A method for one-step propagation of haematococcus pluvialis with apparent zero biomass
By modifying the culture medium and using a phased light intensity control method, Haematococcus pluvialis was propagated in one step in the same container, solving the problem of gradually increasing the volume, improving propagation efficiency and reducing the risk of contamination, and achieving efficient microscopic appearance zero biomass propagation.
Patent Information
- Application Number
- CN202611290024.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-25
- Publication Date
- 2026-11-06
AI Technical Summary
In existing technologies, the propagation of Haematococcus pluvialis requires gradually increasing the propagation volume, which is a complicated process with a long cycle and the risk of contamination. It is impossible to achieve successful propagation with zero biomass as observed under a microscope.
By using a modified culture medium and a phased light intensity control method, one-step propagation of Haematococcus pluvialis with zero apparent biomass under microscopic examination was achieved in the same container. The modified culture medium consisted of glacial acetic acid, D-biotin, and vitamin B12. The light intensity was gradient controlled at different stages, and CO2 was introduced to maintain the pH value of the culture system.
The propagation cycle was shortened from 90 days to 15 days, and the cell density was increased from 200,000-300,000/mL to 700,000-800,000/mL, improving propagation efficiency and reducing the risk of contamination.
Smart Images
Figure CN123042834A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microalgae culture technology, specifically, it relates to a one-step propagation method for Haematococcus pluvialis with zero apparent biomass under microscopic examination. Background Technology
[0002] Haematococcus pluvialis is a core germplasm resource for the synthesis of natural astaxanthin, and the proliferation efficiency of its motile cells directly determines the benefits of subsequent industrial production. The propagation of Haematococcus pluvialis begins indoors. Due to possible reasons such as population signal dependence, nutrient sensing lockout, and the incomplete cell membrane structure of newly formed motile cells in Haematococcus pluvialis with apparent zero biomass, even under conditions of sufficient nutrition and suitable light, it is impossible to achieve successful one-step propagation of Haematococcus pluvialis with apparent zero biomass under microscopic examination. Currently, the propagation of Haematococcus pluvialis with apparent zero biomass under microscopic examination must be carried out by gradually increasing the propagation volume to maintain the algal species at a suitable population concentration for successful propagation; otherwise, the biomass will be extremely low and the activity poor, failing to meet the production requirements. Currently, the indoor propagation of Haematococcus pluvialis mainly uses the following stepwise culture method: plate - 96-well plate - 24-well plate - 15ml test tube - 50ml Erlenmeyer flask - 250ml Erlenmeyer flask - 250ml Erlenmeyer flask - 1L Erlenmeyer flask. This propagation method is cumbersome, has a long culture cycle of about 90 days, and carries a high risk of contamination due to the need for multiple transfers. Summary of the Invention
[0003] To address the problems existing in the background technology, this invention provides a one-step propagation method for Haematococcus pluvialis with apparent zero biomass under microscopic examination. This method eliminates the need for gradually increasing the propagation volume and for transferring propagation containers step-by-step. It achieves the propagation of Haematococcus pluvialis with apparent zero biomass under microscopic examination in a single container in one step. This solves the technical problem that existing methods for propagating Haematococcus pluvialis require gradually increasing the propagation volume to achieve successful propagation. Furthermore, it shortens the propagation cycle to 15 days and increases the cell density from 200,000-300,000 / mL in traditional 90-day progressive propagation to 700,000-800,000 / mL in 15 days. This significantly reduces the indoor propagation time of Haematococcus pluvialis, improves propagation efficiency, and avoids the contamination risks associated with the need for multiple container transfers.
[0004] To achieve the above objectives, the present invention is implemented through the following technical solution:
[0005] The one-step propagation and culture method for Haematococcus pluvialis with zero apparent biomass under microscopic examination uses a modified culture medium for propagation. The modified culture medium is prepared by adding glacial acetic acid, D-biotin and vitamin B12 to the basic culture medium. The concentration of glacial acetic acid is 40-45 ml / L, the concentration of D-biotin is 0.05-0.06 g / L and the concentration of vitamin B12 is 0.0045-0.005 g / L.
[0006] Furthermore, the basal culture medium is BG-11 medium, composed of the following substances at the following concentrations: sodium nitrate 76 g / L, magnesium sulfate heptahydrate 11.4 g / L, anhydrous sodium carbonate 22.4 g / L, disodium ethylenediaminetetraacetate dihydrate 1.09 g / L, ammonium sulfate hydrochloride 0.18 g / L, boric acid 2.6 g / L, manganese chloride tetrahydrate 1.66 g / L, zinc sulfate heptahydrate 0.55 g / L, sodium molybdate dihydrate 0.36 g / L, copper sulfate pentahydrate 0.073 g / L, cobalt nitrate hexahydrate 0.20 g / L, citric acid monohydrate 5.6 g / L, ferric ammonium citrate 8.35 g / L, anhydrous calcium chloride 16.55 g / L, and dipotassium hydrogen phosphate trihydrate 73.6 g / L.
[0007] Furthermore, the initial pH of the modified culture medium is ≤6.0.
[0008] Furthermore, purified Haematococcus pluvialis colonies from plates were picked up using a sterile inoculation loop and directly inoculated into the modified culture medium. In the initial inoculation stage, no algal cells were detected by multi-field microscopy using a hemocytometer, indicating the biomass was below the microscopic detection limit and was recorded as 0. After inoculation, the culture was carried out in the following stages.
[0009] During the recovery and activation period, days 0-3, the light intensity should be 700-1000 lux.
[0010] During the physiological recovery period, days 4-7, the light intensity should be 1000-3000 lux.
[0011] During the energy storage preparation period, on days 8-9, the light intensity is 3000-4000 lux.
[0012] During the logarithmic growth phase, days 10-15, the light intensity is 4000-4500 lux;
[0013] All the above cultures were conducted under 24-hour light.
[0014] Furthermore, the pH value of the stable culture system was maintained at 7.5-8.5 during the cultivation process.
[0015] Furthermore, the pH of the culture system was maintained by introducing CO2. When the pH of the algal solution increased by ≥0.5 per day, the CO2 introduction rate was increased; when the pH fluctuation was <0.5, the CO2 introduction rate was maintained.
[0016] Furthermore, the temperature of the culture system is controlled at 22-25℃.
[0017] The present invention also provides a culture medium for propagating Haematococcus pluvialis, wherein the culture medium is prepared by adding glacial acetic acid, D-biotin and vitamin B12 to a basal culture medium, wherein the concentration of glacial acetic acid is 40-45 ml / L, the concentration of D-biotin is 0.05-0.06 g / L and the concentration of vitamin B12 is 0.0045-0.005 g / L.
[0018] Furthermore, the basal culture medium is BG-11 medium.
[0019] The present invention also provides the application of the above-mentioned culture medium in the propagation of Haematococcus pluvialis.
[0020] The beneficial effects of this invention are:
[0021] The method of this invention solves the technical problem that the propagation volume must be gradually increased in order to successfully propagate Haematococcus pluvialis with zero apparent biomass.
[0022] This invention eliminates the need for gradually expanding the propagation container, allowing direct propagation from plate to large-volume flask. It achieves the propagation of Haematococcus pluvialis with zero apparent biomass under microscopic examination in one step, shortening the propagation time of Haematococcus pluvialis from 90 days to 15 days and increasing the cell density from 200,000-300,000 / mL to 700,000-800,000 / mL. This not only greatly improves the propagation efficiency and shortens the propagation time but also reduces or avoids the risk of contamination caused by multiple container transfers. Attached Figure Description
[0023] Figure 1 This is a comparison of the cell states of Haematococcus pluvialis after propagation in Example 1 and Comparative Example 1, as observed under a microscope;
[0024] Figure 2 The image shows a comparison of the cell states of Haematococcus pluvialis after propagation in Example 1 and Comparative Example 2, as observed under a microscope.
[0025] Figure 3 The comparison of Haematococcus pluvialis cell states after propagation in Example 2 and Comparative Examples 3 to 6 is observed under a microscope.
[0026] Figure 4 This is a comparison of Haematococcus pluvialis cell biomass at different propagation times in Examples 1 and 2 and Comparative Examples 1 to 6 of the present invention. Detailed Implementation
[0027] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.
[0028] The one-step propagation method for Haematococcus pluvialis with zero biomass described in this invention eliminates the need for stepwise propagation of plate-purified Haematococcus pluvialis colonies. Instead, plate-purified Haematococcus pluvialis colonies can be directly inoculated from the purification plate into a large-volume flask and cultured in culture medium. The initial state of the algal solution after inoculation is that no algal cells are visible under a hemocytometer, and the apparent biomass is 0. After inoculation, the algal solution is cultured in stages in a fixed container, with different culture conditions for each stage.
[0029] During the recovery and activation period, days 0-3, the light intensity should be 700-1000 lux.
[0030] During the physiological recovery period, days 4-7, the light intensity should be 1000-3000 lux.
[0031] During the energy storage preparation period, on days 8-9, the light intensity is 3000-4000 lux.
[0032] During the logarithmic growth phase, days 10-15, the light intensity is 4000-4500 lux;
[0033] After a total of about 15 days of cultivation, Haematococcus pluvialis with a cell density of 700,000-800,000 / mL can be obtained, which increases the proliferation efficiency by about 18 times. It also effectively avoids the cumbersome operation of gradually expanding the culture volume in the existing indoor culture process. This not only simplifies the indoor propagation process of Haematococcus pluvialis, but also reduces the probability of contamination caused by multiple transfers of culture containers.
[0034] The culture medium used in this invention is a modified BG-11 medium, specifically prepared by adding glacial acetic acid, D-biotin, and vitamin B12 to BG-11 medium. The concentration of glacial acetic acid is 40-45 ml / L, the concentration of D-biotin is 0.05-0.06 g / L, and the concentration of vitamin B12 is 0.0045-0.005 g / L.
[0035] The BG-11 culture medium used in this invention is composed of the following substances at the following concentrations: sodium nitrate 76 g / L, magnesium sulfate heptahydrate 11.4 g / L, anhydrous sodium carbonate 22.4 g / L, disodium ethylenediaminetetraacetate dihydrate 1.09 g / L, ammonium sulfate hydrochloride 0.18 g / L, boric acid 2.6 g / L, manganese chloride tetrahydrate 1.66 g / L, zinc sulfate heptahydrate 0.55 g / L, sodium molybdate dihydrate 0.36 g / L, copper sulfate pentahydrate 0.073 g / L, cobalt nitrate hexahydrate 0.20 g / L, citric acid monohydrate 5.6 g / L, ferric ammonium citrate 8.35 g / L, anhydrous calcium chloride 16.55 g / L, and dipotassium hydrogen phosphate trihydrate 73.6 g / L.
[0036] During the cultivation process, the pH value of the cultivation system is maintained by introducing CO2. When the pH of the algal solution rises by ≥0.5 per day, the CO2 introduction rate is increased; when the pH fluctuation is <0.5, the CO2 introduction rate is maintained.
[0037] To illustrate the present invention more clearly, the following embodiments will be described in detail.
[0038] Example 1
[0039] Under the aseptic conditions of a laminar flow hood, purified Haematococcus pluvialis colonies were picked up with a sterile inoculation loop and directly inoculated into 800 mL of modified culture medium in a 1 L bottle. After inoculation, the initial state of the algal solution was that no algal cells were visible in the hemocytometer counting frame and the apparent biomass was 0. The entire process was carried out under aseptic conditions to avoid contamination by other microorganisms.
[0040] The modified culture medium is BG-11 basal culture medium with the addition of glacial acetic acid, D-biotin and vitamin B12, wherein the concentration of glacial acetic acid is 42.75 ml / L, the concentration of D-biotin is 0.058 g / L and the concentration of vitamin B12 is 0.0045 g / L.
[0041] It uses a single white LED light source, providing continuous 24-hour illumination, with light intensity adjustable in four stages.
[0042] (1) Resuscitation and activation period, days 0-3, light intensity 700-1000 lux; low light protects newly formed fragile algal cells, avoids light damage, and completes the transformation of algal cells from dormancy to activation;
[0043] (2) Physiological recovery period, days 4-7, light intensity 1000-3000 lux; initial cell division;
[0044] (3) Energy storage preparation period, day 8-9, light intensity 3000-4000 lux; rapid cell division and proliferation.
[0045] (4) During the logarithmic growth phase, on days 10-15, the light intensity is 4000-4500 lux; the cell density continues to increase, with virtually no sedimentation or aging.
[0046] In the four stages described above, the pH of the culture system is maintained by introducing CO2. When the pH of the algal solution rises by ≥0.5 per day, the CO2 introduction rate is increased; when the pH fluctuation is <0.5, the CO2 introduction rate is maintained.
[0047] After a total of 15 days of culture, the proliferation of Haematococcus pluvialis in the culture medium was tested: the number of motile cells increased to about 700,000-800,000 / ml, most of the cells were pear-shaped or oval, with good activity, few immobile cells and dead cells, accounting for about 0.01%, and large periplasmic space.
[0048] Comparative Example 1
[0049] Except for the use of BG-11 basal medium, everything else was the same as in Example 1. The results showed that the number of motile cells increased to 70,000-100,000 / ml. The cells were mostly oval or round, with poor activity, many immobile and dead cells, and the center of the spores turned red. The periplasmic space of the cell membrane was small.
[0050] After propagation, the algal solution was thoroughly stirred and a suitable sample was taken. The cell state was observed under a microscope. The cell number and state of Example 1 and Comparative Example 1 are shown in the attached figure. Figure 1 As shown, Figure 1 In the diagram, A and C represent the cell states of Example 1, while B and D represent the cell states of Comparative Example 1.
[0051] Comparative Example 2
[0052] Propagation was performed using BG-11 basal medium and conventional culture methods, as follows:
[0053] Under the aseptic conditions of a laminar flow hood, a sterile inoculation loop was used to pick up the purified Haematococcus pluvialis colonies from the plate and inoculate them into a 96-well plate for purification. After 7 days of culture in the 96-well plate, an appropriate amount of the algal supernatant was transferred to a 24-well plate. After about 10 days of culture in the 24-well plate, an appropriate amount of the algal supernatant was transferred to a 15ml test tube. After about 10 days of culture in the 15ml test tube, an appropriate amount of the algal supernatant was transferred to a 50ml Erlenmeyer flask. After about 12 days of culture in the 50ml Erlenmeyer flask, an appropriate amount of the algal supernatant was transferred to a 250ml Erlenmeyer flask. The 250ml Erlenmeyer flask was transferred three more times, for a total of about 39 days. Finally, it was transferred to a 1L bottle and cultured for 12 days.
[0054] After approximately 90 days of propagation, the propagation of Haematococcus pluvialis in the culture medium was tested: the number of motile cells increased to approximately 200,000-300,000 / ml, the cells were mostly pear-shaped or elliptical, with good activity, few immobile cells and dead cells, accounting for approximately 0.01%, and a large periplasmic space.
[0055] Figure 2 These are microscope images of Haematococcus pluvialis cells after propagation, for Examples 1 and 2. Figure 2 In the diagram, A and C represent the cell states of Example 1, while B and D represent the cell states of Comparative Example 1.
[0056] Example 2
[0057] Under the sterile environment of the ultra-clean workbench, purified Haematococcus pluvialis colonies were picked up with a sterile inoculation loop and directly inoculated into 800 mL of modified culture medium. After inoculation, the initial state of the algal solution was that no algal cells were visible under a microscope, the apparent biomass was 0, and no algae were visible to the naked eye. The entire process was carried out under sterile conditions to avoid contamination by other microorganisms.
[0058] The improved culture medium is BG-11 basal culture medium with the addition of glacial acetic acid, D-biotin and vitamin B12, wherein the concentration of glacial acetic acid is 45 ml / L, the concentration of D-biotin is 0.05 g / L and the concentration of vitamin B12 is 0.005 g / L.
[0059] It uses a single white LED light source, providing continuous 24-hour illumination, with light intensity adjustable in four stages.
[0060] (1) Resuscitation and activation period, days 0-3, light intensity 700-1000 lux; low light protects newly formed and fragile algal cells, avoids light damage, and completes the transformation of algal cells from dormancy to activation.
[0061] (2) Physiological recovery period, days 4-7, light intensity 1000-3000 lux; during this stage, cells begin to divide.
[0062] (3) Energy storage preparation period, day 8-9, light intensity 3000-4000 lux; during this stage, cells rapidly divide and proliferate.
[0063] (4) Logarithmic growth phase, days 10-15, light intensity 4000-4500 lux; cell density continues to increase during this phase, with virtually no sedimentation or aging.
[0064] In the four stages described above, the pH of the culture system is maintained by introducing CO2. When the pH of the algal solution rises by ≥0.5 per day, the CO2 introduction rate is increased; when the pH fluctuation is <0.5, the CO2 introduction rate is maintained.
[0065] After a total of 15 days of culture, the proliferation of Haematococcus pluvialis in the culture medium was tested: the number of motile cells increased to about 700,000-800,000 / ml, most of the cells were pear-shaped or oval, with good activity, few immobile cells and dead cells, accounting for about 0.01%, and large periplasmic space.
[0066] Comparative Example 3
[0067] The only difference between this comparative example and Example 2 is that glacial acetic acid was not added to the basal culture medium.
[0068] After a total of 15 days of culture, the proliferation of Haematococcus pluvialis in the culture medium was tested: the number of motile cells increased to about 400,000-500,000 / ml, the cells were mostly oval or round, there were more spores and dead debris, accounting for about 10% of the cells in the entire field of view, and the periplasmic space was small.
[0069] Comparative Example 4
[0070] The only difference between this comparative example and Example 2 is that D-biotin was not added to the basal culture medium.
[0071] After a total of 15 days of culture, the proliferation of Haematococcus pluvialis in the culture medium was tested: the number of motile cells increased to about 500,000 / ml, most of the cells were oval or round, a small number of dead cells accounted for about 5% of the cells in the entire field of view, and the periplasmic space was small.
[0072] Comparative Example 5
[0073] The only difference between this comparative example and Example 2 is that vitamin B12 was not added to the basal culture medium.
[0074] After a total of 15 days of culture, the proliferation of Haematococcus pluvialis in the culture medium was tested: the number of motile cells increased to about 500,000-600,000 / ml, most of the cells were oval or round, a small number of dead cells accounted for about 3% of the total cells in the field of view, and the periplasmic space was small.
[0075] Comparative Example 6
[0076] The difference between this comparative example and Example 2 is that the light intensity is not changed during the cultivation process, as detailed below:
[0077] In a sterile environment under a laminar flow hood, purified Haematococcus pluvialis colonies were picked up with a sterile inoculation loop and directly inoculated into 800 mL of modified culture medium. The initial biomass of the algal solution after inoculation was about 60,000-80,000. The entire process was carried out under sterile conditions to avoid contamination by other microorganisms.
[0078] Using a single white LED light source and continuous full illumination for 24 hours, the following culture was carried out:
[0079] The entire process uses a light intensity of 1000 lux and is cultured for 15 days. The cell proliferation is about 200,000-300,000 / ml, which is relatively slow. Most of the cells are oval or round, with few immobile and dead cells, accounting for about 0.01%. The periplasmic space is small.
[0080] It is evident that gradually increasing light intensity can promote cell proliferation and further enhance the proliferation effect.
[0081] During the cultivation process, the pH value of the cultivation system is maintained by introducing CO2. When the pH of the algal solution rises by ≥0.5 per day, the CO2 introduction rate is increased; when the pH fluctuation is <0.5, the CO2 introduction rate is maintained.
[0082] Figure 3 The images show the cell states after propagation in Examples 2 and Comparative Examples 3 to 6 (microscope 10×10). Figure 3 In the diagram, Figure A shows the cell state of Example 2, and Figures B to E show the cell state of Comparative Examples 3 to 6, respectively.
[0083] The biomass statistics for different propagation times in Examples 1, 2, and Comparative Examples 1 to 6 are shown in the table below.
[0084] Table 1. Biomass statistics of different embodiments and comparative examples of the present invention at different propagation times.
[0085]
[0086] Figure 4 This is a comparison chart of biomass at different propagation times for Examples 1, 2, and Comparative Examples 1 to 6.
[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A method for one-step scale-up of Haematococcus pluvialis with apparent zero biomass by microscopy, characterized in that, Propagation was carried out using a modified culture medium, which was prepared by adding glacial acetic acid, D-biotin and vitamin B12 to a basal culture medium, wherein the concentration of glacial acetic acid was 40-45 ml / L, the concentration of D-biotin was 0.05-0.06 g / L and the concentration of vitamin B12 was 0.0045-0.005 g / L.
2. The Haematococcus pluvialis one-step biomass production method for microscope examination according to claim 1, characterized in that, The basal culture medium is BG-11 medium, which is composed of the following substances at the following concentrations: sodium nitrate 76 g / L, magnesium sulfate heptahydrate 11.4 g / L, anhydrous sodium carbonate 22.4 g / L, disodium ethylenediaminetetraacetate dihydrate 1.09 g / L, ammonium sulfate hydrochloride 0.18 g / L, boric acid 2.6 g / L, manganese chloride tetrahydrate 1.66 g / L, zinc sulfate heptahydrate 0.55 g / L, sodium molybdate dihydrate 0.36 g / L, copper sulfate pentahydrate 0.073 g / L, cobalt nitrate hexahydrate 0.20 g / L, citric acid monohydrate 5.6 g / L, ferric ammonium citrate 8.35 g / L, anhydrous calcium chloride 16.55 g / L, and dipotassium hydrogen phosphate trihydrate 73.6 g / L.
3. The one-step propagation method for Haematococcus pluvialis with apparent zero biomass as described in claim 1, characterized in that, The initial pH of the modified culture medium is ≤6.
0.
4. The method of claim 1 to 3, wherein the method is a one-step method of scale-up of Haematococcus pluvialis with apparent zero biomass. 5 Purified Haematococcus pluvialis colonies from plates were picked up using a sterile inoculation loop and directly inoculated into modified culture medium. In the initial inoculation stage, no algal cells were detected by multi-field microscopy using a hemocytometer, indicating the biomass was below the microscopic detection limit; therefore, the apparent biomass was recorded as 0. After inoculation, the culture was divided into the following stages. During the recovery and activation period, days 0-3, the light intensity should be 700-1000 lux. During the physiological recovery period, days 4-7, the light intensity should be 1000-3000 lux. During the energy storage preparation period, on days 8-9, the light intensity is 3000-4000 lux. During the logarithmic growth phase, days 10-15, the light intensity is 4000-4500 lux; All the above cultures were conducted under 24-hour light.
5. The method of claim 4, wherein the method is a one-step method of scaling up the biomass of H. pluvialis to a level of appearance zero. The pH value of the stable culture system during the cultivation process is 7.5-8.
5. 6. The method of claim 5, wherein the method is a one-step method of scaling up the biomass of H. pluvialis to a level of appearance zero. 6 The pH of the culture system is maintained by introducing CO2. When the pH of the algal solution rises by ≥0.5 per day, the CO2 introduction rate is increased; when the pH fluctuation is <0.5, the CO2 introduction rate is maintained.
7. The method of claim 4, wherein the method is a one-step method of scaling up the biomass of H. pluvialis to a level of appearance zero. The temperature of the culture system is controlled at 22-25℃. 8. A culture medium for the propagation of Haematococcus pluvialis, characterized in that, The culture medium is prepared by adding glacial acetic acid, D-biotin and vitamin B12 to the basal culture medium, wherein the concentration of glacial acetic acid is 40-45 ml / L, the concentration of D-biotin is 0.05-0.06 g / L and the concentration of vitamin B12 is 0.0045-0.005 g / L.
9. The H. pluvialis propagation medium according to claim 8, characterized in that, The basal culture medium is BG-11 medium.
10. The application of the culture medium as described in claim 8 or 9 in the propagation of Haematococcus pluvialis.