A 3D microbial printed body and its preparation method and application

Abstract

The present invention discloses a 3D microbial printed body and its preparation method and application. The preparation method of the 3D microbial printed body includes: step 1, using dual-network biological ink to perform vertical alternating 3D printing, fixing different microorganisms with quorum sensing effects in different printing layers to obtain a 3D microbial printed body; step 2, pre-crosslinking the 3D microbial printed body and post-crosslinking in a crosslinking solution to obtain a structurally stable 3D microbial printed body. The present invention uses a dual-network printing ink with high affinity and high mechanical properties to manufacture highly functional 3D microbial materials, so that it can ensure the high metabolic activity of bacteria in a pre-designed structure, and fix microorganisms with quorum sensing benefits in different areas by designing a specific structure, thereby improving the efficiency of bioremediation while having resistance to complex environmental impacts.

Description

Technical Field

[0001] The present invention belongs to the technical field of three-dimensional bioprinting, and specifically relates to a 3D microbial printed body and a preparation method and application thereof. Background Art

[0002] 3D bioprinting, also known as bioadditive manufacturing, has been recognized as a new method for preparing biomaterials. It is mainly used and developed in the fields of tissue engineering, biomedicine, etc. Based on the mature research results of 3D bioprinting technology in these fields, combined with the powerful biochemical mechanisms of bacteria, creating "microbial materials" with controllable 3D shapes, microstructures and dynamic metabolic reactions is a research direction worth exploring.

[0003] Current research indicates that many synergistic microorganisms exist in bioremediation technologies, promoting the growth and activity of functional bacterial communities by releasing signaling molecules. However, the commonly used collaborative bacterial species actually compete to a certain extent, competing for substrate in the same space. Therefore, quorum sensing has limitations in practical applications and requires technical improvements. Immobilizing collaborative bacterial species in different compartments can achieve the goal of efficient collaborative bioremediation while weakening or even avoiding interspecies competition. The gradient printing characteristics of 3D bioprinting technology provide a possible solution to this problem.

[0004] In bioprinting technology, the most critical breakthrough is improving the quality and efficiency of printing inks. Microbial 3D printing, especially the printing of bioactive materials for complex environmental pollution treatment, places high demands on the mechanical properties and biocompatibility of printing inks. When using single-component hydrogels, it is difficult for a single structure to simultaneously possess the physicochemical properties required for 3D printing and the biochemical properties required for encapsulating organisms. Therefore, it is necessary to explore multi-component printing inks with high biocompatibility and mechanical properties.

[0005] Currently, research on 3D microbial materials is very scarce, and there is a gap in the field of pollution treatment. Therefore, it is of great significance to develop a 3D quorum sensing functional microbial printed body based on highly functional double-network printing ink. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a method for preparing a dual-network bioprinting ink. The 3D microbial printed body constructed by 3D bioprinting using the dual-network bioprinting ink provided by the present invention has good physical properties and excellent biological activity, and has good application prospects in the field of bioprocessing for environmental pollution treatment.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing a 3D microbial printed body comprises the following steps:

[0009] Step 1: A first printing ink is obtained by mixing sodium alginate, gelatin, functional bacterial flora and water, and printing an x-direction printing layer with a printing angle of 0°;

[0010] Step 2: Sodium alginate, gelatin, a synergistic bacterial flora solution, and water are mixed to obtain a second printing ink, and a y-direction printing layer is printed on the upper surface of the x-direction printing layer. The printing angle of the y-direction printing layer is 90°.

[0011] Step 3: Printing a layer of the x-direction printing layer on the upper surface of the y-direction printing layer using the first printing ink;

[0012] Step 4: Printing a layer of the y-direction printing layer on the x-direction printing layer using the second printing ink;

[0013] Step 5: Repeat steps 3 and 4 in sequence until a 3D microbial printed body with a 3D structure is printed.

[0014] Preferably, in the first printing ink, the mass ratio of the sodium alginate, the gelatin, the functional bacterial liquid and the water is 2:(4-8):(10-20):100.

[0015] Preferably, the functional bacterial community includes one or more functional bacterial species such as denitrification bacteria and phosphorus removal bacteria extracted from activated sludge, landfill leachate or soil.

[0016] Preferably, in the second printing ink, the mass ratio of the sodium alginate, the gelatin, the synergistic bacterial liquid and the water is 2:(4-8):(10-20):100.

[0017] Preferably, the synergistic bacterial community includes one or more synergistic bacterial species that can release substances that promote the growth and biological activity of the functional bacterial community.

[0018] Preferably, the printing pressure of the first printing ink and the second printing ink is 10-500 kPa, the printing speed is 3-6 mm / s, the printing nozzle diameter is 0.41 mm, the extruder head temperature is 0-37° C., and the printing table temperature is 0-37° C.

[0019] Preferably, the obtained first printing ink and second printing ink are placed in a low temperature environment for pre-crosslinking, and then the 3D microorganism printed body that has completed the pre-crosslinking and printing process is placed in a crosslinking solution for post-crosslinking.

[0020] Preferably, the temperature range of the low-temperature environment is -20°C to 4°C, and the cross-linking solution is a calcium chloride solution.

[0021] Preferably, the mass fraction of the calcium chloride solution is 2%.

[0022] A second object of the present invention is to provide a 3D microbial printed body prepared by the above-mentioned preparation method.

[0023] A third object of the present invention is to provide an application of a 3D microbial printed body, wherein the 3D microbial printed body prepared by the above-mentioned preparation method is applied to environmental pollution treatment.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects:

[0025] (1) The bioprinting ink of the present invention uses sodium alginate as a matrix. The viscosity of the printing ink is enhanced by introducing gelatin with thermosensitive properties and optimizing its content. This allows the 3D microbial printed body to maintain its structure in a stable state before cross-linking after printing, thereby improving the extrusion molding ability of the printed body. After cross-linking, the printed body has both the rigidity of the gelatin network and the toughness of the sodium alginate network, significantly improving the mechanical properties of the printed body.

[0026] (2) In the 3D microbial printed body of the present invention, the functional bacterial community and the collaborative bacterial community are distributed in different printing layers, which weakens or even completely avoids the interspecies competition between the two to a certain extent, thereby maximizing their efficiency;

[0027] (3) Due to the complex structure of the 3D printing design and the specific ink structure of the configuration, the 3D microbial printed body has a good mass transfer effect. The signal molecules released by the collaborative bacterial community printing layer can be transferred to the functional bacterial community printing layer at an extremely fast speed. At the same time, the functional bacterial community and the external environment also have an excellent material exchange rate, which enables it to efficiently complete the collaborative bioremediation process;

[0028] (4) The sodium alginate and gelatin used in the present invention are both natural polymer hydrogels with good biocompatibility. They can improve the activity of the encapsulated microorganisms after molding while protecting the microorganisms from extreme external conditions and will not destroy the treatment environment of bioremediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the formability of Comparative Example 1 of the present application.

[0030] Figure 2 This is the macroscopic structure of Comparative Example 1 of the present application under an optical microscope.

[0031] Figure 3 This is the surface and cross-sectional micromorphology of Comparative Example 1 of the present application.

[0032] Figure 4 This is the surface and cross-sectional micromorphology of Comparative Example 2 of the present application.

[0033] Figure 5 This is the fluorescent image of bacteria alive and dead in Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0034] To make the technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0035] Example 1

[0036] A method for preparing a 3D printed body comprises the following steps:

[0037] (1) Prepare sodium alginate solution: accurately weigh 2 g of sodium alginate and add 50 mL of ultrapure water in a beaker. After mixing, transfer to a constant temperature water bath at 70°C and stir for 1.5 h. After the solution is completely dissolved, let it stand for 30 min to eliminate bubbles before use.

[0038] (2) Prepare gelatin solution: Accurately weigh 4 g of gelatin particles and add 50 mL of ultrapure water in a beaker. Mix the mixture and transfer to a constant temperature water bath at 40°C. Stir for 0.5 h to completely dissolve the gelatin particles. Allow to stand for 30 min to eliminate bubbles before use.

[0039] (3) Prepare dual-network printing ink: Mix the prepared sodium alginate and gelatin solution in a beaker and move to a constant temperature water bath. Set the temperature to 37°C and stir in the water bath for 1.5 hours to completely dissolve the mixture. Then let it stand for 30 minutes to eliminate bubbles and set aside for use.

[0040] (4) Pre-crosslinking: Install the printing needle (0.41 mm diameter, plastic needle) on the printing syringe (10 cc), and load the double network printing ink into the two printing syringes respectively. Let it stand for 30 minutes with the needle tip facing down to remove bubbles, and then move it to a refrigerator at 4°C and store it for more than 24 hours before use;

[0041] (5) 3D printing: Two printing syringes were installed in the dual channels of a pneumatic extrusion bioprinter for alternating layered printing. The printing size was 20 mm*20 mm*4 mm, and the printing parameters were adjusted as follows: printing interval 4 mm, layer height 0.4 mm, printing speed 4 mm / s, extruder temperature 25 °C, printing table temperature 25 °C, extrusion pressure 30-50 kPa, printing angle of the first channel was 0°, and printing angle of the second channel was 90°;

[0042] (6) Post-crosslinking: The printed product was immersed in a 2% calcium chloride solution for crosslinking for 6 h to obtain a 3D printed body.

[0043] Example 2

[0044] A method for preparing a 3D microbial printed body comprises the following steps:

[0045] (1) The method for preparing sodium alginate solution and gelatin solution is the same as that in Example 1;

[0046] (2) The first printing ink is prepared in the same manner as in Example 1, and a predetermined amount of functional bacterial liquid is added, with the liquid accounting for 20% of the ink;

[0047] (3) The second printing ink is prepared in the same manner as in Example 1, and a predetermined amount of synergistic bacterial liquid is added, with the liquid accounting for 20% of the ink.

[0048] (4) The pre-crosslinking, 3D printing and post-crosslinking methods are the same as in Example 1.

[0049] In the first printing ink, the functional bacteria include but are not limited to one or more functional bacteria species such as denitrification bacteria and phosphorus removal bacteria extracted from activated sludge, landfill leachate, and soil.

[0050] In the second printing ink, the synergistic bacterial community includes but is not limited to one or more synergistic bacterial species that can release and promote the growth and biological activity of the functional bacterial community.

[0051] Comparative Example 1

[0052] The method for preparing the gelatin solution is as follows: based on Example 1, the mass of gelatin added is set to 1g, 2g, 4g, 6g, and 8g, and the extrusion pressure in the corresponding 3D printing method is set to 0-10kPa, 0-30kPa, 30-50kPa, 80-100kPa, and 180-200kPa. The remaining steps are the same as Example 1.

[0053] Because sodium alginate requires calcium cross-linking after extrusion to achieve a stable gel structure, the presence of a gelatin system and low-temperature pre-cross-linking imparts specific rheological properties to the ink, enabling smooth extrusion and molding. The present invention compared and analyzed the ink material properties by configuring the aforementioned dual-network printing inks with varying gelatin concentrations.

[0054] Formability analysis results are as follows Figure 1As shown in the figure, different gelatin concentrations can lead to differences in the printing process and the printed body's formation. When the gelatin concentration is too low (w / v = 1%, 2%), the printing ink structure is too loose, close to liquid, making the printing process unable to form smoothly. At moderate gelatin concentrations (w / v = 2%, 4%), the printing ink has good rheological properties, allowing for smooth extrusion and stable forming. When the gelatin concentration is too high (w / v = 8%), the excessive gelatinization causes the ink shape to be closer to a solid state. Although printing can be formed, smooth extrusion is not possible.

[0055] Figure 2 、 Figure 3 Shown are comparisons of the macrostructures of three groups of prints that were successfully formed in the comparative example, as viewed under an optical microscope, and their microstructures under a scanning electron microscope. The images clearly show that the macrostructure of the 4% gelatin concentration is significantly clearer and smoother under the microscope. The bubbles and voids in the two groups with increasing gelatin content become increasingly severe, and resolution decreases, consistent with the comparison of ink formation during printing. This suggests that excessive cross-linking caused by high gelatin concentrations can lead to deviations and irregular stacking during the printing process, resulting in an irregular and loose internal structure and significantly reduced forming performance.

[0056] Therefore, the optimal ink composition obtained after comprehensive evaluation of ink performance, that is, the ink system with a gelatin concentration of 4%, is used as the dual-network bio-ink composition of the present invention.

[0057] Comparative Example 2

[0058] The method for preparing the first printing ink and the second printing ink is based on Example 2, except that the proportion of the added bacterial liquid is set to 0%, 10%, 20%, and 30%. The remaining steps are the same as Example 2.

[0059] Printing verification shows that when the bacterial liquid concentration is greater than 20%, the printing ink will lose some viscosity and mechanical strength due to the dilution effect, resulting in the inability to be smoothly extruded and molded. After the ink with bacterial liquid concentrations of 0%, 10%, and 20% was successfully printed and molded, in order to explore the microstructure of the 3D microbial print body and the growth of microorganisms inside the print body, scanning electron microscope and laser confocal microscope were used to observe it.

[0060] Figure 4 The surface and cross-sectional microstructures of 3D microbial prints prepared with 0%, 10%, and 20% bacterial solution concentrations were shown. The results showed that the 0% group exhibited smooth surfaces and cross-sections, while the microbial-loaded 3D prints exhibited partial bacterial structures, embedded in the cross-sections and surfaces of the prints, with a uniform distribution and high density. As the bacterial solution concentration increased, the density of the bacterial colonies immobilized within the prints also increased, confirming successful bacterial immobilization.

[0061] Figure 5 The fluorescence images of bacteria in the prepared 3D microbial prints with 0% / 10% / 20% bacterial solution concentrations are shown. It can be clearly seen from the images that in the early stage of the print formation, the 3D microbial prints with 10% and 20% bacterial solution concentrations both show very strong green fluorescence intensity (living cells) and very weak red fluorescence intensity (apoptotic cells), while the 4% The Gel group showed almost no fluorescence, directly proving that the printed body had successfully loaded the bacterial community and maintained extremely high biological activity. According to the results of the live-dead cell ratio analysis, the proportion of live cells accounted for over 90%. To verify the protection of microbial activity by the 3D microbial print and the preservation of the microbial print under refrigerated conditions, the print was refrigerated at 4°C and the fluorescence images of the print were observed on the 10th and 20th days. The images show that even without the necessary nutrients for the microbial print, the material still exhibited a certain degree of biological activity. However, over time, the red fluorescence intensity gradually increased, indicating that apoptosis had occurred on the 10th day, and apoptotic cells accounted for over 30% of the total bacteria. On the 20th day, the overall fluorescence intensity decreased. This phenomenon may be caused by the lysis of the bacterial cell wall under long-term freezing conditions, which in turn led to the destruction of bacterial RNA and prevented the successful nucleic acid staining of PI. The green fluorescence in the image indicates that although a large number of bacteria died at this stage, a certain number of viable bacteria still survived, which to some extent proves that the 3D microbial print has a good effect on maintaining its biological activity.

[0062] Based on the disclosure and teachings of the above description, those skilled in the art will be able to make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the above specific embodiments. Any obvious improvements, substitutions, or modifications made by those skilled in the art based on the present invention fall within the scope of protection of the present invention. In addition, although certain specific terms are used in this description, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. A method for preparing a 3D microbial printed body for use in environmental pollution treatment, characterized in that: The following steps are involved: Step 1: A first printing ink is obtained by mixing sodium alginate, gelatin, a functional bacterial flora solution, and water, wherein the functional bacterial flora includes denitrification bacteria and phosphorus removal bacteria extracted from activated sludge, landfill leachate, or soil, and the mass ratio of the sodium alginate, the gelatin, the functional bacterial flora solution, and the water is 2:(4-8):(10-20):

100. A single x-direction printing layer is printed, and the printing angle of the x-direction printing layer is 0°. Step 2: Sodium alginate, gelatin, a synergistic bacterial flora solution, and water are mixed to obtain a second printing ink, wherein the synergistic bacterial flora includes synergistic bacteria that can release synergistic bacteria that promote the growth and biological activity of the functional bacterial flora, and the mass ratio of the sodium alginate, the gelatin, the synergistic bacterial flora solution, and the water is 2:(4-8):(10-20):

100. A y-direction printing layer is printed on the upper surface of the x-direction printing layer, and the printing angle of the y-direction printing layer is 90°. Step 3: Printing a layer of the x-direction printing layer on the upper surface of the y-direction printing layer using the first printing ink; Step 4: Printing a layer of the y-direction printing layer on the x-direction printing layer using the second printing ink; Step 5: Repeat steps 3 and 4 in sequence until a 3D microbial printed body with a 3D structure is printed.

2. The method for preparing a 3D microbial printed body for environmental pollution treatment according to claim 1, characterized in that: The printing pressure of the first printing ink and the second printing ink is 10-500 kPa, the printing speed is 3-6 mm / s, the printing nozzle diameter is 0.41 mm, the extrusion head temperature is 0-37° C., and the printing table temperature is 0-37° C.

3. The method for preparing a 3D microbial printed body for environmental pollution treatment according to claim 1, characterized in that: The obtained first printing ink and second printing ink are placed in a low temperature environment for pre-crosslinking, and then the 3D microorganism printed body that has completed the pre-crosslinking and printing process is placed in a crosslinking solution for post-crosslinking.

4. The method for preparing a 3D printed microbial stamp for environmental pollution treatment according to claim 3, characterized in that: The temperature range of the low-temperature environment is -20°C to 4°C, and the cross-linking solution is a 2 wt% calcium chloride solution.

5. A 3D microbial printed body for use in environmental pollution treatment, characterized in that: Prepared by the preparation method according to any one of claims 1 to 4.

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