3D bioprinted engineering living material of bacteria-algae symbiosis and preparation method and application thereof

Abstract

The application discloses a 3D bioprinting bacteria-algae symbiotic engineering living material and a preparation method and application thereof. The method takes chlorella as a biological oxygen supply source, takes alkali-producing bacteria as phenol-degrading bacteria, takes sodium alginate and carboxymethyl cellulose as carriers, and respectively prepares bacterial bio-ink and algal bio-ink added with silicon dioxide, so as to build bacteria-algae symbiotic engineering living materials with stripe and layer configurations through a 3D bioprinting mechanism. The bacteria-algae symbiotic engineering living material realizes three-dimensional accurate arrangement and functional partition of bacteria and algae, and exhibits excellent degradation performance and photosynthetic performance under different phenol concentrations, and has application prospects in the biological degradation treatment of organic pollutants.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of environmental remediation materials and 3D bioprinting technology, and relates to a 3D bioprinted living material for bacterial-algae symbiotic engineering, its preparation method and application. Background Technology

[0002] With industrial development, organic pollutants such as phenol are increasingly polluting the water environment. Bioremediation technology has become an important direction for pollutant treatment due to its advantages such as environmental friendliness and low cost. Existing anaerobic-aerobic engineered living materials (AA-ELMs) achieve trans-aerobic metabolic degradation of pollutants through chemically released oxygen sources. However, chemical oxygen supply has problems such as the accumulation of toxic byproducts, unsustainable oxygen supply, and limited application scenarios, which leads to easy deactivation of materials and difficulty in stable operation.

[0003] Algal-microbe symbiotic systems utilize algal photosynthesis to produce oxygen, while bacteria degrade pollutants and release CO2 to support algal growth, forming a closed oxygen-carbon cycle—a sustainable bio-oxygenation strategy. However, traditional algal-microbe co-cultivation technologies face several bottlenecks: First, traditional suspension culture or immobilization methods struggle to precisely control the spatial arrangement of algae and bacteria, leading to limited light exposure for algae and uneven oxygen diffusion, thus affecting bacterial bioremediation efficiency. Second, algae are sensitive to the toxicity of pollutants; organic pollutants such as phenol may inhibit algal growth, impacting the long-term stability of the symbiotic system. Third, common immobilization strategies struggle to optimize metabolic collaboration between algae and bacteria, lacking systematic optimization of different spatial structures, making it difficult to precisely regulate algal-microbe interactions and improve degradation efficiency. Therefore, a construction technology capable of precisely building spatial structures, enhancing mass transfer processes, and improving algal-microbe synergy is urgently needed to overcome the bottlenecks in structural design and functional synergy of existing algal-microbe co-cultivation systems.

[0004] 3D bioprinting technology can achieve precise arrangement and functional partitioning of bacteria and algae in three-dimensional space. Through structural optimization, it is expected to improve the light utilization rate and oxygen supply efficiency of algae, enhance the system's tolerance to pollutant stress, and promote synergistic metabolism between bacteria and algae, thereby constructing more efficient and stable bacterial-algae engineered living materials. 3D printed bacterial-algae symbiotic materials encapsulate "energy-supplying" microalgae and "functional" bacteria together in a hydrogel matrix to construct a self-circulating living micro-ecosystem. Current preparation methods mainly use extrusion printing combined with ion or photocrosslinking technology. Reference 1 preliminarily verified the feasibility of constructing bacterial-algae symbiotic systems using 3D bioprinting, but lacked a systematic evaluation of spatial configuration optimization, synergistic mechanisms, and pollutant degradation performance (F.He, Y. Ou, J. Liu, Q. Huang, B. Tang, F. Xin, J. Zhang, M. Jiang, S. Chen, Z.Yu, 3D printed biocatalytic living materials with dual-network reinforcedbioinks, Small, 18 (2022) 2104820.). Summary of the Invention

[0005] The purpose of this invention is to provide a 3D bioprinted bacterial-algae symbiotic engineering living material, its preparation method and application, aiming to solve the problems of byproduct accumulation and unsustainable oxygen supply in existing chemical oxygen-supplying engineering living materials, as well as the bottlenecks of uncontrolled spatial arrangement, poor algal tolerance and low synergistic efficiency in traditional bacterial-algae co-culture systems, and to construct a highly efficient and stable bacterial-algae engineering living material for the synergistic degradation of phenol.

[0006] The technical solution for achieving the objective of this invention is as follows:

[0007] A method for preparing 3D bioprinted living materials for bacterial-algal symbiotic engineering includes the following steps:

[0008] (1) Sodium alginate (SA) and carboxymethyl cellulose (MC) were dissolved in TSB medium and a suspension of alkali-producing bacteria (Alcaligenes faecalis subsp. phenolicus) was added to prepare bacterial bio-ink. The concentration of sodium alginate was 3% w / v and the concentration of carboxymethyl cellulose was 9% w / v.

[0009] (2) Sodium alginate, carboxymethyl cellulose and silica were dissolved in TP medium and Chlorella vulgaris algal solution was added to prepare algal bio-ink. The concentration of sodium alginate was 3% w / v, the concentration of carboxymethyl cellulose was 9% w / v, and the concentration of silica was 0.5%~5% w / v. The silica was nano silica (nSiO2) or micro silica (µSiO2).

[0010] (3) Using a dual-nozzle 3D bioprinter, bacterial bio-ink and algal bio-ink are loaded respectively, and 3D printing is performed to obtain striped or layered 3D bioprinted bacterial-algal symbiotic engineering living materials (BA-ELMs).

[0011] Furthermore, in step (1), the alkali-producing bacteria are alkali-producing bacteria with the ability to degrade organic pollutants, including but not limited to phenol, azo dyes, etc.

[0012] Further, in step (1), the composition of the TSB medium is: 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate, pH=7.3.

[0013] Furthermore, in step (2), the concentration of silicon dioxide is 1% w / v.

[0014] Further, in step (2), the TP culture medium consists of: 20 g / L 1M Tris-Base stock solution, 10 g / L salt stock solution, 1 g / L phosphate buffer, and 1 g / L trace element stock solution; the salt stock solution consists of: 10 g / L MgSO4·7H2O, 5 g / L CaCl2·2H2O, and 75 g / L NaNO3; the phosphate buffer solution consists of: 108 g / L K2HPO4 and 56 g / L KH2PO4; the trace element stock solution consists of: 50 g / L disodium ethylenediaminetetraacetate, 0.20 g / L ferrous sulfate heptahydrate, 0.10 g / L zinc sulfate, 0.03 g / L manganese chloride tetrahydrate, 0.20 g / L cobalt chloride hexahydrate, 0.01 g / L copper chloride dihydrate, and 0.03 g / L sodium molybdate dihydrate.

[0015] Furthermore, in step (3), the 3D printing conditions are: the 3D printing nozzle size is 20 G, the air pressure is 250 kPa, and the printing speed is 6 mm / s.

[0016] Further, in step (3), the method for constructing striped BA-ELMs is as follows: bacterial bio-ink and algal bio-ink are arranged alternately along the same plane to form a striped interwoven composite structure; the method for constructing layered BA-ELMs is as follows: bacterial bio-ink is deposited first, and then algal bio-ink is deposited vertically above it to form an alternating layered structure; in both configurations of BA-ELMs, the filament spacing is 2.4 mm and the substrate area is 12 × 12 mm. 2 Each layer of thin strips is 90 o Alternating orientations were used to print a total of 6 layers.

[0017] This invention provides 3D bioprinting living materials for bacterial-algae symbiotic engineering prepared by the above-described method.

[0018] This invention also provides the application of the above-mentioned 3D bioprinted bacterial-algae symbiotic engineering living materials in the synergistic degradation of organic pollutants.

[0019] Furthermore, the organic pollutants are phenol or azo dyes.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) This invention provides continuous oxygen based on the photosynthesis of Chlorella, avoiding the problems of toxic accumulation of by-products and unsustainability in chemical oxygen supply methods, and realizes a closed oxygen-carbon cycle, making the system more economical, sustainable and environmentally friendly.

[0022] (2) This invention achieves precise arrangement and functional partitioning of bacteria and algae in three-dimensional space through 3D bioprinting, which improves the light utilization rate and oxygen supply efficiency of algae and enhances the metabolic synergy between bacteria and algae.

[0023] (3) The present invention developed a photosynthetic bio-ink with good printing adaptability. Adding 1% w / v nSiO2 significantly improved the effective quantum yield of algae (Y(II) increased from 0.50 to 0.55), and the photosynthetic electron transfer rate (ETR) increased to 5.0. At this concentration, the synergistic effect of photochemical reaction and electron transfer chain reached the optimal state.

[0024] (4) The 3D bioprinted bacterial-algae symbiotic engineering living materials with different configurations in this invention exhibit functional differentiation characteristics under pollutant concentration gradients. The striped BA-ELMs have higher degradation efficiency under 100 mg / L phenol conditions, while the layered BA-ELMs are more effective in maintaining algal photosynthetic activity under 200 mg / L phenol conditions. Attached Figure Description

[0025] Figure 1The effects of different concentrations and particle sizes of SiO2 on the photosynthetic performance parameters of Algae-ELMs are shown; among which (a) effective quantum yield Y(II) and (b) photosynthetic electron transport rate ETR are shown.

[0026] Figure 2 The effects of different concentrations and particle sizes of SiO2 on the photoprotection and energy dissipation parameters of Algae-ELMs are shown; (a) non-photochemical quenching quantum yield Y(NPQ) and (b) non-regulated energy dissipation quantum yield Y(NO).

[0027] Figure 3 The degradation effects of BA-ELMs with different configurations on phenol are shown; (a) under 100 mg / L phenol conditions, and (b) under 200 mg / L phenol conditions.

[0028] Figure 4 The photosynthetic performance was analyzed under different configurations and phenol conditions; (a) statistical analysis of Y(II) in different experimental groups, and (b) statistical analysis of Fv / Fm in different experimental groups. Detailed Implementation

[0029] To further understand the present invention, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0030] In the following examples, the alkali-producing bacteria used were alkali-producing bacteria capable of degrading phenol. The TSB medium consisted of: 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L glucose, 5 g / L sodium chloride, and 2.5 g / L potassium dihydrogen phosphate, pH 7.3. The TP medium consisted of: 20 g / L 1M Tris-Base stock solution, 10 g / L salt stock solution, 1 g / L phosphate buffer, and 1 g / L trace element stock solution. The salt stock solution consisted of: 10 g / L MgSO4·7H2O, 5 g / L CaCl2·2H2O, and 75 g / L NaNO3. The phosphate buffer solution consisted of: 108 g / L K2HPO4 and 56 g / L KH2PO4. The trace element stock solution consists of: 50 g / L disodium ethylenediaminetetraacetate, 0.20 g / L ferrous sulfate heptahydrate, 0.10 g / L zinc sulfate, 0.03 g / L manganese chloride tetrahydrate, 0.20 g / L cobalt chloride hexahydrate, 0.01 g / L copper chloride dihydrate, and 0.03 g / L sodium molybdate dihydrate.

[0031] Example 1

[0032] Sodium alginate and carboxymethyl cellulose were dissolved in TP medium, and different concentrations of nSiO2 or µSiO2 were added. Chlorella extract was also added to prepare algal bioink. The concentrations of sodium alginate (3% w / v), carboxymethyl cellulose (9% w / v), and nSiO2 or µSiO2 (0.5%, 1%, 2%, and 5% w / v) were used. The algal bioink was loaded onto a 3D bioprinter, extruded through a 20 G nozzle at a pressure of 250 kPa and a printing speed of 6 mm / s. The substrate area was 16 × 16 mm². 2 The filament spacing is 3 mm, the number of printing layers is 5, and the interlayer orientation is 90°. o Algae-ELMs were constructed. The effects of SiO2 concentration and particle size on the photosynthetic performance of 3D bioprinted Algae-ELMs were investigated by monitoring changes in the photosystem II effective quantum yield Y(II), photosynthetic electron transport rate ETR, non-photochemical quenching coefficient Y(NPQ), and unregulated energy dissipation ratio Y(NO).

[0033] like Figure 1 As shown, when the amount of nSiO2 added is 1%, Y(II) reaches a peak value of 0.55, and ETR increases to 5.0 simultaneously, indicating that the synergistic effect of photochemical reaction and electron transport chain reaches the optimal state at this concentration. When the concentration is higher than 1%, Y(II) and ETR gradually decrease, which is speculated to be due to particle aggregation or compression of the internal pore structure of the material, resulting in obstruction of light absorption efficiency and electron transport.

[0034] like Figure 2 As shown, Y(NPQ) continuously decreases, indicating that the introduction of nanoparticles significantly reduces the photoprotection requirement; the synchronous increase of Y(NO) indicates that high concentration of nSiO2 may trigger a new imbalance in light energy distribution, leading to an increase in ineffective energy dissipation.

[0035] Compared to 1% nSiO2, all μSiO2 treatment groups showed a weaker effect on increasing Y(II), and Y(II) decreased with increasing μSiO2 concentration. Although the addition of μSiO2 kept Y(NPQ) at a low level, indicating a reduced need for photoprotection, Y(NO) increased significantly with increasing μSiO2 concentration, suggesting that micron particles may have limited photoconversion efficiency and increased ineffective energy dissipation due to uneven light distribution, pore blockage, and mechanical stress interference.

[0036] Example 2

[0037] Sodium alginate and carboxymethyl cellulose were dissolved in TSB medium, and alkali-producing bacteria suspension was added to prepare bacterial bioink, with sodium alginate concentration of 3% w / v and carboxymethyl cellulose concentration of 9% w / v. Sodium alginate and carboxymethyl cellulose were also dissolved in TP medium, and different concentrations of nSiO2 and Chlorella extract were added to prepare algal bioink, with sodium alginate concentration of 3% w / v, carboxymethyl cellulose concentration of 9% w / v, and nSiO2 concentrations of 0.5%, 1%, 2%, and 5% w / v, respectively. Using a dual-nozzle 3D bioprinter, bacterial bioink and algal bioink were loaded separately to construct two different configurations of BA-ELMs: striped (3DPC-Striped) and layered (3DPC-Layered). The striped BA-ELMs were constructed by arranging the two inks alternately along the same plane to form an interwoven stripe composite structure. The construction of layered BA-ELMs involved first depositing bacterial bioink, followed by vertically depositing algal bioink on top, forming an alternating layered structure. The filament spacing between the two configurations was 2.4 mm, and the substrate area was 12 × 12 mm. 2 Each layer of thin strips is 90 o Alternating orientations were used to print a total of 6 layers.

[0038] Two different configurations of BA-ELMs were placed in phenol at concentrations of 100 mg / L and 200 mg / L, and samples were taken every 2 h. After studying the effect of different bacterial and algal printed structures on the phenol degradation capacity, the maximum photochemical quantum yield Fv / Fm and effective quantum yield Y(II) of all printed structures were further analyzed to reveal the effect of different configurations on algal photosynthetic activity during phenol degradation.

[0039] like Figure 3 As shown, striped BA-ELMs exhibited the fastest degradation rate under 100 mg / L phenol conditions; while layered BA-ELMs exhibited the fastest degradation rate under 200 mg / L phenol conditions.

[0040] like Figure 4 As shown, under 100 mg / L phenol conditions, the photosynthetic efficiency of striped BA-ELMs was slightly higher than that of layered configurations; under 200 mg / L phenol conditions, the Fv / Fm and Y(II) values ​​of layered BA-ELMs were significantly higher, which is more effective in maintaining algal photosynthetic activity.

[0041] In summary, this invention optimizes the composition of bio-ink, introduces nSiO2 or µSiO2 to enhance algal photosynthetic efficiency, and utilizes dual-nozzle 3D printing to achieve precise arrangement of bacteria and algae in three-dimensional space, constructing two types of bacterial-algae symbiotic engineering living materials: striped and layered. Experimental results show that the prepared materials exhibit excellent synergistic degradation ability and photosynthetic performance at different phenol concentrations. The striped structure demonstrates higher degradation efficiency at low pollutant concentrations, while the layered structure better preserves algal photosynthetic function under high concentration conditions. This invention overcomes the problems of byproduct accumulation and unsustainable oxygen supply in traditional chemical oxygen supply materials, and also breaks through the technical bottlenecks of uncontrolled spatial arrangement, poor tolerance, and low synergistic efficiency in traditional bacterial-algae co-culture systems, providing support for the optimization and application of biological oxygen supply materials.

Claims

1. A method for preparing 3D bioprinted living materials for bacterial-algae symbiotic engineering, characterized in that, Includes the following steps: (1) Sodium alginate and carboxymethyl cellulose were dissolved in TSB medium and alkali-producing bacteria suspension was added to prepare bacterial bio-ink. The concentration of sodium alginate was 3% w / v and the concentration of carboxymethyl cellulose was 9% w / v. (2) Sodium alginate, carboxymethyl cellulose and silica were dissolved in TP medium and Chlorella liquid was added to prepare algal bio-ink. The concentration of sodium alginate was 3% w / v, the concentration of carboxymethyl cellulose was 9% w / v, and the concentration of silica was 0.5%~5% w / v. The silica was nano silica or micron silica. (3) Using a dual-nozzle 3D bioprinter, bacterial bio-ink and algal bio-ink are loaded respectively, and 3D printing is performed to obtain striped or layered 3D bioprinted bacterial-algal symbiotic engineering living materials.

2. The preparation method according to claim 1, characterized in that, In step (1), the alkali-producing bacteria are alkali-producing bacteria with the ability to degrade organic pollutants, and the organic pollutants are phenol or azo dyes.

3. The preparation method according to claim 1, characterized in that, In step (1), the TSB medium consists of: 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate, and pH=7.

3.

4. The preparation method according to claim 1, characterized in that, In step (2), the concentration of silica is 1% w / v.

5. The preparation method according to claim 1, characterized in that, In step (2), the TP culture medium consists of: 20 g / L 1M Tris-Base stock solution, 10 g / L salt stock solution, 1 g / L phosphate buffer, and 1 g / L trace element stock solution; the salt stock solution consists of: 10 g / L MgSO4·7H2O, 5 g / L CaCl2·2H2O, and 75 g / L NaNO3; the phosphate buffer solution consists of: 108 g / L K2HPO4 and 56 g / L KH2PO4; and the trace element stock solution consists of: 50 g / L disodium ethylenediaminetetraacetate, 0.20 g / L ferrous sulfate heptahydrate, 0.10 g / L zinc sulfate, 0.03 g / L manganese chloride tetrahydrate, 0.20 g / L cobalt chloride hexahydrate, 0.01 g / L copper chloride dihydrate, and 0.03 g / L sodium molybdate dihydrate.

6. The preparation method according to claim 1, characterized in that, In step (3), the 3D printing conditions are: 3D printing nozzle size is 20 G, air pressure is 250 kPa, and printing speed is 6 mm / s.

7. The preparation method according to claim 1, characterized in that, In step (3), the construction method of the striped 3D bioprinting bacterial-algae symbiotic engineering living material is as follows: bacterial bio-ink and algae bio-ink are arranged alternately along the same plane to form a strip-like interwoven composite structure; the construction method of the layered 3D bioprinting bacterial-algae symbiotic engineering living material is as follows: bacterial bio-ink is deposited first, and then algae bio-ink is deposited vertically above it to form an alternating layered structure; in both configurations of the 3D bioprinting bacterial-algae symbiotic engineering living material, the filament spacing is 2.4 mm and the substrate area is 12 × 12 mm. 2 Each layer of thin strips is 90 o Alternating orientations were used to print a total of 6 layers.

8. 3D bioprinted bacterial-algae symbiotic engineering living material prepared by any one of the preparation methods according to claims 1 to 7.

9. The application of the 3D bioprinted bacterial-algae symbiotic engineering living material according to claim 8 in the synergistic degradation of organic pollutants.

10. The application according to claim 9, characterized in that, The organic pollutants are phenol or azo dyes.

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