Method for controlling spatial distribution of microbial community by adopting core-shell hydrogel microspheres

Abstract

The invention discloses a method for controlling spatial distribution of microbial communities by adopting core-shell hydrogel microspheres, which comprises the following steps: controllably packaging microbial components by adopting core-shell structure hydrogel microspheres, and constructing the core-shell structure hydrogel microspheres by utilizing a liquid-liquid phase separation mechanism; the spatial distribution of microbial communities is regulated and controlled by utilizing the difference of a nuclear-phase polymer solution on the aggregation mode and degree of microorganisms. The hydrogel microsphere with the core-shell structure has good mass transfer performance of nutrient substances and regulatory factors, has good biocompatibility, is suitable for growth and reproduction of microorganisms and maintains the activity of the microorganisms. The nuclear phase material of the hydrogel microsphere with the core-shell structure is easy to replace, so that the spatial distribution of the microflora in a microenvironment can be regulated and controlled. The method can be used as a micro-reactor platform for researching interaction of materials and microorganisms, quorum sensing, a microbial community forming process and regulation and control, and has huge development potential in the application fields of green biological manufacturing, engineering living body materials, plant growth promotion, environmental bioremediation, biomedicine and the like.

Description

Technical Field

[0001] This application belongs to the cross - field of biomaterials and microorganisms, and particularly relates to a method for controlling the spatial distribution of microbial communities using core - shell hydrogel microspheres. Background Art

[0002] Microorganisms usually live in dense and diverse communities, and different microbial populations can interact with each other and jointly form a structural unit with specific functions, "microbial community". These microbial communities support various processes, including sewage bioremediation, plant growth promotion, green biomanufacturing, chronic infections, and industrial biological pollution. In particular, due to biological and abiotic influences, these microbial communities form specific community spatial structures. Compared with suspended microorganisms, microorganisms living in a specific spatial structure can affect each other's evolutionary fitness through close interactions, thus having stronger adaptive advantages and playing specific functions.

[0003] The reproduction process of bacteria is often accompanied by environments rich in polymer materials such as extracellular matrix, mucus, and natural polymer solutions. For example, bacteria growing in soil, eutrophic water bodies, and animal host secretion environments all have a large amount of polymer environments. Bacteria have two ways to form bacterial aggregates in such polymer environments: bridging aggregation and depletion aggregation. Polymer bridging aggregation occurs when polymers adsorb to multiple bacteria and hold them together. On the other hand, depletion aggregation operates in an environment where non - adsorbed polymers and bacteria are present at high concentrations. In this case, the polymers in the region between adjacent bacteria become restricted. Since polymers are usually more abundant and more dynamic than bacteria, this constraint reduces the total entropy of the system, and depletion aggregation occurs to increase the entropy of the bacteria aggregates and thus increase the total entropy of the system.

[0004] Currently, due to the complexity of microbial communities and the limitations of related research methods, the research on the spatial structure of microbial communities is still in its infancy, lacking effective experimental platforms and environments to study the influence of different culture environments on the spatial distribution of microbial communities. Summary of the Invention

[0005] Object of the Invention: This application provides a method for controlling the spatial distribution of microbial communities using core - shell hydrogel microspheres, which can be used to study the spatial distribution of microbial communities at the micro - scale. Based on the different characteristics of different core - phase polymer solutions on microbial aggregation behavior, using the characteristics of polymer liquid - liquid phase separation and microfluidic technology, core - shell hydrogel microspheres with different core - phase liquid properties are prepared, and microorganisms are encapsulated to obtain loaded microbial core - shell hydrogel microspheres that can regulate the spatial distribution of microorganisms. This method can also be used as a research platform to explore the influence of different culture environments on the spatial distribution of microbial communities.

[0006] To this end, in one aspect of the present application, a method for controlling the spatial distribution of microbial communities using core-shell hydrogel microspheres is provided, including the following steps: (1) Resuspend different types of microorganisms in different core-phase polymer solutions, combine with a functionally modified shell-phase polymer solution, and use microfluidics to prepare core-shell hydrogel microspheres loaded with microorganisms; (2) Transfer the microspheres to a culture medium for microbial culture, and based on the regulation of the core-phase polymer solution on the microbial growth process, achieve the construction of a specific spatial distribution of microbial communities in the core-shell hydrogel microspheres; wherein, the core-phase polymer solution and the shell-phase polymer solution form a liquid-liquid phase separation system.

[0007] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the microorganisms include one or more of Escherichia coli, Bacillus subtilis, Lactobacillus plantarum, Agrobacterium, Bacillus pasteurii, cyanobacteria, and Chlorella vulgaris.

[0008] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the regulation of the core-phase polymer solution on the microbial growth process includes the influence of the polymer solution on the aggregation and dispersion behaviors of the microbial growth process; wherein, the bridging and depletion aggregation effects between the core-phase polymer and the microorganisms cause the microorganisms to grow in aggregates in the solution, showing the aggregation behavior of the microorganisms; or, when the core-phase polymer solution cannot bridge the microorganisms or one or more of its molecular weight, concentration, viscosity, microbial size, and density do not meet the conditions for depletion aggregation, the microorganisms grow dispersedly, showing the dispersion behavior of the microorganisms.

[0009] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the core-phase polymer solution capable of aggregating microorganisms includes gelatin (Gel) that undergoes bridging and sodium carboxymethyl cellulose (CMC), sodium alginate (Alg), and sodium hyaluronate (HA) that undergo depletion aggregation; the core-phase polymer solution capable of dispersing microorganisms includes dextran (Dex).

[0010] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the functionally modified shell-phase polymer solution in step (1) can polymerize to form a hydrogel without affecting phase separation, including any one of methacrylated gelatin (GelMA), acrylated polyether F127 (F127DA), methacrylated sodium alginate (AlgMA), methacrylated dextran (DexMA), polyethylene glycol dimethacrylate (PEGDA), and hyperbranched polyethylene glycol dimethacrylate (HB-PEGDA).

[0011] Optionally, in combination with any of the above aspects, in another implementation of this aspect, when the concentration of two incompatible polymers in the aqueous solvent in step (2) exceeds the threshold, a two-aqueous-phase system is formed, resulting in liquid-liquid phase separation. Thus, core-shell structured hydrogel microspheres with a liquid core environment are prepared by microfluidic means, including any one of methacrylated gelatin and sodium carboxymethyl cellulose (GelMA@CMC), methacrylated gelatin and sodium methyl cellulose (GelMA@MC), methacrylated gelatin and dextran (GelMA@Dex), methacrylated gelatin and sodium alginate (GelMA@Alg), methacrylated sodium alginate and gelatin (AlgMA@Gel), methacrylated dextran and sodium carboxymethyl cellulose (DexMA@CMC), acrylated polyether F127 and dextran (F127DA@Dex), acrylated polyether F127 and sodium carboxymethyl cellulose (F127DA@CMC), polyethylene glycol dimethacrylate and dextran (PEGDA@Dex), hyperbranched polyethylene glycol dimethacrylate and dextran (HB-PEGDA@Dex).

[0012] Optionally, in combination with any of the above aspects, in another implementation of this aspect, in the core-shell hydrogel microspheres loaded with microorganisms in step (1), the initial density of the microorganisms is in the range of OD value from 0.05 to 0.20, and the diameter of the core-shell structured hydrogel microspheres is 10 - 1000 μm, where the diameter of the core phase is 5 - 990 μm.

[0013] Optionally, in combination with any of the above aspects, in another implementation of this aspect, in step (2), the spatial distribution of the microbial community includes two spatial distribution patterns: spatial separation and spatial mixing. Among them, spatial separation means that the microorganisms are unevenly distributed in space, forming obvious spatial partitions or microhabitats; while spatial mixing is manifested as the unclear distribution boundary of the microorganisms in space, approaching a relatively uniform state.

[0014] Compared with the prior art, the present application has the following beneficial effects: (1) The core-shell hydrogel microspheres loaded with microorganisms prepared in the present application not only provide a good microhabitat for the reproduction of microorganisms, but also can regulate the spatial distribution of the microbial community by regulating the core-phase polymer. (2) The method for controlling the spatial distribution of the microbial community by using the core-shell hydrogel microspheres in the present application is novel, and the formation and evolution of the microscopic community spatial distribution can be visually observed through a microscope. (3) The core-shell hydrogel microspheres loaded with microorganisms in the present application can be used as a research platform to deeply study the bacterial aggregation behavior, the formation process of the microbial community, and the formation and regulation of a specific community space from the micro perspective. Description of the Drawings

[0015] Figure 1Fluorescence images of the growth states of E. coli in water-in-oil microdroplets of different nuclear-phase polymer solutions, where A and B are shown respectively; Figure 2 Distribution map for controlling the spatial separation state of the E. coli community in the core-shell hydrogel microspheres; Figure 3 Distribution map for controlling the spatial mixing state of the E. coli community in the core-shell hydrogel microspheres; Specific embodiments

[0016] The present application will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0017] The growth and aggregation of microorganisms in different polymer solutions are closely related to the molecular weight, concentration, viscosity, charge, functional groups, bacterial density, and bacterial surface properties of the polymer. For example, as Figure 1 shown, we found that E. coli is dispersed and grows in a spatially mixed manner in water-in-oil microdroplets of Dex solution, while E. coli aggregates and shows a tendency to grow in a spatially separated manner in water-in-oil microdroplets of CMC solution. Utilizing this, the aggregation behavior of bacteria can be regulated by polymer materials, thereby achieving the purpose of regulating the spatial distribution of bacterial communities.

[0018] In addition, for polymer solutions, when the concentrations of two incompatible polymers in an aqueous solvent exceed a threshold, a two-aqueous-phase system will be formed, resulting in liquid-liquid phase separation. By virtue of the phase separation generated by the two water-soluble polymers, a unique compartmental structure is obtained to study the regulatory effects of polymer solutions on microbial growth and microbial communities. Moreover, one of the polymers in the two-aqueous-phase is subjected to double-bond modification treatment so that it can form a gel network through photo-crosslinking with a photoinitiator. The double-bond-modified polymer serves as the shell phase, and the other polymer that is phase-separated from it serves as the core phase to load microorganisms. By using microfluidic technology, core-shell microspheres encapsulating microorganisms in the liquid core can be prepared to construct a microhabitat suitable for microbial growth, so as to deeply study microbial behavior at the microscopic level. Examples

[0019] Preparation of core-shell hydrogel microspheres with spatially separated E. coli communities using the GelMA@CMC system (1) Preparation of the GelMA shell-phase solution for core-shell microspheres: Weigh 0.24 g of the functionalized modified polymer material GelMA and 20 mg of the LAP blue-light initiator, and add them to 2 mL of sterilized LB medium. Dissolve the above materials at 30 °C and filter and sterilize them using a 0.22 μm filter head. The resulting homogeneous solution is the shell-phase precursor solution, which is equally filled into two 3 mL syringes connected with a PE tube for standby.

[0020] (2) Preparation of the CMC core-phase solution: Weigh 20 mg of CMC powder, dissolve it in 2 mL of sterilized LB medium to prepare a 1% (w / v) CMC solution, and filter and sterilize it with a 0.22 μm filter head for later use. Take 10 μL each of the red fluorescent Escherichia coli and green fluorescent Escherichia coli bacterial solutions that have been activated for 12 h and add them to 1 mL of the filtered CMC solution, resuspend and mix evenly, and load them into a 3 mL syringe connected to a PE tube for later use.

[0021] (3)Preparation of GelMA@CMC-loaded bacteria core-shell hydrogel microspheres by microfluidics Fix the syringes containing the shell-phase solution and the core-phase solution on the syringe pump, connect the microfluidic chip and set the parameters. The shell-phase is advanced at a total flow rate of 8 μL / min, the core-phase passes through the middle phase at a flow rate of 2 μL / min, and the continuous phase is sheared by fluorinated oil 7500 containing 2% surfactant at a flow rate of 40 μL / min. Use a microfluidic chip with dimensions of 150*150 μm and h = 75 μm, and perform spot blue light curing for 10 s in the collection channel to obtain core-shell hydrogel microspheres encapsulating two kinds of fluorescent Escherichia coli.

[0022] (4)Preparation of core-shell hydrogel microspheres with spatially separated Escherichia coli communities.

[0023] Remove the continuous phase from the bacteria-loaded core-shell hydrogel microspheres prepared in (3) using a syringe, add 10% perfluorohexyl ethanol (PFO) to demulsify, then wash 5 times with fluorinated oil 7500, and then disperse the core-shell microspheres in sterilized LB medium and let it stand to remove a small amount of fluorinated oil 7500. Repeat 3 times and then place them in an LB medium at 37 °C and shake culture at 160 rpm for 8 h to obtain core-shell hydrogel microspheres with spatially separated Escherichia coli communities. As Figure 2 shown, the fluorescence microscope images of Escherichia coli and the Gray Value-Distance curve graphs clearly reflect that the red fluorescent Escherichia coli and the green fluorescent Escherichia coli show spatially separated characteristics in the CMC solution. Therefore, the GelMA@CMC-loaded microorganism core-shell hydrogel microspheres have high value for studying the spatial separation distribution of microbial communities. Example

[0024] Use the F127DA@Dex system to prepare core-shell hydrogel microspheres with spatially mixed Escherichia coli communities.

[0025] (1)Preparation of the F127DA core layer solution: Weigh 0.4 g of the functionalized modified polymer material F127DA and 20 mg of the LAP blue light initiator, and add them to 2 mL of sterilized LB medium. Dissolve the above materials at 4 °C and filter and sterilize them using a 0.22 μm filter head. The resulting homogeneous solution is the shell phase precursor solution, which is equally loaded into two 3 mL syringes connected to a PE tube for standby.

[0026] (2)Prepare the Dex core phase solution: Weigh 120 mg of Dex powder, dissolve it in 2 mL of sterilized LB medium to prepare a 6% (w / v) Dex solution, and filter and sterilize it using a 0.22 μm filter head for standby. Take 10 μL each of the red fluorescent Escherichia coli and green fluorescent Escherichia coli bacterial solutions activated for 12 h and add them to 1 mL of the filtered Dex solution, resuspend and mix evenly, and load them into a 3 mL syringe connected to a PE tube for standby.

[0027] (3)Prepare F127DA@Dex-loaded bacterial core-shell hydrogel microspheres by microfluidics Fix the syringes containing the shell phase solution and the core phase solution on the syringe pump, connect the microfluidic chip and set the parameters. The shell phase is advanced at a total flow rate of 4 μL / min, the core phase travels in the middle phase at a flow rate of 1 μL / min, and the continuous phase is sheared by fluorinated oil 7500 containing 2% surfactant at a flow rate of 32 μL / min. Use a microfluidic chip with dimensions of 150*150 μm and h = 75 μm, and perform spot blue light curing for 5 s in the collection channel to obtain core-shell hydrogel microspheres encapsulating two kinds of fluorescent Escherichia coli.

[0028] (4)Prepare core-shell hydrogel microspheres with a spatially mixed Escherichia coli community.

[0029] Remove the continuous phase of the core-shell microspheres prepared in (3) using a syringe, add 10% perfluorohexyl ethanol (PFO) to demulsify, then wash with fluorinated oil 7500 five times, and then disperse the core-shell microspheres in sterilized LB medium and let them stand to remove a small amount of fluorinated oil 7500. Repeat this three times and then place them in an LB medium at 37 °C and shake culture at 160 rpm for 8 h to obtain core-shell hydrogel microspheres with a spatially mixed Escherichia coli community. As Figure 3 shown, the fluorescence microscope images of Escherichia coli and the Gray Value-Distance curve graphs clearly reflect the spatial mixing characteristics of red fluorescent Escherichia coli and green fluorescent Escherichia coli in the Dex solution.

[0030] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. In addition, it should be understood that although this specification is described according to the embodiments, it does not only contain one technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for controlling the spatial distribution of microbial communities using core-shell hydrogel microspheres, characterized in that: The method comprises the following steps: (1) resuspending different types of microorganisms in different core-phase polymer solutions, combining them with functionally modified shell-phase polymer solutions, and using microfluidics to prepare core-shell hydrogel microspheres loaded with microorganisms; (2) transferring the microspheres to a culture medium for microbial culture, and based on the regulation of the core-phase polymer solution on the microbial growth process, constructing a specific spatial distribution of microbial communities in the core-shell hydrogel microspheres; wherein the core-phase polymer solution and the shell-phase polymer solution constitute a liquid-liquid phase separation system.

2. The method according to claim 1, characterized in that: The microorganisms include one or more of Escherichia coli, Bacillus subtilis, Lactobacillus plantarum, Agrobacterium, Bacillus pasteurianus, cyanobacteria, and Chlorella.

3. The method according to claim 1, characterized in that: The regulation of the microbial growth process by the core phase polymer solution includes the influence of the polymer solution on the aggregation behavior and dispersion behavior of the microbial growth process; wherein, the core phase polymer and the microorganisms produce bridging and exhaustion aggregation effects, so that the microorganisms grow in agglomeration in the solution, showing the aggregation behavior of the microorganisms; or, when the core phase polymer solution cannot bridge the microorganisms or one or more of its molecular weight, concentration, viscosity, microbial size and density cannot meet the exhaustion aggregation, the microorganisms grow in a dispersed manner, showing the dispersion behavior of the microorganisms.

4. The method according to any one of claims 1 to 3, characterized in that: The core phase polymer solution capable of aggregating microorganisms includes bridging gelatin (Gel) and depletion aggregation sodium carboxymethyl cellulose (CMC), sodium alginate (Alg), and sodium hyaluronate (HA); the core phase polymer solution capable of dispersing microorganisms includes dextran (Dex).

5. The method according to claim 1, characterized in that The functionally modified shell phase polymer solution in the step (1) can be polymerized to form a hydrogel without affecting phase separation, including any one of methacryloyl gelatin (GelMA), acrylated polyether F127 (F127DA), methacryloyl sodium alginate (AlgMA), methacryloyl dextran (DexMA), polyethylene glycol dimethacrylate (PEGDA), and hyperbranched polyethylene glycol dimethacrylate (HB-PEGDA).

6. The method according to claim 1, characterized in that In the step (2), the liquid-liquid phase separation system is a system in which the concentration of two incompatible polymers in a water-based solvent exceeds a threshold value, forming a two-phase system, resulting in liquid-liquid phase separation, thereby preparing a core-shell structure hydrogel microsphere with a core phase in a liquid environment by microfluidic means, including methacrylated gelatin and sodium carboxymethyl cellulose (GelMA@CMC), methacrylated gelatin and sodium methyl cellulose (GelMA@MC), methacrylated gelatin and dextran (GelMA@Dex), methacrylated gelatin and sodium alginate (GelM Any one of the following: A@Alg), methacryloyl sodium alginate and gelatin (AlgMA@Gel), methacryloyl dextran and sodium carboxymethyl cellulose (DexMA@CMC), acrylated polyether F127 and dextran (F127DA@Dex), acrylated polyether F127 and sodium carboxymethyl cellulose (F127DA@CMC), polyethylene glycol dimethacrylate and dextran (PEGDA@Dex), and hyperbranched polyethylene glycol dimethacrylate and dextran (HB-PEGDA@Dex).

7. The method according to claim 1, characterized in that In the core-shell hydrogel microspheres loaded with microorganisms in step (1), the initial density of the microorganisms is an OD value in the range of 0.05-0.20, the diameter of the core-shell hydrogel microspheres is 10-1000 μm, and the diameter of the core phase is 5-990 μm.

8. The method according to claim 3, characterized in that In step (2), the spatial distribution of the microbial community includes two spatial distribution modes: spatial separation and spatial mixing, wherein spatial separation means that the microorganisms are unevenly distributed in space, forming obvious spatial partitions or microhabitats; Spatial mixing is manifested as the fact that the spatial distribution boundaries of microorganisms are not obvious and tend to be relatively uniform.

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