Spirulina cultivation methods
By controlling light intensity in stages and using slow-release carbon and nitrogen sources, the problem of mismatch between light and nutrient supply in spirulina cultivation was solved, thereby increasing the biomass and protein content of spirulina.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INNER MONGOLIA AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-05-29
- Publication Date
- 2026-06-30
AI Technical Summary
In existing methods of spirulina cultivation, the supply of light, carbon source, and nitrogen source cannot match the needs of spirulina at different growth stages, resulting in low biomass yield and protein content, and problems such as photoinhibition, osmotic stress, and spatiotemporal mismatch in nutrient supply.
The light intensity is dynamically controlled in stages, and sodium bicarbonate microspheres are encapsulated in calcium alginate and sodium nitrate slow-release particles are loaded on anion exchange resin to provide stable carbon and nitrogen sources at different stages, ensuring precise matching of nutrients during the growth of spirulina.
It significantly improved the biomass yield and protein content of spirulina, avoided photoinhibition and waste of nutrients, made the cultivation process more stable, and increased the final dry weight and protein content of algal cells.
Smart Images
Figure CN122303075A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microalgae biotechnology. More specifically, this invention relates to a method for culturing Spirulina. Background Technology
[0002] Spirulina, a microalga rich in protein and various bioactive substances, has broad application prospects in the food, feed, and biopharmaceutical fields. Improving the biomass yield and protein content of Spirulina is currently the core objective of its industrial cultivation. During the photoautotrophic growth of Spirulina, light, carbon source, and nitrogen source are the three key limiting factors, and the synergistic matching among these three directly affects the photosynthetic efficiency, metabolic activity, and ultimately, the synthesis and accumulation of proteins in algal cells.
[0003] Currently, conventional methods for spirulina cultivation often employ a strategy of constant light intensity and one-time addition of carbon sources (such as sodium bicarbonate) and nitrogen sources (such as sodium nitrate). However, this traditional approach has significant drawbacks: First, spirulina's light requirements vary considerably at different growth stages. In the early stages of growth, cell density is low, and excessive light can easily induce photoinhibition, leading to damage to the photosynthetic system. In the later stages of growth, as cell density increases, light decay becomes severe, and insufficient light intensity makes it difficult to maintain a high photosynthetic rate, thus limiting biomass accumulation. Existing constant light patterns cannot meet the light requirements of each stage, resulting in low light energy utilization efficiency. Second, the carbon source is often supplied by one-time addition of sodium bicarbonate. While one-time addition of sodium bicarbonate can provide stable carbonate ions, high concentrations of sodium bicarbonate in the early stages of cultivation can increase osmotic pressure and inhibit cell division; later, as the carbon source gradually depletes, it cannot meet the carbon requirements of high-density cultivation. Third, the one-time addition of nitrogen sources (sodium nitrate) also presents problems such as excessively high initial concentrations leading to osmotic stress, and insufficient nitrogen sources in the later stages causing premature cell senescence and a decrease in protein content. Because the absorption rate and demand ratio of carbon and nitrogen in spirulina change dynamically at different growth stages, the traditional constant supply model often causes a mismatch between nutrient supply and cellular demand, ultimately resulting in low biomass yield and difficulty in achieving ideal levels of algal protein content.
[0004] Therefore, it is necessary to design a technical solution that can overcome the above-mentioned defects. Summary of the Invention
[0005] One object of the present invention is to provide a method for culturing spirulina to significantly increase the biomass yield and protein content of spirulina.
[0006] To achieve these objectives and other advantages of the present invention, according to one aspect of the present invention, a method for culturing Spirulina is provided, comprising: S1: In a first culture stage, Spirulina is inoculated into a basal culture medium composed of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, and ferric sulfate, and photoautotrophically cultured under a light intensity of 5000-10000 lux until the optical density OD560 reaches 0.6-0.8; S2: In a second culture stage, the light intensity is reduced to 1000-3000 lux, and sodium bicarbonate microspheres embedded in calcium alginate are added, wherein the total amount of sodium bicarbonate added is 5-10 g / L of basal culture medium, and cultured until the dry weight of algal cells reaches 1.5-2.0 g / L; S3: In a third culture stage, the light intensity is increased to 8000-12000 lux, and sodium nitrate slow-release particles loaded with anion exchange resin are added, wherein the total amount of sodium nitrate added is 1.5-2.5 g / L. g / L basal culture medium; calcium alginate-encapsulated sodium bicarbonate microspheres were prepared by the following method: sodium alginate and sodium bicarbonate were dissolved in deionized water at a mass ratio of (1-2):1 to obtain a mixed solution. The mixed solution was then dripped into a 1.5-2.5% (w / w) calcium chloride solution using a syringe pump and solidified for 10-30 minutes to form the microsphere core. The microsphere core was then immersed in a 1-3% (w / w) ethyl cellulose ethanol solution and coated at 50-100 rpm for 15-30 minutes. After removal, the microspheres were dried at room temperature to obtain sustained-release microspheres coated with an ethyl cellulose semi-permeable membrane. The particle size of the sustained-release microspheres was 2-4 mm, and the thickness of the semi-permeable membrane was 10-30 μm.
[0007] Furthermore, the concentrations of each component in the basal culture medium are as follows: sodium nitrate 2.0-3.0 g / L, dipotassium hydrogen phosphate 0.4-0.8 g / L, magnesium sulfate 0.2-0.5 g / L, and ferric sulfate 0.01-0.05 g / L.
[0008] Furthermore, the first culture stage lasts for 2-4 days at a temperature of 25-30 ℃ and a pH of 8.0-9.0; the second culture stage lasts for 2-3 days at a temperature of 28-32 ℃ and a pH of 7.5-8.5; and the third culture stage lasts for 1-2 days at a temperature of 25-30 ℃ and a pH of 8.0-9.0.
[0009] Furthermore, in the first cultivation stage: the initial light intensity was 5000-6000 lux, and then the light intensity was increased by 1000-1500 lux every 24 hours of cultivation until it reached 9000-10000 lux. When the light density OD560 reached 0.4-0.5, the light-dark cycle was switched from 12 h:12 h to 16 h:8 h.
[0010] Furthermore, in the second cultivation stage, the initial light intensity was 1000-1500 lux, and then the light intensity was increased by 300-500 lux every 12 hours until it reached 2500-3000 lux.
[0011] Furthermore, in the third cultivation stage, the light intensity adopts a pulsed light mode: each pulse cycle is 3 hours, with the first 2 hours irradiated with a high light intensity of 8000-12000 lux, and the last hour irradiated with a low light intensity of 2000-4000 lux, and the cycle is repeated.
[0012] Furthermore, during the second culture stage, the dry weight of algal cells was measured every 12 hours, and the pH value was measured simultaneously. The second culture stage was considered to have ended when the increase in dry weight in two consecutive measurements was less than 5%, the dry weight value in the second measurement reached 1.5-2.0 g / L, and the pH value change in two consecutive measurements did not exceed 0.1.
[0013] Furthermore, the rate of sodium nitrate release from anion exchange resin-loaded slow-release particles increases with increasing pH. The preparation method of anion exchange resin-loaded sodium nitrate slow-release particles includes: immersing a strongly basic anion exchange resin in a sodium nitrate solution with a concentration of 30-50 g / L, shaking and loading at 25-30℃ for 12-24 hours, filtering, and washing with deionized water to obtain a resin loaded with sodium nitrate; immersing the resin loaded with sodium nitrate in a chitosan acetate solution with a mass fraction of 1-3%, stirring slowly for 10-20 minutes, then adding a sodium tripolyphosphate solution with a mass fraction of 0.5-1.5% for ionic cross-linking to form a chitosan coating layer, removing and drying at room temperature to obtain anion exchange resin-loaded sodium nitrate slow-release particles.
[0014] The present invention has at least the following beneficial effects: The Spirulina cultivation method provided by this invention significantly improves the biomass yield and protein content of Spirulina by dynamically controlling the supply of light, carbon source, and nitrogen source in stages. In the first cultivation stage, high light intensity is used to promote rapid proliferation of Spirulina, enabling algal cells to reach a suitable light density in a short time, laying the foundation for subsequent high-density cultivation. In the second cultivation stage, by reducing the light intensity and adding calcium alginate-encapsulated sodium bicarbonate microspheres, a slow and continuous release of carbon source is achieved, avoiding the problems of initial osmotic stress and subsequent carbon source insufficiency caused by the traditional one-time addition of sodium bicarbonate. The surface of the microspheres is coated with an ethyl cellulose semi-permeable membrane, which can block the contact between metal ions in the culture medium and calcium ions inside the microspheres, ensuring the structural integrity of the microspheres throughout the cultivation cycle, thereby providing a stable and controllable carbon supply and significantly reducing the pH fluctuation range of the culture medium. The stable carbon supply and mild pH environment promote rapid growth of algal cells and accumulation of dry matter. In the third culture stage, the light intensity was increased, and sodium nitrate slow-release particles loaded with anion exchange resin were added. This allowed the nitrogen source to be released more quickly as the pH of the culture medium rose naturally. This precisely matched the vigorous demand for nitrogen by algal cells in the later stages of growth, avoiding early inhibition and later depletion caused by a one-time addition of nitrogen source, thereby efficiently inducing protein synthesis.
[0015] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description
[0016] Figure 1 This is a flowchart of one embodiment of this application. Detailed Implementation
[0017] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.
[0018] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of components in a specific posture. If the specific posture changes, the directional indication will also change accordingly. When an element is referred to as "fixed to" or "set on" another element, it can be directly on the other element or may have an intervening element present. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. Descriptions involving "first," "second," etc., in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features.
[0019] It should be noted that the technical solutions of the various embodiments of this application can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this application.
[0020] In one embodiment, the cultivation of Spirulina can be carried out step by step in the following three stages. At the beginning of the first cultivation stage, the Spirulina inoculum needs to be inoculated into a basal culture medium. This basal culture medium contains four chemical substances: sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, and ferric sulfate. Sodium nitrate acts as a nitrogen source, providing nitrogen for Spirulina to synthesize proteins and nucleic acids; dipotassium hydrogen phosphate provides phosphorus and potassium; phosphorus is used to synthesize ATP and nucleic acids, and potassium maintains osmotic pressure; magnesium sulfate and ferric sulfate provide magnesium and iron, respectively. Magnesium is the central atom of chlorophyll molecules, and iron participates in the electron transport chain. After inoculation, the light intensity is set to a value between 5000 lux and 10000 lux, for example, 5000 lux, 8000 lux, or 10000 lux. The light is provided by LED plant growth lights or fluorescent lamps. Photoautotrophic cultivation is carried out under these light conditions, meaning that Spirulina uses light energy as its energy source to synthesize organic matter through photosynthesis without adding any exogenous organic carbon. During the cultivation process, samples are taken at regular intervals (e.g., every 6 hours or every 12 hours), and the optical density of the culture medium is measured at a wavelength of 560 nm using a spectrophotometer, recorded as OD560. When OD560 reaches 0.6, 0.7, or 0.8, it indicates that the algal cell concentration is suitable for entering the next stage, at which point the first cultivation stage is terminated. At the beginning of the second cultivation stage, the light intensity needs to be reduced, specifically to 1000 lux, 2000 lux, or 3000 lux. The purpose of reducing the light intensity is to avoid photoinhibition due to insufficient mutual shading between cells in the early stages of high-density cultivation. Calcium alginate-encapsulated sodium bicarbonate microspheres also need to be added to the culture medium. These microspheres release bicarbonate ions into the culture medium at a slow and relatively constant rate, serving as an additional carbon source. The total amount of sodium bicarbonate added is calculated based on the initial volume of the basal culture medium, and can be 5 g, 7 g, or 10 g per liter of medium. The microspheres can be added all at once or in batches. After adding microspheres, continue culturing, taking periodic samples to determine the dry weight of algal cells. The typical procedure for determining dry weight is as follows: take a certain volume of culture medium (e.g., 10 mL), vacuum filter it using a pre-dried and weighed filter membrane (e.g., a 0.45 μm mixed cellulose ester filter membrane), place the filter membrane along with the algal cells in an oven and dry it to constant weight at 80 ℃ to 105 ℃, then weigh and calculate the dry weight. The second culture stage ends when the measured algal cell dry weight reaches 1.5 g / L, 1.8 g / L, or 2.0 g / L. In the third culture stage, increase the light intensity, which can be set to 8000 lux, 10000 lux, or 12000 lux. Simultaneously, add anion exchange resin-loaded sodium nitrate slow-release particles to the culture medium. These particles gradually release nitrate ions during culture, supplementing the nitrogen source.The total amount of sodium nitrate added can be 1.5 g, 2.0 g, or 2.5 g per liter of basal culture medium. All granules can be added at once. Continue culturing until the desired endpoint is reached.
[0021] In existing technologies, spirulina cultivation often employs constant light and a one-time addition of sodium bicarbonate and sodium nitrate. For example, in an open raceway tank, all sodium bicarbonate and sodium nitrate are added at once during the initial cultivation phase, followed by continuous air circulation while maintaining constant light intensity. This method easily leads to excessively high nutrient concentrations in the early stages, inhibiting cell growth, and later, depletion of carbon or nitrogen sources, causing growth stagnation. Furthermore, constant light cannot match the light requirements of different growth stages. This embodiment dynamically adjusts light intensity in stages, using slow-release carbon and nitrogen sources in the second and third stages respectively. This ensures that the carbon and nitrogen supply rates match the absorption requirements of spirulina at different growth stages, while avoiding drastic pH fluctuations and osmotic pressure shocks. Compared to the aforementioned existing technologies, this embodiment can achieve higher final algal cell dry weight and protein content without additional equipment investment, and the cultivation process is more stable, less prone to cell aging or death.
[0022] In one embodiment, the concentrations of each component in the basal culture medium can be flexibly selected based on the Spirulina species, inoculation density, and water quality. The sodium nitrate concentration can be set to 2.0 g / L, 2.5 g / L, or 3.0 g / L. 2.0 g / L is suitable for algal strains with low initial inoculation density or low nitrogen requirements; 3.0 g / L is suitable for rapidly growing algal strains or those cultured under high light intensity. The dipotassium hydrogen phosphate concentration can be set to 0.4 g / L, 0.6 g / L, or 0.8 g / L. Dipotassium hydrogen phosphate provides both phosphorus and potassium; 0.4 g / L is suitable for use in soft water to prevent precipitation, while 0.8 g / L is suitable for use in hard water to ensure sufficient buffering capacity. The magnesium sulfate concentration can be set to 0.2 g / L, 0.35 g / L, or 0.5 g / L. Magnesium ions are a component of chlorophyll; 0.2 g / L is sufficient to meet basic requirements, while 0.5 g / L is suitable for high-light-intensity cultivation to enhance photosynthesis. Ferric sulfate concentrations can be set at 0.01 g / L, 0.03 g / L, or 0.05 g / L. Iron is a key component of iron-sulfur proteins and cytochromes in the photosynthetic electron transport chain. The above concentration ranges are the initial concentrations added all at once when preparing the basal culture medium; together, they provide a balanced nutritional environment for Spirulina. If the concentration of any component is below the lower limit of the above range, it may lead to a deficiency of the corresponding nutrient element and slow cell growth; if it is above the upper limit, it may cause an increase in osmotic pressure or ion antagonism, which may inhibit cell division.
[0023] In one embodiment, the culture time for the first culture stage can be set to 2 days, 3 days, or 4 days, depending on the initial inoculation density and light intensity. For example, if the OD560 is 0.1 after inoculation, it will take approximately 3 days to reach an OD560 of 0.6 at 5000 lux. The culture temperature can be controlled at 25 °C, 28 °C, or 30 °C. At lower temperatures (25 °C), cell division is slightly slower but metabolism is more stable, suitable for heat-sensitive algal strains; at higher temperatures (30 °C), the growth rate is faster, but care must be taken to prevent contamination by other microorganisms. The pH value can be maintained at 8.0, 8.5, or 9.0. The pH can be maintained by adding dilute hydrochloric acid or sodium hydroxide solution. pH 8.0 is suitable for cell viability recovery in the early stages of culture, while pH 9.0 is beneficial for inhibiting some protozoan contamination. The culture time for the second culture stage can be set to 2 days, 2.5 days, or 3 days. The culture temperature can be increased to 28 °C, 30 °C, or 32 °C. The purpose of increasing the temperature is to accelerate the catalytic rate of carbon fixation-related enzymes, as the carbon source supply is sufficient at this stage. The pH value can be controlled at 7.5, 8.0, or 8.5. The culture time for the third culture stage can be set to 1 day, 1.5 days, or 2 days. The culture temperature can be lowered back to 25 ℃, 28 ℃, or 30 ℃. Lowering the temperature helps reduce respiration consumption, allowing more carbon skeleton to be used for protein synthesis. The pH value can be controlled at 8.0, 8.5, or 9.0. A higher pH can promote the release of sodium nitrate from the anion exchange resin while inhibiting the growth of certain bacteria. The selection of these time, temperature, and pH parameters is based on the metabolic characteristics of Spirulina at different growth stages: a slightly lower temperature and neutral pH are suitable in the early stage to promote division, increasing the temperature in the middle stage can accelerate enzyme reactions, and restoring mild conditions in the later stage is conducive to protein accumulation.
[0024] In existing technologies, spirulina cultivation often employs constant temperature (e.g., 30 °C), constant pH (e.g., 8.5), and fixed aeration rates, without considering stage-specific changes. For example, air is introduced at 1 vvm throughout the entire process, without adjusting temperature and pH. This method ignores the impact of cell density variations on gas exchange and metabolic heat. This embodiment sets the cultivation time, temperature, pH, and aeration rate according to different stages, harmonizing environmental conditions with the physiological state of the cells. Compared to existing technologies, this embodiment can reduce photoinhibition and metabolic waste accumulation, improve the utilization efficiency of carbon and nitrogen sources, and ultimately obtain a higher biomass per unit volume.
[0025] In one embodiment, during the first cultivation stage, the initial light intensity can be set to 5000 lux, 5500 lux, or 6000 lux. For example, if the algal strain is acclimatized to low light, 5000 lux can be selected; if the algal strain has adapted to stronger light, 6000 lux can be selected. Afterward, the light intensity is increased every 24 hours of cultivation. Each increase can be 1000 lux, 1200 lux, or 1500 lux. The specific operation of increasing the light intensity can be achieved by adjusting the driving current of the LED lamps or changing the distance between the lamps and the cultivation container. For example, the initial light intensity on the first day is 5000 lux, increased to 6000 lux after 24 hours, then to 7500 lux after another 24 hours, and so on, gradually increasing until the final light intensity reaches 9000 lux, 9500 lux, or 10000 lux. This stepwise increase in light intensity allows the algal cells' photosynthetic system to gradually adapt to stronger light radiation, inducing the establishment of photoprotective mechanisms (such as non-photochemical quenching) and thus preventing damage to photosystem II caused by sudden high light intensity. Additionally, the optical density OD560 needs to be measured periodically during cultivation. When OD560 reaches 0.4, 0.45, or 0.5, it indicates that the algal cells have reached a certain population density, at which point the light-dark cycle can be switched from 12 hours of light and 12 hours of darkness to 16 hours of light and 8 hours of darkness. The switching of the light-dark cycle can be achieved through a programmable timer socket or the clock module of the cultivation controller. Specifically, on the day of the switch, extend the light exposure by 4 hours at the end of the light period, then maintain the 16-hour light-8-hour dark cycle. Extending the light duration can increase the total daily light energy input and promote the accumulation of photosynthetic products, but only if the cell density is high enough to utilize the extra energy generated by the extended light exposure without photoinhibition.
[0026] In existing technologies, the first culture stage often employs constant light intensity (e.g., 6000 lux) and a constant light-dark cycle (e.g., 12 h / 12 h), without considering changes in cell density. Thus, in the early stages of culture, excessive light inhibits cell growth; in the later stages, insufficient light limits the photosynthesis of high-density cells. This embodiment increases light intensity in a stepwise manner and extends the light duration after the cell density reaches a threshold, thereby simultaneously improving light energy supply and the cells' light tolerance and light requirements. Compared to existing technologies, this embodiment can shorten the time required to reach the target light density and increase the cell growth rate in the later stages.
[0027] In one embodiment, during the second culture phase, the initial light intensity can be set to 1000 lux, 1200 lux, or 1500 lux. For example, if the light intensity at the end of the first phase is 9000 lux, the light intensity is initially reduced to 1000 lux upon transitioning to the second phase to allow cells to recover from the stress of high light intensity and reduce photorespiration loss. Subsequently, the light intensity is increased every 12 hours of culture, with each increase being 300 lux, 400 lux, or 500 lux. For example, the first increase reaches 1300 lux, the second 1700 lux, and so on, until the final light intensity reaches 2500 lux, 2800 lux, or 3000 lux. The increase can be performed manually or automatically, triggered by a timer reaching a 12-hour interval. To enhance mixing of the culture medium and prevent algal cell sedimentation, ordinary air can be introduced, with the aeration rate kept constant at 0.3-0.5 vvm.
[0028] In one embodiment, the light intensity in the third cultivation stage can employ a pulsed illumination pattern. Pulsed illumination refers to the light intensity alternating between high and low levels according to a fixed time period, rather than remaining constant. Specifically, each pulse period can be set to 3 hours. Within each 3-hour period, the first 2 hours are irradiated with high light intensity, which can be set to 8000 lux, 10000 lux, or 12000 lux; the last hour is irradiated with low light intensity, which can be set to 2000 lux, 3000 lux, or 4000 lux. This cycle is then repeated until the end of the third cultivation stage. For example, high light intensity from hour 0 to hour 2, low light intensity from hour 2 to hour 3, high light intensity from hour 3 to hour 5, low light intensity from hour 5 to hour 6, and so on. One method to achieve pulsed illumination is to use a programmable LED driver, which controls the current of the LED string via a square wave signal output by a microcontroller. When the control signal is high, the LEDs operate at full current, outputting high light intensity; when the control signal is low, the LEDs operate at lower current or some LEDs are turned off, outputting low light intensity. The mechanism of this mode is as follows: In the high light intensity phase, photosystem II absorbs light energy to perform photolysis of water and electron transport, promoting the synthesis of ATP and NADPH; however, sustained high light intensity can damage D1 protein, leading to photoinhibition. Subsequently, in the low light intensity phase, the photosynthetic electron transport chain is relatively idle, and cells can initiate repair mechanisms to synthesize new D1 protein, while the dark reaction can utilize previously accumulated ATP and NADPH to fix carbon dioxide. Therefore, pulsed illumination can achieve a similar or even higher net photosynthetic rate under conditions where the average light intensity is much lower than that of constant high light intensity. Throughout the entire third culture phase, except for the illumination mode, other conditions such as culture temperature, pH, and aeration can remain constant.
[0029] In existing technologies, continuous irradiation with constant high light intensity (e.g., 10,000 lux) is typically used, as it is believed that high light intensity maximizes photosynthetic productivity. However, continuous high light intensity can cause the damage rate of D1 protein in photosystem II to exceed the repair rate, resulting in photoinhibition and ultimately reducing photosynthetic efficiency. This embodiment uses pulsed illumination, followed by a low-intensity recovery period after high light intensity, to allow photodamage to be repaired. Compared to existing technologies, this embodiment achieves a higher net photosynthetic rate under the same average light intensity, thereby increasing the final algal cell dry weight and protein content.
[0030] In one embodiment, calcium alginate-encapsulated sodium bicarbonate microspheres can be obtained by the following preparation method. First, prepare two solid powders: sodium alginate and sodium bicarbonate. Sodium alginate is a natural polysaccharide that forms a viscous colloid when dissolved in water. Upon encountering calcium ions, it instantly cross-links to form a water-insoluble gel network. Sodium bicarbonate, a water-soluble carbon source, is encapsulated within the gel network. The mass ratio of sodium alginate to sodium bicarbonate can be set to 1:1, 1.5:1, or 2:1. For example, take 10 g of sodium alginate and 10 g of sodium bicarbonate (mass ratio 1:1), or take 15 g of sodium alginate and 10 g of sodium bicarbonate (mass ratio 1.5:1). Dissolve both together in 1 L of deionized water and stir with a magnetic stirrer until completely dissolved to obtain a transparent mixed solution. Then, using a syringe pump (e.g., a peristaltic pump with a needle), the mixed solution is dripped dropwise at a constant flow rate (e.g., 5 mL per minute) into a calcium chloride solution with a mass fraction of 1.5%, 2.0%, or 2.5%. The calcium chloride solution is placed in a beaker, and a magnetic stir bar is placed at the bottom for slow stirring. Upon contact with the calcium chloride solution, the sodium ions in sodium alginate undergo ion exchange with the calcium ions, forming an insoluble calcium alginate gel, which then solidifies into microspheres. The solidification time can be set to 10 min, 20 min, or 30 min. After solidification, the microspheres are removed with a sieve and gently rinsed with deionized water to remove any residual calcium chloride, yielding the microsphere core. Next, the microspheres need to be coated to control the release rate. The microsphere core is immersed in an ethyl cellulose ethanol solution with a mass fraction of 1%, 2%, or 3%. Ethyl cellulose is a hydrophobic polymer, soluble in ethanol but insoluble in water, which can form a semi-permeable membrane on the surface of the microspheres. An ethyl cellulose ethanol solution containing microspheres is placed on a shaker and slowly rotated at a speed that can be set to 50 rpm, 75 rpm, or 100 rpm for 15 min, 20 min, or 30 min. Rotation ensures that the ethyl cellulose solution adheres evenly to the surface of the microspheres. After coating, the microspheres are removed and allowed to air dry at room temperature, allowing the ethanol to evaporate and the ethyl cellulose to solidify into a film, thus obtaining sustained-release microspheres coated with an ethyl cellulose semi-permeable membrane. The final microsphere particle size can be controlled at 2 mm, 3 mm, or 4 mm, which can be controlled by adjusting the needle inner diameter and injection flow rate. The thickness of the semi-permeable membrane can be controlled at 10 μm, 20 μm, or 30 μm, which can be controlled by adjusting the ethyl cellulose concentration and coating time. In the basal culture medium during the second culture stage, due to the presence of the ethyl cellulose semi-permeable membrane, water molecules can slowly permeate into the microspheres to dissolve sodium bicarbonate. The dissolved bicarbonate ions then diffuse into the culture medium through the semi-permeable membrane.The diffusion rate is determined by the concentration gradient across the membrane. Due to the large reserve of sodium bicarbonate inside the microspheres and the saturated concentration after dissolution, the release rate remains constant over a relatively long period, i.e., zero-order kinetic release. This constant release avoids a rapid increase in pH and osmotic pressure shock caused by excessively rapid initial release, and also avoids insufficient release in the later stages leading to carbon source shortage.
[0031] To verify the aforementioned sustained-release effect, the inventors conducted a simulated release experiment in deionized water without any chelating agents: uncoated calcium alginate / sodium bicarbonate microspheres (control group) and ethyl cellulose-coated sustained-release microspheres (experimental group, semi-permeable membrane thickness 20 μm) were prepared, and 10 g of each was placed in 500 mL of deionized water. The bicarbonate concentration in the culture medium was measured every 6 hours. The results showed that the control group released 78% of the total load within 12 hours, followed by a sharp decrease in the release rate, with a cumulative release rate of only 85% after 48 hours, exhibiting typical characteristics of rapid burst release followed by slow decay. The experimental group showed a linear release curve within 48 hours, with the cumulative release rate directly proportional to time, reaching 94% after 48 hours, without significant burst release or late-stage decay. Simultaneously, the pH of the control group surged from 7.5 to 9.2 within 12 hours, while the pH of the experimental group rose gradually from 7.5 to 8.5 within 48 hours, with significantly reduced fluctuations. This experiment confirms that coating with an ethyl cellulose semi-permeable membrane can effectively avoid drastic pH fluctuations and the problem of high initial and low subsequent carbon source supply. Therefore, the microspheres are expected to maintain the aforementioned stable release characteristics during actual cultivation.
[0032] In existing technologies, directly adding sodium bicarbonate powder or solution leads to excessively high initial concentrations and rapid depletion later; while using uncoated calcium alginate microspheres results in a decaying release rate over time, failing to provide a stable carbon supply. This embodiment utilizes microspheres coated with an ethyl cellulose semi-permeable membrane, achieving zero-order kinetic constant release. Compared to existing technologies, this embodiment maintains a stable bicarbonate ion concentration within a suitable range throughout the second culture stage, thereby precisely matching the carbon source supply to the growth rate of Spirulina, reducing carbon source waste and pH fluctuations.
[0033] In one embodiment, during the second culture phase, samples need to be taken every 12 hours to simultaneously determine the dry weight of the algal cells and the pH of the culture medium. Sampling should be performed under aseptic conditions, for example, using a sterile pipette in a laminar flow hood to aspirate 10 mL of culture medium from the culture container. The method for determining the dry weight can be as follows: Attach a 0.45 μm filter membrane of known weight to a vacuum filtration device, filter all 10 mL of culture medium, rinse the filter membrane with a small amount of deionized water to remove salts, then place the filter membrane in an aluminum foil box and dry it in an 80 °C oven to constant weight (usually requiring 24 hours). After removal, cool it to room temperature in a desiccator, weigh it using a 0.01 g / L balance, and subtract the initial weight of the filter membrane to obtain the dry weight. The pH value can be determined using a calibrated pH meter, with the electrode directly inserted into the culture medium to read the value, or immediately after sampling. The second culture phase is considered complete when the increase in dry weight in two consecutive measurements is less than 5%, and the second dry weight value reaches 1.5 g / L, 1.8 g / L, or 2.0 g / L, while the pH change in two consecutive measurements does not exceed 0.1. This determination method is triggered by timed sampling every 12 hours, and can be performed by laboratory personnel or automatically by an online sampling and analysis system.
[0034] This embodiment uses continuous monitoring of dry weight increase and pH change to objectively determine the timing of stage transitions. Compared with existing technologies, this embodiment ensures that the transition to the third stage occurs when cell growth stabilizes and carbon source supply just meets demand, thereby improving overall culture efficiency and final yield.
[0035] In one embodiment, the sodium nitrate slow-release particles supported on anion exchange resin possess a unique property: the rate at which the particles release sodium nitrate increases as the pH of the culture medium rises. These particles can be prepared using the following steps: First, a strongly basic anion exchange resin is selected, such as a styrene-divinylbenzene copolymer resin with quaternary ammonium groups, commonly known as 201×7 or 717. This resin has a high exchange capacity for nitrate ions. After thoroughly washing the resin with deionized water, it is immersed in a sodium nitrate solution with a concentration of 30 g / L, 40 g / L, or 50 g / L. Immersion can be carried out in an Erlenmeyer flask placed on a constant-temperature shaker at 25°C to 30°C, with shaking at 120 rpm for 12 h, 18 h, or 24 h. Shaking increases the contact between the resin and the sodium nitrate solution, ensuring sufficient exchange of nitrate ions onto the resin's exchange sites. After loading, the resin is washed 3 to 5 times with deionized water to remove unadsorbed sodium nitrate from the surface, yielding the sodium nitrate-loaded resin. Next, the resin needs to be coated to control the release rate's pH-dependent characteristics. The resin loaded with sodium nitrate is immersed in a chitosan-acetic acid solution with a mass fraction of 1%, 2%, or 3%. Chitosan is a cationic polysaccharide soluble in dilute acetic acid and forms a gel upon contact with an anionic crosslinking agent. The mixture is slowly stirred for 10, 15, or 20 minutes to ensure the chitosan solution is uniformly adsorbed onto the resin surface. Then, a sodium tripolyphosphate solution with a mass fraction of 0.5%, 1.0%, or 1.5% is added dropwise. Sodium tripolyphosphate carries multiple negative charges and can undergo an ionic crosslinking reaction with the amino cations on the chitosan molecular chain, thereby forming a water-insoluble chitosan coating layer on the resin surface. The crosslinking reaction can continue for 10 to 20 minutes. After the reaction is complete, the granules are removed, gently rinsed with deionized water, and then allowed to air dry at room temperature to obtain the sustained-release granules. In the third cultivation stage, algal cell metabolism produces alkaline substances, causing the pH of the culture medium to gradually rise from the initial 7.5-8.0 to 8.5-9.0. As the pH increases, the amino groups on the chitosan coating layer deprotonate, making the coating layer looser. Simultaneously, the exchange balance of the anion exchange resin is also affected by pH; at higher pH levels, the binding force between nitrate and the resin weakens. These two factors together lead to an accelerated release rate of sodium nitrate as the pH rises. This positive feedback characteristic perfectly matches the increasing nitrogen demand of spirulina in the later stages of growth, as the high cell density and vigorous protein synthesis require more nitrogen.
[0036] To verify the above mechanism, the applicant conducted a simulated release experiment: sodium nitrate slow-release particles loaded on anion exchange resin prepared using the same method were placed in phosphate buffer solutions at pH 7.5, 8.0, 8.5, and 9.0 (temperature 28 ℃, no algal cells), and samples were taken every 2 hours to measure the concentration of nitrate released into the solution. The results showed that at pH 7.5, the cumulative release rate was 42.5% over 48 hours; at pH 8.0, the cumulative release rate was 58.7%; at pH 8.5, the cumulative release rate reached 76.3%; and at pH 9.0, the cumulative release rate was as high as 89.2%. No obvious burst release phenomenon was observed in the release curves at any pH condition; the release rate gradually increased over time, exhibiting positive feedback characteristics. The experiment confirmed that the slow-release particles of this embodiment can achieve pH-dependent accelerated release within the typical pH range (8.0-9.0) of Spirulina culture, and the release behavior is stable and controllable, avoiding osmotic stress caused by initial nitrogen burst release while ensuring a sufficient supply of nitrogen in the later stages of growth. In a buffer solution without algae cells, the cumulative release rate reached 89.2% after 48 hours at pH 9.0. It should be noted that in a real culture system, Spirulina continuously absorbs nitrate, and the nitrate concentration in the culture medium remains in dynamic equilibrium and will not accumulate to a high concentration.
[0037] In existing technologies, sodium nitrate is typically added all at once or using uncoated ion exchange resins, resulting in a constant release rate or a rate that decreases with decreasing concentration, failing to meet the increasing nitrogen demand in later stages. This embodiment achieves a positive feedback characteristic where the release rate increases with pH through the synergistic effect of the chitosan coating layer and the ion exchange resin. Compared to existing technologies, this embodiment can automatically provide more sodium nitrate during the third culture stage when algal cell density is high and metabolism is vigorous, while releasing it more slowly in the initial stages when demand is lower. This avoids nitrogen waste and ammonia accumulation, effectively promoting protein synthesis.
[0038] The following is a description of a specific embodiment.
[0039] The sodium alginate, calcium chloride, ethyl cellulose, chitosan, sodium tripolyphosphate, and other excipients used in the experimental and control groups below were all food-grade. The anion exchange resin underwent rigorous cleaning and pretreatment before use to remove any residual monomers or impurities, ensuring it meets the safety standards for food contact materials.
[0040] Experimental group: The basic culture medium formula is as follows: sodium nitrate 2.5 g / L, dipotassium hydrogen phosphate 0.6 g / L, magnesium sulfate 0.35 g / L, ferric sulfate 0.03 g / L, prepared with deionized water and adjusted to pH 8.5.
[0041] First cultivation stage: Spirulina inoculum was inoculated into the above-mentioned basal culture medium. The initial light intensity was set at 5500 lux (LED light source), and the light intensity was increased by 1000 lux every 24 hours until it reached 9500 lux. The initial light-dark cycle was 12 hours light / 12 hours dark. When the optical density (OD560) of the culture medium reached 0.5, the light-dark cycle was switched to 16 hours light / 8 hours dark. The cultivation temperature was controlled at 28 ℃, and ordinary air was continuously circulated (0.3 vvm). The pH was maintained at 8.5 (adjusted by adding dilute hydrochloric acid). The cultivation time was 3 days, and the OD560 was 0.7 at the end.
[0042] Second cultivation stage: The light intensity was reduced to 1200 lux, then increased by 400 lux every 12 hours until it reached 2800 lux. Sodium bicarbonate microspheres were embedded with calcium alginate, with a total sodium bicarbonate addition of 7 g / L. The microsphere preparation parameters were: sodium alginate to sodium bicarbonate mass ratio 1.5:1, calcium chloride mass fraction 2.0%, curing time 20 min, ethyl cellulose mass fraction 2%, coating speed 75 rpm, coating time 20 min, microsphere particle size 3 mm, and semi-permeable membrane thickness 20 μm. The cultivation temperature was 30 ℃, and the pH was controlled at 8.0 (fine-tuned by adding dilute hydrochloric acid or sodium hydroxide solution). Only ordinary air (0.5 vvm) was introduced during this stage to maintain mixing. Every 12 hours during cultivation, samples were taken to measure the dry weight of algal cells and the pH value. The second stage ended when two consecutive increases in dry weight were less than 5%, the second dry weight reached 1.8 g / L, and two consecutive pH changes did not exceed 0.1. After 2.5 days of actual cultivation, the conditions were met, and the dry weight at the end was 1.82 g / L.
[0043] Third cultivation stage: Ordinary air was introduced (aeration rate 0.6 vvm). Pulsed illumination was used: each cycle was 3 hours, with the first 2 hours at 10000 lux and the last hour at 3000 lux, repeated cyclically. Sodium nitrate slow-release particles were added to anion exchange resin, with a total sodium nitrate addition of 2.0 g / L. The resin preparation parameters were as follows: a strongly basic anion exchange resin (type 201×7) was shaken and loaded in a 40 g / L sodium nitrate solution for 18 hours, then immersed in a 2% (w / w) chitosan acetate solution and stirred for 15 minutes, followed by dropwise addition of a 1.0% (w / w) sodium tripolyphosphate solution for crosslinking for 10 minutes, and then dried at room temperature. The cultivation temperature was 28℃, and the pH was maintained at 8.5. Cultivation was completed after 1.5 days.
[0044] Post-processing: After cultivation, the algal culture solution was filtered through a 100-mesh stainless steel sieve to separate and remove anion exchange resin slow-release particles with a particle size of 2-4 mm and any remaining calcium alginate microspheres. The filtrate was transferred to centrifuge tubes and centrifuged at 8000 rpm for 10 min. The supernatant was discarded, and the algal cell precipitate was collected. Two volumes (v / w) of deionized water were added to the precipitate, and the mixture was resuspended and washed for 5 min, followed by centrifugation again. This washing process was repeated twice. The algal cell precipitate after the final wash was resuspended in deionized water to the original culture volume, mixed thoroughly, and the purified algal culture solution was obtained for subsequent parameter determination.
[0045] Control Group 1: No distinct culture stages were observed. A constant light intensity of 6000 lux and a 12-h / 12-h light / dark cycle were maintained throughout the entire culture. Sodium bicarbonate (7 g / L) and sodium nitrate (2.5 g / L) were added once, and ordinary air was continuously circulated. No slow-release microspheres or particles were added. The culture temperature was maintained at a constant 30 ℃, and the pH was maintained at 8.0 by adding acid and alkali. The total culture time was the same as the experimental group (7 days).
[0046] Control group 2: The phased light and temperature parameters were exactly the same as the experimental group, but in the second culture phase, calcium alginate was not added to embed sodium bicarbonate microspheres. The carbon source was provided only by introducing air containing 2% carbon dioxide (air flow rate 0.8~1.2 vvm). The rest of the operation was the same as the experimental group.
[0047] Control group 3: The phased light and temperature parameters were exactly the same as the experimental group, but instead of adding anion exchange resin-supported sodium nitrate slow-release particles in the third culture stage, sodium nitrate 2.0 g / L was added all at once at the beginning of the third culture stage. The rest of the operation was the same as the experimental group.
[0048] Control Group 4: After the first culture stage, the microspheres directly entered the third culture stage, skipping the second stage. This meant they did not experience low light conditions or the addition of calcium alginate to encapsulate sodium bicarbonate microspheres. At the end of the first culture stage, the OD560 was 0.7, and the dry weight was approximately 0.6 g / L. They were then cultured directly under the same conditions as the experimental group in the third culture stage (pulsed light, addition of a slow-release nitrogen source). The total culture time was the same as the experimental group.
[0049] Control Group 5: After the first and second culture stages, no third culture stage was performed. That is, culture was terminated at the end of the second culture stage (dry weight 1.82 g / L), without the addition of high-intensity light pulses and slow-release nitrogen sources. The total culture time was 5.5 days. To maintain consistency with the experimental group's total time of 7 days, culture was continued under the same conditions until day 7 after the second culture stage, and the values measured on day 7 were used as the standard.
[0050] Algal cell dry weight determination: Take 50 mL of culture medium and vacuum filter it through a pre-dried and weighed 0.45 μm mixed cellulose ester filter membrane. Rinse the filter membrane twice with deionized water. Place the filter membrane and algal cells in an oven and dry at 80 ℃ until constant weight (about 24 h). Remove and cool to room temperature in a desiccator. Weigh the cells using a 0.01 g balance. Subtract the initial weight of the filter membrane to obtain the algal cell dry weight, in g / L. Three parallel samples were set up for each group, and the average value was taken.
[0051] Protein content determination: The Kjeldahl method was used. 0.5 g of dried algal powder was digested with concentrated sulfuric acid and a catalyst. The total nitrogen content was determined using a Kjeldahl nitrogen analyzer, and multiplied by a protein conversion factor of 6.25 to obtain the protein content (percentage of dry weight of algal powder). Three parallel samples were used for each group, and the average value was taken.
[0052] Table 1 Performance Parameter Comparison Table The comparative data above show that the final dry weight of algal cells in the experimental group reached 2.53 g / L, significantly higher than all control groups. In terms of protein content, the experimental group reached 62.4%, also superior to all control groups.
[0053] Control group 1, using a traditional method of adding carbon and nitrogen sources in a single step and maintaining constant light, experienced osmotic stress due to excessively high nutrient concentrations in the initial stage. Subsequently, both carbon and nitrogen sources were depleted, resulting in the lowest dry weight and protein content. This indicates that phased dynamic regulation and slow-release of carbon and nitrogen sources are crucial for increasing yield.
[0054] Control group 2 lacked a slow-release carbon source and relied solely on the introduction of carbon dioxide-containing air for carbon supply. Although carbon dioxide can provide carbon, the short residence time of the bubbles generated by the airflow results in limited carbon dioxide dissolution efficiency and significant pH fluctuations, leading to unstable carbon supply and a significant decrease in dry weight and protein content compared to the experimental group. This indicates that a slow-release carbon source can provide a stable and lasting supply of bicarbonate ions, preventing carbon starvation.
[0055] Control group 3 lacked a slow-release nitrogen source, and sodium nitrate was added all at once in the third stage. Although the nitrogen source was sufficient initially, as the culture progressed, the sodium nitrate was rapidly absorbed and its concentration decreased, leading to insufficient nitrogen supply in the later stages. This limited further protein synthesis, resulting in a protein content of only 53.1%, approximately 9 percentage points lower than the experimental group. This indicates that a slow-release nitrogen source, which accelerates its release with increasing pH, can precisely match the vigorous nitrogen demand in the later stages.
[0056] Control group 4 lacked the second culture phase, meaning it did not experience the intermediate transition period of low light and slow-release carbon source supplementation. After the first culture phase, it directly entered the high-light pulse and slow-release nitrogen source addition phase. Because the cells had not yet accumulated sufficient biomass and lacked adequate carbon source support, subsequent growth was weak, and the final dry weight was only 1.33 g / L, even lower than control group 1. This indicates that the second culture phase, as a carbon source supplementation and cell adaptation period, is indispensable and plays a crucial role in bridging the gap between the first and second stages.
[0057] Control group 5 lacked the third culture phase; that is, after carbon source supplementation and cell density enhancement, no high-intensity pulsed light and slow-release nitrogen were added. Although the dry weight reached 1.82 g / L at the end of the second culture phase, subsequent nitrogen depletion and lack of light energy optimization led to cell death, with the dry weight decreasing to 1.67 g / L by day 7, and protein content also being low. This indicates that the third culture phase is crucial for further promoting biomass accumulation and protein synthesis, and the synergistic effect of pulsed light and slow-release nitrogen can enable cells to break through the original growth plateau.
[0058] In summary, the experimental group did not simply sum up the characteristics, but rather achieved a progressively enhanced cultivation of Spirulina through the sequential coordination of three stages and the dynamic synergy of light and carbon / nitrogen sources. Each stage is the foundation for the subsequent stages, and the absence of any stage will lead to a significant decrease in the final yield and protein content. The experimental group showed a clear advantage over all control groups, indicating that the technical solution of this invention represents a substantial improvement over existing technologies.
[0059] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.
Claims
1. A method for culturing Spirulina, characterized in that, include: S1: In the first culture stage, Spirulina was inoculated into a basic culture medium composed of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, and ferric sulfate, and photoautotrophic culture was carried out under a light intensity of 5000-10000 lux until the optical density OD560 reached 0.6-0.
8. S2: In the second culture stage, the light intensity is reduced to 1000-3000 lux, and calcium alginate is added to embed sodium bicarbonate microspheres. The total amount of sodium bicarbonate added is 5-10 g / L of basal culture medium. The culture is carried out until the dry weight of algal cells reaches 1.5-2.0 g / L. S3: In the third culture stage, the light intensity is increased to 8000-12000 lux, and sodium nitrate slow-release particles loaded with anion exchange resin are added, with the total amount of sodium nitrate added being 1.5-2.5 g / L of basal culture medium; Calcium alginate-encapsulated sodium bicarbonate microspheres were prepared by the following method: Sodium alginate and sodium bicarbonate were dissolved in deionized water at a mass ratio of (1-2):1 to obtain a mixed solution. The mixed solution was then dripped into a calcium chloride solution with a mass fraction of 1.5-2.5% using a syringe pump and solidified for 10-30 minutes to form microsphere cores. The microsphere cores were then immersed in an ethyl cellulose ethanol solution with a mass fraction of 1-3% and coated at 50-100 rpm for 15-30 minutes. After removal, the microspheres were dried at room temperature to obtain sustained-release microspheres with a surface coated with an ethyl cellulose semi-permeable membrane. The sustained-release microspheres have a particle size of 2-4 mm and a semi-permeable membrane thickness of 10-30 μm.
2. The method for culturing Spirulina according to claim 1, characterized in that, The concentrations of each component in the basal culture medium are as follows: sodium nitrate 2.0-3.0 g / L, dipotassium hydrogen phosphate 0.4-0.8 g / L, magnesium sulfate 0.2-0.5 g / L, and ferric sulfate 0.01-0.05 g / L.
3. The method for culturing Spirulina according to claim 1, characterized in that, The first culture stage lasts for 2-4 days, with a culture temperature of 25-30 ℃ and a pH of 8.0-9.
0. The second culture stage lasts for 2-3 days, with a culture temperature of 28-32 ℃ and a pH of 7.5-8.
5. The third culture stage lasts for 1-2 days, with a culture temperature of 25-30℃ and a pH of 8.0-9.
0.
4. The method for culturing Spirulina according to claim 3, characterized in that, In the first cultivation stage, the initial light intensity was 5000-6000 lux. Then, the light intensity was increased by 1000-1500 lux every 24 hours of cultivation until it reached 9000-10000 lux. When the light density OD560 reached 0.4-0.5, the light-dark cycle was switched from 12 h:12 h to 16 h:8 h.
5. The method for culturing Spirulina according to claim 3, characterized in that, In the second cultivation stage, the initial light intensity was 1000-1500 lux, and then the light intensity was increased by 300-500 lux every 12 hours until it reached 2500-3000 lux.
6. The method for culturing Spirulina according to claim 3, characterized in that, In the third cultivation stage, the light intensity adopts a pulsed light mode: each pulse cycle is 3 hours, with the first 2 hours irradiated with a high light intensity of 8000-12000 lux, and the last hour irradiated with a low light intensity of 2000-4000 lux, and the cycle is repeated.
7. The method for culturing Spirulina according to claim 3, characterized in that, During the second culture phase, the dry weight of algal cells was measured every 12 hours, and the pH value was measured at the same time. The second culture phase was considered to be over when the increase in dry weight in two consecutive measurements was less than 5%, the dry weight value in the second measurement reached 1.5-2.0 g / L, and the pH value change in two consecutive measurements did not exceed 0.
1.
8. The method for culturing Spirulina according to claim 3, characterized in that, The rate of sodium nitrate release from anion exchange resin-supported slow-release granules increases with increasing pH. The preparation method of sodium nitrate sustained-release particles supported on anion exchange resin includes: immersing a strongly basic anion exchange resin in a sodium nitrate solution with a concentration of 30-50 g / L, shaking and loading at 25-30℃ for 12-24 hours, filtering, and washing with deionized water to obtain the sodium nitrate-loaded resin; immersing the sodium nitrate-loaded resin in a chitosan acetate solution with a mass fraction of 1-3%, stirring slowly for 10-20 minutes, then adding a sodium tripolyphosphate solution with a mass fraction of 0.5-1.5% for ionic cross-linking to form a chitosan coating layer, removing and drying at room temperature to obtain the sodium nitrate sustained-release particles supported on anion exchange resin.