Method for stress microalgae in solid state and application

Abstract

The invention discloses a method for solid stress of microalgae and application, and belongs to the technical field of microalgae culture, and the method comprises the following steps: preparing a solid culture medium; preparing the microalgae bio-ink; carrying out 3D printing on the microalgae bio-ink; and carrying out solid culture and stress on the microalgae. Based on a solid-state culture technology, a solid-state stress method which is simple to operate is developed, and efficient and rapid accumulation of microalgae metabolites can be realized. In addition, through the 3D printing technology, modular, low-cost, rapid and large-area industrial manufacturing can be achieved, the defects existing in traditional microalgae stress are overcome, and wide application prospects are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microalgae cultivation, and particularly relates to a method for solid-state stress microalgae and application. BACKGROUND

[0002] As a photosynthetic autotrophic organism widely distributed in natural waters, microalgae have extremely high biological value and can be induced by stress to produce various biomass products such as polysaccharides, pigments, proteins and oils. These biomasses have potential application prospects in the production of industrial products such as biofuels, raw materials, food additives, cosmetics and medicines, and can be used as substitutes for synthetic or animal-derived products. However, traditional liquid medium induction stress requires tedious steps such as centrifugal collection and replacement of liquid medium to realize the growth of microalgae and accumulation of stress metabolic products, which consumes a large amount of water resources, energy and land resources, limiting its practical application value.

[0003] A microalgae production method is disclosed in Chinese Patent No. CN112322610A, published on February 5, 2021, which comprises the following steps S1-S5: S1, preparing microalgae hydrogel particles; S2, microalgae culture: placing the microalgae hydrogel particles into a microalgae culture system, controlling the volume ratio of the culture medium to the added microalgae hydrogel particles to be 1:1-10:1, and culturing at 20-35℃ and 5000-100000 lux of light for 1-30 days, until the color of the microalgae hydrogel particles deepens and the number of microalgae cells and the dry weight of microalgae in each microalgae hydrogel particle increase to the stable period, and then entering step S3 induction stage; S3, induction: converting the culture environment of step S2 into a stress environment with a predetermined nutrient deficiency, and maintaining for 1-20 days to induce the production of additional products including oils or polysaccharides; when the content of the additional products increases to more than 25% of the dry weight of microalgae, entering step S4 harvesting stage; S4, harvesting: separating the microalgae hydrogel particles from the microalgae culture system to collect the microalgae hydrogel particles; S5, drying: drying the collected microalgae hydrogel particles to reduce the moisture content of the microalgae hydrogel particles to below 10%, and obtaining the microalgae product. The microalgae production method has complex induction stress process, large resource consumption, and is difficult to realize industrialization manufacturing. SUMMARY

[0004] The present application aims to solve the problems of the prior art and provides a method for solid-state stress microalgae and application. Based on solid-state culture technology, a simple solid-state stress method is developed, which can realize efficient and rapid accumulation of microalgae metabolic products. In addition, through 3D printing technology, modularization, low cost and rapid large-area industrialization manufacturing can be realized, overcoming the defects of traditional microalgae stress, and having wide application prospects.

[0005] The present application is realized by the following technical solutions: A method for solid-state stress microalgae, comprising the following steps: Step one, preparation of solid medium: the liquid medium is sterilized after adding agar, and the mixture containing agar is fixed in a sterile culture dish using the inverted plate method to obtain the solid medium; Step two, preparation of microalgae bio-ink: dissolve sodium alginate in deionized water to obtain a sodium alginate solution, mix the concentrated algal solution with the sodium alginate solution uniformly, and add a certain amount of rheological modifier to obtain the bio-ink; Step three, 3D printing of microalgae bio-ink: transfer the bio-ink to the printing cartridge and load it into the 3D printer; use the software of the printer to build the model structure, and adjust the diameter of the single filament by switching the needle; according to the set parameters, 3D printing is carried out, and after the model is printed, it is further solidified and formed by crosslinking with calcium chloride; Step four, solid-state culture and stress of microalgae: the microalgae gel after printing and solidification is directly placed on the agar solid medium and placed in an incubator for culture; the solid-state stress under different conditions is carried out by replacing the bottom solid medium or changing the external conditions.

[0006] Preferably, in step one, the liquid medium adopts BG11 medium, f / 2 medium, nitrogen-free BG11 medium, phosphorus-free BG11 medium, high-salt BG11 medium, nitrogen-free f / 2 medium, phosphorus-free f / 2 medium or high-salt f / 2 medium.

[0007] Preferably, in step two, the concentration of the sodium alginate solution is 3wt%-6wt%.

[0008] Preferably, in step two, the amount of algal solution added is 20-150mL / L.

[0009] Preferably, in step two, the rheological modifier adopts carrageenan, methyl cellulose or guar gum, and the amount of the rheological modifier is 2wt%-10wt%.

[0010] Preferably, in step three, the printing parameters are: layer height: 0.1-1cm; number of layers: 4-15 layers; printing filling, printing contour, starting point is random; model: quadrilateral; speed: 10-20mm / s; filament breaking lifting: 0.2-0.8mm; air pressure: 0.2-0.4MPa; filament advance: 200-400ms; filament closing: 0.1-0.4mm; filling mode: linear filling; filling method: linear filling; filling offset: 0.7mm; filling spacing: 2mm; rotation angle: 90°; number of rotations: 2 times.

[0011] Preferably, in step three, the ion crosslinking time is 4-15min.

[0012] Preferably, in the step three, the calcium chloride is 2wt%-7wt% of the bio-ink.

[0013] Preferably, in the step four, the temperature of the incubator is 20-25℃, the relative humidity of the incubator is 60%-90%, and the light intensity of the incubator is 40-100μmol / (m²·s).

[0014] Preferably, in the step four, the changed external conditions include temperature and light intensity.

[0015] The application of the method for solid-state stress microalgae, which includes but is not limited to Nannochloropsis, Chlorella, Chlorella pyrenoidosa, Porphyridium or diatom.

[0016] The application of the method for solid-state stress microalgae in microalgae oil production, microalgae astaxanthin accumulation, microalgae starch accumulation or microalgae phycocyanin accumulation.

[0017] Compared with the prior art, the present application has the following advantages and beneficial effects: Firstly, the method for solid-state stress microalgae provided by the present application can realize stress on microalgae by replacing the bottom medium; compared with the traditional liquid microalgae stress method, the solid-state stress system is more convenient to operate, and the cumbersome centrifugal collection step is avoided; the 3D printing technology can be used for large-area batch production, and has the potential for industrial application. Secondly, compared with liquid culture, the method for solid-state stress microalgae provided by the present application can obtain higher biomass and metabolic product accumulation, has higher oil production efficiency, and can accumulate more oil in a shorter time.

[0018] Thirdly, the method for solid-state stress microalgae provided by the present application can avoid the waste of a large amount of water resources and equipment resources in the traditional microalgae stress process. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the growth condition of Nannochloropsis in Example 1 under solid-state culture in different media; Figure 2 is the solid-state stress oil production condition of Chlorella in Example 1 by replacing the medium; Figure 3 is the growth condition of Chlorella vulgaris in Example 2 under solid-state culture in different media; Figure 4 is the cell flow analysis and stress culture carotenoid accumulation condition of Chlorella vulgaris in Example 2 under solid-state culture in different media. DETAILED DESCRIPTION

[0020] The application will be further described in detail below with reference to examples, but the embodiments of the application are not limited thereto.

[0021] Example 1 As Figure 1 and Figure 2 A method for producing oil by solid-state stress microalgae is shown in the following specific implementation method: I. Preparation of solid culture medium of Nannochloropsis oceanica: The microalgae culture medium of Nannochloropsis oceanica is obtained by optimizing the f / 2 culture medium sold in the market. It is divided into biomass accumulation culture medium LDM and stress oil accumulation culture medium NSM.

[0022] The LDM culture medium is configured as follows: NaCl 10 g / L, NaNO3 2 g / L; No. 1-9 stock solutions are configured as follows: No. 1 stock solution Na2SiO3·9H2O 30 g / L, No. 2 stock solution MgSO4·7H2O 217.6 g / L, No. 3 stock solution CaCl2·2H2O 267.6 g / L, No. 4 stock solution KCl 267.6 g / L, NH4Cl 11.9 g / L, No. 5 stock solution Tris-buffer 89.2 g / L, No. 6 stock solution KH2PO4 44.6 g / L, K2HPO4·3H2O 9.8 g / L, No. 7 stock solution concentrated H2SO4, 8.92 mL / L, H3BO3 30.56 g / L, CoCl2·6H2O 10.8 mg / L, ZnCl2 281 mg / L, (NH4)6MO7O 24 ·4H2O 278 mg / L, MnCl2·4H2O 246 mg / L, Na2MoO4·2H2O 24 mg / L, No. 8 stock solution Na2EDTA 3.136 g / L, FeCl3·6H2O 275 mg / L, No. 9 stock solution vitamin B12 0.5 mg / L, vitamin B1 0.1 g / L, biotin 0.5 mg / L. 1 mL of No. 1 stock solution, 10 mL of No. 2 stock solution, 1 mL of No. 3 stock solution, 2 mL of No. 4 stock solution, 10 mL of No. 5 stock solution, 1 mL of No. 6 stock solution, 1 mL of No. 7 stock solution, 10 mL of No. 8 stock solution are added to the mixed solution of NaCl and NaNO3, 1.5% agar is added, sterilized at 121 degrees for 20 minutes, cooled to about 60 degrees (agar does not coagulate), 1 mL of No. 9 stock solution filtered with a 0.22 um sterile filter is added, shaken uniformly and then poured into a plate.

[0023] The NSM culture medium is configured as follows: (1) artificial seawater: NaCl 21.22 g / L, NaHCO3 0.174 g / L, MgCl2.6H2O 9.0342 g / L, CaCl2 1.03344 g / L, Na2SO4 3.407 g / L, KCl 0.3577 g / L, KBr 0.0862 g / L, H3BO3 0.023 g / L, (2) 1-5 mother liquor: 1# mother liquor NaNO3 75 g / L, 2# mother liquor NaH2PO4 5 g / L, 3# mother liquor Na2SiO3 30 g / L, 4# mother liquor FeCl3.6H2O 3.15 g / L, EDTA.2Na 4.36 g / L, CuSO4 0.0098 g / L, Na2MoO4 0.0063 g / L, ZnSO4 0.022 g / L, CoCl2.6H2O 0.01 g / L, MnCl2.4H2O 0.18 g / L, 5# mother liquor vitamin B12 0.001 g / L, vitamin B1 0.2 g / L, biotin 0.001 g / L. Add 1# mother liquor 8 mL / L, 2# mother liquor 4 mL / L, 3# mother liquor 1 mL / L, 4# mother liquor 1 mL / L to the artificial seawater, add 1.5% agar, sterilize at 121 degrees for 20 minutes, cool to about 60 degrees (agar does not coagulate), add 0.5 mL / L 5# mother liquor filtered by 0.22 um sterile filter, shake well and pour flat.

[0024] II. Preparation of microalgae bio-ink: 1.5 g of sodium alginate is dissolved in 50 mL of deionized water, and finally 250 mL of OD 680 0.8 microalgae solution is centrifuged at 5000g for 4 minutes, and then the precipitate is collected and mixed uniformly with the system to obtain the bio-ink; Three, 3D printing microalgae bio-ink: the bio-ink prepared in step one is transferred to the printing cartridge and loaded into the 3D printer (Bio-Architect® SR, Regenovo, Hangzhou, China). The model structure is built using the software of the printer, and the diameter of the single filament is adjusted by switching the needle, and 3D printing is carried out according to the set parameters. After the model is printed, it is further solidified and shaped by calcium chloride crosslinking. The specific printing parameters are: layer height: 1 mm; layer number: 8 layers; printing filling, printing contour, starting point random; model: quadrilateral; speed: 20 mm / s; filament lifting: 0.5 mm; air pressure: 0.25 MPa; filament advance: 200 ms; filament off: 0.5 mm; filling mode: linear filling; filling method: linear filling; filling offset: 0.7 mm; filling interval: 2 mm; rotation angle: 90°; rotation number: 2 times. The printed microalgae gel is further crosslinked in a 5wt% calcium chloride solution for 5 min to obtain a shaped microalgae gel.

[0025] Four, solid stress microalgae oil production: the printed microalgae gel is placed in LDM solid medium for culture, and this cycle is to accumulate biomass; after the accumulation of biomass is completed, the bottom medium is replaced with NSM medium, and the microalgae oil accumulation is induced by stress. The culture temperature is 25°C, the relative humidity is 70%, the light intensity is 50 μmol photons m -2 s -1 .

[0026] Example 2 As shown in Figure 3 and Figure 4 , a method for accumulating astaxanthin in microalgae by solid stress, the specific implementation method is as follows: One, configuration of solid culture medium of Chromochloris zofingiensis: the microalgae culture medium of Chromochloris zofingiensis includes the following two: biomass accumulation medium BG11 and stress accumulation astaxanthin medium BG11 (+ NaCl).

[0027] The BG11 medium is configured as follows: NaNO31.5 g / L; 1-7 mother liquor configuration: 1 mother liquor K2HPO4.3H2O 40 g / L, 2 mother liquor MgSO4.7H2O 75 g / L, 3 mother liquor CaCl2.2H2O 36 g / L, 4 mother liquor citric acid 6 g / L, ammonium iron citrate 6 g / L, 5 mother liquor Na2EDTA 1 g / L, 6 mother liquor Na2CO320 g / L; 7 mother liquor H3BO32.86 g / L, MnCl2.H2O 1.81 g / L, ZnSO4.7H2O 222 mg / L, CuSO4.5H2O 79 mg / L, Na2MoO4.2H2O 390 mg / L, CoCl2.6H2O 49 mg / L. Add 1-8 mother liquor 1 mL / L to the NaNO3 solution, add 1.5% agar, sterilize at 121 degrees for 20 min, shake well and pour flat.

[0028] BG11(+NaCl) culture medium is configured as: NaCl 20 g / L; 1-7 mother liquor configuration: 1 mother liquor K2HPO4.3H2O 40 g / L, 2 mother liquor MgSO4.7H2O 75 g / L, 3 mother liquor CaCl2.2H2O 36 g / L, 4 mother liquor citric acid 6 g / L, ammonium iron citrate 6 g / L, 5 mother liquor Na2EDTA 1 g / L, 6 mother liquor Na2CO320 g / L; 7 mother liquor H3BO32.86 g / L, MnCl2.H2O 1.81 g / L, ZnSO4.7H2O 222 mg / L, CuSO4.5H2O 79 mg / L, Na2MoO4.2H2O 390 mg / L, CoCl2.6H2O 49 mg / L. Add 1-8 mother liquor 1 mL / L to the NaCl-free solution, add 1.5% agar, sterilize at 121 degrees for 20 min, shake well and pour flat.

[0029] II. Preparation of microalgae bio-ink: 1.5 g of sodium alginate was dissolved in 50 mL of deionized water, and finally 50 mL of OD 680 0.7 of Zofniglens green algae liquid was centrifuged at 5000 g for 4 minutes, and then the precipitate was collected and mixed uniformly with the system to obtain the bio-ink; Three, 3D printing microalgae bio-ink: the bio-ink prepared in step one is transferred to the printing cartridge and loaded into the 3D printer (Bio-Architect® SR, Regenovo, Hangzhou, China). The model structure is built using the software of the printer, and the diameter of the single filament is adjusted by switching the needle, and 3D printing is carried out according to the set parameters. After the model is printed, it is further solidified and shaped by calcium chloride crosslinking. The specific printing parameters are: layer height: 1 mm; number of layers: 8 layers; printing fill, printing contour, starting point random; model: quadrilateral; speed: 20 mm / s; filament breaking height: 0.5 mm; air pressure: 0.34 MPa; filament advance: 200 ms; filament off: 0.5 mm; fill mode: linear fill; fill method: linear fill; fill offset: 0.1 mm; fill spacing: 2 mm; rotation angle: 90°; number of rotations: 2 times. The printed microalgae gel is further crosslinked in a 2 wt% calcium chloride solution for 5 min to obtain a shaped microalgae gel.

[0030] Four, astaxanthin production by solid stress Chlorella zofinii: the printed microalgae gel is placed in BG11 solid medium for culture, and this cycle is for biomass accumulation; after the biomass accumulation is completed, the bottom medium is replaced with BG11 (NaCl) medium, and the microalgae are induced to accumulate astaxanthin. The culture temperature is 25°C, the relative humidity is 70%, the light intensity is 50 μmol photons m -2 s -1 .

[0031] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change made on the basis of the technical essence of the present application to the above embodiment falls within the protection scope of the present application.

Claims

1. A method of solid state stressing microalgae, characterized in that, The method comprises the following steps: Step 1: preparing a solid culture medium: sterilizing the liquid culture medium after adding agar, fixing the mixture containing agar in a sterile culture dish by using an inverted plate method, and obtaining the solid culture medium; Step 2: preparing a microalgae bio-ink: dissolving sodium alginate in deionized water to obtain a sodium alginate solution, uniformly mixing the concentrated algal liquid with the sodium alginate solution, and adding a certain amount of a rheological modifier to obtain the bio-ink; Step 3: 3D printing the microalgae bio-ink: transferring the bio-ink to a printing cartridge and loading it into a 3D printer; using the software of the printer to construct a model structure, adjusting the diameter of the single filament by switching the needle, and performing 3D printing according to the set parameters; after the model is printed, further solidification and molding are performed by cross-linking with calcium chloride; Step 4: solid-state culture and stress of microalgae: directly placing the microalgae gel after printing and solidification and molding into the agar solid culture medium, and placing it in an incubator for static culture; the solid-state stress under different conditions is performed by replacing the bottom solid culture medium or changing the external conditions.

2. A method of stress microalgae in solid state according to claim 1, characterized in that, In step 1, the liquid culture medium is BG11 medium, f / 2 medium, nitrogen-deficient BG11 medium, phosphorus-deficient BG11 medium, high-salt BG11 medium, nitrogen-deficient f / 2 medium, phosphorus-deficient f / 2 medium, or high-salt f / 2 medium.

3. The method of claim 1, wherein the microalgae are subjected to a stress in the form of a solid. In step 2, the concentration of the sodium alginate solution is 3wt%-6wt%.

4. The method of claim 1, wherein the microalgae are subjected to a stress. In step 2, the addition amount of the algal liquid is 20-150mL / L.

5. The method of claim 1, wherein the microalgae are subjected to a stress. In step 2, the rheological modifier is carrageenan, methyl cellulose, or guar gum, and the amount of the rheological modifier is 2wt%-10wt%.

6. The method of claim 1, wherein, In step 3, the printing parameters are as follows: layer height: 0.1-1cm; number of layers: 4-15 layers; printing filling, printing contour, and starting point are random; model: quadrilateral; speed: 10-20mm / s; filament breaking lifting: 0.2-0.8mm; air pressure: 0.2-0.4MPa; filament lifting in advance: 200-400ms; filament closing in advance: 0.1-0.4mm; filling mode: linear filling; filling method: linear filling; filling offset: 0.7mm; filling interval: 2mm; rotation angle: 90°; number of rotations: 2 times. In step 3, the ion cross-linking time is 4-15min. In step 3, the calcium chloride is 2wt%-7wt% of the bio-ink. In step 4, the temperature of the incubator for static culture is 20-25℃, the relative humidity of the incubator for static culture is 60%-90%, and the light intensity of the incubator for static culture is 40-100μmol / (m²·s). In step 4, the changed external conditions include temperature and light intensity.

7. The method of claim 1, wherein the microalgae are subjected to a stress. The method is applied to microalgae cultivation, and the microalgae include but are not limited to Nannochloropsis, Chlorella, Chlorella pyrenoidosa, Porphyridium, or diatoms.

8. The method of claim 1, wherein the microalgae are subjected to a solid state stress. The method is applied to microalgae oil production, microalgae astaxanthin accumulation, microalgae starch accumulation, or microalgae phycocyanin accumulation.

9. The method of claim 1, wherein the microalgae are subjected to a solid state stress. ​ 10. The method of claim 1, wherein the microalgae are subjected to a solid state stress. ​ 11. A method of stress microalgae in solid state according to any one of claims 1 to 10, characterized in that, ​ 12. A method of stress microalgae in solid state according to any one of claims 1-10, characterized in that, ​

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