3D printing bio-ink as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to 3D printing bio-ink and a preparation method and application thereof. The 3D printing bio-ink provided by the invention has good printability, biocompatibility and cascade metabolism function promotion effect, can be applied to rapid construction of a double-bacterium cascade degradation triclocarban engineering living body material which is high in bacterium content, stable in long-term operation and capable of efficiently degrading triclocarban (TCC), and is further applied to environmental restoration. The construction scheme of the artificial microbial community is widened, and a new thought is provided for environmental microbial remediation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to 3D printing bio-inks, their preparation methods, and applications. Background Technology

[0002] As an emerging manufacturing technology, 3D bioprinting can rapidly construct artificial microbial communities and precisely control their spatial structure, demonstrating attractive potential for regulating community functions such as cascade metabolism. For example, 3D bioprinting can be used to regulate the spatial structure of artificial microbial communities, enhance microbial cascade metabolism, and thus strengthen their environmental remediation capabilities.

[0003] However, the 3D printing of artificial microbial communities places extremely high demands on both the printing performance of bio-inks and their internal microenvironment. The former requires bio-inks to possess excellent rheological properties and mechanical strength to ensure smooth printing, printing accuracy, and long-term stability; while the latter requires bio-inks to have a sufficiently mild and suitable microenvironment to meet the basic life activities and metabolic enhancement needs of microorganisms. Therefore, designing bio-inks that simultaneously meet the requirements of 3D bioprinting and cascade metabolic regulation remains a crucial problem that urgently needs to be solved in regulating the cascade metabolism of artificial microbial communities through 3D bioprinting. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide 3D printing bio-ink, its preparation method and application. The present invention provides a method for preparing bio-ink using strain Ochrobactrum sp. TCC-2 and strain Diaphorobacter sp. LD72, providing a new approach for environmental microbial remediation.

[0005] This invention provides the application of a strain combination in the preparation of 3D printing bio-ink, the strain combination comprising strain Ochrobactrum sp. TCC-2 and strain Diaphorobacter sp. LD72.

[0006] In some embodiments, the strains Ochrobactrum sp. TCC-2 and Diaphorobacter sp. LD72 were provided by Professor Liang Bin of Harbin Institute of Technology (Shenzhen) and published in 2020 in "Bioaugmentation of trichocarban and its dechlorinated congeners contaminated soil with functional degraders and the bacterial community response".

[0007] In some embodiments, the OD of the strains Ochrobactrum sp. TCC-2 and Diaphorobacter sp. LD72 600 The ratio of values ​​is (0.1~10):(0.1~10).

[0008] In some specific embodiments, the OD values ​​of the strains Ochrobactrum sp. TCC-2 and Diaphorobacters sp. LD72 are... 600 The ratio is 1:1.

[0009] This invention provides a 3D printing bio-ink, comprising a matrix material and a composite microbial culture, wherein:

[0010] The matrix material includes polyether F127 diacrylate, κ-carrageenan, nano-silica, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0011] The composite microbial culture solution includes strain Ochrobactrum sp. TCC-2, strain Diaphorobacters sp. LD72, and liquid culture medium.

[0012] In some embodiments, the matrix material comprises polyether F127 diacrylate, κ-carrageenan, nano-silica, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in a mass ratio of (0.5~4):(0.05~0.4):(0.05~0.4):(0.005~0.05).

[0013] In some embodiments, the OD values ​​of the strains Ochrobactrum sp. TCC-2 and Diaphorobacter sp. LD72 in the composite microbial culture are... 600 The ratio of values ​​is (0.1~10):(0.1~10).

[0014] In some embodiments, the 3D printing bio-ink comprises liquid culture medium, 0.05~0.4 g / mL polyether F127 diacrylate, 0.005~0.04 g / mL κ-carrageenan, 0.005~0.04 g / mL nano-silica, 0.0005~0.005 g / mL lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, and OD. 600 strains Ochrobactrum sp. TCC-2 and OD values ​​ranging from 0.1 to 10 600 The strain Diaphorobacter sp. LD72 has a value of 0.1 to 10.

[0015] In some specific embodiments, the 3D printing bio-ink comprises liquid culture medium, 0.2 g / mL polyether F127 diacrylate, 0.02 g / mL κ-carrageenan, 0.02 g / mL nano-silica, 0.0025 g / mL lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, and OD. 600 The strain Ochrobactrum sp. TCC-2 with an OD value of 4.0 600 The strain Diaphorobacter sp. LD72 has a value of 4.0.

[0016] The present invention provides a method for preparing the 3D printing bio-ink, comprising mixing the matrix material and the composite microbial liquid to obtain the 3D printing bio-ink.

[0017] This invention provides engineered living materials, including the 3D printing bio-ink described above and / or the 3D printing bio-ink prepared by the preparation method described above.

[0018] In some embodiments, the engineered living material has a mesh-like structure.

[0019] The present invention provides a method for preparing the aforementioned engineered living material, comprising printing and cross-linking the 3D printing bio-ink to obtain the engineered living material.

[0020] In some embodiments, the crosslinking includes photocrosslinking and ionic crosslinking.

[0021] This invention provides the application of the 3D printing bio-ink, the 3D printing bio-ink prepared by the preparation method, the engineered living material, and / or the engineered living material prepared by the preparation method in the degradation of triclocarban.

[0022] The present invention provides a method for degrading triclocarban, characterized in that it includes mixing the engineered living material and / or the engineered living material prepared by the preparation method with the contaminant.

[0023] Compared with existing technologies, the 3D printing bio-ink provided by this invention has excellent printability, biocompatibility, and cascade metabolic function promotion effects. It can be used to rapidly construct engineered living materials with high bacterial content, long-term stable operation, and efficient triclocarban (TCC) degradation through a dual-bacterial cascade, and then applied to environmental remediation. This invention broadens the construction schemes for artificial microbial communities and provides new ideas for environmental microbial remediation. Attached Figure Description

[0024] Figure 1The rheological properties of the hydrogel matrix components used in different bio-inks are analyzed. Among them, (a) shows the changes of storage modulus and energy dissipation modulus with shear strain, (b) shows the changes of storage modulus and energy dissipation modulus with shear angular frequency, (c) shows the curve of apparent viscosity with applied shear rate, and (d) shows the curve of apparent viscosity with shear stress.

[0025] Figure 2 The Herschel-Bulkley model rheological parameters of the hydrogel matrix components used in different bioinks are shown in Figure 1. (a) is the power law exponent n, (b) is the yield stress τ0, and (c) is the consistency coefficient K.

[0026] Figure 3 The image shows SEM images of TCC-Cascade ELMs and their embedded bacteria, where (a) is an SEM image of F-127DA, (b) is an SEM image of TCC-Cascade ELMs, (c) is an SEM image of embedded Ochrobactrum sp. TCC-2, and (d) is an SEM image of embedded Diaphorobacter sp. LD72.

[0027] Figure 4 The figure shows the degradation of TCC and the dynamic changes of its metabolites in different degradation systems. (a) shows the change of TCC concentration over time, (b) shows the change of 4CA concentration over time, and (c) shows the change of DCA concentration over time.

[0028] Figure 5 This study demonstrates the adsorption behavior of TCC on the blank TCC-Cascade ELMs scaffold and changes in metabolites.

[0029] Figure 6 The degradation efficiency of TCC in the TCC-Cascade ELMs cyclic degradation experiment is shown.

[0030] Figure 7 The degradation of TCC and the dynamic changes of metabolites in the degradation experiments of TCC-Cascade ELMs with different configurations are shown. Among them, (a) shows the change of TCC concentration over time, (b) shows the change of 4CA concentration over time, and (c) shows the change of DCA concentration over time. Detailed Implementation

[0031] This invention provides 3D printing bio-inks, their preparation methods, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0032] The bacteria used in this invention are *Ochrobactrum* sp. TCC-2 and *Diaphorobacter* sp. LD72. Strain TCC-2 catalyzes the hydrolysis of the diamide bond in TCC, producing two intermediates: 4-chloroaniline (4CA) and 3,4-dichloroaniline (DCA). Strain LD72 further degrades the intermediates DCA and 4CA. These strains were provided by Professor Liang Bin of Harbin Institute of Technology (Shenzhen) and were published in 2020 in *Bioaugmentation of triclocarban and its dechlorinated congeners contaminated soil with functional degraders and thebacterial community response*.

[0033] All materials used in this invention are commercially available products. The polyether F127 diacrylate (F-127DA), the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl)ate (LAP), and the portable photocurable light source (EFL-LS-1601) used in this invention were all purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. (EFL). κ-carrageenan (κCA), nano-silica (nSi), and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The invention is further illustrated below with reference to examples.

[0034] Example 1

[0035] Preparation method and printability characterization of NICE-bioink bio-ink.

[0036] (1) Two strain culture methods

[0037] Two bacterial strains stored at -80℃ were removed and inoculated onto solid LB agar plates. After revival by incubation at 30℃, they were stored at 4℃ for later use. Fresh single colonies were picked and inoculated onto LB liquid medium, and incubated overnight at 30℃ and 150 rpm in a shaker to activate the bacteria. Subsequently, the bacteria were isolated, washed with mineral salt medium, and resuspended to the desired bacterial concentration to obtain Ochrobactrum sp. TCC-2 and Diaphorobacter sp. LD72 bacterial suspensions for later use.

[0038] The mineral salt culture medium contains the following components: 0.46 g / L ammonium chloride, 0.225 g / L ammonium sulfate, 0.123 g / L magnesium sulfate heptahydrate, 2.788 g / L sodium lactate, 11.91 g / L HEPES buffer, and 10 mL / L trace element stock solution. The trace element stock solution consists of the following components: 1.5 g / L hyponitrotriacetic acid, 0.1 g / L manganese chloride tetrahydrate, 0.3 g / L ferrous sulfate heptahydrate, 0.17 g / L cobalt chloride hexahydrate, 0.1 g / L zinc chloride, 0.04 g / L copper sulfate pentahydrate, 0.005 g / L potassium aluminum sulfate dodecahydrate, 0.005 g / L boric acid, 0.09 g / L sodium molybdate dihydrate, 0.12 g / L nickel chloride hexahydrate, 0.02 g / L sodium tungstate dihydrate, and 0.1 g / L disodium selenate. Mineral salt culture media must be autoclaved before use.

[0039] (2) Preparation of NICE-bioink

[0040] NICE-bioink includes composite microbial solutions containing matrix materials, specifically grouped as follows:

[0041] The experimental group F-127DA / κCA / nSi included 20% w / v polyether F127 diacrylate (F-127DA), 2% w / v κ-carrageenan (κCA), 2% w / v nano silica (nSi), 0.25% w / v lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), and OD. 600 The strain Ochrobactrum sp. TCC-2 with an OD value of 4.0 600 The strain Diaphorobacter sp. LD72 with a value of 4.0

[0042] The following control groups were set up respectively:

[0043] The control group F-127DA lacks the κ-carrageenan and nano-silica found in the experimental group F-127DA / κCA / nSi.

[0044] The control group F-127DA / κCA lacked the nano-silica found in the experimental group F-127DA / κCA / nSi.

[0045] The control group F-127DA / nSi lacks the nano-silica found in the experimental group F-127DA / κCA / nSi.

[0046] The preparation method is selected from the following methods 1 and 2:

[0047] Method 1: Pre-activated Ochrobactrum sp. TCC-2 and Diaphorobacter sp. LD72 bacterial suspensions were mixed, and the concentration of each bacterium was 4.0 (based on OD600, prepared or diluted using mineral salt medium) after mixing all bioink components. This mixture was then combined with other NICE-bioink components and homogenized using a vortex mixer. To ensure sterility, all solutes, solvents, and experimental containers were autoclaved at 121°C for 20 min. All solutions used were prepared or diluted using mineral salt medium.

[0048] Method 2: First, dissolve F-127DA in a 0.5% w / v LAP solution, then adjust the final LAP concentration to 0.25% w / v. After mixing, incubate at 4°C for 1 hour, then sterilize using a 0.22 µm aqueous filter membrane. Subsequently, uniformly disperse κCA and nSi into the F-127DA solution, mix thoroughly, and then add Ochrobactrum sp. TCC-2 and Diaphorobacter sp. LD72 bacterial cultures. The combined bacterial culture contains 4.0 g of each bacteria (in OD200). 600 (Calculation), to form the final different NICE-bioinks. To ensure sterility, all solutes, solvents, and experimental containers were autoclaved at 121°C for 20 min. All solutions used were prepared or diluted using mineral salt culture media.

[0049] (3) Testing the rheological properties of the NICE-bioink hydrogel matrix components

[0050] The rheological properties of the NICE-bioink hydrogel matrix F-127DA / κCA / nSi (experimental group) and its control group without bacterial components were characterized using a Bohlin INSTRUMENTS rheometer (CVOR200) ​​at 25 °C. The storage modulus and dissipation modulus of the hydrogel were evaluated using a conical-plate geometry. Its gelation kinetics were tested, and its self-supporting ability was investigated. The test results are shown below. Figure 1As shown in Figures (a) and (b). Subsequently, the shear rate and the relationship between shear stress and viscosity of the hydrogel matrix were measured, its shear thinning characteristics were tested, and its printability was studied. The test results are as follows: Figure 1 As shown in Figures (c) and (d).

[0051] Figure 1 Figure (a) shows the changes in storage modulus and dissipation modulus of different hydrogel matrix formulations within the shear strain range. The results indicate that in the low shear strain range, the storage modulus of all formulations is higher than the dissipation modulus, exhibiting elastic-dominated behavior, suggesting strong structural stability of the hydrogel at low strain. With increasing shear strain, the storage modulus gradually decreases, while the dissipation modulus gradually increases, reflecting the disruption of the internal network structure of the material. Notably, the experimental group F-127DA / κCA / nSi maintains a high storage modulus even under high shear strain, indicating that this hydrogel matrix possesses superior elasticity and resistance to deformation, reflecting that bio-inks using this hydrogel matrix are suitable for 3D bioprinting of complex structures. Figure 1 Figure (b) shows the changes in storage modulus and dissipation modulus with oscillation frequency scanning, further supporting the above conclusions. All hydrogels exhibit predominantly elastic behavior, suitable for maintaining shape stability under dynamic external forces. Furthermore, the storage modulus of the experimental group F-127DA / κCA / nSi is significantly higher than that of other hydrogels, demonstrating its stronger elastic properties.

[0052] Figure 1 Figure (c) shows that all formulations exhibited significant shear-thinning behavior in the shear rate scan test; that is, the viscosity of the hydrogel decreased significantly with increasing shear rate, facilitating printing. Furthermore, the hydrogel rapidly recovered its viscosity after shearing ceased, maintaining its post-printing stability. This shear-thinning behavior is crucial for ensuring the stability and printing accuracy of the hydrogel during the printing process. The experimental group F-127DA / κCA / nSi showed significantly improved elasticity and viscosity, exhibiting better flowability and interlayer stability during printing.

[0053] Figure 1 Figure (d) illustrates the analysis of the hydrogel's yield stress. Within the low shear stress range, the viscosity of all formulations remained stable, indicating good structural stability. When the shear stress reached the yield point, the viscosity decreased rapidly, indicating that the hydrogel entered a flow state. The experimental group F-127DA / κCA / nSi exhibited the highest yield point and a significant decrease in viscosity, indicating stronger resistance to structural damage and better maintenance of precise interlayer deposition during printing.

[0054] The Herschel–Bulkley (HB) model was used to fit and analyze its rheological properties. This model can characterize the shear thinning and yielding behavior of non-Newtonian fluids, and its mathematical expression is as follows: τ = τ0 + K(γ) n Where τ is the apparent stress, τ0 is the yield stress, γ is the shear rate, K is the consistency coefficient, and n is the power-law exponent, representing the non-Newtonian properties of the fluid. The HB model fitting parameters for the experimental group F-127DA / κCA / nSi and its two control groups (F-127DA / κCA and F-127DA) are shown below. Figure 2 .

[0055] Figure 2 In Figure (a), the HB model parameter n < 1 reflects the shear-thinning property of the hydrogel. The parameter n of the experimental group F-127DA / κCA / nSi is significantly lower than that of the control group (F-127DA). Therefore, the experimental group F-127DA / κCA / nSi enhances the shear-thinning effect, reduces flow resistance at high shear rates, and is beneficial for improving printing smoothness. Furthermore, compared to the control group, the experimental group F-127DA / κCA / nSi increases the consistency coefficient K and yield stress τ0, indicating that the bioink NICE-bioink using this hydrogel matrix possesses higher initial viscoelasticity, which helps maintain structural stability after printing, inhibits interlayer diffusion, and ensures printing accuracy.

[0056] Example 2

[0057] 3D printing construction and morphology testing of engineering living materials.

[0058] (1) 3D construction of engineered living materials (TCC-Cascade ELMs) for the degradation of triclocarban by two bacteria.

[0059] TCC-Cascade ELMs are built using a customized 3D bioprinting platform. First, a 3D model is created using computer-aided design and saved as an STL file. Then, the model is sliced ​​using the open-source software Ultimaker Cura and converted into G-code that the 3D printer can recognize. Finally, the generated G-code is imported into the 3D bioprinter for printing preparation.

[0060] NICE-bioink uses a 22G needle to extrude continuous hydrogel filaments during 3D bioprinting, and rapidly returns to a gel state after deposition, ensuring precise shaping of the printed structure. After printing, 405 nm blue light (25 mW / cm²) is used. 2The structure was irradiated for 30 seconds to induce free radical polymerization of the acrylate groups in F-127DA, forming an initial covalent cross-linked network that provides basic mechanical strength. Subsequently, the printed scaffold was immersed in a 5% w / v potassium chloride (KCl) solution for ionic cross-linking, forming a secondary cross-linked network, thereby further enhancing the mechanical properties and structural stability of the hydrogel. Through this dual cross-linking mechanism, NICE-bioink successfully constructed structurally stable and high-performance TCC-Cascade ELMs.

[0061] (2) Morphology test of hydrogels and TCC-Cascade ELMs.

[0062] The internal structure of the hydrogel and the distribution of bacteria in TCC-Cascade ELMs were analyzed using scanning electron microscopy (SEM, JEOL 6700F, JEOL Ltd., Japan). Samples were first fixed overnight in 2.5% glutaraldehyde, then dehydrated using a gradient of alcohols. Next, the samples were frozen in liquid nitrogen and dried in a freeze-drying oven for 24 hours. The dried samples were then fixed onto the sample stage with conductive adhesive and subjected to gold deposition via plasma sputtering for 60 s, observed at an accelerating voltage of 10 kV. The test results are as follows: Figure 3 As shown.

[0063] TCC-Cascade ELMs built with NICE-bioink Figure 3 (b) Figure 1 compared to the control group Figure 3 Figure (a) shows a more dense and complex three-dimensional porous network with a more uniform pore distribution and a significantly increased average pore size. These pore characteristics not only improve the mass transfer efficiency of TCC-Cascade ELMs but also provide a good spatial basis for bacterial colonization and activity maintenance. Figure 3 Figures (c) and (d) show that the functional bacterial strains *Ochrobactrum* sp. TCC-2 and *Diaphorobacter* sp. LD72 are uniformly distributed within the material and structurally intact in TCC-Cascade ELMs, indicating that the bacteria maintain a good physiological state after printing. These results demonstrate that NICE-bioink not only endows TCC-Cascade ELMs with excellent structural support properties but also provides a suitable microenvironment for bacterial embedding and long-term survival, thus providing both structural and biological safeguards for synergistic metabolism of the two bacteria and efficient degradation of pollutants.

[0064] Example 3

[0065] Pollutant degradation performance and cyclic testing of TCC-Cascade ELMs.

[0066] This experiment used homogeneous mesh-like TCC-Cascade ELMs for cyclic experiments to test the ability of TCC-Cascade ELMs to enhance bacterial degradation of TCC.

[0067] The method for printing homogeneous mesh-like TCC-Cascade ELMs is as follows: Pre-activated Ochrobactrum sp. TCC-2 bacterial suspension and Diaphorobacter sp. LD72 bacterial suspension are mixed, with each bacterial species comprising 4.0 (OD) units. 600 The bio-ink was mixed with other components of NICE-bioink and homogenized using a vortex mixer. The mixed bio-ink was then placed in a 4°C refrigerator to eliminate air bubbles. Subsequently, the bio-ink was transferred to a sterile 10 mL syringe, loaded into the material cartridge of a 3D bioprinter (Allevi 2, USA), and printed using a 22G needle at a speed of 70–80 mm / s at 25°C. Finally, TCC-Cascade ELMs were prepared through photocrosslinking and ionic crosslinking.

[0068] In the cyclic experiment, TCC-Cascade ELMs were removed after each test, rinsed clean, and reused for the next test. Each degradation cycle lasted 48 hours, and six cycles were performed consecutively. The experimental procedure was as follows: Homogeneous mesh-like TCC-Cascade ELMs were placed in a reactor containing simulated TCC wastewater and degraded TCC (0.5 mg / L) at 30℃ and 150 rpm for 48 hours. During this period, samples were taken periodically to detect the TCC removal, the concentration of intermediate products 4CA, and DCA. Free Ochrobactrum sp. TCC-2 single-bacterial group (TCC-2-Free) and free mixed-bacterial group (TCC-2+LD72-Free) were set as controls. The test results are shown in […]. Figure 4 As shown. To further clarify the ratio of adsorption to metabolism in pollutant removal, an adsorption experiment was conducted using a blank scaffold. The test results are shown below. Figure 5 As shown in the figure. The 24-hour removal rate after six cycles of the cyclic experiment is as follows. Figure 6 As shown.

[0069] The TCC simulated wastewater was prepared by mixing 0.5 mg / L of pollutant TCC with a mineral salt culture medium.

[0070] The TCC simulated wastewater was prepared by mixing 0.5 mg / L of pollutant TCC with a mineral salt culture medium. The mineral salt culture medium contained the following components: 0.46 g / L ammonium chloride, 0.225 g / L ammonium sulfate, 2.788 g / L sodium lactate, 11.91 g / L HEPES buffer, and 10 mL / L trace element stock solution. The trace element stock consists of the following components: 1.5 g / L hyponitrotriacetic acid, 0.1 g / L manganese chloride tetrahydrate, 0.3 g / L ferrous sulfate heptahydrate, 0.17 g / L cobalt chloride hexahydrate, 0.1 g / L zinc chloride, 0.04 g / L copper sulfate pentahydrate, 0.005 g / L potassium aluminum sulfate dodecahydrate, 0.005 g / L boric acid, 0.09 g / L sodium molybdate dihydrate, 0.12 g / L nickel chloride hexahydrate, 0.02 g / L sodium tungstate dihydrate, and 0.1 g / L disodium selenate. The stock solution concentration of calcium chloride and magnesium sulfate is 100 times. To prevent precipitation, the calcium chloride and magnesium sulfate solutions are autoclaved separately before being mixed with other components.

[0071] TCC was detected using a sacrificial sampling method. The sample was mixed with methanol at a 1:1 ratio and vortexed for 30 seconds. Degradation products were filtered through a 0.22 μm organic filter membrane and then directly injected for detection. TCC and its products were determined by high-performance liquid chromatography (HPLC, 1260 Infinity, Agilent Inc., USA). The instrument was equipped with an Agilent HC-C18 reversed-phase column (5 µm, 4.6 mm × 250 mm) and a UV-DAD detector. The mobile phase was acetonitrile and water containing 0.1% formic acid (60:40 v / v), the flow rate was 0.8 mL / min, and the injection volume was 10 µL. TCC was detected at 275 nm, and degradation products were detected at 240 nm. The column temperature was maintained at 30 °C.

[0072] Figure 4 Figure (a) shows that the TCC degradation rate of the free Ochrobactrum sp. TCC-2 single-cell group (TCC-2-Free) was significantly lower than that of the other three co-culture groups. Meanwhile, from Figure 4 Chinese (b) map Figure 4 As shown in Figure (c), during the 48-hour reaction, the degradation intermediates 4CA and DCA in the single-strain control group (TCC-2-Free) continued to accumulate (peak concentrations of 0.15 mg / L and 0.17 mg / L, respectively), while in the co-culture group, both intermediates were completely degraded within 36 hours, indicating that the introduction of LD72 achieved efficient removal of 4CA and DCA. Figure 4As shown in Figure (a), the degradation rate of the 3D bioprinted TCC-Cascade ELMs group (TCC+LD72-3D1) was lower than that of the free mixed-culture control group (TCC-2+LD72-Free) in the first cycle, which is presumably due to the limited diffusion within the ELMs and insufficient initial adaptation of the microorganisms. In the second cycle experimental group (TCC+LD72-3D2), the TCC degradation rate was significantly increased, surpassing that of the free mixed-culture control group. Meanwhile, Figure 4 Chinese (b) map Figure 4 Figure (c) shows that the peak concentrations of intermediate products 4CA and DCA in the secondary cycle experimental group were reduced by 34% and 32% respectively compared to the free co-culture group. This indicates that after the initial cycle, the microbial community formed a stable metabolic microenvironment within the NICE-bioink, with significantly enhanced metabolic activity. These results demonstrate that the 3D bioprinting system significantly optimized the metabolic activity and spatial distribution of the microbial community within the scaffold during the secondary cycle, forming a stable degradation microenvironment. Despite the performance limitations of the initial cycle, the increased degradation rate and efficient removal of intermediate products in the secondary cycle validate the potential of TCC-Cascade ELMs for long-term contaminant remediation.

[0073] Figure 5 The results showed that the TCC concentration remained relatively stable at 0.43–0.45 mg / L over 48 hours, and no intermediate products 4CA and DCA were detected. This indicates that the TCC-Cascade ELMs themselves have a weak physical adsorption capacity for TCC, and the significant removal of pollutants mainly depends on the metabolic degradation process of the two strains.

[0074] Figure 6 Experimental results show that TCC can be completely degraded within 24 hours in both the first and subsequent cycles, with no 4CA detected in all 6 cycles, and only a small amount of DCA detected in the 5th and 6th cycles. Therefore, the TCC-Cascade ELMs constructed by NICE-bioink can maintain excellent degradation efficiency under repeated use conditions, demonstrating their excellent structural stability and functional continuity.

[0075] Example 4

[0076] Pollutant degradation performance of different printing configurations of TCC-Cascade ELMs.

[0077] This experiment compared the degradation performance of three printing configurations on TCC: the homogeneous mesh structure experimental group (Lattice-ELMs), the homogeneous cylindrical control group (Bulk-ELMs), and the concentric cylindrical control group (Outer / Inner-ELMs). The experimental method was the same as in Example 3, and the test results are shown in [Figure 3]. Figure 7 As shown.

[0078] The experimental group Lattice-ELMs with the homogeneous grid structure and the control group Bulk-ELMs with the homogeneous block structure used the same bio-ink as described in Example 3. Both were printed as grids and blocks of the same volume. The control group Outer / Inner-ELMs with the dual-bacterial isolation structure used two bio-inks containing different single bacteria and were printed as concentric cylinders, with Diaphorobacter sp. LD72 inside and Ochrobactrum sp. TCC-2 outside.

[0079] Figure 7 The test results in Figure (a) show that the homogeneous grid-structured ELMs exhibited the highest TCC removal efficiency, followed by the concentric cylindrical ELMs, while the homogeneous cylindrical ELMs had the lowest removal efficiency. These experimental results demonstrate that the bio-ink provided by this invention can significantly enhance the cascade metabolic capacity of microorganisms for substrates such as TCC in water by constructing different spatial structures, optimizing strain distribution, and expanding the contact surface area, and can be applied in the field of environmental remediation. Figure 7 The concentration changes of DCA and 4CA in Figures (b) and (c) also show that the homogeneous mesh structure of ELMs resulted in the fastest metabolic removal, further confirming the above conclusion.

[0080] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of bacterial strain combinations in the preparation of 3D printing bio-ink, characterized in that, The strain combination includes strain Ochrobactrum sp. TCC-2 and strain Diaphorobacter sp. LD72.

2. The application according to claim 1, characterized in that, The OD values ​​of the strains Ochrobactrum sp. TCC-2 and Diaphorobacter sp. LD72 600 The ratio of values ​​is (0.1~10):(0.1~10). 3.3D printing bio-ink, characterized in that, Includes matrix materials and composite microbial solutions, wherein: The matrix material includes polyether F127 diacrylate, κ-carrageenan, nano-silica, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate. The composite microbial culture solution includes strain Ochrobactrum sp. TCC-2, strain Diaphorobacter sp. LD72, and liquid culture medium.

4. The 3D printing bio-ink according to claim 3, characterized in that, The matrix material comprises polyether F127 diacrylate, κ-carrageenan, nano-silica, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in a mass ratio of (0.5~4):(0.05~0.4):(0.05~0.4):(0.005~0.05).

5. The 3D printing bio-ink according to claim 3, characterized in that, In the composite microbial culture, the OD values ​​of strains Ochrobactrum sp. TCC-2 and Diaphorobacter sp. LD72 were... 600 The ratio of values ​​is (0.1~10):(0.1~10).

6. The 3D printing bio-ink according to any one of claims 3 to 5, characterized in that, The 3D printing bio-ink comprises liquid culture medium, 0.05~0.4 g / mL polyether F127 diacrylate, 0.005~0.04 g / mL κ-carrageenan, 0.005~0.04 g / mL nano-silica, 0.0005~0.005 g / mL lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, and OD. 600 strains Ochrobactrum sp. TCC-2 and OD values ​​ranging from 0.1 to 10 600 The strain Diaphorobactersp. LD72 has a value of 0.1 to 10.

7. The 3D printing bio-ink according to claim 6, characterized in that, The 3D printing bio-ink comprises liquid culture medium, 0.2 g / mL polyether F127 diacrylate, 0.02 g / mL κ-carrageenan, 0.02 g / mL nano-silica, 0.0025 g / mL lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, and OD. 600 The strain Ochrobactrum sp. TCC-2 with an OD value of 4.0 600 The strain Diaphorobacter sp. LD72 has a value of 4.

0.

8. The method for preparing the 3D printing bio-ink according to any one of claims 3 to 7, characterized in that, The method involves mixing the matrix material and the composite microbial liquid to obtain the 3D printing bio-ink.

9. An engineering living material, characterized in that, This includes the 3D printing bio-ink as described in any one of claims 3 to 7 and / or the 3D printing bio-ink prepared by the preparation method described in claim 8.

10. The method for preparing the engineered living material according to claim 9, characterized in that, This includes obtaining the engineered living material by printing and cross-linking the 3D printing bio-ink.

11. The application of the 3D printing bio-ink according to any one of claims 3 to 7, the 3D printing bio-ink prepared by the preparation method according to claim 8, the engineered living material according to claim 9, and / or the engineered living material prepared by the preparation method according to claim 10 in the degradation of triclocarban.

12. A method for degrading triclocarban, characterized in that, This includes mixing the engineered living material of claim 9 and / or the engineered living material prepared by the preparation method of claim 10 with contaminants.

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