Method for efficiently culturing food new resource chlorella

Abstract

The invention discloses a method for efficiently culturing food new resource chlorella, and belongs to the technical field of food new resource mining. According to the chlorella autotrophic culture method, the chlorella is cultured by monitoring the growth condition of the chlorella in real time and dynamically adjusting the ventilation strategy. By monitoring the growth state of the chlorella in real time and dynamically adjusting the ventilation volume, sufficient supply of CO2 is guaranteed, cell damage and energy consumption increase caused by excessive ventilation are avoided, finally, efficient and sustainable accumulation of chlorella biomass and intracellular carbohydrates of the chlorella is achieved, the production cost is reduced, and the culture efficiency is improved.

Description

Technical Field

[0001] The present invention relates to a method for efficiently culturing a new food resource, Chlorella, and belongs to the technical field of new food resource mining. Background Art

[0002] As one of the most photosynthetically efficient organisms in nature, microalgae can effectively alleviate food crises and energy shortages. At the same time, microalgae are rich in nutrients such as protein, carbohydrates and lipids, and are a sustainable food source with functional properties and nutritional value. Chlorella belongs to the Chlorophyta phylum among microalgae, and is characterized by balanced nutrition, making it a new resource food. Chlorella powder, chlorella protein and chlorella polysaccharides are widely used in the general food and health food markets. In order to meet the market demand for the biomass and a certain component of Chlorella, laboratories often use the method of changing the culture conditions of Chlorella to adjust its biomass and nutrient composition, so as to achieve the purpose of increasing biomass and enriching a certain component.

[0003] The determination of the culture conditions of Chlorella often involves multiple parameter variables such as light intensity, ventilation volume, selection of culture medium, and the content of various elements in the culture medium. Under autotrophic culture conditions, Chlorella relies on photosynthesis to convert inorganic carbon (mainly CO2) into organic matter. This process not only requires suitable lighting conditions, but is also highly dependent on the gas exchange efficiency in the culture system, especially the ventilation volume of the air. Laboratories usually use sterilized conical flasks as culture containers for Chlorella and screen their culture parameters by regularly sampling. However, the volume of conical flasks is limited, and there are problems such as the inability to achieve simultaneous culture of multiple culture containers, low degree of automation in the culture process, and time-consuming subsequent algal harvesting, which hinder the development of subsequent culture experiments.

[0004] At the same time, existing autotrophic cultivation methods of Chlorella face many challenges in improving biomass accumulation, growth cycle and biomass productivity of Chlorella. Traditional culture methods usually adopt a fixed aeration strategy, which fails to fully consider the dynamic demand for CO2 at different stages of Chlorella growth, resulting in low culture efficiency and limited biomass accumulation: Research on active substances such as polysaccharides and proteins of Chlorella (Chlorella sorokiniana C74) has disclosed the optimal culture medium conditions, culture environment, and the structure and activity of polysaccharides and proteins of Chlorella. The growth curve, harvested biomass, polysaccharide and protein content were measured at ventilation rates of 24L / h, 32L / h, 40L / h, 48L / h and 60L / h, respectively. However, its fixed aeration strategy has the characteristics of low culture efficiency in the initial growth stage and the end of the growth exponential period until the stable period, and it is impossible to increase the algal cell concentration in the growth exponential period from the perspective of ventilation control (Research on active substances such as polysaccharides and proteins of Chlorella (Chlorella sorokiniana C74) [D]. Hainan University, 2020.); autotrophic culture usually requires a longer culture cycle. The optimization of the cultivation process of Chlorella in an internal light source airlift reactor disclosed the optimal light source wavelength, light intensity, light-dark ratio and ventilation volume for Chlorella cultivation. The optimal cultivation environment was screened through single factor experiments and response surface optimization. The dry weight of Chlorella after 15 days of cultivation was 1.21g / L, and the carbon dioxide ventilation volume during the 15-day cultivation period was 30L / h. The cultivation process has the problems of long growth cycle, low yield and high carbon source cost (Optimization of the cultivation process of Chlorella in an internal light source airlift reactor [J]. Chemical Industry Progress, 2018, 37(02): 525-532.);

[0005] Some researchers have attempted to increase the growth rate and biomass accumulation of Chlorella by adding additional carbon sources or introducing a certain concentration of CO2 gas. For example, patent application CN116515599A discloses a mixotrophic culture reactor for Chlorella vulgaris containing an illumination unit, an aeration unit, and a culture unit. It also proposes a culture method that uses carbon dioxide and glucose as additional carbon sources during the culture process. This patent provides a basic culture system and uses it to screen culture methods. However, it is unable to monitor the growth period of Chlorella vulgaris or adjust culture parameters in real time based on growth curve feedback. These methods are not only complex, increasing operational difficulty, equipment investment, and costs, but may also introduce contamination risks, affecting the stability and sustainability of the culture system.

[0006] Existing technologies show that under normal growth conditions, the carbohydrate content of Chlorella cells is 12% to 17%. Under nitrogen starvation conditions, the carbohydrate content of Chlorella pyrenoidosa is nearly 20% (Study on Enzymatic Extraction of Polysaccharides from Chlorella Algal Residue. Fisheries Science, 2018, 37(1):38-44.). The biomass productivity of Chlorella under autotrophic conditions is limited. Considering the growth characteristics of Chlorella and the physical properties of the culture system, developing an economical, efficient, and high-yield Chlorella biomass autotrophic culture method has become a hot topic and a challenge in current research. Summary of the Invention

[0007] Traditional Chlorella cultivation methods often adopt a fixed aeration strategy, ignoring the important effect of ventilation volume on the growth rate and biomass accumulation of Chlorella.

[0008] To address these issues, the present invention provides an innovative, high-yield autotrophic cultivation method for Chlorella based on dynamic ventilation control. This method monitors the growth status of Chlorella in real time and dynamically adjusts the ventilation strategy to promote nutrient absorption and utilization, ultimately achieving efficient and sustainable accumulation of Chlorella biomass and intracellular carbohydrates. Compared to existing technologies, this method eliminates the need for adding an additional carbon source or introducing high-concentration CO2 gas, reducing production costs and operational complexity. Furthermore, it shortens the cultivation cycle and improves the stability and sustainability of the cultivation system.

[0009] The present invention provides a method for autotrophic cultivation of high-yield Chlorella biomass, wherein the method dynamically adjusts the ventilation volume according to the growth of Chlorella on the basis of an autotrophic culture medium:

[0010] Dynamic ventilation adjustment strategy 1: The ventilation rate is 0.75-1.5 L / min at the initial stage of culture, and the ventilation rate is adjusted to 3-6 L / min from the exponential growth phase until the growth plateau phase;

[0011] Alternatively, dynamic ventilation adjustment strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, 3-6 L / min from the beginning of the exponential growth phase, and 0.75-1.5 L / min in the late exponential phase until the stable growth phase.

[0012] In one embodiment of the present invention, according to dynamic ventilation adjustment strategy 1, preferably, the dynamic ventilation adjustment is: the ventilation volume at the initial stage of culture is 1.5 L / min, and the ventilation volume is adjusted to 3-5 L / min from the growth exponential phase to the growth stable phase.

[0013] In one embodiment of the present invention, according to dynamic ventilation adjustment strategy 2, preferably, the dynamic ventilation adjustment is: the ventilation volume at the initial stage of culture is 1.5 L / min, the ventilation volume is adjusted to 4-6 L / min from the beginning of the growth exponential phase, and the ventilation volume is adjusted to 1.5 L / min from the late exponential phase to the stable growth phase;

[0014] More preferably, the dynamic ventilation adjustment is as follows: the ventilation volume at the initial stage of culture is 1.5 L / min, the ventilation volume is adjusted to 5 L / min at the beginning of the growth exponential phase, and the ventilation volume is adjusted to 1.5 L / min in the late exponential phase until the growth stable phase.

[0015] In one embodiment of the present invention, the Chlorella is Chlorella pyrenoidosa.

[0016] In one embodiment of the present invention, according to the above-mentioned dynamic ventilation adjustment strategy 2, the method includes the following steps:

[0017] (1) Add the Chlorella mother liquid and culture medium into the column reactor; turn on the air pump and light, adjust the initial gas flow rate to 0.75-1.5 L / min, mix the Chlorella and culture medium, and start the culture;

[0018] (2) Maintaining light intensity, when Chlorella begins to grow in the exponential phase, increase the ventilation rate to 3-6 L / min, and when Chlorella grows to the late exponential phase, reduce the ventilation rate to 0.75-1.5 L / min;

[0019] (3) stopping the cultivation during the stable growth period of Chlorella and harvesting the Chlorella;

[0020] Preferably, in step (2), the ventilation rate is increased to 4-6 L / min when the growth of Chlorella begins in the exponential phase, and the ventilation rate is reduced to 1.5 L / min when the growth of Chlorella reaches the late exponential phase;

[0021] More preferably, in step (2), the ventilation rate is increased to 5 L / min when the Chlorella starts to grow in the exponential phase, and the ventilation rate is reduced to 1.5 L / min when the Chlorella grows to the late exponential phase.

[0022] In one embodiment of the present invention, the addition ratio of the Chlorella mother solution and the culture medium in step (1) is (0.8-1):(8-12) (v / v); preferably, the ratio is 1:10 (v / v).

[0023] In one embodiment of the present invention, the Chlorella mother solution in step (1) is in a logarithmic growth phase, which is specifically determined by measuring the absorbance; if the absorbance continues to increase, it indicates that the cell concentration increases, and it is considered to be in a logarithmic growth phase.

[0024] In one embodiment of the present invention, the biomass density of Chlorella is 0.06-0.09 g / L after the Chlorella mother liquid and culture medium are added to the column reactor in step (1).

[0025] In one embodiment of the present invention, the culture temperature in step (1) is 24-28° C.; and the culture medium is TAP culture medium.

[0026] In one embodiment of the present invention, the initial gas flow rate in step (1) is 1.5 L / min.

[0027] In one embodiment of the present invention, the illumination intensity of the illumination is 5000-7000 Lux, and the light-to-dark ratio is (8-12)h:(8-12)h.

[0028] In one embodiment of the present invention, OD 680 The detection method is:

[0029] 200 μL of Chlorella fermentation broth was added to a 96-well plate and placed in a microplate reader to detect absorbance at 680 nm;

[0030] Alternatively, the live cell concentration sensor is used to detect the Chlorella fermentation broth.

[0031] In one embodiment of the present invention, the OD of the Chlorella when the Chlorella starts to grow in the exponential phase in step (2) is 680 0.35~0.90; OD of Chlorella in late exponential phase 680 It is 1.5 to 2.2.

[0032] In one embodiment of the present invention, the OD of Chlorella during the growth stabilization period in step (3) is 680 It is 1.7 to 2.3.

[0033] In one embodiment of the present invention, the harvesting method in step (3) is electrochemical harvesting, and the harvesting is stopped when the culture medium and the Chlorella algae are clearly separated; the electrochemical harvesting stage lasts for 30 to 50 minutes.

[0034] In one embodiment of the present invention, the cycle of autotrophic culture is 5 to 10 days.

[0035] In one embodiment of the present invention, according to the above-mentioned dynamic ventilation adjustment strategy 1, the method includes the following steps:

[0036] (1) Add the Chlorella mother liquid and culture medium into the column reactor; turn on the air pump and light, adjust the initial gas flow rate to 0.75-1.5 L / min, mix the Chlorella and culture medium, and start the culture;

[0037] (2) Maintain light intensity. When the Chlorella enters the exponential growth phase, increase the ventilation rate to 3-6 L / min.

[0038] (3) stopping the cultivation during the stable growth period of Chlorella and harvesting the Chlorella;

[0039] Preferably, in step (2), when the growth of Chlorella begins in the exponential phase, the ventilation rate is increased to 4-6 L / min;

[0040] More preferably, in step (2), the ventilation rate is increased to 5 L / min when the Chlorella is in the exponential growth phase.

[0041] In one embodiment of the present invention, the addition ratio of the Chlorella vulgaris and the culture medium in step (1) is (0.8-1):(8-12) (v / v); preferably, the ratio is 1:10 (v / v).

[0042] In one embodiment of the present invention, the Chlorella mother solution in step (1) is in a logarithmic growth phase, which is specifically determined by measuring the absorbance; if the absorbance continues to increase, it indicates that the cell concentration increases, and it is considered to be in a logarithmic growth phase.

[0043] In one embodiment of the present invention, the biomass density of Chlorella is 0.06-0.09 g / L after the Chlorella mother liquid and culture medium are added to the column reactor in step (1).

[0044] In one embodiment of the present invention, the culture temperature in step (1) is 24-28° C.; and the culture medium is TAP culture medium.

[0045] In one embodiment of the present invention, the initial gas flow rate in step (1) is 1.5 L / min.

[0046] In one embodiment of the present invention, the illumination intensity of the illumination is 5000-7000 Lux, and the light-to-dark ratio is (8-12)h:(8-12)h.

[0047] In one embodiment of the present invention, the OD of the Chlorella when the Chlorella starts to grow in the exponential phase in step (2) is 680 It is 0.35~0.90.

[0048] In one embodiment of the present invention, OD 680 The detection method is:

[0049] 200 μL of Chlorella fermentation broth was added to a 96-well plate and placed in a microplate reader to detect absorbance at 680 nm;

[0050] Alternatively, the live cell concentration sensor is used to detect the Chlorella fermentation broth.

[0051] In one embodiment of the present invention, the OD of Chlorella in the stable growth period of Chlorella in step (3) is 680 It is 1.7 to 2.3.

[0052] In one embodiment of the present invention, the harvesting method in step (3) is electrochemical harvesting, and the harvesting is stopped when the culture medium and the Chlorella algae are clearly separated.

[0053] In one embodiment of the present invention, the electrochemical recovery stage lasts for 30 to 50 minutes.

[0054] In one embodiment of the present invention, the cycle of autotrophic culture is 5 to 10 days.

[0055] In one embodiment of the present invention, the autotrophic cultivation method for high-yield Chlorella biomass comprises the following steps:

[0056] The mother solution of Chlorella and TAP medium were added to the column reactor at a ratio of 1:10 (v / v), so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.08 g / L; the air pump and light were turned on, and the gas flow rate was adjusted to 1.5 L / min, the light-dark ratio was 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C, and the autotrophic culture of Chlorella was started; when the Chlorella growth began to enter the exponential phase (at this time OD 680 0.57±0.03), increase the ventilation rate to 5L / min, when Chlorella grows to the late exponential phase (OD 680 When the growth of Chlorella vulgaris was in the stable period (OD 680 When the pH value was 2.15±0.11), the culture was stopped and electrochemical harvesting was performed. When the culture medium and the algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, and the cultured Chlorella was collected.

[0057] In one embodiment of the present invention, the method utilizes a device for efficient cultivation of Chlorella to cultivate Chlorella; the device for efficient cultivation of Chlorella comprises: an aeration unit, a lighting unit, a temperature control unit, a culture and harvesting unit, and a control unit;

[0058] The aeration unit includes an aeration pump, a vent pipe and an aeration plate. A sterilizing filter is provided at the air inlet of the aeration pump. The aeration pump is connected to the vent pipe. The vent pipe enters the air inlet at the top of the column reactor and is connected to the aeration plate inside the reactor.

[0059] The illumination unit comprises a series of full-light-path culture light strips, which are installed in a light strip frame;

[0060] The temperature control unit includes a heating tube and a temperature sensor, the heater and the temperature sensor are respectively located on the outer side and inner side wall of the column reactor, and the temperature sensor is fixed through the connection site at the top of the column reactor;

[0061] The culture and harvesting unit includes 2 to 6 column reactors, a live cell concentration sensor, and an electrochemical harvesting electrode. The column reactor is made of transparent material and is cylindrical. The top cover of the reactor is provided with an air inlet, an exhaust port for installing a filter membrane, and four connection sites. The live cell concentration sensor is located at the top cover of the column reactor and immersed in the culture medium for real-time monitoring of the growth curve of Chlorella vulgaris. The electrochemical harvesting electrode is connected to the column reactor cover and is located in the column reactor in a plate-like manner.

[0062] The control unit includes a PLC control system and a controller. The control points of the PLC control system are the air pump, light strip, heating tube, temperature sensor, living cell concentration sensor and electrochemical harvesting electrode; the controller is electrically connected to the air pump, light strip, heating tube, temperature sensor, living cell concentration sensor and electrochemical harvesting electrode respectively.

[0063] The present invention also provides a method for increasing the biomass of Chlorella through autotrophic culture. The method dynamically adjusts the ventilation volume according to the growth of Chlorella on the basis of the autotrophic culture medium:

[0064] Dynamic ventilation adjustment strategy 1: The ventilation rate is 0.75-1.5 L / min at the initial stage of culture, and the ventilation rate is adjusted to 3-6 L / min from the exponential growth phase until the growth plateau phase;

[0065] Alternatively, dynamic ventilation adjustment strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, 3-6 L / min from the beginning of the exponential growth phase, and 0.75-1.5 L / min in the late exponential phase until the stable growth phase.

[0066] In one embodiment of the present invention, according to dynamic ventilation adjustment strategy 1, preferably, the dynamic ventilation adjustment is: the ventilation volume at the initial stage of culture is 1.5 L / min, and the ventilation volume is adjusted to 3-5 L / min from the growth exponential phase to the growth stable phase.

[0067] In one embodiment of the present invention, according to dynamic ventilation adjustment strategy 2, preferably, the dynamic ventilation adjustment is: the ventilation volume at the initial stage of culture is 1.5 L / min, the ventilation volume is adjusted to 4-6 L / min from the beginning of the growth exponential phase, and the ventilation volume is adjusted to 1.5 L / min from the late exponential phase to the stable growth phase;

[0068] More preferably, the dynamic ventilation adjustment is as follows: the ventilation volume at the initial stage of culture is 1.5 L / min, the ventilation volume is adjusted to 5 L / min at the beginning of the growth exponential phase, and the ventilation volume is adjusted to 1.5 L / min in the late exponential phase until the growth stable phase.

[0069] The present invention also provides a method for increasing the proportion of carbohydrates in Chlorella cells through autotrophic culture. The method is based on the autotrophic culture medium and dynamically adjusts the ventilation volume according to the growth of Chlorella:

[0070] Dynamic ventilation adjustment strategy 1: The ventilation rate is 0.75-1.5 L / min at the initial stage of culture, and the ventilation rate is adjusted to 3-6 L / min from the exponential growth phase until the growth plateau phase;

[0071] Alternatively, dynamic ventilation adjustment strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, 3-6 L / min from the beginning of the exponential growth phase, and 0.75-1.5 L / min in the late exponential phase until the stable growth phase.

[0072] In one embodiment of the present invention, according to dynamic ventilation adjustment strategy 1, preferably, the dynamic ventilation adjustment is: the ventilation volume at the initial stage of culture is 1.5 L / min, and the ventilation volume is adjusted to 3-5 L / min from the growth exponential phase to the growth stable phase.

[0073] In one embodiment of the present invention, according to dynamic ventilation adjustment strategy 2, preferably, the dynamic ventilation adjustment is: the ventilation volume at the initial stage of culture is 1.5 L / min, the ventilation volume is adjusted to 4-6 L / min from the beginning of the growth exponential phase, and the ventilation volume is adjusted to 1.5 L / min from the late exponential phase to the stable growth phase;

[0074] More preferably, the dynamic ventilation adjustment is as follows: the ventilation volume at the initial stage of culture is 1.5 L / min, the ventilation volume is adjusted to 5 L / min at the beginning of the growth exponential phase, and the ventilation volume is adjusted to 1.5 L / min in the late exponential phase until the growth stable phase.

[0075] Beneficial effects:

[0076] Ventilation not only directly affects the CO2 concentration in the culture medium, and thus the photosynthesis rate, but also indirectly affects the metabolic activity and cell health of Chlorella by regulating the dissolved oxygen level and pH value of the culture medium. Optimizing the ventilation strategy is crucial for increasing the biomass of Chlorella. To this end, the present invention provides an innovative, high-yield autotrophic cultivation method for Chlorella based on dynamic ventilation control. This method monitors Chlorella growth in real time and adjusts the ventilation strategy to respond to the actual needs of Chlorella during growth, providing technical support for achieving efficient and sustainable production of Chlorella biomass.

[0077] (1) The method of the present invention uses a two-stage aeration strategy, and the biomass of Chlorella obtained by autotrophic culture can reach more than 0.7 g / L, and the proportion of intracellular carbohydrates can reach more than 23%;

[0078] (2) The method of the present invention uses a three-stage aeration strategy, and the biomass of Chlorella obtained by autotrophic culture can reach more than 0.8 g / L, and the proportion of intracellular carbohydrates can reach more than 24%;

[0079] (3) The dynamically adjusted ventilation strategy in the method of the present invention not only ensures an adequate supply of CO2, but also avoids cell damage and increased energy consumption caused by excessive ventilation, thereby increasing biomass and intracellular carbohydrates of Chlorella. The technical effect is superior to the fixed ventilation strategy, while reducing production costs and improving culture efficiency.

[0080] (4) The Chlorella cultivation equipment of the present invention has the technical advantages of simple operation, high degree of automation, sterile cultivation environment, and low economic investment. It can automatically monitor the growth curve of Chlorella and realize automatic harvesting. When using the method of the present invention to cultivate Chlorella, the cultivation cycle is short, only 5 to 10 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is a front view of the column reactor culture structure, including: 1. Ventilation pump; 2. Ventilation tube; 3. Aeration plate; 4. Column reactor; 5. Full-light culture light strip; 6. Light strip frame; 7. Heating tube; 8. Temperature sensor; 9. Viable cell concentration sensor; 10. Electrochemical harvesting electrode; 11. Electrochemical power supply; 12. Exhaust port; 13. Drain pipe.

[0082] Figure 2 This is a top view of the column reactor culture structure, including: 1, ventilation pump; 4, column reactor; 5, full-light path culture light strip; 6, light strip frame; 9, live cell concentration sensor; 10, electrochemical harvesting electrode; 12, exhaust port; 14, air inlet;

[0083] Figure 3 This is a simplified diagram of the culture condition screening system architecture, in which: 15, a control system, the culture container is separately connected to the control system end;

[0084] Figure 4 This is a growth curve diagram of different culture media during the dynamic adjustment of ventilation volume;

[0085] Figure 5 This is the growth curve of Chlorella under different ventilation conditions. DETAILED DESCRIPTION

[0086] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0087] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0088] The chlorella used in the following examples is Chlorella pyrenoidosa, purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences, with the number FACHB-11.

[0089] The culture medium involved in the following examples is as follows:

[0090] BG-11 culture medium was purchased from Haibo Biotechnology;

[0091] The preparation method of BBM medium is as follows:

[0092] (1) Trace element stock solution composition: ZnSO4·7H2O 8.82 g / L, MnCl2·4H2O 1.44 g / L, MoO3 0.71 g / L, CuSO4·5H2O 1.57 g / L, Co(NO3)2·6H2O 0.49 g / L;

[0093] (2) Solution 1 components: Na2EDTA 50 g / L, KOH 3.1 g / L;

[0094] (3) Solution 2 components: FeSO4 4.98g / L H2SO4 1mL / L;

[0095] (4) BBM medium components:

[0096] KH2PO4 175mg / L, CaCl2·2H2O 25mg / L, MgSO4·7H2O 75mg / L, NaNO3 250mg / L, K2HPO475mg / L, NaCl 25mg / L, H3BO3 11.42mg / L, trace element stock solution 1mL / L, solution 1 1mL / L, solution 21mL / L.

[0097] The preparation method of TAP medium is as follows:

[0098] (1) Preparation of TAP salt: 15 g NH4Cl, 4 g MgSO4·7H2O, 2 g CaCl2·2H2O, add water to 1 L;

[0099] (2) Preparation of phosphate solution: K2HPO4 28.8g, KH2PO4 14.4g, add water to 100mL;

[0100] (3) Preparation of trace elements:

[0101] EDTAdisodium salt 50g 250mL, ZnSO4·7H2O 22g 100mL, H3BO3 11.4g 200mL, MnCl2·4H2O 5.06g 50mL, CoCl2·6H2O 1.61g 50mL, CuSO4·5H2O 1.57g 50mL, (NH4)6MO7O 24 4H2O (1.10 g, 50 mL) and FeSO4·7H2O (4.99 g, 50 mL). Dissolve EDTA in boiling water, and prepare the FeSO4 last to prevent oxidation. Combine all solutions except the EDTA, bring to a boil, and then add the EDTA solution, which will turn the mixture green. Once dissolved, cool to 70°C. Maintaining the temperature at 70°C, add 85 mL of hot 20% KOH solution (20 g / 100 mL) to bring the total volume of the final solution to 1 L. Stopper the flask with a cotton plug and let it sit for 1 week, shaking it daily. The solution should eventually turn purple and leave a rust-brown precipitate. Refrigerate at 4°C.

[0102] (4) Mix the following substances to obtain the final TAP medium:

[0103] 2.42 g of Tris, 1 mL of glacial acetic acid, 25 mL of the TAP salt prepared in step (1), 0.375 mL of the phosphate solution prepared in step (2), and 1.0 mL of the trace elements prepared in step (3) were added to 1 L of water to obtain TAP culture medium.

[0104] The Chlorella mother solution in the logarithmic growth phase mentioned in the following examples is specifically determined by measuring absorbance. During the culture process, the algae seed stock solution is simultaneously expanded. Specifically, when adding expansion medium at a ratio of 1:10 by volume, 100 mL of the algae seed solution is simultaneously retained and expanded to 1 L, which serves as the mother solution for the next culture. During this period, the absorbance of the mother solution is measured. If the absorbance continues to increase, it indicates an increase in cell concentration and is considered to be in the logarithmic growth phase.

[0105] The methods involved in the following embodiments are as follows:

[0106] 1. Drying method of chlorella:

[0107] The harvested chlorella was washed with water and centrifuged at 4000 r / min for 10 minutes, repeated three times, and then pre-frozen in a -80°C refrigerator, followed by freeze-drying to obtain chlorella powder.

[0108] 2. Calculation of biomass:

[0109] The freeze-dried Chlorella powder was weighed to obtain the biomass.

[0110] 3. Determination of intracellular carbohydrate content: phenol-sulfuric acid method;

[0111] (1) Determination of standard curve:

[0112] Dry about 0.1g of standard glucose at 105℃, wait for it to cool, accurately weigh 25mg of constant weight glucose, add distilled water to dissolve, and dilute to 250mL, shake well, and prepare a 0.1mg / mL standard glucose solution. Pipette 0, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9mL of standard solution in sequence, and add deionized water to 1.0mL. Add 0.5mL of 5% phenol solution respectively, and quickly add 2.5mL of concentrated sulfuric acid, vortex for 20s to mix it, react at room temperature for 20min, and measure the absorbance at 490nm. Draw a standard curve with A490 as the ordinate and glucose content (mg) as the abscissa;

[0113] (2) Determination of intracellular carbohydrate content in samples:

[0114] Weigh 20 mg of algal powder and suspend it in 1 mL of 6 mol / L hydrochloric acid and 2.5 mL of distilled water. Boil for 30 minutes until all carbohydrates are digested into glucose. Pipette the reaction solution and dilute it to 1 mL. Add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid, mix well, and let it rest for 20 minutes. Measure the OD at 490 nm and calculate the intracellular carbohydrate content using a standard curve.

[0115] The chlorella cultivation method of the following embodiment uses a device for efficient chlorella cultivation to cultivate chlorella; the device includes: an aeration unit, a lighting unit, a temperature control unit, a culture and harvesting unit, and a control unit.

[0116] like Figure 1 and Figure 2 As shown, the aeration unit includes an aeration pump 1, a ventilation pipe 2 and an aeration plate 3. A 0.22 μm filter is provided at the air inlet of the ventilation pump. The ventilation pipe 2 is a rubber hose. The micropores of the aeration plate disperse the incoming sterile air into a large number of small bubbles that mix with the culture medium, which is conducive to the growth of Chlorella. The ventilation volume required for the culture is adjusted by the output of the control unit to the air pump control end.

[0117] The lighting unit is a full-light-path culture light strip 5 connected in series and fixed in a light strip frame. The control unit adjusts the light intensity, light-dark ratio and light cycle required for culture by outputting the light control end. The light strip frame is fixed around the outside of the column reactor.

[0118] The temperature control unit includes a heating tube 7 and a temperature sensor 8, which are respectively located on the outer side and inner wall side of the column reactor. The temperature sensor is fixed through the connection site at the top of the column reactor. The control unit input terminal monitors and receives the temperature sensor signal in real time, and the output terminal starts or shuts down the heater to maintain the required culture temperature.

[0119] The culture and harvesting unit includes 2 to 6 column reactors 4, a viable cell concentration sensor 9, an electrochemical harvesting electrode 10, an electrochemical power supply 11, and a drainage pipe 13. The column reactor is made of transparent material with a height-to-diameter ratio of 2:1. The total reactor volume is 5L, and the effective culture volume is 4.5L. The top cover of the column reactor is made of tempered material and includes an air inlet 14, an exhaust port 12, and three connection points. The vent pipe enters the column reactor from the air inlet and connects to the aeration disk at the bottom of the column reactor. Filters are installed at the air inlet and exhaust ports to intercept bacteria and impurities in the air, ensuring sterile conditions during the culture process. The exhaust port is equipped with a 0.22μm filter membrane.

[0120] The three connection sites include a live cell concentration sensor connection site and two electrochemical harvesting electrode sites. The live cell concentration sensor connection site has a power supply path connection containing a high-frequency transformer, which applies a real-time changing voltage to its sensor, forming a high-frequency changing electric field, thereby realizing real-time concentration monitoring. The two connection sites of the electrochemical harvesting electrode are respectively connected to the positive and negative poles of the power supply. During the incubation phase, the path switch is open; during the harvesting phase, the path switch is closed, starting electrochemical flotation harvesting. The electrode materials used in the electrochemical harvesting electrodes are all non-sacrificial carbon electrodes.

[0121] A drainage pipe is provided at the bottom of the column reactor. The drainage pipe is assembled from two segmented detachable pipes. A 2μm filter membrane is connected between the two pipes to separate the Chlorella algae and the culture medium.

[0122] The control unit includes a PLC control system and a controller. The control points of the PLC control system are the air pump 1, the light strip 5, the heating tube 7, the temperature sensor 8, the living cell concentration sensor 9 and the electrochemical harvesting electrode 10; the controller is electrically connected to the air pump 1, the light strip 5, the heating tube 7, the temperature sensor 8, the living cell concentration sensor 9 and the electrochemical harvesting electrode 10 respectively for adjusting the culture parameters.

[0123] Synchronous culture in multiple culture tanks Figure 3 shown.

[0124] The application method of culturing Chlorella using the above-mentioned device comprises the following steps:

[0125] (1) Training stage:

[0126] Pour the sterilized culture medium and Chlorella into the sterilized column reactor 4 at a volume ratio of 10:1. The column reactor lid is then securely closed. The total volume of the column reactor is 5 L, and the usable volume is 90% of the total volume, meaning that 4.5 L can be used for Chlorella cultivation. Turn on the air pump 1 from the control system and adjust the air flow to the appropriate level. The light intensity, light-dark ratio, photoperiod, and culture temperature are also set on the control system.

[0127] Temperature sensor 8 monitors the culture system's temperature in real time. The control unit's input monitors and receives signals from temperature sensor 8 in real time, while its output activates or deactivates the heater to maintain the desired culture temperature. A viable cell concentration sensor 9 monitors the Chlorella cell concentration within the reactor in real time and generates a corresponding growth curve on the control system.

[0128] (2) Harvesting stage:

[0129] When the Chlorella vulgaris reaches a stable growth phase, the viable cell concentration sensor power supply is turned off, and the electrochemical harvesting power supply 11 is turned on to begin flotation harvesting of the algae. The electrochemical harvesting phase lasts for 30 to 50 minutes and stops when the culture medium and algae are clearly separated. The algae are lifted above the culture medium by the gases generated during the electrochemical process and the sterile air from the aeration disk 3. The harvesting power supply 11 is then turned off, and the drain valve of the drain pipe 13 is opened. The culture medium flows out of the pipe, while the algae are trapped within it. The pipe is then disassembled and the resulting Chlorella vulgaris is collected.

[0130] Example 1: Two-stage aeration culture method for Chlorella

[0131] This embodiment uses the above-mentioned equipment to conduct experiments, including the following steps:

[0132] Clean and sterilize the column reactor, rubber hose, aeration plate and other components related to microalgae cultivation; connect the four column reactors to a control system separately, and independently adjust the reactor's ventilation volume, light intensity and cycle, cultivation temperature and other parameters on the control system;

[0133] The column reactor 1 was added with a 1:10 (v / v) ratio of Chlorella mother solution in the logarithmic growth phase and TAP medium, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.09 g / L; the air pump and light switch were turned on at the control system end, the initial ventilation volume was adjusted to 0.75 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, the culture temperature was 27°C, and the culture was started; the growth curve of Chlorella was monitored by a viable cell concentration sensor, as shown in FIG. Figure 5 As shown; when the growth begins to enter the exponential phase (OD 680was 0.73±0.1), the ventilation volume was increased to 3L / min; when the growth was in the stable period (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0134] The mother solution of Chlorella vulgaris in the logarithmic growth phase and TAP medium were added to the column reactor 2 at a ratio of 1:10 (v / v), so that the Chlorella vulgaris and the medium were evenly mixed without precipitation, and the Chlorella vulgaris biomass density was 0.09 g / L; the air pump and light switch were turned on at the control system end, the initial ventilation volume was adjusted to 0.75 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, the culture temperature was 27°C, and the culture was started; the growth curve of Chlorella vulgaris was monitored by the viable cell concentration sensor, as shown in FIG. Figure 5 As shown; when the growth begins to enter the exponential phase (OD 680 was 0.71±0.08), the ventilation volume was increased to 5L / min; when the growth was in the stable period (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0135] The column reactor 3 was added with a 1:10 (v / v) ratio of Chlorella mother solution in the logarithmic growth phase and TAP medium, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.09 g / L; the air pump and light switch were turned on at the control system end, the initial ventilation volume was adjusted to 1.5 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, the culture temperature was 27°C, and the culture was started; the growth curve of Chlorella was monitored by the viable cell concentration sensor, as shown in FIG. Figure 5 As shown; when the growth begins to enter the exponential phase (OD 680 0.75±0.11), increase the ventilation volume to 3L / min; when the growth is in the stable period (at this time OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0136] The column reactor 4 was added with a 1:10 (v / v) ratio of Chlorella mother solution in the logarithmic growth phase and TAP medium, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.09 g / L; the air pump and light switch were turned on at the control system end, the initial ventilation volume was adjusted to 1.5 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, the culture temperature was 27°C, and the culture was started; the growth curve of Chlorella was monitored by the viable cell concentration sensor, as shown in FIG. Figure 5 As shown; when the growth begins to enter the exponential phase (OD 680 was 0.72±0.10), the ventilation volume was increased to 5L / min; when the growth was in the stable period (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0137] As shown in Tables 1 and 2, the dry weight of Chlorella cultured with two-stage aeration reached over 0.7 g / L, and the intracellular carbohydrate content of Chlorella reached over 23%. The dry weight and intracellular carbohydrate content of Chlorella increased significantly during the two-stage aeration culture with an initial aeration rate of 1.5 L / min and an exponential aeration rate of 5 L / min.

[0138] Table 1 Dry weight of Chlorella after two-stage aeration harvest

[0139]

[0140] Note: Ventilation is expressed as "initial ventilation - exponential ventilation"; different superscript letters in the numbers indicate significant differences between the data (p < 0.05).

[0141] Table 2 Intracellular carbohydrate content of Chlorella after two-stage aeration harvest

[0142]

[0143] Note: Ventilation is expressed as "initial ventilation - exponential ventilation"; different superscript letters in the numbers indicate significant differences between the data (p < 0.05).

[0144] Example 2: Three-stage aeration culture method for Chlorella

[0145] This embodiment uses the above-mentioned equipment to conduct experiments, including the following steps:

[0146] Clean and sterilize the microalgae cultivation-related components, including the column reactor, rubber hose, and aeration disk; connect each of the six column reactors to a separate control system, which can independently adjust parameters such as the reactor's ventilation volume, light intensity and cycle, and cultivation temperature;

[0147] The column reactor 1 was added with a 1:10 ratio (v / v) of Chlorella mother solution in the logarithmic growth phase and TAP medium, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.08 g / L; the air pump and light switch were turned on at the control system end, and the gas flow rate was adjusted to 1.5 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C; the growth curve of Chlorella was monitored by a live cell concentration sensor, and when the growth began to enter the exponential phase (OD 680 When the growth reaches the late exponential phase (OD 680 When the growth was in the stable phase (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0148] The mother solution of Chlorella in the logarithmic growth phase and TAP medium in a ratio of 1:10 (v / v) were added to the column reactor 2, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.08 g / L; the air pump and the light switch were turned on at the control system end, and the gas flow rate was adjusted to 1.5 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C; the growth curve of Chlorella was monitored by a live cell concentration sensor. When the growth began to enter the exponential phase (OD 680 When the growth reached the late exponential phase (OD 680 When the growth was in the stable phase (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0149] The mother solution of Chlorella in the logarithmic growth phase and TAP medium in a ratio of 1:10 (v / v) were added to the column reactor 3, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.08 g / L; the air pump and the light switch were turned on at the control system end, and the gas flow rate was adjusted to 1.5 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C. The growth curve of Chlorella was monitored by the viable cell concentration sensor. When the growth began to enter the exponential phase (OD 680 When the growth reached the late exponential phase (OD 680 When the growth was in the stable phase (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0150] The column reactor 4 was added with a 1:10 ratio (v / v) of Chlorella mother solution in the logarithmic growth phase and TAP medium, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.08 g / L; the air pump and light switch were turned on at the control system end, and the gas flow rate was adjusted to 1.5 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C; the growth curve of Chlorella was monitored by a viable cell concentration sensor, and when the growth began to enter the exponential phase (at this time OD 680 When the growth reached the late exponential phase (OD 680 When the growth was in the stable phase (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were obviously separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0151] The column reactor 5 was added with a 1:10 ratio (v / v) of Chlorella mother solution in the logarithmic growth phase and TAP medium, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.08 g / L; the air pump and light switch were turned on at the control system end, and the gas flow rate was adjusted to 0.75 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C; the growth curve of Chlorella was monitored by a viable cell concentration sensor, and when the growth began to enter the exponential phase (at this time OD 680 When the growth reaches the late exponential phase (OD 680 When the growth was in the stable phase (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0152] The column reactor 6 was added with a 1:10 ratio (v / v) of Chlorella mother solution in the logarithmic growth phase and TAP medium, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.08 g / L; the air pump and light switch were turned on at the control system end, the gas flow rate was adjusted to 0.75 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, the culture temperature was 27°C, and the growth curve of Chlorella was monitored by a viable cell concentration sensor. When the growth began to enter the exponential phase (OD 680 When the growth reaches the late exponential phase (OD 680 When the growth was in the stable phase (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0153] Tables 3 and 4 show that, in this example, the three-stage dynamic regulation of aeration rate on TAP basal medium resulted in a Chlorella dry weight exceeding 0.8 g / L and a 24% or greater intracellular carbohydrate content. Chlorella biomass reached its maximum when aeration rates were set at 1.5 L / min in the early growth phase, adjusted to 5 L / min during the exponential phase, and then 1.5 L / min during the late exponential phase. The harvested biomass reached a dry weight of 0.90 ± 0.01 g. This strategy also achieved the highest intracellular carbohydrate content of 25.31 ± 1.73% after harvest.

[0154] Table 3 Dry weight of Chlorella after three-stage aeration harvest

[0155]

[0156] Note: Ventilation is expressed as "initial ventilation - exponential ventilation - post-exponential ventilation"; different superscript letters in the numbers indicate significant differences between the data (p < 0.05).

[0157] Table 4 Intracellular carbohydrate content of Chlorella after three-stage aeration harvest

[0158]

[0159] Note: Ventilation is expressed as "initial ventilation - exponential ventilation - post-exponential ventilation"; different superscript letters in the numbers indicate significant differences between the data (p < 0.05).

[0160] Example 3: Screening of autotrophic culture medium for high-yield Chlorella pyrenoidosa biomass

[0161] This embodiment uses the above-mentioned equipment to conduct experiments, including the following steps:

[0162] The column reactor, rubber tube, aeration plate and other microalgae culture-related components are cleaned and sterilized; the three column reactors are separately connected to a control system, and the control system can independently adjust the reactor's ventilation volume, light intensity and cycle, culture temperature and other parameters.

[0163] The mother solution of Chlorella vulgaris in the logarithmic growth phase and BBM culture medium in a ratio of 1:10 (v / v) were added to the column reactor 1, so that the Chlorella vulgaris and the culture medium were evenly mixed without precipitation, and the Chlorella vulgaris biomass density was 0.09 g / L; the air pump and the light switch were turned on at the control system end, and the gas flow rate was adjusted to 0.75 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C; the growth curve of Chlorella vulgaris was monitored by a viable cell concentration sensor; when the growth was in the exponential phase (OD 680When the growth reaches the late exponential phase (OD 680 When the growth is in the stable period (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, and the cultured Chlorella vulgaris was collected. Its biomass was recorded through subsequent drying treatment.

[0164] The mother solution of Chlorella vulgaris in the logarithmic growth phase and BG-11 culture medium in a ratio of 1:10 (v / v) were added to the column reactor 2, so that the Chlorella vulgaris and the culture medium were evenly mixed without precipitation, and the Chlorella vulgaris biomass density was 0.09 g / L; the air pump and the light switch were turned on at the control system end, and the gas flow rate was adjusted to 0.75 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C; the growth curve of Chlorella vulgaris was monitored by a viable cell concentration sensor; when the growth was in the exponential phase (OD 680 When the growth reached the late exponential phase (OD 680 When the growth is in the stable period (OD 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were obviously separated, the electrochemical harvesting was stopped, the drain valve was opened, and the cultured Chlorella vulgaris was collected. Its biomass was recorded through subsequent drying treatment.

[0165] The mother solution of Chlorella in the logarithmic growth phase and TAP medium in a ratio of 1:10 (v / v) were added to the column reactor 3, so that the Chlorella and the medium were evenly mixed without precipitation, and the Chlorella biomass density was 0.09 g / L; the air pump and the light switch were turned on at the control system end, and the gas flow rate was adjusted to 0.75 L / min, the light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C; the growth curve of Chlorella was monitored by a viable cell concentration sensor; when the growth was in the exponential phase (OD 680 When the growth reaches the late exponential phase (OD 680 When the growth is in the stable period (OD 680The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were obviously separated, the electrochemical harvesting was stopped, the drain valve was opened, and the cultured Chlorella vulgaris was collected. Its biomass was recorded through subsequent drying treatment.

[0166] Table 5 Dry weight of harvested Chlorella cultured in different autotrophic media

[0167]

[0168] Note: Different superscript letters in the numbers indicate significant differences between the data (p < 0.05).

[0169] From Table 5 and Figure 4 It can be seen that: among the three autotrophic culture media used, the Chlorella cultured in TAP medium grew rapidly, with a biomass of 0.67 g / L after 10 days of culture, which is high.

[0170] Comparative Example 1: Method for culturing Chlorella using a fixed aeration strategy

[0171] This embodiment uses the above-mentioned equipment to conduct experiments, including the following steps:

[0172] Clean and sterilize the microalgae cultivation-related components, including the column reactor, rubber hose, and aeration disk; connect the five column reactors to a separate control system, which can independently adjust the reactor's ventilation volume, light intensity and cycle, and cultivation temperature.

[0173] The mother solution of Chlorella vulgaris in the logarithmic growth phase and TAP medium were added to five column reactors at a ratio of 1:10 (v / v), and the biomass density of Chlorella vulgaris was 0.08 g / L. The air pump and light switch were turned on at the control system end, and the ventilation volume was adjusted to 0 L / min (column reactor 1), 0.75 L / min (column reactor 2), 1.5 L / min (column reactor 3), 3 L / min (column reactor 4), and 5 L / min (column reactor 5), respectively. The light-dark ratio was adjusted to 12h:12h, the light intensity was 5000 Lux, and the culture temperature was 27°C.

[0174] The growth curve of Chlorella was monitored by using a live cell concentration sensor. Figure 5 When the growth is in the stable phase (at this time, the OD 680 The OD in column reactor 2 was 1.27±0.01; 680 The OD in column reactor 3 was 1.54±0.05; 680 The OD in column reactor 4 was 1.82±0.15; 680The OD in column reactor 5 was 2.25±0.06; 680 The culture was stopped and electrochemical harvesting was performed. When the culture medium and the Chlorella algae were obviously separated, the electrochemical harvesting was stopped, the drain valve was opened, the cultured Chlorella algae were collected, and their biomass was recorded through subsequent drying treatment.

[0175] Depend on Figure 5 The results show that in this example, the aeration rate of the TAP basal medium was adjusted. There is a growth threshold during the initial growth process. That is, when the initial aeration rate is sufficient to support the growth and division of all Chlorella in the system, the initial growth stage of Chlorella does not increase with the increase of the aeration rate. The biomass of the Chlorella finally harvested is relatively small, at 0.5-0.75 g / L, and the proportion of intracellular carbohydrates is low (<19%) (Tables 6 and 7).

[0176] Table 6 Dry weight of Chlorella after harvest at different ventilation rates

[0177]

[0178] Note: Different superscript letters in the numbers indicate significant differences between the data (p < 0.05).

[0179] Table 7 Intracellular carbohydrate content of Chlorella after harvest at different ventilation rates

[0180]

[0181] Note: Different superscript letters in the numbers indicate significant differences between the data (p < 0.05).

[0182] In summary, based on the data of Example 1, Example 2 and Comparative Example 1, it can be found that compared with not adjusting the ventilation rate (fixed ventilation rate) in Comparative Example 1, dynamically adjusting the ventilation rate to cultivate Chlorella can increase the dry weight of the harvested Chlorella and the proportion of intracellular carbohydrates in the Chlorella to a certain extent.

[0183] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for the autotrophic cultivation of high-yield Chlorella biomass, characterized in that: The method is based on the autotrophic culture medium and dynamically adjusts the ventilation volume according to the growth of Chlorella: Dynamic ventilation adjustment strategy 1: The ventilation rate is 0.75-1.5 L / min at the initial stage of culture, and the ventilation rate is adjusted to 3-6 L / min from the exponential growth phase until the growth plateau phase; Alternatively, dynamic ventilation adjustment strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, 3-6 L / min from the beginning of the exponential growth phase, and 0.75-1.5 L / min in the late exponential phase until the stable growth phase.

2. The autotrophic culture method according to claim 1, wherein According to the dynamic ventilation adjustment strategy 1, the dynamic ventilation adjustment is as follows: the ventilation volume is 1.5 L / min at the initial stage of culture, and the ventilation volume is adjusted from the exponential growth phase to 3-5 L / min until the growth stability phase; According to dynamic ventilation adjustment strategy 2, the dynamic ventilation adjustment is as follows: the ventilation volume is 1.5 L / min at the beginning of the culture, the ventilation volume is adjusted to 4-6 L / min at the beginning of the growth exponential phase, and the ventilation volume is adjusted to 1.5 L / min in the late exponential phase until the growth stable phase; Preferably, the ventilation volume is dynamically adjusted as follows: the ventilation volume at the initial stage of culture is 1.5 L / min, the ventilation volume is adjusted to 5 L / min at the beginning of the growth exponential phase, and the ventilation volume is adjusted to 1.5 L / min in the late exponential phase until the growth stable phase.

3. The autotrophic culture method according to claim 1 or 2, wherein According to Strategy 2 of dynamically adjusting ventilation, the method comprises the following steps: (1) Add the Chlorella mother liquid and culture medium into the column reactor, turn on the air pump and light, adjust the initial gas flow rate to 0.75-1.5 L / min, mix the Chlorella and culture medium, and start autotrophic cultivation; (2) Maintaining light intensity, when Chlorella begins to grow in the exponential phase, increase the ventilation rate to 3-6 L / min, and when Chlorella grows to the late exponential phase, reduce the ventilation rate to 0.75-1.5 L / min; (3) stopping the cultivation during the stable growth period of Chlorella and harvesting the Chlorella; Preferably, in step (2), the ventilation rate is increased to 4-6 L / min when the Chlorella starts to grow in the exponential phase, and the ventilation rate is reduced to 1.5 L / min when the Chlorella grows to the late exponential phase; More preferably, in step (2), the ventilation rate is increased to 5 L / min when the Chlorella starts to grow in the exponential phase, and is reduced to 1.5 L / min when the Chlorella grows to the late exponential phase.

4. The autotrophic culture method according to claim 3, wherein The OD of Chlorella when the growth starts in the exponential phase in step (2) 680 The OD of Chlorella in the late exponential phase is 0.35-0.

90. 680 1.5 to 2.2; OD of Chlorella during the growth stabilization period in step (3) 680 It is 1.7 to 2.

3.

5. The autotrophic culture method according to claim 1 or 2, wherein According to Strategy 1 of dynamically adjusting ventilation, the method comprises the following steps: (1) Adding the Chlorella mother liquid and culture medium in the logarithmic growth phase to the column reactor; turning on the air pump and light, adjusting the initial gas flow rate to 0.75-1.5 L / min, mixing the Chlorella and culture medium, and starting the culture; (2) Maintain light intensity. When the Chlorella enters the exponential growth phase, increase the ventilation rate to 3-6 L / min. (3) stopping the cultivation during the stable growth period of Chlorella and harvesting the Chlorella; Preferably, in step (2), when the growth of Chlorella begins in the exponential phase, the ventilation rate is increased to 4-6 L / min; More preferably, in step (2), the ventilation rate is increased to 5 L / min when the growth of Chlorella begins to enter the exponential phase.

6. The autotrophic culture method according to claim 3, wherein The OD of Chlorella when the growth starts in the exponential phase in step (2) 680 0.35 to 0.90; OD of Chlorella during the growth stabilization period in step (3) 680 It is 1.7 to 2.

3.

7. The method according to any one of claims 1 to 6, characterized in that: The culture medium is TAP culture medium; the autotrophic culture period is 5 to 10 days.

8. The method according to any one of claims 1 to 7, characterized in that: The method specifically comprises the following steps: The Chlorella mother solution and TAP medium were added to the column reactor at a ratio of 1:10 (v / v) to ensure that the Chlorella and the medium were evenly mixed without precipitation. The Chlorella biomass density was 0.08 g / L. Turn on the air pump and light, adjust the gas flow rate to 1.5 L / min, the light-dark ratio to 12h:12h, the light intensity to 5000 Lux, and the culture temperature to 27°C to start the autotrophic culture of Chlorella vulgaris; When the growth of Chlorella begins to enter the exponential phase (OD 680 0.57±0.03), increase the ventilation rate to 5L / min, when Chlorella grows to the late exponential phase (OD 680 When the growth of Chlorella vulgaris was in the stable period (OD 680 When the pH value was 2.15±0.11), the culture was stopped and electrochemical harvesting was performed. When the culture medium and the algae were clearly separated, the electrochemical harvesting was stopped, the drain valve was opened, and the cultured Chlorella was collected.

9. A method for increasing the biomass of Chlorella by autotrophic cultivation, characterized in that: The method is based on the autotrophic culture medium and dynamically adjusts the ventilation volume according to the growth of Chlorella: Dynamic ventilation adjustment strategy 1: The ventilation rate is 0.75-1.5 L / min at the initial stage of culture, and the ventilation rate is adjusted to 3-6 L / min from the exponential growth phase until the growth plateau phase; Alternatively, dynamic ventilation adjustment strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, 3-6 L / min from the beginning of the exponential growth phase, and 0.75-1.5 L / min in the late exponential phase until the stable growth phase.

10. A method for increasing the intracellular carbohydrate content of Chlorella by autotrophic culture, characterized in that: The method is based on the autotrophic culture medium and dynamically adjusts the ventilation volume according to the growth of Chlorella: Dynamic ventilation adjustment strategy 1: The ventilation rate is 0.75-1.5 L / min at the initial stage of culture, and the ventilation rate is adjusted to 3-6 L / min from the exponential growth phase until the growth plateau phase; Alternatively, dynamic ventilation adjustment strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, 3-6 L / min from the beginning of the exponential growth phase, and 0.75-1.5 L / min in the late exponential phase until the stable growth phase.

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