Algae / bacterium symbiotic hydrogel with core-shell structure and preparation method of algae / bacterium symbiotic hydrogel

Abstract

The invention discloses a core-shell structure algae / bacteria symbiotic hydrogel and a preparation method thereof, the core-shell structure algae / bacteria symbiotic hydrogel (algae is a core and bacteria are a shell) is prepared by using a 3D printing technology, the algae and the bacteria are separated from each other in space, and a customized latticed structure can effectively disperse light, so that microalgae in a bracket can receive the light, and the algae / bacteria symbiotic hydrogel is prepared. The problem of low hydrogen production efficiency caused by serious mutual shielding among cells, low light energy utilization rate, nutrient substance competition and the like is solved, the system almost eliminates the requirement on a liquid medium, and growth of microalgae and bacteria in the hydrogel can be supported only by forming a layer of solid culture medium at the bottom of the printed hydrogel; water resources are saved; and the space utilization rate is improved.

Description

[0001] manual Technical Field

[0002] The invention relates to a biological hydrogen production technology, in particular to a method for improving photosynthetic hydrogen production by using a core-shell structured algae / bacteria symbiotic hydrogel. Background Art

[0003] As a clean, efficient and renewable energy source, hydrogen energy has great potential and application prospects. It only produces water after combustion without any pollutants, and is considered to be an important pillar in the future energy structure. However, in the field of hydrogen energy production, despite its broad prospects, biohydrogen production technology, especially microalgae hydrogen production, still faces many limitations and challenges.

[0004] Microalgae produce oxygen and hydrogen during photosynthesis. Through specific technical means, the hydrogen produced by microalgae can be extracted, thus achieving the goal of producing hydrogen directly from water.

[0005] At present, the main technologies for using microalgae for photosynthetic hydrogen production include sulfur-deficient culture, genetic engineering, adding oxygen scavengers, cell aggregation, and direct mixed culture of microalgae and bacteria. Sulfur-deficient culture, cell aggregation, and the use of genetic engineering methods to construct high-hydrogen-producing algae strains can improve the hydrogen production efficiency of microalgae, but their operations are complex and costly; adding oxygen scavengers can make green algae easier to enter the photosynthetic hydrogen production process, but the requirements for added chemical reagents are high, and there is still a certain gap from large-scale application. In the algae / bacteria co-culture hydrogen production system, the oxygen released by the microalgae is consumed by the bacteria through respiration. As the oxygen in the environment decreases, the oxygen in the microalgae cells is quickly released, and the hydrogen production system reaches an anaerobic state in a short time, ensuring the high activity of the reversible hydrogen production enzyme, and realizing the improvement of the hydrogen production efficiency of the microalgae and the extension of the hydrogen production time. The respiration of bacteria not only promotes the consumption of oxygen, but also participates in the carbon cycle of microalgae photosynthesis by producing carbon dioxide, which is beneficial to the accumulation of microalgae biomass. However, during the algae / bacteria symbiosis, when cultured at high density, cells shield each other severely, and the light intensity inside the reactor is too low to maintain the microalgae photolysis of water to produce hydrogen, resulting in a decrease in hydrogen production capacity. In addition, long-term high-density culture easily accumulates a large amount of toxic metabolites in the system, causing certain reactions in the cells to fail to proceed normally. At the same time, the increase in cell density gradually increases the amount of hydrogen produced in the system, and the excessively high hydrogen partial pressure prompts the hydrogenase to absorb hydrogen, causing the catalytic reaction to proceed in reverse, and the hydrogen production rate drops rapidly. As the substrate is insufficient, substrate competition inhibition will also occur between algae and bacteria. Summary of the invention

[0006] The purpose of the present invention is to solve the problem of low hydrogen production efficiency caused by severe mutual shielding between cells, competition for nutrients and the like in algae / bacteria co-culture hydrogen production, and to provide a core-shell structure algae / bacteria symbiotic hydrogel and a preparation method thereof.

[0007] To achieve the above-mentioned purpose, the present invention provides a core-shell structure algae / bacteria symbiotic hydrogel, characterized in that the gel is composed of a core layer structure and a shell layer structure, the core layer structure includes sodium alginate (SA), a rheology modifier and microalgae, and the shell layer structure includes polyethylene glycol diacrylate (PEGDA), sodium alginate (SA), a rheology modifier and fungi.

[0008] Furthermore, the rheology modifier is carrageenan.

[0009] Furthermore, the microalgae may be any one of Chlamydomonas reinhardtii, Chlorella vulgaris or Chlorella pyrenoidosa, or a combination of at least two thereof.

[0010] Furthermore, the fungi may be aerobic bacteria such as Bacillus subtilis and Escherichia coli.

[0011] The present invention also provides a method for preparing a core-shell structure algae / bacteria symbiotic hydrogel, which is characterized by:

[0012] (1)Configure printing ink

[0013] Ink preparation for the core structure: Weigh a certain amount of sodium alginate (SA) and add it to the TAP medium and stir evenly. Add an appropriate amount of rheology modifier, and finally add a certain amount of Chlamydomonas reinhardtii, stir evenly and set aside.

[0014] Ink configuration of shell structure: Weigh a certain amount of polyethylene glycol diacrylate (PEGDA) and add it to deionized water and stir it evenly to obtain PEGDA solution. Weigh a certain amount of sodium alginate SA and add it to it, heat it in a 50°C water bath until it is completely dissolved, then add 0.1% initiator and an appropriate amount of rheology modifier, and finally add a certain amount of Bacillus subtilis, stir it evenly and set aside.

[0015] (2) 3D printing and cross-linking

[0016] The core-shell structure ink configured as above is added into the two-component nozzle barrel of the 3D printer respectively, the printing equipment is built, the parameters of the two-component nozzle are set, the print head moves along the preset path, and prints layer by layer under ultraviolet light.

[0017] (3) Cross-linking

[0018] After printing, the hydrogel is placed in a calcium chloride solution for cross-linking.

[0019] Furthermore, in step (1), the concentration of sodium alginate (SA) in the core structure ink is 1%-10%, and the amount of Chlamydomonas reinhardtii is 1×10 6 -1×10 8 Pieces / ml.

[0020] Furthermore, in step (1), the concentration of polyethylene glycol diacrylate (PEGDA) in the shell structure ink is 1%-10%, the concentration of sodium alginate (SA) is 1%-10%, and the amount of Bacillus subtilis is 1×10 6 -1×10 9 Pieces / ml.

[0021] Furthermore, the 3D printing parameters in step (2) are set as follows: nozzle size: 1.2 mm, printing speed: 15 mm / min, number of layers: 6 layers, early wire output: 200 ms, early wire shut-off: 200 ms.

[0022] Furthermore, in step (3), the cross-linking time is 1-10 minutes, and the curing time under ultraviolet light is 20-60 seconds.

[0023] The present invention has the following beneficial effects:

[0024] The core-shell algae / bacteria symbiotic hydrogel (algae as the core and bacteria as the shell) was prepared using 3D printing technology, which separated algae and bacteria in space. The customized grid structure can effectively disperse light, allowing the microalgae in the scaffold to receive light, solving the problem of low hydrogen production efficiency caused by serious mutual shielding between cells, low light energy utilization, and competition for nutrients. The system almost eliminates the need for liquid media. It only needs to form a layer of solid culture medium at the bottom of the printed hydrogel to support the growth of microalgae and bacteria in the hydrogel, saving water resources and improving space utilization. In short, the core-shell algae / bacteria symbiotic hydrogel not only improves the hydrogen production efficiency of microalgae, but also achieves resource conservation and environmental sustainability by reducing the use of liquid media, providing an innovative and efficient solution for the development of green energy in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Core-shell structured gels can be printed into different shapes.

[0026] Figure 2 Example 4: Different layers of algae and bacteria fluorescence in the core-shell structure gel are distributed in layers. The red fluorescent part in the figure is Chlamydomonas reinhardtii, and the green fluorescent part is Bacillus subtilis. This shows that Chlamydomonas reinhardtii and Bacillus subtilis are distributed in layers.

[0027] Figure 3 : Fluorescence distribution of core-shell structure gels of different sizes in Examples 3-5. The red fluorescent part in the figure is Chlamydomonas reinhardtii, and the green fluorescent part is Bacillus subtilis, indicating that different printing parameters can successfully achieve layered distribution of algae and bacteria.

[0028] Figure 4 : The effect of different algae-bacteria ratios on hydrogen production in Examples 1-5. As can be seen from the figure, the hydrogen production is highest when the algae-bacteria ratio is 1:15.

[0029] Figure 5 : Example 4 Core-shell structure gel confocal microscopy image, from which it can be seen that in the subsequent culture cycle, algae and bacteria achieve layered growth in the gel. Using microscope and confocal microscopy to explore the growth of algae and bacteria in the gel

[0030] Figure 6 : Comparison diagram of the core-shell structure gel in Example 4 and the non-core-shell structure gel in the comparative example. It can be seen from the figure that the biomass of the core-shell structure gel is higher than that of the direct mixed printing. The microalgae began to turn yellow on the 4th day of mixed printing. Due to the high-density culture, some toxic metabolites of bacteria are not conducive to the growth of microalgae. Specific embodiments

[0031] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0032] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the art can be used. If no manufacturer is specified for the raw materials used, they are all conventional reagents that can be purchased commercially or prepared according to the literature.

[0033] Embodiment 1:

[0034] A core-shell structure algae / bacteria symbiotic hydrogel preparation for photosynthetic hydrogen production, the specific implementation method is as follows:

[0035] (1) Printing ink configuration

[0036] Ink preparation for core structure: Weigh 0.3 g SA and add it to 10 mL TAP medium and stir evenly. Add 1 g rheology modifier carrageenan and finally add 1×10 6 Add 1000 cells of Chlamydomonas reinhardtii into it, stir well and set aside.

[0037] Ink preparation for shell structure: Weigh 2g PEGDA and add it to 10mL deionized water, stir well to obtain PEGDA solution. Weigh 0.5g SA and add it to the above solution, heat it in a 50℃ water bath until it is completely dissolved, then add 100μg initiator LAP and 1g rheology modifier carrageenan, stir well and set aside.

[0038] (2) 3D printing and cross-linking

[0039] The core-shell structure ink configured above is added to the two syringes of the print head respectively, the printing equipment is set up, the parameters of the two-component nozzle are set, and the printing parameters are set as nozzle size: 1.2mm, printing speed: 15mm / min, number of layers: 6 layers, early wire: 200ms, early wire closing: 200ms. The print head moves along the preset path and prints layer by layer.

[0040] (3) Cross-linking

[0041] After printing, the hydrogel was placed in a calcium chloride solution for cross-linking for 3 minutes and cured under UV for 30 seconds.

[0042] Embodiment 2:

[0043] A core-shell structure algae / bacteria symbiotic hydrogel preparation for photosynthetic hydrogen production, the specific implementation method is as follows:

[0044] (1) Printing ink configuration

[0045] Ink preparation for core structure: Weigh 0.5 g SA and add it to 10 mL TAP medium and stir evenly. Add 1 g rheology modifier carrageenan and finally add 1×10 8 Add 1000 cells of Chlamydomonas reinhardtii into it, stir well and set aside.

[0046] Ink preparation for shell structure: 3 g PEGDA was weighed and added to 10 mL deionized water, and stirred to obtain PEGDA solution. 0.5 g SA was weighed and added to the above solution, and heated in a 50 °C water bath until completely dissolved. Then 100 μg initiator LAP and 1 g rheology modifier carrageenan were added, and finally 5 × 10 8 Add Bacillus subtilis, stir well and set aside.

[0047] (2) 3D printing and cross-linking

[0048] The core-shell structure ink configured above is added to the two syringes of the print head respectively, the printing equipment is set up, the parameters of the two-component nozzle are set, and the printing parameters are set as nozzle size: 1.2mm, printing speed: 15mm / min, number of layers: 6 layers, early wire: 200ms, early wire closing: 200ms. The print head moves along the preset path and prints layer by layer.

[0049] (3) Cross-linking

[0050] After printing, the hydrogel was placed in a calcium chloride solution for cross-linking for 3 minutes and cured under UV for 30 seconds.

[0051] Embodiment 3:

[0052] A core-shell structure algae / bacteria symbiotic hydrogel preparation for photosynthetic hydrogen production, the specific implementation method is as follows:

[0053] (1) Printing ink configuration

[0054] Ink preparation for core structure: Weigh 0.5 g SA and add it to 10 mL TAP medium and stir evenly. Add 1 g rheology modifier carrageenan and finally add 1×10 8 Add 1000 cells of Chlamydomonas reinhardtii into it, stir well and set aside.

[0055] Ink configuration for shell structure: 3 g PEGDA was weighed and added to 10 mL deionized water, and stirred to obtain a PEGDA solution. 0.5 g SA was weighed and added to the above solution, and heated in a 50 °C water bath until completely dissolved. Then 100 μg initiator LAP and 1 g rheology modifier carrageenan were added, and finally 1×10 9 Add Bacillus subtilis, stir well and set aside.

[0056] (2) 3D printing and cross-linking

[0057] The core-shell structure ink configured above is added to the two syringes of the print head respectively, the printing equipment is set up, the parameters of the two-component nozzle are set, and the printing parameters are set as nozzle size: 1.2mm, printing speed: 15mm / min, number of layers: 6 layers, early wire: 200ms, early wire closing: 200ms. The print head moves along the preset path and prints layer by layer.

[0058] (3) Cross-linking

[0059] After printing, the hydrogel was placed in a calcium chloride solution for cross-linking for 3 minutes and cured under UV for 30 seconds.

[0060] Embodiment 4:

[0061] A core-shell structure algae / bacteria symbiotic hydrogel preparation for photosynthetic hydrogen production, the specific implementation method is as follows:

[0062] (1) Printing ink configuration

[0063] Ink preparation for core structure: Weigh 0.5 g SA and add it to 10 mL TAP medium and stir evenly. Add 1 g rheology modifier carrageenan and finally add 1×10 8 Add 1000 cells of Chlamydomonas reinhardtii into it, stir well and set aside.

[0064] Ink configuration for shell structure: 3 g PEGDA was weighed and added to 10 mL deionized water, and stirred to obtain a PEGDA solution. 0.5 g SA was weighed and added to the above solution, and heated in a 50 °C water bath until completely dissolved. Then 100 μg initiator LAP and 1 g rheology modifier carrageenan were added, and finally 1.5 × 10 9 Add Bacillus subtilis, stir well and set aside.

[0065] (2) 3D printing and cross-linking

[0066] The core-shell structure ink configured above is added to the two syringes of the print head respectively, the printing equipment is set up, the parameters of the two-component nozzle are set, and the printing parameters are set as nozzle size: 1.0mm, printing speed: 15mm / min, number of layers: 6 layers, early wire: 200ms, early wire closing: 200ms. The print head moves along the preset path to print layer by layer.

[0067] (3) Cross-linking

[0068] After printing, the hydrogel was placed in a calcium chloride solution for cross-linking for 3 minutes and cured under UV for 30 seconds.

[0069] Embodiment 5:

[0070] A core-shell structure algae / bacteria symbiotic hydrogel preparation for photosynthetic hydrogen production, the specific implementation method is as follows:

[0071] (1) Printing ink configuration

[0072] Ink preparation for core structure: Weigh 0.5 g SA and add it to 10 mL TAP medium and stir evenly. Add 1 g rheology modifier carrageenan and finally add 1×10 8 Add 1000 cells of Chlamydomonas reinhardtii into it, stir well and set aside.

[0073] Ink configuration for shell structure: 3 g PEGDA was weighed and added to 10 mL deionized water, and stirred to obtain a PEGDA solution. 0.5 g SA was weighed and added to the above solution, and heated in a 50 °C water bath until completely dissolved. Then 100 μg initiator LAP and 1 g rheology modifier carrageenan were added, and finally 2.5 × 10 9 Add Bacillus subtilis, stir well and set aside.

[0074] (2) 3D printing and cross-linking

[0075] The core-shell structure ink configured above is added to the two syringes of the print head, the printing equipment is set up, the parameters of the two-component nozzle are set, and the printing parameters are set as nozzle size: 0.8mm, printing speed: 15mm / min, number of layers: 6 layers, early wire: 200ms, early wire closing: 200ms. The print head moves along the preset path to print layer by layer.

[0076] (3) Cross-linking

[0077] After printing, the hydrogel was placed in a calcium chloride solution for cross-linking for 3 minutes and cured under UV for 30 seconds.

[0078] Embodiment 6:

[0079] A core-shell structure algae / bacteria symbiotic hydrogel preparation for photosynthetic hydrogen production, the specific implementation method is as follows:

[0080] (1) Printing ink configuration

[0081] Ink preparation for core structure: Weigh 0.5 g SA and add it to 10 mL TAP medium and stir evenly. Add 1 g rheology modifier carrageenan and finally add 1×10 8 Add 1000 cells of Chlamydomonas reinhardtii into it, stir well and set aside.

[0082] Ink configuration for shell structure: 3 g PEGDA was weighed and added to 10 mL deionized water, and stirred to obtain a PEGDA solution. 0.5 g SA was weighed and added to the above solution, and heated in a 50 °C water bath until completely dissolved. Then 100 μg initiator LAP and 1 g rheology modifier carrageenan were added, and finally 2 × 10 9 Add Bacillus subtilis, stir well and set aside.

[0083] (2) 3D printing and cross-linking

[0084] The core-shell structure ink configured above is added to the two syringes of the print head respectively, the printing equipment is set up, the parameters of the two-component nozzle are set, and the printing parameters are set as nozzle size: 1.2mm, printing speed: 15mm / min, number of layers: 6 layers, early wire: 200ms, early wire closing: 200ms. The print head moves along the preset path and prints layer by layer.

[0085] (3) Cross-linking

[0086] After printing, the hydrogel was placed in a calcium chloride solution for cross-linking for 3 minutes and cured under UV for 30 seconds.

[0087] Comparative Example 1:

[0088] A preparation method of algae-bacteria blended hydrogel for photosynthetic hydrogen production is as follows:

[0089] (1) Printing ink configuration

[0090] Weigh 0.3 g SA and 2 g PEGDA into 20 mL TAP medium and stir evenly. Add 100 μg initiator LAP and 1 g rheology modifier carrageenan, and finally add 1×10 8 Chlamydomonas reinhardtii cells and 1×10 9 Add Bacillus subtilis, stir well and set aside.

[0091] (2) 3D printing and cross-linking

[0092] Add the above-configured inks into the syringes respectively, build the printing equipment, set the parameters, move the print head along the preset path, and print layer by layer.

[0093] (3) Cross-linking

[0094] After printing, the hydrogel was placed in a calcium chloride solution for crosslinking for 3 minutes and cured under UV for 30 seconds.

[0095] Add 5 ml of LB solid culture medium to a 100 ml hydrogen production bottle. After the culture medium cools and solidifies, spread the printed gel on top of the solid culture medium. Install the device and purge with nitrogen for three minutes to eliminate the inhibitory effect of oxygen in the top air on hydrogenase. Next, place the sample in an incubator at 25°C and a light intensity of 50E·m-2·s-1, and regularly measure the amount of hydrogen released in the headspace of the hydrogen production bottle by gas chromatography.

[0096] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A core-shell structure algae / bacteria symbiotic hydrogel, characterized in that: The gel is composed of a core layer structure and a shell layer structure. The core layer structure comprises sodium alginate (SA), a rheology modifier and microalgae, and the shell layer structure comprises polyethylene glycol diacrylate (PEGDA), sodium alginate (SA), a rheology modifier and fungi.

2. The core-shell algae / bacteria symbiotic hydrogel according to claim 1, characterized in that: The rheology modifier is carrageenan.

3. The core-shell algae / bacteria symbiotic hydrogel according to claim 1, characterized in that: The microalgae may be any one of Chlamydomonas reinhardtii, Chlorella vulgaris or Chlorella pyrenoidosa, or a combination of at least two thereof.

4. The core-shell structure algae / bacteria symbiotic hydrogel according to claim 1, characterized in that: The fungi can be aerobic bacteria such as Bacillus subtilis and Escherichia coli.

5. The method for preparing a core-shell structure algae / bacteria symbiotic hydrogel according to any one of claims 1 to 4, characterized in that: (1)Configure printing ink Ink preparation for the core structure: Weigh a certain amount of sodium alginate (SA) and add it to the TAP medium and stir evenly. Add an appropriate amount of rheology modifier, and finally add a certain amount of Chlamydomonas reinhardtii, stir evenly and set aside. Ink configuration for shell structure: Weigh a certain amount of polyethylene glycol diacrylate (PEGDA) and add it to deionized water and stir evenly to obtain a PEGDA solution. Weigh a certain amount of sodium alginate SA and add it into the solution. Heat in a 50°C water bath until completely dissolved, then add 0.1% initiator and an appropriate amount of rheology modifier, and finally add a certain amount of Bacillus subtilis, stir evenly and set aside. (2) 3D printing and cross-linking The core-shell structure ink configured as above is added into the two-component nozzle barrel of the 3D printer respectively, the printing equipment is built, the parameters of the two-component nozzle are set, the print head moves along the preset path, and prints layer by layer under ultraviolet light. (3) Cross-linking After printing, the hydrogel is placed in a calcium chloride solution for cross-linking.

6. The method for preparing the core-shell structure algae / bacteria symbiotic hydrogel according to claim 5, characterized in that: In step (1), the concentration of sodium alginate (SA) in the core structure ink is 1%-10%, and the amount of Chlamydomonas reinhardtii is 1×10 6 -1×10 8 Pieces / ml.

7. The method for preparing the core-shell structure algae / bacteria symbiotic hydrogel according to claim 5, characterized in that: In step (1), the concentration of polyethylene glycol diacrylate (PEGDA) in the shell structure ink is 1%-10%, the concentration of sodium alginate (SA) is 1%-10%, and the amount of Bacillus subtilis is 1×10 6 -1×10 9 Pieces / ml.

8. The method for preparing the core-shell structure algae / bacteria symbiotic hydrogel according to claim 5, characterized in that: The 3D printing parameters in step (2) are set as follows: nozzle size: 1.2 mm, printing speed: 15 mm / min, number of layers: 6 layers, early wire drawing: 200 ms, early wire closing: 200 ms.

9. The method for preparing the core-shell structure algae / bacteria symbiotic hydrogel according to claim 5, characterized in that: The cross-linking time in step (3) is 1-10 minutes, and the curing under ultraviolet light is 20-60 seconds.

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