A multi-layer shallow tray electrochemical bioreactor and its application

Abstract

The present invention discloses a multi-layer shallow dish electrochemical bioreactor and its application, which relates to the field of microbial fermentation and microbial electrosynthesis. The multi-layer shallow dish electrochemical bioreactor comprises a shallow dish culture module, the shallow dish culture module comprises multiple layers of shallow dishes stacked on each other, an overflow baffle is arranged on the shallow dish, the overflow baffle separates the shallow dish into a culture area and an overflow area, a first electrode is arranged on the bottom of the dish in the culture area, the first electrode is connected to the electrochemical regulation system through a wire, an independent electrode cavity is also fixedly installed in the culture area, a single electrode is arranged in the independent electrode cavity, the single electrode is connected to the electrochemical regulation system through a wire, and one side of the independent electrode cavity is connected to the culture area through a proton membrane, and an overflow port is arranged in the overflow area. The present invention has the advantages of efficient and controllable fermentation, diverse functions, economical space utilization, low energy consumption, simple control, convenient operation and easy separation of products.

Description

Technical Field

[0001] The invention relates to the fields of microbial fermentation and microbial electrosynthesis, and in particular to a multi-layer shallow-disk type electrochemical bioreactor and application thereof. Background Art

[0002] Liquid deep fermentation is a fermentation method commonly used in the modern bio-industry. At present, various biological products such as antibiotics, amino acids, enzyme preparations, etc. are mostly produced by liquid deep fermentation. Although liquid deep fermentation has developed into a mature process, it still has its shortcomings. For example, when cultivating aerobic microorganisms, liquid deep fermentation requires stirring, aeration and other methods to increase dissolved oxygen in order to meet the growth needs of aerobic microorganisms. These aeration methods not only have potential pollution risks, but also bring shear forces to the bacteria, especially when cultivating filamentous fungi, the shear forces caused by stirring will cause serious damage to the mycelium. It can be said that there is a contradiction between the mass transfer effect and the shear force in liquid deep fermentation, and it is difficult to ensure a high dissolved oxygen content without causing shear stress.

[0003] One idea to solve the contradiction between mass transfer and shear force is to adopt a static fermentation method, using a shallow dish reactor to increase the gas-liquid contact area while maintaining static state to maintain the natural growth of structures such as mycelium. Ventilation above the shallow dish accelerates gas exchange, which consumes less energy than stirring and other methods, and will not be affected by changes in the viscosity of the reaction system. The shallow dish reactor is a very classic bioreactor, and the reaction matrix that can be used includes solid and liquid. Among them, the solid-state fermentation process is often used for the cultivation of filamentous fungi. Domestic patents CN 112481140 A and CN 104531521 A design a multi-layer shallow dish reactor for solid-state microbial fermentation. However, the solid-state fermentation system cannot ensure that the bacteria and nutrients are fully mixed and may bring difficulties to downstream processes such as product separation.

[0004] Redox potential regulation is an emerging means of regulating cell metabolism. As a complex redox system, organisms carry out countless redox reactions in cells all the time. Many substances, such as NADH / NAD+, undergo dynamic changes in cells in different redox states. By changing the redox balance in cells, the redox pairs in cells, especially the ratio of NADH / NAD+ and NADPH / NADP+, can be adjusted, thereby further changing the direction of cell metabolism. The redox potential regulation methods applied to microbial fermentation can be divided into two categories: strain modification and process engineering. Strain modification usually means directly regulating the concentration of cofactors in cells. If the microorganisms themselves cannot balance the redox changes caused by fermentation production, genetic engineering can directly make the strains more robust. However, strain modification is complex and time-consuming, and changes in a metabolic pathway often cause fluctuations in the entire metabolic network, which makes the results of the modification often difficult to predict. At the same time, the metabolic burden brought by the modification to the strain cannot be ignored. On the contrary, process engineering is simple and controllable, and it comprehensively changes the redox potential of the cell environment, so there is no need to worry about cell metabolic disorders caused by regulation. Process engineering methods for regulating redox potential include ventilation, addition of redox reagents and electrochemical regulation. Generally speaking, the additional cost required for process engineering optimization is easy to calculate, and the optimal solution can be directly applied to industrial production after it is determined based on technical and economic performance.

[0005] Electrochemical regulation in microbial fermentation is also known as microbial electrosynthesis (MES). Its basic regulation method is to add electrodes and other components to the fermentation system to construct an electrochemical reaction device, form current through external controllable voltage, and use direct and indirect electron transfer to allow microbial cells to receive or output electrons, thereby completing redox potential regulation, promoting microbial growth or adjusting microbial metabolic flow to increase product yield. In electrochemical regulation, electrodes, especially the contact area between electrodes and fermentation systems, have a key influence on the efficiency of regulation. However, in deep liquid fermentation, there is often a large amount of liquid space that cannot contact the electrodes, and the dynamic culture method is not only easy to destroy the attachment of bacteria to the electrodes, but also may cause damage to the electrodes themselves.

[0006] Therefore, in order to avoid various unfavorable factors in liquid deep fermentation, technicians in this field are committed to developing a shallow plate reaction device combined with electrochemical regulation to achieve efficient oxygen transfer, efficient space utilization and static culture under efficient electrochemical regulation, which is an urgent problem to be solved. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a shallow dish type reaction device combined with electrochemical regulation to achieve efficient oxygen transfer, efficient space utilization and static culture under efficient electrochemical regulation. In addition, in addition to the above-mentioned electrochemically regulated aerobic static culture, the present invention can also perform multiple functions such as continuous culture, anaerobic fermentation, and microbial electricity generation.

[0008] In order to achieve the above technical objectives, the present invention provides a multi-layer shallow tray electrochemical bioreactor, including a shallow tray culture module, the shallow tray culture module includes multiple layers of shallow trays stacked on each other, an overflow baffle is provided on the shallow tray, the overflow baffle separates the shallow tray into a culture area and an overflow area, a first electrode is provided on the bottom of the tray in the culture area, the first electrode is connected to an electrochemical regulation system through a wire, an independent electrode cavity is also fixedly installed in the culture area, a single electrode is provided in the independent electrode cavity, the single electrode is connected to the electrochemical regulation system through a wire, and one side of the independent electrode cavity is connected to the culture area through a proton membrane, and an overflow port is provided in the overflow area.

[0009] Preferably, the overflow baffle is a serrated overflow baffle.

[0010] In the present invention, the shallow tray culture module also includes an outer shell installed on the outside of the multi-layer shallow tray, a feed inlet is arranged above the outer shell, the feed inlet is connected to the culture area on the shallow tray, an air inlet is arranged on one side of the bottom of the outer shell, a feed outlet is arranged on the other side, and an air outlet is arranged above the outer shell.

[0011] Furthermore, the multiple layers of shallow trays are stacked alternately in reverse order, so that the sample flows downward in a serpentine shape between the shallow trays during batch sampling or continuous fermentation, and the gas flows upward in a serpentine shape between the shallow trays.

[0012] Furthermore, a multifunctional channel is provided on one side of the shallow dish, and a variety of sensors are inserted in the multifunctional channel. One end of the sensor is located in the culture area of ​​the shallow dish, and the other end is connected to the data analyzer.

[0013] Preferably, the sensors include a temperature sensor, a pH sensor and an ORP sensor.

[0014] In the present invention, a shell sensor channel and a shell feed sampling channel are also provided on one side of the shell, the sensor is connected to the data analyzer through the shell sensor channel, and the shell feed sampling channel is connected to the feed sampling channel on one side of the shallow plate.

[0015] Furthermore, a total anode and a total cathode are also provided on one side of the shell, and the total anode and the total cathode are respectively connected to the first electrode at multiple shallow dish culture areas or to a single electrode at multiple independent electrode cavities, and the total anode and the total cathode are respectively connected to the electrochemical regulation system through wires.

[0016] The present invention also provides the use of the above-mentioned multi-layer shallow dish type electrochemical bioreactor in static fermentation on the surface of liquid, microbial electrosynthesis, preparation of microbial fuel cells and cultivation of electricity-producing microorganisms.

[0017] Specifically, as one of the embodiments of the present invention, the multi-layer shallow tray type electrochemical bioreactor is used to culture Gluconacetobacter xylosus to produce bacterial cellulose.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention combines electrochemical regulation with a shallow dish reactor, which can not only avoid the shear force damage caused by dynamic culture, but also, due to the structural characteristics of the shallow dish, only a layer of carbon cloth is needed to be laid on the bottom of the dish to achieve a very large electrode-liquid contact area, thereby achieving efficient electrochemical regulation, and having the advantages of efficient and controllable fermentation, diverse functions, economical space utilization, low energy consumption, simple control, convenient operation, and easy product separation.

[0020] 2. Liquid static culture of aerobic microorganisms can be performed in the shallow plate culture module, where aerobic microorganisms include Acetobacter xylinum that produces bacterial cellulose and Trichoderma reesei with mycelium structure. The shallow plate provides a larger gas-liquid interface for the culture system, which is conducive to the growth of aerobic microorganisms. Liquid static culture does not have the shear force caused by dynamic culture, and can ensure the integrity of structures such as mycelium. Liquid static culture is different from solid-state culture. The culture system is liquid rather than solid, which is conducive to the separation and purification of the final product.

[0021] 3. The multi-layer shallow-plate electrochemical bioreactor can also be used for anaerobic fermentation when nitrogen is introduced and air is exhausted or the air inlet is closed. When the discharge port is opened, continuous fermentation similar to a fluidized bed reactor can be performed.

[0022] 4. The redox potential of microbial growth is regulated by an electrochemical control system to adjust the metabolic flow of microorganisms to increase the yield of target products and realize microbial electrosynthetic production; the electrodes in the shallow dish are located at the bottom of the dish, and the area is almost the same as the shallow dish to achieve more efficient electron transfer.

[0023] 5. The multilayer shallow-plate electrochemical bioreactor can not only be used for microbial electrosynthesis, but also can cultivate electricity-producing microorganisms under the condition of a suitable culture system, and the entire system can operate as a microbial fuel cell.

[0024] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Overall schematic diagram of multi-layer shallow tray electrochemical bioreactor;

[0026] Figure 2 Schematic diagram of the multi-layer shallow tray module inside the shell;

[0027] Figure 3 A single shallow dish top view;

[0028] Figure 4 Front view of the multi-layer shallow tray module and the housing;

[0029] Figure 5 Side view of the multi-layer shallow tray module and housing;

[0030] Among them, 1-total anode; 2-total cathode; 3-conductor; 4-electrochemical control system; 5-feed port; 6-gas outlet; 7-gas inlet; 8-shallow plate culture module; 9-shallow plate; 10-shell sensor channel; 11-shell feeding sampling channel; 12-multi-function channel; 13-sensor; 14-feed sampling channel; 15-first electrode; 16-independent electrode cavity; 17-single electrode; 18-proton membrane; 19-overflow baffle; 20-overflow port; 21-discharge port; 22-air flow direction diagram. DETAILED DESCRIPTION

[0031] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0032] In the drawings, components with the same structure are indicated by the same numerical reference numerals, and components with similar structures or functions are indicated by similar numerical reference numerals. The size and thickness of each component shown in the drawings are arbitrarily shown, and the present invention does not limit the size and thickness of each component. In order to make the illustration clearer, the thickness of the components is appropriately exaggerated in some places in the drawings.

[0033] Example 1

[0034] like Figure 1-Figure 5As shown, this embodiment provides a multi-layer shallow dish electrochemical bioreactor, which can not only operate a small reactor in the laboratory, but also be enlarged for industrial production, including a shallow dish culture module, and the shallow dish culture module 8 is composed of shallow dishes 9 stacked alternately in reverse so that the overflow port 20 of each layer of shallow dishes corresponds to the lower layer culture block, thereby ensuring that the culture fluid can enter the lower layer only after filling the upper layer. Among them, the number of shallow dishes for small reactors is not less than 6 layers, and the number of shallow dishes for large reactors is not less than 15 layers. Each layer of shallow dishes 9 is rectangular in shape, with an aspect ratio of less than 4:1, and the height of the side wall of the shallow dish is less than 10% of the length and not higher than 20 cm.

[0035] A serrated overflow baffle 19 is provided on the shallow tray 9, and the overflow baffle 19 divides the shallow tray into a culture area and an overflow area. The height of the overflow baffle 19 is 50%-80% of the height of the side wall of the shallow tray 9, which can control the depth of the culture solution and leave the upper space of the culture solution as a gas channel. After the gas enters from the bottom air inlet 7, it passes through the top of each layer of culture solution, and then flows upward through each layer of overflow 20, and finally flows out from the top layer of the outlet 6 to achieve efficient gas exchange. Before the system starts working, the fermentation system after inoculation and mixing is introduced in batches or continuously from the feed port 5 above the shallow tray module. After the fermentation solution of the upper layer reaches the height of the overflow baffle 19, it enters the next layer through the overflow port 20, thereby completing the inoculation and cultivation of each layer at one time or achieving continuous cultivation.

[0036] Among them, a first electrode 15 is arranged on the bottom of the culture area. Specifically, the first electrode 15 can be composed of a first electrode 15 made of carbon cloth or other materials such as graphite, carbon felt or steel mesh laid on the bottom of the dish and a wire 3 to achieve efficient electron transfer in the microbial electrochemical system. The area of ​​the first electrode 15 is larger than 70% of the bottom area of ​​the shallow dish 9 and the thickness is less than 80% of the liquid depth. It can work as an anode or a cathode according to needs.

[0037] The first electrode 15 is connected to the electrochemical regulation system 4 through the wire 3. An independent electrode cavity 16 is also fixedly installed in the culture area. The independent electrode cavity 16 includes a single electrode 17 and an electrolyte enclosed in the cavity. The single electrode 17 is connected to the outside world through the wire 3 passing through the cavity. The cavity is partially open but closed by a proton membrane 18. The electrolyte exchanges protons with the external liquid through the proton membrane 18, and finally a current loop can be formed.

[0038] Each shallow dish 9 has an independent electrode cavity 16, which is placed in the culture area. After inoculation with culture fluid, the independent electrode cavity 16, the culture fluid in the shallow dish 9 and the electrode form an anode-cathode type double-cavity electrochemical reaction device.

[0039] The shallow dish 9 is also provided with a multifunctional channel 12, a sensor 13 and a feed sampling channel 14. Among them, the sensor 13 integrates sensors such as temperature, pH, ORP, etc., and is connected to an additional data analysis instrument through the housing sensor channel 10 for monitoring the culture process.

[0040] The feed sampling channel 14 is connected to the shell feed sampling channel 11, and can be used as a sampling port for sampling and analysis during the culture process and an independent discharge port after the fermentation is completed, and can also be used as a sampling port for adding chemical reagents when it is necessary to adjust parameters such as pH and ORP. In order to facilitate the modularization of the shallow dish 9, the multifunctional channel 12 can be used as a feed sampling channel 14 and a wire channel for other components such as the sensor 13, the first electrode 15 or the independent electrode cavity 16.

[0041] The shallow plate culture module 8 is encapsulated as a whole in the outer shell to ensure a sterile environment. The outer shell is provided with a total anode 1, a total cathode 2, a feed port 5, an air outlet 6, an air inlet 7, and a discharge port 21 to ensure material exchange and signal transmission between the culture area and the outside; the bottom shallow plate 9 has a total discharge port and is connected to the discharge port 21 on the outer shell. During batch fermentation, the discharge port is closed. After the fermentation is completed, the discharge port is opened to collect the product. During the continuous fermentation process, the discharge port is open to continuously discharge the fermentation liquid.

[0042] The feed port 5 is located at the top of the multi-layer shallow dish culture module 8, and the air inlet 7 is located at the bottom of the shallow dish culture module 8. The air flow direction diagram 22 after ventilation is serpentine, flowing from bottom to top through the overflow port 20 of each shallow dish, and flowing out from the topmost air outlet 6. The shallow dish 9 on the bottom layer has an opening in the culture block, which is connected to the opening on the outer shell as the total discharge port 21. During batch fermentation, the discharge port 21 is closed, and can be opened as a discharge port to collect the product after the fermentation is completed. During continuous fermentation, the discharge port 21 is opened as a continuous discharge port. The independent electrode cavity 16 is fixed inside the shallow dish 9 and is connected to the space inside the shallow dish 9 through a proton membrane 18. The cavity includes a single electrode 17, which can work as an anode or cathode according to needs.

[0043] The electrochemical control system 4 outputs a stable voltage to control the fermentation according to the culture requirements, wherein the electrochemical control system 4 is common knowledge of those skilled in the art and is not further explained here. It is connected to the total anode 1 and the total cathode 2 of the reactor through a wire 3, and the total anode 1 and the total cathode 2 are respectively connected to the first electrode 15 at multiple shallow plate culture areas or to a single electrode 17 at multiple independent electrode cavities 16 (that is, when the first electrode 15 is set as a cathode, it is connected to the total cathode, and at this time, the single electrode is connected to the total anode as an anode; when the first electrode 15 is set as an anode, it is connected to the total anode, and at this time, the single electrode is connected to the total cathode as a cathode), so that it can be connected to each electrode unit through the internal wire 3.

[0044] Example 2

[0045] In this example, a multi-layer shallow dish electrochemical bioreactor was used to culture Gluconobacter xylosus ( Gluconacetobacter xylinus ) to produce bacterial cellulose. Xylinum Acetobacter is cultured in a shallow dish 9, and the first electrode at the feeder of the shallow dish 9 is set as a cathode, which is connected to the negative electrode of the electrochemical control system 4. The culture medium for culturing Xylinum Acetobacter is HS culture medium, and the components of HS culture medium include 2.5% glucose, 1% peptone, 0.5% yeast extract, 0.27% disodium hydrogen phosphate, 0.115% citric acid, and 0.025% magnesium sulfate. The single electrode at the independent electrode chamber 16 is used as an anode, which is connected to the total anode of the electrochemical control system 4, and the electrolyte is a red blood salt solution. Under static culture conditions, Xylinum Acetobacter will form a dense cellulose film at the gas-liquid interface, and a sterile airflow passes above the liquid surface of the shallow dish to provide oxygen for Xylinum Acetobacter, and the cathode carbon cloth outputs electrons to provide reducing power for Xylinum Acetobacter, thereby ultimately increasing the yield of bacterial cellulose.

[0046] The specific application steps are as follows:

[0047] Seed culture, culture medium preparation and sterilization, reactor packaging and sterilization, under sterile operation, the inoculated and mixed culture medium is slowly injected into the reactor through the feed port 5, and the feed port 5 is closed after each layer is filled with liquid, and sterile air is introduced from the air inlet 7. The air inlet 7 and the air outlet 6 are sealed with a sterilizing filter to ensure that there is no risk of contamination, and the electrochemical control system 4 is connected to control the voltage, temperature and other conditions for static fermentation.

[0048] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A multi-layer shallow tray electrochemical bioreactor, characterized in that: The shallow tray culture module comprises a shallow tray that is stacked with multiple layers of shallow trays, an overflow baffle is provided on the shallow tray, and the overflow baffle separates the shallow tray into a culture area and an overflow area, a first electrode is provided on the bottom of the tray in the culture area, and the first electrode is connected to an electrochemical regulation system through a wire, an independent electrode cavity is fixedly installed in the culture area, a single electrode is provided in the independent electrode cavity, and the single electrode is connected to the electrochemical regulation system through a wire, and one side of the independent electrode cavity is connected to the culture area through a proton membrane, and an overflow port is provided in the overflow area; The shallow tray culture module also includes a shell installed outside the multi-layer shallow tray, a feed inlet is arranged on the top of the shell, the feed inlet is connected to the culture area on the shallow tray, an air inlet is arranged on one side of the bottom of the shell, a material outlet is arranged on the other side, and an air outlet is arranged on the top of the shell; The multiple layers of shallow plates are stacked in reverse and alternately, so that the sample flows downward in a serpentine shape between the shallow plates during batch sampling or continuous fermentation, and the gas flows upward in a serpentine shape between the shallow plates; A total anode and a total cathode are also provided on one side of the shell. The total anode and the total cathode are respectively connected to the first electrode level at multiple shallow dish culture areas or to a single electrode at multiple independent electrode cavities. The total anode and the total cathode are respectively connected to the electrochemical regulation system through wires.

2. The multi-layer shallow tray electrochemical bioreactor according to claim 1, characterized in that: The overflow baffle is a sawtooth overflow baffle.

3. The multi-layer shallow tray electrochemical bioreactor according to claim 1, characterized in that: A multifunctional channel is also arranged on one side of the shallow dish, and a variety of sensors are inserted in the multifunctional channel. One end of the sensor is located in the culture area of ​​the shallow dish, and the other end is connected to the data analyzer.

4. The multi-layer shallow tray electrochemical bioreactor according to claim 3, characterized in that: The sensors include a temperature sensor, a pH sensor and an ORP sensor.

5. The multi-layer shallow tray electrochemical bioreactor according to claim 3, characterized in that: A shell sensor channel and a shell feed sampling channel are also provided on one side of the shell. The sensor is connected to the data analyzer through the shell sensor channel, and the shell feed sampling channel is connected to the feed sampling channel on one side of the shallow dish.

6. Use of the multi-layer shallow dish electrochemical bioreactor according to any one of claims 1 to 5 in static fermentation on the surface of liquid, microbial electrosynthesis, preparation of microbial fuel cells and cultivation of electricity-producing microorganisms.

7. The use according to claim 6, characterized in that: The multi-layer shallow dish type electrochemical bioreactor is used for culturing Gluconacetobacter xylosus to produce bacterial cellulose.

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