A culture method for increasing the content of 24-methylenecolesterol in nannochloropsis

Abstract

The application relates to the technical field of microbial culture, and specifically discloses a culture method for improving the content of 24-methylenecholesterol of Nannochloropsis, which involves key substances including a nitrogen source, a phosphorus source, salicylic acid, an organic carbon source and L-methionine; the preparation method is as follows: first, the Nannochloropsis is allowed to accumulate biomass through photoautotrophic culture; then, a target product synthesis path is activated through nitrogen and phosphorus double restriction and light compound stress; finally, the product accumulation is strengthened through exogenous carbon source and L-methionine cooperation. The method can be used for large-scale culture of the Nannochloropsis, and can be used for efficiently obtaining the 24-methylenecholesterol and is suitable for the fields of medicines, health products and the like, and has the advantages of high target product synthesis efficiency; in addition, the preparation method is designed through three-stage cooperation, the process is clear, the operation is controllable, the growth of the Nannochloropsis and the product synthesis can be balanced, the preparation method is suitable for industrial production requirements, and has the advantages of strong practicability.

Description

Technical Field

[0001] This application relates to the field of microbial culture technology, and more specifically, to a method for culturing *Microcystis globulus* to increase the content of 24-methylene cholesterol. Background Technology

[0002] Microalgae *Nyctocele* is a microalga rich in bioactive substances. Its synthesized 24-methylene cholesterol has significant applications in medicine, health products, and other fields. Therefore, developing efficient cultivation methods to increase the 24-methylene cholesterol content of *Nyctocele* has become a key research focus in this field. Current technologies for increasing cholesterol content in *Nyctocele* often employ a two-stage design of vegetative growth and single stress induction. The functional roles of each stage are vague, and a clear division of labor and cooperation between biomass accumulation, stress induction, and product synthesis has not been established.

[0003] Traditional processes lack a synergistic mechanism that links each step together, causing the biomass accumulation of *Microcystis aeruginosa* and the 24-methylene cholesterol synthesis process to mutually restrict each other, making it impossible to achieve efficient connection between each step. Ultimately, this results in a low overall synthesis efficiency of the target product, which is difficult to meet the needs of large-scale production in practical applications. Therefore, there is an urgent need for a cultivation method that can improve the 24-methylene cholesterol synthesis efficiency of *Microcystis aeruginosa* through staged functional synergy. Summary of the Invention

[0004] To address the problem of low 24-methylene cholesterol synthesis efficiency in existing technologies, this application provides a cultivation method for increasing the 24-methylene cholesterol content of *Microcystis aeruginosa*.

[0005] A method for cultivating *Microcystis globulus* to increase the content of 24-methylene cholesterol includes the following steps performed in sequence:

[0006] S1. Vegetative growth stage: Microcystis globulus was photosynthetically autotrophically cultured in a culture medium containing nitrogen and phosphorus sources. The culture conditions were controlled as follows: light intensity 50 to 150 μmol photons / (m²·s), temperature 20 to 28℃, and a mixture of sterile air and carbon dioxide was continuously introduced at a rate of 100 to 300 mL / min·L. The culture period was 3 to 7 days.

[0007] S2, Nitrogen and Phosphorus Dual Limitation and Light Combined Stress Stage: After the vegetative growth stage, salicylic acid at a concentration of 0.05 to 0.2 mmol / L is added to the culture medium, and a combined stress including nitrogen limitation, phosphorus limitation and moderate light intensity is applied to the algal solution; wherein, the residual concentration of nitrate nitrogen in the culture medium is controlled to be 5% to 25% of the initial concentration, and the residual concentration of phosphate is controlled to be 5% to 25% of the initial concentration.

[0008] S3. External carbon source enhancement and recovery stage: After the completion of the combined stress stage, add organic carbon source to the algal solution and simultaneously supplement with 0.1 to 0.5 g / L of L-methionine, and then continue the co-trophic culture.

[0009] By employing the above-mentioned technical solutions, providing nitrogen and phosphorus sources during the vegetative growth stage, and simultaneously regulating light intensity, temperature, aeration rate, and culture cycle within specific ranges, a stable and suitable photosynthetic autotrophic environment is created for *Microcystis aeruginosa*, promoting rapid cell proliferation and accumulating sufficient biomass. Adding salicylic acid during the combined stress stage, combined with nitrogen and phosphorus restriction and moderate light intensity synergistic stress, enhances the stress response of algal cells and activates metabolic pathways related to 24-methylene cholesterol synthesis. Adding organic carbon sources and simultaneously supplementing L-methionine during the enhanced recovery stage, combined with a multitrophic culture mode, continuously provides carbon skeletons and methyl donors for the synthesis of the target product, extending the efficient synthesis process. This forms a coherent regulatory system from biomass accumulation to synthesis pathway activation and product enhancement, achieving the effect of promoting the orderly accumulation of 24-methylene cholesterol.

[0010] Preferably, in step S1, the initial concentration of the nitrogen source in the culture medium is 3 to 20 mmol / L, and the initial concentration of the phosphorus source is 0.3 to 2.0 mmol / L.

[0011] By adopting the above technical solution, and by limiting the initial concentrations of nitrogen and phosphorus sources in the culture medium in step S1 to a specific range, a precise and suitable nutrient supply is provided for the photosynthetic autotrophic growth of *Microcystis aeruginosa*. This avoids the slow growth and insufficient biomass accumulation of algal cells due to excessively low nitrogen and phosphorus concentrations, or the waste of nutrients and nutrient excess during subsequent stress induction due to excessively high concentrations. In this way, the algal cells can steadily proliferate and accumulate sufficient and active biomass, providing a solid cellular foundation for the metabolic shift and 24-methylene cholesterol synthesis during the subsequent compound stress stage.

[0012] Preferably, in step S2, the applied light intensity is 150 to 300 μmol photons / (m²·s), and the culture time is 2 to 5 days.

[0013] By adopting the above technical solution, and limiting the light intensity and culture time of the combined stress in step S2 to a specific range, a suitable light environment and sufficient response time are provided for the synergistic stress of nitrogen and phosphorus dual restriction and salicylic acid regulation. This avoids the effects of insufficient light intensity failing to effectively trigger the metabolic shift of algal cells, excessive light intensity causing damage to algal cells, insufficient stress induction due to excessively short culture time, and decreased algal cell viability due to excessively long culture time. In this way, the 24-methylene cholesterol synthesis pathway is stably activated, ensuring that algal cells complete metabolic adjustment in an orderly manner under stress conditions.

[0014] Preferably, in step S3, the conditions for the co-culture include: light intensity of 100 to 200 μmol photons / (m²·s), temperature of 22 to 26°C, and culture time of 12 to 72 hours.

[0015] By adopting the above technical solution, and by limiting the light intensity, temperature, and continued culture time in the co-trophic culture in step S3 to a specific range, a suitable metabolic environment and sufficient synthesis cycle are provided for the synergistic effect of organic carbon source and L-methionine. This avoids the effects of improper light intensity on the synergistic effect of photosynthesis and heterotrophic metabolism in algal cells, temperature deviation leading to fluctuations in metabolic enzyme activity, and insufficient product accumulation due to excessively short culture time or nutrient depletion due to excessively long culture time. In this way, the effect of ensuring that algal cells can stably utilize carbon source and methyl donor under co-trophic conditions and continuously promote the synthesis and accumulation of 24-methylene cholesterol is achieved.

[0016] Preferably, in step S3, the organic carbon source is any one or more combinations of sodium acetate, acetic acid, glycerol, or glucose.

[0017] By adopting the above technical solution, by clarifying the specific types and combinations of organic carbon sources in step S3, and selecting carbon source types that are compatible with the metabolic pathway of Micrococcus pluvialis, the use of unsuitable carbon sources can avoid the low absorption and utilization efficiency of algal cells and the increase in metabolic burden caused by the use of unsuitable carbon sources. At the same time, multiple optional carbon sources and combinations can flexibly adapt to the metabolic needs under different culture scenarios, thereby enabling algal cells to efficiently convert carbon sources into the carbon skeleton required for the synthesis of 24-methylene cholesterol, and ensuring the continuous and stable progress of product synthesis in the exogenous carbon source enhancement stage.

[0018] Preferably, the organic carbon source is sodium acetate, and its concentration is 0.5 to 5.0 g / L.

[0019] By adopting the above technical solution, sodium acetate is selected as the organic carbon source in step S3, and its addition concentration is limited to the range of 0.5 to 5.0 g / L. The characteristics of sodium acetate that match the carbon metabolism preference of Micrococcus filamentosa can be utilized to avoid the lack of sufficient carbon skeleton in product synthesis due to the carbon source concentration being too low, and the osmotic pressure stress or increased metabolic burden on algal cells caused by the concentration being too high. At the same time, the high compatibility of sodium acetate can improve the carbon source absorption and conversion efficiency, thereby enabling algal cells to continuously and efficiently utilize carbon sources to synthesize 24-methylene cholesterol, and ensuring the stable progress of product accumulation in the exogenous carbon source enhancement stage.

[0020] Preferably, in step S3, the duration of the continued co-culture is 24 to 48 hours.

[0021] By adopting the above technical solution, and limiting the co-culture time in step S3 to 24 to 48 hours, the synergistic metabolic cycle of organic carbon source and L-methionine is precisely matched. This avoids insufficient conversion of carbon source and methyl donor due to excessively short culture time, resulting in incomplete accumulation of target product. At the same time, it prevents excessively long culture time from causing nutrient depletion of culture medium and decreased algal cell vitality, thereby affecting metabolic efficiency. This allows algal cells to continuously and efficiently promote the synthesis of 24-methylene cholesterol within the optimal time window, ensuring stable and controllable product accumulation during the exogenous carbon source enhancement stage.

[0022] Preferably, in step S1, the nitrogen source is sodium nitrate or potassium nitrate.

[0023] By adopting the above technical solution, and by explicitly selecting sodium nitrate or potassium nitrate as the nitrogen source in step S1, the characteristics of both being easily absorbed and utilized nitrate nitrogen sources by *Microcystis aeruginosa* and suitable for its nitrogen metabolism pathway during photosynthetic autotrophic stage are utilized. This avoids the use of poorly absorbed nitrogen sources that would lead to insufficient nutrient supply and limited growth rate of algal cells. At the same time, the choice of the two nitrogen sources can be flexibly adapted to different culture medium formulations and culture environment requirements, thereby ensuring that algal cells efficiently obtain nitrogen nutrition, steadily complete biomass accumulation, and provide a sufficient material basis for subsequent complex stress induction and product synthesis.

[0024] Preferably, in step S1, the phosphorus source is potassium dihydrogen phosphate or dipotassium hydrogen phosphate.

[0025] By adopting the above technical solution, and by explicitly selecting potassium dihydrogen phosphate or dipotassium hydrogen phosphate as the phosphorus source in step S1, the characteristics of both being easily absorbed and utilized phosphorus forms in the photosynthetic autotrophic stage of *Microcystis aeruginosa* and being suitable for its phosphorus metabolism pathway, can avoid the use of poorly soluble or poorly absorbed phosphorus sources, which would lead to insufficient phosphorus nutrition supply and hindered growth and metabolism in algal cells. At the same time, the choice of the two phosphorus sources can flexibly adapt to different culture medium systems and culture conditions, thereby ensuring that algal cells efficiently obtain phosphorus nutrition, maintain normal physiological metabolism and proliferation activity, and steadily accumulate sufficient biomass.

[0026] Preferably, in step S1, the microbiocyst is marine microbiocyst.

[0027] By adopting the above technical solution, and by clearly selecting marine microalgae in step S1, we can leverage its growth characteristics adapted to nitrogen- and phosphorus-containing culture media, its strong photosynthetic autotrophic capacity, and its natural compatibility with the 24-methylene cholesterol synthesis pathway. This avoids the disconnect between growth metabolism and culture process, and the obstruction of biomass accumulation caused by the selection of incompatible microalgae. At the same time, the physiological characteristics of marine microalgae are highly compatible with the regulatory requirements of subsequent stages such as compound stress induction and carbon source enhancement. This ensures the stable proliferation of algal cells during the vegetative growth stage, providing a highly compatible cellular basis for subsequent metabolic shifts and target product synthesis.

[0028] In summary, this application has the following beneficial effects:

[0029] 1. This application adopts a three-stage sequential culture process design of vegetative growth, compound stress and carbon source enhancement. Since each stage undertakes the core functions of biomass accumulation, stress induction and product synthesis, a synergistic mechanism is formed, which achieves the technical effect of significantly improving the overall synthesis efficiency of 24-methylene cholesterol in Micrococcus microcarpa.

[0030] 2. In this application, a synergistic approach is preferred, which simultaneously implements nitrogen and phosphorus dual restriction and adds salicylic acid during the compound stress stage. Since the synergistic restriction of nitrogen and phosphorus nutrients and the signal induction effect of salicylic acid jointly enhance the stress response intensity, the technical effect of more effectively activating the target product biosynthetic pathway is obtained.

[0031] 3. The method of this application, by simultaneously supplementing L-methionine and organic carbon source during the carbon source enhancement stage, achieves a significant technical effect of promoting the final synthesis and accumulation of 24-methylene cholesterol, since L-methionine provides an essential precursor for sterol methylation and organic carbon source provides sufficient carbon skeleton and energy for the synthesis process. Attached Figure Description

[0032] Figure 1 This is a flowchart of a cultivation method for increasing the 24-methylene cholesterol content of *Microcystis aeruginosa*, as provided in this application. Detailed Implementation

[0033] The present application will be further described in detail below with reference to embodiments and comparative examples. Unless otherwise specified, the experimental methods used below are conventional methods. Unless otherwise specified, the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels or prepared according to literature methods.

[0034] Technical concept:

[0035] In existing technologies, the cultivation of 24-methylene cholesterol from *Nyctaginus* often employs a two-stage process, which suffers from low efficiency in the synthesis of the target product. The core reason is that this type of process does not clearly define the functional stages of biomass accumulation, stress induction, and product synthesis, lacking a synergistic regulatory mechanism that links each stage together. This leads to mutual constraints between the growth process of *Nyctaginus* and the cholesterol synthesis process, resulting in poor coordination between the various stages and an inability to fully activate relevant metabolic pathways and ensure continuous product accumulation.

[0036] This technical solution addresses this problem through a precise three-stage sequential cultivation design: First, photosynthetic autotrophic cultivation provides suitable nutrients and environment for *Microcystis aeruginosa*, efficiently accumulating biomass; second, through dual nitrogen and phosphorus limitation, light stress, and the synergistic effect of salicylic acid, the target product synthesis pathway is directionally activated; finally, the synergistic enhancement of a suitable organic carbon source and L-methionine provides continuous material support for product synthesis. Simultaneously, by clearly defining the selection, concentration, and environmental parameters of key substances at each stage, a well-defined and highly efficient regulatory system is constructed to achieve a balanced optimization of *Microcystis aeruginosa* growth and product synthesis.

[0037] Example 1

[0038] This application provides a method for increasing the 24-methylene cholesterol content of *Microcystis aeruginosa*, comprising the following steps performed in sequence:

[0039] S1. Vegetative growth stage: Microcystis globulus was photosynthetically autotrophically cultured in a culture medium containing nitrogen and phosphorus sources. The culture conditions were controlled as follows: light intensity 100 μmol photons / (m²·s), temperature 24℃, and a mixture of sterile air and carbon dioxide was continuously introduced at a rate of 200 mL / min·L. The culture period was 5 days.

[0040] The initial concentration of nitrogen source in the culture medium was 11.5 mmol / L, and the initial concentration of phosphorus source was 1.15 mmol / L.

[0041] The nitrogen source is sodium nitrate;

[0042] The phosphorus source is potassium dihydrogen phosphate;

[0043] Among them, *Microcystis globulus* is a marine microcystis.

[0044] S2, Nitrogen and Phosphorus Dual Limitation and Light Combined Stress Stage: After the vegetative growth stage, salicylic acid at a concentration of 0.125 mmol / L was added to the culture medium, and the algal solution was subjected to combined stress including nitrogen limitation, phosphorus limitation, and moderate light intensity; the residual concentration of nitrate nitrogen and the residual concentration of phosphate in the culture medium were controlled to be 15% of the initial concentration.

[0045] The applied light intensity was 225 μmol photons / (m²·s), and the culture time was 3.5 days.

[0046] S3, Exogenous Carbon Source Enhancement and Recovery Stage: After the combined stress stage, add organic carbon source to the algal solution and simultaneously supplement with 0.3 g / L L-methionine, and then continue the co-trophic culture;

[0047] The organic carbon source is sodium acetate, and its concentration is 2.75 g / L.

[0048] The conditions for the mixed culture included: light intensity of 150 μmol photons / (m²·s), temperature of 24℃, and a culture time of 42 hours.

[0049] The period of continued mixed-culture training is 36 hours.

[0050] Example 2

[0051] This application provides a method for increasing the 24-methylene cholesterol content of *Microcystis aeruginosa*, comprising the following steps performed in sequence:

[0052] S1. Vegetative growth stage: Microcystis globulus was photosynthetically autotrophically cultured in a culture medium containing nitrogen and phosphorus sources. The culture conditions were controlled as follows: light intensity 50 μmol photons / (m²·s), temperature 20℃, and a mixture of sterile air and carbon dioxide was continuously introduced at a rate of 100 mL / min·L. The culture period was 3 days.

[0053] The initial concentration of nitrogen source in the culture medium was 3 mmol / L, and the initial concentration of phosphorus source was 0.3 mmol / L.

[0054] The nitrogen source is potassium nitrate;

[0055] The phosphorus source is dipotassium hydrogen phosphate;

[0056] Among them, *Microcystis globulus* is a marine microcystis.

[0057] S2, Nitrogen and Phosphorus Dual Limitation and Light Combined Stress Stage: After the vegetative growth stage, salicylic acid at a concentration of 0.05 mmol / L was added to the culture medium, and a combined stress including nitrogen limitation, phosphorus limitation, and moderate light intensity was applied to the algal solution; the residual concentration of nitrate nitrogen and the residual concentration of phosphate in the culture medium were controlled to be 5% of the initial concentration.

[0058] The applied light intensity was 150 μmol photons / (m²·s), and the culture time was 2 days.

[0059] S3, Exogenous Carbon Source Enhancement and Recovery Stage: After the combined stress stage, add organic carbon source to the algal solution and simultaneously supplement with 0.1 g / L L-methionine, and then continue the co-trophic culture.

[0060] The organic carbon source is acetic acid, and its concentration is 0.5 g / L.

[0061] The conditions for the combined culture included: light intensity of 100 μmol photons / (m²·s), temperature of 22℃, and a culture time of 12 hours.

[0062] The period of continued mixed-culture training is 24 hours.

[0063] Example 3

[0064] This application provides a method for increasing the 24-methylene cholesterol content of *Microcystis aeruginosa*, comprising the following steps performed in sequence:

[0065] S1. Vegetative growth stage: Microcystis globulus was photosynthetically autotrophically cultured in a culture medium containing nitrogen and phosphorus sources. The culture conditions were controlled as follows: light intensity 150 μmol photons / (m²·s), temperature 28℃, and a mixture of sterile air and carbon dioxide was continuously introduced at a rate of 300 mL / min·L. The culture period was 7 days.

[0066] The initial concentration of nitrogen source in the culture medium was 20 mmol / L, and the initial concentration of phosphorus source was 2.0 mmol / L.

[0067] The nitrogen source is sodium nitrate;

[0068] The phosphorus source is potassium dihydrogen phosphate;

[0069] Among them, *Microcystis globulus* is a marine microcystis.

[0070] S2, Nitrogen and Phosphorus Dual Limitation and Light Combined Stress Stage: After the vegetative growth stage, salicylic acid at a concentration of 0.2 mmol / L was added to the culture medium, and the algal solution was subjected to combined stress including nitrogen limitation, phosphorus limitation, and moderate light intensity; wherein, the residual concentration of nitrate nitrogen and the residual concentration of phosphate in the culture medium were controlled to be 25% of the initial concentration;

[0071] The applied light intensity was 300 μmol photons / (m²·s), and the culture time was 5 days.

[0072] S3, Exogenous Carbon Source Enhancement and Recovery Stage: After the combined stress stage, add organic carbon source to the algal solution and simultaneously supplement with 0.5 g / L of L-methionine, and then continue the co-trophic culture.

[0073] The organic carbon source is glucose, and its concentration is 5.0 g / L.

[0074] The conditions for the mixed culture included: light intensity of 200 μmol photons / (m²·s), temperature of 26℃, and a culture time of 72 hours.

[0075] The period of continued mixed-culture training is 48 hours.

[0076] Comparative Example 1

[0077] The only difference between this comparative example and Example 1 is that salicylic acid is not added to the culture medium in step S2. The remaining steps and parameters are exactly the same as in Example 1.

[0078] Comparative Example 2

[0079] The only difference between this comparative example and Example 1 is that L-methionine is not supplemented in step S3, while the other conditions are exactly the same as in Example 1.

[0080] Comparative Example 3

[0081] The only difference between this comparative example and Example 1 is that in step S3, the organic carbon source sodium acetate is replaced with an equal mass of ethanol, while the other conditions are exactly the same as in Example 1.

[0082] Comparative Example 4

[0083] The only difference between this comparative example and Example 1 is that in step S2, only nitrogen restriction is applied, and phosphorus restriction is not applied; the other conditions are exactly the same as in Example 1.

[0084] Comparative Example 5

[0085] The only difference between this comparative example and Example 1 is that the S3 exogenous carbon source enhancement and recovery stage is omitted. That is, after the S2 combined stress stage, the algal solution is harvested directly, and the other conditions are exactly the same as in Example 1.

[0086] Comparative Example 6

[0087] This comparative example uses existing conventional culture methods. The core difference between this example and Example 1 is that it adopts a two-stage process of vegetative growth and natural depletion of nitrogen source, without phosphorus restriction, combined stress and exogenous carbon source enhancement stages; it uses general BG-11 medium and does not control the initial and residual concentrations of nitrogen and phosphorus; and it does not add salicylic acid and L-methionine during the culture process.

[0088] Test Item 1: Determination of 24-methylene cholesterol content in *Microcystis aeruginosa*

[0089] The testing standard adopted was GB / T22220-2008, "Determination of Cholesterol in Food - Gas Chromatography," adapted to the requirements for microalgae sample testing. 50 mL of algal culture from Examples 1-3 and Comparative Examples 1-6 was collected after cultivation and centrifuged at 8000 rpm for 10 minutes to collect the algal cells. The algal cells were washed three times with deionized water and then dried to constant weight at -50°C in a freeze dryer. 0.5 g of dried algal powder was accurately weighed and placed in a 50 mL centrifuge tube. 10 mL of anhydrous ethanol was added, and the mixture was refluxed in a 60°C water bath for 2 hours. The mixture was then cooled to room temperature. The supernatant was collected after centrifugation at 4000 rpm for 15 minutes and transferred to a rotary evaporator, where it was concentrated to near dryness at 45°C. 2 mL of n-hexane was added to dissolve the residue, followed by 1 mL of trimethylsilylating agent. The mixture was derivatized at 70°C for 30 minutes, cooled, and filtered through a 0.22 μm organic phase filter membrane. Gas chromatography was used for detection. An HP-5 capillary column was selected, with a length of 30 m, an inner diameter of 0.32 mm, and a film thickness of 0.25 μm. The column temperature program was as follows: initial temperature 180 °C, held for 2 minutes, then ramped to 280 °C at a rate of 10 °C / min and held for 15 minutes. The injection port temperature was 290 °C, the detector temperature was 300 °C, the carrier gas was high-purity nitrogen, the flow rate was 1.0 mL / min, the injection volume was 1 μL, and the split ratio was 10:1. Three parallel experiments were set up for each sample. The content of the target product in each sample was calculated based on a standard curve plotted using 24-methylene cholesterol standards, expressed in mg / g dry weight. The results were taken as the average of the parallel experiments.

[0090] Test Item 2: Determination of Dry Weight of Microcystis aeruginosa Biomass

[0091] The testing standard referenced HY / T058-2010, the Technical Specification for Marine Microalgae Cultivation. 100 mL of algal culture from each of Examples 1-3 and Comparative Examples 1-6 was collected after cultivation. Quantitative filter paper was pre-dried to constant weight in a 105℃ oven, and its mass (m1) was recorded. The algal culture was then vacuum-filtered through the quantitative filter paper to collect the algae. The algae were washed twice with deionized water to remove residual salts from the culture medium. The quantitative filter paper containing the algae was dried in a 105℃ oven for 4 hours, then removed and cooled to room temperature in a desiccator. The total mass (m2) of the filter paper and algae was accurately weighed. Three parallel experiments were set up for each sample. The biomass of each sample was calculated using the formula: biomass dry weight (g / L) = (m2 - m1) × 10. The test results were the average of the parallel experiments. This index reflects the growth and accumulation capacity of *Microcystis aeruginosa* under each cultivation method, providing basic data for subsequent synthetic efficiency analysis.

[0092] Test Item 3: Determination of 24-methylene cholesterol synthesis efficiency

[0093] The testing standard defines the synthesis efficiency as the product of the 24-methylene cholesterol content and the biomass, expressed in mg / L. This indicator comprehensively reflects the total yield capacity of the target product per unit volume of the culture system. Based on the 24-methylene cholesterol content (mg / g dry weight) of Examples 1-3 and Comparative Examples 1-6 obtained from Test Item 1, and the biomass dry weight (g / L) obtained from Test Item 2, the synthesis efficiency of each sample was calculated using the formula: "Synthesis efficiency (mg / L) = 24-methylene cholesterol content (mg / g dry weight) × biomass dry weight (g / L)". The synthesis efficiency of each sample was calculated based on the content and biomass data from three parallel experiments, and the average value was taken. A higher synthesis efficiency indicates a more significant yield advantage of this culture method in practical production applications.

[0094] The experimental data of Examples 1-3 and Comparative Examples 1-6 are shown in Table 1.

[0095] Table 1:

[0096] Sample Name 24-Methylene cholesterol content (mg / g dry weight) Biomass dry weight (g / L) 24-Methylene cholesterol synthesis efficiency (mg / L) Example 1 12.94±0.08 4.82±0.04 62.33±0.39 Example 2 9.38±0.04 3.62±0.04 33.94±0.21 Example 3 11.61±0.04 5.22±0.04 60.48±0.51 Comparative Example 1 10.28±0.04 4.75±0.03 48.85±0.24 Comparative Example 2 9.90±0.04 4.68±0.03 46.30±0.17 Comparative Example 3 8.78±0.04 4.52±0.04 39.64±0.27 Comparative Example 4 8.26±0.04 4.45±0.03 36.74±0.15 Comparative Example 5 7.61±0.04 4.19±0.04 31.93±0.15 Comparative Example 6 4.35±0.03 3.15±0.03 13.70±0.11

[0097] Note: The "±" value after the average is the standard deviation, which is calculated from three sets of parallel experimental data. Its value is much smaller than the average, indicating that the experiment has good repeatability and the data is accurate and reliable.

[0098] As can be seen from Examples 1-3 and Comparative Example 1, and Table 1, salicylic acid plays a crucial role in the combined stress stages of nitrogen and phosphorus limitation and light exposure. Salicylic acid enhances the response of *Microcystis globulus* cells to combined stress, activates metabolic pathways related to 24-methylene cholesterol synthesis, and exhibits a synergistic induction effect with nitrogen and phosphorus limitation and light stress, thereby influencing the accumulation process of the target product. The absence of salicylic acid leads to insufficient targeting and intensity of stress induction, preventing algal cells from fully initiating the relevant mechanisms for cholesterol synthesis.

[0099] As can be seen from Examples 1-3 and Comparative Example 2, and in conjunction with Table 1, L-methionine plays a crucial auxiliary role in the exogenous carbon source enhancement and recovery stage in step S3. As a methyl donor, it provides the necessary material basis for the methylation reaction in the synthesis of 24-methylene cholesterol, forming a functional synergy with the organic carbon source to jointly supplement key raw materials for the synthesis of the target product. Without the supplementation of L-methionine, the methylation step in cholesterol synthesis would lack sufficient support, leading to a decrease in the efficiency of the synthetic pathway and ultimately affecting the accumulation of the target product.

[0100] As shown in Examples 1-3 and Comparative Example 3, and in conjunction with Table 1, the type of organic carbon source has a significant impact on the accumulation of 24-methylene cholesterol in *Microcystis aeruginosa*. Different organic carbon sources exhibit different metabolic pathways and efficiencies in absorption and utilization by algal cells. Suitable organic carbon sources can more efficiently provide the carbon skeleton for algal cells, meeting the carbon source requirements during cholesterol synthesis, and simultaneously synergistically promote the metabolic activities of algal cells with other nutrients. When replaced with unsuitable organic carbon sources, the carbon metabolism process of algal cells is affected, failing to provide sufficient and appropriate carbon source support for cholesterol synthesis, thus impacting product accumulation and synthesis efficiency.

[0101] Combining Examples 1-3 and Comparative Example 4 with Table 1, it can be seen that dual nitrogen and phosphorus restriction plays a crucial synergistic role in the induction stage of combined stress. Nitrogen restriction alone cannot generate a precise stress signal, while dual nitrogen and phosphorus restriction can jointly regulate the metabolic direction of *Microcystis aeruginosa*, prompting algal cells to shift metabolic resources from biomass accumulation to the synthesis of secondary metabolites. Combined with moderate light intensity stress, this creates multidimensional induction conditions. When only nitrogen restriction is applied, the stress signal is not comprehensive enough, and the metabolic regulation of algal cells cannot reach its optimal state, thus affecting the induction effect of 24-methylene cholesterol synthesis.

[0102] As can be seen from Examples 1-3 and Comparative Example 5, and Table 1, the exogenous carbon source enhancement and recovery stage is a key step in increasing the content of 24-methylene cholesterol. After the combined stress stage, the algal cells have initiated the cholesterol synthesis pathway. At this time, adding an exogenous carbon source can provide a continuous carbon source supplement for the subsequent synthesis process, prolong the synthesis cycle, and promote the further accumulation of the target product. Without this stage, the algal cells lack the necessary carbon source support after stress induction, and the cholesterol synthesis process cannot continue to advance, only maintaining the synthesis level of the stress stage, making it difficult to further increase the product content.

[0103] As can be seen from Examples 1-3 and Comparative Example 6, and Table 1, the three-stage culture process and the synergistic effect of key parameters and functional adjuvants in this application are the core of improving product performance. Traditional two-stage processes use universal culture media, lack precise nitrogen-phosphorus concentration ratios, rely solely on a single nitrogen source for induction, and lack complex stress design and functional adjuvant additions, thus failing to achieve balanced regulation of algal cell growth and cholesterol synthesis. This application, through precise culture to accumulate biomass in the vegetative growth stage, multi-dimensional induction to initiate the synthetic pathway in the complex stress stage, and supplementation to promote product accumulation in the exogenous carbon source enhancement stage, combined with the synergistic effect of functional substances such as salicylic acid and L-methionine, forms a complete regulatory system that can more efficiently promote the synthesis and accumulation of 24-methylene cholesterol.

[0104] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for cultivating *Microcystis globulus* to increase the content of 24-methylene cholesterol, characterized in that, The steps are performed in the following order: S1. Vegetative Growth Stage: Marine Micrococcus pluvialis was photosynthetically autotrophically cultured in a culture medium containing nitrogen and phosphorus sources, wherein the nitrogen source was sodium nitrate and the phosphorus source was potassium dihydrogen phosphate; the initial concentration of nitrogen source in the culture medium was 11.5–20 mmol / L and the initial concentration of phosphorus source was 1.15–2.0 mmol / L; the culture conditions were controlled as follows: light intensity 100–150 μmol photons / (m²·s), temperature 24–28℃, and a mixture of sterile air and carbon dioxide was continuously introduced at a rate of 200–300 mL / min·L; the culture period was 5–7 days. S2, Nitrogen and Phosphorus Dual Limitation and Light Combined Stress Stage: After the vegetative growth stage, salicylic acid at a concentration of 0.125–0.2 mmol / L was added to the culture medium, and the algal solution was subjected to combined stress including nitrogen limitation, phosphorus limitation, and moderate light intensity; wherein, the residual concentration of nitrate nitrogen in the culture medium was controlled at 15%–25% of the initial concentration, the residual concentration of phosphate was controlled at 15%–25% of the initial concentration, the applied light intensity was 225–300 μmol photons / (m²·s), and the culture time was 3.5–5 days; S3. External carbon source enhancement and recovery stage: After the completion of the combined stress stage, an organic carbon source, namely sodium acetate or glucose, is added to the algal solution at a concentration of 2.75–5.0 g / L, and 0.3–0.5 g / L of L-methionine is added simultaneously. Then, the co-trophic culture is continued. The conditions for the co-trophic culture are: light intensity of 150–200 μmol photons / (m²·s), temperature of 24–26℃, and culture time of 36–48 hours.

2. The method according to claim 1, characterized in that, In step S2, the residual concentration of nitrate nitrogen in the culture medium is controlled to be 15% of the initial concentration, and the residual concentration of phosphate is controlled to be 15% of the initial concentration.

3. The method according to claim 1, characterized in that, In step S3, the organic carbon source is sodium acetate at a concentration of 2.75 g / L, the concentration of L-methionine is 0.3 g / L, and the culture time is 36 hours.

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