Method for cultivating high content of beta-carotene in dujia salt algae based on stress induction

Abstract

This invention discloses a stress-induced method for cultivating high-content β-carotene algae, comprising three sequentially executed stages: a balanced growth stage I, used to obtain highly active algal cells; a synergistic induction stage II, to trigger β-carotene synthesis; and an enhanced accumulation stage III, which applies intermittent strong light pulses and temperature oscillations synchronized with the light-dark cycle to increase product accumulation. Stages II and III are implemented in a closed photobioreactor. This invention, through phased, multi-stress synergy and closed-loop precise control, directionally activates and enhances the β-carotene biosynthetic pathway while maintaining high cell activity and biomass. Ultimately, it achieves a volumetric yield ≥50% higher than the traditional high-salt stress method, with cell dry weight not less than 85% of the control, resolving the contradiction between algal cell growth and product accumulation, and possesses promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of algae cultivation technology, and more specifically, to a method for cultivating Dunaliella salina with high β-carotene content based on stress induction. Background Technology

[0002] Beta-carotene is a natural carotenoid with important physiological functions. It is not only a precursor to vitamin A, but also has broad application prospects in the food, health product, cosmetic, and pharmaceutical fields due to its excellent antioxidant, immune-enhancing, and chronic disease-preventing properties. Dunaliella salina, a single-celled eukaryotic green algae, is recognized as one of the best biological sources for the commercial production of natural beta-carotene due to its unique physiological characteristic of accumulating large amounts of beta-carotene intracellularly (up to 14% or more of the cell's dry weight) under abiotic stress.

[0003] Currently, the technical approaches to increasing β-carotene content in Dunaliella salina based on the stress-induced principle can be mainly divided into the following categories, but each has its own limitations: 1. Traditional Environmental Stress Culture Method: This is the most classic production method, the core of which lies in applying single or combined stress conditions such as high salt, high light, and nutrient restriction (e.g., nitrogen deficiency) to algal cells to activate their carotene synthesis pathway. For example, existing technologies disclose induction by transferring algal cells to high-salinity (e.g., 3.5 mol / L NaCl) or zero-nitrogen medium. However, this type of method has significant drawbacks: Intense stress conditions, while inducing the accumulation of the target product, can severely inhibit the growth of algal cells and the accumulation of biomass, making it difficult to improve the overall yield.

[0004] Many processes employ a simple "one-step" cultivation method, blurring the boundaries between the growth phase and the stress-induced phase, making it impossible to separately optimize the two conflicting objectives (maximizing biomass and maximizing product).

[0005] Traditional large-scale production heavily relies on salt lake areas with abundant sunshine and high evaporation rates, relying on natural conditions to provide a high-light, high-salinity environment. The production cycle is long (usually several weeks) and is easily affected by weather and biological pollution (such as algae and protozoa), limiting production capacity and stability.

[0006] 2. Optimized Two-Stage Cultivation Method: To overcome the shortcomings of traditional methods, a "two-stage cultivation method" was subsequently developed, which physically or nutritionally separates the algal cell growth stage from the product accumulation stage. For example, the invention patent entitled "A Method for Stressed Dunaliella salina Accumulating β-Carotene" (application publication number CN104450849A) improved this method by, for instance, replacing the culture medium with a nitrogen-free, high-salt stress medium in the later stages of cultivation and gradually increasing the light and temperature, reporting a 70% increase in β-carotene content. Another Chinese invention patent entitled "An Optimized Cultivation Method for Dunaliella salina" (authorization announcement number CN105199957B) achieves a β-carotene content of over 14.45% by precisely controlling the culture medium composition and cultivation conditions (temperature 23-25℃, light 2000-3000 lx) in a closed photobioreactor.

[0007] While the two-stage method represents a significant advancement, its core inducing factors remain limited to traditional physicochemical stresses such as salinity, light, and nutrient restriction. These stresses exert a broad, non-specific physiological pressure on algal cells, inevitably leading to significant metabolic burdens such as photosynthetic inhibition and growth arrest while effectively inducing the target product. Therefore, how to more precisely and efficiently trigger and enhance β-carotene synthesis without severely sacrificing growth viability is a key bottleneck facing existing stress culture techniques.

[0008] 3. Chemical Inducer-Assisted Stress Method: To more precisely intervene in metabolism, researchers have begun exploring methods involving the addition of exogenous chemical inducers. For example, an invention patent (authorization announcement number CN108949888B) entitled "A Method for Promoting the Accumulation of Carotenoids and β-Carotene in Dunaliella using β-Ionone" discloses that treating Dunaliella with β-Ionone (β-iOnOne) can increase the total carotenoid and β-carotene contents by 9.3-33.0% and 16.2-39.3%, respectively, within a short period of time (6 hours to 4 days). The advantage of this method is that it provides a new induction pathway beyond traditional environmental stress and may work by mimicking or interfering with endogenous signaling pathways. However, its application still faces challenges: First, the induction effect is extremely sensitive to the concentration of the compound; excessively high concentrations (such as 50 mg / L) can lead to a decrease in yield due to cytotoxicity; second, the exogenous addition of high-value fine chemicals can significantly increase production costs and may raise compliance considerations for downstream extraction, purification, and product safety, which is not conducive to large-scale industrial application.

[0009] In summary, there is a need for a method to cultivate Dunaliella salina with high β-carotene content that can overcome the problem that the core inducing factors are still limited by traditional physical and chemical stresses such as salinity, light, and nutrients, and that is stable, environmentally friendly, and can more accurately and efficiently coordinate the contradiction between Dunaliella salina growth and β-carotene accumulation. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to provide a method for cultivating Dunaliella salina with high β-carotene content based on stress induction.

[0011] To achieve the above objectives, the present invention provides the following technical solution: A method for cultivating Dunaliella salina with high β-carotene content based on stress-induced growth is characterized by comprising the following sequentially executed stages: Balanced growth stage I: Used to obtain highly active algal cells, grown in a primary culture medium containing basal nutrients at 100-250 μmol·m⁻¹ -2 ·s -1 Cultured continuously or in 16h:8h light-dark cycles at 0.5-1.5mol / L NaCl, pH 7.5-8.5, and 20-30℃ until the logarithmic growth phase; Synergistic induction phase II: This phase is used to trigger and enhance the synthesis and accumulation of β-carotene. When the maximum quantum efficiency Fv / Fm obtained from the online monitoring of algal cells in equilibrium growth phase I is ≥0.70, the cells are transferred to the second culture medium and the synergistic induction system is started. The synergistic induction system includes the following three components: Mild oxidative stress: Add 0.5-2.0 mM H2O2 to the second culture medium using pulsed supply, replenishing 10-20% of the initial amount every 12-24 h, and maintaining the system ORP ≤ +180 mV; Precise osmotic stress: at 0.03-0.08 mol·L⁻¹ -1 ·h -1 A constant slope was used to slowly increase the salinity of NaCl from the first culture medium to 1.8-2.8 mol / L, with the salinity not exceeding 3.5 mol / L throughout the entire salinity increase process; Carbon source and pH: Used to supplement the second culture medium with inorganic carbon source to maintain a stable concentration of dissolved inorganic carbon and to stably control the pH value of the culture medium within the range of 7.8-8.2; In the second culture medium of the synergistic induction phase II, the molar ratio of carbon to nitrogen (C:N) is maintained at ≥10; Enhanced Accumulation Phase III: This phase, building upon the synergistic induction phase II, applies intermittent, intense light pulses synchronized with the light-dark cycle within a set light-dark period. The cycle is 12 hours, consisting of a 2-3 hour peak period and a 4-6 hour recovery period, with a peak light intensity of 400-800 μmol·m⁻¹. -2 ·s -1 The recovery light intensity is 150-300 μmol·m⁻¹ -2 ·s -1Simultaneously perform 24-hour cycle temperature oscillation, with an amplitude of ±2-3℃ and an upper limit not exceeding 30℃; Both the synergistic induction phase II and the enhanced accumulation phase III are implemented in a closed photobioreactor equipped with online Fv / Fm, ORP, pH, dissolved oxygen (DO), and dissolved inorganic carbon (DIC) sensors, as well as automatic dosing, carbon supplementation, salt control, light control, and temperature control modules.

[0012] Further configured, in the mild oxidation stress, when the online monitoring shows that Fv / Fm drops by ≥0.05 relative to the current stage's highest value or ORP ≥+200 mV, the H2O2 pulse supply is suspended or reduced; when Fv / Fm recovers to ≥0.68 and ORP ≤+170 mV, the supply is restored to the set amount.

[0013] The setting is further configured such that the slope of the gradual increase in salinity during the precise osmotic stress is 0.03-0.08 mol·L⁻¹. -1 ·h -1 The slope is determined by the following formula: Slope = Target increase ΔNaCl / Gradual increase time t, where the target increase ΔNaCl refers to the amount of increase in salinity from the initial salinity to the target salinity, and the gradual increase time t refers to the time taken to reach the target salinity. Furthermore, the slope must satisfy ΔNaCl / t ≤ 0.08 mol·L⁻¹. -1 ·h -1 Furthermore, the gradual rise time t ≥ 6h.

[0014] Further configured, the preferred salinity gradient is 0.05 mol·L⁻¹. -1 ·h -1。

[0015] Further configured, in the precise osmotic stress, after each level of salinity is increased during the slow salinity increase process, the salinity is maintained at that level for ≥6 hours to achieve a steady state, and the next increase is carried out only after Fv / Fm, ORP, pH, DO, and DIC have stabilized; when Fv / Fm < 0.68 or ORP > +190 mV, the salinity increase operation is paused until Fv / Fm ≥ 0.70 and ORP ≤ +180 mV before continuing.

[0016] Further configured, the second culture medium of the synergistic induction phase II is a nitrogen-limiting culture medium obtained by adjusting the NaNO3 concentration to 0.2-0.4 g / L on the basis of the first culture medium, and by adding NaNO3 to control the concentration of dissolved inorganic carbon so that the carbon-nitrogen molar ratio (C:N) in the culture medium is maintained above 10:1 (mol / mol).

[0017] Further configured, in the enhanced accumulation stage III, the intermittent strong light pulse and temperature oscillation are controlled in phase: the temperature setpoint corresponding to the high light period is 1-2℃ higher than the base temperature, and the temperature setpoint corresponding to the recovery light period is 1-2℃ lower than the base temperature, and the temperature is controlled within the range of 22-30℃ throughout the process.

[0018] The first culture medium is prepared from artificial seawater or seawater filtered through 0.22 μm, containing basic nutrients (in g / L): NaNO3 1.5 g / L, KH2PO4 0.0256 g / L, Fe-citrate 0.001 g / L, with the remainder being deionized water and seawater matrix.

[0019] By adopting the above technical solution, the beneficial effects of the present invention are as follows: 1. This invention employs a design of "balanced growth stage I → synergistic induction stage II → enhanced accumulation stage III," first in a first culture medium at 100-250 μmol·m -2 ·s -1 High activity and high biomass were obtained by using 0.5-1.5 mol / L NaCl, pH 7.5-8.5, and 20-30℃. Then, using Fv / Fm ≥ 0.70 as the entry criterion, the cells were transferred to a second culture medium and subjected to "mild oxidative stress (0.5-2.0 mM h₂O₂ pulse, ORP ≤ +180 mV) + precise osmotic stress (slow salinity increase of 0.03-0.08 mol·L⁻¹)". -1 ·h -1 The synergistic induction of "1.8-2.8 mol / L (≤3.5 mol / L throughout) + stable carbon and stable pH (10-50 mg / L NaHCO3, pH 7.8-8.2)" is finally superimposed with intermittent strong light pulses (400-800 / 150-300 μmol·m) synchronized with light and dark. -2 ·s -1 The process involves 2-3 h / 4-6 h of temperature oscillation (±2-3℃, upper limit ≤30℃). This pathway, without excessively inhibiting growth, directionally activates carotenoid synthesis and storage pathways, significantly increasing β-carotene content and yield. Compared to the control without synergistic induction and enhanced accumulation, the β-carotene yield per unit volume of culture medium is increased by ≥50%, and the cell dry weight is not less than 85% of the control, achieving simultaneous optimization of "content increase + yield increase". Compared to the traditional one-step method of "high salt / high light / limited nitrogen", this invention overcomes the contradiction between Dunaliella salina growth and β-carotene accumulation in traditional methods through staged culture (balanced growth → synergistic induction → enhanced accumulation) and decoupling stress (mild oxidation + precise osmosis + stable carbon and pH).

[0020] 2. By "gradually increasing salinity (0.03-0.08 mol·L⁻¹)"-1 ·h -1 The "t≥6h)" and "step steady-state" strategies control osmotic shock at a sub-inhibition level, avoiding growth arrest and photosynthetic collapse caused by a single high-salt transition, thus ensuring the continuity and repeatability of the induction period; the "H2O2 pulse supply" is triggered by a reduction / pause when Fv / Fm drops ≥0.05 from its peak or ORP ≥+200 mV, and recovers to Fv / Fm ≥0.68 and ORP ≤+170. The mV recovery supply establishes a hysteresis-controlled anti-vibration mechanism that is both precise and robust. The "nitrogen-limited + carbon-stabilized" window (second medium NaNO3 0.2-0.4 g / L, C:N ≥ 10 (mol:mol)) inhibits nitrogen-driven rapid growth while maintaining DIC and pH stability, ensuring carbon skeleton and reducing power supply and avoiding the mismatch of "strong induction - weak assimilation." "Photothermal-phase" control (high light period corresponding to +1-2℃, recovery light period corresponding to −1-2℃, set point 22-30℃) synergizes with intermittent strong light pulses to improve the balance between light energy utilization and photoprotection, promoting the directional accumulation and intracellular storage of β-carotene. Compared to single-factor stress, this invention, with its core of gradual salinity increase, H2O2 pulses, and precise synergy of carbon and pH stabilization, significantly reduces and avoids metabolic imbalances caused by excessive or single stress, improves induction efficiency and process window controllability, and possesses better industrial feasibility and scale-up consistency.

[0021] 3. Using this method, the critical process window for continuous operation at a scale of ≥10 L for ≥7 days was: Fv / Fm≥0.65, ORP≤+190 mV, pH7.8-8.2, and cell viability≥90%, demonstrating steady-state, healthy, and scalable process control capabilities.

[0022] 4. After cultivating Dunaliella salina using this method, compared with the control without synergistic induction and enhanced accumulation, the yield of β-carotene per unit volume of culture medium increased by ≥50%, and the cell dry weight was not less than 85% of the control, achieving simultaneous optimization of "content improvement + yield improvement". Attached Figure Description

[0023] Figure 1 This is a process flow diagram of the present invention (Stage I → Stage II → Stage III). Figure 2 The logical flow diagram for the online monitoring and control system (Fv / Fm, ORP, pH, DO, DIC) Figure 3 Example curves for slow salinity increase and step steady-state (time-salinity-ORP) Figure 4 Example curves showing intermittent intense light and temperature oscillations in phase (time—light intensity—temperature—Fv / Fm) Figure 5This is the logic diagram for the H2O2 pulse hysteresis control.

[0024] Figure 6 This is a schematic diagram of a closed photobioreactor.

[0025] Figure 6 The diagram shows a typical structure of a stirred photobioreactor, including: 1-reactor body; 2-multi-layer stirring paddles; 3-motor; 4-distributed LED light source; 5-dosing / carbon replenishment interface; 6-sensor integration port (Fv / Fm, ORP, pH, DO, DIC); 7-temperature jacket; 8-air inlet distributor; 9-exhaust port. Detailed Implementation

[0026] Reference Figures 1 to 6 The embodiments of the present invention will be further described below.

[0027] A stress-induced method for cultivating high-β-carotene content algae includes a sequential execution of three phases: a balanced growth phase I for obtaining highly active algal cells, a synergistic induction phase II for triggering and enhancing β-carotene synthesis and accumulation, and an enhanced accumulation phase III for increasing the accumulation amount. Balanced growth stage I: Dunaliella salina inoculates the algal strain into the first culture medium containing basal nutrients, under a light intensity of 100-250 μmol·m⁻¹. -2 ·s -1 The algal cells were cultured under continuous light or a 16-hour light / 8-hour dark cycle, with a salinity of 0.5-1.5 mol / L, a pH of 7.5-8.5, and a temperature of 20-30℃ until they entered the logarithmic growth phase. The first culture medium was prepared from artificial seawater or seawater filtered through 0.22 μm, containing basic nutrients (in g / L): NaNO3 1.5 g / L (≈17.6 mmol), KH2PO4 0.0256 g / L (≈0.19 mmol), Fe-citrate 0.001 g / L, with the remainder being deionized water and seawater matrix. Synergistic Induction Phase II: When the maximum quantum efficiency Fv / Fm obtained from online monitoring of algal cells in Equilibrium Growth Phase I is ≥0.70, the cells are transferred to the second culture medium and the synergistic induction system is started. The synergistic induction system includes the following three components: Mild oxidative stress: Add 0.5-2.0 mM H2O2 to the second culture medium using pulsed supply, replenishing 10-20% of the initial amount every 12-24 h, and maintaining the system ORP ≤ +180 mV. By controlling the dosage and frequency of addition, a moderate oxidative stress environment is created in the system to stimulate β-carotene synthesis. Precise osmotic stress: at 0.03-0.08 mol·L⁻¹ -1 ·h -1A constant slope is used to slowly increase the NaCl salinity in the second culture medium from that in the first culture medium to 1.8-2.8 mol / L. During this slow increase, the salinity does not exceed 3.5 mol / L. By gradually increasing the salinity of the culture system at a specific slope, the algal cells are placed in an environment with appropriate osmotic pressure changes, thereby promoting β-carotene accumulation. The slope of the slow increase is determined by the following formula: Slope = Target increase ΔNaCl / Slow increase time t, where ΔNaCl is the increase in salinity from the initial salinity to the target salinity, and t is the time taken to reach the target salinity. Furthermore, the slope must satisfy ΔNaCl / t ≤ 0.08 mol·L. -1 ·h -1 The salinity should be gradually increased, with a duration of t ≥ 6 hours. During the gradual increase in salinity, after each salinity level is increased, the salinity should be maintained in a steady state for ≥ 6 hours within that salinity level. The next salinity increase should only proceed after all indicators have stabilized. When online monitoring shows Fv / Fm < 0.68 or ORP > +190mV, the salinity increase operation should be suspended until Fv / Fm recovers to ≥ 0.70 and ORP recovers to ≤ +180mV before continuing the salinity increase.

[0028] Carbon source and pH: Used to add 10-50 mg / L NaHCO3 to the second culture medium to maintain a stable concentration of dissolved inorganic carbon (DIC) and to keep the pH of the culture medium stable within the range of 7.8-8.2. When the pH or DIC deviates from the set point, it will automatically replenish carbon or finely adjust the aeration / alkali solution.

[0029] In the synergistic induction phase II, 0.5-2.0 mM H2O2 was added to the second culture medium using a pulsed supply method. When the online monitoring showed that the Fv / Fm ratio dropped by ≥0.05 relative to the current phase's highest value or the redox potential (ORP) was ≥+200 mV, the H2O2 pulsed supply was reduced or paused. When the Fv / Fm recovered to ≥0.68 and the ORP was ≤+170 mV, the H2O2 pulsed supply was restored to the set supply amount, thus forming a hysteresis control anti-vibration mechanism to regulate the addition of H2O2. The H2O2 pulsed control adopted hysteresis control logic to avoid frequent start-stop; the salinity was gradually increased using a step-by-step increase strategy, with each steady state lasting ≥6 hours; pH and DIC were maintained stable by automatically adding NaHCO3 or fine-tuning the aeration rate.

[0030] In the synergistic induction system, mild oxidative stress activates the expression of genes related to β-carotene synthesis in algal cells by generating a moderate oxidative stress environment; precise osmotic stress affects intracellular substance transport and metabolic pathways by changing the osmotic pressure of algal cells, synergistically promoting β-carotene synthesis in conjunction with mild oxidative stress; the stability of carbon source and pH provides the necessary carbon skeleton and suitable acid-base environment for the metabolic activities of algal cells, ensuring the activity of enzymes related to β-carotene synthesis; mild oxidative stress and precise osmotic stress work together, combined with the stability of carbon source and pH, to synergistically trigger and enhance the accumulation of β-carotene synthesis.

[0031] The criterion for entering the co-induction phase II was chosen because Fv / Fm ≥ 0.70 is an important indicator of algal cell photosynthetic function. When Fv / Fm ≥ 0.70, it indicates that the algal cells are in a good photosynthetic state, possessing strong metabolic activity and growth capacity. At this point, entering the co-induction phase II allows for a better response to external stress stimuli and promotes the synthesis and accumulation of β-carotene. In the co-induction phase II, the H2O2 concentration was set in the range of 0.5-2.0 mM, and the salinity increase slope was set in the range of 0.03-0.08 mol·L⁻¹. -1 ·h -1 The range was determined through cultivation experiments on Dunaliella salina under different H2O2 concentrations and salinity gradients. The experiments showed that when the H2O2 concentration and salinity gradient were within this range, the expression of genes related to β-carotene synthesis could be effectively activated without excessively inhibiting algal cell growth, resulting in a significant increase in β-carotene content and yield.

[0032] The H2O2 pulse supply in this synergistic induction phase II is reduced or suspended when Fv / Fm drops by ≥0.05 relative to the current phase's highest value or ORP ≥+200 mV. When Fv / Fm recovers to ≥0.68 and ORP ≤+170 mV, the supply is restored to the set amount.

[0033] The second culture medium in the synergistic induction phase II is a nitrogen-limited medium obtained by adjusting the NaNO3 concentration to 0.2-0.4 g / L (≈2.35-4.70 mmol / L) based on the first culture medium. The concentration of dissolved inorganic carbon is controlled by adding NaNO3 to maintain the carbon-nitrogen molar ratio (C:N) in the culture medium above 10:1 (mol / mol). This ensures that dissolved inorganic carbon (DIC) and pH are stable without providing too much nitrogen source to drive rapid growth, thus ensuring the supply of carbon skeleton and reducing power and avoiding the mismatch of "strong induction-weak assimilation".

[0034] Enhanced Accumulation Phase III: Building upon the synergistic induction phase II, intermittent high-intensity light pulses synchronized with the light-dark cycle are applied within a set light-dark cycle, with a period of 12 hours. This includes 2-3 hours of high-intensity light and 4-6 hours of recovery light, with a high-intensity light of 400-800 μmol·m⁻¹. -2 ·s -1 The recovery light intensity is 150-300 μmol·m⁻¹ -2 ·s -1 Simultaneously perform 24-hour cycle temperature oscillation, with an amplitude of ±2-3℃ and an upper limit not exceeding 30℃; The intermittent high-intensity light pulses and temperature oscillations in the enhanced accumulation phase III are controlled in phase: the high-intensity period corresponds to a high-temperature setpoint (+1-2℃), and the recovery period corresponds to a low-temperature setpoint (-1-2℃). The high-intensity setpoint corresponding to the high-intensity period refers to the high light intensity (400-800 μmol·m⁻¹) of the intermittent high-intensity light pulses. -2 ·s -1 During the irradiation period, the temperature inside the closed photobioreactor is set 1-2°C higher than the baseline temperature (e.g., 25°C); the low-temperature setpoint corresponding to the recovery period refers to the recovery light (150-300 μmol·m⁻¹) temperature. -2 ·s -1 During the irradiation period, the temperature is set to be 1-2℃ lower than the base temperature, and the temperature set point throughout the process does not exceed the range of 22-30℃. This in-phase control method improves the balance between light energy utilization and photoprotection, and promotes the directional accumulation and intracellular storage of β-carotene.

[0035] Both the synergistic induction phase II and the enhanced accumulation phase III are implemented in a closed photobioreactor. This closed photobioreactor can be based on commonly used airlift or stirred photobioreactors (such as those from ApplikOn, SartOrius, and INFORS), equipped with an Fv / Fm fluorescence spectrometer, ORP electrodes, a DIC probe, and corresponding automatic dosing, carbon supplementation, and light control modules, enabling closed-loop operation of synergistic induction and enhanced accumulation. For large-scale cultivation, to ensure uniform mixing within the closed photobioreactor, multi-layered impellers or optimized aeration methods can be used to ensure thorough mixing of the culture medium. Sensors collect data every 30-60 minutes and perform automatic drift compensation. For precise control of dissolved oxygen and pH, in addition to online sensors and an automatic control system, periodic manual monitoring and adjustments can be made to ensure the stability of cultivation conditions. Simultaneously, based on the actual conditions of large-scale production, the volume and flow rate of the culture medium are rationally adjusted to ensure that the algal cells receive sufficient nutrients and light.

[0036] A method for cultivating Dunaliella salina with high β-carotene content based on stress-induced growth includes the following steps: Step 1: Materials and Equipment (1.1) Algal strains and culture medium Algal strain: Dunaliella salina working library strain, purified by monoclonal method, uncontaminated, and in the logarithmic growth phase for later use.

[0037] The first culture medium (for balanced growth, example formulation): prepared with artificial seawater or seawater filtered through 0.22 μm, containing basic nutrients (g / L): NaNO3 1.5 g (≈17.6 mmol), KH2PO4 0.0256 g / L (≈0.19 mmol), Fe-citrate 0.001 g / L; pH adjusted to 7.5-8.5 with NaOH / HCl; the remainder is deionized water and seawater substrate. This formulation is beneficial for obtaining high biomass, serving as the basic nutrient window for stage I.

[0038] Second culture medium (for synergistic induction, nitrogen-limited): Based on the first culture medium, remove or reduce the nitrogen source and adjust to NaNO3 0.2-0.4 g / L (≈2.35-4.70 mmol / L), while keeping other macro and micro elements unchanged; used for the "nitrogen-limited + stable carbon and stable pH" window of stage II.

[0039] (1.2) Reactor and Online Monitoring A closed photobioreactor (such as an airlift or stirred reactor) with a working volume ≥10 L; equipped with online / bypass sensors: Fv / Fm (pulse-modulated chlorophyll fluorescence instrument), ORP (platinum / reference electrode), pH (pH electrode), DO (dissolved oxygen), DIC (total alkalinity / CO2 probe or offline titration); and configured with automatic dosing (H2O2 / reducing agent), carbon supplementation (NaHCO3 solution), salt control (NaCl solution), light / temperature control modules, and a data recording system.

[0040] The closed photobioreactor used in this invention has an optional implementation structure as follows: Figure 6 As shown.

[0041] 1. Reactor body and mixing system: The reactor body is a columnar or flat container made of transparent material (such as glass or polycarbonate), with a working volume of 10-200 L, preferably 20-50 L for pilot testing.

[0042] Hybrid systems can be implemented in one of the following two ways: Airlift type: A porous distributor is installed at the bottom of the reactor to achieve liquid circulation by adjusting the flow rate of sterile air or CO2 mixed gas.

[0043] Stirred type: The reactor is equipped with a multi-layer paddle agitator (preferably 3-4 layers, with the paddle spacing being 1 / 3-1 / 2 of the reactor height), driven by a top motor with an adjustable speed range of 50-200 rpm.

[0044] 2. Light source system layout: The light source uses an LED array, and the light intensity can be independently adjusted (0-1000 μmol·m). -2 ·s -1 ) and light quality (white light or supplemental blue / red light).

[0045] Arrangement method: External type: LED panels are arranged parallel to each other on both sides of the reactor, 5-15 cm away from the reactor wall, to ensure uniform illumination.

[0046] Built-in type: LED light strips or modules are inserted into the reactor through a sealed interface and distributed in 2-4 layers along the height of the reactor.

[0047] The light source is driven by a programmable controller to achieve continuous illumination, light-dark cycle, and intermittent strong light pulses.

[0048] 3. Sensor Installation and Interface: Fv / Fm sensor (pulse-modulated chlorophyll fluorescence meter): Its optical probe is inserted into the reactor through a flange or threaded sealed interface, located in the lower part of the reactor (10-20 cm below the liquid surface), and avoids direct light exposure to avoid interference.

[0049] ORP electrode, pH electrode, DO probe: Integrated into the side or top cover of the reactor via standard IngOld or MettlertOledO type multi-port electrode sheaths, with the probe submerged below the liquid surface and connected to the transmitter.

[0050] DIC (Dissolved Inorganic Carbon) monitoring: Any of the following methods can be used: Online probes: such as CO2 partial pressure (pCO2) electrodes, installed in the same way as pH electrodes.

[0051] Bypass circulation system: The culture medium is circulated to an external flow tank via a peristaltic pump. The tank integrates pH and conductivity sensors, and DIC is calculated using an algorithm.

[0052] Temperature sensor: PT100 platinum resistance thermometer inserted inside the reactor or attached to the jacket of the reactor wall.

[0053] 4. Actuator Connection: Dosing / Carbon Supplementation / Salt Control Module: Connected to the dosing port at the top of the reactor via a peristaltic pump or metering pump. The pump is controlled by a PLC and operates based on sensor feedback signals.

[0054] Temperature control system: The reactor is equipped with a temperature jacket that is connected to a constant temperature water bath circulation system to achieve precise temperature control and oscillation.

[0055] Ventilation system: The gases (air, CO2, N2) are mixed by the mass flow controller and then enter the reactor through the bottom air inlet distributor.

[0056] 5. Control system integration: All sensor signals are connected to a PLC (such as Siemens S7-1200) or an industrial PC.

[0057] The control program (such as using LabVIEW, WinCC or custom software) performs data acquisition, logical judgment (such as hysteresis control, step easing) and outputs control signals to the actuator.

[0058] Human-machine interface (HMI) is used to set parameters, display real-time curves, and alarms.

[0059] (1.3) Conventional equipment and consumables Spectrophotometer, centrifuge, clean bench, sterile inoculation tools, 0.22 μm filter, peristaltic pump / syringe pump, online electrode calibration solution and standard solution, etc.

[0060] Step 2: Cultivation Process and Parameter Setting (2.1) Balanced growth stage I Inoculation and conditions: Inoculate the algal solution in the logarithmic growth phase into the first culture medium at a concentration of 10-20% (v / v); light intensity: 100-250 μmol·m -2 ·s -1 (Continuous or 16h:8h light / dark), temperature 20-30℃, pH 7.5-8.5, ventilation and circulation to maintain DO and uniform mixing.

[0061] Operation and transition criteria: Continue cultivation until the logarithmic growth phase, and if the online monitoring shows Fv / Fm ≥ 0.70, it is determined to enter stage II; if Fv / Fm does not meet the standard, maintain the stage I conditions and optimize light, temperature, nutrition and mixing until the standard is met.

[0062] (2.2) Synergistic Induction Phase II Culture medium switching: Transfer the algal solution to the second culture medium (nitrogen-limited), maintain the temperature at 20-30℃ and the pH at 7.8-8.2; immediately start the following synergistic modules: Mild oxidative stress: The initial dose of H2O2 is 0.5-2.0 mM, administered via pulse, with 10-20% of the initial dose replenished every 12-24 hours; the target is online ORP ≤ +180 mV. If the ORP is higher than the target, the dose will be automatically reduced or the medication will be temporarily discontinued.

[0063] Precise osmotic stress (gradual salinity increase): at a constant slope of 0.03-0.08 mol·L⁻¹ -1 ·h -1 The NaCl salinity was gradually increased from stage I to 1.8-2.8 mol / L; the salinity throughout the process was ≤3.5 mol / L. A "step-by-step gradual increase + steady-state" strategy is recommended: after each salinity increase, maintain a steady-state state for ≥6 hours (Fv / Fm, ORP, pH, DO, and DIC are all stable) before proceeding to the next step; if online monitoring shows Fv / Fm <0.68 or ORP > +190 mV, immediately stop increasing the salinity until the indicators recover to Fv / Fm ≥0.70 and ORP ≤ +180 mV before continuing.

[0064] Carbon source and pH stabilization: Add 10-50 mg / L NaHCO3 to maintain DIC and pH stability (pH setpoint 7.8-8.2); when pH or DIC deviates from the setpoint, automatically replenish carbon or finely adjust ventilation / alkali solution.

[0065] Operation and linkage control: Phase II is implemented entirely within a closed PBR, with dissolved oxygen and pH controlled online in a closed loop; H2O2 pulse supply and salinity gradual increase are both constrained by Fv / Fm and ORP windows, forming a hysteresis control logic that is resistant to jitter.

[0066] (2.3) Strengthening the accumulation stage III In-phase control of light and temperature: intermittent strong light pulses are applied synchronously with the light-dark cycle, with a period of 12 hours, including 2-3 hours of high light and 4-6 hours of recovery light; the intensity of the high light is 400-800 μmol·m. -2 ·s -1 Recovery light 150-300 μmol·m -2 ·s -1 Simultaneously perform 24-hour temperature oscillation with an amplitude of ±2-3℃ and an upper limit of ≤30℃. The high-brightness period corresponds to a setpoint of +1-2℃, and the recovery period corresponds to a setpoint of −1-2℃ (the basic setpoint is recommended to be 25-28℃).

[0067] Process constraints: Stage III uses the H2O2 pulse and ORP / Fv / Fm window from Stage II, and maintains pH at 7.8-8.2; if any key indicator exceeds the limit, automatic degradation (reducing the high light duty cycle, reducing H2O2 supplementation, slowing down the increase in salinity, or temporarily pausing the drug) will be implemented to ensure cell health and process homeostasis.

[0068] Step 3: Online monitoring, control logic, and anomaly handling (3.1) Sensing and Calibration Fv / Fm: Automatically measured every 10-15 min (pulse measurement after 10-15 min of dark adaptation); ORP, pH, DO, and DIC are collected online at 30-60 min intervals and drift is automatically compensated; all electrodes are calibrated at two or more points daily.

[0069] (3.2) Control Logic and Thresholds H2O2 pulse supply: When Fv / Fm drops ≥0.05 from its peak or ORP ≥+200 mV, the supply is reduced / paused; when Fv / Fm ≥0.68 and ORP ≤+170 mV, the supply is restored to the set amount (hysteresis control to prevent frequent jitter).

[0070] Salinity increase slowly: Stop increasing salinity when Fv / Fm < 0.68 or ORP > +190 mV; continue after Fv / Fm ≥ 0.70 and ORP ≤ +180 mV; after each increase, maintain steady state for ≥ 6 hours before proceeding to the next step.

[0071] pH and DIC: When pH deviates from 7.8-8.2, NaHCO3 (10-50 mg / L in steps) or trace amounts of acid / base will be automatically added; when DIC decreases, carbon will be added first, followed by fine-tuning of ventilation and circulation intensity.

[0072] DO and mixing: When DO is below the set lower limit, the ventilation and circulation ratio is automatically increased; when it is too high, the ventilation is reduced and the aeration distribution is optimized to avoid shear damage.

[0073] The logic flow of the online monitoring and control system of this invention is as follows: Figure 2 As shown, in specific implementation: The sensor collects Fv / Fm, ORP, pH, DO, and DIC signals in real time, which are then converted into 4-20 mA or digital signals (such as MOdbus) by the transmitter and uploaded to the controller.

[0074] The controller compares the measured value with the set value and determines whether to trigger operations such as H2O2 dosing, salinity increase, carbon supplementation, light adjustment, and temperature adjustment according to the preset logic (as described in claims 2-5).

[0075] Control commands are sent to the corresponding actuators (pumps, valves, light source drivers, temperature controllers) to complete closed-loop regulation.

[0076] All process data is stored in a database for traceability and optimization.

[0077] (3.3) Abnormal Degradation and Security The failure of any critical sensor (signal loss / over-range) will trigger the "safety mode": the high-light duty cycle drops to ≤50%, H2O2 replenishment is suspended, salinity remains unchanged, carbon replenishment and mixing are increased, the event is recorded and an alarm is triggered; after the fault is cleared, the system will be gradually restored to the set operating conditions according to the procedure.

[0078] Step 4: Sampling, Testing, and Data Processing (4.1) Sampling and Pretreatment Sampling was performed every 24 hours in Phase I; and every 12 hours in Phases II and III. Immediately after sampling, the samples were aseptically filtered at 0.22 μm or centrifuged (4℃, 5,000 g, 5 min) and aliquoted. One portion was used for viability indicators (Fv / Fm, ORP, pH, DO, DIC), and the other portion was used for dry weight and β-carotene analysis.

[0079] (4.2) Index Measurement Cell density and dry weight: Cell density is expressed as OD 684 Conversion (provide the standard curve equation and R²); cell dry weight (DCW) is calculated based on drying at 105°C to constant weight.

[0080] β-Carotene content: Algal mud was extracted with 90% acetone (0.1% BHT can be added for antioxidant effect), sonicated for 10-15 min, and shaken overnight in the dark. The extracts were combined and A was determined. 453 The content can be converted according to the standard curve (based on cell dry weight, % DW); at the same time, the yield per unit volume (mg / (L·d)) can be calculated.

[0081] Process health indicators: Fv / Fm according to the standard dark adaptation pulse method; ORP, pH, DO, and DIC according to online instrument readings; cell viability is recommended to be measured using FDA / PI double staining or dehydrogenase activity method, and thresholds and judgment methods are given.

[0082] (4.3) Statistics and Judgment Each experiment had n ≥ 3 replicates, and the results were expressed as mean ± SD; significance was determined by independent samples t test or ANOVA + Tukey (p < 0.05); yield improvement and process window compliance were determined by batch mean and RSD (recommended ≤ 10%).

[0083] Example 1: Low light - low slope - low H2O2 - mild photothermal oscillation Phase I: 150 μmol·m -2 ·s -1 , 12h: 12h, 1.0 mol / L NaCl, 20℃, pH 7.8; Transfer conditions: Fv / Fm ≥ 0.70.

[0084] Phase II: Salinity gradually increases from 1.0 to 2.2 mol / L, with a slope of 0.03 mol·L⁻¹. -1 ·h -1Steady-state condition ≥ 6 hours per stage; H2O2 0.5mM pulse (10% replenished every 24 hours), ORP maintained ≤ +180 mV; NaHCO3 20 mg / L; intermittent intense light: 600 μmol·m -2 ·s -1 ×2.5h / 300×4.5h; temperature oscillation ±2℃, upper limit 28℃.

[0085] Experimental results: Fv / Fm ≥0.65 throughout; β-carotene content and yield increased by ≥18.1% compared with the control (evidence of lower limit of window).

[0086] Example 2 (Highlight—Medium Slope—Medium H2O2—In-Phase Light Temperature) Phase I: 250 μmol·m -2 ·s -1 , 16h:8h, 1.5 mol / L NaCl, 25℃, pH 8.0; Transfer conditions: Fv / Fm ≥ 0.70.

[0087] Phase II: Salinity gradually increases from 1.5 to 2.8 mol / L. In this embodiment, the preferred salinity increase slope is 0.05 mol·L. -1 ·h -1 H2O2 1.0mM pulse (20% replenished every 12h), ORP maintained ≤+180 mV; NaHCO3 30 mg / L; intermittent intense light: 800×3h / 150×6h; temperature oscillation ±3℃, upper limit 30℃, high light period setpoint +2℃, recovery period −2℃.

[0088] Experimental results: Fv / Fm ≥ 0.68; β-carotene content and yield increased by ≥ 50% compared to the control (evidence of target value). Example 3 (Medium light—high slope—high H2O2—limiting light temperature) Phase I: 200 μmol·m -2 ·s -1 , 12h:12h, 1.2 mol / L NaCl, 23℃, pH 8.0; Transfer conditions: Fv / Fm≥0.70.

[0089] Phase II: Salinity gradually increased from 1.2 to 2.8 mol / L, with a slope of 0.08 mol·L⁻¹. -1 ·h -1 H2O2 2.0mM pulse (20% replenished every 12h), ORP maintained ≤+180 mV; NaHCO3 50 mg / L; intermittent intense light: 800×3h / 150×6h; temperature oscillation ±3℃, upper limit 30℃.

[0090] Experimental results: Fv / Fm≥0.65; demonstrating that high slope and higher H2O2 are still feasible under stable carbon, stable pH and ORP control, but the yield may be slightly lower than that of medium slope (balanced evidence).

[0091] Example 4 (Nitrogen-limiting window and C:N boundary) The difference between this embodiment and Example 2 is that in Stage II, the nitrogen limit levels are 0.2 / 0.3 / 0.4 g / L NaNO3, combined with 10-50 mg / L NaHCO3 to maintain DIC. The verification results are shown in Table 1. Table 1 shows the nitrogen limiting window and C:N boundary data for Example 4 (mean ± SD, n = 3).

[0092] As shown in Table 1, when C:N≥10 (mol:mol), the content and yield of β-carotene increased by ≥40-60% compared with the control; 0.2 g / L and 0.4 g / L are the upper and lower limits of the window.

[0093] Example 5 (Scale-up and Steady-State Operation) Scale: 10 L, 30 L, 50 L closed-loop PBR; operation ≥7 days; online Fv / Fm, ORP, pH, DO, DIC closed-loop monitoring; judgment criteria: Fv / Fm≥0.65, ORP≤+190 mV, pH7.8-8.2, cell viability≥90%; percentage of time within the target window = time within the window / total steady-state time × 100%; batch-to-batch RSD (yield and content)≤10%. The validation results are shown in Table 2, which presents the scale-up and steady-state operation data for Example 5 (10 / 30 / 50 L, mean ± SD, n=3).

[0094] As shown in Table 2, the key process indicators at each scale steady-state stage all fell within the target window (Fv / Fm≥0.65, ORP≤+190 mV, pH7.8-8.2, cell viability≥90%), with batch-to-batch RSD (yield / content) ≤2.9% / 1.5%, and the window compliance time percentage ≥97%, demonstrating good scalability and consistency of the process. When this method was used continuously for ≥7 days at a scale of ≥10L, the key process indicators met the following requirements: maximum quantum efficiency Fv / Fm≥0.65, redox potential ORP≤+190mV, pH 7.8-8.2, and cell viability≥90%, reflecting steady-state, healthy, and scalable process control capabilities.

[0095] To maintain Fv / Fm ≥ 0.65, ORP ≤ +190mV, pH 7.8–8.2, and cell viability ≥ 90% during continuous operation at a scale of ≥ 10L for ≥ 7 days, these indicators were monitored in real time using an online monitoring system. When Fv / Fm deviated from ≥ 0.65, adjustments were made by varying parameters such as light intensity and oxidative stress. When ORP exceeded ≤ +190mV, the pulsed supply of H2O2 was controlled. When pH exceeded the 7.8–8.2 range, pH stability was maintained by adding NaHCO3 or adjusting aeration. Cell viability ≥ 90% was ensured by optimizing culture conditions, such as providing sufficient nutrients, suitable light, and temperature.

[0096] Example 6 (Control vs. Single Factor Control) Control A: Phase II without any induction (simply transferred to high-salt medium 2.8 mol / L), without other stress treatments; Control B: Nitrogen only (0.2 g / L); Control C: High-light pulse only (800×3h / 150×6h); Control D: H2O2 only (1.0mM).

[0097] In this example, all control groups shared the same conditions as in Example 2 (Stage II: salinity 2.8 mol / L, temperature 25℃, pH 8.0; Stage III: intermittent strong light 800×3h / 150×6h, temperature oscillation ±3℃; Stages II-III: online closed-loop control of Fv / Fm / ORP / pH / DO / DIC). Each control group was subjected to only a single variable, with all other conditions remaining consistent. The percentage increase compared to control A was calculated as: Percentage increase = (β-carotene content in this example - β-carotene content in control A) / β-carotene content in control A × 100%. The validation results are shown in Table 3, which presents the data for the control group and the single-factor control group in Example 6 (mean ± SD, n = 3).

[0098] Table 4 compares the final product and yield (Note: Control A is "Stage II without any induction (only transitioned to high salt 2.8 mol / L)"; Example 2 is the "target window"; Example 5 is a scale-up batch (50 L, ≥7 d). Yield increase = (Process group − Control A) / Control A × 100%).

[0099] As shown in Table 3, the synergistic induction (Example 2) was significantly superior to any single factor and control A in terms of β-carotene content and yield (p < 0.05), supporting the "synergistic necessity" and the robust reproducibility of the technical effect.

[0100] As shown in Table 4, after cultivating Dunaliella salina using the method of the present invention (Example 2), compared with the control without synergistic induction and enhanced accumulation (i.e., only transferred to the high-salt 2.8 mol / L culture method), the yield of β-carotene per unit volume of culture medium increased by ≥50%, and the cell dry weight was not less than 85% of the control, achieving simultaneous optimization of "content improvement + yield improvement".

[0101] Table 5 shows the comprehensive process parameters and performance data for Examples 1-3 (mean ± SD, n = 3).

[0102] The specific process parameter settings, process health indicators, and final product data for Examples 1-3 are detailed in Table 5 (Comprehensive Process Parameters and Performance Data Table). As shown in Table 5, Example 2 (optimal window) uses a moderate slope salinity increase (0.05 mol·L⁻¹) during the synergistic induction stage. -1 ·h -1 By applying a medium-concentration H2O2 pulse (1.0 mM) and implementing photothermal phase control during the enhanced accumulation phase, the target of increasing β-carotene yield by +50.0% compared to control A was ultimately achieved. Examples 1 (low stress) and 3 (high stress) verified the feasibility of the lower limit of the parameter window and the trade-off effect under high stress conditions, respectively, with yield increases of +18.1% and +26.0%, and key process health indicators (Fv / Fm, ORP, pH, cell viability) all remained within the set window.

[0103] In determining the parameters such as light intensity, temperature, and salinity in Examples 1-3, the growth characteristics and β-carotene synthesis patterns of Dunaliella salina were studied, and the parameters were screened and optimized based on the results of previous preliminary experiments. In the preliminary experiments, different light intensities (e.g., 100-250 μmol·m⁻¹) were set. -2 ·s -1 By combining different parameters such as temperature (e.g., 20-30℃) and salinity (e.g., 0.5-1.5mol / L), the growth of algal cells (e.g., cell density, biomass) and the amount of β-carotene synthesis (e.g., β-carotene content, yield) were observed. Finally, the specific values ​​of each parameter in this embodiment were determined to maximize the β-carotene content and yield without excessively inhibiting the growth of algal cells.

[0104] In this invention, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," "link," and "fix" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection, a direct connection, or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0105] The control method of this invention is automatic control through a controller. The control circuit of the controller can be implemented by simple programming by those skilled in the art. The power supply is also common knowledge in the art. Furthermore, since this invention is mainly used to protect mechanical devices, the control method and circuit connection will not be explained in detail here.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any ordinary changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for cultivating Dunaliella salina with high β-carotene content based on stress-induced growth, characterized in that, This includes the following stages executed sequentially: Balanced growth stage I: Used to obtain highly active algal cells, grown in a primary culture medium containing basal nutrients at 100-250 μmol·m⁻¹ -2 ·s -1 Cultured continuously or in 16h:8h light-dark cycles at 0.5-1.5mol / L NaCl, pH 7.5-8.5, and 20-30℃ until the logarithmic growth phase; Synergistic Induction Phase II: This phase is used to trigger and enhance the synthesis and accumulation of 9-cis-β-carotene. When the maximum quantum efficiency Fv / Fm obtained from the online monitoring of algal cells in the equilibrium growth phase I is ≥0.70, the cells are transferred to the second culture medium and the synergistic induction system is initiated. The synergistic induction system includes the following three components: Mild oxidative stress: Add 0.5-2.0 mM H2O2 to the second culture medium using pulsed supply, replenishing 10-20% of the initial amount every 12-24 h, and maintaining the system ORP ≤ +180 mV; Precise osmotic stress: at 0.03-0.08 mol·L⁻¹ -1 ·h -1 A constant slope was used to slowly increase the salinity of NaCl from the first culture medium to 1.8-2.8 mol / L, with the salinity not exceeding 3.5 mol / L throughout the entire salinity increase process; Carbon source and pH: Used to supplement the second culture medium with inorganic carbon source to maintain a stable concentration of dissolved inorganic carbon and to stably control the pH value of the culture medium within the range of 7.8-8.2; In the second culture medium of the synergistic induction phase II, the molar ratio of carbon to nitrogen (C:N) is maintained at ≥10; Enhanced Accumulation Phase III: This phase, building upon the synergistic induction phase II, applies intermittent, intense light pulses synchronized with the light-dark cycle within a set light-dark period. The cycle is 12 hours, consisting of a 2-3 hour peak period and a 4-6 hour recovery period, with a peak light intensity of 400-800 μmol·m⁻¹. -2 ·s -1 The recovery light intensity is 150-300 μmol·m⁻¹ -2 ·s -1 Simultaneously perform 24-hour cycle temperature oscillation, with an amplitude of ±2-3℃ and an upper limit not exceeding 30℃; Both the synergistic induction phase II and the enhanced accumulation phase III are implemented in a closed photobioreactor equipped with online Fv / Fm, ORP, pH, dissolved oxygen (DO), and dissolved inorganic carbon (DIC) sensors, as well as automatic dosing, carbon supplementation, salt control, light control, and temperature control modules.

2. The method for cultivating Dunaliella salina with high β-carotene content based on stress induction according to claim 1, characterized in that, In the mild oxidation stress, when the online monitoring shows that Fv / Fm drops by ≥0.05 relative to the current stage's highest value or ORP ≥+200 mV, the H2O2 pulse supply is suspended or reduced; when Fv / Fm recovers to ≥0.68 and ORP ≤+170 mV, the supply is restored to the set amount.

3. The method for cultivating Dunaliella salina with high β-carotene content based on stress induction according to claim 1, characterized in that, The slope of the gradual increase in salinity under the precise osmotic stress is 0.03-0.08 mol·L⁻¹. -1 ·h -1 The slope is determined by the following formula: Slope = Target increase ΔNaCl / Gradual increase time t, where the target increase ΔNaCl refers to the amount of increase in salinity from the initial salinity to the target salinity, and the gradual increase time t refers to the time taken to reach the target salinity. Furthermore, the slope must satisfy ΔNaCl / t ≤ 0.08 mol·L⁻¹. -1 ·h -1 Furthermore, the gradual rise time t ≥ 6h.

4. The method for cultivating Dunaliella salina with high β-carotene content based on stress induction according to claim 1, characterized in that, The preferred salinity gradient is 0.05 mol·L⁻¹. -1 ·h -1 .

5. The method for cultivating Dunaliella salina with high β-carotene content based on stress induction according to claim 1, characterized in that, In the precise osmotic stress process, after each level of salinity is increased during the gradual increase in salinity, the salinity is maintained at that level for ≥6 hours to achieve steady state. The next increase is carried out only after Fv / Fm, ORP, pH, DO, and DIC have stabilized. When Fv / Fm < 0.68 or ORP > +190 mV, the salinity increase operation is paused until Fv / Fm ≥ 0.70 and ORP ≤ +180 mV before continuing.

6. The method for cultivating Dunaliella salina with high β-carotene content based on stress induction according to claim 1, characterized in that, The second culture medium in the synergistic induction phase II is a nitrogen-limiting culture medium obtained by adjusting the NaNO3 concentration to 0.2-0.4 g / L on the basis of the first culture medium, and by adding NaNO3 to control the concentration of dissolved inorganic carbon, the carbon-nitrogen molar ratio (C:N) in the culture medium is maintained above 10:1 (mol / mol).

7. The method for cultivating Dunaliella salina with high β-carotene content based on stress induction according to claim 1, characterized in that, In the enhanced accumulation stage III, the intermittent strong light pulse and temperature oscillation are controlled in phase: the temperature setpoint corresponding to the high light period is 1-2℃ higher than the base temperature, and the temperature setpoint corresponding to the recovery light period is 1-2℃ lower than the base temperature. The temperature is controlled within the range of 22-30℃ throughout the process.

8. The method for cultivating Dunaliella salina with high β-carotene content based on stress induction according to claim 1, characterized in that, The first culture medium is prepared from artificial seawater or seawater filtered through 0.22 μm, containing basic nutrients (in g / L): NaNO3 1.5 g / L, KH2PO4 0.0256 g / L, Fe-citrate 0.001 g / L, with the remainder being deionized water and seawater matrix.

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