A gel-based method for preserving high-concentration microalgae
Through the gel-based high-concentration microalgae preservation method, the problem of difficulty in maintaining activity during microalgae transportation and preservation is solved, and high-concentration long-term preservation and low-cost transportation are achieved, and both biomass and activity are significantly improved.
Patent Information
- Application Number
- CN202411254108.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-09
- Publication Date
- 2025-04-01
AI Technical Summary
The prior art has problems such as difficulty in maintaining activity, high transportation costs, and susceptibility to contamination in the transportation and preservation of microalgae, which makes it difficult to achieve long-term preservation and high-concentration transportation of microalgae.
A gel-based high-concentration microalgae preservation method is used to form a solution by dissolving sodium alginate and agar under high temperature conditions, adding microalgae cells and carrageenan powder after cooling to form a hydrogel solution, and then 3D printing is used to cross-link it in calcium chloride solution, and finally culture on solid culture medium.
The high concentration of microalgae is achieved for long-term freshness, with biomass up to 19.7g/L, stored for at least 72 days, and the transportation cost is reduced and the activity maintenance effect of microalgae is improved.
Smart Images
Figure CN119736165A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a microalgae preservation method, and more particularly to a gel-based high-concentration microalgae preservation method. Background Art
[0002] Microalgae can be used as feed, growth promoter and immunostimulant in aquaculture. Microalgae can reproduce rapidly after entering the aquaculture water, forming a water body with single-cell microalgae as the dominant species, providing fish, shrimp, crab, shellfish and other aquatic animals with rich protein, unsaturated fatty acids, vitamins, minerals, chlorophyll, algae polysaccharides, nucleic acids and other nutrients, promoting growth, reducing costs and improving the survival rate of aquatic animals. In the early stage of aquaculture, using microalgae and fertilizers at the same time can play a role in rapid water fertilization. Using microalgae alone during high temperature periods can also achieve water quality regulation, reduce ammonia nitrogen and nitrite, inhibit blue algae, and improve the water environment.
[0003] Although solid culture medium can preserve unicellular algae at present, it also has the disadvantages of drying and dehydration of culture medium, weak genetic stability of germplasm, and low density of algae species. There are also technologies that mention that algae powder and algae flakes can be made, but during the manufacturing process, its nutrients will be lost in large quantities and lose vitality, and it no longer has the function of regulating and purifying water quality. In addition, some substances harmful to the body may be added during the production process. Therefore, the current industrial microalgae culture adopts liquid immersion culture, and the transportation of algae cells dispersed in a large amount of culture medium water is mainly carried out in closed containers (barrels or tanks). There is no gas exchange in this process, and algae cells die of lack of oxygen. Some high-concentration algae mud is also transported by freezing, which has high transportation costs and high energy consumption. Low-concentration algae liquid is transported at room temperature, and the transportation cost is high due to the low density, and the microalgae in this process are easily contaminated by bacteria, fungi and other bacteria, and the purity of algae cells is low. Therefore, the existing methods are not conducive to maintaining the activity of microalgae during transportation. Therefore, at this stage, there is an urgent need for a transportation method that is not restricted by seasons and can both conveniently transport and preserve the vitality of microalgae for a long time. Summary of the invention
[0004] The purpose of the present invention is to provide a gel-based high-concentration microalgae preservation method to solve the current problems of transportation and preservation of industrialized microalgae.
[0005] To achieve the above object, the cultivation method provided by the present invention mainly comprises the following steps:
[0006] (1) Sodium alginate and agar are dissolved separately under high temperature conditions to form a solution, and after cooling to room temperature, microalgae cells are added thereto, carrageenan powder is added thereto after mixing evenly, and the mixture is allowed to stand for a while after mixing evenly to form a hydrogel solution.
[0007] (2) The hydrogel is 3D printed using a 3D printer, placed in a calcium chloride solution for cross-linking, and then placed on a solid culture medium for culture.
[0008] Furthermore, the high temperature in step (1) is 85-95°C.
[0009] Furthermore, the microalgae described in step (1) are Chlorella, Chlorella pyrenoidosa, or Chlamydomonas reinhardtii;
[0010] Furthermore, the concentration of sodium alginate in step (1) is 3.75-22.5 g / L;
[0011] Furthermore, the concentration of the agar in step (1) is 3.75-22.5 g / L;
[0012] Furthermore, the carrageenan in step (1) is 45-90 g / L;
[0013] Furthermore, the 3D printing described in step (2) can be a hexagonal prism, a cylinder, a triangular prism, or a hand shape.
[0014] Furthermore, the 3D printing parameters described in step (2) are: needle diameter 0.34 mm, printing pressure 0.2 MPa, printing speed 16 mm / s, and printing layer height 0.25 mm.
[0015] The present invention has the following advantages:
[0016] (1) Agar is added to the hydrogel of the present invention, so that the hydrogel has improved gelling properties at a lower solid content, further increasing the content of microalgae in the gel, while reducing the amount of carrageenan used, further saving costs.
[0017] (2) Its formula is simple and its design is scientific, which enables high-density cultivation of microalgae and long-term preservation of high-concentration microalgae;
[0018] (3) The biomass of microalgae can reach 19.7 g / L, which is much higher than the biomass of traditional liquid culture and photosynthetic autotrophic microalgae reported in existing literature.
[0019] (4) Achieve long-term preservation of high-concentration microalgae for at least 72 days. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a rheological comparison diagram of the hydrogel without adding agar in Example 1-2;
[0021] Figure 2 This is a rheological comparison diagram of the hydrogels with agar added in Example 3-5;
[0022] Figure 3 Rheological properties of hydrogels with different microalgae added for Examples 6-10;
[0023] Figure 4The tensile mechanical properties of the hydrogels with different microalgae added in Examples 6-10;
[0024] Figure 5 The porosity of the hydrogels with different microalgae added for Examples 6-10;
[0025] Figure 6 Printing microalgae hydrogels of different shapes for Examples 6-10;
[0026] Figure 7 The activity of microalgae during the hydrogel culture process in Example 10;
[0027] Figure 8 The activity of microalgae during the hydrogel culture process in Example 10;
[0028] Fig. 9 This is a fluorescent image of microalgae during hydrogel culture in Example 10;
[0029] Fig.10 The viability of microalgae preserved for different days in Example 11 and Example 12;
[0030] Fig.11 The microalgae biomass preserved for different days in Example 11 and Example 12;
[0031] Fig.12 Microscopic and SEM images of microalgae extracted from the gels of Example 11 and Example 12; DETAILED DESCRIPTION
[0032] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.
[0033] Embodiment 1:
[0034] Add 500 mg of sodium alginate (SA) to 50 mL of sterile water, add 2250 mg of carrageenan powder after it is fully dissolved, stir evenly and let stand for 10 minutes to ensure uniformity. The solid content of the hydrogel is 6%. Figure 1 As shown, although this formula can be 3D printed, its modulus is low, which is not conducive to the implementation of 3D printing.
[0035] Embodiment 2:
[0036] Add 1500 mg of sodium alginate (SA) to 50 mL of sterile water, add 4500 mg of carrageenan powder after it is fully dissolved, stir evenly and let stand for 10 minutes to ensure uniformity. The solid content of the hydrogel is 12%. Figure 1 As shown, this formula can be 3D printed with high modulus and optimal mechanical properties, but the high solid content increases the cost.
[0037] Embodiment three:
[0038] Add 562.5 mg sodium alginate and 187.5 mg agar to 50 mL sterile water, heat at 85°C to dissolve, add 2250 mg carrageenan powder after fully dissolved, stir evenly and let stand for 10 minutes to ensure uniformity, the solid content of the hydrogel is 6%, recorded as MPH-0.0.
[0039] Embodiment 4:
[0040] Add 375 mg of sodium alginate and 375 mg of agar to 50 mL of sterile water, heat at 95° C. to dissolve, add 2250 mg of carrageenan powder after it is fully dissolved, stir evenly and let stand for 10 minutes to ensure uniformity. The solid content of the hydrogel is 6%.
[0041] Embodiment five:
[0042] Add 187.5 mg of sodium alginate and 562.5 mg of agar to 50 mL of sterile water, heat at 95° C. to dissolve, add 2250 mg of carrageenan powder after it is fully dissolved, stir evenly and let stand for 10 minutes to ensure uniformity. The solid content of the hydrogel is 6%.
[0043] like Figure 2 As shown, after adding agar, Examples 3, 4 and 5 can all achieve smooth 3D printing at a solid content of 6%, and the mechanical properties are: Example 3> Example 4> Example 5, and the formability is: Example 3> Example 4> Example 5.
[0044] Embodiment six:
[0045] Add 562.5 mg of sodium alginate (SA) and 187.5 mg of agar to 50 mL of sterile water, heat to dissolve at 95 ° C, cool to room temperature, add 0.05 g of dry weight of active protein core chlorella (Chlorella pyrenoidosa, CP), stir well, add 2250 mg of carrageenan powder, stir well and let stand for 10 minutes to ensure uniformity, recorded as MPH-0.1. Put 50 mL of MPH-0.1 into a 50 mL syringe, and use 3D printing to print hexagonal microalgae hydrogels. The printing parameters are set as follows: needle diameter 0.34 mm, printing pressure 0.2 MPa, printing speed 16 mm / s, printing layer height 0.25 mm, and then cross-link the print with 200 mM calcium chloride solution for 1 hour.
[0046] Embodiment seven:
[0047] Add 562.5 mg of sodium alginate (SA) and 187.5 mg of agar to 50 mL of sterile water, heat at 95 ° C to dissolve, cool to room temperature, add 0.25 g of dry weight of active protein core chlorella (Chlorella pyrenoidosa, CP) of microalgae, stir evenly, add 2250 mg of carrageenan powder, stir evenly and let stand for 10 minutes to ensure uniformity, recorded as MPH-0.5. Put 50 mL of MPH-0.5 into a 50 mL syringe, and use 3D printing to print a triangular prism-shaped microalgae hydrogel. The printing parameters are set as follows: needle diameter 0.34 mm, printing pressure 0.2 MPa, printing speed 20 mm / s, printing layer height 0.25 mm, and then cross-link the print with 200 mM calcium chloride solution for 1 hour and take it out.
[0048] Embodiment eight:
[0049] Add 562.5 mg of sodium alginate (SA) and 187.5 mg of agar to 50 mL of sterile water, heat to dissolve at 95 ° C, cool to room temperature, add 0.5 g of dry weight of active Chlamydomonas reinhardtii, stir well, add 2250 mg of carrageenan powder, stir well and let stand for 10 minutes to ensure uniformity, recorded as MPH-1.0. Put 50 mL of MPH-1.0 into a 50 mL syringe, and use 3D printing to print cylindrical microalgae hydrogels. The printing parameters are set as follows: needle diameter 0.60 mm, printing pressure 0.3 MPa, printing speed 30 mm / s, printing layer height 0.30 mm, and then cross-link the print with 200 mM calcium chloride solution for 1 hour.
[0050] Embodiment nine:
[0051] Add 562.5 mg of sodium alginate (SA) and 187.5 mg of agar to 50 mL of sterile water, heat to dissolve at 95 ° C, cool to room temperature, add 1.5 g of dry weight of active protein core chlorella (Chlorella pyrenoidosa, CP), stir well, add 2250 mg of carrageenan powder, stir well and let stand for 10 minutes to ensure uniformity, recorded as MPH-3.0. Put 50 mL of MPH-3.0 into a 50 mL syringe, and use 3D printing to print hand-shaped microalgae hydrogels. The printing parameters are set as follows: needle diameter 0.75 mm, printing pressure 0.4 MPa, printing speed 50 mm / s, printing layer height 0.35 mm, and then cross-link the print with 200 mM calcium chloride solution for 1 hour.
[0052] Embodiment ten:
[0053] Add 562.5 mg of sodium alginate (SA) and 187.5 mg of agar to 50 mL of sterile water, heat to dissolve at 95 ° C, cool to room temperature, add 2.5 g of dry weight of active Chlorella, stir well, add 2250 mg of carrageenan powder, stir well and let stand for 10 minutes to ensure uniformity, record as MPH-5.0. Put 50 mL of MPH-5.0 into a 50 mL syringe, and use 3D printing to print hexagonal microalgae hydrogels. The printing parameters are set as follows: needle diameter 0.75 mm, printing pressure 0.4 MPa, printing speed 60 mm / s, printing layer height 0.40 mm, and cross-linking with 200 mM calcium chloride solution for 1 hour.
[0054] Embodiment eleven:
[0055] Add 562.5 mg of sodium alginate (SA) and 187.5 mg of agar to 50 mL of sterile water, heat to dissolve at 95 ° C, cool to room temperature, add 3 g of dry weight of active protein core chlorella (Chlorella pyrenoidosa, CP), stir evenly, add 2250 mg of carrageenan powder, stir evenly and let stand for 10 minutes to ensure uniformity, record as MPH-6. Put 50 mL of MPH-6 into a 50 mL syringe, and use 3D printing to print hexagonal microalgae hydrogels. The printing parameters are set as follows: needle diameter 0.75 mm, printing pressure 0.4 MPa, printing speed 60 mm / s, printing layer height 0.40 mm, and cross-linking with 200 mM calcium chloride solution for 1 hour.
[0056] Embodiment 12:
[0057] Add 562.5 mg of sodium alginate (SA) and 187.5 mg of agar to 50 mL of sterile water, heat to dissolve at 95 ° C, cool to room temperature, add 6 g of dry weight of active protein core chlorella (Chlorella pyrenoidosa, CP), stir evenly, add 2250 mg of carrageenan powder, stir evenly and let stand for 10 minutes to ensure uniformity, recorded as MPH-12. Put 50 mL of MPH-12 into a 50 mL syringe, and use 3D printing to print hexagonal microalgae hydrogels. The printing parameters are set as follows: needle diameter 0.75 mm, printing pressure 0.4 MPa, printing speed 60 mm / s, printing layer height 0.40 mm, and cross-linked with 200 mM calcium chloride solution for 1 hour.
[0058] like Figure 3 The shear thinning ability of the hydrogel after adding agar and microalgae can be used for extrusion 3D printing, and the addition of different amounts of microalgae will not significantly affect the rheological properties of the hydrogel.
[0059] like Figure 4 The tensile properties of the hydrogel after adding agar and microalgae prove that the mechanical properties are suitable for the reproduction of microalgae.
[0060] like Figure 5 The porosity of the hydrogel after adding agar and microalgae reached 86.99%, proving that there is enough space for the microalgae to survive.
[0061] like Figure 6 The hydrogels with different formulas shown can be successfully printed according to different printing parameters.
[0062] like Figure 7 As shown, the viability of microalgae in MPH-0.1 hydrogel was maintained above 90% during 14 days of microalgae cultivation.
[0063] like Figure 8 As shown, the biomass of microalgae in MPH-0.1 hydrogel continued to grow during 14 days of microalgae cultivation and was maintained at 19.7 g / L after 7 days.
[0064] like Fig. 9 As shown, the fluorescent image of microalgae culture in MPH-5.0 hydrogel, red represents living microalgae, green represents dead microalgae, and the red microalgae increased significantly after 7 days.
[0065] like Fig.10 As shown, the microalgae activity of MPH-6.0 and MPH-12.0 was maintained at about 85% after being stored in the microalgae hydrogel for 72 days.
[0066] like Fig.11As shown, the microalgae biomass of MPH-6.0 and MPH-12.0 remained above 100 g / L and 50 g / L, respectively, after being stored in the microalgae hydrogel for 72 days.
[0067] like Fig.12 As shown, the microalgae morphology and vitality of MPH-6.0 and MPH-12.0 remained normal after being stored in microalgae hydrogel for 72 days.
Claims
1. A gel-based high-concentration microalgae preservation method, characterized in that The following steps are involved: (1) Sodium alginate and agar are dissolved separately under high temperature conditions to form a solution, and after cooling to room temperature, microalgae cells are added thereto, carrageenan powder is added thereto after mixing evenly, and the mixture is allowed to stand for a while after mixing evenly to form a hydrogel solution. (2) The hydrogel is 3D printed using a 3D printer, placed in a calcium chloride solution for cross-linking, and then placed on a solid culture medium for culture.
2. The method for preserving high-concentration microalgae based on gel according to claim 1, characterized in that: The high temperature in step (1) is 85-95°C.
3. The method for preserving high-concentration microalgae based on gel according to claim 1, characterized in that: The microalgae described in step (1) are Chlorella, Chlorella pyrenoidosa, and Chlamydomonas reinhardtii.
4. The method for preserving high-concentration microalgae based on gel according to claim 1, characterized in that: The concentration of sodium alginate in step (1) is 3.75-22.5 g / L.
5. The gel-based high-concentration microalgae preservation method according to claim 1, characterized in that: The concentration of the agar in step (1) is 3.75-22.5 g / L.
6. The gel-based high-concentration microalgae preservation method according to claim 1, characterized in that: The carrageenan described in step (1) is 45-90 g / L.
7. The gel-based high-concentration microalgae preservation method according to claim 1, characterized in that: The 3D printing described in step (2) can be a hexagonal prism, a cylinder, a triangular prism, or a hand shape.
8. The gel-based high-concentration microalgae preservation method according to claim 1, characterized in that: The 3D printing parameters described in step (2) are: needle diameter 0.34 mm, printing pressure 0.2 MPa, printing speed 16 mm / s, and printing layer height 0.25 mm.
9. The gel-based high-concentration microalgae preservation method according to claim 1, characterized in that: The cross-linking time in step (2) is 5 min-1 h.