A 3D printing bio-ink, a preparation method thereof, a 3D printing living functional material, a preparation method and application thereof

Abstract

The present application provides a kind of 3D printing biological ink, it includes: methacrylated gelatin, polyethylene glycol diacrylate, cellulase, lactobacillus pentosus and liquid medium.The present application also provides the preparation method of 3D printing biological ink.The present application also provides a kind of 3D printing functional living body material.The 3D printing biological ink provided by the present application uses methacrylated gelatin as the biological ink matrix, uses polyethylene glycol diacrylate as toughening agent, uses cellulase and lactobacillus pentosus as biological catalytic active component, and obtains the biological ink with good synergistic catalytic performance;On this basis, relying on the technical advantages of 3D biological printing, the biological ink is quickly, accurately and controllably constructed into functional living body material with good spatial configuration and higher synergistic catalytic efficiency, and the efficient conversion of cellulose to lactic acid can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cellulose resource utilization, and particularly relates to a 3D printing biological ink, a preparation method thereof, a 3D printing living body functional material and a preparation method and application thereof. BACKGROUND

[0002] The resource utilization of cellulose has great significance in reducing global carbon emissions and alleviating resource crisis, and is one of the difficulties that need to be overcome in the field of waste resource utilization at the present stage.

[0003] In recent years, the research on the resource utilization of cellulose into lactic acid by using the simultaneous saccharification and fermentation technology of enzymes and microorganisms has attracted much attention. However, the simultaneous saccharification and fermentation technology freely mixes enzymes and microorganisms in the same reactor, and due to the different optimal reaction conditions and the mutual influence between enzymes and microorganisms, the production efficiency of the system is reduced. Therefore, how to controllably construct a living body functional material with high enzyme-microorganism synergistic biological catalytic efficiency to intensify the conversion of cellulose into lactic acid in a short time is particularly urgent. SUMMARY

[0004] The technical problem solved by the present application is to provide a 3D printing biological ink, a preparation method thereof, a 3D printing living body functional material and a preparation method and application thereof. The living body functional material constructed by using the biological ink provided by the present application has good spatial configuration and high synergistic catalytic efficiency, and can efficiently convert cellulose into lactic acid.

[0005] Therefore, the present application provides a 3D printing biological ink, which comprises: methacrylated gelatin, polyethylene glycol diacrylate, cellulase, lactobacillus pentosus and a liquid culture medium.

[0006] Preferably, the use amount ratio of the methacrylated gelatin, the polyethylene glycol diacrylate and the liquid culture medium is (0.03-0.12) g:(0.018-0.06) g:1.2 mL.

[0007] The bacterial cell density OD of the lactobacillus pentosus in the liquid culture medium is 2-4; and the enzyme loading of the cellulase in the liquid culture medium is 1200-4000 U / ml. 600

[0008] Preferably, the liquid culture medium is a PBS solution, and the PBS solution further comprises 0.2-0.3% (w / v) of a blue light photoinitiator phenyl 2,4,6-trimethylbenzoyl lithium phosphate.

[0009] The present application further provides a preparation method of the 3D printing biological ink, which comprises the following steps:

[0010] ​Methacrylamide gelatin, polyethylene glycol diacrylate, cellulase, culture medium of Lactobacillus pentosus, and liquid culture medium were mixed to obtain 3D printing bio-ink;

[0011] The culture medium for Lactobacillus pentosus includes Lactobacillus pentosus and liquid culture medium.

[0012] Preferably, the preparation method of the 3D printing bio-ink is as follows:

[0013] Methacrylamide gelatin, polyethylene glycol diacrylate, cellulase and liquid culture medium were mixed to obtain 3D printing bio-ink No. 1;

[0014] Methacrylamide gelatin, polyethylene glycol diacrylate, and Lactobacillus pentosus culture medium were mixed with liquid culture medium to obtain 3D printing bio-ink No. 2.

[0015] This application also provides a method for preparing 3D printed functional living materials, including:

[0016] 3D printing bio-ink is added to a 3D bioprinter for 3D printing, and then photocrosslinking is performed to obtain 3D printed functional living material. The 3D printing bio-ink is the 3D printing bio-ink described above or the 3D printing bio-ink prepared by the preparation method described above.

[0017] Preferably, the preparation method of the 3D printed functional living material is as follows:

[0018] Methacrylamide gelatin, polyethylene glycol diacrylate, cellulase and liquid culture medium were mixed to obtain 3D printing bio-ink No. 1;

[0019] Methacrylamide gelatin, polyethylene glycol diacrylate, and Lactobacillus pentosus culture medium were mixed with liquid culture medium to obtain 3D printing bio-ink No. 2;

[0020] The 3D printing bio-ink No. 1 and the 3D printing bio-ink No. 2 are respectively loaded into the No. 1 and No. 2 material cylinders of the 3D bioprinter, and 3D printing is performed according to the preset spatial structure. The 3D printing bio-ink No. 1 is printed on the inner layer and the 3D printing bio-ink No. 2 is printed on the outer layer, and then photocrosslinking is performed under blue light.

[0021] This application also provides a 3D printed functional living material, which is obtained by sequentially 3D printing and cross-linking treatment of the 3D printed bio-ink prepared by the aforementioned 3D printed bio-ink or the 3D printed bio-ink prepared by the aforementioned preparation method.

[0022] Preferably, the 3D printed functional living material has a concentric cylindrical structure with inner and outer layers, the outer layer having a radius of 5-6 mm, the inner layer having a radius of 2-3 mm, and a height of 2-5 mm.

[0023] This application also provides a method for the resource utilization of cellulose, including:

[0024] The fermentation broth is mixed with a 3D printing functional living material for processing; the 3D printing functional living material is prepared by the aforementioned preparation method or is the 3D printing functional living material described above; the fermentation broth contains cellulose.

[0025] This invention provides a 3D printing bio-ink, its preparation method, a 3D printing functional living material, its preparation method, and its application. The 3D printing bio-ink provided by this invention comprises: methacryloyl gelatin, polyethylene glycol diacrylate, cellulase, Lactobacillus pentosus, and liquid culture medium. The 3D printing functional living material provided by this invention is made from the 3D printing bio-ink through 3D printing and cross-linking treatment. The 3D printing bio-ink provided by this invention uses methacryloyl gelatin (GelMA) as the bio-ink matrix, polyethylene glycol diacrylate (PEGDA) as a toughening agent, and cellulase and Lactobacillus pentosus as biocatalytic active ingredients, thus obtaining a bio-ink with good synergistic catalytic performance. Based on this, relying on the technological advantages of 3D bioprinting, this bio-ink is rapidly, precisely, and controllably constructed into a functional living material with good spatial configuration and high synergistic catalytic efficiency, enabling the efficient conversion of cellulose to lactic acid. Experimental results show that the functional living material with concentric cylindrical structure of inner and outer layers can efficiently convert microcrystalline cellulose into lactic acid. Using 17.5 g / L microcrystalline cellulose as substrate, the highest lactic acid yield can reach 6.55 g / L within 120 h. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the preparation process of the 3D printing functional living material provided in Embodiment 1 of the present invention;

[0027] Figure 2 This is a synergistic pathway diagram of cellulase and lactic acid bacteria provided by the present invention;

[0028] Figure 3 This is the frequency-modulus diagram of the bio-ink provided in Embodiment 2 of the present invention;

[0029] Figure 4 This is a shear rate-viscosity diagram of the bio-ink provided in Embodiment 2 of the present invention;

[0030] Figure 5This is the temperature-modulus diagram of the bio-ink provided in Embodiment 2 of the present invention;

[0031] Figure 6 This is a scanning electron microscope image of the cellulase portion contained in the functional living material provided in Embodiment 3 of the present invention;

[0032] Figure 7 This is a scanning electron microscope image of Lactobacillus pentosus contained in the functional living material provided in Embodiment 3 of the present invention;

[0033] Figure 8 These are model diagrams and digital photos of 3D printed functional living materials with different configurations provided in Embodiment 4 of the present invention;

[0034] Figure 9 This is a time-yield graph showing the final product lactic acid during the conversion of cellulose into lactic acid using 3D printing functional living materials with different configurations provided in Embodiment 4 of the present invention.

[0035] Figure 10 This is a time-yield graph of the intermediate product cellobiose during the conversion of cellulose into lactic acid using 3D-printed functional living materials with different configurations provided in Embodiment 4 of the present invention.

[0036] Figure 11 This is a time-yield graph showing the final product lactic acid during the conversion of cellulose into lactic acid using 3D functional living materials with different cellulase contents provided in Example 5 of the present invention.

[0037] Figure 12 This is a time-yield graph showing the intermediate product cellobiose during the conversion of cellulose into lactic acid using 3D functional living materials with different cellulase contents provided in Example 5 of the present invention. Detailed Implementation

[0038] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0039] Given the existing technology's need for efficient conversion of cellulose into lactic acid using living functional materials, this application provides a 3D printing bio-ink that uses cellulase and Lactobacillus pentosaceus as biocatalytic active ingredients, exhibiting good synergistic catalytic performance (e.g., ...). Figure 2 As shown, it can efficiently catalyze the conversion of cellulose into lactic acid. Specifically, this invention first provides a 3D printing bio-ink, which includes: methacrylamide gelatin, polyethylene glycol diacrylate, cellulase, Lactobacillus pentosus, and liquid culture medium.

[0040] In the 3D printing bio-ink provided by this invention, the methacryloyl gelatin (GelMA) serves as the bio-ink matrix; the methacryloyl gelatin is a commercially available product or prepared according to methods well known to those skilled in the art; specifically, the methacryloyl gelatin is preferably provided by Suzhou Yongqinquan Intelligent Equipment Co., Ltd., with product number EFL-GM-60; the preferred ratio of the methacryloyl gelatin to the liquid culture medium is (0.03-0.12) g: 1.2 ml, specifically 0.03 g: 1.2 ml, 0.04 g: 1.2 ml, 0.05 g: 1.2 ml, 0.06 g: 1.2 ml, 0.07 g: 1.2 ml, 0.08 g: 1.2 ml, 0.09 g: 1.2 ml, 0.1 g: 1.2 ml, 0.11 g: 1.2 ml, or 0.12 g: 1.2 ml, with the most preferred ratio being 0.06 g: 1.2 ml.

[0041] In the 3D printing bio-ink provided by this invention, polyethylene glycol diacrylate (PEGDA) is used as a toughening agent; the polyethylene glycol diacrylate is a commercially available product or prepared according to methods well known to those skilled in the art; specifically, the polyethylene glycol diacrylate is preferably provided by Suzhou Yongqinquan Intelligent Equipment Co., Ltd., with product number: EFL-PEGDA; the preferred ratio of polyethylene glycol diacrylate to liquid culture medium is (0.018~0.06) g: 1.2 ml, specifically it can be 0. 0.18g: 1.2ml, 0.021g: 1.2ml, 0.024g: 1.2ml, 0.027g: 1.2ml, 0.03g: 1.2ml, 0.033g: 1.2ml, 0.036g: 1.2ml, 0.039g: 1.2ml, 0.042g: 1.2ml, 0.045g: 1.2ml, 0.048g: 1.2ml, 0.051g: 1.2ml, 0.054g: 1.2ml, 0.057g: 1.2ml or 0.06g: 1.2ml, with 0.036g: 1.2ml being the most preferred.

[0042] In the 3D printing bio-ink provided by this invention, the cellulase is an active substance that decomposes cellulose into soluble sugars; the enzyme loading value of the cellulase in the liquid culture medium is preferably 1250-3750 U / ml, specifically 1375, 1500, 1625, 1750, 1875, 2000, 2125, 2250, 2375, 2500, 2625, 2750, 2875, 3000, 3125, 3250, 3375, 3500, 3625 or 3750 U / ml.

[0043] In the 3D printing bio-ink provided by this invention, Lactobacillus pentosus is used as a lactic acid-producing bacterium; the OD600 value of the bacterial cell density of Lactobacillus pentosus in liquid culture medium is preferably 2 to 4, specifically 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4, with 3 being the most preferred.

[0044] In the 3D bioprinting ink provided by this invention, the liquid culture medium can maintain the activity of cellulase and Lactobacillus pentosus without interfering with the photocrosslinking of the bioprinting ink matrix; the liquid culture medium is preferably a PBS solution, which uses deionized water as a solvent, and its solute components include: potassium dihydrogen phosphate (KH2PO4) 0.27 g / L, disodium hydrogen phosphate (Na2HPO4) 1.42 g / L, sodium chloride (NaCl) 8 g / L, potassium chloride (KCl) 0.2 g / L, wherein 2.5 g / L of blue light photoinitiator phenyl 2,4,6-trimethylbenzoyl phosphate lithium salt (LAP) is added.

[0045] This invention also provides a method for preparing the 3D printing bio-ink described in the above technical solution, comprising the following steps:

[0046] Methacrylamide gelatin, polyethylene glycol diacrylate, cellulase, culture medium of Lactobacillus pentosus, and liquid culture medium were mixed to obtain 3D printing bio-ink;

[0047] The culture medium for Lactobacillus pentosus includes Lactobacillus pentosus and liquid culture medium.

[0048] More specifically, the preparation method of the 3D printing bio-ink includes the following steps:

[0049] Methacrylamide gelatin, polyethylene glycol diacrylate, cellulase and liquid culture medium were mixed to obtain 3D printing bio-ink No. 1;

[0050] Methacrylamide gelatin, polyethylene glycol diacrylate, and the culture medium of Lactobacillus pentosus were mixed with liquid culture medium to obtain 3D printing bio-ink No. 2.

[0051] The culture medium of Lactobacillus pentosus includes Lactobacillus pentosus and liquid culture medium;

[0052] The liquid culture medium is preferably a PBS solution, which uses deionized water as a solvent. The solute components include: potassium dihydrogen phosphate (KH2PO4) 0.27 g / L, disodium hydrogen phosphate (Na2HPO4) 1.42 g / L, sodium chloride (NaCl) 8 g / L, potassium chloride (KCl) 0.2 g / L, and 2.5 g / L of blue light photoinitiator phenyl 2,4,6-trimethylbenzoyl phosphate lithium salt (LAP) is added.

[0053] In the preparation method of bio-ink No. 1 provided by the present invention, before the culture medium of Lactobacillus pentosus is mixed, the original LB culture medium of Lactobacillus pentosus is first centrifuged to remove the LB medium used for culture, and then the obtained bacteria are resuspended in fresh PBS medium; the LB liquid medium uses deionized water as solvent, and its solute components include: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride (NaCl).

[0054] In the preparation method of bio-ink No. 1 provided by the present invention, the preferred mixing process is to first mix methacrylamide gelatin and polyethylene glycol diacrylate, add them to PBS solution, then bathe them in a water bath at 70°C for 30 minutes, and after they melt, bathe them in a water bath at 37°C for 10 minutes, and then mix them with the culture medium of Lactobacillus pentosus.

[0055] In the preparation method of bio-ink No. 2 provided by the present invention, the preferred mixing process is to first mix methacrylamide gelatin and polyethylene glycol diacrylate, add them to PBS solution, then bathe them in a water bath at 70°C for 30 minutes, and after they melt, bathe them in a water bath at 37°C for 10 minutes, and then mix them with cellulase.

[0056] This invention also provides a method for preparing 3D printed functional living materials, comprising:

[0057] The 3D printing bio-ink is added to a 3D bioprinter for 3D printing, and then photocrosslinked to obtain a 3D printed functional living material. The 3D printing bio-ink is the 3D printing bio-ink described in the above scheme.

[0058] To improve the performance of the 3D printed functional living material, this application preferably utilizes the aforementioned 3D printing bio-ink No. 1 and 3D printing bio-ink No. 2 to prepare the 3D printed functional living material, specifically as follows:

[0059] The 3D printing bio-inks No. 1 and No. 2 described in the above technical solution are respectively loaded into the No. 1 and No. 2 material cylinders of the 3D bioprinter. 3D printing is carried out according to the preset spatial structure, with bio-ink No. 1 printed on the inner layer and bio-ink No. 2 printed on the outer layer. Finally, the 3D printed material is photocrosslinked under blue light to obtain 3D printed functional living material.

[0060] In the method for preparing biocathode materials provided by the present invention, the 3D bioprinter is preferably provided by Allevi; the specific method of 3D printing is performed in accordance with the methods known to those skilled in the art, and there are no particular limitations in this application.

[0061] In the method for preparing functional living materials provided by this invention, the crosslinking treatment is preferably performed at 405 nm with a light intensity of 25 mW / cm². 2 Blue light crosslinking; the crosslinking time is preferably 0.5 to 5 min, specifically 0.5 min, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min or 5 min, and most preferably 2 min.

[0062] The present invention also provides a 3D printing functional living material, which is made from the 3D printing bio-ink described in the above technical solution after 3D printing and cross-linking treatment.

[0063] In the functional living material provided by the present invention, the 3D printed functional living material is preferably a concentric cylindrical structure with inner and outer layers. The inner layer radius of the mesh structure is preferably 1 to 4 mm, specifically 1 mm, 1.15 mm, 1.3 mm, 1.45 mm, 1.6 mm, 1.75 mm, 1.9 mm, 2.05 mm, 2.2 mm, 2.35 mm, 2.5 mm, 2.65 mm, 2.8 mm, 2.95 mm, 3.1 mm, 3.25 mm, 3.4 mm, 3.55 mm, 3.7 mm, 3.85 mm or 4 mm, with 2.5 mm being the most preferred.

[0064] This invention also provides a method for cellulose resource utilization, comprising:

[0065] The fermentation broth is mixed with a 3D printing functional living material for processing; the 3D printing functional living material is the 3D printing functional living material described in the above scheme, and the fermentation broth contains cellulose.

[0066] In the cellulose resource utilization method provided by the present invention, the treatment specifically uses a 75ml cylindrical reactor, and then nitrogen gas is blown into it to remove oxygen from the system, thereby creating anaerobic conditions for lactic acid bacteria fermentation.

[0067] In the cellulose resource utilization method provided by the present invention, the cellulose contained in the fermentation broth is preferably microcrystalline cellulose (MCC); the concentration of microcrystalline cellulose in the fermentation broth is preferably 7.5-40 g / L, specifically 7.5 g / L, 9.5 g / L, 11.5 g / L, 13.5 g / L, 15.5 g / L, 17.5 g / L, 19.5 g / L, 21.5 g / L, 23.5 g / L, 25.5 g / L, 27.5 g / L, 29.5 g / L, 31.5 g / L, 33.5 g / L, 35.5 g / L, 37.5 g / L, or 40 g / L.

[0068] The 3D-printed functional living material provided by this invention is made from the 3D-printed bio-ink through 3D printing and cross-linking treatment. The technical solution provided by this invention uses gelatin methacryloyl (GelMA) as the bio-ink matrix, polyethylene glycol diacrylate (PEGDA) as a toughening agent, and cellulase and Lactobacillus pentosus as biocatalytic active ingredients to obtain a bio-ink with good synergistic catalytic performance. Based on this, relying on the technological advantages of 3D bioprinting, this bio-ink is rapidly, precisely, and controllably constructed into a functional living material with good spatial configuration and high synergistic catalytic efficiency, enabling the efficient conversion of cellulose to lactic acid. Experimental results show that the functional living material with a concentric cylindrical structure of inner and outer layers can efficiently convert microcrystalline cellulose into lactic acid. Using 17.5 g / L microcrystalline cellulose as a substrate, the highest lactic acid yield can reach 6.55 g / L within 120 hours.

[0069] To further understand the present invention, the following detailed description, in conjunction with embodiments, provides the 3D printing bio-ink, its preparation method, 3D printing living functional materials, their preparation methods, and applications provided by the present invention. The scope of protection of the present invention is not limited by the following embodiments.

[0070] Example 1

[0071] according to Figure 1 The process shown is for preparing 3D printed functional living materials. The specific process is as follows:

[0072] First, mix 0.015g of methacrylamide gelatin and 0.009g of polyethylene glycol diacrylate, add them to 0.3ml of PBS solution, then incubate in a water bath at 70℃ for 30min. After melting, incubate in a water bath at 37℃ for 10min, then add 0.018g of cellulase, mix well and the resulting bio-ink No. 1 is prepared.

[0073] First, centrifuge the purchased Lactobacillus pentosus culture medium at 25°C and 4000 rpm for 8 minutes to remove the LB medium used for culture. Then, resuspend the bacteria in PBS liquid medium and adjust the OD. 600 The value was 6.0; then 0.045g of methacrylamide gelatin and 0.027g of polyethylene glycol diacrylate were mixed and added to 0.45ml of PBS solution. The mixture was then placed in a water bath at 70℃ for 30min. After melting, it was placed in a water bath at 37℃ for 10min. Finally, it was mixed with 0.45ml of resuspended Lactobacillus pentosus culture medium to prepare bio-ink No. 2.

[0074] Bio-inks 1 and 2 were transferred to 10mL sterile syringes and added to a 3D bioprinter (ALLEVI, USA) equipped with a 25G nozzle (0.26mm filament diameter) for 3D printing (Bio-ink 1 was printed on the inner layer and Bio-ink 2 on the outer layer, or Bio-ink 1 on the outer layer and Bio-ink 2 on the inner layer), resulting in a functional living material with a concentric cylindrical structure (overall material radius of 5mm, with an inner layer radius of 2.5mm and an overall height of 3mm); the semi-finished product was then exposed to light at a wavelength of 405nm and an intensity of 25mW / cm². 2 Photocrosslinking treatment under blue light source for 2 minutes yields 3D printed functional living material.

[0075] Example 2

[0076] Rheological characterization of the two bio-inks prepared, GelMA / PEGDA (with the same component content as in Example 1, but without cellulase and Lactobacillus pentosus) and GelMA (with the same component content as in Example 1, but without polyethylene glycol diacrylate, cellulase, and Lactobacillus pentosus), was performed using a rheometer. The results are as follows: Figures 3-5 As shown. Figure 3 This is a frequency-modulus diagram of two bio-inks, GelMA / PEGDA and GelMA, provided in Embodiment 2 of the present invention. Figure 4 This is a shear rate-viscosity diagram of two bio-inks, GelMA / PEGDA and GelMA, provided in Example 2 of this invention. Figure 5 This is a temperature-modulus diagram of two bio-inks, GelMA / PEGDA and GelMA, provided in Embodiment 2 of the present invention. Figure 3It can be seen that the storage modulus (G') of both GelMA / PEGDA and GelMA bio-inks is higher than their loss modulus (G'). The addition of PEGDA increases the storage modulus of the bio-ink, indicating that PEGDA enhances its strength. Figure 4 It was found that both GelMA / PEGDA and GelMA bio-inks exhibited shear-thinning behavior with varying viscosities over a wide range of shear rates. Furthermore, no hysteresis was observed in the increase or decrease of shear rate, indicating that the viscoelasticity of the bio-ink formulated with PEGDA recovered rapidly after printing, ensuring the accuracy of 3D printing and the survival rate of cells. Figure 5 It is known that both GelMA / PEGDA and GelMA bio-inks exhibit thermosensitivity, with a phase transition temperature of 25℃. They exhibit solid behavior at temperatures below 25℃ and liquid behavior at temperatures above 25℃. Furthermore, the addition of PEGDA does not affect the phase transition temperature of the ink, indicating that 3D printing can be performed at 25℃, at which point the ink is in a gel state.

[0077] Example 3

[0078] Scanning electron microscopy analysis was performed on the 3D-printed living functional material prepared using bio-ink No. 1 in Example 1. The results are as follows: Figure 6 As shown, Figure 6 This is a scanning electron microscope (SEM) image of the inner layer of the functional living material provided in Embodiment 3 of the present invention. (The image is obtained through...) Figure 6 It can be seen that the internal pore structure of the living functional material is a macroporous structure, and the cellulase material is uniformly distributed in the internal pores of the material.

[0079] Scanning electron microscopy analysis was performed on the 3D-printed living functional material prepared using bio-ink No. 2 in Example 1. The results are as follows: Figure 7 As shown, Figure 7 This is a scanning electron microscope (SEM) image of the functional living material provided in Embodiment 3 of the present invention. (The image is obtained through...) Figure 7 It can be seen that bacteria thrive in the internal porous structure of the living functional material.

[0080] Example 4

[0081] The 3D-printed functional living material prepared in Example 1 was added to a 75 ml reactor for cellulose resource recovery. The reactor contained a microcrystalline cellulose solution. 0.875 g of microcrystalline cellulose was weighed and dispersed in 50 ml of water to obtain a simulated fermentation broth with a concentration of 17.5 g / L. The reactor was placed in a shaker at 37°C and 200 r / min for fermentation, and the timer was started. Every 24 h, 0.5 ml of the reaction solution was taken, filtered through an aqueous membrane (0.22 μm), and the supernatant was obtained. 0.3 ml of the supernatant was taken, and 15 μL of 72% (v / v) H2SO4 was added to acidify the sample. The concentrations of glucose, cellobiose, and lactic acid in the fermentation broth were tested by high performance liquid chromatography (HPLC). The separation was performed using an HPX-87H column at a column temperature of 55°C, a mobile phase of 5 mL H2SO4, and a mobile phase flow rate of 0.6 ml / min. The RID detector was used for detection at 35°C.

[0082] The functional living materials with different configurations prepared in Example 1 are respectively labeled as Bacteria inside (inner bacterial layer, cellulase printed on the outer layer, and lactic acid bacteria printed on the inner layer) and Bacteria outside (outer bacterial layer, cellulase printed on the inner layer, and lactic acid bacteria printed on the outer layer). Their physical images and model diagrams are shown below. Figure 8 As shown, lactic acid production is as follows Figure 9 As shown, the yield of the intermediate product cellobiose is as follows: Figure 10 As shown. (Through) Figure 9 It can be seen that the lactate-producing effect of the Bacteria outside configuration is about 5 times that of the Bacteria inside configuration. Figure 10 It can also be seen that the accumulation of cellobiose also shows the same trend. The possible reason is that the lactic acid bacteria are printed on the outer layer, which effectively protects the activity of cellulase in the inner layer, maintains the efficient conversion of cellulose into soluble sugar, and thus increases the lactic acid production of lactic acid bacteria.

[0083] Example 5

[0084] Optimizing cellulase loading is crucial for enhancing fermentation efficiency, saving material costs, and improving practical applicability. Example 5 aims to optimize the cellulase loading of the Bacteria outside structure 3D-printed functional living material prepared in Example 1. The lactic acid yield was tested within the enzyme loading range of 15–45 U / ml using the Bacteria outside structure 3D-printed functional living material prepared in Example 1. Specific sampling and testing procedures are detailed in Example 4. Lactic acid yield is as follows: Figure 11 As shown, the yield of the intermediate product cellobiose is as follows: Figure 12 As shown. (Through) Figure 11It can be seen that with the increase of cellulase loading, lactic acid production first increases and then decreases, reaching its highest level at an enzyme loading of 35 U / ml. Figure 12 It can be seen that the accumulation of cellobiose increases with the increase of cellulase loading. When the enzyme loading reaches 45 U / ml, the accumulation of cellobiose increases sharply, producing a substrate inhibition effect, which leads to a decrease in lactic acid production in the entire system.

[0085] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0086] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for the resource utilization of cellulose, comprising: The fermentation broth is mixed with 3D printed functional living material for processing; the fermentation broth contains cellulose; The specific method for preparing the 3D printing functional living material is as follows: Methacrylamide gelatin, polyethylene glycol diacrylate, cellulase and liquid culture medium were mixed to obtain 3D printing bio-ink No. 1; Methacrylamide gelatin, polyethylene glycol diacrylate, and Lactobacillus pentosus culture medium were mixed with liquid culture medium to obtain 3D printing bio-ink No. 2; The liquid culture medium also includes the blue light photoinitiator lithium phenyl 2,4,6-trimethylbenzoyl phosphate; The culture medium for *Lactobacillus pentosus* includes *Lactobacillus pentosus* and PBS liquid culture medium, and the preparation method is as follows: The Lactobacillus pentosaceus culture medium was centrifuged at 25°C and 4000 rpm for 8 min to remove the LB medium used for culture. The resulting bacteria were then resuspended in PBS liquid medium, and the OD was adjusted. 600 The value is 2-4; the enzyme loading of the cellulase in the liquid culture medium is 1200-4000 U / ml; The 3D printing bio-ink No. 1 and the 3D printing bio-ink No. 2 are respectively loaded into the No. 1 and No. 2 material cylinders of the 3D bioprinter, and 3D printing is performed according to the preset spatial structure. The 3D printing bio-ink No. 1 is printed on the inner layer and the 3D printing bio-ink No. 2 is printed on the outer layer, and then photocrosslinking is performed under blue light. The ratio of methacrylamide gelatin, polyethylene glycol diacrylate, and liquid culture medium is (0.03~0.12) g : (0.018~0.06) g : 1.2 mL; The 3D printed functional living material has a concentric cylindrical structure with inner and outer layers. The outer layer has a radius of 5-6 mm, the inner layer has a radius of 2-3 mm, and the height is 2-5 mm. Cellulase is printed in the inner layer, and Lactobacillus pentosus is printed in the outer layer.

2. The method according to claim 1, characterized in that, The liquid culture medium is a PBS solution, which further contains 0.2-0.3% w / v of phenyl 2,4,6-trimethylbenzoyl phosphate lithium salt, a blue light photoinitiator.

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