CN111500463A - Method for continuously culturing chrysophyceae - Google Patents
Method for continuously culturing chrysophyceae Download PDFInfo
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- CN111500463A CN111500463A CN202010285759.4A CN202010285759A CN111500463A CN 111500463 A CN111500463 A CN 111500463A CN 202010285759 A CN202010285759 A CN 202010285759A CN 111500463 A CN111500463 A CN 111500463A
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
- C12M37/04—Seals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/10—Separation or concentration of fermentation products
Abstract
A method for continuously culturing chrysophyceae comprises the following steps of, firstly, building a continuous culture reactor; step two, algae seed expanding culture; step three, carrying out algae seed expanding culture in a reactor; and step four, harvesting and re-culturing the algae. The invention provides a method for continuously culturing chrysophyceae, which realizes stable and efficient continuous production of the chrysophyceae, and finally realizes continuous industrial production of the chrysophyceae through algae seed pure culture → secondary algae seed expanding culture → chrysophyceae closed circulating reactor industrial culture.
Description
Technical Field
The invention belongs to the field of microalgae industrial culture, and relates to a method for continuously culturing chrysophyceae.
Background
The xanthomonas (tribonema.sp) is a kind of yellow green algae with plant bodies being non-branched filaments, belongs to the xanthophyta, and mostly grows in a pond and a humid fresh water body environment, so that the environmental adaptability is extremely strong, and in addition, the xanthomonas has good industrial properties of high oil yield (50-60 percent of oil content), pollution resistance and easy collection, and is an ideal material for producing biodiesel. The xanthomonas has good nitrogen and phosphorus absorption capacity, can be applied to sewage treatment, in addition, the grease extracted from the xanthomonas contains higher content of palmitoleic acid and EPA (docosapentaenoic acid), the fatty acid belongs to omega-7 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 palmitoleic acid is an indispensable nutrient component for normal growth and development of aquatic animal seedlings, and is an indispensable important nutrient which can not be synthesized by human bodies, so the xanthomonas is called as essential fatty acid for human bodies. Palmitoleic acid helps to reduce the content of cholesterol and triglyceride and promote the metabolism of saturated fatty acid in vivo, thereby reducing blood viscosity, promoting blood circulation, and improving oxygen supply of tissues 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, there is no method for continuously culturing xanthoceras fulva, and the prior culture method, for example, the invention patent application, "xanthoceras fulva culture device and culture method" (application number: 201811426390.3), discloses a method for culturing xanthoceras fulva by using a raceway pond device, which can realize the low-cost high-density culture of xanthoceras fulva, and the produced xanthoceras fulva is easy to separate and collect, labor-saving and can be repeatedly cultured, but the continuous and cyclic culture can not be realized, multiple times of inoculation are required, and the culture efficiency is low.
Disclosure of Invention
In order to solve the problems of no continuous culture and low culture efficiency in the prior art, the invention provides a method for continuously culturing the chrysophyceae, which realizes stable and efficient continuous production of the chrysophyceae, and realizes the continuous industrial production of the chrysophyceae through algae seed pure culture → secondary algae seed expansion culture → chrysophyceae closed circulating reactor industrial culture, and finally realizes the continuous industrial production of the chrysophyceae.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for continuously culturing Bothrina includes the following steps,
step one, building a continuous culture reactor: the reactor comprises a nutrient solution storage tank, a plurality of closed glass plates, a collecting plate, a filtering device, a circulating pump, a fan, a nutrient solution pipeline and a ventilation pipeline; the nutrient solution storage tank is connected with one ends of the plurality of closed glass flat plates through nutrient solution pipelines respectively, the other ends of the plurality of closed glass flat plates are connected with the collecting flat plate through nutrient solution pipelines respectively, the other end of the collecting flat plate is sequentially connected with a filtering device and a circulating pump through nutrient solution pipelines, the circulating pump is also connected with the nutrient solution storage tank, the fan is connected with the nutrient solution storage tank through a ventilation pipeline, and the nutrient solution pipeline and the ventilation pipeline are both provided with control valves;
step two, algae seed expanding culture: firstly, carrying out monoclonal culture on a chrysophyceae strain, carrying out pure culture on a sterilization culture medium after the monoclonal phycostrain grows out, carrying out primary algae strain expansion culture on the obtained pure algae strain, and then carrying out secondary algae strain expansion culture;
step three, algae seed expanding culture in the reactor: injecting nutrient solution into the nutrient solution storage tank, starting a fan to introduce required gas into the nutrient solution storage tank, injecting the nutrient solution in the nutrient solution storage tank into the plurality of closed glass plates after the secondary algae seeds are expanded and cultured in the second closed glass plate inoculation step, continuously injecting the nutrient solution into the plurality of closed glass plates until the rear collecting plates and the nutrient solution pipeline are filled with the nutrient solution, and starting a circulating pump to pump the nutrient solution back to the nutrient solution storage tank;
step four, harvesting and re-culturing algae: after the algae seeds are cultured in the reactor for a certain time, the filter device connected behind the collecting flat plate is filled with the algae seeds, at the moment, the circulating pump and the control valve on the nutrient solution pipeline are closed, the chrysophyceae algae seeds are taken out of the filter device, then the control valve is opened to restart the circulating pump, and the chrysophyceae is continuously cultured.
Further, the monoclonal culture process of the chrysophyceae strain in the first step is that 1% of agarose content is added into sterilized fresh water containing BG11 culture medium, the chrysophyceae plate is scribed or coated in a super clean bench, and the sterile culture medium pure culture is carried out after the monoclonal algae strain in the plate grows out;
the first-stage algae strain expanding culture process comprises the steps of carrying out 250ml conical flask strain expanding on pure-strain chrysophyceae obtained by pure-strain culture according to the inoculation amount of 1-3 g/L, wherein a culture medium is a sterilized BG11 culture medium, and carrying out shake flask culture for 10-12 days at a constant temperature of 23 ℃ for 24 hours under a 3000lx artificial led light source;
the secondary algae seed expanding culture process comprises inoculating Aphanizomenon flavidus grown in an erlenmeyer flask according to an initial inoculation amount of 1-3 g/L, culturing with BG11 culture medium, and introducing CO at 20-25 deg.C for 24 hr under L ED light source2The culture was carried out with a gas flow of 200 ml/min.
Further, the culture conditions in the third step are that BG11 culture medium is adopted as nutrient solution, and the specific components are as follows: c6H12O63-7g/L、NaNO30.5-0.7g/L、KH2PO40.3-0.5g/L、CaCl20.05-0.1g/L、MgSO4·7H2O0.075-0.09g/L、C6H8FeNO70.006-0.008g/L、C6H12O60.5-1.5 g/L, and culturing in greenhouse at pH 7-7.5 under illumination of 3000lx and full illumination period.
Preferably, the gas introduced by the fan is mixed with CO2Of air of (2), wherein CO2The volume ratio is 2-4.
Preferably, a 100-300 mesh filter screen is installed in the filter device.
The invention has the beneficial effects that:
the method for continuously culturing the chrysophyceae realizes stable and efficient continuous production of the chrysophyceae, and finally realizes continuous industrial production of the chrysophyceae through pure culture of algae seeds → second-level algae seed culture expansion → industrialized culture of the chrysophyceae in a closed circulating reactor. The culture method can reduce the inoculation times of the chrysophyceae in the industrialized expansion and preparation, realizes the continuous circulating industrialized production of the chrysophyceae by a culture mode of feeding and supplementing materials, and compared with the traditional open batch chrysophyceae industrialized culture, the culture method adopts closed culture, can avoid the problem of protozoan pollution of the chrysophyceae in the expansion and culture process, and the chrysophyceae closed circulating reactor adopts closed circulating supplementing materials to operate, and can greatly prolong the growth period of the chrysophyceae in the expansion and culture process. The method has the advantages that the biomass of the chrysophyceae in the production process can be remarkably improved and the collection process is simplified by adding the filtering device, the production efficiency of the chrysophyceae is greatly improved by improving the culture process and upgrading the production mode, the activity of the chrysophyceae cells is enhanced, the stable and efficient industrial production of the chrysophyceae fronds is finally realized, the manpower and material resources are greatly saved, and the powerful guarantee is provided for the industrial production of the chrysophyceae.
Drawings
FIG. 1 is a schematic view of a continuous culture reactor according to the present invention;
FIG. 2 is a schematic view of a filter device according to the present invention;
in the figure: the nutrient solution storage tank 1, the nutrient solution pipeline 2, 3 closed glass plates, 4 flow controllers, 5 collecting plates, 6 filtering devices, 7 circulating pumps, 8 control valves, 9 fans, 10 filtering device valves and 11 inner filter screens.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and embodiments.
Please refer to the attached drawings. A method for continuously culturing Bothrina includes the following steps,
step one, building a continuous culture reactor: the reactor comprises a nutrient solution storage tank 1, a plurality of closed glass plates 3, a collection plate 5, a filtering device 6, a circulating pump 7, a fan 9, a nutrient solution pipeline and a ventilation pipeline, wherein the nutrient solution storage tank 1 can be used for supplementing nutrient solution, and the closed glass plates 3 can be opened. Nutrient solution storage tank 1 links to each other with 3 one ends of a plurality of closed glass flat plates through the nutrient solution pipeline respectively, 3 other ends of a plurality of closed glass flat plates respectively through the nutrient solution pipeline with collect dull and stereotyped 5 and link to each other, it is similar with 3 structures of closed glass flat plates to collect dull and stereotyped 5, it has connected gradually filter equipment 6, circulating pump 7 to collect the dull and stereotyped 5 other end through the nutrient solution pipeline, circulating pump 7 still connects nutrient solution storage tank 1, fan 9 passes through the ventilation pipe and connects nutrient solution storage tank 1, fan 9 is customization constant voltage breather, lets in nutrient solution storage tank 1 and reactor main part with gas in, provides the necessary mist of growth for the cultivation of yellow silk algae. The nutrient solution pipeline and the ventilation pipeline are both provided with control valves 8, the filtering device 6 is preferably of a barrel-shaped structure, a water flow control valve 8, namely a filtering device valve 10, between the filtering device 6 and the collection flat plate 5 can block the nutrient solution flow through a water flow control method, the front section of the filtering device 6 is of a hollow structure and is used for concentrating and collecting the chrysophyceae algae, and the rear end of the filtering device is provided with 1-3 layers of inner filter screens 11 with the aperture of 100 plus 300. The circulating pump 7 is preferably a diaphragm pump, and the pump body has little damage to algae and is more suitable for being used as the circulating pump 7 for the circulating culture of the chrysophyceae. Flow controllers 4 are arranged on nutrient solution pipelines at the front ends of the closed glass plates 3 and used for controlling the flow of nutrient solution flowing through each closed glass plate 3 and accurately regulating and controlling the flow speed of the nutrient solution in the process of culturing the chrysophyceae. The circulating pump 7 can output in the forward direction or the reverse direction, the forward output is the circulation flow of the nutrient solution when the normal culture is carried out, and the reverse output can be carried out when the filtering device 6 is blocked, so that the filtering device 6 is washed.
And step two, algae seed expanding culture, namely, purifying the xanthoceras sp, namely adding the xanthoceras sp into sterilized fresh water containing a BG11 culture medium according to the agarose content of 1 percent, carrying out plate scribing or coating on the xanthoceras sp in a super clean workbench, carrying out sterile culture medium pure seed culture after a monoclonal algae strain in the plate grows out, carrying out 250ml conical flask expansion on the obtained pure culture xanthoceras sp according to the inoculation amount of 1-3 g/L, carrying out shake flask culture on the culture medium which is a sterilized BG11 culture medium for 10-12 days at the constant temperature of 23 ℃ for 24 hours under a 3000lx artificial led light source, and carrying out shake flask culture on the xanthoceras sp for 10-12 daysCulturing the obtained Bothrina fulva according to initial inoculation amount of 1-3 g/L with BG11 culture medium under artificial light source (L ED light source) for 24 hr at 20-25 deg.C mixed with CO2(CO22-4% of air volume) at a flow rate of 200 ml/min.
Step three, algae seed expanding culture in the reactor, namely, culturing water is connected into the reactor through a nutrient solution storage tank 1, a fan 9 is used for introducing mixed gas for culturing into the nutrient solution storage tank 1 through a ventilation pipeline, the gas enters the reactor along with the nutrient solution, the nutrient solution in the reactor is disinfected through a chemical reagent method, then, the nutrient salt of the chrysophyceae is added into the reactor and is circulated, when the nutrient salt is circulated uniformly in a water body, the chrysophyceae algae (with the algae density of 3 g/L) is inoculated, after inoculation, the algae solution passes through a flow controller 4 through a nutrient solution pipeline, a circulating closed glass plate 3 is circulated and collected into a collection plate 5 after culture, the chrysophyceae enters a filtering device 6 to be blocked, the nutrient solution returns to the nutrient solution storage tank 1 through a circulating pump 7 to finish the reactor circulating culture of the chrysophyceae, the chrysophyceae seeds are produced in large quantities and are collected in the filtering device 6 along with the culture, and the culture adopts an improved industrial culture medium formula C6H12O63-7g/L、NaNO30.5-0.7g/L、KH2PO40.3-0.5g/L、CaCl20.05-0.1g/L、MgSO4·7H2O 0.075-0.09g/L，C6H8FeNO70.006-0.008g/L C6H12O6Culturing in 0.5-1.5 g/L culture medium under the conditions of greenhouse, pH 7-7.5, illumination 3000lx, and full illumination period.
Step four, harvesting and re-culturing algae: after the algae seeds are cultured in the reactor for a certain time, the filter device 6 connected behind the collecting plate 5 is filled with the algae seeds, at the moment, the circulating pump 7 and the control valve 8 on the nutrient solution pipeline are closed, the chrysophyceae fronds are taken out from the filter device 6, then the control valve 8 is opened, the circulating pump 7 is restarted, and the chrysophyceae are continuously cultured.
It should be noted that in this cultivation process, all containers need to be sterilized, and the nutrient solution needs to be sterilized.
The method can greatly prolong the inoculation period of the Aphanizomenon flavus, and the period is prolonged from the original single batch of 7-10 days to the continuous culture period of 330-350 days, so that the Aphanizomenon flavus can reach the large-scale production state of collecting and culturing.
Claims (5)
1. A method for continuously culturing chrysophyceae is characterized in that: comprises the following steps of (a) carrying out,
step one, building a continuous culture reactor: the reactor comprises a nutrient solution storage tank, a plurality of closed glass plates, a collecting plate, a filtering device, a circulating pump, a fan, a nutrient solution pipeline and a ventilation pipeline; the nutrient solution storage tank is connected with one ends of the plurality of closed glass flat plates through nutrient solution pipelines respectively, the other ends of the plurality of closed glass flat plates are connected with the collecting flat plate through nutrient solution pipelines respectively, the other end of the collecting flat plate is sequentially connected with a filtering device and a circulating pump through nutrient solution pipelines, the circulating pump is also connected with the nutrient solution storage tank, the fan is connected with the nutrient solution storage tank through a ventilation pipeline, and the nutrient solution pipeline and the ventilation pipeline are both provided with control valves;
step two, algae seed expanding culture: firstly, carrying out monoclonal culture on a chrysophyceae strain, carrying out pure culture on a sterilization culture medium after the monoclonal phycostrain grows out, carrying out primary algae strain expansion culture on the obtained pure algae strain, and then carrying out secondary algae strain expansion culture;
step three, algae seed expanding culture in the reactor: injecting nutrient solution into the nutrient solution storage tank, starting a fan to introduce required gas into the nutrient solution storage tank, injecting the nutrient solution in the nutrient solution storage tank into the plurality of closed glass plates after the secondary algae seeds are expanded and cultured in the second closed glass plate inoculation step, continuously injecting the nutrient solution into the plurality of closed glass plates until the rear collecting plates and the nutrient solution pipeline are filled with the nutrient solution, and starting a circulating pump to pump the nutrient solution back to the nutrient solution storage tank;
step four, harvesting and re-culturing algae: after the algae seeds are cultured in the reactor for a certain time, the filter device connected behind the collecting flat plate is filled with the algae seeds, at the moment, the circulating pump and the control valve on the nutrient solution pipeline are closed, the chrysophyceae algae seeds are taken out of the filter device, then the control valve is opened to restart the circulating pump, and the chrysophyceae is continuously cultured.
2. The method of claim 1, wherein the method comprises the steps of: adding the 1% agarose content into sterilized fresh water containing BG11 culture medium, carrying out plate scribing or coating of the Aphanizomenon flavivirus in a super clean bench, and carrying out pure culture of the sterilized culture medium after a monoclonal algae strain grows out in the plate;
the first-stage algae strain expanding culture process comprises the steps of carrying out 250ml conical flask strain expanding on pure-strain chrysophyceae obtained by pure-strain culture according to the inoculation amount of 1-3 g/L, wherein a culture medium is a sterilized BG11 culture medium, and carrying out shake flask culture for 10-12 days at a constant temperature of 23 ℃ for 24 hours under a 3000lx artificial led light source;
the secondary algae seed expanding culture process comprises inoculating Aphanizomenon flavidus grown in an erlenmeyer flask according to an initial inoculation amount of 1-3 g/L, culturing with BG11 culture medium, and introducing CO at 20-25 deg.C for 24 hr under L ED light source2The culture was carried out with a gas flow of 200 ml/min.
3. The method of claim 1, wherein the method comprises the steps of: the culture conditions in the third step are that BG11 culture medium is adopted as nutrient solution, and the specific components are as follows: c6H12O63-7g/L、NaNO30.5-0.7g/L、KH2PO40.3-0.5g/L、CaCl20.05-0.1g/L、MgSO4·7H2O 0.075-0.09g/L、C6H8FeNO70.006-0.008g/L、C6H12O60.5-1.5 g/L, and culturing in greenhouse at pH 7-7.5 under illumination of 3000lx and full illumination period.
4. The method of claim 1, wherein the method comprises the steps of: the gas introduced by the fan is mixed with CO2Of air of (2), wherein CO2The volume ratio is 2-4.
5. The method of claim 1, wherein the method comprises the steps of: the filter device is internally provided with a filter screen with 100 meshes and 300 meshes.
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