A system and cultivation method for the dual-trophic culture of Chlorella proteoglycans

Abstract

This invention provides a system and method for the co-trophic cultivation of Chlorella proteoglycans. The system includes an aeration unit, a cultivation unit, and a lighting unit. The aeration unit includes a carbon dioxide cylinder, a mixing tank, and a porous distributor. The carbon dioxide cylinder is connected to the mixing tank via a gas pipe, and an adjustable gas pump is installed on the gas pipe. The cultivation unit includes 1 to 20 columnar reactors made of transparent material. The lighting unit includes LED light columns and a photoelectric control system. The LED light columns are installed within a light column frame, and casters are installed at the bottom of the light column frame to adjust the relative position of the light source and the cultivation unit. This invention provides a high biomass cultivation system and method for Chlorella proteoglycans in a co-trophic cultivation mode, and has the technical advantages of precise light and carbon regulation, simple operation, low economic investment, and short cultivation cycle.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a microalgae omniculture system and a matching culture method. Background Technology

[0002] Chlorella pyrenoidosa is a ubiquitous single-celled green alga belonging to the Chlorella genus of the Chlorophyta phylum. It is a spherical freshwater algae with dual attributes of plants and microorganisms. It has high photosynthetic efficiency, strong carbon fixation ability, and short growth cycle. It is rich in nutrients such as protein, vitamins, chlorophyll, and unsaturated fatty acids, and has been widely used in many fields such as biofuel, natural pigments, and feed.

[0003] Microalgae primarily exhibit three nutritional modes: autotrophic, heterotrophic, and multitrophic. In photosynthetic autotrophy, microalgae use carbon dioxide as their carbon source and light as their energy source, synthesizing organic matter and energy by fixing inorganic carbon. Most microalgae are photosynthetic autotrophs. Microalgal heterotrophy refers to microalgae growing entirely in a dark environment, relying solely on organic carbon sources for both carbon and energy. Multitrophic microalgae cultivation combines autotrophic and heterotrophic metabolic capabilities, utilizing light and CO2 as energy and carbon sources for photosynthesis while simultaneously using organic carbon as a carbon source and energy source for heterotrophic growth—a mixed nutritional model. Currently, most commercial-scale microalgae cultivation employs photosynthetic autotrophic production, which is highly dependent on light conditions and CO2 supply, resulting in low biomass yield, long growth cycles, and extremely unstable cultivation returns. While completely dark fermenter-based heterotrophic microalgae cultivation can achieve high-density cultivation, it involves significant equipment investment, high energy consumption, poor pollution resistance, and extremely high cultivation costs. Microalgal megatrophic culture combines the advantages of autotrophic and heterotrophic processes, simultaneously generating ATP through photophosphorylation and oxidative phosphorylation, and synthesizing the carbon skeleton from inorganic and organic carbon. The synergistic mechanism of photochemical processes and organic carbon metabolism significantly reduces microalgae's dependence on light and makes them less susceptible to limitations imposed by CO2 or organic carbon concentrations, offering greater flexibility in energy and carbon source acquisition compared to autotrophic and heterotrophic methods. Due to these synergistic advantages, megatrophy is currently the microalgal culture method with the highest known biomass.

[0004] While microalgal métrophic culture can achieve large-scale biomass accumulation in a short period, high-density algal solutions can cause a self-shading effect, reducing the average received irradiance and photosynthetic efficiency, thus lowering light energy utilization efficiency. Furthermore, existing culture devices often have incompatible reactors and aeration systems, failing to achieve uniform mixing of air, algae, and nutrients, resulting in inefficient utilization of organic matter and hindering high biomass growth. Meanwhile, light and carbon are the decisive factors for energy generation and carbon skeleton formation in microalgal métrophic culture, respectively, especially in environments with high light attenuation. The interaction between light and carbon creates a synergistic effect of oxidative phosphorylation and photosynthetic phosphorylation, ultimately promoting efficient métrophic growth of microalgae. However, research on the optimal light-to-carbon ratio in microalgal métrophic culture is still insufficient. These are all problems that need to be addressed in the métrophic culture of microalgae. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention designs a culture system for Chlorella proteoglycans in a co-trophic mode, and proposes a system for the co-trophic culture of Chlorella proteoglycans.

[0006] This invention verifies the optimal photoperiod, light intensity, and carbon source ratio through numerous experiments, optimizes the corresponding culture methods, and proposes a culture method for the dual-trophic culture of Chlorella proteoglycans.

[0007] The technical solution for achieving the above-mentioned objective of this invention is as follows:

[0008] A system for the dual-trophic culture of Chlorella proteoglycans, the system comprising an aeration unit, a culture unit, and a lighting unit.

[0009] The aeration unit includes a carbon dioxide cylinder, a gas mixing tank, and a porous distributor; the carbon dioxide cylinder is connected to the gas mixing tank via a gas pipe, and an air pump with an adjustable gas output is installed on the gas pipe; a filter is installed at the outlet of the gas mixing tank; the outlet of the gas mixing tank is connected to the culture unit via a gas delivery pipe.

[0010] The culture unit includes 1 to 20 column reactors, which are made of transparent material;

[0011] The illumination unit includes LED light columns and a photoelectric control system. The LED light columns are installed inside the light column frame, and casters are installed at the bottom of the light column frame to adjust the relative position of the light source and the cultivation unit.

[0012] Furthermore, the filter is a vacuum filter, and the filter element inside the filter is a 0.1-0.4μm filter element.

[0013] A pore size of less than 0.4μm is sufficient to meet filtration requirements, and a pore size of 0.2 to 0.3μm is preferred for filter elements.

[0014] A vacuum filter with a pore size of 0.1–0.4 μm is installed at the outlet of the gas mixing tank. This effectively blocks impurities and bacteria in the mixed gas, preventing contamination of microalgae by miscellaneous bacteria in the gas. And / or

[0015] The illumination unit uses stepless dimming LED warm light.

[0016] When microalgal biomass is low, light penetration is strong, leading to photoinhibition damage to the microalgal photosynthetic system. As the microalgal biomass gradually increases to higher concentrations, a light attenuation effect begins to appear due to the self-shading effect between algal cells. Microalgae farther from the light source receive less light energy, resulting in a decrease in photosynthetic efficiency. Therefore, stepless dimming LED warm light lamps are preferred to meet the different light source requirements of microalgae at different growth stages, which is more conducive to microalgal growth and the accumulation of active substances.

[0017] The gas mixing tank has a gas outlet connected to a perforated distributor and a flow meter. The gas delivery pipe is connected to the perforated distributor. The perforated distributor has multiple independent gas outlets, each of which is connected to a gas delivery pipe. Each gas delivery pipe is connected to the top of each columnar reactor, and a flow meter is installed on the gas delivery pipe. The gas delivery pipe extends from the top of the reactor and exits from the bottom. A fixing bolt is installed at the end of the gas delivery pipe to fix the end of the gas delivery pipe to the bottom of the columnar reactor.

[0018] Specifically, the gas supply pipe is inserted through the air inlet hole of the plexiglass cover and then through to the cold-plated fixing bolt at the bottom of the cone-shaped reactor to fix the air outlet, and then aeration is carried out; a cold-plated steel round hole fixing bolt is set 1-2cm above the bottom of the reactor, and the gas supply pipe is inserted into the round hole of the fixing bolt to stabilize the aeration position and prevent it from being disturbed by the aerated airflow.

[0019] The main body of the columnar reactor is cylindrical, and the lower part of the cylinder is conical; the height-to-diameter ratio of the cylindrical part is 7 to 9:1.

[0020] More preferably, the cylindrical and conical portions of the columnar reactor are made of glass, and the reactor top cover is made of plexiglass. The plexiglass cover is provided with an air inlet and an air outlet. Because the input gas will not be fully utilized, an air outlet is needed to discharge excess gas. More preferably, a filter membrane is installed on the air outlet to reduce losses.

[0021] Based on the optimization and comparison of aeration conditions and effective culture volume, and through extensive experimental verification, the optimal height-to-diameter ratio of the reactor was determined to be 7–9:1. This design achieves a throughput of 1 L·min -1 Under aeration conditions, algae are thoroughly mixed with gases and nutrients. High-density algae alternately contact the wall surface to absorb photon energy, improving the efficiency of carbon source and light energy utilization, and promoting the growth of microalgae and the accumulation of active substances.

[0022] In this invention, the reactor body is preferably made of 3.3 glass, a clear, transparent, colorless glass with extremely low ferrous ion content. It exhibits excellent transmittance in the ultraviolet and visible-near-infrared light range, making it an ideal material for light-transmitting instruments, floodlights, and light-illuminating experimental equipment. Using 3.3 glass as the reactor wall material achieves high light transmittance, improves light energy utilization efficiency, and promotes the further growth of high-concentration microalgae.

[0023] A method for the dual-trophic culture of Chlorella proteoglycans includes the following steps:

[0024] (1) Add Chlorella proteoglycans in the logarithmic growth phase to a column reactor along with BG11 medium, and add 5-30 g·L glucose. -1 The biomass density of *Chlorella proteoglycans* in the early stage of cultivation was 0.08–0.15 g·L⁻¹. -1 The incubation temperature is 24–28℃;

[0025] (2) Turn on the carbon dioxide cylinder and adjustable air pump, control the CO2 content to 1-6%, mix the CO2 with air in the mixing tank, and adjust the aeration rate of the reactor body to 0.8-1.2 L / min using a glass rotor flow meter. -1 This ensures that the algae are evenly mixed with the culture medium without precipitation.

[0026] (3) Move the lamp post frame to position so that the light source is directly facing the reactor body, with a spacing of 18–22 mm and a light intensity of 80–150 μmol·m. -2 ·s -1 To conduct commensal culture of Chlorella proteoglycans.

[0027] Preferably, in step (1), the amount of glucose added is 10-15 g·L. -1 The biomass density of *Chlorella proteoglycans* during the initial culture stage was 0.10–0.12 g·L⁻¹. -1 .

[0028] In step (2), the CO2 content is controlled at 4-6%, and no CO2 is supplied at night.

[0029] In step (2), the aeration rate is 1 L·min. -1 Under these conditions, the CO2 content is controlled at 4-5%. The gas flow ratio of a single reactor can be 0.1-0.5 vvm.

[0030] In step (3), the light intensity is controlled to be 100-120 μmol·m. -2 ·s -1 The light exposure time ranges from 15 to 17 hours, while the corresponding darkness time ranges from 9 to 7 hours.

[0031] The following is a preferred embodiment of the present invention: the method for the dual-trophic culture of Chlorella proteoglycans includes the following steps:

[0032] (1) Add Chlorella proteoglycans in the logarithmic growth phase to a column reactor along with BG11 medium, and add 11-13 g·L⁻¹ of glucose. -1 The biomass density of *Chlorella proteoglycans* in the early stage of cultivation was 0.10–0.11 g·L⁻¹. -1 The temperature is 25-27℃;

[0033] (2) Turn on the carbon dioxide cylinder and adjustable air pump, control the CO2 content to 4-5%, mix the CO2 with air in the mixing tank, and adjust the aeration rate of the reactor body to 1 L·min using a glass rotor flow meter. -1 This ensures that the algae are evenly mixed with the culture medium without precipitation.

[0034] (3) Move the lamp post frame to position it so that the light source is directly facing the reactor body, with a spacing of 18-22 mm. Adjust the light intensity to 100-120 μmol·m using the light control. -2 ·s -1 The light duration is 15–17 hours for the co-trophic culture of Chlorella proteoglycans.

[0035] (4) The culture time is 11 to 15 days. The growth curve is plotted based on the growth of Chlorella proteoglycans. Microalgal biomass is obtained during the plateau period and then transferred to an outdoor culture reactor for further expansion.

[0036] The beneficial effects of this invention are as follows:

[0037] To address the problems of poor aeration, low efficiency of light and carbon source utilization, and insignificant biomass growth under mixed-culture conditions in existing microalgae cultivation systems and methods, this invention aims to provide a low-cost, high-yield, simple, and widely applicable cultivation system for Chlorella proteoglycans under mixed-culture conditions, along with a matching cultivation method.

[0038] This invention provides a high biomass culture system and method for Chlorella proteoglycans in a mixed-culture mode, which has the technical advantages of precise light and carbon regulation, simple operation, low economic investment and short culture cycle.

[0039] This invention focuses on key technologies for the co-culture of Chlorella proteoglycans, investigating metabolic mechanisms, carbon source supply, and light conditions. Extensive experiments were conducted to verify the optimal photoperiod, light source, and carbon source ratio, achieving high biomass growth of Chlorella proteoglycans under synergistic light-carbon effects. Ultimately, the optimal aeration conditions, CO2 ratio, and glucose dosage of 15-20 g·L⁻¹ were determined. -1That is, the carbon content is 6-8 g·L -1 Photoperiod, etc. Under the optimal conditions proposed in this invention, *Chlorella proteoglycans* can achieve optimal growth. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the aquaculture system structure;

[0041] Figure 2 This is a schematic diagram of the lighting unit structure;

[0042] Figure 3 A top-view diagram showing the relative positions of the culture unit and the illumination unit;

[0043] Figure 4 This is a top view of the plexiglass lid of a columnar reactor.

[0044] The correspondence between components and their numbers in the diagram is as follows:

[0045] 1. Carbon dioxide cylinder; 2. Pressure reducing valve; 3. Air pump; 4. Plastic gas tubing; 5. Gas mixing tank; 6. Vacuum filter; 7. Porous distributor; 8. Silicone tubing; 9. Flow meter; 10. Cold-plated fixing bolts.

[0046] Columnar reactor 11, plexiglass cover 12, upper perforated groove 13, air pipe fixing hole 14, upper support plate 15, PMMA glass bracket 16, sampling valve 17, lower support plate 18, lower perforated groove 19, rotating screw 20, warm light lamp column 21, LED bulbs constituting the lamp column 22, lighting control 23, casters 24, lamp column frame 25, hollow crossbeam 26, air inlet 27, 28 are 0.2μm air filter membranes, exhaust port 29. Detailed Implementation

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0048] Unless otherwise specified, all methods used in this specification are existing techniques in the field.

[0049] As described in the background section, algal cultures with high biomass growth exhibit a self-shading effect, resulting in lower average received irradiance and reduced photosynthetic efficiency, thus lowering light energy utilization efficiency. Existing cultivation devices often have mismatched reactors and aeration systems, failing to achieve uniform mixing of air, algae, and nutrients, leading to inefficient utilization of organic matter. Furthermore, light and carbon are the decisive factors for energy generation and carbon skeleton formation in microalgae co-culture, respectively, especially in environments with high light attenuation. However, research on the optimal ratio of light intensity to carbon source in the co-culture of microalgae is still insufficient. Therefore, this invention proposes a cultivation system and matching cultivation method for the co-culture of Chlorella proteoglycans, which will be further described below with reference to the accompanying drawings and specific embodiments.

[0050] The *Chlorella pyrenoidosa* strain used in this embodiment was isolated and purified from the water of Xianlin Lake in Qixia District, Nanjing, and deposited on March 14, 2023, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO.45509.

[0051] Test case

[0052] To explore the optimal aeration conditions, several columnar reactors with height-to-diameter ratios ranging from 5 to 12:1 were designed based on experimental requirements. Aeration was conducted using silicone tubing with an outer diameter of 10 mm, resulting in bubble diameters of approximately 0.2–0.6 mm. By examining the bubble buoyancy and the degree of mixing between algae and the culture medium during aeration, it was found that when the reactor's height-to-diameter ratio was between 7 and 9:1, the bubbles achieved uniform, straight upward movement, with most of the algae remaining suspended. Furthermore, there was no adhesion to the reactor walls or air flotation, indicating an ideal aeration state.

[0053] Subsequent experiments used a column reactor with a height-to-diameter ratio of 8:1.

[0054] Example 1

[0055] This embodiment proposes a system for the dual-trophic culture of Chlorella proteoglycans, which includes three units: an aeration unit, a culture unit, and a lighting unit.

[0056] See Figure 1The aeration unit includes a carbon dioxide cylinder 1, a mixing tank 5, and a porous distributor 7. The carbon dioxide cylinder is connected to the mixing tank 5 via a gas pipe, and an adjustable gas pump 3 is installed on the gas pipe. A vacuum filter 6 is installed at the outlet of the mixing tank. The outlet of the mixing tank 5 is connected to the culture unit via a gas delivery pipe. In this embodiment, a silicone tubing 8 is used as the gas delivery pipe. In this embodiment, the gas pump 3 is a 40W AC / DC gas pump with a knob for adjusting the gas output, which can be used to adjust the aeration rate as needed.

[0057] The culture unit includes 1 to 10 column reactors 11, which are made of transparent material: the cylindrical and conical parts are made of 3.3 glass, and the top cover of the reactor is made of plexiglass; specifically in this embodiment, a total of 10 reactors are provided.

[0058] See Figure 2 The illumination unit includes an LED lamp post and a photoelectric control system. The LED lamp post is installed within a lamp post frame, and casters 24 are installed at the bottom of the lamp post frame to adjust the relative position of the light source and the culture unit. A rotating screw 20 is used to rotate the lamp post, which is cylindrical with two sides; the front side is illuminated, and the back side is a protective shell. Rotation via the bottom rotating screw 20 adjusts the position of the light source irradiating the reactor. In this embodiment, the illumination unit uses a stepless dimming LED warm light lamp post 21, equipped with LED bulbs 22 that make up the lamp post.

[0059] In this embodiment, the filter element inside the vacuum filter 6 is a 0.2μm FC200 soft filter element.

[0060] The outlet of the mixing tank is connected to a porous distributor 7 and a flow meter 9, and the gas delivery pipe is connected to the porous distributor 7. The porous distributor has multiple independent gas outlets, each connected to a gas delivery pipe. Each gas delivery pipe is connected to the top of each columnar reactor, and a flow meter is installed on the gas delivery pipe. The gas delivery pipe passes through the air inlet of the plexiglass cover at the top of the reactor and is fixed at the position of the cold-plated fixing bolt 10 at the bottom of the conical reactor before aeration. The flow meter 9 is a glass rotor flow meter.

[0061] The application method of the system in this embodiment is as follows:

[0062] First, open the carbon dioxide cylinder 1, adjust the CO2 output through the pressure reducing valve 2, and start the adjustable air pump 3 to mix in air. The mixed gas is connected to the mixing tank 5 through the plastic air tube 4. The outlet of the mixing tank 5 is connected to a vacuum filter equipped with a 0.2μm FC200 soft filter element and a 10mm outer diameter claw connector. This filter is then connected to the porous distributor 7 through a 10mm outer diameter plastic air tube, and finally connected to the distributor and flow meter 9 through a 10mm outer diameter silicone hose 8. This design allows for real-time and precise control of the aeration rate and precise adjustment of the CO2 ratio.

[0063] See Figure 4 The mixed gas, passing through the flow meter 9, flows through the silicone hose 8 and through the plexiglass cover 12 with an air inlet 27, into the body of the column reactor 11. The plexiglass cover has an air inlet 27 and an exhaust port 29, and a 0.2μm air filter membrane 28 is installed on the exhaust port. A cold-plated fixing bolt 10 is installed at the end of the gas supply pipe to prevent turbulence from disturbing the gas supply. All column reactors are placed in a PMMA glass support 16 with upper and lower perforated slots 13 and 19, with a height-to-diameter ratio of 8:1. In this embodiment, the column reactor is designed with a diameter of 100mm, a height of 800mm, and a bottom funnel-shaped constriction with a diameter of 20mm and a height of 15mm. Sampling is performed periodically through the bottom sampling valve 17. This design allows for thorough mixing of algae with gas and nutrients under aeration conditions, and high-density algae alternately contacting the wall surface to absorb photon energy, improving the utilization efficiency of carbon and light sources.

[0064] When the light source is switched on, the LED light column, embedded in the hollow crossbeam 26 with a rotating screw 20, vertically illuminates the reactor. The hollow crossbeam 26 houses a photoelectric control system that allows adjustment of the light cycle and intensity. The light column frame 25 is equipped with a gas pipe fixing hole 14, an upper support plate 15, and a lower support plate 18 for securing the reactor bottom and housing a flow meter. The grounding end of the light column frame 25 is fitted with four casters 24, allowing the light source position to be moved according to cultivation conditions and stages. This design enables real-time adjustment of the light cycle, intensity, and position of the light source, meeting the light requirements of *Chlorella proteoglycans* at different growth stages.

[0065] Example 2

[0066] Using the system proposed in Example 1, this example presents a method for the dual-trophic culture of Chlorella proteoglycans, comprising the following steps:

[0067] Multiple independent column reactors are set up, and the effective culture volume, carbon source supply, aeration rate, and light conditions can be adjusted individually.

[0068] (1) First, the reactor body, silicone gas pipe, plexiglass cover and other microalgae culture-related components are cleaned and sterilized.

[0069] (2) Chlorella proteoglycans in the logarithmic growth phase were added to the reactor body in proportion to BG11 medium (formula shown in Table 1) that had been sterilized (121℃, 205.8kPa for 30 min). The glucose dosage was 10 g·L⁻¹. -1 The biomass density of *Chlorella proteoglycans* in the early stage of cultivation was 0.12 g·L⁻¹. -1 The effective culture volume of a single reactor is 5L, and the temperature is 26.5±0.5℃.

[0070] Table 1. BG11 Culture Medium Formulation

[0071]

[0072]

[0073] Note: stock: mother liquor; Ferric ammonium citrate: ferric ammonium citrate.

[0074] (3) Turn on the carbon dioxide cylinder and the adjustable air pump. Mix 2% CO2 with air in the mixing tank until homogeneous. Adjust the aeration rate of the reactor body to 1 L / min using a glass rotor flow meter. -1 This ensures that the algae are evenly mixed with the culture medium without precipitation.

[0075] (4) Move the lamp post frame to face the reactor body, with a spacing of 20±1mm, and set the light intensity to 80μmol·m⁻². -2 ·s -1 By adjusting the photoperiod through the light control, gradually increasing the light ratio, and selecting the most suitable light time, the commensal culture of Chlorella proteoglycans was carried out.

[0076] (5) During the cultivation process, samples are taken periodically through the sampling valve to determine the biomass of Chlorella proteoglycans.

[0077] (6) Draw a growth curve based on the growth of Chlorella proteoglycans, obtain microalgal biomass during the plateau phase, and transfer it to an outdoor culture reactor for further expansion of cultivation.

[0078] The cotrophic culture of *Chlorella proteoglycans* was conducted according to the photoperiod regulation strategy in Table 2. After 12 days of culture, the highest microalgal biomass concentrations were observed in treatment groups A-15, A-16, and A-17, reaching 6.52 g·L⁻¹. -1 6.81 g·L -1 6.43 g·L -1 This embodiment verifies that, in the culture system of the present invention, the optimal light duration for *Chlorella proteoglycans* under co-trophic culture conditions is 15–17 h, and the corresponding dark duration is 9–7 h.

[0079] Table 2 Treatment groups and biomass yield under photoperiod regulation strategy.

[0080]

[0081]

[0082] Example 3:

[0083] Unlike Example 2, the amount of glucose added and the light intensity were varied, with the glucose addition ranging from 5 to 30 g / L. -1 The light intensity adjustment range is 20–200 μmol·m⁻². -2 ·s -1 Treatment groups were set up according to the control strategies in Table 2. The algal strain added was *Chlorella proteoglycans* in its logarithmic growth phase, the nutrient source was BG11 medium, and the initial biomass density was 0.11 g·L⁻¹. -1 The effective culture volume of a single reactor is 5L, the temperature is 26.5±0.5℃, the distance between the light source and the reactor is 20±1mm, the light-dark ratio is 16:8, and the aeration rate is 1L·min. -1 The co-trophic culture of Chlorella proteoglycans was carried out under conditions where the CO2 content was 2%.

[0084] The results are shown in Table 3. After 12 days of cultivation, the highest concentrations of Chlorella proteoglycans biomass were observed in treatment groups B-28, B-29, B-36, and B-37, at 11.85 g·L⁻¹. -1 12.14 g·L -1 12.19 g·L -1 12.35 g·L -1 However, the biomass yield of glucose per unit in treatment groups B-36 and B-37 was 0.61 and 0.62, respectively, which was much lower than that in treatment groups B-20 and B-21 (0.75 and 0.81), indicating lower carbon source utilization.

[0085] This embodiment verifies that, in the culture system of the present invention, the optimal culture conditions for *Chlorella proteoglycans* to achieve high biomass growth while also having high carbon source utilization efficiency in a multi-trophic culture mode are a glucose dosage of 10-15 g·L⁻¹. -1 The light intensity is 100-120 μmol·m -2 ·s -1 Furthermore, this embodiment demonstrates that during the co-culture of Chlorella proteoglycans, as the amount of glucose added gradually increases, the biomass yield also increases to some extent. However, excessive glucose input will lead to inefficient utilization of the carbon source, resulting in a decrease in the biomass yield per unit area.

[0086] Table 3. Treatment groups and biomass under light intensity and glucose dosage regulation strategies.

[0087]

[0088]

[0089] Example 4:

[0090] The difference from Example 3 lies in the change in the amount and method of CO2 supply in the gas mixture. The added algae was *Chlorella proteoglycans* in its logarithmic growth phase, the nutrient source was BG11 medium, and the initial biomass density was 0.10 g·L⁻¹. -1 The glucose dosage is 15 g / L. -1 The effective culture volume of a single reactor is 5 L, the temperature is 26.5 ± 0.5 °C, and the light intensity is 110 μmol·m⁻². -2 ·s -1 The distance between the light source and the reactor is 20±1mm, and the light-to-dark ratio is 16:8. The air flow ratio of a single reactor is 0.2vvm, which means the aeration rate is 1L·min. -1 An orthogonal experiment was conducted using CO2 percentage and whether CO2 was supplied during darkness as variables. The CO2 percentage was controlled within a range of 1–6%, as shown in Table 4.

[0091] After 9 days of cultivation, treatment groups C-8 and C-10 had the highest biomass densities, at 13.17 g·L⁻¹. -1 13.55 g·L -1 This embodiment verifies that, in the culture system of the present invention, the aeration rate is 1 L·min. -1 Under these conditions, with the CO2 content controlled at 4-5% and no CO2 supply provided at night, the maximum biomass growth of *Chlorella protozoa* in the commensal culture mode can be achieved. This is likely because photosynthesis of *Chlorella protozoa* ceases under light-free conditions, and the presence of inorganic carbon sources inhibits its utilization of organic carbon sources. Therefore, stopping CO2 supply in the dark and using only air with a suitable aeration ratio to prevent algal seed deposition at the bottom of the reactor can improve the biomass yield of *Chlorella protozoa*.

[0092] Table 4. Treatment groups and biomass under CO2 percentage, supply mode regulation strategies, and biomass production.

[0093]

[0094] Example 5:

[0095] Using the system proposed in Example 1, this example presents a method for the dual-trophic culture of Chlorella proteoglycans, comprising the following steps:

[0096] (1) Add Chlorella proteoglycans in the logarithmic growth phase to a column reactor along with BG11 medium and add 12 g·L⁻¹ glucose. -1 The biomass density of *Chlorella proteoglycans* in the early stage of cultivation was 0.10 g·L⁻¹. -1 The temperature was 26.5±0.5℃.

[0097] (2) Turn on the carbon dioxide cylinder and adjustable air pump, control the CO2 content at 4-5%, and do not supply CO2 at night; mix CO2 with air in the mixing tank, and adjust the aeration rate of the reactor body to 1 L·min using a glass rotor flow meter. -1 This ensures that the algae are evenly mixed with the culture medium without precipitation.

[0098] (3) Move the lamp post frame to position it so that the light source is directly facing the reactor body, and set the light intensity to 110 μmol·m⁻¹. -2 ·s -1 The distance between the light source and the reactor was 20±1mm, and the light-dark ratio was 16:8, for the co-culture of Chlorella proteoglycans.

[0099] (4) The culture period is 12 days. During the plateau period, the microalgal biomass is obtained and then transferred to an outdoor culture reactor for further expansion.

[0100] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from its spirit and essence should fall within the protection scope of the present invention.

Claims

1. A cultivation method for mixotrophic cultivation of Chlorella pyrenoidosa, characterized by, The system for the dual-trophic culture of Chlorella proteoglycans includes an aeration unit, a culture unit, and a lighting unit. The aeration unit includes a carbon dioxide cylinder and a gas mixing tank; the carbon dioxide cylinder is connected to the gas mixing tank via a gas pipe, and an air pump with an adjustable gas output is installed on the gas pipe; a filter is installed at the outlet of the gas mixing tank; the outlet of the gas mixing tank is connected to the culture unit via a gas delivery pipe. The culture unit includes 1 to 20 column reactors, which are made of transparent material. The main body of the column reactor is cylindrical, and the lower part of the cylinder is conical. The height-to-diameter ratio of the cylindrical part is 7 to 9:

1. The cylindrical and conical parts of the column reactor are made of glass, and the top cover of the reactor is made of plexiglass. The illumination unit includes LED light columns and a photoelectric control system. The LED light columns are installed inside the light column frame, and the bottom of the light column frame is equipped with casters to adjust the relative position of the light source and the culture unit. The illumination unit uses stepless dimming LED warm light lamps. The cultivation method includes the following steps: (1) Add Chlorella proteoglycans in the logarithmic growth phase to a column reactor along with BG11 medium, and add 10-15 g·L glucose. -1 The biomass density of *Chlorella proteoglycans* during the initial culture stage was 0.10–0.12 g·L⁻¹. -1 The incubation temperature is 24~28℃; (2) Turn on the carbon dioxide cylinder and adjustable air pump to control the CO2 content to 4-6%, and do not supply CO2 at night; mix CO2 with air in the mixing tank, and adjust the aeration rate of the reactor body to 0.8-1.2 L·min using a glass rotor flow meter. -1 This ensures that the algae are evenly mixed with the culture medium without precipitation. (3) Move the lamp post frame to position it so that the light source is directly facing the reactor body, with a spacing of 18-22 mm and a light intensity of 100-120 μmol·m. -2 ·s -1 The light duration ranged from 15 to 17 hours, and the corresponding dark duration ranged from 9 to 7 hours, for the co-culture of Chlorella proteoglycans.

2. The cultivation method of the parikarya cultivation of Chlorella pyrenoidosa according to claim 1, characterized by, The filter is a vacuum filter, and the filter element inside the filter is a 0.1~0.4 μm filter element.

3. The cultivation method of the Chlorella pyrenoidosa mixotrophic cultivation according to claim 1, characterized by, The outlet of the mixing tank is connected to a perforated distributor and a flow meter. The gas delivery pipe is connected to the perforated distributor. The perforated distributor has multiple independent outlets, each of which is connected to a gas delivery pipe. Each gas delivery pipe is connected to the top of each columnar reactor, and a flow meter is installed on the gas delivery pipe. The gas delivery pipe extends from the top of the reactor and exits from the bottom. A fixing bolt is installed at the end of the gas delivery pipe to fix the end of the gas delivery pipe to the bottom of the columnar reactor.

4. The cultivation method of the parikarya Chlorella vulgaris mixotrophic cultivation according to claim 1, characterized in that, In step (2), the aeration amount is 1 L·min -1 Under the condition that the proportion of CO2 is controlled to be 4-5%.

5. The method for the dual-trophic culture of Chlorella proteoglycans according to any one of claims 1 to 4, characterized in that, Including the following steps: (1) Add Chlorella proteoglycans in the logarithmic growth phase to a column reactor along with BG11 medium, and add 11-13 g·L glucose. -1 The biomass density of *Chlorella proteoglycans* during the initial culture stage was 0.10–0.11 g·L⁻¹. -1 The temperature is 25~27℃; (2) Turn on the carbon dioxide cylinder and the adjustable air pump, control the CO2 content to 4-5%, mix the CO2 with the air in the mixing tank, and adjust the aeration rate of the reactor body to 1 L·min using a glass rotor flow meter. -1 This ensures that the algae are evenly mixed with the culture medium without precipitation. (3) Move the lamp post frame to position the light source directly facing the reactor body, with a spacing of 18~22 mm. Adjust the light intensity to 100~120 μmol·m using the light control. -2 ·s -1 The light duration was 15-17 h for the commensal culture of Chlorella proteoglycans. (4) The culture time is 11 to 15 days. The growth curve is plotted based on the growth of Chlorella proteoglycans. Microalgal biomass is obtained during the plateau period and then transferred to an outdoor culture reactor for further expansion.

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