Culture method of microalgae with high starch content

Abstract

The invention discloses a culture method of microalgae with high starch content, and belongs to the technical field of new food resource mining. Microalgae are cultured in two stages, the ventilation capacity is fixed in the first stage, the ventilation capacity is dynamically adjusted in the second stage, the ventilation capacity in the early stage of culture is 0.75-1.5 L / min, the ventilation capacity is adjusted to 3-6 L / min when the microalgae start to grow in the exponential stage, and the ventilation capacity is adjusted to 0.75-1.5 L / min when the microalgae start to grow in the later stage of the exponential stage until culture is finished. The high-efficiency culture of the microalgae with high starch content is realized by dynamically adjusting the ventilatory capacity of the second culture stage, so that the microalgae cell powder with high starch content is obtained, and the maximum starch content can reach 61% or above. The method has the advantages of short required culture period, simplicity and convenience, no pollution to the environment in the culture process, and important significance for expanding the microalgae as a new resource food and expanding a new starch resource.

Description

Technical Field

[0001] The invention relates to a method for culturing microalgae with high starch content, and belongs to the technical field of new food resource mining. Background Art

[0002] Microalgae have the advantages of fast growth, high photosynthetic efficiency, strong environmental adaptability, no occupation of traditional crop land, and easy artificial control of growth status. Microalgae are rich in biomass such as protein, starch and oil, and are a high-quality new food resource. As a nutritious and fast-growing microalgae, Chlorella has broad application prospects in food, feed, bioenergy and environmental remediation. Most microalgae are photoautotrophic organisms, which mainly rely on photosynthesis to convert inorganic carbon in the air into glucose. In addition to being used for growth and metabolism, the remaining glucose is mainly stored in the form of starch and oil.

[0003] Under suitable culture conditions, the biomass of microalgae is high, but the starch content is low. Microalgae often accumulate and store more starch under stressful growth conditions. However, while stressful growth conditions promote the accumulation of starch in microalgae, they significantly inhibit the growth of microalgae. At present, a two-stage culture method of autotrophic-nitrogen or sulfur deficiency is often used to promote the accumulation of starch in microalgae cells. That is, in the first stage of autotrophic process, sufficient nutrients are provided for the accumulation of microalgae biomass. When the microalgae grows to the stable period and its biomass reaches the maximum, the algae cells are collected and cultured in a nitrogen or sulfur-deficient culture medium to accumulate starch. However, it takes a long time to grow to the stable period, and the process of collecting algae cells and then replacing the culture medium to stress the algae cells is relatively complicated, and the growth of algae cells will be significantly inhibited in the nitrogen or sulfur deficiency stage. Therefore, how to efficiently cultivate and improve the starch content in Chlorella is one of the key challenges facing the current field of microalgae biotechnology.

[0004] In order to improve the starch content and growth efficiency of microalgae, researchers have tried a variety of improvement methods, but all of them have certain limitations. "Identification of a microalgae and research on the culture conditions for improving its total sugar and starch content" reported a method of using nutritional stress to improve the starch accumulation of Scenedesmus: first, sufficient nutrients are provided to allow Scenedesmus to accumulate biomass, and then the Scenedesmus cell fluid is inoculated into a culture medium with nitrogen removed, and the inoculated Scenedesmus cell fluid accounts for 10% of the total culture medium volume (Identification of a microalgae and research on the culture conditions for improving its total sugar and starch content [D]. Nanjing Agricultural University, 2014.). The nutritional stress condition of removing nitrogen elements promoted the accumulation of starch in Scenedesmus cells. On the second day, the starch content reached the highest level of 21.1%. However, after the second day, the starch content in Scenedesmus cells decreased significantly, and by the fourth day it had decreased to 14.5%. And on the second day, the biomass of Scenedesmus was still low. Therefore, the starch content in the algae cells cultured by this method was low, and the biomass of microalgae in the stress stage was not considered to be increased. "Comparative transcriptome analysis of the molecular regulatory mechanism of nitrogen limitation on the accumulation of oil and starch in microalgae Ankistrodesmus sp. HJ12" reported that under the nutritional stress condition of nitrogen removal, the starch content of microalgae Ankistrodesmus sp. HJ12 reached 32.6% after 14 days of growth, and the biomass was 1.53g / L. The starch content in the microalgae cells cultured by this method is still low, and the culture cycle is long. The Chinese invention patent with publication number CN118702831A discloses a method for increasing the starch content in microalgae cells by two-stage culture: first, sufficient nutrients are provided to allow Spirulina to accumulate biomass, and then the Spirulina cells grown for 8 days are collected by centrifugation, and then the collected Spirulina cells are inoculated in a culture medium removed from nitrogen for 7 days. The starch content of Spirulina cultured by this method is 56%. Although this method obtains microalgae cells with a higher starch content, the culture cycle of this method is long, and the biomass of microalgae in the stress stage is not considered to be increased. In addition, the operation of collecting algae cells and replacing the culture medium is complicated, and the culture facilities are not systematic enough. Therefore, there is a need for a cultivation method that can achieve the cultivation of microalgae cells with a high starch content and promote biomass accumulation, as well as a cultivation device for two-stage continuous cultivation of microalgae cells with a high starch content.

[0005] The traditional method of increasing the biomass accumulation of microalgae mainly adopts fixed aeration strategies and increases the supply of carbon dioxide, but fails to fully consider the dynamic demand for carbon dioxide at different stages of the growth of Chlorella, resulting in low culture efficiency and limited biomass accumulation. Therefore, considering the growth characteristics of Chlorella and the physical characteristics of the culture system, developing an economical and efficient method for cultivating microalgae with high starch content has become a hot spot and difficulty in current research. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for cultivating microalgae with high starch content. Green swimming microalgae cells are continuously cultivated by providing sufficient macroelements and trace elements, and then the efficient cultivation of microalgae with high starch content is achieved by dynamically regulating the ventilation volume and limiting the nitrogen source in the culture medium and adding sodium acetate, thereby obtaining microalgae cell powder with high starch content. The method requires a short cultivation cycle, a simple cultivation method, and does not cause pollution to the environment during the cultivation process. It is of great significance to expand microalgae as a new resource food and expand new starch resources.

[0007] The present invention provides a method for culturing microalgae with a high starch content, wherein the method is divided into two stages of cultivation; the first stage is to fix the ventilation volume, and the second stage is to dynamically adjust the ventilation volume:

[0008] The second stage of dynamic ventilation adjustment strategy 1: the ventilation volume at the initial stage of cultivation is 0.75-1.5 L / min, and the ventilation volume is adjusted to 3-6 L / min from the exponential growth period of microalgae until the end of cultivation;

[0009] Or, in the second stage, the ventilation volume is dynamically adjusted. Strategy 2: the ventilation volume in the initial stage of culture is 0.75-1.5 L / min, the ventilation volume is adjusted to 3-6 L / min at the beginning of the exponential growth period of microalgae, and the ventilation volume is adjusted to 0.75-1.5 L / min in the late exponential period until the end of culture.

[0010] In one embodiment, the method is divided into two stages of cultivation; the first stage has a fixed ventilation volume, and the second stage has a dynamically adjusted ventilation volume:

[0011] The second stage of dynamic ventilation adjustment strategy 1: the ventilation rate at the beginning of the culture is 1.5L / min, and the ventilation rate is adjusted to 5L / min from the exponential growth period of the microalgae until the end of the culture;

[0012] Or, in the second stage, the ventilation rate is dynamically adjusted by strategy 2: the ventilation rate is 1.5 L / min at the beginning of the culture, the ventilation rate is adjusted to 5 L / min at the beginning of the exponential growth period of the microalgae, and the ventilation rate is adjusted to 1.5 L / min in the late exponential period until the end of the culture.

[0013] In one embodiment, the microalgae is Chlorella; preferably, the microalgae is Chlorella sorokiniana.

[0014] In one embodiment, the method comprises the following steps:

[0015] (1) First stage of cultivation: Add the microalgae seed solution and culture medium into the column reactor, mix well, turn on the air pump and light, adjust the ventilation flow rate to 0.75-6 L / min, and cultivate for 96-168 h;

[0016] (2) Second stage of cultivation: the microalgae liquid obtained in step (1) is directly inoculated into a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 24 to 120 hours; the initial ventilation flow rate is 0.75 to 1.5 L / min, and when the growth begins to enter the exponential phase, the ventilation volume is increased to 3 to 6 L / min. When the growth is in the late exponential phase, the ventilation volume is changed to 0.75 to 1.5 L / min; after the cultivation is completed, the microalgae cell fluid is collected.

[0017] In one embodiment, the OD value after the microalgae seed solution and the culture medium are evenly mixed in step (1) is 680 The content of microalgae in the culture medium is 0.08-0.1, and the biomass of microalgae is 0.05-0.2 g / L culture medium; preferably, the biomass of microalgae is 0.1 g / L culture medium.

[0018] In one embodiment, the culture temperature in steps (1) and (2) is 26-30°C; the light intensity is 0-15000 lux, 12-24 h light, 0-12 h dark; preferably, the culture temperature is 26°C, the light intensity is 5000 lux, 12 h light conditions, 12 h dark conditions.

[0019] In one embodiment, the gas ventilated in steps (1) and (2) is a mixture of carbon dioxide and air, wherein the volume of carbon dioxide accounts for 1-5% of the volume of air; preferably, the volume of carbon dioxide accounts for 2% of the volume of air.

[0020] In one embodiment, the culture medium in step (1) is BG11 culture medium, BBM culture medium or TAP culture medium; preferably, the culture medium is TAP culture medium.

[0021] In one embodiment, the nitrogen-reduced medium containing sodium acetate in step (2) is a BG11 medium, a BBM medium or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; the nitrogen source is NH4Cl;

[0022] Preferably, the nitrogen-reduced culture medium containing sodium acetate is a TAP culture medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added; and the nitrogen source is NH4Cl.

[0023] In one embodiment, after the microalgae and the culture medium are uniformly mixed in step (2), the biomass of the microalgae is 0.05-0.2 g / L of the culture medium; preferably, the biomass of the microalgae is 0.1 g / L of the culture medium.

[0024] In one embodiment, the OD of the microalgae when the growth starts in the exponential phase in step (2) is 680It is 0.2~0.5.

[0025] In one embodiment, the OD of the microalgae when the growth starts in the late exponential phase in step (2) is 680 It is 1.1 to 1.5.

[0026] In one embodiment, the method comprises the following steps:

[0027] (1) First stage of cultivation: Add the microalgae seed solution and culture medium into the column reactor, mix well, turn on the air pump and light, adjust the ventilation flow rate to 0.75-6 L / min, and cultivate for 96-168 h;

[0028] (2) Second stage of cultivation: the microalgae liquid obtained in step (1) is directly inoculated into a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 24 to 120 hours; the initial ventilation flow rate is 0.75 to 1.5 L / min, and when the growth begins to enter the exponential phase, the ventilation volume is increased to 3 to 6 L / min; after the cultivation is completed, the microalgae cell liquid is collected.

[0029] In one embodiment, the OD value after the microalgae seed solution and the culture medium are evenly mixed in step (1) is 680 The content of microalgae in the culture medium is 0.08-0.1, and the biomass of microalgae is 0.05-0.2 g / L culture medium; preferably, the biomass of microalgae is 0.1 g / L culture medium.

[0030] In one embodiment, the culture temperature in steps (1) and (2) is 26-30°C; the light intensity is 0-15000 lux, 12-24 h light, 0-12 h dark; preferably, the culture temperature is 26°C, the light intensity is 5000 lux, 12 h light conditions, 12 h dark conditions.

[0031] In one embodiment, the gas ventilated in steps (1) and (2) is a mixture of carbon dioxide and air, wherein the volume of carbon dioxide accounts for 1-5% of the volume of air; preferably, the volume of carbon dioxide accounts for 2% of the volume of air.

[0032] In one embodiment, the culture medium in step (1) is BG11 culture medium, BBM culture medium or TAP culture medium; preferably, the culture medium is TAP culture medium.

[0033] In one embodiment, the nitrogen-reduced medium containing sodium acetate in step (2) is a BG11 medium, a BBM medium or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; the nitrogen source is NH4Cl;

[0034] Preferably, the nitrogen-reduced culture medium containing sodium acetate is a TAP culture medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added; and the nitrogen source is NH4Cl.

[0035] In one embodiment, after the microalgae and the culture medium are uniformly mixed in step (2), the biomass of the microalgae is 0.05-0.2 g / L of the culture medium; preferably, the biomass of the microalgae is 0.1 g / L of the culture medium.

[0036] In one embodiment, the OD of the microalgae when the growth starts in the exponential phase in step (2) is 680 It is 0.2~0.5.

[0037] In one embodiment, the method specifically comprises the following steps:

[0038] (1) First stage of culture: inoculate the seed solution of Chlorella sorokinensis into TAP medium and mix evenly. 680 is 0.08-0.1, and the biomass of Chlorella is about 0.1g / L; turn on the air pump and light, adjust the ventilation flow rate to 5L / min, the culture temperature to 26℃, the carbon dioxide concentration to 2% v / v, 12h light condition, 12h dark condition, light intensity to 5000lux, and culture for 96h;

[0039] (2) Second stage of cultivation: The Chlorella sorokinensis algae solution obtained in step (1) was directly inoculated into TAP medium containing 5 g / L sodium acetate and the nitrogen source mass volume concentration was reduced to 1 / 15 of the original, with a biomass of 0.1 g / L of the medium, mixed evenly, and the air pump and light were turned on. The culture temperature was 26°C, the carbon dioxide concentration was 2% v / v, the light was continuously illuminated for 24 h, the light intensity was 5000 lux, and the culture was carried out for 72 h. The ventilation strategy was as follows: the initial ventilation flow rate was 1.5 L / min, and when the Chlorella growth began to enter the exponential phase (at this time OD 680 When the aeration rate was increased to 5 L / min, the growth of Chlorella began to enter the late exponential phase (OD 680 When the temperature was 1.4±0.05), the ventilation rate was changed to 1.5 L / min; after the culture was completed, the cell fluid of Chlorella vulgaris with high starch content obtained by the culture was collected.

[0040] In one embodiment, the method utilizes a culture device for culturing microalgae with high starch content to culture microalgae; the device mainly comprises: an aeration unit, a lighting unit, and a culture unit;

[0041] The aeration unit includes an aeration pump, a vent pipe, and a flow meter. A sterilizing filter is provided at the air inlet of the aeration pump. The aeration pump is connected to the flow meter and the vent pipe. The vent pipe enters from the air inlet at the top of the column reactor.

[0042] The illumination unit comprises a series of full-spectrum culture light strips, which are installed in a light strip frame, and the light strip frame is placed around the culture unit;

[0043] The culture unit includes three column reactors, which are made of transparent material and are cylindrical. The top cover of the reactor is provided with an inlet, a sampling port, and an exhaust port with a filter membrane installed; the three column reactors of the culture unit include two small column reactors on the upper layer (a first column reactor and a second column reactor) and a large column reactor on the lower layer (a third column reactor), wherein the two small column reactors are connected in series with each other, a first valve is provided between the series connection, the small column reactor on the upper layer is connected in series with the large column reactor on the lower layer, a second valve and a third valve are provided between the series connection, and a fourth valve is provided at the bottom of the large column reactor on the lower layer for rapid material discharge.

[0044] In one embodiment, based on the above device, the method comprises the following steps:

[0045] (1) adding the microalgae seed solution and culture medium into the first column reactor, mixing them evenly, turning on the air pump and light, adjusting the ventilation flow rate to 0.75-6 L / min, and culturing for 96-168 h;

[0046] (2) When the microalgae are cultured in the first column reactor for 72 to 168 hours, the first valve is opened, and the microalgae cells in the first column reactor are inoculated as seed liquid into the second column reactor containing the culture medium, mixed evenly, the air pump and the light are turned on, the ventilation flow rate is adjusted to 0.75 to 6 L / min, and the culture is continued for 96 to 168 hours;

[0047] (3) After the microalgae are cultured in the first column reactor for 96 to 168 hours, the second valve is opened, and the microalgae solution in the first column reactor is inoculated into the third column reactor containing a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 72 to 144 hours; the initial ventilation flow rate is 0.75 to 1.5 L / min, and when the growth begins to enter the exponential phase, the ventilation volume is increased to 3 to 6 L / min, and when the growth is in the late exponential phase, the ventilation volume is changed to 0.75 to 1.5 L / min; after the cultivation is completed, the fourth valve is opened to collect the microalgae cell solution cultured in the third column reactor;

[0048] (4) When the microalgae in the second column reactor are cultured for 72 to 168 hours, the first valve is opened, and the microalgae cells in the second column reactor are inoculated into the first column reactor containing the culture medium as a seed solution, mixed evenly, and the air pump and light are turned on. The initial ventilation flow rate is 0.75 to 6 L / min, and the culture is carried out for 96 to 168 hours;

[0049] (5) After the microalgae in the second column reactor have been cultured for 96 to 168 hours, open the third valve, and directly inoculate the microalgae solution cultured in the second column reactor into the third column reactor containing nitrogen-reduced culture medium containing sodium acetate, mix evenly, turn on the air pump and light, and culture for 72 to 144 hours; the initial ventilation flow rate is 0.75 to 1.5 L / min. When the growth begins to enter the exponential phase, the ventilation volume is increased to 3 to 6 L / min, and when the growth is in the late exponential phase, the ventilation volume is changed to 0.75 to 1.5 L / min; after the cultivation is completed, open the fourth valve to collect the microalgae cell solution cultured in the third column reactor;

[0050] (6) Repeating the culture steps of (2), (3), (4), and (5) can achieve efficient continuous culture of microalgae cells with high starch content.

[0051] In one embodiment, steps (1), (2) and (4), i.e., the OD after the microalgae seed solution and the culture medium are uniformly mixed in the first column reactor and the second column reactor, 680 The content of microalgae in the culture medium is 0.08-0.1, and the biomass of microalgae is 0.05-0.2 g / L culture medium; preferably, the biomass of microalgae is 0.1 g / L culture medium.

[0052] In one embodiment, the culture temperature in steps (1) to (5), i.e., the first column reactor, is 26 to 30°C; the light intensity is 0 to 15,000 lux, 12 to 24 h of light, and 0 to 12 h of darkness; preferably, the culture temperature is 26°C, the light intensity is 5,000 lux, 12 h of light conditions, and 12 h of dark conditions.

[0053] In one embodiment, the gas ventilated in steps (1) to (5), i.e., the first column reactor, is a mixed gas of carbon dioxide and air, wherein the volume of carbon dioxide accounts for 1 to 5% of the volume of air; preferably, the volume of carbon dioxide accounts for 2% of the volume of air.

[0054] In one embodiment, the culture medium in steps (1), (2) and (4), i.e., the first column reactor and the second column reactor, is BG11 culture medium, BBM culture medium or TAP culture medium; preferably, the culture medium is TAP culture medium.

[0055] In one embodiment, the nitrogen-reduced medium containing sodium acetate in steps (3) and (5), i.e., the third column reactor, is a BG11 medium, a BBM medium, or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; the nitrogen source is NH4Cl;

[0056] Preferably, the nitrogen-reduced culture medium containing sodium acetate is a TAP culture medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added; and the nitrogen source is NH4Cl.

[0057] In one embodiment, after the microalgae and the culture medium are uniformly mixed in steps (3) and (5), the biomass of the microalgae is 0.05-0.2 g / L of culture medium; preferably, the biomass of the microalgae is 0.1 g / L of culture medium.

[0058] In one embodiment, the OD of the microalgae at the beginning of the exponential growth phase in the third column reactor is 680 It is 0.2~0.5.

[0059] In one embodiment, the OD of the microalgae in steps (3) and (5), i.e., the growth in the third column reactor at the late exponential phase, is 680 It is 1.1 to 1.5.

[0060] In one embodiment, based on the above device, the method comprises the following steps:

[0061] (1) Adding the microalgae seed solution and culture medium into the first column reactor, mixing them evenly, turning on the air pump and light, adjusting the ventilation flow rate to 0.75-6 L / min, and culturing for 96 h;

[0062] (2) When the microalgae are cultured in the first column reactor for 72 hours, the first valve is opened, and the microalgae cells in the first column reactor are inoculated as seed liquid into the second column reactor containing the culture medium, mixed evenly, the air pump and light are turned on, the ventilation flow rate is adjusted to 0.75-6 L / min, and the culture is continued for 96 hours;

[0063] (3) After the microalgae are cultured in the first column reactor for 96 hours, the second valve is opened, and the microalgae solution in the first column reactor is inoculated into the third column reactor containing a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 72 hours; the initial ventilation flow rate is 0.75-1.5 L / min, and when the growth begins to enter the exponential phase, the ventilation volume is increased to 3-6 L / min, and when the growth is in the late exponential phase, the ventilation volume is changed to 0.75-1.5 L / min; after the cultivation is completed, the fourth valve is opened to collect the microalgae cell solution cultured in the third column reactor;

[0064] (4) When the microalgae in the second column reactor are cultured for 72 hours, the first valve is opened, and the microalgae cells in the second column reactor are inoculated into the first column reactor containing the culture medium as a seed solution, mixed evenly, and the air pump and light are turned on. The initial ventilation flow rate is 0.75 to 6 L / min, and the culture is carried out for 96 hours;

[0065] (5) After the microalgae in the second column reactor have been cultured for 96 hours, open the third valve, and directly inoculate the microalgae solution cultured in the second column reactor into the third column reactor containing nitrogen-reduced culture medium containing sodium acetate, mix evenly, turn on the air pump and light, and culture for 72 hours; the initial ventilation flow rate is 0.75-1.5L / min. When the growth begins to enter the exponential phase, the ventilation volume is increased to 3-6L / min, and when the growth is in the late exponential phase, the ventilation volume is changed to 0.75-1.5L / min; after the cultivation is completed, open the fourth valve to collect the microalgae cell solution cultured in the third column reactor;

[0066] (6) Repeating the culture steps of (2), (3), (4), and (5) can achieve efficient continuous culture of microalgae cells with high starch content.

[0067] In one embodiment, steps (1), (2) and (4), i.e., the OD after the microalgae seed solution and the culture medium are uniformly mixed in the first column reactor and the second column reactor, 680 The content of microalgae in the culture medium is 0.08-0.1, and the biomass of microalgae is 0.05-0.2 g / L culture medium; preferably, the biomass of microalgae is 0.1 g / L culture medium.

[0068] In one embodiment, the culture temperature in steps (1) to (5), i.e., the first column reactor, is 26 to 30°C; the light intensity is 0 to 15,000 lux, 12 to 24 h of light, and 0 to 12 h of darkness; preferably, the culture temperature is 26°C, the light intensity is 5,000 lux, 12 h of light conditions, and 12 h of dark conditions.

[0069] In one embodiment, the gas ventilated in steps (1) to (5), i.e., the first column reactor, is a mixed gas of carbon dioxide and air, wherein the volume of carbon dioxide accounts for 1 to 5% of the volume of air; preferably, the volume of carbon dioxide accounts for 2% of the volume of air.

[0070] In one embodiment, the culture medium in steps (1), (2) and (4), i.e., the first column reactor and the second column reactor, is BG11 culture medium, BBM culture medium or TAP culture medium; preferably, the culture medium is TAP culture medium.

[0071] In one embodiment, the nitrogen-reduced medium containing sodium acetate in steps (3) and (5), i.e., the third column reactor, is a BG11 medium, a BBM medium, or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; the nitrogen source is NH4Cl;

[0072] Preferably, the nitrogen-reduced culture medium containing sodium acetate is a TAP culture medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added; and the nitrogen source is NH4Cl.

[0073] In one embodiment, after the microalgae and the culture medium are uniformly mixed in steps (3) and (5), the biomass of the microalgae is 0.05-0.2 g / L of culture medium; preferably, the biomass of the microalgae is 0.1 g / L of culture medium.

[0074] In one embodiment, the OD of the microalgae at the beginning of the exponential growth phase in the third column reactor is 680 It is 0.2~0.5.

[0075] In one embodiment, the OD of the microalgae in steps (3) and (5), i.e., the growth in the third column reactor at the late exponential phase, is 680 It is 1.1 to 1.5.

[0076] The present invention also provides a method for increasing the starch content in microalgae cells, which is divided into two stages of cultivation: the first stage is to fix the ventilation volume, and the second stage is to dynamically adjust the ventilation volume:

[0077] The second stage of dynamic ventilation adjustment strategy 1: the ventilation volume at the initial stage of cultivation is 0.75-1.5 L / min, and the ventilation volume is adjusted to 3-6 L / min from the exponential growth period of microalgae until the end of cultivation;

[0078] Or, in the second stage, the ventilation volume is dynamically adjusted. Strategy 2: the ventilation volume in the initial stage of culture is 0.75-1.5 L / min, the ventilation volume is adjusted to 3-6 L / min at the beginning of the exponential growth period of microalgae, and the ventilation volume is adjusted to 0.75-1.5 L / min in the late exponential period until the end of culture.

[0079] In one embodiment, the method is divided into two stages of cultivation; the first stage has a fixed ventilation volume, and the second stage has a dynamically adjusted ventilation volume:

[0080] The second stage of dynamic ventilation adjustment strategy 1: the ventilation rate at the beginning of the culture is 1.5L / min, and the ventilation rate is adjusted to 5L / min from the exponential growth period of the microalgae until the end of the culture;

[0081] Or, in the second stage, the ventilation rate is dynamically adjusted by strategy 2: the ventilation rate is 1.5 L / min at the beginning of the culture, the ventilation rate is adjusted to 5 L / min at the beginning of the exponential growth period of the microalgae, and the ventilation rate is adjusted to 1.5 L / min in the late exponential period until the end of the culture.

[0082] In one embodiment, the microalgae is Chlorella; preferably, the microalgae is Chlorella sorokiniana.

[0083] In one embodiment, the method comprises the following steps:

[0084] (1) First stage of cultivation: Add the microalgae seed solution and culture medium into the column reactor, mix well, turn on the air pump and light, adjust the ventilation flow rate to 0.75-6 L / min, and cultivate for 96-168 h;

[0085] (2) Second stage of cultivation: the microalgae liquid obtained in step (1) is directly inoculated into a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 24 to 120 hours; the initial ventilation flow rate is 0.75 to 1.5 L / min, and when the growth begins to enter the exponential phase, the ventilation volume is increased to 3 to 6 L / min. When the growth is in the late exponential phase, the ventilation volume is changed to 0.75 to 1.5 L / min; after the cultivation is completed, the microalgae cell fluid is collected.

[0086] In one embodiment, the OD value after the microalgae seed solution and the culture medium are evenly mixed in step (1) is 680 The content of microalgae in the culture medium is 0.08-0.1, and the biomass of microalgae is 0.05-0.2 g / L culture medium; preferably, the biomass of microalgae is 0.1 g / L culture medium.

[0087] In one embodiment, the culture temperature in steps (1) and (2) is 26-30°C; the light intensity is 0-15000 lux, 12-24 h light, 0-12 h dark; preferably, the culture temperature is 26°C, the light intensity is 5000 lux, 12 h light conditions, 12 h dark conditions.

[0088] In one embodiment, the gas ventilated in steps (1) and (2) is a mixture of carbon dioxide and air, wherein the volume of carbon dioxide accounts for 1-5% of the volume of air; preferably, the volume of carbon dioxide accounts for 2% of the volume of air.

[0089] In one embodiment, the culture medium in step (1) is BG11 culture medium, BBM culture medium or TAP culture medium; preferably, the culture medium is TAP culture medium.

[0090] In one embodiment, the nitrogen-reduced medium containing sodium acetate in step (2) is a BG11 medium, a BBM medium or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; the nitrogen source is NH4Cl;

[0091] Preferably, the nitrogen-reduced culture medium containing sodium acetate is a TAP culture medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added; and the nitrogen source is NH4Cl.

[0092] In one embodiment, after the microalgae and the culture medium are uniformly mixed in step (2), the biomass of the microalgae is 0.05-0.2 g / L of the culture medium; preferably, the biomass of the microalgae is 0.1 g / L of the culture medium.

[0093] In one embodiment, the OD of the microalgae when the growth starts in the exponential phase in step (2) is 680 It is 0.2~0.5.

[0094] In one embodiment, the OD of the microalgae when the growth starts in the late exponential phase in step (2) is 680 It is 1.1 to 1.5.

[0095] In one embodiment, the method comprises the following steps:

[0096] (1) First stage of cultivation: Add the microalgae seed solution and culture medium into the column reactor, mix well, turn on the air pump and light, adjust the ventilation flow rate to 0.75-6 L / min, and cultivate for 96-168 h;

[0097] (2) Second stage of cultivation: the microalgae liquid obtained in step (1) is directly inoculated into a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 24 to 120 hours; the initial ventilation flow rate is 0.75 to 1.5 L / min, and when the growth begins to enter the exponential phase, the ventilation volume is increased to 3 to 6 L / min; after the cultivation is completed, the microalgae cell liquid is collected.

[0098] In one embodiment, the OD value after the microalgae seed solution and the culture medium are evenly mixed in step (1) is 680 The content of microalgae in the culture medium is 0.08-0.1, and the biomass of microalgae is 0.05-0.2 g / L culture medium; preferably, the biomass of microalgae is 0.1 g / L culture medium.

[0099] In one embodiment, the culture temperature in steps (1) and (2) is 26-30°C; the light intensity is 0-15000 lux, 12-24 h light, 0-12 h dark; preferably, the culture temperature is 26°C, the light intensity is 5000 lux, 12 h light conditions, 12 h dark conditions.

[0100] In one embodiment, the gas ventilated in steps (1) and (2) is a mixture of carbon dioxide and air, wherein the volume of carbon dioxide accounts for 1-5% of the volume of air; preferably, the volume of carbon dioxide accounts for 2% of the volume of air.

[0101] In one embodiment, the culture medium in step (1) is BG11 culture medium, BBM culture medium or TAP culture medium; preferably, the culture medium is TAP culture medium.

[0102] In one embodiment, the nitrogen-reduced medium containing sodium acetate in step (2) is a BG11 medium, a BBM medium or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; the nitrogen source is NH4Cl;

[0103] Preferably, the nitrogen-reduced culture medium containing sodium acetate is a TAP culture medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added; and the nitrogen source is NH4Cl.

[0104] In one embodiment, after the microalgae and the culture medium are uniformly mixed in step (2), the biomass of the microalgae is 0.05-0.2 g / L of the culture medium; preferably, the biomass of the microalgae is 0.1 g / L of the culture medium.

[0105] In one embodiment, the OD of the microalgae when the growth starts in the exponential phase in step (2) is 680 It is 0.2~0.5.

[0106] In one embodiment, the method specifically comprises the following steps:

[0107] (1) First stage of culture: inoculate the seed solution of Chlorella sorokinensis into TAP medium and mix evenly. 680 is 0.08-0.1, and the biomass of Chlorella is about 0.1g / L; turn on the air pump and light, adjust the ventilation flow rate to 5L / min, the culture temperature to 26℃, the carbon dioxide concentration to 2% v / v, 12h light condition, 12h dark condition, light intensity to 5000lux, and culture for 96h;

[0108] (2) Second stage of cultivation: The Chlorella sorokinensis algae solution obtained in step (1) was directly inoculated into TAP medium containing 5 g / L sodium acetate and the nitrogen source mass volume concentration was reduced to 1 / 15 of the original, with a biomass of 0.1 g / L of the medium, mixed evenly, and the air pump and light were turned on. The culture temperature was 26°C, the carbon dioxide concentration was 2% v / v, the light was continuously illuminated for 24 h, the light intensity was 5000 lux, and the culture was carried out for 72 h. The ventilation strategy was as follows: the initial ventilation flow rate was 1.5 L / min, and when the Chlorella growth began to enter the exponential phase (at this time OD 680 When the aeration rate was increased to 5 L / min, the growth of Chlorella began to enter the late exponential phase (OD 680 When the temperature was 1.4±0.05), the ventilation rate was changed to 1.5 L / min; after the culture was completed, the cell fluid of Chlorella vulgaris with high starch content obtained by the culture was collected.

[0109] The present invention also provides a method for increasing the biomass of microalgae by culturing microalgae, wherein the method is divided into two stages of cultivation; the first stage is to fix the ventilation volume, and the second stage is to dynamically adjust the ventilation volume:

[0110] The second stage of dynamic ventilation adjustment strategy 1: the ventilation volume at the initial stage of cultivation is 0.75-1.5 L / min, and the ventilation volume is adjusted to 3-6 L / min from the exponential growth period of microalgae until the end of cultivation;

[0111] Or, in the second stage, the ventilation volume is dynamically adjusted. Strategy 2: the ventilation volume in the initial stage of culture is 0.75-1.5 L / min, the ventilation volume is adjusted to 3-6 L / min at the beginning of the exponential growth period of microalgae, and the ventilation volume is adjusted to 0.75-1.5 L / min in the late exponential period until the end of culture.

[0112] In one embodiment, the method is divided into two stages of cultivation; the first stage has a fixed ventilation volume, and the second stage has a dynamically adjusted ventilation volume:

[0113] The second stage of dynamic ventilation adjustment strategy 1: the ventilation rate at the beginning of the culture is 1.5L / min, and the ventilation rate is adjusted to 5L / min from the exponential growth period of the microalgae until the end of the culture;

[0114] Or, in the second stage, the ventilation rate is dynamically adjusted by strategy 2: the ventilation rate is 1.5 L / min at the beginning of the culture, the ventilation rate is adjusted to 5 L / min at the beginning of the exponential growth period of the microalgae, and the ventilation rate is adjusted to 1.5 L / min in the late exponential period until the end of the culture.

[0115] In one embodiment, the microalgae is Chlorella; preferably, the microalgae is Chlorella sorokiniana.

[0116] In one embodiment, the method comprises the following steps:

[0117] (1) First stage of cultivation: Add the microalgae seed solution and culture medium into the column reactor, mix well, turn on the air pump and light, adjust the ventilation flow rate to 0.75-6 L / min, and cultivate for 96-168 h;

[0118] (2) Second stage of cultivation: the microalgae liquid obtained in step (1) is directly inoculated into a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 24 to 120 hours; the initial ventilation flow rate is 0.75 to 1.5 L / min, and when the growth begins to enter the exponential phase, the ventilation volume is increased to 3 to 6 L / min. When the growth is in the late exponential phase, the ventilation volume is changed to 0.75 to 1.5 L / min; after the cultivation is completed, the microalgae cell fluid is collected.

[0119] In one embodiment, the OD value after the microalgae seed solution and the culture medium are evenly mixed in step (1) is 680 The content of microalgae in the culture medium is 0.08-0.1, and the biomass of microalgae is 0.05-0.2 g / L culture medium; preferably, the biomass of microalgae is 0.1 g / L culture medium.

[0120] In one embodiment, the culture temperature in steps (1) and (2) is 26-30°C; the light intensity is 0-15000 lux, 12-24 h light, 0-12 h dark; preferably, the culture temperature is 26°C, the light intensity is 5000 lux, 12 h light conditions, 12 h dark conditions.

[0121] In one embodiment, the gas ventilated in steps (1) and (2) is a mixture of carbon dioxide and air, wherein the volume of carbon dioxide accounts for 1-5% of the volume of air; preferably, the volume of carbon dioxide accounts for 2% of the volume of air.

[0122] In one embodiment, the culture medium in step (1) is BG11 culture medium, BBM culture medium or TAP culture medium; preferably, the culture medium is TAP culture medium.

[0123] In one embodiment, the nitrogen-reduced medium containing sodium acetate in step (2) is a BG11 medium, a BBM medium or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; the nitrogen source is NH4Cl;

[0124] Preferably, the nitrogen-reduced medium containing sodium acetate is a TAP medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added, and the nitrogen source is NH4Cl.

[0125] In one embodiment, after the microalgae and the culture medium are uniformly mixed in step (2), the biomass of the microalgae is 0.05-0.2 g / L of the culture medium; preferably, the biomass of the microalgae is 0.1 g / L of the culture medium.

[0126] In one embodiment, the OD of the microalgae when the growth starts in the exponential phase in step (2) is 680 It is 0.2~0.5.

[0127] In one embodiment, the OD of the microalgae when the growth starts in the late exponential phase in step (2) is 680 It is 1.1 to 1.5.

[0128] In one embodiment, the method comprises the following steps:

[0129] (1) First stage of cultivation: Add the microalgae seed solution and culture medium into the column reactor, mix well, turn on the air pump and light, adjust the ventilation flow rate to 0.75-6 L / min, and cultivate for 96-168 h;

[0130] (2) Second stage of cultivation: the microalgae liquid obtained in step (1) is directly inoculated into a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 24 to 120 hours; the initial ventilation flow rate is 0.75 to 1.5 L / min, and when the growth begins to enter the exponential phase, the ventilation volume is increased to 3 to 6 L / min; after the cultivation is completed, the microalgae cell liquid is collected.

[0131] In one embodiment, the OD value after the microalgae seed solution and the culture medium are evenly mixed in step (1) is 680 The content of microalgae in the culture medium is 0.08-0.1, and the biomass of microalgae is 0.05-0.2 g / L culture medium; preferably, the biomass of microalgae is 0.1 g / L culture medium.

[0132] In one embodiment, the culture temperature in steps (1) and (2) is 26-30°C; the light intensity is 0-15000 lux, 12-24 h light, 0-12 h dark; preferably, the culture temperature is 26°C, the light intensity is 5000 lux, 12 h light conditions, 12 h dark conditions.

[0133] In one embodiment, the gas ventilated in steps (1) and (2) is a mixture of carbon dioxide and air, wherein the volume of carbon dioxide accounts for 1-5% of the volume of air; preferably, the volume of carbon dioxide accounts for 2% of the volume of air.

[0134] In one embodiment, the culture medium in step (1) is BG11 culture medium, BBM culture medium or TAP culture medium; preferably, the culture medium is TAP culture medium.

[0135] In one embodiment, the nitrogen-reduced medium containing sodium acetate in step (2) is a BG11 medium, a BBM medium or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; the nitrogen source is NH4Cl;

[0136] Preferably, the nitrogen-reduced culture medium containing sodium acetate is a TAP culture medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added; and the nitrogen source is NH4Cl.

[0137] In one embodiment, after the microalgae and the culture medium are uniformly mixed in step (2), the biomass of the microalgae is 0.05-0.2 g / L of the culture medium; preferably, the biomass of the microalgae is 0.1 g / L of the culture medium.

[0138] In one embodiment, the OD of the microalgae when the growth starts in the exponential phase in step (2) is 680 It is 0.2~0.5.

[0139] In one embodiment, the method specifically comprises the following steps:

[0140] (1) First stage of culture: inoculate the seed solution of Chlorella sorokinensis into TAP medium and mix evenly. 680 is 0.08-0.1, and the biomass of Chlorella is about 0.1g / L; turn on the air pump and light, adjust the ventilation flow rate to 5L / min, the culture temperature to 26℃, the carbon dioxide concentration to 2% v / v, 12h light condition, 12h dark condition, light intensity to 5000lux, and culture for 96h;

[0141] (2) Second stage of cultivation: The Chlorella sorokinensis algae solution obtained in step (1) was directly inoculated into TAP medium containing 5 g / L sodium acetate and the nitrogen source (NH4Cl) concentration was reduced to 1 / 15 of the original concentration at a biomass of 0.1 g / L of the medium, mixed evenly, and the air pump and light were turned on. The culture temperature was 26°C, the carbon dioxide concentration was 2% v / v, the light was continuously illuminated for 24 h, the light intensity was 5000 lux, and the culture was carried out for 72 h. The ventilation strategy was as follows: the initial ventilation flow rate was 1.5 L / min, and when the Chlorella growth began to enter the exponential phase (at this time OD 680 When the aeration rate was increased to 5 L / min, the growth of Chlorella began to enter the late exponential phase (OD 680 When the temperature was 1.4±0.05), the ventilation rate was changed to 1.5 L / min; after the culture was completed, the cell fluid of Chlorella vulgaris with high starch content obtained by the culture was collected.

[0142] Beneficial effects:

[0143] Optimizing aeration strategy is of great significance for regulating the biomass and intracellular starch content of Chlorella culture. By adjusting the ventilation volume, the growth rate and biological composition of Chlorella can be controlled, thereby optimizing the intracellular starch content.

[0144] (1) Using the method and apparatus of the present invention, three column reactors are used for two-stage cultivation, which can achieve efficient continuous cultivation of microalgae cells with high starch content;

[0145] (2) The method of the present invention dynamically adjusts the ventilation strategy of the second culture stage (adjusting the ventilation volume in two or three stages), and the biomass of the cultivated Chlorella can reach more than 1.35 g / L, the proportion of intracellular starch can reach more than 55%, and the highest proportion of intracellular starch can reach more than 61%;

[0146] (3) The method of the present invention increases biomass and intracellular carbohydrates of Chlorella by adjusting the aeration strategy in the second culture stage, and the technical effect is better than that of the fixed aeration strategy, while reducing production costs and improving culture efficiency;

[0147] (4) The method of the present invention requires a short culture period, is simple in culture method, and does not cause environmental pollution during the culture process, which is of great significance for expanding microalgae as a new resource food and expanding new starch resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 This is a structural diagram of the microalgae cultivation equipment, in which: 1. ventilation pump; 2. ventilation pipe; 3. flow meter; 4. full-spectrum cultivation light strip; 5. injection port; 6. sampling port; 7. exhaust port; 8. first valve; 9. second valve; 10. third valve; 11. fourth valve; 12. first column reactor; 13. second column reactor; 14. third column reactor. DETAILED DESCRIPTION

[0149] The following examples are used to illustrate the present invention, but are not intended to limit the scope of use of the present invention.

[0150] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0151] The chlorella used in the following examples is Chlorella sorokiniana, which was purchased from the Freshwater Algae Seed Bank of the National Aquatic Biological Germplasm Resource Bank and is numbered FACHB-2217.

[0152] The culture medium involved in the following examples is as follows:

[0153] 1. TAP medium:

[0154] (1) Preparation of TAP salt:

[0155] 15g NH4Cl, 4g MgSO4·7H2O, 2g CaCl2·2H2O, add water to 1L;

[0156] (2) Preparation of phosphate solution:

[0157] K2HPO4 28.8g, KH2PO4 14.4g, add water to 100mL;

[0158] (3) Preparation of trace elements:

[0159] EDTA disodium salt 50g dissolved in 250mL water, ZnSO4·7H2O 22g dissolved in 100mL water, H3BO3 11.4g dissolved in 200mL water, MnCl2·4H2O 5.06g dissolved in 50mL water, CoCl2·6H2O 1.61g dissolved in 50mL water, CuSO4·5H2O 1.57g dissolved in 50mL water, (NH4)6MO7O 24 ·4H2O 1.10g dissolved in 50mL water, FeSO4·7H2O 4.99g dissolved in 50mL water; Among them, EDTA is dissolved in boiling water, and FeSO4 is prepared last to avoid oxidation; Mix all the above prepared solutions except EDTA, boil, and then add EDTA solution, the mixture turns green. When all substances are dissolved, cool to 70℃, keep the temperature at 70℃, add 85mL of hot 20% KOH solution (20g / 100mL), and increase the total volume of the final solution to 1L. Plug the flask with cotton plugs and let it stand for 1 week, shaking once a day. The solution should eventually turn purple and leave a rust-brown precipitate, refrigerate at 4℃.

[0160] (4) Mix the following substances to obtain the final TAP medium:

[0161] 2.42 g Tris, 1 mL glacial acetic acid, 25 mL TAP salt prepared in step (1), 0.375 mL phosphate solution prepared in step (2), 1.0 mL trace elements prepared in step (3), add water to 1 L to obtain TAP culture medium.

[0162] 2. TAP medium with nitrogen (NH4Cl) reduced to 1 / 15 of the original and containing 5g / L sodium acetate:

[0163] (1) Preparation of TAP salt:

[0164] NH4Cl 1g, MgSO4·7H2O 4g, CaCl2·2H2O 2g, add water to 1L;

[0165] (2) Preparation of phosphate solution:

[0166] K2HPO4 28.8g, KH2PO4 14.4g, add water to 100mL;

[0167] (3) Preparation of trace elements:

[0168] EDTA disodium salt 50g dissolved in 250mL water, ZnSO4·7H2O 22g dissolved in 100mL water, H3BO3 11.4g dissolved in 200mL water, MnCl2·4H2O 5.06g dissolved in 50mL water, CoCl2·6H2O 1.61g dissolved in 50mL water, CuSO4·5H2O 1.57g dissolved in 50mL water, (NH4)6MO7O 24 ·4H2O 1.10g dissolved in 50mL water, FeSO4·7H2O 4.99g dissolved in 50mL water; Among them, EDTA is dissolved in boiling water, and FeSO4 is prepared last to avoid oxidation; Mix all the above prepared solutions except EDTA, boil, and then add EDTA solution, the mixture turns green. When all substances are dissolved, cool to 70℃, keep the temperature at 70℃, add 85mL of hot 20% KOH solution (20g / 100mL), and increase the total volume of the final solution to 1L. Plug the flask with cotton plugs and let it stand for 1 week, shaking once a day. The solution should eventually turn purple and leave a rust-brown precipitate, refrigerate at 4℃.

[0169] (4) Mix the following substances to obtain the final TAP medium:

[0170] 2.42 g Tris, 1 mL glacial acetic acid, 5 g sodium acetate, 25 mL TAP salt prepared in step (1), 0.375 mL phosphate solution prepared in step (2), 1.0 mL trace elements prepared in step (3), add water to 1 L to obtain TAP culture medium.

[0171] 3. TAP solid slant medium:

[0172] Add 20g / L agar to TAP liquid medium, boil and dissolve, and transfer to a test tube. Place the test tube containing the medium at 121°C and 103.4kPa for 20 minutes for high temperature and high pressure sterilization. Tilt the sterilized test tube containing the medium and cool it to obtain TAP solid slant medium.

[0173] The methods involved in the following embodiments are as follows:

[0174] 1. Drying method of Chlorella:

[0175] The harvested Chlorella liquid was centrifuged at 8000r / min and 4°C for 5 minutes, and the precipitated Chlorella cells were washed with distilled water. After repeated centrifugation and washing twice, the cells were pre-frozen in a -80°C refrigerator. The cells were then freeze-dried to obtain Chlorella powder.

[0176] 2. Calculation of Chlorella biomass:

[0177] The freeze-dried Chlorella powder obtained by the above method 1 (drying treatment method of Chlorella) is weighed to obtain the Chlorella biomass.

[0178] 3. Determination of starch content in Chlorella cells:

[0179] (1) Drawing of standard curve:

[0180] Dry about 0.1g of standard glucose at 105°C, 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 turn, and add deionized water to 1.0mL. Add 0.5mL of 5% (w / v, g / mL) phenol solution respectively, and quickly add 2.5mL of concentrated sulfuric acid. After reacting for 20min at room temperature in the dark, measure the absorbance at 490nm. Draw a standard curve with the absorbance value at 490nm as the ordinate and the glucose content (mg) as the abscissa;

[0181] (2) Determination of starch content in Chlorella cells:

[0182] Weigh 20 mg of dried algae powder, grind it with liquid nitrogen to break the wall, and then decolorize the algae powder with 2 mL of 80% (v / v) ethanol solution. After decolorization, centrifuge to remove the supernatant, and use 2 mL of 30% (v / v) perchloric acid to shake and extract the sample to fully hydrolyze the starch. Then combine the extracts, react with 0.5 mL of 5% (w / v, g / mL) phenol solution and 2.5 mL of concentrated sulfuric acid, and measure the absorbance at 490 nm. The starch content in the algae cells can be calculated according to the standard curve.

[0183] The gas used in the following examples is air rich in carbon dioxide, wherein the concentration of carbon dioxide is 2% (v / v), and the preparation method is as follows:

[0184] Connect the carbon dioxide cylinder and the air pump, mix the gases in the mixing chamber and then pump them into the column reactor through the ventilation pump.

[0185] The preparation method of the Chlorella sorokinensis seed solution mentioned in the following examples is:

[0186] A small amount of algal cells were picked from the TAP solid slant medium, inoculated into 20 mL of TAP medium, and cultured in a light incubator at 26°C, 12 h of light conditions, 12 h of dark conditions, a light intensity of 5000 lux, and an oscillation rate of 120 rpm / min. After 7 days of culture, the cultured Chlorella sorokinensis algae solution was inoculated into 200 mL of TAP medium as a Chlorella sorokinensis seed solution, which was cultured in a light incubator at 26°C, 12 h of light conditions, 12 h of dark conditions, a light intensity of 5000 lux, and an oscillation rate of 120 rpm / min.

[0187] The Chlorella mother solution mentioned in the following examples that is in the logarithmic growth phase is specifically determined by measuring the absorbance. During the culture process, the algae seed mother solution is expanded synchronously, that is, when the expansion culture medium is added at a volume ratio of 1:10, 100 mL of the algae seed is simultaneously left to expand to 1 L, which is used 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 means that the cell concentration increases, and it is considered to be in the logarithmic growth phase.

[0188] Wait for the absorbance OD 680 When the pH is between 0.2 and 0.5, Chlorella begins to enter the exponential growth phase.

[0189] The following examples describe the determination method of Chlorella in the late exponential growth phase:

[0190] Take 150 μL of Chlorella solution and add it to a 96-well plate. Place it in an ELISA reader and measure the absorbance at 680 nm. 680 When it is 1.1-1.5, Chlorella begins to enter the late exponential growth phase.

[0191] Example 1: Cultivation method of Chlorella

[0192] It is divided into two stages of cultivation. The specific steps are as follows:

[0193] (1) First stage training:

[0194] 200 mL of Chlorella sorokinensis seed solution was inoculated into a column reactor containing 2000 mL of TAP medium to mix the Chlorella sorokinensis and the medium evenly (OD 680 is 0.08~0.1), the biomass of Chlorella is about 0.1g / L; turn on the air pump and light, adjust the ventilation flow rate to 5L / min, the culture temperature to 26℃, the carbon dioxide concentration to 2% (v / v), 12h light conditions, 12h dark conditions, the light intensity to 5000lux, and culture for 96h.

[0195] (2) Second stage of training:

[0196] The Chlorella Sorokinensis algae solution obtained by culturing in step (1) is directly inoculated into a 20L column reactor containing a TAP medium in which nitrogen is reduced to 1 / 15 of the original amount and contains 5g / L sodium acetate, so that the Chlorella Sorokinensis and the medium are evenly mixed, and the Chlorella biomass is about 0.1g / L; the air pump and light are turned on, the culture temperature is 26°C, the carbon dioxide concentration is 2% (v / v), the light is continuously illuminated for 24h, the light intensity is 5000lux, and the culture is carried out for 72h; wherein the ventilation strategy is: the initial ventilation flow rate is 1.5L / min, when the Chlorella growth begins to enter the exponential phase (at this time OD 680 When the aeration rate was increased to 5 L / min, the growth of Chlorella was in the late exponential phase (OD 680 When the aeration rate was 1.4±0.05), the ventilation rate was changed to 1.5 L / min. After the culture was completed, the cultured microalgae cell fluid with high starch content was collected.

[0197] The collected Chlorella was dried, and its biomass was 1.54 g / L. The starch content in the Chlorella cells was measured to be 61.56%.

[0198] Example 2: Cultivation method of Chlorella

[0199] The specific implementation method is the same as Example 1, except that the ventilation flow rate strategy in step (2) is adjusted as follows: the initial ventilation flow rate is 0.75 L / min, when the growth begins to enter the exponential phase (at this time OD 680 When the growth rate was in the late exponential phase (OD 680 When the pressure drop is 1.1±0.05), change the ventilation volume to 0.75L / min.

[0200] The collected Chlorella was dried and its biomass was 1.37 g / L. The intracellular starch content of Chlorella was measured to be 55.12%.

[0201] Example 3: Cultivation method of Chlorella

[0202] The specific implementation method is the same as Example 1, except that the ventilation flow rate strategy in step (2) is adjusted as follows: the initial ventilation flow rate is 1.5 L / min, when the growth begins to enter the exponential phase (at this time OD 680 When the growth rate is in the late exponential phase (OD 680 When the pressure drop is 1.35±0.05), change the ventilation volume to 1.5L / min.

[0203] The collected Chlorella was dried, and its biomass was 1.42 g / L. The starch content in the Chlorella cells was measured to be 57.16%.

[0204] Example 4: Cultivation method of Chlorella

[0205] The specific implementation method is the same as Example 1, except that the ventilation flow rate strategy in step (2) is adjusted as follows: the initial ventilation flow rate is 1.5 L / min, when the growth begins to enter the exponential phase (at this time OD 680 When the growth rate was in the late exponential phase (OD 680 When the pressure drop is 1.1±0.05), change the ventilation volume to 1.5L / min.

[0206] The collected Chlorella was dried and its biomass was 1.43 g / L. The intracellular starch content of Chlorella was measured to be 59.26%.

[0207] Example 5: Cultivation method of Chlorella

[0208] The specific implementation method is the same as Example 1, except that the ventilation flow adjustment strategy in the adjustment step (2) is: the initial ventilation flow is 0.75 L / min, when the growth begins to enter the exponential phase (at this time OD 680 When the pressure drop was 0.27±0.05), the ventilation volume increased to 3L / min.

[0209] The collected Chlorella was dried, and its biomass was 1.35 g / L. The starch content in the Chlorella cells was measured to be 54.79%.

[0210] Example 6: Cultivation method of Chlorella

[0211] The specific implementation method is the same as Example 1, except that the ventilation flow adjustment strategy in the adjustment step (2) is: the initial ventilation flow is 1.5 L / min, when the growth begins to enter the exponential phase (at this time OD 680 When the pressure drop was 0.3±0.05), the ventilation volume increased to 4L / min.

[0212] The collected Chlorella was dried, and its biomass was 1.37 g / L. The starch content in the Chlorella cells was measured to be 55.72%.

[0213] Example 7: Cultivation method of Chlorella

[0214] The specific implementation method is the same as Example 1, except that the ventilation flow adjustment strategy in the adjustment step (2) is: the initial ventilation flow is 1.5 L / min, when the growth begins to enter the exponential phase (at this time OD 680 When the pressure drop is 0.3±0.05), the ventilation volume increases to 5L / min.

[0215] The collected Chlorella was dried, and its biomass was 1.48 g / L. The starch content in the Chlorella cells was measured to be 59.89%.

[0216] Comparative Example 1: Cultivation Method of Chlorella

[0217] The specific implementation method is the same as that of Example 1, except that the fixed ventilation flow rate of the entire culture process in step (2) is adjusted to 1.5 L / min.

[0218] The collected Chlorella was dried, and its biomass was 1.21 g / L. The starch content in the Chlorella cells was measured to be 50.93%.

[0219] Comparative Example 2: Cultivation Method of Chlorella

[0220] The specific implementation method is the same as that of Example 1, except that the fixed ventilation flow rate of the entire culture process in step (2) is adjusted to 5 L / min.

[0221] The collected Chlorella was dried, and its biomass was 1.34 g / L. The starch content in the Chlorella cells was measured to be 53.83%.

[0222] Example 7: Continuous culture method of Chlorella

[0223] The continuous culturing method of Chlorella in this embodiment uses a Chlorella culturing device to cultivate Chlorella Sorokinensis;

[0224] The device comprises:

[0225] Figure 1 This is a structural diagram of a microalgae cultivation device, in which: 1. aeration pump; 2. ventilation tube; 3. flow meter; 4. full-spectrum cultivation light strip; 5. injection port; 6. sampling port; 7. exhaust port; 8. first valve; 9. second valve; 10. third valve; 11. fourth valve; 12. first column reactor; 13. second column reactor; 14. third column reactor;

[0226] There are two parallel column reactors above the third column reactor (14), namely the first column reactor (12) and the second column reactor (13); the third column reactor (14) and the first column reactor (12) are connected by a second valve (9); the third column reactor (14) and the second column reactor (13) are connected by a third valve (10); and the first column reactor (12) and the second column reactor (13) are connected by a first valve (8);

[0227] Each column reactor is connected with ventilation equipment, which includes a ventilation pipe (2), a flow meter (3) and a ventilation pump (1) in sequence; a sterilizing filter is provided at the air inlet of the ventilation pump, the ventilation pump is connected with the flow meter and the ventilation pipe, and the ventilation pipe enters from the air inlet at the top of the column reactor;

[0228] The left side of the first column reactor (12), the right side of the second column reactor (13), and both sides of the third column reactor (14) are all connected with full-spectrum culture light strips (4), and the full-spectrum culture light strips are installed in the light strip frame;

[0229] The top cover of each column reactor is provided with a sample inlet (5), a sampling port (6) and an exhaust port (7) equipped with a filter membrane; the bottom of the third column reactor (14) is provided with a fourth valve (11) for quickly emptying the material.

[0230] The method comprises the following steps:

[0231] (1) 200 mL of Chlorella sorokinensis seed solution was inoculated into the first column reactor containing 2000 mL of TAP medium to mix the Chlorella sorokinensis and the medium evenly (OD 680 The biomass of Chlorella vulgaris was about 0.1 g / L. The culture temperature was 26°C, the carbon dioxide concentration was 2% (v / v), the light condition was 12 hours, the dark condition was 12 hours, the light intensity was 5000 lux, and the culture was 96 hours. The ventilation flow rate was 5 L / min.

[0232] (2) After the Chlorella was cultured in the first column reactor for 72 hours, the first valve was opened, and 200 mL of the Chlorella Sorokinensis cells in the first column reactor were inoculated into the second column reactor containing 2000 mL of TAP medium as seed liquid, so that the Chlorella Sorokinensis and the medium were evenly mixed, and the Chlorella biomass density was 0.1 g / L. The culture temperature was 26° C., the carbon dioxide concentration was 2% (v / v), the light condition was 12 hours, the dark condition was 12 hours, the light intensity was 5000 lux, and the culture was carried out for 96 hours. The ventilation flow rate was 5 L / min.

[0233] (3) After culturing Chlorella in the first column reactor for 96 hours, open the second valve and directly inoculate the remaining Chlorella Sorokinensis liquid cultured in the first column reactor into a third column reactor containing 20L of TAP medium reduced to 1 / 15 of the original nitrogen and containing 5g / L sodium acetate, so that the Chlorella Sorokinensis and the medium are evenly mixed, and the Chlorella biomass density is 0.1g / L. The culture temperature is 26°C, the carbon dioxide concentration is 2% (v / v), the light is continuously illuminated for 24 hours, the light intensity is 5000lux, and the culture is carried out for 72 hours. The initial ventilation flow rate is 1.5L / min. When the growth begins to enter the exponential phase (at this time OD 680When the growth rate is in the late exponential phase (OD 680 When the temperature is 1.4±0.05), the ventilation rate is changed to 1.5 L / min. After the culture is completed, the fourth valve is opened to collect the microalgae cell fluid with high starch content cultured in the third column reactor.

[0234] (4) When the Chlorella in the second column reactor was cultured for 72 hours, the first valve was opened, and 200 mL of Chlorella Sorokinensis cells in the second column reactor were inoculated into the first column reactor containing 2000 mL of TAP medium as seed liquid, so that the Chlorella Sorokinensis and the medium were evenly mixed, and the Chlorella biomass density was 0.1 g / L. The culture temperature was 26° C., the carbon dioxide concentration was 2% (v / v), the light condition was 12 hours, the dark condition was 12 hours, the light intensity was 5000 lux, and the culture was carried out for 96 hours. The ventilation flow rate was 5 L / min.

[0235] (5) After the Chlorella in the second column reactor has been cultured for 96 hours, the third valve is opened, and the Chlorella Sorokinensis liquid cultured in the second column reactor is directly inoculated into the third column reactor containing 20L of TAP culture medium with a nitrogen source reduced to 1 / 15 of the original amount and containing 5g / L sodium acetate, so that the Chlorella Sorokinensis and the culture medium are evenly mixed, and the Chlorella biomass density is 0.1g / L. The culture temperature is 26°C, the carbon dioxide concentration is 2% (v / v), the light is continuously illuminated for 24 hours, the light intensity is 5000lux, and the culture is carried out for 72 hours. The initial ventilation flow rate is 1.5L / min. When the growth begins to be in the exponential phase (at this time OD 680 When the growth rate is in the late exponential phase (OD 680 When the temperature is 1.4±0.05), the ventilation rate is changed to 1.5 L / min. After the culture is completed, the fourth valve is opened to collect the high starch content Chlorella cell liquid cultured in the third column reactor.

[0236] (6) Repeating the culture steps (2), (3), (4), and (5) can achieve efficient continuous culture of Chlorella cells with high starch content.

[0237] The collected Chlorella was dried and its biomass was measured to be 1.54 g / L. The intracellular starch content of Chlorella was measured to be 61.56%.

[0238] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 culturing microalgae with high starch content, characterized in that: The method is divided into two stages of cultivation; the first stage has a fixed ventilation volume, and the second stage has a dynamic ventilation volume adjustment: The second stage of dynamic ventilation adjustment strategy 1: the ventilation volume at the beginning of the culture is 0.75-1.5 L / min, and the ventilation volume is adjusted to 3-6 L / min when the microalgae growth begins to enter the exponential phase until the end of the culture; Or, in the second stage, the ventilation volume is dynamically adjusted by strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, the ventilation volume is adjusted to 3-6 L / min when the microalgae growth begins to enter the exponential phase, and the ventilation volume is adjusted to 0.75-1.5 L / min when the microalgae growth begins to enter the late exponential phase until the end of culture.

2. The culture method according to claim 1, characterized in that The method comprises the following steps: (1) First stage training: Inoculate the microalgae seed solution into the culture medium, mix well, turn on the air pump and light, adjust the ventilation flow rate to 0.75-6L / min, and culture for 96-168h; (2) Second stage of training: The microalgae liquid obtained by culturing in step (1) is directly inoculated into a nitrogen-reduced culture medium containing sodium acetate, mixed evenly, and the air pump and light are turned on for 24 to 120 hours; wherein the ventilation strategy is as follows: the initial ventilation flow rate is 0.75 to 1.5 L / min, when the microalgae growth begins to be in the exponential phase, the ventilation volume is increased to 3 to 6 L / min, and when the microalgae growth begins to be in the late exponential phase, the ventilation volume is changed to 0.75 to 1.5 L / min; after the cultivation is completed, the microalgae cell fluid is collected.

3. The culture method according to claim 2, characterized in that The culture medium in step (1) is BG11 culture medium, BBM culture medium or TAP culture medium; Preferably, the culture medium is TAP medium.

4. The culture method according to claim 2, characterized in that The nitrogen-reduced medium containing sodium acetate in step (2) is a BG11 medium, a BBM medium or a TAP medium in which the nitrogen source mass volume concentration is reduced to 1 / 20 to 1 / 7.5 and 1 to 10 g / L of sodium acetate is additionally added; Preferably, the nitrogen-reduced medium containing sodium acetate is a TAP medium in which the mass volume concentration of the nitrogen source is reduced to 1 / 15 and 5 g / L sodium acetate is additionally added.

5. The culture method according to claim 1 or 2, characterized in that: The OD of the microalgae when the microalgae growth begins to enter the exponential phase 680 0.2 to 0.5; the OD of the microalgae when the growth begins in the late exponential phase 680 It is 1.1 to 1.

5.

6. The culture method according to claim 2, characterized in that: The gas ventilated in steps (1) and (2) is a mixture of carbon dioxide and air; wherein the volume of carbon dioxide accounts for 1 to 5% of the volume of air; Preferably, the volume of carbon dioxide accounts for 2% of the volume content of air.

7. The culture method according to claim 2, characterized in that: In step (1) and step (2), the light intensity is 0 to 15000 lux, 12 to 24 hours of light, and 0 to 12 hours of darkness; Preferably, in step (1), the illumination intensity is 5000 lux, with 12 h of light and 12 h of dark conditions; and in step (2), the illumination intensity is 5000 lux, with 24 h of continuous illumination.

8. The culture method according to claim 2, characterized in that: The culture time in step (1) is 96 hours, and the culture time in step (2) is 72 hours.

9. A method for increasing the starch content in microalgae cells, characterized in that: The method is divided into two stages of cultivation; the first stage has a fixed ventilation volume, and the second stage has a dynamic ventilation volume adjustment: The second stage of dynamic ventilation adjustment strategy 1: the ventilation volume at the beginning of the culture is 0.75-1.5 L / min, and the ventilation volume is adjusted to 3-6 L / min when the microalgae growth begins to enter the exponential phase until the end of the culture; Or, in the second stage, the ventilation volume is dynamically adjusted by strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, the ventilation volume is adjusted to 3-6 L / min when the microalgae growth begins to enter the exponential phase, and the ventilation volume is adjusted to 0.75-1.5 L / min when the microalgae growth begins to enter the late exponential phase until the end of culture.

10. A method for increasing microalgae biomass by culturing microalgae, characterized in that: The method comprises two stages of cultivation; The ventilation volume is fixed in the first stage and dynamically adjusted in the second stage: The second stage of dynamic ventilation adjustment strategy 1: the ventilation volume at the beginning of the culture is 0.75-1.5 L / min, and the ventilation volume is adjusted to 3-6 L / min when the microalgae growth begins to enter the exponential phase until the end of the culture; Or, in the second stage, the ventilation volume is dynamically adjusted by strategy 2: the ventilation volume is 0.75-1.5 L / min at the initial stage of culture, the ventilation volume is adjusted to 3-6 L / min when the microalgae growth begins to enter the exponential phase, and the ventilation volume is adjusted to 0.75-1.5 L / min when the microalgae growth begins to enter the late exponential phase until the end of culture.

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