Method for continuously culturing and stably harvesting chlorella

Abstract

The invention discloses a method for continuously culturing and stably harvesting chlorella, and relates to the technical field of biology. Chlorella is cultured in a mode of combining mixed nutrition and continuous culture, nutrition input is accurately controlled, and organic carbon sources and nitrogen sources, such as glucose and urea, in a specific proportion are continuously input, so that nutrients of chlorella can be fully utilized, and continuous production of biomass is realized; the enrichment of different products of chlorella is controlled by changing the dilution rate and supplying organic carbon sources and nitrogen sources with different concentrations; a specific continuous culture unit is constructed and comprises a culture medium storage tank, a culture container and a collection container, the culture medium storage tank, the culture container and the collection container are connected through a double-channel peristaltic pump and a sterilization silicone tube, and the specific continuous culture unit design ensures stable supply of a culture medium, full mixing of algae and timely collection of products and creates good conditions for growth of chlorella.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a method for continuous culture and stable harvesting of Chlorella. BACKGROUND

[0002] Microalgae have been widely used in aquaculture, nutritional food, pharmaceuticals, wastewater treatment, and renewable energy due to their short production cycle, wide environmental adaptability, and diverse bioactive substances. Among them, Chlorella has great commercial potential due to its excellent performance in biosynthesis of bioactive substances and growth rate. However, large-scale commercial cultivation faces many challenges, including low productivity, uncertainty, and poor uniformity.

[0003] To overcome these challenges, researchers have tried various strategies, such as optimizing nutritional modes and cultivation methods. Mixotrophy, as a nutritional mode that combines the advantages of photosynthetic autotrophy and heterotrophy, can significantly improve biomass and active substance accumulation. In addition, the strategy of mixotrophy does not strictly depend on photosynthesis, which can reduce the cost of light in actual production.

[0004] In addition to the optimization of nutritional strategies, the cultivation mode also significantly affects the production of active substances. In addition, compared with traditional batch culture, continuous culture system can maintain cells in exponential growth phase, ensure high yield and product consistency, and reduce production preparation time. At the same time, the constant cell state provides an excellent condition for studying the physiological response and metabolism of algae under specific conditions.

[0005] Although previous studies have shown that mixotrophy and continuous culture have great potential, there are still few studies on microalgae continuous culture. For example, the invention patent with the application number CN202011156157 proposes a culture medium for large-scale cultivation of Chlorella and its cultivation method, aiming to improve the biomass and active substance accumulation of Chlorella. The patent proposes an optimized culture medium formula, including ammonium bicarbonate, potassium dihydrogen phosphate, magnesium sulfate, and other inorganic salts, and adjusts the nitrogen-phosphorus ratio to optimize the growth of Chlorella. The experimental results show that under the conditions of light intensity of 3000-5000 Lux and temperature of 25-30℃, the biomass of Chlorella is significantly improved. This method significantly improves the biomass of Chlorella by optimizing the culture medium formula, but the energy consumption is large under high light intensity, increasing the operating cost. In addition, this method mainly focuses on the improvement of biomass, and does not discuss in detail how to optimize the accumulation of amino acids and other active substances.

[0006] Meanwhile, the invention patent with application number CN202111365382 proposes a food-grade Chlorella cultivation medium and method, providing a cultivation method based on food-grade photoautotrophic medium, with discarded yeast liquid and washing liquid from the beer brewing process as organic nitrogen source and carbon source. This patent emphasizes that under the light intensity of 5000-10000 Lux, through the mixotrophic mode of photosynthetic autotrophy and heterotrophy, food-grade Chlorella with high biomass, high protein, and no pollution can be obtained. However, maintaining such high light intensity will significantly increase energy consumption and production cost. In contrast, the present invention achieves efficient accumulation of amino acids, lipids, and pigments under lower light intensity, significantly reducing energy consumption and operating cost.

[0007] Further, the invention patent with application number CN202411394257 proposes a method for strengthening Chlorella protein production through heterotrophic fermentation, using glucose as an organic carbon source for heterotrophic fermentation to enhance the protein content of Chlorella. This method significantly improves the protein yield of Chlorella by using glucose as the main carbon source under lightless conditions. Under optimal conditions, the protein content reaches more than 60%. However, this method completely relies on organic carbon sources, lacks photosynthesis under light conditions, limits the accumulation of other metabolic products (such as lipids, pigments, etc.), and increases the cost of organic carbon sources.

[0008] Therefore, the present invention aims to provide an economical and sustainable continuous cultivation method of Chlorella to improve the productivity of Chlorella, increase the biomass productivity, and achieve efficient accumulation of bioactive compounds. SUMMARY

[0009] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the commercial-scale cultivation of Chlorella has low productivity, the accumulation amount and accumulation rate control method of target products are not clear, such as the production and accumulation control of nutritional pigments, lipids, and amino acids are unstable; traditional cultivation modes cannot keep cells in active exponential growth phase, resulting in poor yield and product consistency, and long production cycle.

[0010] The present study constructs a continuous culture system of Chlorella, taking photosynthetic autotrophy as the baseline, adding organic carbon and nitrogen sources, and setting different dilution rates to achieve accurate regulation of organic nutrition. At the same time, the chlorophyll fluorescence is used to explore the changes of photosynthesis of algae under different nutritional strategies. The fatty acid and amino acid spectrum is used as an index to evaluate the nutritional value of Chlorella, providing theoretical and practical basis for the stable acquisition of high-value active substances. Due to the availability and low cost of organic carbon sources (such as glucose, acetate, glycerol) and organic nitrogen sources (such as urea), compared with traditional batch culture, continuous culture is accompanied by the addition of fresh nutrients and the outflow of mature algal cells, which keeps the cells in active exponential growth phase, ensures high yield and product consistency, and reduces the steady-state establishment time. At the same time, the constant cell state provides an excellent condition for studying the physiological response and metabolism of algae under specific conditions.

[0011] The present application adopts the combination of mixed nutrition and continuous culture to culture Chlorella, accurately controls the nutrition input, continuously inputs specific proportion of glucose and urea, so that Chlorella can fully utilize the nutrients and realize the continuous production of biomass; the dilution rate is changed, different concentrations of glucose and urea are supplied to control the enrichment of different products of Chlorella; a specific continuous culture unit is constructed, including a culture medium storage tank, a culture container and a collection container, which are connected through a double-channel peristaltic pump and sterilized silica gel pipe, and the specific continuous culture unit design ensures the stable supply of culture medium, the sufficient mixing of algae and the timely collection of products, thereby creating good conditions for the growth of Chlorella.

[0012] To achieve the above object, the present application provides a method for continuous culture and stable harvesting of Chlorella, which comprises the following steps: (1) Set up a continuous culture unit: each unit is composed of a culture medium storage tank, a culture container and a collection container, which are connected through a double-channel peristaltic pump and several sterilized silica gel pipes; (2) Sterilize the continuous culture unit: rinse the culture medium storage tank, heat the purified water to boiling, boil for a period of time, introduce into the culture container for sterilization, and empty the culture container; (3) Continuous culture: configure the culture medium in the sterilized culture medium storage tank, heat to boiling and keep warm for 20 minutes, then continue to boil for 10 minutes, repeat this operation before adding culture medium each time, introduce into the culture container, and cool to 25℃ with ice water; set the dilution rate, supplement fresh culture medium every day, and discharge the same volume of culture solution; accurately control the proportion of added nutrients according to different culture stages; add Chlorella to the culture container, control the initial concentration of Chlorella at 1×1 The cell density is 1.0 x 106 cells / ml, sterile air filtered by 0.22 μm syringe filter is continuously supplied to the bottom of the culture vessel to prevent the algae from sinking, and a magnetic stirrer is used to stir the algae to make them uniformly receive light. The light intensity is 2500 Lux, and the light cycle is 12 hours: 12 hours (light: dark). The temperature is maintained at 28°C. (4) Detection analysis: Fresh algae samples are taken from each culture system every day for detection analysis.

[0013] Further, the culture medium in step (3) is sterilized modified BG11 medium as the base culture medium, and the composition is: sodium nitrate 1.2 g / L, sodium carbonate 0.02 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, calcium chloride 0.03 g / L, magnesium sulfate 0.03 g / L, dipotassium hydrogen phosphate 0.04 g / L, EDTA 0.001 g / L; trace elements: boric acid 2.86 μg / L, manganese chloride 1.18 μg / L, zinc sulfate 0.22 μg / L, copper sulfate 0.08 μg / L, sodium molybdate 0.39 μg / L, cobalt nitrate 0.05 μg / L. Further, the dilution rate in step (3) is 0.1-0.8. Further, the nutrients added in different culture stages in step (3) are specific proportions of organic carbon source and nitrogen source, such as glucose and urea. Further, the different culture stages in step (3) are divided into four stages. In the first stage (S1), the algae are subjected to photoautotrophy, and fresh BG11 medium is used. In the second stage (S2), 1 g / L of glucose is supplemented daily. In the third stage (S3), 2 g / L of glucose is supplemented daily. In the fourth stage (S4), 2 g / L of glucose is supplied daily, and the nitrogen source (17.6 mM) is replaced with urea of the same concentration. Further, the Chlorella is first subjected to pre-culture, and then the different culture stages are started in the continuous culture mode. Preferably, the pre-culture time is 6 days. Preferably, the dilution rate in step (3) is 0.1, 0.3, or 0.5. Preferably, the continuous culture unit in step (3) is 3, and the initial pH of the culture medium is 7. Further, the parameters for detection analysis in step (4) include the daily change rate of cell density, pH value, and photosynthetic parameters. When the daily change rate of cell density, pH value, and photosynthetic parameters is within ± 5 %, it is determined that the culture system has reached a stable state, at which time the dilution rate is equal to the growth rate μ. Each stable state lasts for 3-9 days to harvest sufficient samples for detection analysis.

[0014] Technical effects

[0015] 1、The present application realizes efficient biomass and accumulation of various active substances at lower light intensity (2500 Lux) by optimizing dilution rate, glucose and urea supply, significantly reducing energy consumption and operating cost. Specifically, the present application adopts a continuous culture system with different dilution rates (such as D=0.1, D=0.3, D=0.5), and adjusts the input of glucose and urea as needed. Studies have shown that low dilution rate (D=0.1) is conducive to the accumulation of lipids and pigments, while high dilution rate (D=0.5) is more suitable for efficient synthesis of amino acids. Glucose supplementation improves photosynthesis efficiency and biomass productivity, while urea effectively promotes protein synthesis under low nitrogen conditions. These strategies achieve efficient and stable accumulation of nutritional pigments, lipids and amino acids, suitable for industrial production. The present application can realize precise regulation of Chlorella growth and product accumulation, and can enrich different active substances according to needs. For example, by promoting mixed nutrition Chlorella lipid yield through nitrogen limitation, Chlorella fatty acid yield is highest up to 10.34 mg / L when 3.20 mg / L urea is added.

[0016] 2、Under mixed nutrition, Chlorella can not only perform photosynthesis, but also metabolize organic carbon. Photosynthetic autotrophy and heterotrophy synergize to improve biomass and active substance content. Continuous culture keeps cells in active exponential growth phase, with high reproduction efficiency. Changing dilution rate and nutrient concentration can adjust Chlorella growth environment to meet its nutritional needs at different growth stages, significantly improving Chlorella biomass productivity, up to 0.429 g / L / d; promoting bioactive substance accumulation, such as total amino acid yield up to 0.231 g / L / d.

[0017] 3、Specific continuous culture unit design ensures stable supply of culture medium, sufficient mixing of algae and timely collection of products, creating good conditions for Chlorella growth. The culture system has been running stably for up to 3 months, ensuring the continuity and stability of production, which is conducive to large-scale production. DETAILED DESCRIPTION

[0018] The following describes several preferred embodiments of the present application to make its technical content clearer and easier to understand. The present application can be embodied in many different forms, and the scope of protection of the present application is not limited to the embodiments described herein.

[0019] 1. Material preparation: Chlorella (Chlorella vulgaris) from Shanghai Guangyu Biotechnology Co., Ltd. was used. Chlorella pyrenoidosaGY-D12 algal strain. Prepare sterilized modified BG11 medium as the basal medium, with the following composition: sodium nitrate 1.2 g / L, sodium carbonate 0.02 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, calcium chloride 0.03 g / L, magnesium sulfate 0.03 g / L, dipotassium hydrogen phosphate 0.04 g / L, EDTA 0.001 g / L; trace elements: boric acid 2.86 μg / L, manganese chloride 1.18 μg / L, zinc sulfate 0.22 μg / L, copper sulfate 0.08 μg / L, sodium molybdate 0.39 μg / L, cobalt nitrate 0.05 μg / L. Simultaneously, prepare glucose stock solution (100 g / L) and urea stock solution (50 g / L). Adjust the initial pH to 7.0. A light incubator (TH1200N, Shanghai Jinwen Instrument Equipment Co., Ltd.) was used, with the temperature set to 28 °C, the illuminance to 2500 Lux, and the photocycle to be 12 hours of light followed by 12 hours of darkness.

[0020] 2. Construction of Continuous Culture Units: Three continuous culture units were constructed. Each unit consisted of a culture medium storage tank, a 1-liter working volume culture container, and a collection container. The containers were connected by a dual-channel peristaltic pump and several sterile silicone tubing. The initial concentration of Chlorella was controlled at 1×10⁻⁶. Cells / mL, sterile air filtered through a 0.22 μm syringe filter (approximately 5 mL / s) is continuously introduced into the bottom of the culture container to prevent algae from sinking; at the same time, a magnetic stirrer (150 rpm / min) is used to stir the algae so that they are evenly exposed to light.

[0021] 3. Cultivation Process: Pre-culturing for 6 days, followed by continuous cultivation. The dilution ratios for the three units were set at 0.1, 0.3, and 0.5. In stage 1 (S1), the algae underwent photoautotrophic growth using fresh BG11 medium; in stage 2 (S2), 1 g / L glucose was added; in stage 3 (S3), 2 g / L glucose was added; in stage 4 (S4), while supplying 2 g / L glucose, the nitrogen source (17.6 mM) was replaced with urea of ​​the same concentration. The culture system was considered to have reached a steady state when the daily variation rates of cell density, pH, and photosynthetic parameters were within ±5%, at which point the dilution ratio equaled the growth rate (μ). Each steady state lasted 3–9 days to allow for the harvesting of sufficient samples for analysis.

[0022] 4. Detection and Analysis: Fresh algae samples were taken daily from each culture system, and the absorbance (OD) at 680 nm was measured using a UV-Vis spectrophotometer (Cary300, Agilent Technologies). 680pH value of the culture medium was determined using a pH meter; a certain volume of algal sample was filtered, dried at 60°C to constant weight, and the dry weight (DW) was determined, and the biomass productivity was calculated. The photosynthetic activity was determined using Phyto-Pam II, including parameters such as maximum photochemical quantum yield (Fv / Fm). The contents of photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), fatty acid composition, amino acid composition and residual nutrients (NO3-N, PO4-P, glucose and urea) were determined by specific methods and formulas. Statistical analysis was performed using IBM SPSS 24, two-way ANOVA and Duncan's method were used for post-hoc test (P<0.05), and principal component analysis (PCA) and data visualization were performed using related software packages in Rstudio.

[0023] To investigate the effects of different dilution rates on the content of pigments in Chlorella vulgaris at different continuous culture stages, the present application sets four culture stages S1: photoautotrophic, using fresh BG11 medium; S2: supplementing 1 g / L glucose daily; S3: supplementing 2 g / L glucose daily; S4: supplementing 2 g / L glucose and replacing nitrogen source with urea of the same concentration. Three groups of parallel experiments were set for each culture stage, with dilution rates of D=0.1, 0.3 and 0.5 respectively. Each steady state lasted for 3-9 days to harvest enough samples for detection and analysis. The content of different pigments (chlorophyll a, chlorophyll b and carotenoids) in Chlorella vulgaris under different culture stages and different dilution rates was determined, and the results showed that the content of pigments was significantly affected by dilution rate and organic carbon and nitrogen source ( p <0.05); except for S3, the content of photosynthetic pigments in the other stages decreased with the increase of dilution rate ( p <0.05); 2 g / L glucose could promote the production of photosynthetic pigments in the groups of D=0.3 and D=0.5 to some extent, especially in the group of D=0.3, the promoting effect of 2 g / L glucose was more obvious, and the content of photosynthetic pigments decreased at a lower growth rate (D=0.1); the addition of urea could also promote the production of photosynthetic pigments, especially for the group of D=0.1.

[0024] To investigate the influence of different lipid contents in Chlorella under different culture stages and different dilution rates. The present application sets 4 culture stages S1: photoautotrophic, using fresh BG11 medium; S2: daily supplement of 1g / L glucose; S3: daily supplement of 2g / L glucose; S4: supplement of 2g / L glucose and nitrogen source is replaced with the same concentration of urea, each culture stage sets 3 groups of parallel experiments, and the dilution rates are D=0.1, 0.3 and 0.5 respectively. Each steady state lasts for 3-9 days in order to harvest enough samples for detection analysis, and the content of various lipids (including PUFA polyunsaturated fatty acids, MUFA monounsaturated fatty acids, SFA saturated fatty acids, and 14:0, 16:1, 18:3 specific fatty acids) in Chlorella under different culture stages and different dilution rates is determined. The result data shows that the fatty acid content of Chlorella under mixed nutrition (S2-S4) is significantly higher than that under photoautotrophic (S1); the fatty acid content at each stage decreases with the increase of dilution rate, and low dilution rate (D=0.1) is more conducive to accumulation (especially PUFA); the addition of urea in S4 stage can significantly promote the enrichment of fatty acids in D=0.1 group, which provides a basis for lipid directional culture.

[0025] To investigate the influence of different amino acid contents in Chlorella under different culture stages and different dilution rates. The present application sets 4 culture stages S1: photoautotrophic, using fresh BG11 medium; S2: daily supplement of 1g / L glucose; S3: daily supplement of 2g / L glucose; S4: supplement of 2g / L glucose and nitrogen source is replaced with the same concentration of urea, each culture stage sets 3 groups of parallel experiments, and the dilution rates are D=0.1, 0.3 and 0.5 respectively. Each steady state lasts for 3-9 days in order to harvest enough samples for detection analysis, and the content of various lipids (including PUFA polyunsaturated fatty acids, MUFA monounsaturated fatty acids, SFA saturated fatty acids, and 14:0, 16:1, 18:3 specific fatty acids) in Chlorella under different culture stages and different dilution rates is determined. The result data shows that the fatty acid content of Chlorella under mixed nutrition (S2-S4) is significantly higher than that under photoautotrophic (S1); the fatty acid content at each stage decreases with the increase of dilution rate, and low dilution rate (D=0.1) is more conducive to accumulation (especially PUFA); the addition of urea in S4 stage can significantly promote the enrichment of fatty acids in D=0.1 group, which provides a basis for lipid directional culture.

[0026] Amino acids are different: the types of amino acids are divided into "essential amino acids (Essential)" and "non-essential amino acids (Non-Essential)", essential amino acids (EAA) include isoleucine Ile, valine Val, lysine Lys, etc., non-essential amino acids (NEAA) include alanine Ala, glutamic acid Glu, glycine Gly, etc.

[0027] The culture stages are different: S1 is the photoautotrophic stage (only using BG11 medium), S2 is the daily supplement of 1g / L glucose stage, S3 is the daily supplement of 2g / L glucose stage, and S4 is the daily supplement of 2g / L glucose and the nitrogen source is replaced with the same concentration of urea stage, and there are obvious differences in the amino acid content at different stages, such as the total amino acid and essential amino acid content at each dilution rate in S4 is higher and the difference between groups is small.

[0028] Dilution rate impact: The amino acid content of S1 stage low dilution rate (D=0.1) is significantly higher than that of high dilution rate (D=0.3, D=0.5); S2-S3 stage is the opposite, high dilution rate (D=0.5) is more conducive to amino acid enrichment; Overall, the supply of nutrients by adjusting the dilution rate and the culture stage can realize the regulation of Chlorella amino acid (especially essential amino acid) accumulation, and provide data support for targeted production of high amino acid content Chlorella.

[0029] Each experimental group EAA / NEAA is greater than 0.6, and EAA / TAA is greater than 0.4, indicating that Chlorella can provide high-quality protein. The experimental results show that: (1) except for S3 D=0.1 group, adding organic carbon source and nitrogen source can significantly promote the yield of total amino acid and essential amino acid; (2) in the photosynthetic autotrophic stage (S1), the amino acid content of the low growth rate group (D=0.1) is significantly higher than that of the higher growth rate group (D=0.3, D=0.5, p <0.05); while in the mixed nutrition stage (S2-S3), Chlorella with high growth rate can often enrich more amino acids p <0.05); (3) for essential amino acids, in the photosynthetic autotrophic stage, the content of D=0.1 and D=0.3 groups is significantly higher than that of D=0.5 group p <0.05); and after adding urea (S2-S3), D=0.5 often produces more essential amino acids; (4) after adding urea, the total amino acid content (49.6997-50.3735 g / 100g) and essential amino acid content (20.8915-21.4835 g / 00g) of Chlorella under each growth rate are high, and there is no significant difference between groups (p>0.05).

[0030] To investigate the influence of different culture stages and dilution rates on Chlorella biomass productivity and dry weight. The four culture stages S1: photoautotrophic, using fresh BG11 medium; S2: 1g / L glucose is added daily; S3: 2g / L glucose is added daily; S4: 2g / L glucose + nitrogen source is replaced with urea of the same concentration, each culture stage sets 3 groups of parallel experiments, and the dilution rates are D=0.1, 0.3 and 0.5 respectively. Each steady state lasts for 3-9 days in order to harvest enough samples for detection and analysis, the biomass productivity of Chlorella in different culture stages and different dilution rates is measured, which is g / L / d, reflecting the growth efficiency of Chlorella biomass per unit time per unit volume; at the same time, the dry weight of Chlorella under different culture stages and different dilution rates is measured. The dilution rate is the volume ratio of fresh medium added daily to the discharged culture solution in continuous culture.

[0031] In the same cultivation stage, the biomass productivity increased with the increase of dilution rate (e.g. in S3 stage, the productivity of D=0.5 group was significantly higher than that of D=0.1 group); With the progress of cultivation stage (from S1 to S3), the addition of glucose gradually increased the biomass productivity of each dilution rate group, and reached a higher level in S3 stage (2 g / L glucose); After entering S4 stage (addition of glucose + urea replacing nitrogen source), the biomass productivity of some dilution rate groups (e.g. D=0.3, D=0.5) was slightly adjusted, but the overall remained in a higher range, reflecting the influence of nutrient regulation on Chlorella biomass production.

[0032] The experimental results showed that the dry weight was also affected by dilution rate and organic carbon and nitrogen source; (1) In any stage, the dry weight decreased with the increase of dilution rate, and the productivity increased with the increase of dilution rate; (2) The addition of glucose significantly promoted the dry weight and productivity of each group, and reached a productivity of 428.75 mg / L / d in the 2 g / L glucose group; (3) Urea could promote the dry weight of D=0.1 group, but not significantly, and the dry weight and yield of the other two groups decreased.

[0033] In summary, it can be considered to cultivate lipids and pigments under low dilution rate and low organic carbon source conditions, to cultivate amino acids under high dilution rate and high organic carbon source and / or organic carbon and nitrogen source conditions, and to conduct comprehensive cultivation under medium dilution rate conditions.

[0034] Example 1: Cultivation of lipids and pigments under low dilution rate (D=0.1)

[0035] The culture medium tank was rinsed clean, 60 L of purified water was used to heat to boiling and boil for 20 minutes, and was introduced into the culture container for sterilization for 30 minutes, and the culture container was emptied. The air filter device was sterilized by steam at high temperature. In the sterilized culture medium tank, 50 L of BG11 culture medium was configured, heated to boiling and kept for 20 minutes, and then boiled for another 10 minutes. Before adding the culture medium each time, the operation was repeated, and the culture container was introduced and cooled to 25°C with ice water. The dilution rate was set to 0.1, and fresh BG11 culture medium was added daily, while the same volume of culture solution was discharged. The light intensity was 2500 Lux, and the light cycle was 12 hours:12 hours (light:dark), and the temperature was maintained at 28°C. 1 g / L of glucose was added daily, and no urea was added.

[0036] The results showed that after the end of the cultivation period, the cell density reached 0.1 g / L / d, the total lipid content reached 10.34 mg / L, and the carotenoids were significantly enriched, reaching 12.3 mg / L.

[0037] Example 2: Cultivation of amino acids under high dilution rate (D=0.5)

[0038] The medium tank was rinsed clean, 60 L purified water was heated to boiling and boiled for 20 minutes, introduced into the culture vessel for 30 minutes of sterilization, and the culture vessel was emptied. The air filter device was sterilized using steam. 50 L of BG11 medium was prepared in the sterilized medium tank, heated to boiling and maintained for 20 minutes, and then boiled for another 10 minutes. This operation was repeated before each addition of medium, introduced into the culture vessel, and cooled to 25°C with ice water. The dilution rate was set to 0.5, and fresh BG11 medium was added daily while the same volume of culture solution was discharged. The light intensity was 2500 Lux, and the light cycle was 12 hours: 12 hours (light: dark), with the temperature maintained at 28°C. 2 g / L of glucose was added daily, and the same concentration of urea was used to replace the nitrogen source.

[0039] The results showed that after the end of the culture period, the cell density reached 0.429 g / L / d, the total amino acid content reached 53.87 g / 100g DW, and the essential amino acid content reached 20.8915 g / 100g DW.

[0040] Example 3: Medium dilution rate (D = 0.3) comprehensive culture

[0041] The medium tank was rinsed clean, 60 L purified water was heated to boiling and boiled for 20 minutes, introduced into the culture vessel for 30 minutes of sterilization, and the culture vessel was emptied. The air filter device was sterilized using steam. 50 L of BG11 medium was prepared in the sterilized medium tank, heated to boiling and maintained for 20 minutes, and then boiled for another 10 minutes. This operation was repeated before each addition of medium, introduced into the culture vessel, and cooled to 25°C with ice water. The dilution rate was set to 0.3, and fresh BG11 medium was added daily while the same volume of culture solution was discharged. The light intensity was 2500 Lux, and the light cycle was 12 hours: 12 hours (light: dark), with the temperature maintained at 28°C. 1 g / L of glucose was added daily, and the same concentration of urea was used to replace the nitrogen source.

[0042] The results showed that after the end of the culture period, the cell density reached 0.3 g / L / d. The total lipid content reached 9.8 mg / L, with significant enrichment of polyunsaturated fatty acids (PUFA). The chlorophyll a and carotenoid contents were 6.5 mg / L and 4.2 mg / L, respectively. The amino acid content: the total amino acid content reached 51.2 g / 100g DW, and the essential amino acid content reached 20.5 g / 100g DW. Under medium dilution rate, combined with glucose and urea supply, comprehensive optimization of lipids, pigments, and amino acids was achieved, with high practicality and industrial application potential

[0043] Comparative Example 1: Traditional photoautotrophic culture

[0044] The culture medium tank was rinsed clean, 60 L of purified water was heated to boiling and boiled for 20 minutes, introduced into the culture container for 30 minutes of sterilization, and the culture container was emptied. The air filter device was sterilized using steam. In the sterilized culture medium tank, 50 L of BG11 culture medium was prepared, heated to boiling and kept for 20 minutes, and then boiled for another 10 minutes. This operation was repeated before each addition of culture medium, introduced into the culture container, and cooled to 25°C with ice water. Only BG11 culture medium was used daily, without adding any organic carbon source or urea. The light intensity was maintained at 2500 Lux, and the light cycle was 12 hours:12 hours (light:dark), with the temperature maintained at 28°C.

[0045] The results show that after the end of the culture period, the cell density is only 0.05 g / L / d, and the lipid, pigment, and amino acid contents are low, showing the limitations of traditional photoautotrophic culture methods.

[0046] Comparative Example 2: Single nutrient source culture

[0047] The culture medium tank was rinsed clean, 60 L of purified water was heated to boiling and boiled for 20 minutes, introduced into the culture container for 30 minutes of sterilization, and the culture container was emptied. The air filter device was sterilized using steam. In the sterilized culture medium tank, 50 L of BG11 culture medium was prepared, heated to boiling and kept for 20 minutes, and then boiled for another 10 minutes. This operation was repeated before each addition of culture medium, introduced into the culture container, and cooled to 25°C with ice water. Only BG11 culture medium was used daily, with 1 g / L of glucose added, without urea or other nitrogen sources. The light intensity was maintained at 2500 Lux, and the light cycle was 12 hours:12 hours (light:dark), with the temperature maintained at 28°C.

[0048] After the end of the culture period, although the lipid content was improved, the amino acid and pigment contents were still lower than expected, showing that a single nutrient source cannot achieve optimal biomass productivity and active substance accumulation.

[0049] Through the comparison of the above examples and comparative examples, it can be clearly seen that the method of continuous culture and harvesting of Chlorella proposed in the present application can significantly improve the accumulation efficiency and quality of target products under different dilution rates and nutrient supply conditions. These data verify the practicality and feasibility of the method, which is suitable for industrial large-scale production.

[0050] Industrial applicability

[0051] 1. Economic benefits

[0052] Cost-effectiveness: By using a mixed nutrient strategy, inexpensive and easily available organic carbon sources (such as glucose) and nitrogen sources (such as urea) can significantly reduce production costs. Compared to traditional photoautotrophic culture, mixed nutrition not only improves biomass productivity, but also increases the yield of high-value active substances.

[0053] Resource Utilization: The continuous culture system can operate stably for months, ensuring efficient resource utilization and reducing waste through precise control of nutrient inputs.

[0054] 2. Operational Simplicity

[0055] Modular Design: The three independent continuous culture units can be flexibly configured by adjusting the dilution rate and nutrient supply to adapt to different product requirements. This modular design makes operation more simple, easy to maintain and expand.

[0056] High Degree of Automation: The replenishment and discharge of nutrient solution are automatically controlled by peristaltic pumps and sensors, reducing manual intervention and improving system stability and repeatability.

[0057] 3. Product Diversity and Quality

[0058] Diverse Product Portfolio: By adjusting the dilution rate and nutrient supply, a variety of target products (such as lipids, pigments, and amino acids) can be enriched in the same system, meeting different market demands.

[0059] Stable Product Quality: The continuous culture system can keep cells in the exponential growth phase for a long time, ensuring product consistency and high quality.

[0060] 4. Environmental Friendliness

[0061] Low Carbon Footprint: The mixed nutrient strategy reduces dependence on light, reducing energy consumption. At the same time, through efficient carbon dioxide fixation mechanisms, it helps to reduce greenhouse gas emissions.

[0062] Wastewater Treatment Potential: Microalgae have good wastewater purification capacity, and this system not only can be used for producing high-value-added products, but also can be used as part of wastewater treatment, further enhancing its environmental friendliness.

[0063] 5. Industrial Feasibility

[0064] Large-scale Production: This method has been verified at laboratory scale and has good scaling potential. Through optimization of reactor design and operating parameters, large-scale industrial production can be realized.

[0065] Broad Market Prospects: With the increasing global demand for healthy foods, functional ingredients, and sustainable energy, the market prospects for Chlorella and its derivative products are broad, with great commercial potential.

[0066] In summary, from the technical advantages, the present application breaks through the limitations of traditional Chlorella cultivation, and the combination of mixed nutrition and continuous cultivation greatly improves the growth efficiency and active substance accumulation of Chlorella. In terms of performance indicators, the key indicators such as biomass productivity and amino acid yield are excellent, far exceeding the traditional cultivation method. From the perspective of production implementation, the cost of the culture equipment and nutrients (such as glucose and urea) used is low and easy to obtain, and the culture process is relatively simple to operate. It can be modified on the basis of existing fermentation equipment, and is convenient for large-scale industrial application.

[0067] This technical scheme has broad market prospects in the microalgae-related industries such as food, feed, pharmaceuticals, etc., and is expected to significantly reduce production costs, improve product quality and yield, and enhance industry competitiveness.

[0068] The above describes the preferred embodiments of the present application in detail. It should be understood that those skilled in the art can make many modifications and changes to the present application without creative labor based on the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment based on the existing technology according to the concept of the present application shall be within the protection scope determined by the claims.

Claims

1. A method for continuous cultivation and stable harvesting of Chlorella, comprising the following steps: (1) Constructing a continuous culture unit: Each unit consists of a culture medium storage tank, a culture container and a collection container, and the containers are connected by a dual-channel peristaltic pump and several sterile silicone tubes; (2) Sterilization of continuous culture unit: Rinse the culture medium storage tank clean, heat purified water to boiling for a period of time, pour it into the culture container for heat preservation and sterilization, and empty the culture container; (3) Continuous culture: Prepare the culture medium in the sterilized culture medium storage tank, heat it to boiling and keep it warm for 20 minutes, then continue to boil for 10 minutes. Repeat this operation before each addition of culture medium, pour it into the culture container, and cool it down to 25°C with ice water; set the dilution rate, add fresh culture medium daily, and drain the same volume of culture medium at the same time; precisely control the proportion of added nutrients according to different culture stages. Add Chlorella to the culture container, controlling the initial concentration of Chlorella at 1×1 Cells / mL, sterile air filtered through a 0.22 μm syringe filter was continuously introduced into the bottom of the culture container to prevent the algae from sinking; at the same time, a magnetic stirrer was used to stir the algae to ensure that they were evenly exposed to light; the light intensity was 2500 Lux, the photoperiod was 12 hours:12 hours (light:dark), and the temperature was maintained at 28°C. (4) Detection and analysis: Fresh algae samples are taken from each culture system every day for detection and analysis.

2. The method for continuous cultivation and stable harvesting of Chlorella according to claim 1, characterized in that, The culture medium mentioned in step (3) is a sterilized and modified BG11 medium as the basic culture medium, which consists of: sodium nitrate 1.2 g / L, sodium carbonate 0.02 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, calcium chloride 0.03 g / L, magnesium sulfate 0.03 g / L, dipotassium hydrogen phosphate 0.04 g / L, EDTA 0.001 g / L; and trace elements: boric acid 2.86 μg / L, manganese chloride 1.18 μg / L, zinc sulfate 0.22 μg / L, copper sulfate 0.08 μg / L, sodium molybdate 0.39 μg / L, and cobalt nitrate 0.05 μg / L.

3. The method for continuous cultivation and stable harvesting of Chlorella according to claim 1, characterized in that: The dilution rate set in step (3) is 0.1-0.

8.

4. The method for continuous cultivation and stable harvesting of Chlorella according to claim 1, characterized in that, The nutrients added in different cultivation stages in step (3) are organic carbon sources and nitrogen sources in specific proportions.

5. The method for continuous cultivation and stable harvesting of Chlorella according to claim 1, characterized in that, The different cultivation stages in step (3) are divided into four stages. In the first stage (S1), the algae are photoautotrophic and fresh BG11 medium is used. In the second stage (S2), 1 g / L glucose is supplemented daily. In the third stage (S3), 2 g / L glucose is supplemented daily. In the fourth stage (S4), while supplying 2 g / L glucose daily, the nitrogen source (17.6 mM) is replaced with urea of ​​the same concentration.

6. The method for continuous cultivation and stable harvesting of Chlorella according to claim 1, characterized in that, In step (3), the Chlorella is first pre-cultured, and then a continuous culture mode with different culture stages is started.

7. The method for continuous cultivation and stable harvesting of Chlorella according to claim 6, characterized in that, The pre-culture time of Chlorella in step (3) is 6 days.

8. The method for continuous cultivation and stable harvesting of Chlorella according to claim 1, characterized in that, The dilution rates in step (3) are 0.1, 0.3, and 0.

5.

9. The method for continuous cultivation and stable harvesting of Chlorella according to claim 1, characterized in that, In step (3), there are 3 continuous culture units, the initial pH of the culture medium is 7, and the added nutrients are glucose and urea in a specific ratio.

10. The method for continuous cultivation and stable harvesting of Chlorella according to claim 1, characterized in that, The parameters detected and analyzed in step (4) include the daily variation rate of cell density, pH value and photosynthetic parameters. When the daily variation rate of cell density, pH value and photosynthetic parameters is within ±5%, the culture system is considered to have reached a stable state. At this time, the dilution rate is equal to the growth rate μ. Each stable state lasts for 3-9 days in order to harvest enough samples for detection and analysis.