An array type microfluidic chip for continuous cultivation of microorganisms and its application

Abstract

The present invention discloses an array-type microfluidic chip for continuous culture of microorganisms and its application, belonging to the field of bioengineering and synthetic biology. The microfluidic chip includes an upper channel structure and an underlying support structure supporting the upper channel structure. The upper channel structure includes a main channel, a cell trap, an injection channel and a recovery channel; the main channel is a microreactor chamber for accommodating the circulation of microorganisms and culture medium, the cell traps are evenly distributed in the main channel in an array form, and the injection channel and the recovery channel are respectively arranged at both ends of the main channel for connecting the main channel. Using the microfluidic chip to cultivate Pseudomonas putida KT2440 to produce protocatechuic acid can accurately control fluid flow and culture parameters, and realize long-term continuous culture of microorganisms. Compared with traditional bioreactors, the present invention improves the production efficiency of strains and reduces process costs, provides new ideas and methods for strain culture and metabolite production, and is expected to develop into a new generation of bioreactors.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and synthetic biology, and in particular to an array-type microfluidic chip for continuous culture of microorganisms and applications thereof. Background Art

[0002] Microchannel reactors are reaction equipment with specific microstructures. The microstructure is the core of the reactor. Different forms of microreactors are formed according to different types of microstructures. The obvious characteristics of microfluidic technology are small size, large specific surface area, laminar flow, precise control of residence time, fast mass transfer and heat transfer, increased reaction rate, and no amplification effect. The advantages of microreactors are miniaturization of equipment, convenient and fast operation, green and efficient process, and high safety of use. Therefore, the development of microreactors provides new ideas and methods for cell transformation, microbial culture and product synthesis.

[0003] As an emerging branch of bioengineering in the 21st century, synthetic biology provides the possibility for traditional chemical synthesis to move towards biotransformation of intelligent manufacturing. It is not only an innovative technology that transforms basic research in the field of biotechnology into actual productivity, but also a key driving force for life sciences to move into a new era. Biosynthesis using microorganisms as carriers is often inseparable from bioreactors used for fermentation. At present, the tool for industrial production is large-scale fermentation tanks, but fermentation tanks are plagued by problems such as large size, complex operating procedures, and safety issues. Therefore, it is urgent to develop a new generation of bioreactors to find feasible solutions to the defects of the current microbial fermentation process. Summary of the invention

[0004] The purpose of the present invention is to provide an array-type microfluidic chip for continuous cultivation of microorganisms and its application to solve the problems existing in the above-mentioned prior art. The microfluidic chip of the present invention adopts a microreactor device with an array design. Using the microfluidic chip to cultivate microorganisms can improve the metabolic efficiency of microorganisms and reduce process costs, thereby providing the possibility for continuous production of high-value biological products.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a microfluidic chip, comprising an upper channel structure and an underlying support structure supporting the upper channel structure, wherein the upper channel structure comprises a main channel, a cell trap, an injection channel and a recovery channel;

[0007] The main channel is a microreactor chamber for accommodating the circulation of microorganisms and culture medium, the cell traps are evenly distributed in the main channel in an array form, and the injection channel and the recovery channel are respectively arranged at both ends of the main channel for connecting the main channel.

[0008] Preferably, it also includes an injection chamber and a recovery chamber, the injection channel connects the injection chamber and the main channel, so that the culture medium flows from the injection chamber into the main channel, and the recovery channel connects the recovery chamber and the main channel, and the culture medium flowing out of the main channel flows into the recovery chamber through the recovery channel.

[0009] Preferably, the length of the main channel is set to 16000 μm, the width is set to 8000 μm, and the height is set to 75 μm.

[0010] Preferably, the cell traps are provided in a plurality (the number is selected as required), and the plurality of the cell traps are crescent-shaped groove structures of the same size, which is conducive to the bacteria being captured and trapped in the microfluidic reaction chamber. The advantage is that the bottom of the crescent-shaped structure is an arc, which does not produce a structural dead angle, thereby avoiding the situation where bubbles are trapped in the cell trap or the bacteria cannot contact the culture medium.

[0011] More preferably, the diameter of the crescent-shaped groove structure (i.e., the distance between the two ends of the opening of the crescent-shaped groove structure) is 640 μm, the thickness (i.e., the distance between the side walls of the crescent-shaped groove structure) is 80 μm, and the height (i.e., the distance from the opening end of the crescent-shaped groove structure to the bottom surface) is 75 μm.

[0012] Three 15-25 μm gaps are set on each of the cell traps. More preferably, three 20 μm gaps are set according to the mycelial clumping characteristics of the cultured strain Pseudomonas putida. This design facilitates the circulation of fluids such as culture medium and increases the contact rate between the bacteria in the cell trap and the culture medium; secondly, when the fluid flow is increased, the bacteria in poor condition at the bottom of the cell trap can be flushed out from the gaps to update the bacteria in the microfluidic reaction chamber; thirdly, when the fluid flow direction is changed, the bacteria in the microfluidic reaction chamber can be flushed out in the reverse direction, which is convenient for observing the culture status of the bacteria;

[0013] Preferably, the cell traps are arranged in an array, and the same spacing is set between adjacent cell traps. The array design can enhance the capture efficiency of microorganisms. The same spacing is set between adjacent cell traps. More preferably, the spacing between adjacent cell traps is 400 μm.

[0014] Preferably, the connection between the injection channel and the recovery channel and the main channel is set to a trumpet-shaped structure for the dispersion effect of injection and the convergence effect of recovery. More preferably, the length of the injection channel and the recovery channel is set to 1500μm, the width is set to 400μm, and the height is set to 75μm.

[0015] In order to increase the amount of product synthesis, the present invention also provides a high-throughput microreactor for microbial fermentation, wherein four main reaction channels are connected in parallel (such asFigure 3 shown).

[0016] The present invention also provides the use of the microfluidic chip in any of the following:

[0017] (1) Application in microbial culture;

[0018] (2) Application in the production of protocatechuic acid by fermentation of Pseudomonas putida.

[0019] The present invention also provides a method for culturing Pseudomonas putida to produce protocatechuic acid using the microfluidic chip, comprising the following steps:

[0020] After pre-treating the microfluidic chip, a Pseudomonas putida seed solution is added to the injection chamber. After flowing into the main channel through the injection channel, part of the Pseudomonas putida seed solution is retained by the cell trap, and the Pseudomonas putida seed solution not retained by the cell trap flows out to the recovery chamber through the recovery channel, thereby completing the inoculation;

[0021] The culture medium is added into the injection cavity, and the culture medium flows into the main channel through the injection channel, and evenly perfuses the Pseudomonas putida trapped in the cell trap to cultivate the Pseudomonas putida so that it metabolizes to produce protocatechuic acid, and the protocatechuic acid flows out of the recovery channel with the culture medium and is collected in the recovery cavity.

[0022] Preferably, the pretreatment of the microfluidic chip comprises the following steps:

[0023] A hydrophilic treatment reagent is introduced into the injection cavity of the microfluidic chip to modify the inner surface of the upper channel structure, anhydrous ethanol is introduced into the injection cavity for disinfection and sterilization, and deionized water is added into the injection cavity to clean the upper channel structure.

[0024] Preferably, the conditions for culturing the Pseudomonas putida are: a medium flow rate of 4 μL / min, an inoculation age of 1 h for the secondary seed solution, and an inoculation OD of 600 The value of is 0.03;

[0025] During the cultivation of the Pseudomonas putida, the flow rate of the culture medium in the microfluidic chip is changed to regularly flush out strains in poor condition, thereby maintaining the growth and metabolic activity of the Pseudomonas putida in the microfluidic chip.

[0026] The present invention discloses the following technical effects:

[0027] The microfluidic chip used in the present invention is a microfluidic culture system with a microliter volume, which can accurately control fluid flow and culture parameters to achieve long-term continuous culture of microorganisms and improve product generation efficiency.

[0028] The present invention utilizes the cell trap structure in the microfluidic chip, which can conveniently, effectively and high-throughput trap Pseudomonas putida KT2440 in the main channel of the microreactor, and based on the culture conditions of Pseudomonas putida KT2440, continuously introduce an appropriate amount of culture medium, so that Pseudomonas putida KT2440 can maintain the best state in the microreactor and continuously produce the required collected product protocatechuic acid. The structure of the microfluidic chip provided by the present invention and the method for culturing Pseudomonas putida KT2440 have good universality; and can effectively control the cell growth in the microfluidic chip, while maximizing the yield of each cell. Cultivating Pseudomonas putida KT2440 through the microfluidic chip of the present invention improves the product synthesis efficiency and reduces the process cost, provides a new idea and method for strain culture and metabolite production using microfluidic technology, and is expected to develop into a new generation of bioreactors.

[0029] The microfluidic chip of the present invention facilitates high-throughput biosynthesis culture condition screening analysis, potentially reducing the time required for the scale-up process from laboratory scale to industrial scale. Healthcare can also benefit from the development of microfluidic chips, by creating "organs on a chip" devices, creating very powerful tools for accelerating drug and treatment development, such as factory-scale production of antibiotics using microfluidic chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 It is a schematic diagram of the structure of the microfluidic chip of the present invention;

[0032] Figure 2 It is a schematic diagram of the structure of the cell trap of the present invention;

[0033] The numbers involved in the above figure are: 1: main channel; 2: cell trap; 3: injection channel; 4: recovery channel; 5: injection chamber; 6: recovery chamber; 7: gap;

[0034] Figure 3 A schematic diagram of a high-throughput microfluidic chip device formed by a plurality of side-by-side chips of the present invention;

[0035] Figure 4 This is a diagram showing the experimental results of culturing Pseudomonas putida KT2440-PCA using the microfluidic device of the present invention;

[0036] Figure 5Flow cytometry was used to detect cell viability after 36 h of culture;

[0037] Figure 6 The survival rate of cells trapped by flushing out and retaining cells was measured by flow cytometry;

[0038] Figure 7 This is a graph showing the change in concentration of the target product after continuous culture for 216 hours in the present invention. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.

[0044] Example 1 Microfluidic Chip

[0045] like Figure 1 and Figure 2 As shown, the present invention provides a microfluidic chip, including an upper channel structure and an underlying support structure supporting the upper channel structure, wherein the upper channel structure includes a main channel 1, a cell trap 2, an injection channel 3 and a recovery channel 4;

[0046] Among them, the main channel 1 is a microreactor chamber, which is used to accommodate the circulation of microorganisms and culture medium. The microorganisms maintain a stable state and metabolize products in this structure; a number of cell traps 2 are provided, and the cell traps are crescent-shaped groove structures of the same size, which are evenly distributed in the main channel 1 in the form of an array, and the injection channel 3 and the recovery channel 4 are respectively provided at both ends of the main channel 1 for connecting the main channel 1.

[0047] In a preferred embodiment, it also includes an injection chamber 5 and a recovery chamber 6. The injection channel 3 connects the injection chamber 5 and the main channel 1, so that the culture medium flows from the injection chamber 5 into the main channel 1. The recovery channel 4 connects the recovery chamber 6 and the main channel 1, and the culture medium flowing out of the main channel 1 flows into the recovery chamber 6 through the recovery channel 4.

[0048] In a preferred embodiment, the length of the main channel 1 is set to 16000 μm, the width is set to 8000 μm, and the height is set to 75 μm.

[0049] In a preferred embodiment, the cell trap 2 is configured as a crescent-shaped groove structure of the same size, wherein the diameter of the crescent-shaped groove structure is 640 μm, the thickness is 80 μm, and the height is 75 μm.

[0050] Three slits 7 of 20 μm are arranged on each cell trap 2 , and the same spacing is arranged between adjacent cell traps 2 .

[0051] In a preferred embodiment, the connections between the injection channel 3 and the recovery channel 4 and the main channel 1 are configured as trumpet-shaped structures, and the length of the injection channel 3 and the recovery channel 4 is configured to be 1500 μm, the width is configured to be 400 μm, and the height is configured to be 75 μm.

[0052] Microfluidic chip structure number:

[0053] 1: Main channel; 2: Cell trap; 3: Injection channel; 4: Recovery channel; 5: Injection chamber; 6: Recovery chamber; 7: Slit.

[0054] Example 2 Method for using the microfluidic chip

[0055] (1) A hydrophilic treatment reagent is introduced into the microfluidic chip to modify the surface of the upper channel structure of the microfluidic chip. The hydrophilic modification can prevent bubbles from being trapped in the dead volume of the main channel and affecting the growth and metabolism of the bacteria. Anhydrous ethanol is then introduced into the microfluidic chip to complete the disinfection and sterilization operation of the microfluidic chip.

[0056] (2) After cleaning the internal channels of the upper channel structure of the microfluidic chip with deionized water, the seed solution of the bacterial species is added to the injection chamber. After flowing into the main channel through the injection channel, part of the bacterial species is intercepted by the cell trap in the main channel, and the unretained bacterial seed solution flows out through the recovery channel to the recovery chamber. After a period of time, the microfluidic bioreactor completes the inoculation.

[0057] (3) The culture medium for culturing the above-mentioned bacterial strain seed solution is added to the injection chamber, and the culture medium slowly flows into the main channel through the injection channel, evenly perfusing the strain trapped in the cell trap, and fresh culture medium continuously flows in so that the strain obtains rich nutrients and sufficient oxygen required for growth and metabolism. The bacterial strain cultured in the microfluidic chip metabolizes to produce target products, and the target products flow out of the recovery channel with the culture medium and are collected in the recovery chamber.

[0058] (4) By changing the flow rate in the microfluidic chip, strains in poor condition are flushed out regularly to maintain the growth and metabolic vitality of the strains in the microfluidic chip. The culture medium collected in the recovery chamber is used for qualitative and quantitative detection of the target product using a high performance liquid chromatography-mass spectrometer. As a result, the continuous flow of the microfluidic chip improves the reaction rate and product generation efficiency of the microorganisms, ensures the continuity of microbial culture, and makes the yield of the target product stable and efficient.

[0059] Example 3 Using microfluidic chips to culture Pseudomonas putida and ferment to produce protocatechuic acid

[0060] In this example, Pseudomonas putida KT2440-PCA was used as a culture bacterium, and the metabolism of the bacterium was utilized to produce protocatechuic acid.

[0061] Pseudomonas putida KT2440-PCA was modified and preserved by our laboratory (reference: Jin Li, Bang-CeYe. Metabolic engineering of Pseudomonas putida KT2440 for high-yield production of protocatechuic acid, Bioresource Technology. Volume 319, 2021, 124239.)

[0062] The same microfluidic chip was used to culture Pseudomonas putida KT2440 to produce protocatechuic acid. The culture medium flow rate was 4 μL / min and the inoculation age was the secondary seed liquid (obtained by culturing KT2440 in LB liquid medium) for 1 h. The inoculation OD 600 The production of protocatechuic acid was verified under the condition that the value of was 0.03.

[0063] The results are as follows Figure 4As shown in the figure, after experimental verification, the protocatechuic acid production can reach 2.04±0.15g / L at the 36th hour of culture. At the same time, by monitoring the residual sugar content of the recovered fermentation broth, the amount of glucose consumed by bacterial metabolism is the highest at 36 hours, and the target product is in a stage of substantial accumulation at this time. After 36 hours, the bacteria have filled the cell traps, and the newly propagated bacteria will flow out of the reaction chamber with the culture medium. The bacteria at the bottom of the cell traps cannot contact the fresh culture medium and lack of oxygen, so their vitality decreases, and their ability to synthesize protocatechuic acid decreases. Therefore, 36 hours is selected as the time node for updating the strain.

[0064] In order to further improve the yield of protocatechuic acid produced by culturing Pseudomonas putida KT2440 on the microfluidic chip of the present invention and realize long-term cultivation, on the basis of the above-mentioned optimal cultivation method, the speed of the peristaltic pump was increased every 36 hours, and the bacterial survival rate was monitored using the SYTO9 / PI Live / Dead Bacterial Double Stain Kit, as shown in FIG. Figure 5 As shown in the figure, the results of flow cytometry showed that the bacterial survival rate was 24.06% at 36 hours, so the peristaltic pump flow rate was increased appropriately to flush out the bacteria in poor condition at the bottom of the cell trap through three 20μm gaps, leaving only about 25% of the bacteria in the front of the cell trap. Figure 6 The results of flow cytometry showed that the survival rate of the flushed-out bacteria was only 8.64%, while the survival rate of the retained bacteria was 76.83%. This proves that most of the retained bacteria are highly active. The above results show that by increasing the speed of the peristaltic pump, the bacteria in poor condition at the bottom of the cell trap can be flushed out through the three 20μm gaps, and only the bacteria in good condition at the front can be retained to continue to be cultured and produce protocatechuic acid.

[0065] By changing the flow rate in the microfluidic chip, strains in poor condition are flushed out every 36 hours to maintain the growth and metabolic vitality of the strains in the microfluidic chip. Figure 7 As shown in the figure, the product concentration can still be maintained at 2.11±0.19 g / L after 216 hours of cultivation, and there is no product degradation, which realizes the continuous long-term cultivation of Pseudomonas putida. Compared with the batch culture of traditional bioreactors, it saves the complex process of cleaning, sterilization and re-tanking.

[0066] In addition, in actual applications, microfluidic chips containing multiple main channels can be used in parallel, or the microfluidic chips can be directly scaled up proportionally. The actual results will be directly multiplied without the need for parameter optimization experiments after the system is scaled up, which has obvious advantages over traditional bioreactors.

[0067] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for producing protocatechuic acid by culturing Pseudomonas putida using a microfluidic chip, characterized in that: The microfluidic chip comprises an upper channel structure and an underlying support structure supporting the upper channel structure, wherein the upper channel structure comprises a main channel, a cell trap, an injection channel and a recovery channel; Wherein, the main channel is a microreactor chamber for accommodating the circulation of microorganisms and culture medium, the cell traps are evenly distributed in the main channel in the form of an array, and the injection channel and the recovery channel are respectively arranged at both ends of the main channel for connecting the main channel; The microfluidic chip further comprises an injection chamber and a recovery chamber, wherein the injection channel connects the injection chamber and the main channel, so that the culture medium flows from the injection chamber into the main channel, and the recovery channel connects the recovery chamber and the main channel, so that the culture medium flowing out of the main channel flows into the recovery chamber through the recovery channel; A plurality of cell traps are provided, and the plurality of cell traps are crescent-shaped groove structures of the same size, and are used for inoculation and cultivation of microorganisms; Each of the cell traps is provided with three gaps of 15-25 μm for liquid circulation; the spacing between adjacent cell traps is 400 μm; The cell traps are arranged in an array, and the same spacing is set between adjacent cell traps, which is conducive to the capture and retention of microorganisms; The method comprises the following steps: After pre-treating the microfluidic chip, a Pseudomonas putida seed solution is added to the injection chamber. After flowing into the main channel through the injection channel, part of the Pseudomonas putida seed solution is retained by the cell trap, and the Pseudomonas putida seed solution not retained by the cell trap flows out to the recovery chamber through the recovery channel, thereby completing the inoculation; The culture medium is added into the injection cavity, and the culture medium flows into the main channel through the injection channel, and evenly perfuses the Pseudomonas putida trapped in the cell trap to cultivate the Pseudomonas putida so that it metabolizes to produce protocatechuic acid, and the protocatechuic acid flows out of the recovery channel with the culture medium and is collected in the recovery cavity.

2. The method according to claim 1, characterized in that During the cultivation of the Pseudomonas putida, the flow rate of the culture medium in the microfluidic chip is changed to regularly flush out strains in poor condition, thereby maintaining the growth and metabolic activity of the Pseudomonas putida in the microfluidic chip, and Pseudomonas putida can be continuously cultured in the microfluidic chip for more than 216 hours.

3. Application of the microfluidic chip as claimed in any one of claims 1 to 2 in any one of the following: (1) Application in microbial culture; (2) Application in the production of protocatechuic acid by fermentation using Pseudomonas putida.

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