Method for culturing biological cells

Abstract

The invention relates to a novel biological cell packaging technology for efficiently separating fermentation products based on a connected polyhedral framework. In particular, the present disclosure relates to linked polyhedral frameworks that can capture and encapsulate solutions containing biological cells, such as microbial cells, followed by biological fermentation and subsequent efficient product separation.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of biological cell fermentation. In particular, the present invention provides a novel biological cell capture and encapsulation technology that can efficiently isolate fermentation products from biological cells. BACKGROUND

[0002] Biological cell fermentation has a wide range of applications in biomedical, chemical engineering, food industry, agriculture, environmental protection, and other fields. After fermentation is completed, biological cells need to be separated from the fermentation broth, and then target products are extracted from the fermentation biological cells or outside the cells, which is called post-fermentation processing (DSP). The steps and technology selection of DSP play a decisive role in the quality and cost of the harvested fermentation products. However, due to the involvement of complex equipment and processes, including filtration, centrifugation, extraction, and flocculation, etc., the current DSP process is cumbersome and expensive. In some cases, the cost of recovering fermentation products can be as high as 80-90% of the total cost. Due to the random dispersion of biological cells, it is like finding a needle in a haystack to extract high-quality active products from complex fermentation broth containing biological cells, cell fragments, soluble and insoluble components, and fermentation products, which leads to the complexity and high cost of DSP. Therefore, there is an urgent need to develop a technology that can efficiently separate biological cell fermentation products to simplify the DSP process, improve the efficiency and quality of product separation, and reduce production costs. SUMMARY

[0003] The present disclosure provides a novel biological cell encapsulation technology that can efficiently isolate fermentation products in combination with interfacial gel and connected polyhedral frames.

[0004] The present invention generally relates to a method of producing and collecting fermentation products, comprising the steps of:

[0005] i) contacting a device composed of connected polyhedral frames with a solution containing biological cells, thereby capturing the solution containing biological cells within at least one polyhedral frame in the device;

[0006] ii) contacting the device with the solution containing biological cells with a medium capable of forming an interfacial gel with the solution containing biological cells, thereby encapsulating the biological cells within at least one polyhedral frame in the device;

[0007] iii) placing the device encapsulating the biological cells into a bioreactor for biological cell culture fermentation; and

[0008] iv) separating the device and the reactor, and collecting the extracellular products of the biological cells in the reactor; or

[0009] v) removing the device from the reactor, removing the interfacial gel on the device, releasing the biological cells within the polyhedral frames, and collecting the intracellular products;

[0010] in,

[0011] Each polyhedral frame of the device includes multiple ribs (101),

[0012] Two adjacent polyhedral frames are connected by a connecting rod (102);

[0013] In this context, the length of the free interface of each face of each polyhedral frame is within the capillary scale of the corresponding captured solution, thereby enabling each polyhedral frame to capture liquid within the polyhedral frame through capillary forces.

[0014] In some implementations, the interfacial gel is a membrane formed by a semi-permeable hydrogel, which can be generated through a gel reaction between divalent metal ions in a biological cell-containing solution and sodium alginate in a gelation medium. Common divalent metal sources include CaCl2, BaCl2, or ZnCl2. The pore size range of the semi-permeable hydrogel membrane on the framework surface allows small molecules (e.g., gaseous reactant molecules or fermentation product molecules) to enter and exit the polyhedral framework, while the size of micron-scale biological cells is larger than the pore size of the hydrogel membrane and is therefore encapsulated within the polyhedral framework.

[0015] In some embodiments, the biological cells suitable for use in this invention include bacterial cells, fungal cells, plant cells, or animal cells. These cells can be cultured in a bioreactor containing a culture medium suitable for the growth of these biological cells.

[0016] In some implementations, the biological cells captured within the polyhedral framework generate the target fermentation product after fermentation. The extracellular products of the fermenting biological cells can pass through a semi-permeable hydrogel membrane into the reaction solution of the bioreactor, and the target product can be separated and collected from the reaction solution after the device is removed. If the target product is an intracellular product of the fermenting biological cells, the interfacial gel on the polyhedral framework can be removed mechanically or chemically to release the cells within the polyhedral framework, thereby collecting the intracellular product.

[0017] In some implementations, the volume of the polyhedral frame is not particularly limited, as long as it can capture liquid through capillary force. The polyhedral frame can be 3D printed, and its substrate composition does not affect the function of the trap. The material composition can be selected from one or more of the following, depending on the application requirements: metal, polymer, polylactic acid (PLA), or polyurethane (PU). The geometry of the polyhedral frame is flexible and can adopt one or more of the following or combinations thereof: tetrahedral, pentahedral, hexahedral, octahedral, dodecahedral, icosahedral, tridecahedral, etc. Various shapes and spatial distributions of connected devices are formed by connecting these polyhedral frames with single bars. The ribs of the polyhedral frame can be surface modified to improve its wettability. Attached Figure Description

[0018] Figure 1 An example of the polyhedral frame connection of the present invention is shown;

[0019] Figure 2 A schematic diagram of a biological cell capture, encapsulation, and fermentation according to an embodiment of the present invention is shown;

[0020] Figure 3 A schematic diagram of an embodiment of the present invention for separating extracellular products of fermented organisms is shown;

[0021] Figure 4 A schematic diagram of an embodiment of the present invention for separating intracellular products of fermenting organisms is shown;

[0022] Figure 5 a to Figure 5 e illustrates an example of a bacterial encapsulation application for isolating microbial electrosynthetic products according to an embodiment of the present invention; wherein... Figure 5 The diagram shows that after the acetic acid-producing bacteria solution containing CaCl2 is captured by a device containing a polyhedral framework, the device is immersed in a sodium alginate solution to undergo interfacial gelation, thereby successfully encapsulating the acetic acid-producing bacteria in the capture device. Subsequently, the encapsulated capture device is placed in a bioreactor for microbial electrosynthesis, and the target product of the acetic acid-producing bacteria, acetic acid, can pass through the hydrogel semipermeable membrane into the reaction solution. Figure 5 b shows that the trap, which is composed of a connected array of polyhedral frames according to the present invention, successfully captured and encapsulated acetic acid-producing bacteria. Figure 5 c and 5d show the microstructure of the hydrogel semipermeable membrane on the surface of the trap, where the nanopore structure of the hydrogel membrane is shown. Figure 5 e shows a specific example of a reactor according to the invention for electrosynthesis reactions by acetic acid-producing bacteria. Figure 5 In bd, the scale bars represent 5mm, 1μm and 200nm respectively.

[0023] Figure 6 This shows the relationship between the concentrations of acetic acid and suspended bacteria in the solution and time during the electrosynthesis reaction of acetic acid-producing bacteria. Detailed Implementation

[0024] Many aspects of the present invention will be fully set forth below in conjunction with the accompanying drawings. The following description is for illustrative purposes only and should not be construed as limiting the invention.

[0025] The frames, frame units, and polyhedral frames mentioned in this manual are interchangeable.

[0026] The present invention has at least the following advantages:

[0027] • By adjusting the concentration of the encapsulated biological cell solution and the size, geometry, number of units, and spatial arrangement of the connected polyhedral framework (CPF), the CPF can capture biological cells of a specific concentration and amount in its three-dimensional space.

[0028] • Combining CPF and interfacial gelation allows for easy differentiation of biological cells and fermentation products in three-dimensional space. Once the CPF is removed from the bioreactor, the biological cells and the extracellular products of the fermentation organisms can be separated.

[0029] • Because the semi-permeable hydrogel membrane encapsulates biological cells in CPF, the products within fermentation biological cells can be separated efficiently.

[0030] The semi-permeability of hydrogel membranes allows molecules of bio-fermentation reactants to freely enter the membrane and be replenished in a timely manner, while cell products can also diffuse out of the membrane promptly. Therefore, high yields of bio-fermentation products can be guaranteed, while avoiding the loss of bio-cells caused by mechanical stirring in traditional fermentation processes.

[0031] A capture device consisting of a connected polyhedral frame

[0032] Figure 1 This is a schematic diagram of a liquid capture device according to one embodiment. The device includes two cubic frames connected by a single rod. Each cubic frame is composed of mutually fixedly connected ridge bars 101, each ridge bar 101 forming an edge of the resulting cube. The solid surface of the capture device formed by the connected cubic frames has a contact angle of less than 90° with the target liquid. Therefore, when the capture device is lifted from the liquid into the air, the liquid is captured within the capture device due to capillary action. Figure 1 In the example shown, when the two cubic frames are lifted out of the liquid, the two cubic frames are connected to each other by a single rod 102. The connecting rod 102 only serves as a mechanical connection and does not affect the liquid capture function of each frame unit.

[0033] Those skilled in the art will understand that the liquid capture device of the present invention may include any number of polyhedral frames, each polyhedral frame including a plurality of ribs (101) connected as edges of a polyhedron. Multiple polyhedral frame units can be connected to form a set of structures or combined to form a polyhedral frame array structure (see [link]). Figure 5 b). Each set of connected polyhedral frames may include several polyhedral frame units, which are connected in sequence, and adjacent polyhedral frame units are connected by a connecting rod (102).

[0034] As the connected polyhedral frame is slowly lifted from the liquid into the air, the lifted frame units utilize capillary action to trap the liquid within the frame. Those skilled in the art will understand that the connecting rod 102 can be rigid or flexible, depending on the specific implementation.

[0035] In this disclosure, it is preferred to immerse the device containing the polyhedral framework into a solution containing biological cells and then remove the device to capture the biological cell solution. In some embodiments, the solution containing biological cells can be captured by other contact methods, such as pouring or spraying the solution onto the device. These methods can be used to capture and culture biological cells outdoors in non-laboratory settings.

[0036] The polyhedral frame unit in this invention features flexible geometry design. Each connected polyhedral frame geometry can adopt one or more of the following, or a combination thereof: tetrahedral, pentahedral, hexahedral, octahedral, dodecahedral, icosahedral, trisouciral, etc. The CPF of this invention exhibits broad compatibility with different liquids and frame materials. The frame and connecting rods can be made from any malleable material using conventional mechanical manufacturing methods or 3D printing. Materials can be stainless steel and polymeric materials, including polylactic acid (PLA) and polyurethane (PU). CPFs made from these materials all exhibit excellent liquid trapping performance.

[0037] The principle of CPF capturing liquid

[0038] See Figure 1 The CPF consists of interconnected frame units, each of which is a polyhedron at the capillary scale that can be wetted by the liquid. As the frame is slowly lifted out of the liquid, capillary forces, that is, the adhesion force of the solid polyhedral frame to the liquid, can overcome the gravity of the liquid, enabling the frame to capture the liquid.

[0039] The liquid capture function of CPF can be understood as follows: when the CPF is lifted from the liquid, the hydrophilic frame provides capillary force to capture the liquid, while the individual rods between the frames only serve as mechanical connections and do not affect the independent liquid capture behavior of each frame.

[0040] CPF liquid encapsulation

[0041] In one embodiment, a semi-permeable hydrogel membrane formed from sodium alginate is used to encapsulate the liquid captured by the CPF. Sodium alginate is a linear anionic polysaccharide, mainly found in brown algae such as Sargassum and giant kelp, and is one of the main components of the intercellular matrix and cell wall of algae. Sodium alginate has the molecular formula (C6H7NaO6)x and is a linear copolymer composed of two units: α-L-guluronic acid (G unit) and β-D-mannuronic acid (M unit) linked by β-1,4-glycosidic bonds. Its molecular structure is mainly composed of GM, GG, and MM units linked in different sequences in a certain proportion, with molecular sizes ranging from several thousand to hundreds of thousands. The G and M units are spatial isomers and have significantly different properties. Different arrangements and proportions of the G and M units will lead to changes in the stability, viscosity, metal ion selectivity, and gel properties of sodium alginate.

[0042] Sodium alginate forms a gel primarily because its M and G units can bind to divalent metal ions (such as calcium ions), and the affinity of the M and G units for metal ions differs. G units bind more readily to calcium ions, while M units do not distinguish between calcium ions and other metal ions and can bind to any metal ion. The molecular chain segment of the G unit has a double-folded helical conformation, forming serrated hydrophilic spaces in the middle. After the sodium ion and divalent metal ion connected to the carboxyl groups of two adjacent G units undergo a displacement reaction, a tightly packed gel structure is formed. Different divalent metal ions have different affinities for sodium alginate, ranked in order of binding affinity as follows: Pb 2+ >Cu 2+ >Cd 2+ >Ba 2+ >Sr 2+ >Ca 2+ >Zn 2+ >Mn 2+ The pore size of the hydrogel membrane in this invention should be smaller than the size of a biological cell, approximately 50 nm. Since biological cells are in the micrometer range, they cannot pass through the hydrogel membrane to enter the fermentation broth outside the membrane.

[0043] Figure 2This invention illustrates a bio-cell encapsulation technology for the efficient separation of fermentation products according to a certain embodiment. When a hydrophilic CPF (cell-containing polymeric fungicide) is lifted from a cell-containing solution A into the air, the solution within the CPF is successfully captured due to capillary action provided by the solid framework. Next, the CPF containing the cell-containing solution A is immersed in solution B. Gelation at the interface between solutions A and B encapsulates solution A within the CPF, forming a stable liquid core-membrane shell structure. The cell-encapsulated CPF is then placed in a bioreactor for fermentation. Because the semi-permeable hydrogel membrane formed by the interfacial gelation has nanoscale pores, the cell-containing fungicide is confined within the membrane during fermentation, while reactant molecules can enter and replenish the membrane, allowing the bioreaction products to diffuse out.

[0044] In some embodiments, the solution containing biological cells contains divalent metal ions (e.g., CaCl2), which can form an interfacial gel with the sodium alginate solution to encapsulate the biological cell solution within the CPF. In other embodiments, the solution containing biological cells contains sodium alginate, which can contact the CaCl2-containing solution medium to form an interfacial gel to encapsulate the biological cell solution within the CPF. As mentioned above, compounds capable of reacting with sodium alginate to form an interfacial gel film are not limited to CaCl2, but also include other divalent metal ion compounds, such as zinc chloride and barium chloride. In some embodiments, the device encapsulating the cell solution containing sodium alginate can be contacted with a solution containing divalent metal ions (e.g., CaCl2) by any means, such as pouring or spraying, to form a hydrogel film.

[0045] Bio-fermentation

[0046] It should be understood that the term "fermentation" generally refers to the process of culturing microorganisms (bacteria and fungi) and producing a target product, while methods for preparing cell products using plant and animal cells are generally referred to as cell culture / production. In this disclosure, the terms "culture of biological cells" and "fermentation" are used interchangeably to refer to the process of culturing and producing a target product using biological cells.

[0047] Biological cells suitable for use in this invention include bacterial cells, fungal cells, plant cells, and animal cells.

[0048] The bacterial and fungal cells suitable for use in this invention can be derived from the genera *Clostridium*, *Zymonomas*, *Escherichia*, *Salmonella*, *Rhodococcus*, *Pseudomonas*, *Bacillus*, *Lactobacillus*, *Enterococcus*, *Alcaligenes*, *Klebsiella*, *Paenibacillus*, and others. The genera *Arthrobacter*, *Corynebacterium*, *Brevibacterium*, *Pichia*, *Candida*, *Hansenula*, *Synechococcus*, *Synechocystis*, *Anabaena*, *Lactococcus*, *Saccharomyces*, *Brevibacterium*, or *Microbacterium*.

[0049] More specifically, biological cells include, but are not limited to, the following species: *Escherichia coli*, *Alcaligenes eutrophus*, *Bacillus licheniformis*, *Zymonomas mobilis*, *Paenibacillus macerans*, *Rhodococcus erythropolis*, *Pseudomonas putida*, *Bacillus subtilis*, *Lactobacillus plantarum*, *Corynebacterium glutamicum*, *Enterococcus faecium*, *Enterococcus gallinarum*, *Enterococcus faecalis*, *Saccharomyces cerevisiae*, *Synechocystis* sp., and *Anabaena*. The bacteria include *Alcaligenes sp.*, *Ralstonia eutropha*, *Lactococcus lactis*, and *Synechococcus elongatus*.

[0050] The biological cells can also be algal cells such as green algae, red algae, and blue-green algae. In one implementation, the biological cells are Chlorella vulgaris, Porphyridium purpureum, Spirulina platensis, or Synechococcus elongates.

[0051] Plant cells can be derived from freshly prepared plant cells or commercially available plant cell lines. The main plant cell sources are Arabidopsis thaliana, yew, tobacco, alfalfa, rice, tomato, and soybean. Tobacco cell lines BY-2 and NT-1 are the most widely used because their cells are easily transformed and propagated, and they exhibit good growth characteristics. In addition, rice, tomato, and soybean cell lines are more suitable host cells than tobacco cell lines because rice, tomatoes, and soybeans are edible, highly controllable, have high expression levels, can effectively secrete proteins, and have high protein content.

[0052] Biological cells can also be mammalian cells, such as CHO and CHOK1SV. GS-KO, GS-CHO, CHO-K1, BSC-1, NS0 myeloma cells, CV-1, COS-1, COS-7, P3X3Ag8.653, C127, 293EBNA, MSR293, Colo25, U937, SP2 cells, L cells, human embryonic kidney HEK293 cells, young hamster kidney BHK21 cells, African green monkey kidney VERO-76 cells, HELA cells, VERO, BHK, MDCK, WI38 cells, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, CRL7O3O, HsS78Bst cells, PER.C6, SP2 / O-Ag14, myeloma cell lines, hybridoma cell lines, human lung cells W138, retinal cells, human liver cancer cell lines (Hep. G2) and hybridoma cells.

[0053] Biological cell culture media can be selected according to different cell types. The basic components of the culture medium include:

[0054] • Nutritional source – a source of protein, vitamins, minerals, and carbohydrates; primarily a mixture of peptone and meat extracts.

[0055] • Energy source – the source of carbohydrates; primarily glucose

[0056] • Basic minerals – a source of trace and macro minerals; primarily found in peptone and meat extracts.

[0057] • Buffers – reagents that maintain optimal pH levels; primarily specific amino acids, phosphates, citrates, and zwitterions.

[0058] pH indicator – A reagent that indicates changes in the pH of a culture medium through color changes; primarily phenol red.

[0059] • Selective agents – reagents that allow only specific bacteria to grow; such as antibiotics, tellurites, azides, and bile salts.

[0060] • Hardener – A reagent that can keep the culture medium in a solid or semi-solid state when needed; agar is widely used, although gelatin is sometimes used.

[0061] Certain substances, such as growth factors and enzymes, can also be incorporated into specific bacterial culture media.

[0062] Common culture media include: LB medium, basal salt (M9), Hanahan broth (SOB medium), SOC medium, 2X YT medium, NZCYM broth, etc. Common yeast culture media include LB (Luria-Bertani) medium, LLB (Low Salt LB) medium, YPD (also known as YEPD), BMGY medium, BMMY medium, YPDS+Zeocin medium, minimal glycerol medium (MGY), glucose regeneration medium (RD), etc. Common mammalian culture media include BME, DMEM, IMDM, MEM, NEAA, etc.

[0063] Fermentation products can be divided into intracellular products and extracellular products. If the extracellular products (i.e., those that have entered the fermentation broth) are to be collected, the trapping device can be directly removed from the reactor, and the fermentation broth can be collected for downstream processing to collect the products. Figure 3 On the other hand, if the product to be collected is intracellular, the interfacial gel on the CPF can be removed mechanically or with solvents, the biological cells can be released from the CPF, and the biological cells can be processed downstream to collect the product. Figure 4 Various methods for removing interfacial gels already exist in the art. For example, sodium citrate solution can be used to rapidly dissolve calcium alginate hydrogel membranes. The citrate ions of sodium citrate can chelate with calcium ions to form calcium citrate complexes, which have been proven to be an effective method for dissolving alginate hydrogels cross-linked with calcium chloride. In addition, alginate hydrogel membranes can also be dissolved using alginate lyase solution.

[0064] The CPF of this invention can be reused repeatedly. For example, after a single use of the CPF through a biological reaction, it can be sterilized and cleaned at room temperature or high temperature, depending on the material of the CPF.

[0065] Figure 5 a to Figure 5 b illustrates a setup for a bacterial encapsulation application for isolating microbial electrosynthetic products according to an embodiment of the present invention. Figure 5 a. Schematic diagram of bacterial encapsulation using CPF via interfacial gelation. An acetogenic bacteria solution containing calcium chloride is first captured by CPF, then immersed in a sodium alginate solution for interfacial gelation to form a semi-permeable hydrogel membrane, completing the encapsulation of the acetogenic bacteria. The CPF-encapsulated acetogenic bacteria are placed in a classic H-type reactor for microbial electrosynthesis. The acetogenic bacteria are confined within the semi-permeable membrane, while reactants can be replenished by timely entry into the membrane, and reaction products can diffuse out and be separated. Figure 5 b, CPF encapsulated with acetic acid-producing bacteria having a liquid core-hydrogel membrane shell structure. Figure 5 (cd) Microporous structure of semipermeable hydrogel membrane. Figure 5 e, a specific experimental apparatus used for bacterial encapsulation applications.

[0066] The semipermeable membrane exhibits a porous structure with a pore size of approximately 50 nm. Therefore, during microbial electrosynthesis, micron-sized acetic acid-producing bacteria are confined within the membrane, while gaseous reactants such as H2 and CO2 can readily enter and replenish the membrane, allowing cell products like acetic acid to diffuse out. This facilitates the separation of biological cells from reaction products, simplifies the reaction process, and improves cell utilization.

[0067] Bioreactors can contain the culture medium required for cell growth and the instruments needed to monitor the fermentation process (e.g., pH meters), inlet, outlet, and aeration port. Alternatively, the fermentation process can be carried out in a conventional container containing microbial fermentation broth without the need for additional monitoring instruments (see [link to bioreactor]). Figure 5 e).

[0068] Example

[0069] Fabrication and operation of connected polyhedral frames (CPF)

[0070] The CPFs were printed using a commercial resin PlasCLEAR V2 on an Asiga Freeform Pico 2 printer. Stainless steel CPFs were printed using a commercial selective laser melting printer, and PLA and PU CPFs were printed using a commercial fused deposition modeling printer. A lifting system was used to dynamically manipulate the movement of the CPFs. This system included a motorized precision translation stage that drove the printed CPFs along a predetermined path, with precise adjustment for both direction and speed.

[0071] Bacterial encapsulation and fermentation

[0072] (i)Packaging

[0073] The bacterial solution used in the bacterial encapsulation experiment was a mixed culture mainly composed of acetic acid-producing bacteria containing 1 wt.% CaCl2. Prior to bacterial encapsulation, the CPF was treated with oxygen plasma for 2 minutes to obtain a superhydrophilic surface. The bacterial solution containing 1 wt.% CaCl2 was first captured by the CPF, then immersed in a 1 wt.% sodium alginate solution for 2 minutes for interfacial gelation to complete bacterial encapsulation. After rinsing the encapsulated structure with distilled water, it was immersed in a 1 wt.% CaCl2 solution for 2 minutes to stabilize the hydrogel membrane. After rinsing the structure with distilled water, it was immersed in a microbial reaction solution for microbial electrosynthesis. To characterize the microstructure of the semi-permeable hydrogel membrane, the membrane on the CPF was subjected to critical point drying using a K850 critical point dryer and CO2. Ethanol was used as the solvent during dehydration. The microstructure of the dried membrane was characterized by SEM. Figure 5 c, d).

[0074] (ii) Fermentation

[0075] Microbial electrosynthesis reactions are performed using a classic H-type reactor ( Figure 5 e). The bacterial-encapsulated CPF was placed in the cathode reaction chamber. The cathode reaction chamber was separated from the cathode chamber by a cation exchange membrane. Every 24 hours, 5 ml of the catholyte was sampled for pH testing, volatile fatty acid (VFA) analysis, and suspended microbial concentration measurement. After sampling, an equal volume of fresh catholyte was injected, and the pH of the catholyte was maintained at 7 using 1 mol / L NaOH solution. The acetic acid concentration in the solution was obtained by VFA analysis using gas chromatography. VFA in the liquid sample was first extracted with diethyl ether, followed by gas chromatography analysis using a flame ionization detector and capillary column. Nitrogen was used as the carrier gas at a flow rate of 9.4 ml / min, and the injector and detector temperatures were 230 °C. For suspended microbial concentration analysis, the optical density of the liquid sample was measured at 600 nm (OD600) using a UV-Vis spectrophotometer. Changes in OD600 were used as an indicator of whether bacteria had escaped from the CPF.

[0076] The results are as follows Figure 5 As shown in Figure a, when the CaCl2-containing acetic acid-producing bacteria solution captured in the CPF comes into contact with the sodium alginate solution, a semi-permeable hydrogel membrane is formed, encapsulating the acetic acid bacteria within a truncated icosahedral CPF. The microstructure of the semi-permeable hydrogel membrane exhibits a porous structure with a pore size of approximately 50 nm. Figure 6 Therefore, during the microbial electrosynthesis reaction, the micron-sized acetic acid-producing bacteria are confined inside the membrane, while gaseous reactants such as H2 and CO2 can enter the membrane in a timely manner to replenish the product, and the acetic acid product can diffuse out. Figure 3 The experimental results showed that the acetic acid concentration in the reactor continued to increase, while the bacterial concentration (OD600) in the reactor remained almost constant, indicating that the bacteria were successfully encapsulated in the CPF. Figure 4 and ​ After fermentation is complete, the fermentation products can be collected from the culture medium or cells through simple operations. This example demonstrates that the device of the present invention can achieve efficient separation of bacteria and reaction products, simplify the microbial fermentation process, and improve the utilization rate of bacteria.

[0077] Although the invention has been described with reference to specific embodiments, various modifications and alterations may be made without departing from the scope of this disclosure. Therefore, the specification and drawings should be considered illustrative rather than restrictive, and all such modifications are intended to be included within the scope of this disclosure. Any benefit, advantage, or solution to the problem described herein with reference to specific embodiments is not intended to be construed as a key, desired, or essential feature or element of any or all of the claims.

Claims

1. A method for producing and collecting fermented biological cell products, comprising the following steps: i) The device, which is composed of connected polyhedral frames, is brought into contact with a solution containing biological cells, thereby capturing the solution containing biological cells within at least one polyhedral frame in the device. ii) Contact the device containing the biological cell solution with a medium that can form an interfacial gel with the biological cell solution, thereby encapsulating the biological cell within at least one polyhedral frame in the device; iii) The device containing biological cells is placed in a bioreactor for biological cell culture and fermentation; and iv) Separate the apparatus and reactor, and collect the extracellular products of the biological cells in the reactor; or v) Remove the device from the reactor, remove the interfacial gel from the device, release the biological cells within the polyhedral framework, and collect the intracellular products; in, Each polyhedral frame of the device includes multiple ribs (101), Two adjacent polyhedral frames are connected by a connecting rod (102); In this context, the length of the free interface of each face of each polyhedral frame is within the capillary scale of the corresponding captured solution, thereby enabling each polyhedral frame to capture liquid within the polyhedral frame through capillary forces.

2. The method according to claim 1, wherein the interfacial gel is a semi-permeable hydrogel with a pore size in the range of 10-100 nm.

3. The method according to claim 1, wherein the solution containing biological cells contains divalent metal ions.

4. The method according to claim 1, wherein the medium for the interfacial gel reaction with the solution containing biological cells is a sodium alginate solution.

5. The method according to claim 1, wherein the solution containing biological cells contains CaCl2, and the interfacial gelation medium is a sodium alginate solution.

6. The method of claim 1, wherein the biological cell is a bacterium, fungus, plant cell or animal cell.

7. The method according to claim 1, wherein the biological cells are acetic acid-producing bacteria, lactic acid bacteria, acetic acid bacteria, short bacilli, corynebacterium, yeast, Bacillus, lactic acid bacteria, micrococci, streptococci, pedicillin, or Leuconostoc mesenteroides.

8. The method of claim 1, wherein the interfacial gel on the polyhedral framework is removed by mechanical means or chemical reagents.

9. The method of claim 1, further comprising a step of reusing the device containing the polyhedral frame.

10. The method of claim 1, wherein the polyhedral frame is formed by 3D printing.

11. The method of claim 1, wherein the polyhedral frame is made of a metallic material, a polymeric material, polylactic acid, or polyurethane.

12. The method of claim 1, wherein the polyhedral frame comprises one or more of the following or a combination thereof: tetrahedral frame, pentahedral frame, hexahedral frame, octahedral frame, dodecahedral frame, icosahedron, and trichosahedron.

13. The method of claim 1, wherein the ribs of the plurality of polyhedral frames are surface modified to improve their wettability to the liquid to be captured.

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