CN111500462A - Method for industrial culture of nannochloropsis - Google Patents
Method for industrial culture of nannochloropsis Download PDFInfo
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- CN111500462A CN111500462A CN202010285164.9A CN202010285164A CN111500462A CN 111500462 A CN111500462 A CN 111500462A CN 202010285164 A CN202010285164 A CN 202010285164A CN 111500462 A CN111500462 A CN 111500462A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/02—Photobioreactors
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C—CHEMISTRY; METALLURGY
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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Abstract
A method for the industrial culture of nannochloropsis oculata comprises the following steps of, firstly, building a closed continuous nannochloropsis oculata culture bioreactor; step two, the nannochloropsis oculata is inoculated; step three, expanding and culturing nannochloropsis oculata; step four, industrial culture; step five, collecting and re-culturing algae liquid; and step three, continuous culture is carried out in the built closed continuous nannochloropsis oculata culture bioreactor, and semi-continuous culture is carried out in an open raceway pond industrially cultured in step four after the step three. The method is adopted to carry out early-stage efficient continuous nannochloropsis oculata expansion culture through the closed continuous nannochloropsis oculata bioreactor, the expansion method has the advantages of labor saving, high efficiency, sustainability, high reliability and the like, and compared with the traditional one-time step-by-step expansion culture method, the method can realize continuous multiple expansion culture of algae seeds through one-time expansion culture.
Description
Technical Field
The invention belongs to the field of microalgae industrial culture, and particularly relates to a method for the industrial culture of nannochloropsis.
Background
Nannochloropsis sp, belongs to Phaeophyta, Octophyceae and Nannochloropsis, is a kind of spherical or approximately spherical single-cell microalgae, the cells of which are small (usually 2-4 μm), the shape is simple, the sphere is formed, the lipid in the nannochloropsis sp is high, the dry weight of the cells can reach 30-60% according to literature report, wherein the lipid contains high content of eicosapentaenoic acid (EPA), the fatty acid belongs to omega-3 series polyunsaturated fatty acid, the fatty acid has very important function for aquatic animals and human bodies, in the seedling culture of fishes, shrimps and shellfish, the EPA is an indispensable nutrient component for the normal growth and development of aquatic animal seedlings, and is an important nutrient which can not be synthesized but is indispensable for human bodies, so the EPA is called as essential fatty acid for human bodies. EPA has effects of reducing cholesterol and triglyceride, promoting saturated fatty acid metabolism, lowering blood viscosity, promoting blood circulation, and improving oxygen supply of tissue to relieve fatigue. It also can prevent fat deposition on blood vessel wall, prevent atherosclerosis formation and development, and prevent cardiovascular diseases such as cerebral thrombosis, cerebral hemorrhage, hypertension, etc. .
At present, the algae culture mostly adopts a rough grading type expansion method, and has more defects, for example, in the aspect of culture solution, the culture solution is prepared by filtering seawater, so the cost is higher, the process is more complicated, the applicability is not wide, and the formula adjustment is often needed to be carried out aiming at different algae; in the aspect of the culture method, the unreasonable culture process results in long culture period, easy pollution of algae, resource waste and the like.
Disclosure of Invention
The invention aims to provide a method for the industrial culture of nannochloropsis oculata, aiming at the prior extensive grading-type microalgae expansion culture method, the method replaces the conventional grading-type microalgae industrial culture expansion culture mode by a closed continuous nannochloropsis oculata culture bioreactor, so that the early-stage expansion culture efficiency of the microalgae industrial culture is greatly improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for the industrial culture of nannochloropsis includes such steps as,
step one, constructing a closed continuous nannochloropsis oculata culture bioreactor: the bioreactor comprises a three-mouth triangular flask, a three-mouth round-bottom flask and a three-mouth closed glass plate, wherein the three-mouth triangular flask, the three-mouth round-bottom flask and the three-mouth closed glass plate are communicated with each other through pipelines to form a unified whole, and three containers are respectively provided with three openings, namely a nutrient solution injection port, an algae solution outflow port, a ventilation port and an air outlet port; the three containers are respectively in different heights in the horizontal position, the horizontal heights of the three containers are respectively three-mouth triangular flask > three-mouth round-bottom flask > three-mouth closed glass flat plate, and after the different containers reach the rated culture volume, the algae liquid of the previous stage can naturally flow into the next container through a communicating pipe through the liquid level difference;
step two, nannochloropsis oculata inoculation, namely purifying the known nannochloropsis oculata by adopting a plate streaking method to obtain a monoclonal algae strain, then carrying out pure culture, and starting inoculation work after the volume of algae liquid reaches 200ml and the biomass reaches 1 g/L;
step three, the nannochloropsis oculata is expanded and cultivated: inoculating nannochloropsis oculata obtained by culturing into a three-mouth triangular flask, then adding nutrient solution into the three-mouth triangular flask every day for culturing, when the volume of the cultured algae solution reaches the rated volume of the three-mouth triangular flask, feeding the algae solution into a three-mouth round-bottom flask through a pipeline, then adding a certain amount of nutrient solution into the three-mouth round-bottom flask every day, when the volume of the algae solution reaches the rated volume of the three-mouth round-bottom flask, feeding the algae solution into a three-mouth closed glass plate through a pipeline, and continuously adding the nutrient solution into the three-mouth closed glass plate for culturing until the volume of the algae solution reaches the rated volume of the three-mouth closed glass plate;
step four, industrial culture, namely, after completing the nannochloropsis oculata culture in the step three in one period, injecting the algae liquid in the three-port closed glass flat plate into an open raceway pond for culture, and collecting the nannochloropsis oculata after the biomass of the nannochloropsis oculata reaches 1-1.5 g/L after the culture is carried out for 10-12 days;
step five, collecting and re-culturing algae liquid: half of the algae liquid obtained by culturing in the open raceway pond is pumped into a centrifuge through a pipeline to be collected, and after the algae liquid is collected, fresh nutrient solution is supplemented into the open raceway pond and the algae liquid cultured in the three-port closed glass flat plate is subjected to multiple circulating culture and collection.
Preferably, the volume of the three-neck triangular flask is 2L, the volume of the three-neck round-bottom flask is 15L, the volume of the three-neck closed glass plate is 200L, and the volume of the open raceway pond is 5 t.
Further, the initial inoculation density in the nannochloropsis oculata inoculation and propagation stage in the third step is 2 × 107cell/ml。
Further, in the third step, the algae cultivation needs to be controlled by temperature, illumination and ventilation, and the cultivation conditions are as follows: the nutrient solution is f/2 sterilized culture medium with pH of 6-8, the light source is artificial led light source, the illumination intensity is 3000lx, the culture is performed under full illumination for 24h at constant temperature of 23 deg.C for 10-15 days, and gas CO is introduced2The aeration flow rate of the filtered air is 200 ml/min.
Furthermore, in the fourth step, the culture conditions of nannochloropsis oculata are that the nutrient solution is f/2 sterile culture medium with pH value of 6-8, the temperature is 23-25 ℃, the ratio of light to dark cycles is 12:12, and the ventilation volume is 5L/min.
Further, the f/2 sterilization medium consists of: NaNO30.3-0.35g/L，KH2PO40.15-0.2g/L、CaCl20.05-0.1g/L、EDTA-Na24-7mg/L。
Further, CO was introduced2Filtering air of (2), wherein CO2The volume of (a) is 3-5% of the volume of air.
The invention has the beneficial effects that:
the invention relates to a method for the industrialized culture of nannochloropsis oculata, which aims at the prior extensive grading-type microalgae expansion culture method.A closed continuous nannochloropsis oculata culture bioreactor replaces the grading-type expansion culture mode of the conventional industrialized culture of microalgae, so that the early-stage expansion culture efficiency of the microalgae is greatly improved; according to the invention, continuous subculture and amplification of nannochloropsis is carried out through the technical route of pure culture of algae liquid (plate scribing) → closed continuous nannochloropsis culture bioreactor seed expansion → continuous production of nannochloropsis (oval open raceway pond), and finally the industrial stable production of nannochloropsis is realized.
Drawings
FIG. 1 is a schematic view of a culture apparatus according to the present invention;
FIG. 2 is a corresponding relationship between the volume of algae liquid in a three-neck round-bottom flask and the culture time;
FIG. 3 is the corresponding relationship between the volume of algae liquid and the cultivation time in a three-port closed glass plate
In the figure: the reactor comprises a nutrient solution storage tank 1, a three-neck triangular flask 2, a three-neck round-bottom flask 3, a three-neck closed glass plate 4, an open raceway pond 5, a disc centrifuge 6, a fan 7, a reactor ventilation supply pipeline 8 and a reactor nutrient solution supply pipeline 9.
Detailed Description
The invention is illustrated below with reference to specific examples:
firstly, as shown in figure 1, a closed continuous nannochloropsis oculata culture bioreactor is set up, the bioreactor comprises a three-port triangular flask, a three-port round-bottom flask and a three-port closed glass flat plate, the three-port closed glass flat plate is of a cuboid closed container structure and can be opened, inoculation is convenient, and the whole system needs to be disinfected and sterilized before culture. The three containers are respectively provided with three openings, namely a nutrient solution injection port, an algae liquid outflow port, a ventilation port and an air outlet; and the three containers are respectively in different heights in horizontal positions, the horizontal heights of the three containers are respectively three-mouth triangular flask > three-mouth round-bottom flask > three-mouth closed glass flat plate, and after the different containers reach rated culture volumes, the algae liquid of the previous stage can naturally flow into the next container through a communicating pipe through liquid level difference. The three containers are communicated with each other through pipelines, a nutrient solution storage tank is arranged above the three-opening triangular flask, the nutrient solution storage tank is respectively connected with the three containers through reactor nutrient solution supply pipelines, the upper outlets of the three containers are closed, the three containers are connected with a fan through reactor ventilation supply pipelines, and gas introduced into the three containers by the fan is mixed with CO2Filtering air of (2), wherein CO2The volume of (a) is 3-5% of the volume of air. The reactor aeration supply pipeline and the reactor nutrient solution supply pipeline are preferably inserted in a bottom-inserting pipe manner in the three containers. The reactor aeration supply pipeline and the reactor nutrient solution supply pipeline are also provided with control valves for controlling the flow of gas supply and liquid supply to ensure the survival of microalgae. The nutrient solution is f/2 sterilization culture medium with pH value of 6-8, and comprises the following components: NaNO30.3-0.35g/L，KH2PO40.15-0.2g/L、CaCl20.05-0.1g/L、EDTA-Na24-7mg/L。
And step two, nannochloropsis oculata inoculation, namely purifying the known nannochloropsis oculata by adopting a plate streaking method, adding the purified nannochloropsis oculata into sterilized seawater containing f/2 sterilization culture medium according to the agarose content of 1%, streaking or coating the nannochloropsis oculata in an ultraclean workbench, performing pure culture of the sterilization culture medium after a monoclonal algae strain in the plate grows out, and starting to inoculate after the volume of algae liquid reaches 200ml and the biomass reaches 1 g/L.
Step three, performing nannochloropsis oculata cultivation, namely performing 2L three-opening triangular flask cultivation on the obtained pure-cultivated nannochloropsis oculata liquid according to the proportion of 1:5, wherein the inoculation density of nannochloropsis oculata is 2 × 107cell/ml and volume of 200ml, culturing by introducing a 23 ℃ ventilating feeding nutrient solution at a constant temperature for 24h under a 3000lx artificial led light source, controlling the flow rate of a nutrient solution supply pipeline of the reactor through a nutrient solution supply system of the reactor after inoculation, supplementing and supplying 200ml of nutrient solution to the three-mouth triangular flask every day, continuously supplementing 500ml of nutrient solution to the three-mouth triangular flask every day by feeding and culturing for ten days until the rated volume of the three-mouth triangular flask is 2L, continuously feeding 500ml of nutrient solution into the three-mouth triangular flask every day for 15 days for feeding and culturing, and automatically feeding the algae solution cultured in the three-mouth triangular flask into the three-mouth round-bottom flask due to liquid level difference in the process of continuously feeding the nutrient solution.
After the three-mouth triangular flask with 2L mouth reaches the rated volume, 500ml of pure nannochloropsis oculata solution can be provided for the 15L three-mouth round-bottom flask every day by fed-batch culture, at the moment, 500ml of fresh sterilization nutrient solution is directly added into the three-mouth round-bottom flask every day by adjusting a reactor ventilation supply pipeline connected with the three-mouth round-bottom flask and a control valve on a reactor nutrient solution supply pipeline for carrying out expanded culture (the culture conditions are f/2 culture medium, L ED light source for 24h, constant temperature is 23 ℃, and CO is mixed with the culture medium2(CO23-5% of air volume) of the filter gas flow rate is 200ml/min), after 15 days, the algae liquid flowing in through the upstream three-neck triangular flask and the fresh nutrient solution are supplemented, the algae liquid in the three-neck round-bottom flask reaches the rated volume of 15L, and the nannochloropsis oculata biomass in the three-neck round-bottom flask can be cultured to 7 × 108～1.3×109cell/ml. Then adjusting the nutrient solution supply pipeline of the reactorControlling the parameters of the valve, adding 1.5L of fresh sterilization nutrient solution into the three-mouth round-bottom flask every day for supplement, simultaneously continuously supplementing 500ml of fresh sterilization nutrient solution into the upstream three-mouth triangular flask, and flowing into the three-mouth round-bottom flask through the three-mouth triangular flask from the liquid level difference for continuous culture.
After the three-mouth round-bottom flask reaches the rated culture volume of nannochloropsis oculata, continuously adding 1.5L fresh sterilizing nutrient solution and 500ml algae solution flowing from a three-mouth triangular flask, so that the three-mouth round-bottom flask can provide 2L nannochloropsis oculata high-density pure culture algae solution for a 200L three-mouth closed rectangular glass plate every day, inoculating in the three-mouth closed rectangular glass plate, adding 20L fresh sterilizing nutrient solution into the 200L three-mouth closed glass plate every day by a nutrient solution supply system, completing one cycle of nannochloropsis oculata expansion culture after the three-mouth closed glass plate reaches the rated volume for 10 days, then inoculating the cultured 200L nannochloropsis oculata solution into a 5t open raceway pond for semi-continuous culture, completing one cycle of nannochloropsis oculata in a reactor, and realizing continuous culture in the reactor.
Step four, industrial culture: inoculating the algae liquid in the three-opening closed glass plate into a 5t open raceway pond, and culturing under the conditions of room temperature, pH 6-8 and natural light illumination, wherein the culture medium formula is as follows: NaNO30.3-0.35g/L，KH2PO40.15-0.2g/L、CaCl20.05-0.1g/L、EDTA-Na24-7 mg/L, and the culture period is 10-12 days.
Step five, collecting and re-culturing the algae liquid, wherein the biomass density of the algae liquid reaches 5 × 10 after the 5t open raceway pond finishes the industrial culture for 10 to 12 days8-8×108And when cell/ml is needed, extracting 2.5t of algae liquid for disc-type centrifugal collection, supplementing 2.3t of sterilized fresh culture solution after collection and expanding culture in a closed continuous nannochloropsis oculata culture bioreactor to obtain 200L nannochloropsis oculata algae liquid, carrying out next batch culture for 10-12 days, and then carrying out periodic circulation so as to achieve semi-continuous industrial culture of nannochloropsis oculata.
The method is adopted to carry out early-stage efficient continuous nannochloropsis oculata expansion culture through a closed continuous nannochloropsis oculata bioreactor, the expansion method has the advantages of labor saving, high efficiency, sustainability, high reliability and the like, compared with the traditional one-time step-by-step expansion culture method, the method can realize continuous multiple expansion culture of algae seeds through one-time expansion culture, and compared with the traditional open raceway pond nannochloropsis oculata culture, the later-stage semi-continuous industrial culture method of the open raceway pond has the advantages of high culture efficiency and culture cost saving. The invention provides a set of simple and efficient culture method for the industrial culture of nannochloropsis.
Claims (7)
1. A method for the industrial culture of nannochloropsis is characterized in that: comprises the following steps of (a) carrying out,
step one, constructing a closed continuous nannochloropsis oculata culture bioreactor: the bioreactor comprises a three-mouth triangular flask, a three-mouth round-bottom flask and a three-mouth closed glass plate, wherein the three-mouth triangular flask, the three-mouth round-bottom flask and the three-mouth closed glass plate are communicated with each other through pipelines to form a unified whole, and three containers are respectively provided with three openings, namely a nutrient solution injection port, an algae solution outflow port, a ventilation port and an air outlet port; the three containers are respectively in different heights in the horizontal position, the horizontal heights of the three containers are respectively three-mouth triangular flask > three-mouth round-bottom flask > three-mouth closed glass flat plate, and after the different containers reach the rated culture volume, the algae liquid of the previous stage can naturally flow into the next container through a communicating pipe through the liquid level difference;
step two, nannochloropsis oculata inoculation, namely purifying the known nannochloropsis oculata by adopting a plate streaking method to obtain a monoclonal algae strain, then carrying out pure culture, and starting inoculation work after the volume of algae liquid reaches 200ml and the biomass reaches 1 g/L;
step three, the nannochloropsis oculata is expanded and cultivated: inoculating nannochloropsis oculata obtained by culturing into a three-mouth triangular flask, then adding nutrient solution into the three-mouth triangular flask every day for culturing, when the volume of the cultured algae solution reaches the rated volume of the three-mouth triangular flask, feeding the algae solution into a three-mouth round-bottom flask through a pipeline, then adding a certain amount of nutrient solution into the three-mouth round-bottom flask every day, when the volume of the algae solution reaches the rated volume of the three-mouth round-bottom flask, feeding the algae solution into a three-mouth closed glass plate through a pipeline, and continuously adding the nutrient solution into the three-mouth closed glass plate for culturing until the volume of the algae solution reaches the rated volume of the three-mouth closed glass plate;
step four, industrial culture, namely, after completing the nannochloropsis oculata culture in the step three in one period, injecting the algae liquid in the three-port closed glass flat plate into an open raceway pond for culture, and collecting the nannochloropsis oculata after the biomass of the nannochloropsis oculata reaches 1-1.5 g/L after the culture is carried out for 10-12 days;
step five, collecting and re-culturing algae liquid: half of the algae liquid obtained by culturing in the open raceway pond is pumped into a centrifuge through a pipeline to be collected, and after the algae liquid is collected, fresh nutrient solution is supplemented into the open raceway pond and the algae liquid cultured in the three-port closed glass flat plate is subjected to multiple circulating culture and collection.
2. The method of claim 1, wherein the volume of the three-necked flask is 2L, the volume of the three-necked round-bottomed flask is 15L, the volume of the three-necked closed glass plate is 200L, and the volume of the open raceway pond is 5 t.
3. The method for the industrial culture of nannochloropsis oculata as claimed in claim 1, wherein the initial inoculation density in the expansion stage of nannochloropsis oculata in step three is 2 × 107cell/ml。
4. The method for industrial culture of nannochloropsis oculata as claimed in claim 1, wherein: in the third step, the algae cultivation needs to be carried out with temperature, illumination and ventilation control, and the cultivation conditions are as follows: the nutrient solution is f/2 sterilized culture medium with pH of 6-8, the light source is artificial led light source, the illumination intensity is 3000lx, the culture is performed under full illumination for 24h at constant temperature of 23 deg.C for 10-15 days, and gas CO is introduced2The aeration flow rate of the filtered air is 200 ml/min.
5. The method for the industrial culture of nannochloropsis oculata as claimed in claim 1, wherein the nannochloropsis oculata is cultured under the conditions that the nutrient solution is f/2 sterilized medium with pH 6-8, the temperature is 23-25 ℃, the ratio of light to dark cycles is 12:12, and the ventilation rate is 5L/min.
6. The method for industrial culture of nannochloropsis oculata as claimed in claim 4 or 5, wherein: the f/2 sterilization culture medium comprises the following components: NaNO30.3-0.35g/L，KH2PO40.15-0.2g/L、CaCl20.05-0.1g/L、EDTA-Na24-7mg/L。
7. The method for industrial culture of nannochloropsis oculata as claimed in claim 4, wherein: introducing CO2Filtering air of (2), wherein CO2The volume of (a) is 3-5% of the volume of air.
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