A photobioreactor with brush-like carrier and its application in high-density cultivation of chlorella pyrenoidosa

Abstract

The application discloses a light bioreactor with brush-shaped carriers and application of the light bioreactor in high-density cultivation of chlorella pyrenoidosa, and relates to the field of microalgae cultivation. The light bioreactor comprises a support, a reactor, a lamp strip and brush-shaped carriers, the reactor is arranged on the support, the reactor is made of a transparent material, and an aeration disc is arranged at the bottom in the reactor; the lamp strip is arranged on the support and spirally surrounds the periphery of the reactor; and the brush-shaped carriers are arranged in the reactor, the brush-shaped carriers comprise support rods and a plurality of bristle units distributed along the length direction of the support rods, and each bristle unit is formed by bristles which are densely distributed inside and sparsely distributed outside. The brush-shaped carriers are three-dimensional configurations, which are beneficial to high-density cultivation of microalgae; each bristle unit is formed by bristles which are densely distributed inside and sparsely distributed outside, so that the shielding effect of the carriers on light is reduced.

Description

Technical Field

[0001] This application relates to the field of microalgae cultivation, and in particular to a photobioreactor and its application in high-density cultivation of Chlorella proteoglycans. Background Technology

[0002] Microalgae, as a type of photosynthetic autotrophic microorganism, can use light energy to fix carbon dioxide and synthesize biomass, showing broad application prospects in fields such as biological carbon fixation, food, feed, and biofuels. Among them, *Chlorella pyrenoidosa* is considered one of the microalgae species with the greatest potential for industrial application due to its rapid growth rate, strong stress resistance, and high protein content. Achieving high-density cultivation of microalgae is a key prerequisite for promoting the industrial application of microalgae biological carbon fixation technology.

[0003] Currently, microalgae cultivation mainly includes two modes: suspension culture and attached culture. In traditional suspension culture, microalgae cells are dispersed freely in the culture medium, resulting in low cultivation density and low light utilization efficiency, making it difficult to meet the needs of high-density cultivation. Attached culture, by placing a carrier in the reactor, allows microalgae to grow as biofilms attached to the carrier surface. This leads to high cell aggregation and a significantly higher cultivation density compared to suspension culture, and has been proven to be an effective method for achieving high-density microalgae cultivation.

[0004] However, existing attachment culture technologies still face bottlenecks in carrier design, mainly in the following aspects: Firstly, regarding carrier configuration, existing studies mostly employ two-dimensional planar carriers such as paper, cloth, mesh, and membrane. These carriers offer limited attachment area and cannot fully utilize the three-dimensional space inside the reactor, resulting in low reactor space utilization and limited biomass yield per unit volume. Although some studies have attempted to increase the specific surface area of ​​the carrier through surface etching or modification, the improvement effect is limited and has not fundamentally improved the space utilization problem.

[0005] Secondly, regarding the light-shading effect, in attached culture, the carrier itself blocks light. When the amount of carrier is increased to improve biomass yield, light penetration further decreases, leaving deeply attached microalgae in a state of insufficient light, thus inhibiting photosynthesis. To ensure light transmittance, the amount of carrier added in existing attached culture reactors is usually low, which in turn limits further increases in biomass yield.

[0006] Therefore, there is an urgent need to develop a three-dimensional structured carrier with high specific surface area, high space utilization and good light penetration, and a matching photobioreactor, in order to overcome the contradiction between carrier configuration and light utilization efficiency in existing attachment culture technology and achieve high-density cultivation of microalgae. Summary of the Invention

[0007] To address the technical problems of existing microalgae attachment culture carriers, such as low specific surface area, insufficient space utilization, and significant light shading effect, this application proposes a photobioreactor with a brush-like carrier and its application in high-density cultivation of Chlorella proteoglycans.

[0008] In a first aspect, this application provides a photobioreactor with a brush-like carrier, comprising: support; A reactor, mounted on the support, is made of transparent material and has an aeration disc at the bottom inside the reactor; The light strip is mounted on the support and spirals around the periphery of the reactor; A brush-shaped carrier is disposed in the reactor. The brush-shaped carrier includes a support rod and a plurality of bristle units distributed along the length of the support rod. Each bristle unit is formed by bristles that are densely distributed inside and sparsely distributed outside.

[0009] By adopting the above technical solution, the brush-like carrier of this application has a three-dimensional structure, which has a higher specific surface area and space utilization rate compared with the traditional two-dimensional planar carrier. It can provide more attachment sites for Chlorella proteoglycans, make full use of the three-dimensional space inside the reactor, and facilitate high-density cultivation of microalgae. In the brush-like carrier, each bristle unit is formed by bristles that are densely distributed inside and sparsely distributed outside. That is, the bristles are arranged radially from the center outward, with dense bristles inside and sparse bristles outside. This structure is conducive to the propagation and penetration of light from the outside of the reactor into the inside of the brush-like carrier, reducing the shading effect of the carrier itself on light. This allows the microalgae attached inside the carrier to obtain sufficient light, which is beneficial to the photosynthesis of Chlorella proteoglycans, thus maintaining good light utilization efficiency even under high carrier volume conditions.

[0010] The LED light strip is spirally arranged around the reactor, providing 360° uniform illumination and eliminating shadow areas caused by directional light. This ensures that microalgae in all directions receive sufficient light radiation, improving the uniformity and utilization of light. A gap is left between the light strip and the reactor wall to prevent heat generated by the light strip from being directly transferred to the reactor, avoiding localized overheating that could interfere with microalgae growth.

[0011] The reactor is made of a transparent material, which can be either glass or acrylic, with glass being preferred to ensure good light transmittance and chemical stability.

[0012] Optionally, the reactor has a cylindrical structure that is open at one end and hollow. The bristles are made of polyester fiber, the thickness of each bristle unit is 1.0~1.2cm, and the diameter of each bristle unit is adapted to the inner diameter of the reactor, which is 6~8cm.

[0013] By adopting the above technical solution, the cylindrical reactor has the advantages of short optical path and high surface area to volume ratio, which is beneficial to improving photosynthetic efficiency and biomass productivity per unit volume. The bristles are made of polyester fiber, which has the advantages of good chemical stability, resistance to acid and alkali corrosion, and non-degradability. It can maintain the integrity and stability of the structure during long-term cultivation, which is conducive to the stable attachment of Chlorella cells and the formation of biofilm. If the thickness of the bristle unit is too large, it will increase the attenuation path of light within the bristle unit, resulting in insufficient light for deep microalgae; if the thickness is too small, it will reduce the attachment area provided by each bristle unit. By controlling the thickness range of each bristle unit, sufficient attachment space for microalgae can be provided while ensuring light transmittance.

[0014] The diameter of each brush unit is matched to the inner diameter of the reactor, which is 6-8 cm. This means the outer edge of each brush unit is essentially flush with the inner wall of the reactor. This design allows the brush-like carrier to fully utilize the radial space inside the reactor, maximizing the space utilization rate of the carrier within the reactor's cross-section. Simultaneously, because the outer edge of the brush unit is close to the inner wall, the light emitted by the spiral-shaped lamp strip can directly illuminate the sparse outer area of ​​the brush unit and propagate radially inward, reducing ineffective light attenuation in the culture medium.

[0015] Optionally, the weight of the bristles corresponding to each of the bristle units is 1.0~2.5g.

[0016] Preferably, the weight of the bristles corresponding to each bristle unit is 1.5~2.0g. More preferably, the weight of the bristles corresponding to each bristle unit is 2.0g.

[0017] By adopting the above technical solution, excessive weight of a single layer of bristles will result in overly dense bristles, reducing light transmittance; conversely, insufficient weight of a single layer of bristles will reduce the surface area available for microalgae to attach. Controlling the weight of the bristles corresponding to each bristle unit can achieve a better balance between light transmittance and attachment area.

[0018] Optionally, the spacing between two adjacent bristle units is 1.0 to 2.5 cm.

[0019] Preferably, the spacing between two adjacent bristle units is 1.5 cm.

[0020] By adopting the above technical solution, multiple brush units are distributed along the length of the support rod, with a clear gap between adjacent brush units. This layered structure ensures free flow of fluid between layers within the reactor, avoids the formation of dead zones in the flow field, and facilitates the uniform distribution of nutrients, providing a uniform nutrient supply environment for microalgae growth. Simultaneously, appropriate interlayer spacing also promotes the uniform dispersion and rising of bubbles generated during aeration, enhancing gas-liquid mass transfer and improving CO2 supply efficiency. Too small an interlayer spacing will hinder fluid flow and light penetration between layers; too large an interlayer spacing will reduce space utilization and decrease the total amount of carrier material.

[0021] It should be noted that "the spacing between two adjacent bristle units" refers to the distance between the centers of two adjacent bristle units.

[0022] Optionally, the number of bristle units can be 8 to 19.

[0023] Secondly, this application provides an application of the photobioreactor as described in any of the above claims in a high-density cultivation method of Chlorella proteoglycans.

[0024] By adopting the above technical solution, microalgae are cultivated using a photobioreactor with a brush-like carrier. The brush-like carrier can provide a higher specific surface area and space utilization rate, providing more attachment sites for Chlorella proteoglycans, and facilitating the penetration of light and the supply of nutrients, which is conducive to achieving high-density cultivation of microalgae.

[0025] Optionally, the application of the photobioreactor in the high-density cultivation method of Chlorella vulgaris includes the following steps: S1. Provide Chlorella proteoglycans and place them in BG11 medium for large-scale culture to obtain Chlorella proteoglycan solution; S2. Add the culture medium and the Chlorella proteoglycan solution obtained in step S1 to the reactor, and maintain the temperature at 22~28℃ and the light intensity at 400~600μmol / m². 2 High-density cultivation of Chlorella proteoglycans was carried out under the conditions of light-dark ratio of (17~19)h: (5~7)h and aeration rate of 0.04~0.2vvm.

[0026] By adopting the above technical solution, a photobioreactor with a brush-like carrier is used to cultivate microalgae, and parameters such as aeration and light intensity are controlled, providing a suitable cultivation environment for high-density cultivation of Chlorella proteoglycans.

[0027] Optionally, in step S2, the aeration rate is 0.08~0.16vvm, and the gas corresponding to the aeration includes the following components by volume percentage: 6~8% CO2, 3~5% O2, and the balance is N2.

[0028] Preferably, in step S2, the aeration rate is 0.12vvm.

[0029] By adopting the above technical solution and using simulated flue gas containing CO2 for aeration, sufficient carbon source and suitable culture environment are provided for the high-density cultivation of Chlorella proteoglycans.

[0030] Optionally, in step S2, the ratio of the volume of the culture medium, the volume of the Chlorella proteoglycans solution, and the volume of the reactor is (8~9):(1~2):11.5.

[0031] By adopting the above technical solution, and by controlling the volume of the culture medium, the volume of the Chlorella proteoglycans solution, and the reactor volume, suitable conditions are provided for the high-density cultivation of Chlorella proteoglycans.

[0032] Optionally, in step S2, the composition of the culture medium is as follows: NaNO3 7.3~7.7 g / L, K2HPO4·3H2O 1.2~1.4 g / L, KH2PO4 0.6~0.7 g / L, MgSO4·7H2O 0.3~0.4 g / L, CaCl2·2H2O 0.16~0.20 g / L, citric acid 0.02~0.04 g / L, ferric ammonium citrate 0.02~0.04 g / L, EDTA 0.004~0.006 g / L, Na2CO3 0.05~0.15 g / L, H3BO3 0.01~0.02 g / L, MnCl2·H2O 0.008~0.010 g / L, ZnSO4·7H2O 0.0010~0.0013g / L, CuSO4·5H2O 0.0002~0.0005g / L, Na2MoO4·2H2O 0.001~0.003g / L, Co(NO3)2·6H2O 0.0001~0.0003g / L.

[0033] By adopting the above technical solution, the concentration of each component in the improved culture medium is increased by about 5 times compared with the standard BG11 medium. The purpose is to provide sufficient nutrients such as nitrogen, phosphorus, magnesium, calcium, iron and trace elements for the high-density and rapid growth of Chlorella pyrenoidosa, and to avoid the depletion of a certain nutrient element becoming a limiting factor during the culture process.

[0034] The additional phosphate buffer pair (K₂HPO₄·3H₂O / KH₂PO₄) added to the culture medium serves to maintain pH stability. During high-density cultivation of *Chlorella proteoglycans*, photosynthesis by the microalgae causes pH fluctuations in the culture medium. The dissociation equilibrium of the phosphate buffer pair effectively buffers these pH changes, maintaining the pH within a suitable range and providing a suitable environment for the growth of *Chlorella proteoglycans*.

[0035] Optionally, in step S1, the scale-up culture includes multiple rounds of culture. In the first round of culture, the volume ratio of Chlorella proteoglycans to BG11 culture medium is 1:(8.5~9.5). In the next round of culture, the algal solution obtained from the previous round of culture is mixed with BG11 culture medium at a volume ratio of 1:(8.5~9.5) for culture. The culture conditions for each round of cultivation included: temperature of 22–28°C and light intensity of 100–200 μmol / m². 2 •s, light-dark ratio is (17~19)h:(5~7)h, aeration rate is 0.1~0.3vvm, and the gas used for aeration is air.

[0036] By adopting the above technical solution, the required amount of microalgae can be obtained through multiple rounds of cultivation. Furthermore, through multiple rounds of subculturing, algal populations with strong adaptability and vigorous growth can be screened out, which is beneficial for achieving rapid attachment and high-density growth in the reactor.

[0037] In summary, this application includes at least one of the following beneficial technical effects: (1) The brush-like carrier of this application has a three-dimensional structure with a higher specific surface area and space utilization rate, which can provide more attachment sites for Chlorella pyrenoidosa, make full use of the three-dimensional space inside the reactor, and facilitate the high-density cultivation of microalgae.

[0038] (2) In the brush-like carrier of this application, each brush unit is formed by brushes that are densely distributed inside and sparsely distributed outside, that is, the brushes are arranged radially from the center outward, with dense inside and sparse outside. This structural design is conducive to the propagation and penetration of light from the outside of the reactor to the inside of the brush-like carrier, reducing the light-blocking effect of the carrier itself, so that microalgae attached to different depths of the carrier can obtain sufficient light, which is beneficial to the photosynthesis of Chlorella proteoglycans, thus maintaining good light utilization efficiency even under high carrier conditions.

[0039] (3) The brush-like carrier of this application adopts a layered structure design, with multiple brush units distributed along the length of the support rod, and a clear gap between adjacent brush units. This layered structure ensures the free flow of fluid between the layers in the reactor, avoids the formation of dead zones in the flow field, and is conducive to the uniform distribution of nutrients, providing a uniform nutrient supply environment for microalgae growth. At the same time, the appropriate interlayer spacing is also conducive to the uniform dispersion and rise of bubbles generated by aeration, promoting gas-liquid mass transfer and improving CO2 supply efficiency.

[0040] (4) The light strip of this application is spirally wrapped around the outside of the reactor, which can provide uniform lighting conditions for the reactor, eliminate the shadow area caused by the directionality of the light, and enable microalgae in all directions in the reactor to receive sufficient light radiation, thereby improving the uniformity and utilization rate of the light. There is a gap between the light strip and the outer wall of the reactor to prevent the heat generated by the light strip from being directly transferred to the reactor, thus avoiding interference with the growth of microalgae due to local overheating.

[0041] (5) This invention optimizes process parameters such as culture medium composition, aeration gas composition, and aeration rate, and uses simulated flue gas containing CO2 for aeration, providing sufficient carbon source and suitable culture environment for high-density cultivation of Chlorella pyrenoidosa, thus achieving efficient biomass production and carbon fixation. The phosphate buffer added to the culture medium can maintain the stability of the culture medium pH, preventing excessive pH increase caused by high-density microalgae growth, and ensuring the long-term stable operation of the cultivation system. Attached Figure Description

[0042] Figure 1 This application provides a schematic diagram of the structure of a photobioreactor; Figure 2 A schematic diagram of the structure of the brush-like carrier provided in this application; Figure 3 for Figure 2 A magnified structural diagram of the medium-sized brush bristles; Figure 4 A schematic diagram of the structure of the disc-shaped carrier provided in this application; Figure 5 A schematic diagram of the structure of the spiral carrier provided in this application; Figure 6 A schematic diagram of the structure of the spherical carrier provided in this application; Figure 7 This is a schematic diagram of the structure of the cluster-shaped carrier unit provided in this application; Figure 8 Graphs showing biomass production and carbon fixation of Chlorella proteoglycans cultured on different carrier configurations; Figure 9 The graph shows the changes in nitrate concentration, total phosphorus concentration, and pH in reactors with different carrier configurations. Figure 10 Morphology and diameter distribution of Chlorella proteoglycans cultured on different carrier configurations; Figure 11 Figure a shows the biomass production results of Chlorella proteoglycans under different monolayer carrier weights and different culture times; Figure 11 b is a graph showing the biomass production results of Chlorella proteoglycans after cultivation under different monolayer carrier weights; Figure 12Figure a shows the biomass production results of Chlorella proteoglycans under different interlayer spacings on different carriers and at different culture times; Figure 12 b is a graph showing the biomass production results of Chlorella proteoglycans after cultivation under different carrier interlayer spacings; Figure 13 Figure a shows the biomass production of Chlorella proteoglycans under different aeration rates and culture times; Figure 13 Figure b shows the biomass production results of Chlorella proteoglycans after cultivation at different aeration rates.

[0043] In the figure, 1 is the support; 11 is the limiting ring; 2 is the light strip; 3 is the reactor; 31 is the gas pipe; 4 is the brush-shaped carrier; 41 is the support rod; 42 is the brush unit; 421 is the brush bristle; 5 is the disc-shaped carrier; 6 is the spiral carrier; 7 is the spherical carrier; 8 is the cluster carrier; 81 is the cluster carrier unit. Detailed Implementation

[0044] The present application will be further described in detail below with reference to the embodiments. Example 1

[0045] like Figures 1-3 As shown, this embodiment provides a photobioreactor with a brush-like carrier, including a support 1, a light strip 2, a reactor 3, and a brush-like carrier 4.

[0046] The bracket 1 is used to fix the light strip 2 and the limiting reactor 3, and the bottom of the bracket is in contact with the flat ground.

[0047] Reactor 3 is made of transparent glass and has a hollow cylindrical structure with an open top. The reactor has an inner diameter of 7 cm, a wall thickness of 1 mm, and a volume of 1.15 L. A limiting ring 11 is installed at the top of the support 1. The inner diameter of the limiting ring is slightly larger than the outer diameter of the reactor, allowing the reactor to pass through. The bottom of the reactor contacts the ground, thus limiting its movement. An aeration disc is installed at the bottom of the reactor, connected to an external air source via a 4 mm diameter air pipe 31. The aeration disc is made of nano-aeration stone, and the pore size of the aeration holes on the disc is 15~35 μm.

[0048] The light strip 2 is set on the support and spirals around the outside of the reactor with a pitch s of 2cm. The light strip is an LED white light strip, which spirals around the outer wall of the reactor and there is a gap of 3.5cm between the light strip and the outer wall of the reactor. The height of the light strip is matched with the height of the mixture obtained by mixing algae liquid and culture liquid in the reactor to provide sufficient light for the reactor.

[0049] A brush-like carrier 4 is placed inside the reactor. The brush-like carrier includes a support rod 41 and multiple brush units 42 distributed along the length of the support rod 41. The support rod is a plastic rod, vertically arranged along the axial direction of the reactor. The weight of the support rod allows the multiple brush units to be immersed in the mixture obtained by mixing the culture medium and the algal solution. Each brush unit is fixed to the support rod and equidistantly distributed along the length of the support rod, forming a layered structure. The height difference between the center of the lowest brush unit and the bottom end of the support rod is 2 cm. When the brush-like carrier is placed inside the reactor, the bottom end of the support rod contacts the surface of the aeration disc inside the reactor. The diameter of the support rod is small, much smaller than the surface area of ​​the aeration disc, and therefore does not affect the aeration of the aeration disc.

[0050] Each bristle unit 42 is formed by bristles 421 that are densely distributed internally and sparsely distributed externally. Specifically, the bristles in each bristle unit radiate outward from the center of the support rod. Within each bristle unit, the bristles are densely arranged near the center of the support rod and gradually become sparser further away from the center. The bristles are made of polyester fiber with a diameter of 55~70μm, and the diameter d1 of each bristle unit is 7cm. Example 2

[0051] This embodiment provides a method for high-density cultivation of Chlorella vulgaris using the photobioreactor described in Example 1, including the following steps: S1, Large-scale culture of Chlorella proteoglycans Chlorella pyrenoidosa was provided, and the algal strain was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, with the accession number FACHB-9. Chlorella pyrenoidosa was cultured on a scale-up scale in BG11 medium (Qingdao Haibo Biotechnology Co., Ltd.) to obtain Chlorella pyrenoidosa solution.

[0052] The specific steps are as follows: Preparation before culture expansion: Immerse the necessary glassware, such as beakers, glass rods, and Erlenmeyer flasks, in 5wt% dilute nitric acid overnight, then rinse and set aside. Add 1.35L of BG11 culture medium (using deionized water as solvent) to a 2L Erlenmeyer flask, seal the flask opening with sealing film and rubber bands, and place it in a steam sterilizer along with the trachea and air stone (placed in a heat-resistant sterilization bag). Set the sterilization temperature to 121℃ and the sterilization time to 15 minutes. After sterilization, remove and cool to room temperature (25℃).

[0053] Large-scale cultivation process: In a clean bench, after sterilizing with ultraviolet light for 30 minutes, *Chlorella proteoglycans* and BG11 medium were mixed at a volume ratio of 1:9 to obtain an algal solution. 1.5L of this solution was added to a 2L Erlenmeyer flask. An air-supported plate was connected via an air tube and placed into the flask, which was then sealed with sealing film and a rubber band. All operations were performed near an alcohol lamp flame to avoid contamination. The flask was then placed in a constant temperature incubator under the following conditions: temperature 25℃, light intensity 100 μmol / m². 2 With a light-dark ratio of 18h:6h, air was pumped into the algal solution using an air pump at an aeration rate of 0.2 vvm. A 0.45 μm microporous membrane was installed between the air pump and the air tube connecting to the algae-absorbing plate to isolate dust and microorganisms from the air. The flasks were shaken every 12 hours, and the suspension culture was carried out for 5 days to complete the first round of cultivation.

[0054] The algal culture obtained from the first round of cultivation was mixed with fresh BG11 medium at a volume ratio of 1:9, and the second round of cultivation was carried out in the same manner as the first round. Multiple rounds of scale-up cultivation were then carried out in the same manner as the second round, i.e., the algal culture obtained from the previous round was mixed with fresh BG11 medium for the next round of cultivation, until a Chlorella proteoglycans culture with a concentration of approximately 2 g / L was obtained, which was then used for inoculation of the reactor.

[0055] High-density cultivation of S2 and Chlorella pyrenoidosa The culture medium and the *Chlorella vulgaris* solution obtained in step S1 were mixed at a volume ratio of 9:1 to obtain the culture medium. 1 L of the culture medium was added to a 1.15 L reactor. A brush-like carrier was pre-placed in the reactor. The parameters of the brush-like carrier are as follows: the total number of brush unit 42 layers is 10, the weight of each brush unit is 1.5 g, the thickness D of each brush unit is 1 cm, the interlayer spacing L between adjacent brush units is 2 cm, and the total amount of polyester fiber is 15 g. The reactor was operated at a temperature of 25℃ and a light intensity of 500 μmol / m². 2 Under the conditions of light-dark ratio of 18h:6h and aeration rate of 0.08vvm, the gas corresponding to aeration includes the following components by volume percentage: 7% CO2, 4% O2, and the balance being N2. High-density cultivation of Chlorella proteoglycans is carried out, with a cultivation cycle of 5 days.

[0056] The culture medium consisted of the following components: NaNO3 7.5 g / L, K2HPO4·3H2O 1.341 g / L, KH2PO4 0.6805 g / L, MgSO4·7H2O 0.375 g / L, CaCl2·2H2O 0.18 g / L, citric acid 0.03 g / L, ferric ammonium citrate 0.03 g / L, EDTA 0.005 g / L, Na2CO3 0.1 g / L, H3BO3 0.0143 g / L, MnCl2·H2O 0.00905 g / L, ZnSO4·7H2O 0.00111 g / L, CuSO4·5H2O 0.000395 g / L, and Na2MoO4·2H2O. 0.00195g / L, Co(NO3)2·6H2O0.000245g / L. Comparative Examples 1-4

[0057] Comparative Examples 1-4 are based on Example 2, except that in step S2, a carrier with a different configuration is used instead of the brush-like carrier; the other steps remain the same as in Example 2. Specifically, Comparative Example 1: Figure 4 As shown, 5 disc-shaped carriers are used, and there are 10 disc-shaped carriers. The diameter d2 of each disc-shaped carrier is 7cm, the thickness is 1cm, and the weight of polyester fiber in each disc-shaped carrier is 1.5g. Each disc-shaped carrier is inserted into the reactor, and the distance L1 between two adjacent disc-shaped carriers is 2cm. The total amount of polyester fiber used is 15g.

[0058] Comparative Example 2: Figure 5 As shown, a spiral carrier 6 is used, wherein the pitch s1 is 6.5cm, the spiral radius s2 is 3.5cm, the diameter of the polyester wire bundle s3 is 3cm, the total amount of polyester fiber is 15g, and the height h1 of the spiral carrier is the same as the height h0 of the brush carrier.

[0059] Comparative Example 3: Figure 6 As shown, spherical carrier 7 is used, and there are 14 spherical carriers. The diameter d3 of each spherical carrier is 3.5cm, the weight of polyester fiber in each spherical carrier is 1.07g, and the total amount of polyester fiber used is about 15g. The spherical carriers are stacked in the reactor.

[0060] Comparative Example 4: Figure 7 As shown, clustered carrier units 81 are used to replace brush units. The side length c1 of each clustered carrier unit is 3.1cm, the diameter of the cone base c2 is 3.5cm, the cone angle A is 60°, and the height h2 of the clustered carrier 8 is the same as the height h0 of the brush carrier. Performance Test 1

[0061] Samples were taken from the algal solutions obtained in Example 2 and Comparative Examples 1-4 on days 0, 0.25, 0.5, 1, 2, 3, 4, and 5 of cultivation. The biomass, carbon fixation rate, carbon fixation efficiency, nitrate nitrogen concentration, total phosphorus concentration, and pH changes in the reactor were measured. The morphology and size distribution of microalgal cells obtained on day 5 of cultivation were also obtained. Each time a sample was taken, a corresponding volume of fresh culture medium was added to the reactor. The culture medium added had the same composition as the culture medium in step S2 of Example 2.

[0062] Chlorella biomass: The microalgal biomass in the reactor consists of two parts: microalgal biomass suspended in the culture medium and microalgal biomass attached to the carrier. The suspended microalgal biomass was determined spectrophotometrically to obtain the concentration m1 of suspended microalgal biomass in the reactor. The microalgal biomass attached to the carrier was determined using a direct weighing method. The initial wet weight of the carrier and the wet weight of the carrier after attachment and growth were measured using a precision electronic balance. The difference between these two weights is the fresh weight of the algae. The dry weight was then calculated based on the water content, and finally divided by the culture medium volume to obtain the concentration m2 of the attached microalgal biomass. The formula for calculating m2 is as follows: m2 = [m3 × (1 - ω1)] / V; In the formula, m2 is the biomass concentration of the attached microalgae in the reactor, g / L; m3 is the fresh weight of the attached microalgae, g; ω1 is the water content of the algae; and V is the volume of the culture medium in the reactor, L.

[0063] The biomass of Chlorella proteoglycans is m = m1 + m2.

[0064] Carbon fixation rate: The carbon fixation rate reflects the rate at which the reactor fixes CO2 from the inlet gas, and is calculated using the following formula: RCO2=C biomass ×μ L ×M CO2 / Mc; In the formula, RCO2 is the carbon fixation rate of the reactor, g / L / h; C biomass The carbon content in microalgal biomass was determined by an elemental analyzer; μ L The growth rate of microalgae is expressed in g / L / h; M CO2 M is the molar mass of CO2, in g / mol; C ρ is the molar mass of a carbon atom, in g / mol.

[0065] Carbon sequestration efficiency: Carbon sequestration efficiency reflects the reactor's utilization of the total CO2 in the inlet air, and is calculated using the following formula: η CO2 =[(m×C biomass ×M CO2 / M c ) / (Q×C0×ρCO2 ×T)]×100% In the formula, η CO2 ρ is the CO2 utilization efficiency of the reactor; m is the final biomass yield, g / L; Q is the aeration flow rate, L / h; C0 is the CO2 concentration in the aeration, 7%; CO2 ρ represents the CO2 density; T represents the culture period, in hours.

[0066] Nitrate nitrogen concentration: The nitrate nitrogen content of the culture medium was determined by ultraviolet spectrophotometry.

[0067] Total phosphorus concentration: The total phosphorus content of the culture medium was determined using the molybdenum-antimony spectrophotometric method.

[0068] pH value: The pH value of the culture medium was measured using a pH meter.

[0069] Figure 8 The growth patterns and carbon fixation effects of *Chlorella proteoglycans* cultured on different carrier configurations are shown. A1 corresponds to the blank control group (no carrier added); A2 corresponds to Comparative Example 1; A3 corresponds to Example 2; A4 corresponds to Comparative Example 2; A5 corresponds to Comparative Example 3; and A6 corresponds to Comparative Example 4. Figure 8 As can be seen, the *Chlorella proteoglycans* in the suspension culture group (A1) grew slowly during the culture period, with a final biomass yield of only 2.05 g / L, far lower than the biomass productivity range of 5.45-8.94 g / L in the attached culture groups. This indicates that using a three-dimensional carrier for attached culture can significantly increase the cultivation density of *Chlorella proteoglycans*. The carrier provides a more favorable microenvironment for the microalgae's growth, thus the biomass of the attached culture group increased rapidly within the first day. By the third day of culture, the growth of *Chlorella proteoglycans* in all attached culture groups entered a stable period, after which biomass growth essentially stagnated or grew extremely slowly, remaining at a relatively stable level. Furthermore, Figure 8 b. Biomass of each group during the stationary phase was compared. The growth performance of *Chlorella proteoglycans*, from best to worst, was: brush-shaped (8.77 g / L) > cluster-shaped (8.16 g / L) > spiral-shaped (7.45 g / L) > spherical (6.90 g / L) > disc-shaped (5.87 g / L) > suspension culture (1.89 g / L). The biomass of the brush-shaped carrier culture was 7.48%, 17.72%, 27.1%, and 49.4% higher than that of the cluster-shaped, spiral-shaped, spherical, and disc-shaped carriers, respectively. The differences in the physical characteristics of different three-dimensional carrier configurations in the reactor (such as light penetration depth, shear force, gas-liquid mass transfer, etc.) may be an important reason for this result: the configuration affects the light field and flow field in the reactor, thereby changing the microenvironment for *Chlorella proteoglycans* growth. In summary, the difference in carrier configuration significantly affects the growth of *Chlorella proteoglycans*. From the perspective of biomass, the brush-shaped configuration is most conducive to achieving high-density cultivation of *Chlorella proteoglycans*.

[0070] Depend on Figure 8 As shown in c, the carbon fixation rate of each attached culture group reached its peak within the first day of cultivation. The maximum carbon fixation rate of the brush-like carrier group was 0.91 g / L / h, which was 10.89%, 12.4%, 62.57%, and 88.75% higher than that of the clustered, spiral, spherical, and disc-shaped groups, respectively. This is consistent with the biomass growth rate of each group. After reaching the peak carbon fixation rate, the carbon fixation rate of each attached culture group gradually decreased, reaching almost zero by the third day. This suggests the importance of timely harvesting of microalgal biomass within the first day, reducing competition among algal cell groups for survival resources such as light and nutrients, and helping to maintain the reactor's continuous carbon fixation capacity. The difference in carbon fixation rate ultimately reflects the difference in overall carbon fixation efficiency. Figure 8 d). The carbon fixation efficiency of each group in the attached culture followed the same trend as the change in biomass yield throughout the entire culture period. The brush-shaped carrier group had the highest carbon fixation efficiency, reaching 18.72%, while the disc-shaped carrier group had the lowest carbon fixation efficiency, at only 11.39%. Therefore, from the perspective of carbon fixation efficiency, the brush-shaped carrier is the optimal carrier configuration.

[0071] Nitrogen and phosphorus are the most important macronutrients in microalgae biomass production, and the consumption of nutrients in the reactor can indirectly reflect the growth status of Chlorella proteoglycans. Figure 9 a shows the NO3 in the culture medium under different configurations. - Changes in NO3- concentration. Except for the suspension culture group (A1), the NO3- concentrations in each attached culture group... - -N was rapidly consumed, and by the end of the culture, NO3 in the disc-shaped, brush-shaped, spirulina, spherical, and cluster-shaped groups was significantly reduced. - -N consumption was 568 mg / L, 795 mg / L, 724 mg / L, 662 mg / L, and 727 mg / L, respectively, indicating different nitrogen utilization rates among the groups. This is due to differences in the growth rate of *Chlorella proteoglycans*. It is worth noting that NO3- - -N concentration continues to decline at a low rate even after biomass growth ceases. Nitrogen is an essential element for the synthesis of basic biomolecules such as nucleic acids, chlorophyll, and proteins. Although biomass ceases to increase after Chlorella enters its stationary phase, it still needs to maintain basic life activities. The use of absorbed nitrogen shifts from cell proliferation to the accumulation of metabolic products (such as proteins), thus manifesting as NO3 in the culture medium. - -N concentration continued to decrease; differences in the growth rate of Chlorella proteoglycans also led to differences in phosphorus utilization rates among the groups. Figure 9 (b) The TP consumption of the suspended, disc-shaped, brush-shaped, spirulina, spherical, and clustered groups during the entire culture period were 24 mg / L, 76 mg / L, 145 mg / L, 125 mg / L, 112 mg / L, and 134 mg / L, respectively. The changes in TP concentration were correlated with NO3.- -N, similarly, also showed a continuous decreasing trend. Phosphorus content is typically less than 1% of the dry weight of microalgal biomass, yet it can significantly affect the photosynthetic activity of microalgae by participating in ATP synthesis. The modified BG11 medium used in this application was supplemented with phosphate buffer to ensure that phosphorus is in excess and does not become a limiting factor. Among all groups, the brush-carrier group showed the fastest nitrogen and phosphorus consumption, which is consistent with its highest biomass yield. Interestingly, the brush-carrier group showed the highest NO3 content. - The N:TP consumption ratio was approximately 5.48:1, indicating that the amount of phosphorus consumed in the culture medium exceeded the amount assimilated by Chlorella. It is speculated that the Peptidon Chlorella in the brush-carrier group produced more EPS, resulting in phosphorus consumption exceeding assimilation even under excessive conditions.

[0072] The phosphate buffer solution maintained the pH of all groups between 6.8 and 7.5, creating a suitable environment for the growth of Chlorella proteoglycans. From Figure 9 c It can be seen that the pH of each group decreased slightly in the early stage of cultivation, which was caused by the dissolution of CO2 introduced by aeration to generate H2CO3. As the cultivation time increased, the pH of each group slowly increased, and the final values ​​from high to low were: brush-shaped (7.51) > cluster-shaped (7.44) > spiral-shaped (7.37) > spherical (7.32) > disc-shaped (7.22) > suspended (6.90). The pH change range varied due to the differences in growth of Chlorella proteoglycans, and the pH of the group with higher biomass tended to rise faster. The reasons include two aspects: (1) Photosynthesis continuously consumes CO2 and HCO3 in the culture medium. - The faster the carbon fixation rate, the higher the OH content. - The accumulation rate is also higher; (2) the group with higher biomass absorbs and consumes more total phosphorus, which leads to a faster weakening of the pH stabilizing ability of the phosphate buffer. When nitrate is used as the only nitrogen source for high-density microalgae cultivation, if no control measures are taken, the growth of microalgae will cause the pH of the culture medium to rise rapidly to 11 or even higher, eventually leading to the collapse of the reactor. The phosphate buffer added to the culture medium in this application makes the pH rise slowly and always keep it in the near-neutral range, which is an important prerequisite for achieving high biomass.

[0073] Figure 10 SEM images and particle size distribution histograms of *Chlorella proteoglycans* cells cultured on different carrier configurations are presented. By observing cell morphology and size, cell growth and physiological status can be further assessed. Suspension culture group ( Figure 10 The average diameter of algal cells in group a) was 1.8 ± 0.43 μm, smaller than that in the attached culture group. They exhibited a significantly irregular morphology, with contracted folds in the cell walls and some cell damage. This is presumably due to the lack of a carrier in the suspended culture reactor to slow the airflow, resulting in environmental stress damage to the cell membrane caused by the shear force generated by the high-speed fluid. The disc-shaped group (…) Figure 10b) showed a high proportion of cell membrane damage, with a medium diameter (2.10±0.69 μm) and a relatively dispersed distribution. The experiment observed numerous microbubbles being intercepted on the lower surface of the disc carrier, coalescing into large bubbles, indicating poor gas flow and the presence of significant dead zones. The energy released during the rupture of these large bubbles damaged the integrity of the algal cells; brush-like ( Figure 10 c) Spiral ( Figure 10 d) and clustered groups ( Figure 10 The algal cell diameters of f) were 2.17±0.68 μm, 2.08±0.74 μm, and 2.23±0.63 μm, respectively. The diameter ranges of these three groups were relatively concentrated, with a small distribution span and a generally uniform appearance. There were no significant differences in cell morphology; most cells were round and plump without wrinkling, indicating that these three configurations had a strong ability to redistribute fluid evenly, resulting in lower shear forces generated by the fluid within the reactor compared to other groups, thus leading to higher algal cell integrity. The spherical group ( Figure 10 The algal cell diameter in group e) was the smallest among all attached groups (1.89 ± 0.65 μm), closer to that of the suspended group, and showed some morphological damage. This may be related to the fact that the spherical carrier was in motion within the reactor, while other carrier configurations were stationary. The moving spherical carrier caused disordered turbulence and uncontrolled flow within the reactor, negatively impacting algal cell growth. Therefore, from the perspective of cell morphology and size, the suitable carrier configurations are ranked as follows: cluster > brush > spiral > disc > spherical. Examples 3-5

[0074] Examples 3-5 are based on Example 2, with the difference being that in step S2, the thickness of each bristle unit remains constant, the weight of the bristles in each bristle unit is adjusted, the total amount of polyester fiber used changes accordingly, the cultivation period is 3 days, and the other steps are the same as in Example 2. Specifically, In Example 3, the weight of the bristles in each layer of the brush unit is 1g, and the total amount of polyester fiber used is 10g.

[0075] In Example 4, the weight of the bristles in each layer of the brush unit is 2g, and the corresponding total amount of polyester fiber used is 20g.

[0076] In Example 5, the weight of the bristles in each layer of the brush unit is 2.5g, and the corresponding total amount of polyester fiber used is 25g. Performance Test 2

[0077] The biomass of *Chlorella proteoglycans* obtained from the cultivation of microalgae in Examples 3-5 was determined using the method described in Performance Test 1. Figure 11The effect of different carrier amounts on the growth of Chlorella was shown, with 1.5 g of carrier corresponding to Example 2. As the carrier amount in the monolayer increased within the range of 1-2 g, the biomass yield in the reactor gradually increased. When the carrier amount in the monolayer reached 2 g, the biomass yield reached a maximum of 10.23 g / L; further increases to 2.5 g resulted in a decrease in biomass. For brush-like carriers, with a fixed number of brush layers, the carrier amount in the monolayer was directly proportional to the total carrier amount in the reactor. Increasing the carrier amount in the monolayer could increase the total carrier amount, thus promoting further growth in biomass yield in the reactor; however, this was contingent on other conditions such as light and nutrients not being significantly affected. In reality, however, excessively high carrier amounts would reduce the gaps between the polyester filaments, hindering light penetration and thus negatively impacting the growth of Chlorella. Examples 6-8

[0078] Examples 6-8 are based on Example 4, the difference being that in step S2, the spacing between two adjacent layers of bristle units is adjusted, thus changing the number of layers of bristle units; the other steps remain the same as in Example 4. Specifically, In Example 6, the spacing between two adjacent bristle units is 1 cm, and the number of bristle units is 19.

[0079] In Example 7, the spacing between two adjacent bristle units is 1.5 cm, and the number of bristle units is 13.

[0080] In Example 8, the spacing between two adjacent bristle units is 2.5 cm, and the number of bristle units is 8. Performance Test 3

[0081] The biomass of *Chlorella proteoglycans* obtained from the cultivation of microalgae in Examples 6-8 was determined using the method described in Performance Test 1. Figure 12 The effect of carrier interlayer spacing on Chlorella growth is shown, with a 2.0 cm interlayer spacing corresponding to Example 4. As can be seen from the figure, within the interlayer spacing range of 1-2.5 cm, the biomass yield in the reactor first increases and then decreases as the interlayer spacing decreases. The biomass yield reaches its maximum of 12.28 g / L when the interlayer spacing is 1.5 cm. Further reducing the interlayer spacing to 1 cm results in a decrease in biomass yield to 10.62 g / L. This indicates that, with a constant total carrier brush length, reducing the carrier interlayer spacing allows for the inclusion of more layers, thereby increasing the total carrier volume in the reactor and achieving a higher biomass yield. However, similar to the effect of increasing the amount of a single carrier layer, excessively small interlayer spacing hinders light penetration, leading to a decrease in Chlorella yield. Examples 9-12

[0082] Examples 9-12 are based on Example 7, the difference being that in step S2, the aeration rate is adjusted, while the other steps remain the same as in Example 7. Specifically, In Example 9, the aeration rate was 0.04 vvm.

[0083] In Example 10, the aeration rate was 0.12 vvm.

[0084] In Example 11, the aeration rate was 0.16 vvm.

[0085] In Example 12, the aeration rate was 0.20 vvm. Performance Test 4

[0086] The biomass of *Chlorella proteoglycans* obtained from the cultivation of microalgae in Examples 9-12 was determined using the method described in Performance Test 1. Figure 13 The effect of aeration rate on Chlorella growth is shown, with 0.08 vvm corresponding to Example 7. At lower aeration rates, appropriately increasing the aeration rate can accelerate carbon supply, prevent the formation of a "nutrient dead zone" around the biofilm, and slow the accumulation of oxygen bubbles and metabolic products on the biofilm surface. Both of these effects are beneficial to Chlorella growth. Therefore, as shown in the figure, when the aeration rate increased from 0.04 to 0.12 vvm, the Chlorella biomass yield in the reactor increased from 10.06 g / L to 13.25 g / L. However, when the aeration rate increased from 0.12 to 0.2 vvm, the Chlorella yield decreased to 10.69 g / L. This may be because the excessively high aeration rate caused greater shear force in the reactor liquid, damaging the Chlorella cells.

[0087] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the principles of this application should be covered within the scope of protection of this application.

Claims

1. A photobioreactor with a brush-like carrier, characterized in that, include: support; A reactor, mounted on the support, is made of transparent material and has an aeration disc at the bottom inside the reactor; The light strip is mounted on the support and spirals around the periphery of the reactor; A brush-shaped carrier is disposed in the reactor. The brush-shaped carrier includes a support rod and a plurality of bristle units distributed along the length of the support rod. Each bristle unit is formed by bristles that are densely distributed inside and sparsely distributed outside.

2. The photobioreactor with a brush-like carrier according to claim 1, characterized in that, The reactor is a hollow cylindrical structure with one end open. The bristles are made of polyester fiber. The thickness of each bristle unit is 1.0~1.2cm. The diameter of each bristle unit is adapted to the inner diameter of the reactor, which is 6~8cm.

3. The photobioreactor with a brush-like carrier according to claim 2, characterized in that, The weight of the bristles corresponding to each of the bristle units is 1.0~2.5g.

4. The photobioreactor with a brush-like carrier according to claim 2, characterized in that, The spacing between two adjacent bristle units is 1.0~2.5cm.

5. The application of a photobioreactor with a brush-like carrier as described in any one of claims 1 to 4 in a high-density cultivation method of Chlorella proteoglycans.

6. The application of the photobioreactor with a brush-like carrier according to claim 5 in the high-density cultivation method of Chlorella vulgaris, characterized in that, Includes the following steps: S1. Provide Chlorella proteoglycans and place them in BG11 medium for large-scale culture to obtain Chlorella proteoglycan solution; S2. Add the culture medium and the Chlorella proteoglycan solution obtained in step S1 to the reactor, and maintain the temperature at 22~28℃ and the light intensity at 400~600 μmol / m². 2 High-density cultivation of Chlorella proteoglycans was carried out under the conditions of light-dark ratio of (17~19)h: (5~7)h and aeration rate of 0.04~0.2vvm.

7. The application of the photobioreactor with a brush-like carrier according to claim 6 in the high-density cultivation method of Chlorella vulgaris, characterized in that, In step S2, the aeration rate is 0.08~0.16vvm, and the gas corresponding to the aeration includes the following components by volume percentage: 6~8% CO2, 3~5% O2, and the balance is N2.

8. The application of the photobioreactor with a brush-like carrier according to claim 6 in the high-density cultivation method of Chlorella vulgaris, characterized in that, In step S2, the ratio of the volume of the culture medium, the volume of the Chlorella proteoglycans solution, and the volume of the reactor is (8~9):(1~2):11.

5.

9. The application of the photobioreactor with a brush-like carrier according to claim 6 in a high-density cultivation method of Chlorella vulgaris, characterized in that, In step S2, the culture medium composition is as follows: NaNO3 7.3~7.7 g / L, K2HPO4·3H2O 1.2~1.4 g / L, KH2PO4 0.6~0.7 g / L, MgSO4·7H2O 0.3~0.4 g / L, CaCl2·2H2O 0.16~0.20 g / L, citric acid 0.02~0.04 g / L, ferric ammonium citrate 0.02~0.04 g / L, EDTA 0.004~0.006 g / L, Na2CO3 0.05~0.15 g / L, H3BO3 0.01~0.02 g / L, MnCl2·H2O 0.008~0.010 g / L, ZnSO4·7H2O 0.0010~0.0013g / L, CuSO4·5H2O 0.0002~0.0005g / L, Na2MoO4·2H2O 0.001~0.003g / L, Co(NO3)2·6H2O 0.0001~0.0003g / L.

10. The application of the photobioreactor with a brush-like carrier according to claim 6 in the high-density cultivation method of Chlorella vulgaris, characterized in that, In step S1, the scale-up culture includes multiple rounds of culture. In the first round of culture, the volume ratio of Chlorella proteoglycans to BG11 medium is 1:(8.5~9.5). In the next round of culture, the algal solution obtained from the previous round of culture is mixed with BG11 medium at a volume ratio of 1:(8.5~9.5) for culture. The culture conditions for each round of cultivation included: temperature of 22–28°C and light intensity of 100–200 μmol / m². 2 •s, light-dark ratio is (17~19)h:(5~7)h, aeration rate is 0.1~0.3vvm, and the gas used for aeration is air.

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