid separation tank 21, the circulating gas outlet is connected to the air inlet in the water separator body, the water outlet is connected to the fermentation product collection tank 22, and the air outlet is connected to the atmosphere; the circulation of the gas and the circulation of the fermentation liquid are controlled by valve 23 respectively.

[0053] Embodiment 2:

[0054] The above device is used to carry out immobilized Corynebacterium glutamicum fermentation to produce L-lysine, which specifically includes:

[0055] Seed culture and fermentation of Corynebacterium glutamicum:

[0056] (1) Fermentation strain: Wild strain of Corynebacterium glutamicum KFCC 11043, preserved in our laboratory.

[0057] (2) Seed culture medium (g / L) was 20% sucrose, 10% peptone, 5% yeast powder, 3.5% urea, 5% ammonium sulfate, 0.5% MgSO4·7H2O, 4% KH2PO4, 10.5% K2HPO4, 0.5% cysteine, and 10 mL trace element solution a.

[0058] (3) The fermentation medium (g / L) was glucose 80, yeast powder 8, urea 15, ammonium sulfate 13.2, MgSO4·7H2O 0.6, K2HPO4 1.5, FeSO4 0.2, 3-morpholinepropanesulfonic acid 42, and trace element solution b 10 mL.

[0059] (4) Trace element solution a (mg / L) is 2 mg / L of calcium pantothenate, 3 mg / L of nicotinamide, 1.5 mg / L of vitamin B1, and 0.15 mg / L of biotin. It is filtered and sterilized before being added to the seed culture medium.

[0060] (5) Trace element solution b (mg / L) is 0.9 mg / L of copper sulfate, 1 mg / L of zinc sulfate, 1.8 mg / L of biotin, 9 mg / L of vitamin B1, 150 mg / L of manganese sulfate, 9 mg / L of calcium pantothenate, and 60 mg / L of nicotinamide. After filtration and sterilization, it is added to the fermentation medium.

[0061] (6) After the shake flask seed solution was activated at 30°C and 220 rpm for 12 h, the activated Corynebacterium glutamicum seed solution was transferred to seed tank 2 at 80% inoculation rate and cultured at 30°C and 220 rpm until OD 600 It is used for immobilized cell fermentation in the range of 0.3 to 0.4.

[0062] The bioreactor is used for the immobilized fermentation of Corynebacterium glutamicum according to the following process:

[0063] (7) First, wrap cotton fibers of size 70 cm*140 cm on a cylindrical, hollow wire mesh holder at a dosage of 35 g / L and place it in the center of the reaction chamber in the fermentation tank 17. Then, steam sterilize the materials in the seed tank 2, the batching tank 16, and the fermentation tank 17 at 115°C for 30 min for later use.

[0064] (8) Then, after the first activation by shaking bottle and the second transfer to seed tank 2, the seed liquid OD is controlled. 600 In the range of 0.3-0.4, start the oxygen generator 1 and the seed tank 2, and after filtering and sterilizing by the gas filter 3, adjust the oxygen flow rate to 240g / h, and at the same time, the rate at which the seed liquid enters the gas-liquid mixing pump 4 reaches 5m 3 / h, after the action of the gas-liquid mixing pump 4, the oxygen microbubbles are evenly mixed with the seed liquid, and the formed mixed liquid is 1m 3 / h flows into the nano bubble pump 6, and is pressurized twice by the pressure tank 8 to control the pressure to reach 8MPa. Then, the bubble atomizer 9 discharges oxygen micro-nano bubbles. At this time, oxygen micro-nano bubbles carrying Corynebacterium glutamicum are formed, with an average bubble size of 220nm and an existence time of more than 650s.

[0065] (9) Further, the motor 12, the batching tank 16 and the water pump 18 are started so that the stirring speed of the stirring paddle 13 reaches 180 rpm, and the material in the batching tank 16 is stirred at a speed of 6 m 3 / h into the reaction chamber of the fermentation tank 17. During this period, the first gas phase balance pipe 15 is opened to maintain the pressure balance between the batching tank 16 and the fermentation tank 17. At the same time, the precooled 5°C sterile water in the water tank 19 is pumped at a rate of 1m 3 The rate of flowing through the heat exchanger 14 at / h is used to prevent the local temperature from being too high and causing the cell activity to decrease.

[0066] (10) After the temperature of the fermentation liquid in the fermenter 17 is stably maintained at 30° C., the oxygen micro-nano bubbles containing cells prepared in step (8) are flowed into the reaction chamber through the feed port at the lower side of the fermenter 17 at a flow rate of 8 L / min to perform immobilized cell fermentation.

[0067] (11) After the fermentation is completed for 20 hours, the second gas phase balance pipe 20 is opened to put the fermentation tank 17 and the gas-liquid separation tank 21 in a pressure balance state. The fermentation liquid enters the gas-liquid separation tank 21 from the connecting pipe and is discharged into the fermentation liquid collection tank 22 through the separation material port. In addition, in order to prevent the fermentation liquid from being discharged too much and not being discharged in time, and to prevent the entrainment of the fermentation liquid by micro-nano bubbles and causing losses, the separation of the product and the gas is further carried out through the water separator 24.

[0068] (12) The gas in the water separator 24 circulates continuously, and the fermentation liquid is further separated. The corresponding gas and liquid valves are opened, and the remaining fermentation liquid finally flows into the fermentation product collection tank 22 through the water outlet, and the gas is discharged into the atmosphere.

[0069] (13) After all the fermented liquid has flowed out and been collected, a second batch of fermentation is carried out using the immobilized cells on the surface of the cotton fibers in the fermentation tank 17 without the need for re-inoculation. The materials in the batching tank 16 are refilled and steam sterilized for later use. The specific operation process is the same as (9) to (12).

[0070] In the single-batch immobilized cell fermentation process, the fermentation cycle is 20 hours, the L-lysine output is 38 g / L, the L-lysine productivity is 1.9 g / L / h, the L-lysine yield can reach 47.5%, the oxygen micro-nano bubble size is 220 nm, and the existence time can reach more than 630 seconds.

[0071] Furthermore, in the continuous immobilized cell fermentation process, the fermentation cycle was shortened from 20h to 8h. At the 8th time, the L-lysine production was increased to 48g / L, the L-lysine yield was 6g / L / h, and the L-lysine yield could reach 60%. After multiple cycles, the size of oxygen micro-nano bubbles could be reduced to 7-200nm, and the existence time could reach 670s.

[0072] Comparative Example

[0073] The same example was used to activate and culture the Corynebacterium glutamicum strain.

[0074] The fermentation of immobilized cells of Corynebacterium glutamicum was carried out according to the method in the embodiment, except that the entire bioreactor system does not contain the micro-nano bubble generator 5, and the bubbles generated by the gas-liquid mixing pump 4 are directly introduced into the reaction chamber from the feed port at the bottom of the fermentation tank 17.

[0075] In contrast, in the single batch immobilized cell fermentation process, in order to ensure the consistency of the fermentation cycle, the stirring speed corresponding to the stirring paddle 13 needs to be increased to 200 rpm to enhance the dissolved oxygen, the L-lysine production is 36 g / L, the L-lysine yield is 1.8 g / L / h, the L-lysine yield can reach 45%, the oxygen microbubble size is 580 nm, and the existence time is 280 s.

[0076] Furthermore, in the continuous immobilized cell fermentation process, the fermentation cycle was shortened from 20 h to 12 h. At the 10th time, the L-lysine production was increased to 43 g / L, the L-lysine yield was 3.58 g / L / h, the L-lysine yield was 53.75%, and after multiple cycles, the size of the oxygen micron bubbles could be reduced to 290-560 nm, and the existence time could reach 300 s.

[0077] The present invention provides an oxygen micro-nano bubble enhanced aerobic fermentation bioreactor and its application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. Application of oxygen micro-nano bubble enhanced aerobic fermentation bioreactor in the production of L-lysine by immobilized Corynebacterium glutamicum fermentation, characterized in that: The oxygen micro-nano bubble enhanced aerobic fermentation bioreactor comprises an oxygen generator (1), a seed tank (2), a gas-liquid mixing pump (4), a micro-nano bubble generator (5), a fermentation tank (17), a batching tank (16), a gas-liquid separation tank (21) and a fermentation product collection tank (22); The gas-liquid mixing pump (4), the micro-nano bubble generator (5), the fermentation tank (17), the gas-liquid separation tank (21) and the fermentation product collection tank (22) are connected in sequence through pipelines; the batching tank (16) is connected to the fermentation tank (17); The oxygen generator (1) and the seed tank (2) are connected to the first inlet and the second inlet of the gas-liquid mixing pump (4) respectively, and oxygen and seed liquid are sent to the gas-liquid mixing pump (4) for mixing, and then sent to the micro-nano bubble generator (5) to form oxygen micro-nano bubbles through shearing, and then sent to the fermentation tank (17) for fermentation together with the raw materials sent from the batching tank (16), and the fermentation product is separated by the gas-liquid separation tank (21) and sent to the fermentation product collection tank (22); The micro-nano bubble generator (5) comprises a nano bubble pump (6), a safety valve (7), a pressure tank (8) and a bubble atomizer (9); the feed port of the nano bubble pump (6) is connected to the discharge port of the gas-liquid mixing pump (4); the nano bubble pump (6), the pressure tank (8) and the bubble atomizer (9) are connected in sequence; the discharge port of the bubble atomizer (9) is connected to the feed port of the fermentation tank (17); the nano bubble pump (6) and the pressure tank (8) are also connected via a gas circulation pipe to return excess oxygen in the pressure tank (8) to the nano bubble pump (6); the safety valve (7) is installed on the gas circulation pipe; The outlet of the bubble atomizer (9) is connected to the feed inlet at the lower side of the fermentation tank (17), and a water pump (10) is provided on the connecting pipeline; the gas outlet at the top of the fermentation tank (17) is connected to the pressure tank (8) through a gas circulation pipe to return excess oxygen in the fermentation tank (17) to the pressure tank (8), and the gas circulation pipe is provided with an air extraction pump (11); The application of the bioreactor in the production of L-lysine by immobilized Corynebacterium glutamicum fermentation specifically comprises the following steps: (1) steam sterilizing the materials in the seed tank (2), the batching tank (16), and the immobilized carrier in the fermentation tank (17) for later use; (2) The filtered and sterilized oxygen and the activated seed liquid are simultaneously introduced into the micro-nano bubble generator (5), and the oxygen flow rate is controlled at 200-250 g / h to form oxygen micro-nano bubbles containing cells, which are further introduced into the fermentation tank (17) at a flow rate of 6-8 L / min for immobilized cell fermentation; wherein the average size of the oxygen micro-nano bubbles is less than 220 nm and the existence time is more than 630 s; the stirring rate in the fermentation tank (17) is controlled at less than 180 rpm; (3) After the fermentation is completed, the fermentation liquid is circulated and separated using the gas-liquid separation tank (21) and the water separator (24), and the product finally flows into the fermentation product collection tank (22); after the fermentation liquid has all flowed out and has been collected, the second batch of fermentation is carried out using the immobilized cells on the surface of the cotton fibers in the fermentation tank (17) without the need for re-inoculation; The fermentation tank (17) is a mechanical stirring fermentation tank, the feed port at the lower side of the fermentation tank is connected to the micro-nano bubble generator (5), the feed port at the top is connected to the batching tank (16), and the discharge port at the bottom is connected to the gas-liquid separation tank (21); A stirring paddle (13) is provided in the fermentation tank (17), and is connected to a motor (12) located above the top of the fermentation tank (17). The stirring paddle (13) is driven by the motor (12) to stir the material in the fermentation tank (17); A cylindrical, hollow wire mesh holder is provided in the center of the reaction chamber in the fermentation tank (17) for fixing the cotton fibers; a stirring paddle (13) passes through the hollow portion in the middle of the wire mesh holder; The fermentation tank (17) is also provided with a heat exchanger (14), and the temperature of the fermentation liquid in the fermentation tank (17) is adjusted by the heat exchanger (14); The heat exchanger (14) is a built-in multi-group coil heat exchanger fixed on the inner wall of the reaction chamber of the mechanical stirring fermentation tank. The heat exchanger (14) is connected to a water tank (19) outside the fermentation tank via a water pipe. A water pump (18) is provided on the water pipe. The precooled sterile water in the water tank (19) is sent into the coil in the fermentation tank by the water pump (18) to perform heat exchange with the fermentation liquid in the tank. A first gas phase balance pipe (15) is provided on the pipeline connecting the top feed port of the fermentation tank (17) and the batching tank (16); a second gas phase balance pipe (20) is provided on the pipeline connecting the bottom discharge port of the fermentation tank (17) and the gas-liquid separation tank (21); The bottom feed port of the gas-liquid separation tank (21) is connected to the discharge port of the fermentation tank (17), the middle separation port is connected to the fermentation product collection tank (22), and the top gas outlet is connected to the water separator (24); The water separator (24) has a water outlet, an air inlet, an air outlet, a circulating gas outlet and a circulating gas inlet, wherein the circulating gas inlet is connected to the top air outlet of the gas-liquid separation tank (21), the circulating gas outlet is connected to the air inlet in the water separator body, the water outlet is connected to the fermentation product collection tank (22), and the air outlet is connected to the atmosphere; a valve (23) is provided on the water separator (24) for controlling the circulation of gas and the flow of fermentation liquid.

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