Microchip model for microbial visual fermentation and application thereof

Abstract

The invention provides a microchip model for microbial visual fermentation and an application of the microchip model. The chip comprises a ventilation layer, a semi-permeable membrane layer and a culture layer, a gas micro-channel and a gas chamber are arranged on the lower surface of the ventilation layer and are used for providing a gas environment required by microbial fermentation; the culture layer comprises a liquid microchannel and a culture chamber and is used for microorganism culture; the two layers are separated by a semipermeable membrane. The ventilation layer and the semi-permeable membrane layer are arranged above the culture layer to provide a suitable gas environment for microorganisms, meanwhile, the culture chamber is connected with the micro-pressure device to provide sufficient nutrient substances for the microorganisms, and the culture layer is provided with the transparent thin bottom to achieve real-time monitoring of single cell behaviors and the fermentation product synthesis process. The problems that traditional sampling is large in destructiveness and fermentation cannot be monitored in real time are solved, and an efficient research tool is provided for research on the fermentation product synthesis mechanism and in-situ modification of the fermentation product.

Description

Technical Field

[0001] This invention relates to the field of microbial culture and biomanufacturing technology, specifically to a microchip model and its application for visualizing microbial fermentation. Background Technology

[0002] Microbial fermentation refers to the process by which microorganisms (such as bacteria, yeast, or algae) transform substrates into products needed by humans through specific metabolic pathways under suitable conditions. The use of microbial fermentation to prepare materials is not only environmentally friendly but also economically beneficial, and is widely used in medicine, food, energy, chemical industry, and agriculture. The morphology, motility, interfacial distribution characteristics, and product formation of microorganisms directly affect the production efficiency of fermentation processes. However, commonly used research methods still have limitations. For example, judging fermentation status by extracting fermentation broth and detecting microbial cell concentration, substrate concentration, and metabolite accumulation is lagging, while sensor-based detection methods often carry the risk of contamination. Therefore, there is an urgent need for a visual culture device that can directly reflect the state of microbial fermentation and the product synthesis process.

[0003] Microchip technology is an emerging approach that utilizes micro- and nano-fabrication techniques to provide a controllable microenvironment. It allows for precise control of the microenvironment during microbial culture. Furthermore, its high-throughput characteristics significantly reduce labor costs and improve fermentation efficiency, accelerating the progress of related research. The purpose of this invention is to provide a microchip model for long-term monitoring and recording of microbial fermentation processes, which is of great significance for studying microbial fermentation status and product quality at the single-cell scale. Summary of the Invention

[0004] The purpose of this invention is to provide a microchip for visualizing microbial fermentation, which can intuitively monitor and record the fermentation status of microorganisms with long fermentation cycles and the synthesis process of fermentation products.

[0005] The objective of this invention is achieved through the following technical solution: A first aspect of the present invention provides a microchip for visualizing microbial fermentation, comprising, in sequence, an aeration layer, a semi-permeable membrane layer, and a culture layer. The lower surface of the aeration layer is provided with gas microchannels and a gas chamber to provide the gaseous environment required for microbial fermentation. The culture layer includes liquid microchannels and a culture chamber for microbial culture. The two layers are separated by a semi-permeable membrane. By providing a suitable gaseous environment for the microorganisms through the aeration layer and the semi-permeable membrane layer positioned above the culture layer, and simultaneously connecting the culture chamber to a micro-pressure device to provide sufficient nutrients for the microorganisms, wherein… The ventilation layer is located at the top and includes a gas inlet hole, a gas microchannel, two or more gas chambers, and a gas outlet hole, which are used to provide the gaseous environment required for microbial fermentation. The culture layer includes a liquid inlet pore, a liquid microchannel, two or more culture chambers, and a liquid outlet pore, and is used for microbial culture. A semi-permeable membrane is placed between the aeration layer and the culture layer, and is tightly connected to both layers; The upper gas chambers and the lower culture chambers are the same number and corresponding in position. Each gas chamber is located directly above each culture chamber and is separated by a semi-permeable membrane.

[0006] Preferably, the gas chamber is located on the lower surface of the ventilation layer, and the gas inlet and gas outlet holes penetrate the ventilation layer. The gas chamber is connected to the gas inlet and gas outlet holes by gas microchannels.

[0007] Preferably, the culture chamber is located on the upper surface of the culture layer, and the liquid inlet and liquid outlet holes penetrate through the ventilation layer and the semi-permeable membrane layer to the liquid culture layer. Each culture chamber is provided with a separate liquid inlet and liquid outlet hole, which are connected by liquid microchannels.

[0008] Preferably, the culture layer has a transparent thin bottom to enable real-time monitoring of single-cell behavior and the fermentation product synthesis process.

[0009] Preferably, the thickness of the culture chamber is 0.05-0.15 mm.

[0010] Furthermore, the cross-sectional area of ​​the culture chamber is smaller than that of the gas chamber.

[0011] Preferably, the gas inlet is connected to a gas pipeline; the liquid inlet is connected to a micro-pressure device, such as a peristaltic pump or a gravity drive device, to regulate the flow rate and velocity of the inlet and outlet liquids.

[0012] Furthermore, the gravity-driven device includes a liquid injector, a first connecting tube, and a second connecting tube. The liquid injector is connected to the first connecting tube, the first connecting tube is connected to and communicates with the liquid inlet hole of the microchannel structure, and the second connecting tube is connected to and communicates with the liquid outlet hole. The liquid level in the first connecting tube is higher than the height of the microchip, and the liquid level in the second connecting tube is lower than the height of the microchip.

[0013] Preferably, the microchip fabrication method includes, but is not limited to, laser etching, injection molding, and 3D printing.

[0014] Preferably, the ventilation layer and culture layer of the microchip are constructed from materials that are transparent and biocompatible, such as polydimethylsiloxane (PDMS), cycloolefin (COC), and glass.

[0015] Preferably, the semi-permeable membrane is a microporous breathable membrane material including silicone or PDMS.

[0016] Another aspect of the present invention proposes an application of a microchip for visualizing microbial fermentation, comprising the following steps: Step 1: The microchip is sterilized under high temperature and high pressure; Step 2: For aerobic microorganisms, open the gas valve to diffuse gas from the ventilation layer through the semi-permeable membrane layer into the culture chamber; or for anaerobic microorganisms, close the gas valve. Step 3: Under the drive of a micro-pressure device, inject microorganisms and culture medium into the microchip; Step 4: Place the microchip loaded with microorganisms from Step 3 in a suitable temperature for incubation, and continuously acquire images using a microscopic imaging system.

[0017] This invention's chip comprises a ventilation layer, a semi-permeable membrane layer, and a culture layer. The lower surface of the ventilation layer has gas microchannels and a gas chamber to provide the gaseous environment required for microbial fermentation. The culture layer includes liquid microchannels and a culture chamber for microbial culture. The two layers are separated by the semi-permeable membrane. This invention provides a suitable gaseous environment for microorganisms by placing a ventilation layer and a semi-permeable membrane layer above the culture layer. Simultaneously, a micro-pressure device is connected to the culture chamber to provide sufficient nutrients for the microorganisms. The culture layer has a transparent thin bottom to enable real-time monitoring of single-cell behavior and the synthesis process of fermentation products. This invention solves the problems of traditional sampling being highly destructive and unable to monitor fermentation in real time, providing an efficient research tool for studying the synthesis mechanism and in-situ modification of fermentation products.

[0018] The technical solution of the present invention has the following beneficial effects: 1) The chip model for visual fermentation provided by the present invention provides a stable and suitable fermentation microenvironment for microorganisms, and realizes real-time monitoring of fermentation under a microscope; 2) The optical resolution of the chip model for visual fermentation provided by the present invention enables observation of individual microorganisms; 3) The chip for visual fermentation of microorganisms provided by the present invention can provide more intuitive and powerful evidence for the mechanism study of the synthesis process of microbial fermentation products. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a schematic diagram of the microchip structure used for visual culture of microorganisms in Example 1. Figure 2 This is a top view of the microchip structure used for visual culture of microorganisms in Example 1; Figure 3 This is a physical image of the microchip used for visual culture of microorganisms in Example 1; Figure 4The image shows *Acetobacter xylinum* and its products being fermented, collected by a microchip used for microbial visualization culture in Example 1. Explanation of main icon numbers: 1. Ventilation layer; 2. Gas inlet hole; 3. Gas microchannel; 4. Gas chamber; 5. Gas outlet hole; 6. Semi-permeable membrane layer; 7. Culture layer; 8. Liquid inlet hole; 9. Liquid microchannel; 10. Culture chamber; 11. Liquid outlet hole. Detailed Implementation

[0020] The present disclosure will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the disclosure. Furthermore, it should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other. The present disclosure will now be described in detail with reference to the accompanying drawings and embodiments.

[0021] Example 1 A microchip for visualizing microbial culture, its layered schematic diagram is shown below. Figure 1 As shown, the structure sequentially includes an aeration layer 1, a semi-permeable membrane layer 6, and a culture layer 7. The lower surface of the aeration layer 1 is provided with gas microchannels 3 and gas chambers 4 to provide the gaseous environment required for microbial fermentation. The culture layer 7 includes liquid microchannels 9 and a culture chamber 10 for microbial cultivation. The two layers are separated by the semi-permeable membrane layer 6. By setting the aeration layer 1 and the semi-permeable membrane layer 6 above the culture layer 7, a suitable gaseous environment is provided for the microorganisms. Simultaneously, by connecting the culture chamber 10 to a micro-pressure device, sufficient nutrients are provided to the microorganisms. The ventilation layer 1 is located at the top and includes a gas inlet hole 2, a gas microchannel 3, four identical gas chambers 4, and a gas outlet hole 5, which are used to provide the gaseous environment required for microbial fermentation. The culture layer 7 includes a liquid inlet hole 8, a liquid microchannel 9, four identical culture chambers 10, and a liquid outlet hole 11, which are used for microbial culture. The semi-permeable membrane layer 6 is located between the ventilation layer 1 and the culture layer 7, and is tightly connected to both layers; The upper gas chamber 4 and the lower culture chamber 10 are the same in number and in corresponding positions. Each gas chamber is located directly above each culture chamber and is separated by a semi-permeable membrane layer.

[0022] like Figure 1 and Figure 2 As shown, the total thickness of the ventilation layer 1 is 3 mm, the width of the gas microchannel is 1 mm, and four annular gas chambers 4 are located on the lower surface of the ventilation layer 1. Each gas chamber 4 has a cross-section of a 5 mm diameter ring and a thickness of 0.5 mm; the gas inlet hole 2 and the gas outlet hole 5 penetrate the ventilation layer 1 through the gas microchannel 3 and are respectively located at both ends of the gas microchannel 3.

[0023] The total thickness of the culture layer 7 is 0.13 mm, the width of the liquid microchannel 9 is 1 mm, and four identical culture chambers 10 with a cross-sectional diameter of 4 mm are located on the upper surface of the culture layer 7 with a thickness of 0.05 mm. The liquid inlet hole 8 and the liquid outlet hole 11 penetrate the culture layer 7 and are respectively located at both ends of the liquid microchannel 9.

[0024] The microchip used in this embodiment for visualizing microbial culture is constructed from PDMS material and prepared using the following method: Step 1: Preparation of ventilation layer 1 and culture layer 7, specifically including: designing the chip structure and performing 3D modeling; 3D printing the anolyte membrane of ventilation layer 1 and culture layer 7 by photopolymerization; mixing silicon polymer and curing agent at a mass ratio of 10:1, stirring for 5 min and degassing treatment (vacuum degree 50 kPa, time 30 min) to obtain PDMS prepolymer; pouring the PDMS prepolymer into the anolyte membrane and letting it stand at 60℃ for 30 min; Step 2: Drop approximately 200 μL of PDMS prepolymer onto the surface of a glass slide. Using a gyroscope at a speed of 3000 RPM, coat the PDMS prepolymer evenly onto the surface to a thickness of approximately 20 μm. Allow the coating to stand at room temperature for 30 min to obtain a PDMS semi-permeable membrane layer 6. Step 3: After plasma treating the ventilation layer 1 and the PDMS semi-permeable membrane layer 6 at 150 W power and 0.5 mbar pressure for 40 seconds, quickly bond the treated sides together. Use a drill to make through holes at the gas and liquid inlet and outlet positions, with a hole diameter of 0.8 mm; then, After further plasma treatment of the above structure and the microfluidic side of culture layer 7 for 40 seconds (150 W power, 0.5 mbar pressure), the components were aligned and quickly bonded together to complete the assembly of the visualization fermentation microchip. A physical image of the microchip used for microbial visualization culture can be found here. Figure 3 .

[0025] Example 2 An application of a microchip for visual culture of microorganisms is provided, which is based on the aforementioned microchip for visual culture of microorganisms and includes the following steps: Step 1: The microchip is sterilized under high temperature and high pressure; Step 2: Connect the gas inlet hole 2 of the ventilation layer 1 to an external gas pipeline, open the gas valve, and diffuse oxygen from the ventilation layer 1 through the semi-permeable membrane layer 6 into the culture chamber 10. Step 3: Under the drive of a micro-pressure device, microorganisms and culture medium are injected into the microchip. Specifically, the Acetobacter xylinum seed culture is cultured to the logarithmic growth phase and then inoculated into the fermentation culture medium at a rate of 10% to prepare the bacterial solution. Step 4: Place the microchip loaded with microorganisms from Step 3 at a temperature of 29±1℃ and perform continuous imaging using a microscopic imaging system.

[0026] In this embodiment, the micro-pressure device is a liquid injector.

[0027] The microchip is connected to the injector, the first connecting tube and the second connecting tube. Under gravity, culture medium is continuously introduced into the liquid microchannel 9 through the injector containing the bacterial solution and the first connecting tube. The inlet and outlet flow rates are adjusted and controlled by adjusting the height difference between the first connecting tube, the microchip and the second connecting tube. A microchip connected to a gravity-driven device was placed on a microscope stage for fermentation, and continuous imaging was performed at an ambient temperature of 29±1℃ to observe the growth and movement patterns and product synthesis processes during microbial fermentation. The collected images of fermenting *Acetobacter xylinum* and its products are shown below. Figure 4 .

[0028] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A microchip for visualizing fermentation of microorganisms, characterized by, The microchip comprises a ventilation layer, a semi-permeable membrane layer and a culture layer in sequence, the lower surface of the ventilation layer is provided with gas microchannels and gas chambers for providing the gas environment required by microbial fermentation; the culture layer comprises liquid microchannels and culture chambers for microbial culture; the two layers are separated by the semi-permeable membrane; the ventilation layer and the semi-permeable membrane layer are arranged above the culture layer to provide a suitable gas environment for the microorganism, and the culture chambers are connected to the micro-pressure device to provide sufficient nutrients for the microorganism, wherein The ventilation layer is located above and comprises a gas inlet hole, gas microchannels, two or more gas chambers and a gas outlet hole for providing the gas environment required by microbial fermentation; The culture layer comprises a liquid inlet hole, liquid microchannels, two or more culture chambers and a liquid outlet hole for microbial culture; The semi-permeable membrane is arranged between the ventilation layer and the culture layer and is tightly connected to the two layers; The upper gas chambers and the lower culture chambers are the same in number and correspond in position, and each gas chamber is arranged directly above each culture chamber and is separated by the semi-permeable membrane.

2. The microchip for visualizing microbial fermentation according to claim 1, wherein The gas chambers are located on the lower surface of the ventilation layer, the gas inlet hole and the gas outlet hole penetrate through the ventilation layer, and the gas chambers are connected to the gas inlet hole and the gas outlet hole by the gas microchannels; The culture chambers are located on the upper surface of the culture layer, the liquid inlet hole and the liquid outlet hole penetrate through the ventilation layer and the semi-permeable membrane layer to the liquid culture layer, each culture chamber is provided with a separate liquid inlet hole and a liquid outlet hole, and each culture chamber is connected by the liquid microchannels.

3. The microchip for visualizing microbial fermentation according to claim 1, wherein The culture layer is provided with a transparent thin bottom to realize real-time monitoring of single-cell behavior and fermentation product synthesis process.

4. The microchip for visualizing fermentation of microorganisms according to any one of claims 1 to 3, wherein The thickness of the culture chamber is 0.05-0.15 mm.

5. The microchip for visualizing fermentation of microorganisms according to claim 4, wherein The gas chamber is annular.

6. The microchip for visualizing fermentation of microorganisms according to claim 1, wherein The gas inlet hole is connected to a gas pipeline, the liquid inlet hole is connected to a micro-pressure device through a connecting pipe, and the micro-pressure device comprises a peristaltic pump and a gravity-driven device to regulate the flow and flow rate of liquid inlet and outlet.

7. The microchip for visualizing fermentation of microorganisms according to claim 1, wherein The semi-permeable membrane layer is a microporous gas-permeable film comprising silica gel or PDMS.

8. Application of a microchip for visualizing microbial fermentation, which is carried out by the microchip according to any one of claims 1-7 and comprises the following steps: Step one: sterilizing the microchip by high-temperature and high-pressure sterilization; Step two: for aerobic microorganisms, opening the gas valve to diffuse gas from the ventilation layer to the culture chamber through the semi-permeable membrane layer; or for anaerobic microorganisms, closing the gas valve; Step three: injecting microorganisms and culture medium into the chip under the driving of the micro-pressure device; Step four: culturing the microchip loaded with microorganisms at a suitable temperature and continuously collecting images by a microscopic imaging system.

9. Use of a microchip for visualizing fermentation of microorganisms according to claim 8, characterized in that, The micro-pressure device comprises a liquid injector, a first connecting pipe and a second connecting pipe, the liquid injector is connected with the first connecting pipe, the first connecting pipe is connected with and communicates with a liquid inlet hole of the micro-channel structure, the second connecting pipe is connected with and communicates with a liquid outlet hole, a liquid level of the first connecting pipe is higher than a height of the micro-chip, and a liquid level of the second connecting pipe is lower than the height of the micro-chip.

Images