3D printing high-light-transmittance bio-ink, artificial 3D algal-bacterial symbiont and preparation method and application of artificial 3D algal-bacterial symbiont

Abstract

The invention discloses 3D printing high-light-transmittance bio-ink, an artificial 3D algal-bacterial symbiont and a preparation method and application of the artificial 3D algal-bacterial symbiont. The 3D printing high-light-transmittance bio-ink is prepared from polyether F127 diacrylate, polyethylene glycol diacrylate, pyridine degradation special-effect bacterium paracoccus sp. NJUST47, chlorella sorokiniana FACHB-275, a light absorbent and a liquid culture medium containing a photoinitiator, and the 3D printing high-light-transmittance bio-ink is prepared into an artificial 3D bacterium-algae symbiont through one-step 3D printing. The 3D printing high-light-transmittance bio-ink has good synergistic pyridine degradation performance, the constructed artificial 3D algal-bacterial symbiont has high algal-bacterial loading capacity and excellent light utilization rate, enhanced removal of volatile toxic organic pollutant pyridine can be achieved, and degradation stability is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological treatment of environmental organic pollutants, and relates to a 3D printed high-transmittance biological ink, an artificial 3D bacteria-algae symbiont, and a preparation method and application thereof. Background Art

[0002] As an important nitrogen-containing heterocyclic compound, pyridine is widely used in the chemical production of dyes, herbicides, pesticides, pharmaceuticals, coking gas, etc., and is present in the chemical wastewater generated. Biological treatment is an environmentally friendly and cost-effective wastewater treatment technology for treating pyridine-containing wastewater. Among them, anaerobic biological processes are limited by low removal rates and poor process stability, while traditional aerobic biodegradation methods have problems with pollutant volatilization and high energy consumption costs when treating volatile pollutants such as pyridine. In addition, as pyridine degrades, the nitrogen on the benzene ring will eventually be released into the water body in the form of ammonia nitrogen, causing secondary pollution.

[0003] The microalgae-bacteria symbiotic system is a biological treatment technology that uses the synergistic effect of algae and bacteria to improve pollutant removal efficiency and reduce wastewater treatment energy consumption. In this system, microalgae provide oxygen to heterotrophic microorganisms through photosynthesis, enabling aerobic biodegradation and mineralization of organic pollutants without any external oxygen supply. This addresses the volatility of volatile organic compounds (VOCs) and reduces wastewater treatment energy consumption. Furthermore, the microalgae's assimilation of carbon, nitrogen, and phosphorus eliminates the secondary contamination problem of pyridine in traditional aerobic treatment processes.

[0004] In recent years, the application of bacteria-algae symbiotic systems in the treatment of wastewater containing organic compounds has received widespread attention. Chinese patent ZL201911292340.5 discloses a bacteria-algae symbiotic system for enhancing the biodegradation of pyridine under microaerobic conditions. Pyridine-degrading functional bacteria and microalgae are used to construct a bacteria-algae symbiotic system with pyridine degradation function, which can achieve efficient degradation of pyridine without external oxygen supply. However, this free bacteria-algae symbiotic system still has problems such as poor light capture ability, low light utilization rate, uncontrollable spatial microstructure, and easy loss of algae, resulting in low degradation efficiency of toxic organic pollutants in the system. Therefore, how to controllably construct a bacteria-algae symbiosis with high bacteria-algae load and excellent light utilization rate in a relatively short period of time to enhance the removal of volatile toxic organic pollutant pyridine is particularly urgent. Summary of the Invention

[0005] The present invention aims to provide a 3D-printed high-transmittance bio-ink, an artificial 3D bacterial-algal symbiont, and its preparation and application. The bacterial-algal symbiont constructed by 3D printing with the high-transmittance bio-ink of the present invention exhibits a well-defined spatial configuration and high light utilization efficiency, effectively enhancing the removal of pyridine, a volatile toxic organic pollutant.

[0006] The technical solutions for achieving the purpose of the present invention are as follows:

[0007] The 3D-printed high-transmittance bio-ink includes the following components: polyether F127 diacrylate (F127DA), polyethylene glycol diacrylate (PEGDA), pyridine-degrading bacteria Paracoccus sp. NJUST47, Chorella sorokiniana FACHB-275, a light absorber, and a liquid culture medium containing a photoinitiator.

[0008] Preferably, the molecular weight of the polyethylene glycol diacrylate is 400 to 6000, specifically 400, 600, 800, 1000, 2000, 4000 or 6000, with 400 being taken as an example in one embodiment of the present invention; the molecular weight of the polyether F127 diacrylate is 10 to 15 kDa, specifically 10 kDa, 12 kDa or 15 kDa, with 15 kDa being taken as an example in one embodiment of the present invention.

[0009] Preferably, the dosage ratio of the polyether F127 diacrylate, polyethylene glycol diacrylate and liquid culture medium is 0.15g:(0.01-0.075)g:1mL, specifically 0.15g:0.01g:1mL, 0.15g:0.03g:1mL, 0.15g:0.05g:1mL or 0.15g:0.075g:1mL. In one embodiment of the present invention, 0.15g:0.03g:1mL is taken as an example.

[0010] The pyridine-degrading specific bacteria Paracoccus sp. NJUST47 described in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on May 24, 2019. The depository address is Wuhan University, Wuhan City, Hubei Province, China, with a deposit number of CCTCC NO: M2019392. It has been fully disclosed in Chinese patent ZL201911292340.5.

[0011] The Chorella sorokiniana FACHB-275 described in the present invention comes from the Freshwater Algae Bank of the Chinese Academy of Sciences (FACHB).

[0012] Preferably, the MLSS of the pyridine-degrading special bacteria Paracoccus sp. NJUST47 in the liquid culture medium is 7.5-22.5 mg / mL, specifically 7.5 mg / mL, 10 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL or 22.5 mg / mL. In one embodiment of the present invention, 15 mg / mL is taken as an example. The MLSS of the Chlorella sorokiniana FACHB-275 in the liquid culture medium is 0.75-2.25 mg / mL, specifically 0.75 mg / mL, 1.0 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 1.75 mg / mL, 2.0 mg / mL or 2.25 mg / mL. In one embodiment of the present invention, 1.5 mg / mL is taken as an example.

[0013] Preferably, the dosage ratio of the light absorber to the liquid culture medium is (0.002-0.008) g:1 mL, specifically 0.002 g:1 mL, 0.003 g:1 mL, 0.004 g:1 mL, 0.005 g:1 mL, 0.006 g:1 mL, 0.007 g:1 mL or 0.008 g:1 mL. In one embodiment of the present invention, 0.005 g:1 mL is taken as an example.

[0014] The light absorber of the present invention can reduce the scattering of 405nm light, and is preferably Sudan red, β-carotene, curcumin or tartrazine. In one embodiment of the present invention, tartrazine is used as an example.

[0015] Preferably, the liquid culture medium is a PBS solution containing 0.25% (w / v) photoinitiator, and the photoinitiator is 2,4,6-trimethylformyl phosphate lithium salt (LAP) or hydroxyketone photoinitiator Irgacure 2959. In one embodiment of the present invention, LAP is used as an example.

[0016] Preferably, the 3D printing high-transmittance bio-ink consists of bio-ink No. 1, bio-ink No. 2 and a light absorber; the bio-ink No. 1 consists of polyether F127 diacrylate, polyethylene glycol diacrylate, a resuspension of pyridine-degrading special bacteria Paracoccus sp. NJUST47 and a liquid culture medium containing a photoinitiator; the bio-ink No. 2 consists of polyether F127 diacrylate, polyethylene glycol diacrylate, a resuspension of Chorella sorokiniana FACHB-275 and a liquid culture medium containing a photoinitiator.

[0017] The pyridine-degrading specific bacteria Paracoccus sp.NJUST47 resuspension is a resuspension obtained by resuspending the culture solution of the pyridine-degrading specific bacteria Paracoccus sp.NJUST47 after removing the original culture solution by centrifugation.

[0018] The Chorella sorokiniana FACHB-275 resuspension is a resuspension obtained by resuspending the culture solution of the Chorella sorokiniana FACHB-275 after removing the original culture solution by centrifugation.

[0019] The application provides a preparation method of the 3D printing high-transmittance biological ink, which comprises mixing polyether F127 diacrylate, polyethylene glycol diacrylate, pyridine-degrading specific bacteria Paracoccus sp.NJUST47, Chorella sorokiniana FACHB-275, a light absorber and a liquid culture medium containing a photoinitiator.

[0020] Preferably, the application provides a preparation method of the 3D printing high-transmittance biological ink, which specifically comprises the following steps:

[0021] (1) mixing polyether F127 diacrylate and polyethylene glycol diacrylate in a liquid culture medium containing a photoinitiator, and then adding the Paracoccus sp.NJUST47 resuspension to obtain biological ink No.1;

[0022] (2) mixing polyether F127 diacrylate and polyethylene glycol diacrylate in a liquid culture medium containing a photoinitiator, and then adding the Chorella sorokiniana FACHB-275 resuspension to obtain biological ink No.2;

[0023] (3) mixing biological ink No.1 and biological ink No.2, adding a light absorber, and uniformly mixing to obtain the 3D printing high-transmittance biological ink.

[0024] The application provides an artificial 3D bacteria-algae symbiont based on the 3D printing high-transmittance biological ink, which is prepared by photopolymerization 3D printing under light irradiation.

[0025] The application further provides a preparation method of the artificial 3D bacteria-algae symbiont, which comprises the following steps:

[0026] The above-mentioned 3D printed high-transmittance biological ink is dropped into the printing platform of a projection light-curing 3D bioprinter, 3D printed according to the preset spatial structure, and cross-linked under light to produce an artificial 3D bacteria-algae symbiont.

[0027] Preferably, the artificial 3D bacteria-algae symbiont is in a suction cup configuration, with a total length of 40 mm, a width of 12 mm, a bottom plate thickness of 0.5 to 1 mm, and 9 suction cups distributed on the base, with a diameter of 4.66 mm and a height of 2.5 mm.

[0028] Preferably, the light is blue light with a wavelength of 405 nm and a light intensity of 25 mW / cm 2 ; The cross-linking time of a single layer is preferably 2 to 10 s, specifically 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s or 10 s. In one embodiment of the present invention, 5 s is taken as an example.

[0029] Furthermore, the present invention provides the use of the artificial 3D bacteria-algae symbiont in the treatment of pyridine-containing wastewater.

[0030] Preferably, the application method is: mixing pyridine-containing wastewater with the artificial 3D bacteria-algae symbiont to treat the wastewater.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] The 3D-printed high-transmittance bio-ink of this invention uses polyether F127 diacrylate as the bio-ink matrix, polyethylene glycol diacrylate as a toughening agent, and the pyridine-degrading bacteria Paracoccus sp. NJUST47 and Chorella sorokiniana FACHB-275 as pyridine biodegradation active ingredients, resulting in a bio-ink with excellent synergistic pyridine degradation performance. Furthermore, leveraging the technological advantages of projection-based light-curing 3D bioprinting, this high-transmittance bio-ink was rapidly, precisely, and controllably constructed into an artificial 3D bacteria-algae symbiosis with high bacterial and algal loading and excellent light utilization efficiency, which can enhance the removal of the volatile toxic organic pollutant pyridine. Experimental results show that the artificial 3D bacteria-algae symbiosis with a suction cup structure has excellent removal ability for the volatile organic pollutant pyridine. Compared with the free bacteria-algae symbiosis system, the artificial 3D bacteria-algae symbiosis with a suction cup structure can completely degrade 50 mg / L of pyridine within 48 hours, with a faster degradation rate and better degradation stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a flow chart for preparing the artificial 3D bacteria-algae symbiont provided in Example 1;

[0034] Figure 2(a) model and (b) digital photo of the artificial 3D bacterial-algal symbiont in the disc configuration provided in Example 1;

[0035] Figure 3 pyridine degradation effect of the bacterial-algal symbiont in Example 2, the artificial 3D bacterial-algal symbiont and the artificial 3D bacterial-algal symbiont stored at 4℃ for 40d;

[0036] Figure 4 cycling test of pyridine removal of the artificial 3D bacterial-algal symbiont in Example 3 and the artificial 3D bacterial-algal symbiont stored at 4℃ for 40d;

[0037] Figure 5 pyridine degradation effect of the bacterial-algal symbiont in Comparative Example 1, the artificial 3D bacterial-algal symbiont compounded by gelatin methacrylate (GelMA) and polyethylene glycol diacrylate and the artificial 3D bacterial-algal symbiont in the disc configuration provided in Example 1;

[0038] Figure 6 pyridine degradation effect of the bacterial-algal symbiont in Comparative Example 2, the artificial 3D bacterial-algal symbiont compounded by hyaluronic acid methacrylate (HAMA) and polyethylene glycol diacrylate and the artificial 3D bacterial-algal symbiont in the disc configuration provided in Example 1;

[0039] Figure 7 pyridine degradation effect of the bacterial-algal symbiont in Comparative Example 3, the artificial 3D bacterial-algal symbiont compounded by different proportions of polyether F127 diacrylate and polyethylene glycol diacrylate. DETAILED DESCRIPTION

[0040] In order to further understand the present application, the preferred embodiments of the present application are described below in conjunction with the examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application, and are not limitations to the claims of the present application.

[0041] The pyridine-degrading Paracoccus sp. NJUST47 described in the present application was preserved in China Center for Type Culture Collection (CCTCC) on May 24, 2019, and the address of the preservation unit is Wuhan University, Wuhan, Hubei Province, China, and the preservation number is CCTCC NO: M2019392, which has been fully disclosed in Chinese patent ZL201911292340.5.

[0042] The Chorella sorokiniana FACHB-275 described in the present application is from the Freshwater Algae Culture Collection of China (FACHB).

[0043] The projection-type light-curing 3D bioprinter described herein was provided by Suzhou Yongquan Intelligent Equipment Co., Ltd.; the specific printing method described herein is performed according to methods well known to those skilled in the art and is not particularly limited thereto. The polyether F127 diacrylate and polyethylene glycol diacrylate described herein are commercially available products or prepared according to methods well known to those skilled in the art. In the following examples, the polyether F127 diacrylate used was provided by Suzhou Yongquan Intelligent Equipment Co., Ltd. under the product number EFL-F127DA-001; the polyethylene glycol diacrylate was provided by Suzhou Yongquan Intelligent Equipment Co., Ltd. under the product number EFL-PEGDA. The photoinitiator-containing liquid culture medium used was a PBS solution supplemented with 0.25% (w / v) of the blue light photoinitiator LAP. The PBS solution formulation was: 0.36 g / L potassium dihydrogen phosphate dihydrate (KH2PO4·2H2O), 1.32 g / L disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), and deionized water.

[0044] In order to solve the problems of poor light capture ability, low light utilization rate, uncontrollable spatial microstructure, and easy loss of algae in the bacterial-algal symbiotic system, which lead to low efficiency in degrading volatile toxic organic pollutant pyridine, the present invention provides a 3D printing high-transmittance bio-ink, including the following components: polyether F127 diacrylate, polyethylene glycol diacrylate, pyridine-degrading special bacteria Paracoccus sp. NJUST47, Chorella sorokiniana FACHB-275, a light absorber and a liquid culture medium containing a photoinitiator.

[0045] The high-transmittance 3D printing bio-ink provided by the present invention contains the pyridine-degrading bacteria Paracoccus sp. NJUST47 and Chorella sorokiniana FACHB-275 as active pyridine biodegrading ingredients, polyether F127 diacrylate as the bio-ink matrix, and polyethylene glycol diacrylate as a toughening agent. Chorella sorokiniana FACHB-275 provides oxygen to the pyridine-degrading bacteria Paracoccus sp. NJUST47 through photosynthesis. Paracoccus sp. NJUST47 then degrades pyridine by ring-opening under aerobic conditions. Furthermore, Chorella sorokiniana FACHB-275 immobilizes the released ammonia nitrogen, thereby achieving efficient pyridine degradation.

[0046] The liquid culture medium can maintain the activity of the pyridine-degrading specific bacteria Paracoccus sp. NJUST47 and Chorella sorokiniana FACHB-275, and does not interfere with the photocrosslinking of the biological ink matrix.

[0047] The pyridine-degrading specific bacteria Paracoccus sp. NJUST47 resuspension in the 3D printing high-transmittance biological ink is obtained by centrifugation of Luria-Bertan (LB) liquid medium of the pyridine-degrading specific bacteria Paracoccus sp. NJUST47 to remove the LB medium used for culture, and then resuspension of the obtained bacteria with fresh PBS solution; the formula of the LB liquid medium is: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, deionized water.

[0048] The pyridine-degrading specific bacteria Paracoccus sp. NJUST47 resuspension in the 3D printing high-transmittance biological ink is obtained by centrifugation of Luria-Bertan (LB) liquid medium of the pyridine-degrading specific bacteria Paracoccus sp. NJUST47 to remove the LB medium used for culture, and then resuspension of the obtained bacteria with fresh PBS solution; the formula of the LB liquid medium is: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, deionized water.

[0049] Example 1

[0050] According to the process shown in the following scheme, an artificial 3D bacteria-algae symbiotic body is prepared, and the specific process is as follows: Figure 1

[0051] ​(1) The pyridine-degrading special bacteria Paracoccus sp. NJUST47 isolated in the laboratory was first centrifuged at 4°C and 8000 rpm for 5 min to remove the LB culture medium used for cultivation. The obtained bacteria were then resuspended in a PBS solution added with 0.25% (w / v) blue light photoinitiator LAP, and the MLSS was adjusted to 60 mg / mL; then 0.15 g of polyether F127 diacrylate and 0.03 g of polyethylene glycol diacrylate were mixed and added to 0.5 mL of PBS solution added with 0.25% (w / v) blue light photoinitiator LAP, and dissolved at 2-8°C for 30 min, shaken several times during the period, and after it was dissolved, mixed with 0.5 mL of the pyridine-degrading special bacteria Paracoccus sp. NJUST47 resuspension to prepare bio-ink No. 1.

[0052] (2) The purchased Chorella sorokiniana FACHB-275 was first centrifuged at 4°C and 5000 rpm for 8 minutes to remove the BG culture medium used for cultivation. The obtained microalgae was then resuspended in a PBS solution added with 0.25% (w / v) blue light photoinitiator LAP, and its MLSS was adjusted to 6 mg / mL; then 0.15g of polyether F127 diacrylate and 0.03g of polyethylene glycol diacrylate were mixed and added to 0.5mL of PBS solution added with 0.25% (w / v) blue light photoinitiator LAP, and dissolved at 2-8°C for 30 minutes, shaken several times during the period, and after it was dissolved, it was mixed with 0.5mL of Chorella sorokiniana FACHB-275 resuspension to prepare bio-ink No. 2.

[0053] (3) After mixing bio-ink No. 1 and bio-ink No. 2 in a sterile centrifuge tube, 0.01 g of light absorber lemon yellow was added to obtain 3D printing high transmittance bio-ink. 1 mL of ink was dropped into the printing platform of a projection light-curing 3D bio-printer (Suzhou Yongquan Intelligent Equipment Co., Ltd.) using a sterile pipette. Figure 2 (a) The preset spatial structure is 3D printed with a light source wavelength of 405 nm and a light intensity of 25 mW / cm 2 The blue light cross-linking was carried out layer by layer with a thickness of 0.45 μm. Figure 2 (b) Artificial 3D bacteria-algae symbiosis with the suction cup structure shown.

[0054] Example 2

[0055] The artificial 3D bacteria-algae symbiont prepared in Example 1 was adhered to the inside of a 120 mL reaction bottle for degradation of volatile toxic organic pollutants such as pyridine. At the same time, a free bacteria-algae symbiotic system with an MLSS equivalent of 6000 lux was set as a control group. Nitrogen was blown into the reaction bottle to exclude oxygen from the system. It was ensured that the oxygen required for the degradation of pyridine by the pyridine-degrading special bacteria Paracoccus sp. NJUST47 was provided by photosynthesis of Chorella sorokiniana FACHB-275. The reaction bottle was filled with simulated wastewater having a pyridine concentration of 100 mL and no nitrogen source. The reaction bottle was placed in a light incubator for the experiment. The light source was set on the side, using an LED cold light source with a light intensity of 6000 lux, a light cycle of light / darkness = 12 h / 12 ​​h, a temperature of 26-30 ° C, pH = 7.20 ± 0.05, an oscillation speed of 160 rpm / min, and a glass-made transparent serum bottle was used for the reaction bottle. During the reaction, the reaction bottle was placed in a nitrogen operating environment, and the pyridine concentration in the system was monitored during the reaction.

[0056] The simulated wastewater used deionized water as solvent, and its solute components included: pyridine 50 mg / L, Na2HPO4·12H2O 1.53 g / L, KH2PO4 0.38 g / L, KH2PO4·3H2O 40 mg / L, MgCl2·7H2O 0.16 g / L, CaCl2·2H2O 36 mg / L, EDTA 1 mg / L, Na2CO3 20 mg / L, ferric citrate 6 mg / L, ammonium ferric citrate 6 mg / L, trace elements A5+Co 1 mL / L (A5+Co mother liquor is H3BO3 2.86 g / L; MnCl2·4H2O 1.81 g / L; ZnSO4·7H2O 0.222 g / L; CuSO4·5H2O 0.079 g / L; NaMoO4·2H2O 0.390g / L; Co(NO3)2·6H2O 0.0494g / L).

[0057] Figure 3 This is a free bacteria-algae symbiotic system with an MLSS equivalent of the artificial 3D bacteria-algae symbiotic system, and a graph showing the degradation effect of pyridine on the artificial 3D bacteria-algae symbiotic system prepared in Example 1 and the artificial 3D bacteria-algae symbiotic system prepared in Example 1 stored at 4°C for 40 days. Figure 3It can be seen that both the artificial 3D bacteria-algae symbiont and the artificial 3D bacteria-algae symbiont stored at 4°C can effectively degrade pyridine. The artificial 3D bacteria-algae symbiont degrades pyridine faster than the free bacteria-algae symbiont system, completely degrading 50 mg / L of pyridine within 48 hours. The artificial 3D bacteria-algae symbiont stored at 4°C for 40 days still maintains biological activity and is capable of degrading pyridine. The artificial 3D bacteria-algae symbiont enhances light capture by the microalgae, thereby increasing algal photosynthesis and, in turn, increasing dissolved oxygen levels in the system, promoting the degradation of pyridine, a volatile toxic organic pollutant.

[0058] Example 3

[0059] Good repeatability is an important indicator for measuring the practical application of engineering living materials. In this example, the repeatability of the artificial 3D bacteria-algae symbiont prepared in Example 1 was tested. The results are as follows: Figure 4 As shown in the cyclic degradation diagram, the artificial 3D bacteria-algae symbiont can still completely degrade pyridine after 8 consecutive degradation tests; the artificial 3D bacteria-algae symbiont stored at 4°C for 40 days recovers its biological activity after 3 degradation experiments and can completely degrade pyridine.

[0060] Comparative Example 1

[0061] Different from Example 1, the bio-ink components include: methacrylated gelatin (GelMA), polyethylene glycol diacrylate, pyridine-degrading bacteria Paracoccus sp. NJUST47, Chorella sorokiniana FACHB-275, a light absorber, and a liquid culture medium containing a blue light photoinitiator. The specific preparation process is as follows:

[0062] (1) The pyridine-degrading special bacteria Paracoccus sp. NJUST47 isolated from the laboratory was first centrifuged at 4°C and 8000 rpm for 5 min to remove the LB culture medium used for cultivation. The obtained bacteria were then resuspended in a PBS solution added with 0.25% (w / v) blue light photoinitiator LAP, and the MLSS was adjusted to 60 mg / mL; then 0.08 g of methacrylated gelatin and 0.03 g of polyethylene glycol diacrylate were mixed and added to 0.5 mL of PBS solution added with 0.25% (w / v) blue light photoinitiator LAP, and dissolved in a 30°C water bath for 30 min, shaken several times during the period, and after dissolution, mixed with 0.5 mL of the pyridine-degrading special bacteria Paracoccus sp. NJUST47 resuspension to prepare bio-ink No. 1;

[0063] (2) First, the purchased microalgae Chorella sorokiniana FACHB-275 was centrifuged at 4°C and a speed of 5000 rpm for 8 min to remove the BG culture medium used in the culture, and the obtained microalgae was resuspended with a PBS solution added with 0.25% (w / v) of the blue light initiator LAP, and the MLSS thereof was adjusted to 6 mg / mL; then, 0.08 g of methacrylated gelatin and 0.03 g of polyethylene glycol diacrylate were mixed and added to 0.5 mL of the PBS solution added with 0.25% (w / v) of the blue light initiator LAP, and dissolved at 2-8°C for 30 min, during which the solution was shaken several times, and after the solution was dissolved, the solution was mixed with 0.5 mL of the microalgae Chorella sorokiniana FACHB-275 resuspension to prepare bio-ink No. 2;

[0064] (3) After mixing bio-ink No. 1 and bio-ink No. 2 in a sterile centrifuge tube, 0.01 g of the light absorber lemon yellow was added, and the mixture was moved into a 3D bioprinter to print an artificial 3D fungus-algae symbiotic body with the same structure as the sucker structure of Example 1 according to a preset spatial structure.

[0065] The artificial 3D fungus-algae symbiotic body prepared by compounding the methacrylated gelatin and polyethylene glycol diacrylate and the artificial 3D fungus-algae symbiotic body prepared in Example 1 were respectively adhered to the inside of 120 mL reaction bottles for degradation of volatile toxic organic pollutants pyridine, and the reaction bottles were filled with simulated wastewater with a pyridine concentration of 100 mL and without a nitrogen source, and the pyridine concentration of the simulated wastewater was set to 200 mg / L, and the other solute components were the same as in Example 2. The reaction bottles were placed in a light incubator for the experiment, the light source was set on the side, a LED cold light source with a light intensity of 6000 lux was used, a light / dark cycle of 12h / 12h was used, the temperature was set to 26-30°C, the pH was 7.20±0.05, the shaking speed was 160 rpm / min, and a glass transparent serum bottle was used for the reaction bottle. During the reaction, the reaction bottles were in a nitrogen operating environment, and the pyridine concentration in the system was monitored during the reaction.

[0066] Figure 5 is a degradation effect diagram of pyridine by the artificial 3D fungus-algae symbiotic body constructed by compounding the methacrylated gelatin and polyethylene glycol diacrylate (GelMA&PEGDA) and the artificial 3D fungus-algae symbiotic body prepared in Example 1. As Figure 5As shown, the pyridine removal can be achieved by both the artificial 3D bacterial-algal symbiotic body constructed by the bio-ink of methylacrylated gelatin and polyethylene glycol diacrylate (GelMA & PEGDA) and the artificial 3D bacterial-algal symbiotic body constructed by the bio-ink of polyether F127 diacrylate and polyethylene glycol diacrylate (F127DA & PEGDA), and the pyridine degradation rate of the F127DA & PEGDA system is faster, which is reduced to close to 100 mg / L after about 50 hours and still has a slow downward trend, and the structure of the 3D bacterial-algal symbiotic body is intact during the whole experiment, and no sterile algae escape. In comparison, the pyridine degradation rate of the GelMA & PEGDA system is slower, and there is algae escape in the 3D bacterial-algal symbiotic body to the solution, which may lead to partial shading in the system, reduce the light capture of microalgae, and thus reduce the dissolved oxygen concentration in the system, and reduce the aerobic degradation of pyridine in the system.

[0067] The artificial 3D bacterial-algal symbiotic body and the artificial 3D bacterial-algal symbiotic body stored at 4°C can both achieve effective degradation of pyridine; the degradation rate of the artificial 3D bacterial-algal symbiotic body to pyridine is faster than that of the free bacterial-algal symbiotic system, and the complete degradation of 50 mg / L concentration of pyridine can be achieved within 48 h; the artificial 3D bacterial-algal symbiotic body stored at 4°C for 40 d still has biological activity and can degrade pyridine. The artificial 3D bacterial-algal symbiotic body improves the light capture of microalgae, thereby improving the photosynthesis of algae, and thus improving the level of dissolved oxygen in the system, promoting the degradation of volatile toxic organic pollutants pyridine.

[0068] Comparative Example 2

[0069] Different from Example 1, the bio-ink components include: methylacrylated hyaluronic acid (HAMA), polyethylene glycol diacrylate, pyridine degradation specific bacteria Paracoccus sp. NJUST47, Chlorella sorokiniana FACHB-275, light absorber, and liquid medium containing blue light photoinitiator, and the specific process is as follows:

[0070] (1) First, the pyridine-degrading specific bacteria Paracoccus sp. NJUST47 isolated in the laboratory was centrifuged at 4°C and a speed of 8000 rpm for 5 min to remove the LB culture medium used for culture, and the obtained bacteria were resuspended in a PBS solution to which 0.25% (w / v) of the blue light photoinitiator LAP was added, and the MLSS thereof was adjusted to 60 mg / mL; then 0.10 g of methacrylated hyaluronic acid and 0.03 g of polyethylene glycol diacrylate were mixed and added to 0.5 mL of the PBS solution to which 0.25% (w / v) of the blue light photoinitiator LAP was added, and dissolved at 30°C for 30 min with shaking several times during the process, and after the dissolution, 0.5 mL of the pyridine-degrading specific bacteria Paracoccus sp. NJUST47 resuspension was mixed to prepare bio-ink No. 1.

[0071] (2) First, the purchased small ball algae Chorella sorokiniana FACHB-275 was centrifuged at 4°C and a speed of 5000 rpm for 8 min to remove the BG culture medium used for culture, and the obtained microalgae were resuspended in a PBS solution to which 0.25% (w / v) of the blue light photoinitiator LAP was added, and the MLSS thereof was adjusted to 6 mg / mL; then 0.10 g of methacrylated hyaluronic acid and 0.03 g of polyethylene glycol diacrylate were mixed and added to 0.5 mL of the PBS solution to which 0.25% (w / v) of the blue light photoinitiator LAP was added, and dissolved at 2-8°C for 30 min with shaking several times during the process, and after the dissolution, 0.5 mL of the resuspended small ball algae Chorella sorokiniana FACHB-275 culture solution was mixed to prepare bio-ink No. 2.

[0072] (3) After mixing bio-ink No. 1 and bio-ink No. 2 in a sterile centrifuge tube, 0.01 g of the light absorber lemon yellow was added, and then moved to a 3D bio-printer, and an artificial 3D bacteria-algae symbiotic organism with the same structure as the suction cup structure of Example 1 was printed according to the preset spatial structure.

[0073] The artificial 3D bacterial-algal symbiont of methyl methacrylated hyaluronic acid (HAMA) and polyethylene glycol diacrylate compound and the artificial 3D bacterial-algal symbiont prepared in Example 1 were respectively adhered to the inside of a 120 mL reaction bottle for degradation of volatile toxic organic pollutants pyridine. The reaction bottle was filled with simulated wastewater with a pyridine concentration of 100 mL and without nitrogen source, and the simulated wastewater was the same as that in Example 2. The reaction bottle was placed in a light incubator for the experiment. The light source was set on the side, and a LED cold light source with an illumination intensity of 6000 lux was used. The light cycle was light / dark = 12h / 12h. The temperature was set to 26-30°C, pH = 7.20 ± 0.05, and the shaking speed was 160 rpm / min. The reaction bottle was a glass-made fully transparent serum bottle. During the reaction, the reaction bottle was in a nitrogen operating environment, and the pyridine concentration in the system was monitored during the reaction.

[0074] Figure 6 Figure of the degradation effect of pyridine by the artificial 3D bacterial-algal symbiont of methyl methacrylated hyaluronic acid (HAMA) and polyethylene glycol diacrylate compound (GelMA & PEGDA) prepared in Example 1. The artificial 3D bacterial-algal symbiont constructed by the bio-ink of methyl methacrylated hyaluronic acid and polyethylene glycol diacrylate compound (GelMA & PEGDA) and the artificial 3D bacterial-algal symbiont constructed by the bio-ink of polyether F127 diacrylate and polyethylene glycol diacrylate compound (F127DA & PEGDA) can both achieve the removal of pyridine. The pyridine degradation rate of the F127DA & PEGDA system is faster, and it can completely degrade 50 mg / L of pyridine in about 48 hours. The structure of the 3D bacterial-algal symbiont is intact during the entire experimental process, and no sterile algae escape. In comparison, the pyridine degradation rate of the GelMA & PEGDA system is slower, and it needs more than 72 hours to completely degrade 50 mg / L of pyridine. At the end of the experiment, the structure of the 3D bacterial-algal symbiont swells and is damaged, and part of the algae escapes into the solution, which may lead to partial shading of the system, a decrease in light utilization rate of the system, and a decrease in the dissolved oxygen concentration in the system, which is not conducive to the aerobic degradation of pyridine in the system.

[0075] Comparative Example 3

[0076] Different from Example 1, the use amount ratio of the polyether F127 diacrylate, polyethylene glycol diacrylate, and liquid medium was 0.15 g:(0.01-0.15) g:1 mL, specifically 0.15 g:0.01 g:1 mL, 0.15 g:0.03 g:1 mL, 0.15 g:0.05 g:1 mL, 0.15 g:0.075 g:1 mL, 0.15 g:0.10 g:1 mL, and 0.15 g:0.15 g:1 mL. The other conditions were the same, and the artificial 3D bacterial-algal symbiont was prepared according to the process shown in Figure 1 .

[0077] The aforementioned bacterial-algal symbiota, formulated with varying ratios of polyether F127 diacrylate and polyethylene glycol diacrylate, were attached to the interior of a 120 mL reaction flask to degrade the volatile toxic organic pollutant pyridine. A free bacterial-algal symbiota system, with an MLSS equivalent equivalent to that of the artificial 3D bacterial-algal symbiota, was also used as a control. The reaction flask contained simulated wastewater containing 100 mL of pyridine and no nitrogen source. The simulated wastewater composition and experimental conditions were the same as in Example 2. The pyridine concentration in the system was monitored during the reaction.

[0078] Figure 7 This is a graph showing the degradation effect of pyridine on the free algae symbiotic system with an MLSS equivalent of the artificial 3D algae symbiotic system and the algae symbiotic system with different proportions of polyether F127 diacrylate and polyethylene glycol diacrylate. 0.15g:0.01g:1mL, 0.15g:0.03g:1mL, 0.15g:0.05g:1mL, 0.15g:0.075g:1mL, 0.15g:0.10g:1mL and 0.15g:0.15g:1mL correspond to Figure 7 The legend in the figure is F127DA15 PEGDA1, F127DA15PEGDA3, F127DA15 PEGDA5, F127DA15 PEGDA7.5, F127DA15 PEGDA10 and F127DA15PEGDA15; the control group is Control. Figure 7 As shown, the PEGDA content significantly affects the pyridine degradation rate. When the PEGDA content ranged from F127DA15 PEGDA1 to F127DA15 PEGDA7.5, the degradation efficiency of pyridine was stronger than that of the free system. In contrast, systems with lower PEGDA content (e.g., F127DA15 PEGDA1) exhibited poor degradation efficiency, while increasing the PEGDA content (e.g., F127DA15 PEGDA3 to F127DA15 PEGDA7.5) significantly improved the degradation efficiency. This may be related to the fact that increasing the PEGDA content enhanced the mechanical strength of the artificial 3D bacterial-algal symbiosis and reduced bacterial-algal escape. When the PEGDA content continued to increase to F127DA15PEGDA15, the degradation efficiency decreased, possibly because the excessive PEGDA content affected the system's light transmittance, air permeability, and bacterial-algal activity. Therefore, in practical applications, selecting an appropriate PEGDA ratio is very important for improving the degradation efficiency of artificial 3D bacteria-algae symbionts. The ratio of polyether F127 diacrylate, polyethylene glycol diacrylate and liquid culture medium of 0.15 g: (0.01-0.075) g: 1 mL may be more suitable as a system for efficient pyridine degradation.

Claims

1. 3D printing high-transmittance bio-ink, characterized by: Includes the following components: Polyether F127 diacrylate, polyethylene glycol diacrylate, pyridine-degrading bacteria Paracoccus Paracoccus sp. NJUST47, Chlorella Chorella sorokiniana FACHB-275, a light absorber, and a liquid culture medium containing a photoinitiator.

2. The 3D printing high-transmittance bio-ink according to claim 1, characterized in that: The molecular weight of the polyethylene glycol diacrylate is 400 to 6000, the molecular weight of the polyether F127 diacrylate is 10 to 15 kDa, the light absorber is Sudan red, β-carotene, curcumin, or tartrazine, and the liquid culture medium is a PBS solution containing 0.25% (w / v) of a photoinitiator, wherein the photoinitiator is 2,4,6-trimethylformyl phosphate lithium salt or hydroxyketone photoinitiator Irgacure 2959.

3. The 3D printing high-transmittance bio-ink according to claim 1, wherein: The dosage ratio of the polyether F127 diacrylate, polyethylene glycol diacrylate and liquid culture medium is 0.15 g: (0.01-0.075) g: 1 mL, and the pyridine-degrading special bacteria Paracoccus Paracoccus sp. NJUST47 in liquid culture medium MLSS is 7.5 ~ 22.5mg / mL, the Chlorella Chorella sorokiniana The MLSS of FACHB-275 in the liquid culture medium is 0.75-2.25 mg / mL, and the dosage ratio of the light absorber to the liquid culture medium is (0.002-0.008) g:1 mL.

4. The 3D printing high-transmittance bio-ink according to claim 1, wherein: The 3D printing high-transmittance bio-ink is composed of bio-ink No. 1, bio-ink No. 2 and a light absorber; the bio-ink No. 1 is composed of polyether F127 diacrylate, polyethylene glycol diacrylate, pyridine-degrading special bacteria Paracoccus Paracoccus sp. NJUST47 resuspension and a liquid culture medium containing a photoinitiator; the bio-ink No. 2 is composed of polyether F127 diacrylate, polyethylene glycol diacrylate, Chlorella vulgaris Chorella sorokiniana The solution consists of FACHB-275 resuspension and liquid culture medium containing photoinitiator.

5. The method for preparing a 3D printing high-transmittance bio-ink according to any one of claims 1 to 3, wherein: Polyether F127 diacrylate, polyethylene glycol diacrylate, pyridine degradation effect bacteria Paracoccus Paracoccus sp.NJUST47, Chlorella Chorella sorokiniana FACHB-275, a light absorber, and a liquid culture medium containing a photoinitiator are mixed.

6. The method for preparing 3D printing high-transmittance biological ink according to claim 4, characterized in that: The details are as follows: (1) First, polyether F127 diacrylate and polyethylene glycol diacrylate were mixed and dissolved in a liquid culture medium containing a photoinitiator, and then pyridine-degrading bacteria Paracoccus were added. Paracoccus sp. NJUST47 resuspension to obtain bio-ink No. 1; (2) First, dissolve polyether F127 diacrylate and polyethylene glycol diacrylate in a liquid culture medium containing a photoinitiator, and then add Chlorella vulgaris. Chorella sorokiniana FACHB-275 resuspension was used to obtain bio-ink No. 2; (3) After mixing bio-ink No. 1 and bio-ink No. 2, a light absorber is added and mixed evenly to obtain 3D printing high-transmittance bio-ink.

7. An artificial 3D bacteria-algae symbiont based on the 3D printed high-transmittance bio-ink according to any one of claims 1 to 4, characterized in that: The 3D printing high-transmittance biological ink according to any one of claims 1 to 4 is prepared by light-curing 3D printing under light irradiation.

8. The method for preparing an artificial 3D bacteria-algae symbiosis according to claim 7, characterized in that: The following steps are involved: The 3D printing high-transmittance biological ink described in any one of claims 1 to 4 is dropped into the printing platform of a projection light-curing 3D bioprinter, 3D printing is performed according to a preset spatial structure, and cross-linked under light to produce an artificial 3D bacteria-algae symbiont.

9. The artificial 3D bacteria-algae symbiosis according to claim 7, wherein: The artificial 3D bacteria-algae symbiont has a sucker configuration, with a total sucker length of 40 mm, a width of 12 mm, a bottom thickness of 0.5-1 mm, and 9 suckers distributed on the base. The sucker diameter is 4.66 mm and the height is 2.5 mm. The blue light wavelength is 405 nm and the light intensity is 25 mW / cm 2 ; The cross-linking time of a single layer is 2~10 s.

10. Use of the artificial 3D bacteria-algae symbiont according to claim 7 in the treatment of pyridine-containing wastewater.

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