CN113736665A - Haematococcus pluvialis strain breeding method suitable for industrial production and application - Google Patents
Haematococcus pluvialis strain breeding method suitable for industrial production and application Download PDFInfo
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Abstract
The invention discloses a haematococcus pluvialis strain breeding method suitable for industrial production and application, which comprises the following steps: the method comprises the following steps: selecting original algae seeds → purifying original algae seeds → expanding culture of algae seeds → establishing algae seed series → primary screening of algae seeds → secondary screening of algae seeds → establishment and conservation of algae seed → production and application test of algae seeds → determination of excellent algae seeds. The original algae seeds obtained by screening by the method provided by the invention are used for industrial large-scale culture of haematococcus pluvialis, the breeding period is short, and the efficiency for producing astaxanthin is high; in addition, the obtained algae seeds are not easy to degenerate, age and stable in heredity, keep good activity in a growth period, are full in chromoplast and low in mortality, and are directionally bred by using production conditions, so that the success rate is high, the obtained algae seeds can be directly applied to production, the large production risk caused by adaptability of common algae seeds is avoided, the obtained algae seeds are stable in heredity and safe in product, and the control on the production process is improved.
Description
Technical Field
The invention relates to the technical field of microalgae biological breeding, in particular to a haematococcus pluvialis strain breeding method suitable for industrial production application.
Background
Microalgae, as a primary productivity on earth, are themselves capable of synthesizing a variety of specific bioactive substances, such as fatty acids, sugars, pigments, toxins, etc., many of which are not synthesized by other organisms and are ultimately obtained from microalgae only through the food chain. Obtaining valuable secondary metabolites from microalgae by large-scale cultivation has been the focus of algal development.
Haematococcus pluvialis belongs to unicellular freshwater microalgae in the phylum Chlorophyta, and can accumulate a large amount of astaxanthin under adversity stress, wherein cells are changed from green to red, which is called green cell stage and red cell stage for short, and the process of promoting the cells to turn red by artificial stress is called red urging for short. The haematococcus pluvialis changed into red is rich in a large amount of astaxanthin, the astaxanthin is the highest-level product synthesized by carotenoid in cells, has the strongest antioxidant activity, is the strongest natural antioxidant found at present, can be widely applied to food, health care products, feed, cosmetics and the like, and is a novel active substance from algae.
In the process of research, development and application of microalgae, the algae species are the basis for developing all the works and determine the success or failure and efficiency of the subsequent works. The pure and excellent algae species can not only ensure the stability, reliability and repeatability of the result, but also fundamentally ensure the obtaining of valuable positive results. The seed selection method of microalgae generally comprises selective breeding, mutation breeding, genetic engineering breeding and small-amount cell fusion breeding. The selective breeding is the basis of all breeding methods, although the breeding period is long, the method is simple and easy to implement, does not change the original characters of the algae, is high in safety, and avoids uncertain and uncontrollable factors and risks. At present, selective breeding is basically adopted in the breeding of haematococcus pluvialis, but the general haematococcus pluvialis strain breeding method in the prior art has the defects of long breeding period, low efficiency for producing astaxanthin, easy degradation, aging, instable heredity, high death rate, incapability of industrial production and the like.
Disclosure of Invention
The invention aims to provide a haematococcus pluvialis strain breeding method suitable for industrial production and application, and solves the problems.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides a haematococcus pluvialis strain breeding method suitable for industrial production and application, which comprises the following steps: selecting original algae seeds → purifying original algae seeds → expanding culture of algae seeds → establishing algae seed series → primary screening of algae seeds → secondary screening of algae seeds → establishment and conservation of algae seed → production and application test of algae seeds → determination of excellent algae seeds, wherein:
s1, selection of original algae species: samples with outstanding performance are reserved in production application practice through field collection, communication or purchase and are used as original algae seeds of breeding seeds;
s2, purifying original algae species: purifying the original algae species according to the pollution degree of the algae species;
s3, algae seed expanding culture: performing cell culture on an illumination culture rack of an independent breeding room, wherein a sterile BBM culture medium is adopted as a culture medium;
s4, establishing an algae species series: carrying out propagation on the algae obtained after purification in the S2 to reach a certain volume and cell density, and establishing a series which can be used for carrying out selective breeding work under specific production conditions;
s5, primary screening of algae: carrying out primary screening on different series of algae seeds subjected to propagation by using astaxanthin induction conditions of nitrogen deficiency;
s6, secondary screening of algae: carrying out directional screening on algae seeds under the condition of industrial production;
s7, establishing and protecting the seeds in a quality library: preserving and establishing a library for the good algae screened by the simulation experiment;
s8, algal species production application test: testing the algae species in the germplasm library in S7, and detecting the astaxanthin content and dry weight indexes;
s9, determination of excellent algae species: determining the haematococcus pluvialis strain suitable for industrial production.
Preferably, when the field collection is carried out in S1, samples which can realize artificial production and have formed dominant growth of algae seeds in the original environment are selected for collection; when in communication and purchase, the selected algae species with the following characteristics are used as starting algae species of selective breeding, and the starting algae species comprise: the cell growth is rapid, the division is vigorous, the activity is strong, the chromoplast is full, the individual cell is generally large, the stress resistance is strong, and the dominant growth can be formed under the condition that other organisms exist.
Preferably, in S2, the relatively pure stock is not required to be specially purified, and only the fresh sterile BBM culture medium is replaced by a small amount for rejuvenation; purifying algae species with general pollution, including microorganism and protozoan pollution but without other algae or other biological pollution by adopting a centrifugal cleaning and solid plate separation method; for samples with heavy pollution degree and impurities and various organisms in the samples, pure algae cells are obtained by adopting a capillary micro-operation method after pretreatment such as filtration and dilution; the method for purifying the stock with common pollution degree by adopting solid plate separation comprises the following specific steps:
a, performing low-speed centrifugation on original algae seeds, wherein the centrifugation speed is not more than 5000rpm, the time is 3.0min, removing supernatant, retaining bottom sediment, adding fresh sterile culture solution or buffer solution into a centrifuge tube, performing re-suspension dispersion on algae mud, performing centrifugation again after cleaning, collecting sediment, wherein the cleaning operation frequency can be different according to the pollution condition of the original seeds, and finally performing microscopic examination and rough counting on the obtained algae mud in the sterile culture solution;
b, diluting the algae liquid obtained in the step a to enable the final cell density to be about 500-1000cells/ml for later use;
c, adding agar powder into a conventional BBM liquid culture medium according to the proportion of 1.5%, adding antibiotics after sterilization when the culture medium is cooled, wherein the antibiotics consist of nystatin and chloramphenicol, the concentration is 80ppm, uniformly mixing, and immediately pouring the mixture into a flat plate;
d, taking 100 mu L of the cell diluent in the step b, inoculating the cell diluent into a flat plate, lightly and uniformly coating the cell diluent by using a coating rod, turning the culture dish upside down, and putting the culture dish into a constant-temperature illumination box for culture;
e, performing plate culture until macroscopic algae colonies grow out, selecting single algae colonies by using an inoculating loop, inoculating into a test tube, continuously culturing, gradually amplifying, and performing secondary streak inoculation and separation on the algae colonies in a solid culture medium according to the condition; for the original seeds with serious pollution, a capillary microscopy method is adopted for purification, and the specific steps are as follows:
a, filtering original algae seeds to remove impurities and partial pollutants, wherein a bolting silk or a filter membrane is adopted for filtering, the bottom sediment is reserved after the algae liquid is centrifuged at a low speed (5000rpm for 3.0min), fresh sterile culture solution or buffer solution is added into a centrifuge tube, algae mud is uniformly dispersed, cleaned and then centrifuged again, the sediment is collected, the cleaning times are different according to the pollution condition of the original seeds for 3-5 times, and finally the obtained algae mud is re-suspended by the sterile culture solution, and is subjected to microscopic examination and rough counting;
b, diluting the algae liquid obtained in the step a to enable the final cell density to be about 300-500cells/ml for later use;
c, drawing a capillary glass tube at high temperature by using an alcohol blast burner, wherein the aperture of the capillary tube is about 40-100 um;
d, dripping the diluted algae liquid obtained in the step b onto a glass slide sterilized in advance in a microdroplet mode, and quickly performing microscopic examination on the microdroplet size based on the fact that the visual field can be completely covered to determine target cells which are healthy, full in pigment and large in size;
e, sucking the target cells by using the glass capillary tube drawn by the step c under a microscope, transferring the target cells into a 24-hole cell culture plate filled with a sterilized culture medium, repeating the operation, and picking a plurality of cells into the culture plate, wherein one cell is placed into each hole;
f, culturing the cell plate in dark light, volatilizing the supplementary culture medium during the culturing, transferring the cell plate into a test tube for culturing when the color of the cell plate is visible to naked eyes, and gradually culturing and amplifying from a small volume to obtain the purified algae.
Preferably, the nutrient salts with the following concentrations are contained in each L of the algae liquid after the culture medium is added into S3: 1.5 to 2.0 multiplied by 10 to 3g of sodium nitrate, 7.5 to 10.0 multiplied by 10 to 3g of dipotassium phosphate, 1.75 to 2.25 multiplied by 10 to 2g of monopotassium phosphate, 2.5 to 3.5 multiplied by 10 to 2g of sodium chloride, 3.7 to 4.7 multiplied by 10 to 2g of magnesium sulfate, 1.9 to 2.5 multiplied by 10 to 2g of calcium chloride, 1.10 to 1.50 multiplied by 10 to 2g of boric acid, 8.70 to 9.50 multiplied by 10 to 3g of zinc sulfate, 1.40 to 1.60 multiplied by 10 to 3g of manganese chloride, 1.2 to 1.4 multiplied by 10 to 3g of sodium molybdate, 1.50 to 1.80 multiplied by 10 to 3g of copper sulfate, 4.9 to 5.3 multiplied by 10 to 4g of cobalt nitrate, 5.0 to 6.0 multiplied by 10 to 2g of disodium ethylenediaminetetraacetate, 3.0 to 5.0 to 5.10 to 2g of potassium hydroxide, 4.9 to 5.5 multiplied by 10 to 3g of potassium phosphate, and a white light LED light source, the illumination intensity is adjusted according to the cell density, the photoperiod L: D is 12:12, the pH value is controlled to be 7.0-7.5, the temperature is controlled to be 24-26 ℃, the shake flask culture is carried out in a culture volume of 5L or more, the aeration culture is carried out in a volume of 5L or more, and the introduced gas is the mixed gas of compressed air and carbon dioxide which are subjected to sterilization and filtration.
Preferably, the specific operation of S4 is: when the copied original algae species with pure sources reach 1L of volume and the cell density reaches 25x104cells/ml, diluting the algae liquid by 50 times with a fresh culture medium until the cell density is 5x103cells/ml, subpackaging the algae liquid into a plurality of triangular flasks for continuous culture, and when the volume is increased to 2.5L and the cell density is about 15x104cells/ml, using the algae liquid as a screening algae species series for later use, numbering the series and establishing a file.
Preferably, the specific operation method of S5 is: 2.5L of algae solution after each series of expanding culture is settled, filtered and cleaned, after the interference of the original culture medium is removed, the cells are suspended by nitrogen deficiency culture medium, and are subpackaged into 3 1L triangular flasks, wherein each flask is 750ml, and each algae seed is 3 parallel; all the triangular flasks were placed on a culture shelf and kept consistent under the following conditions: the culture medium adopts a nitrogen-deficient culture medium, namely nitrate components are removed from the conventional BBM culture medium, the light source is a special LED lamp tube capable of emitting the following specific spectrum for promoting red blood production by Haematococcus pluvialis, the specific spectrum comprises a first LED light-emitting chip with the wavelength of 370-: 32-50: 66-70, arranging the lamp tubes above and below the culture frame, controlling the illumination intensity of 15000Lux, the light period L: D to be 16:8, the pH value to be 8.5-9.0, controlling the temperature to be 27-29 ℃, and obtaining the algae series with quick astaxanthin accumulation, high astaxanthin accumulation amount in the same time, high cell dry weight, low cell death rate and clean culture system by primary screening.
Preferably, since the industrial equipment in S6 is bulky and is not suitable for the algal species screening experiment, firstly, a simulated production condition needs to be established, and the specific operation method of S6 is as follows: according to the shape, accessories and related equipment of the vertical photobioreactor with built-in light sources adopted in industrial production, a small simulation photobioreactor is manufactured, experimental conditions which are consistent with the red-promoting induction conditions of haematococcus pluvialis adopted in production to the maximum extent are provided, and the simulation photobioreactor has the following characteristics: the capacity is reduced to 15L from 1000L of industrial equipment, the material is changed into acrylic tube from stainless steel, according to the light path of light source for production, 1 special red LED fluorescent tube that urges that is used for red ball algae is put to the simulation reactor, in addition, the monitoring regulation and control system of the aeration system, temperature, PH etc. of the simulation reactor is with industrial equipment, a plurality of simulation reactors are installed and fixed on the support in unison, each container package has the reflective membrane to carry out the isolation of light, guarantee that each container condition is unanimous.
Preferably, the algae liquid used in S7 is an experimental algae or a corresponding algae liquid before an experiment, and the specific operation method of S7 is as follows: the method comprises the following steps of numbering and registering algae seeds in a book, noting the original source, the primary screening result, the production simulation experiment result and other related characters of the algae seeds, dividing the algae seed preservation into three levels, adopting a solid form for primary seed preservation, comprising a solid flat plate and an inclined plane, adopting a liquid small-volume seed preservation for secondary seed preservation, comprising a test tube and a small triangular flask below 150ml, adopting a 250-plus-500-ml triangular flask for the tertiary seed preservation, wherein the primary seed preservation is stored in a refrigerator-20 ℃ after being sealed, the secondary seed preservation is stored in a refrigerator 4 ℃, the tertiary seed preservation is placed in a light incubator or an incubator, and the algae seed nursing period is as follows: the content of the inducer for the first-class seeds is 0.1-0.2g/L, the second-class seeds is 3 months, and the third-class seeds are 45 days.
Preferably, the operation method in S8 is: selecting algae seeds from a germplasm library established in S7, amplifying until the volume of algae liquid is 30L and the cell density is about 50x104cells/ml, inoculating the algae seeds into a 1000L large-scale production photobioreactor, turning on a built-in light source, adjusting the pH value to 8.5-9.0, adjusting the light intensity to 15000Lux, the light period L: D to 16:8, controlling the temperature to 27-29 ℃ and the gas flow to 3m3/h, performing red induction on cells, performing microscopic examination on cell changes every day, starting to analyze the astaxanthin content and dry weight indexes from the 10 th day to the 15 th day, and comparing experimental results.
Preferably, the haematococcus pluvialis strain suitable for industrial production in S9 has the following characteristics: (1) the cells are exuberant in division and fast in growth rate in the green amplification stage; (2) the size of the cells is generally larger, the proportion of green cells is more than 25 mu m is 80%, and the proportion of red cells is more than 4 mu m is 80%; (3) the cells are full of chromosomes, the color is heavy in the green stage, and the color is dark red in the red stage; (4) cells can rapidly turn red under the condition of erythroinduction, and the accumulation of astaxanthin is started earlier; (5) the death rate of the cells is lower under the condition of induced erythropoiesis, and the death rate is lower than 10 percent except for normal death; (6) the cells can continue to increase under the condition of erythropoiesis induction, and the final dry weight of the harvested cells exceeds 0.75 g/L; (7) the cells accumulate astaxanthin rapidly, and the time required for the astaxanthin accumulation amount to reach the same value is shorter under the same induction condition; (8) the astaxanthin content of the cell harvest is more than 4.5%; (9) the algae species are stable in heredity, and show stable performance in production and application, and the results of batch production experiment data are close; further comprising S10, follow-up work: the algae seed performance is monitored at any time in the subsequent production, the algae seed activation work is carried out regularly, and the aging and character degradation of the algae seeds are prevented.
The invention has the beneficial effects that:
1. the original algae species obtained by screening by the method provided by the invention are used for the industrial large-scale culture of haematococcus pluvialis, the breeding period is short, and the efficiency for producing astaxanthin is high; in addition, the obtained algae seeds are not easy to degenerate, age, stable in heredity, good in vitality in the growth period, full in chromoplast and low in mortality.
2. The directional breeding is carried out by utilizing the production conditions, the success rate is high, the obtained algae seeds can be directly applied to production, and the large production risk caused by the adaptability of common algae seeds is avoided;
3. the natural selection method is adopted for breeding, no unsafe and uncertain factors are introduced, the obtained algae species are stable in heredity and safe in product, and the certainty among batch production and the quality of the product are guaranteed;
4. the algae seed breeding process is a careful process of primary screening and secondary screening, the obtained result is reliable, the characters and the performances of the algae seeds are well documented, the characteristics of the algae seeds can be mastered by producers, and the control on the production process is improved;
5. the method is simple and convenient to operate, has low technical threshold, and can be mastered by operators with general algology knowledge in large-scale production;
6. the method can be synchronously realized with production, for example, an important source of the original seeds is a sample which is outstanding in large-scale production, the special breeding time is saved, and the production efficiency is improved;
7. the method can ensure the stability and high efficiency of the large-scale production process from the source of the algae species, and realize the real industrialized process of the algae culture.
8. By utilizing the method, production units can establish a germplasm resource library with rich diversity at the same time, algae seed storage is formed in production, emergency situations in production are prevented, and continuous high-quality production is kept;
the invention provides a simple, high-efficiency and practical method for breeding algae seeds for industrial production.
Drawings
FIG. 1 is a flow chart of the present invention.
Detailed Description
The following further describes embodiments of the present invention with reference to examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Referring to fig. 1, a method for breeding haematococcus pluvialis species suitable for industrial production application includes the following steps: selecting original algae seeds → purifying original algae seeds → expanding culture of algae seeds → establishing algae seed series → primary screening of algae seeds → secondary screening of algae seeds → establishment and conservation of algae seed → production and application test of algae seeds → determination of excellent algae seeds, wherein:
s1, selection of original algae species: samples with outstanding performance are reserved in production application practice through field collection, communication or purchase and are used as original algae seeds of breeding seeds;
s2, purifying original algae species: purifying the original algae species according to the pollution degree of the algae species;
s3, algae seed expanding culture: performing cell culture on an illumination culture rack of an independent breeding room, wherein a sterile BBM culture medium is adopted as a culture medium;
s4, establishing an algae species series: carrying out propagation on the algae obtained after purification in the S2 to reach a certain volume and cell density, and establishing a series which can be used for carrying out selective breeding work under specific production conditions;
s5, primary screening of algae: carrying out primary screening on different series of algae seeds subjected to propagation by using astaxanthin induction conditions of nitrogen deficiency;
s6, secondary screening of algae: carrying out directional screening on algae seeds under the condition of industrial production;
s7, establishing and protecting the seeds in a quality library: preserving and establishing a library for the good algae screened by the simulation experiment;
s8, algal species production application test: testing the algae species in the germplasm library in S7, and detecting the astaxanthin content and dry weight indexes;
s9, determination of excellent algae species: determining the haematococcus pluvialis strain suitable for industrial production.
In the embodiment, when the field collection is carried out in S1, samples which can realize artificial production and have formed dominant growth of algae seeds in the original environment are selected for collection; when in communication and purchase, the selected algae species with the following characteristics are used as starting algae species of selective breeding, and the starting algae species comprise: the cell growth is rapid, the division is vigorous, the activity is strong, the chromoplast is full, the individual cell is generally large, the stress resistance is strong, and the dominant growth can be formed under the condition that other organisms exist.
In this example, the relatively pure elite in S2 need not be purified specially, but only the fresh sterile BBM medium is replaced one by one for rejuvenation; purifying algae species with general pollution, including microorganism and protozoan pollution but without other algae or other biological pollution by adopting a centrifugal cleaning and solid plate separation method; for samples with heavy pollution degree and impurities and various organisms in the samples, pure algae cells are obtained by adopting a capillary micro-operation method after pretreatment such as filtration and dilution; the method for purifying the stock with common pollution degree by adopting solid plate separation comprises the following specific steps:
a, performing low-speed centrifugation on original algae seeds, wherein the centrifugation speed is not more than 5000rpm, the time is 3.0min, removing supernatant, retaining bottom sediment, adding fresh sterile culture solution or buffer solution into a centrifuge tube, performing re-suspension dispersion on algae mud, performing centrifugation again after cleaning, collecting sediment, wherein the cleaning operation frequency can be different according to the pollution condition of the original seeds, and finally performing microscopic examination and rough counting on the obtained algae mud in the sterile culture solution;
b, diluting the algae liquid obtained in the step a to enable the final cell density to be about 500-1000cells/ml for later use;
c, adding agar powder into a conventional BBM liquid culture medium according to the proportion of 1.5%, adding antibiotics after sterilization when the culture medium is cooled, wherein the antibiotics consist of nystatin and chloramphenicol, the concentration is 80ppm, uniformly mixing, and immediately pouring the mixture into a flat plate;
d, taking 100 mu L of the cell diluent in the step b, inoculating the cell diluent into a flat plate, lightly and uniformly coating the cell diluent by using a coating rod, turning the culture dish upside down, and putting the culture dish into a constant-temperature illumination box for culture;
e, performing plate culture until macroscopic algae colonies grow out, selecting single algae colonies by using an inoculating loop, inoculating into a test tube, continuously culturing, gradually amplifying, and performing secondary streak inoculation and separation on the algae colonies in a solid culture medium according to the condition; for the original seeds with serious pollution, a capillary microscopy method is adopted for purification, and the specific steps are as follows:
a, filtering original algae seeds to remove impurities and partial pollutants, wherein a bolting silk or a filter membrane is adopted for filtering, the bottom sediment is reserved after the algae liquid is centrifuged at a low speed (5000rpm for 3.0min), fresh sterile culture solution or buffer solution is added into a centrifuge tube, algae mud is uniformly dispersed, cleaned and then centrifuged again, the sediment is collected, the cleaning times are different according to the pollution condition of the original seeds for 3-5 times, and finally the obtained algae mud is re-suspended by the sterile culture solution, and is subjected to microscopic examination and rough counting;
b, diluting the algae liquid obtained in the step a to enable the final cell density to be about 300-500cells/ml for later use;
c, drawing a capillary glass tube at high temperature by using an alcohol blast burner, wherein the aperture of the capillary tube is about 40-100 um;
d, dripping the diluted algae liquid obtained in the step b onto a glass slide sterilized in advance in a microdroplet mode, and quickly performing microscopic examination on the microdroplet size based on the fact that the visual field can be completely covered to determine target cells which are healthy, full in pigment and large in size;
e, sucking the target cells by using the glass capillary tube drawn by the step c under a microscope, transferring the target cells into a 24-hole cell culture plate filled with a sterilized culture medium, repeating the operation, and picking a plurality of cells into the culture plate, wherein one cell is placed into each hole;
f, culturing the cell plate in dark light, volatilizing the supplementary culture medium during the culturing, transferring the cell plate into a test tube for culturing when the color of the cell plate is visible to naked eyes, and gradually culturing and amplifying from a small volume to obtain the purified algae.
In this example, the nutrient salts were added to S3 at the following concentrations for each L of algal solution: 1.5 to 2.0 multiplied by 10 to 3g of sodium nitrate, 7.5 to 10.0 multiplied by 10 to 3g of dipotassium phosphate, 1.75 to 2.25 multiplied by 10 to 2g of monopotassium phosphate, 2.5 to 3.5 multiplied by 10 to 2g of sodium chloride, 3.7 to 4.7 multiplied by 10 to 2g of magnesium sulfate, 1.9 to 2.5 multiplied by 10 to 2g of calcium chloride, 1.10 to 1.50 multiplied by 10 to 2g of boric acid, 8.70 to 9.50 multiplied by 10 to 3g of zinc sulfate, 1.40 to 1.60 multiplied by 10 to 3g of manganese chloride, 1.2 to 1.4 multiplied by 10 to 3g of sodium molybdate, 1.50 to 1.80 multiplied by 10 to 3g of copper sulfate, 4.9 to 5.3 multiplied by 10 to 4g of cobalt nitrate, 5.0 to 6.0 multiplied by 10 to 2g of disodium ethylenediaminetetraacetate, 3.0 to 5.0 to 5.10 to 2g of potassium hydroxide, 4.9 to 5.5 multiplied by 10 to 3g of potassium phosphate, and a white light LED light source, the illumination intensity is adjusted according to the cell density, the photoperiod L: D is 12:12, the pH value is controlled to be 7.0-7.5, the temperature is controlled to be 24-26 ℃, the shake flask culture is carried out in a culture volume of 5L or more, the aeration culture is carried out in a volume of 5L or more, and the introduced gas is the mixed gas of compressed air and carbon dioxide which are subjected to sterilization and filtration.
In this embodiment, the specific operation of S4 is: when the copied original algae species with pure sources reach 1L of volume and the cell density reaches 25x104cells/ml, diluting the algae liquid by 50 times with a fresh culture medium until the cell density is 5x103cells/ml, subpackaging the algae liquid into a plurality of triangular flasks for continuous culture, and when the volume is increased to 2.5L and the cell density is about 15x104cells/ml, using the algae liquid as a screening algae species series for later use, numbering the series and establishing a file.
In this embodiment, the specific operation method of S5 is as follows: 2.5L of algae solution after each series of expanding culture is settled, filtered and cleaned, after the interference of the original culture medium is removed, the cells are suspended by nitrogen deficiency culture medium, and are subpackaged into 3 1L triangular flasks, wherein each flask is 750ml, and each algae seed is 3 parallel; all the triangular flasks were placed on a culture shelf and kept consistent under the following conditions: the culture medium adopts a nitrogen-deficient culture medium, namely nitrate components are removed from the conventional BBM culture medium, the light source is a special LED lamp tube capable of emitting the following specific spectrum for promoting red blood production by Haematococcus pluvialis, the specific spectrum comprises a first LED light-emitting chip with the wavelength of 370-: 32-50: 66-70, arranging the lamp tubes above and below the culture frame, controlling the illumination intensity of 15000Lux, the light period L: D to be 16:8, the pH value to be 8.5-9.0, controlling the temperature to be 27-29 ℃, and obtaining the algae series with quick astaxanthin accumulation, high astaxanthin accumulation amount in the same time, high cell dry weight, low cell death rate and clean culture system by primary screening.
In this embodiment, since the industrial equipment in S6 is bulky and is not suitable for the algal species screening experiment, a simulated production condition needs to be established first, and the specific operation method of S6 is as follows: according to the shape, accessories and related equipment of the vertical photobioreactor with built-in light sources adopted in industrial production, a small simulation photobioreactor is manufactured, experimental conditions which are consistent with the red-promoting induction conditions of haematococcus pluvialis adopted in production to the maximum extent are provided, and the simulation photobioreactor has the following characteristics: the capacity is reduced to 15L from 1000L of industrial equipment, the material is changed into acrylic tube from stainless steel, according to the light path of light source for production, 1 special red LED fluorescent tube that urges that is used for red ball algae is put to the simulation reactor, in addition, the monitoring regulation and control system of the aeration system, temperature, PH etc. of the simulation reactor is with industrial equipment, a plurality of simulation reactors are installed and fixed on the support in unison, each container package has the reflective membrane to carry out the isolation of light, guarantee that each container condition is unanimous.
In this embodiment, the algae liquid used in S7 is an experimental algae or a corresponding algae liquid before an experiment, and the specific operation method of S7 is as follows: the method comprises the following steps of numbering and registering algae seeds in a book, noting the original source, the primary screening result, the production simulation experiment result and other related characters of the algae seeds, dividing the algae seed preservation into three levels, adopting a solid form for primary seed preservation, comprising a solid flat plate and an inclined plane, adopting a liquid small-volume seed preservation for secondary seed preservation, comprising a test tube and a small triangular flask below 150ml, adopting a 250-plus-500-ml triangular flask for the tertiary seed preservation, wherein the primary seed preservation is stored in a refrigerator-20 ℃ after being sealed, the secondary seed preservation is stored in a refrigerator 4 ℃, the tertiary seed preservation is placed in a light incubator or an incubator, and the algae seed nursing period is as follows: the content of the inducer for the first-class seeds is 0.1-0.2g/L, the second-class seeds is 3 months, and the third-class seeds are 45 days.
In this embodiment, the operation method in S8 is: selecting algae seeds from a germplasm library established in S7, amplifying until the volume of algae liquid is 30L and the cell density is about 50x104cells/ml, inoculating the algae seeds into a 1000L large-scale production photobioreactor, turning on a built-in light source, adjusting the pH value to 8.5-9.0, adjusting the light intensity to 15000Lux, the light period L: D to 16:8, controlling the temperature to 27-29 ℃ and the gas flow to 3m3/h, performing red induction on cells, performing microscopic examination on cell changes every day, starting to analyze the astaxanthin content and dry weight indexes from the 10 th day to the 15 th day, and comparing experimental results.
In this example, the haematococcus pluvialis strain suitable for industrial production in S9 has the following characteristics: (1) the cells are exuberant in division and fast in growth rate in the green amplification stage; (2) the size of the cells is generally larger, the proportion of green cells is more than 25 mu m is 80%, and the proportion of red cells is more than 4 mu m is 80%; (3) the cells are full of chromosomes, the color is heavy in the green stage, and the color is dark red in the red stage; (4) cells can rapidly turn red under the condition of erythroinduction, and the accumulation of astaxanthin is started earlier; (5) the death rate of the cells is lower under the condition of induced erythropoiesis, and the death rate is lower than 10 percent except for normal death; (6) the cells can continue to increase under the condition of erythropoiesis induction, and the final dry weight of the harvested cells exceeds 0.75 g/L; (7) the cells accumulate astaxanthin rapidly, and the time required for the astaxanthin accumulation amount to reach the same value is shorter under the same induction condition; (8) the astaxanthin content of the cell harvest is more than 4.5%; (9) the algae species are stable in heredity, and show stable performance in production and application, and the results of batch production experiment data are close; further comprising S10, follow-up work: the algae seed performance is monitored at any time in the subsequent production, the algae seed activation work is carried out regularly, and the aging and character degradation of the algae seeds are prevented.
Example 1
S1, selection of original algae species: selecting a sample which shows outstanding performance in production application practice as an original algae species, wherein the production information of the original algae species is as follows: collecting 0.95g/L dry weight, 6.1% astaxanthin content, culturing for 5 days in green stage, and inducing for 12 days; intercepting 2L of algae liquid at a harvesting stage, randomly sampling and performing microscopic examination to obtain a result: the algae liquid is clean, the cells are round, the individual is generally large, the size of each cell is more than 45um, all the cells are dark red, the astaxanthin is filled in the cells, the thickness of cell walls is moderate, other pollution is basically not seen in the algae liquid, bacteria are not eliminated, and metabolic waste of the cells is not eliminated;
s2, purifying original algae species: the algae species in the S1 are pure, and only bacteria microorganisms and cell self metabolic wastes cannot be removed, so that 2L of algae liquid of a reserved sample is settled firstly, the redundant liquid is poured, then, a sterile BBM culture medium is added for cleaning and centrifuging to obtain bottom algae mud, the cells are re-suspended by the culture medium, and the cleaning is repeated twice;
s3, algae seed expanding culture: transferring the washed algae liquid into a 5L flask, and adding a fresh sterile BBM culture medium for culture under the culture condition; the culture medium adopts a sterile BBM culture medium, and nutrient salt components with the following concentrations are added into each liter of algae liquid of the culture medium: 1.5X 10-3g of sodium nitrate, 7.5X 10-3g of dipotassium phosphate, 1.75X 10-2g of monopotassium phosphate, 2.5X 10-2g of sodium chloride, 3.7X 10-2g of magnesium sulfate, 1.9X 10-2g of calcium chloride, 1.14X 10-2g of boric acid, 8.82X 10-3g of zinc sulfate, 1.44X 10-3g of manganese chloride, 1.2X 10-3g of sodium molybdate, 1.57X 10-3g of copper sulfate, 4.9X 10-4g of cobalt nitrate, 5.0X 10-2g of disodium ethylenediamine tetraacetic acid, 3.1X 10-2g of potassium hydroxide and 4.98X 10-3g of ferric sulfate. The light source is an LED white light tube, the illumination intensity is 5000Lux, the photoperiod L is 12:12, the pH value is controlled to be 7.0-7.5, the temperature is controlled to be 24-26 ℃, the aeration culture is carried out, the air quantity is 3m3/h, and the introduced air is the mixed gas of sterilized and filtered compressed air and carbon dioxide;
s4, establishing an algae species series: when the cell density is about 25x104cells/ml in S3, diluting the algae liquid with a fresh culture medium to about 5x103cells/ml, then filling the algae liquid into 15 triangular flasks for continuous culture, expanding the algae liquid to about 2.5L volume and 15x104cells/ml, numbering the series and establishing an archive;
s5, primary screening of algae: through 15 bottles of algae species of partial shipment, expand cultivateing and decide to 15 series, subside and wash at first, get rid of former culture medium interference, utilize the astaxanthin induction condition of lack of nitrogen to carry out preliminary screening, 3 parallels are done to every series, adopt 1L triangular flask to carry out the experiment, 750ml of algae liquid of every bottle, on all triangular flasks put into the culture rack, keep the condition unanimous: the culture medium adopts a nitrogen-deficient culture medium, namely nitrate components are removed from the conventional BBM culture medium, a light source is a special LED lamp tube capable of emitting the following specific spectrum for red-forcing of haematococcus pluvialis, the lamp tube is arranged above and below a culture frame, the illumination intensity is 15000Lux, the photoperiod L: D is 16:8, the pH value is controlled to be 8.5-9.0, and the temperature is controlled to be 27-29 ℃. Comparing to obtain the algae species series with fast astaxanthin accumulation, high astaxanthin accumulation amount, high cell dry weight, low cell death rate and clean culture system, entering a secondary sieve, and performing primary sieve to obtain 5 series;
s6, secondary screening of algae: expanding and culturing 5 series of algae seeds subjected to primary screening by S5 to reach the volume of 5L and about 30x104 cells/ml, settling, filtering, cleaning, re-suspending, then connecting the algae seeds into a simulation reactor, cleaning and disinfecting the reactor in advance, adjusting the cell inoculation density to about 15x104cells/ml, turning on a built-in light source of the simulation reactor, adjusting the PH to 8.5-9.0, adjusting the light intensity to 15000Lux, adjusting the light period L to 16:8, controlling the temperature to 27-29 ℃, controlling the gas flow to 3m3/h, wherein the gas is a mixed gas of compressed air and carbon dioxide, and the introduction amount of the carbon dioxide is 2% of the volume of the air. Performing microscopic examination on cell change every day, analyzing astaxanthin content and dry weight indexes every day from 7 th day to 15 th day, selecting 1 series from the series to perform the best performance, wherein the result is that the astaxanthin content reaches 4.5% on the induction day 9, the dry weight is 0.63g/L, the daily index is 15-20% higher than that of other series, the astaxanthin content reaches 6.3% on the culture day 15, the dry weight is 1.11g/L, the cell death rate in the whole induction process is low, and the culture system is clean;
s7, establishing and protecting the seeds in a quality library: the excellent strain screened by the simulation experiment is preserved and registered with the number YZY 0120180509050301. Three forms are manufactured: solid flat plates, inclined planes and liquid, wherein the number of liquid seeds is 3 in 500ml triangular flasks, the liquid seeds are placed on a specified algae seed culture rack, and the solid flat plates and the inclined planes are stored in a refrigerator;
s8, algal species production application test: and (2) expanding the YZY0120180509050301 alga seeds to about 30L of alga liquid volume and 50x104 cell/ml of cell density, inoculating the alga seeds into a 1000L large-scale production photobioreactor, turning on a built-in light source, adjusting the pH to 8.5-9.0, adjusting the light intensity to 15000Lux, the light period L: D to 16:8, controlling the temperature to 27-29 ℃, and performing erythropoiesis induction on the cells at the gas flow rate of 3m 3/h. The cells were examined daily, starting on day 10 and analyzing astaxanthin content and dry weight indices to day 15, with the results shown in the table below;
| days (D) | Astaxanthin content (%) | Dry weight (g/L) |
| 10 | 4.20 | 0.62 |
| 11 | 4.71 | 0.64 |
| 12 | 4.93 | 0.73 |
| 13 | 5.12 | 0.88 |
| 14 | 5.81 | 0.79 |
| 15 | 6.05 | 0.91 |
TABLE 1 algal species YZY0120180509050301 production data
S9, determination of excellent algae species: according to the data in the table, and by combining the whole culture process, the algae species YZY0120180509050301 can be used for storing and producing alternative algae species in a germplasm resource library.
Example 2
S1, selection of original algae species: the original algae are collected in the field, the sampling site is fresh water lake in mansion area, the sampling time is 8 months in 2018, the temperature of the sampling place on the day is 38 ℃ at the highest and 22 ℃ at the lowest, and the local weather is clear and rainless 10 days before sampling;
s2, purifying original algae species: after simple microscopic examination and treatment, the sample is taken back to a laboratory, firstly, the sample is filtered to remove large granular substances and impurities, the sample is transferred to a triangular flask, a halved BBM culture medium is added to carry out adaptive culture of algae species, the change condition of the sample is monitored at any time in the process, the sample is microscopically examined, besides haematococcus pluvialis cells, various protozoa, mixed algae and microbial pollution are also included in a water sample, when the haematococcus pluvialis cells occupy more than half of the number in a culture solution, the water sample is filtered to remove most of protozoa and filamentous mixed algae, the filtered algae solution is subjected to low-speed centrifugation to remove most of mixed algae and microbial pollution smaller than the haematococcus pluvialis, a fresh sterile culture solution is added into a centrifuge tube, the algae mud is uniformly dispersed and is subjected to centrifugation again, the precipitate is collected, finally, the algae mud is re-suspended by the sterile culture solution, microscopic examination and rough counting are carried out, counting only haematococcus pluvialis cells, diluting the algae liquid according to a counting result to obtain a final cell density of about 300cells/ml, dripping the diluted algae liquid onto a glass slide sterilized in advance in a microdroplet mode, quickly performing microscopic examination, and determining target cells which are healthy, full in pigment and large in size. The target cells were rapidly aspirated under a microscope using a previously drawn glass capillary and transferred to a 24-well cell culture plate containing a sterile medium. Repeating the operation, selecting a plurality of cells, putting the cells into a culture plate, putting one cell in each hole, selecting 50 haematococcus pluvialis cells in total, putting the cell plate in dark light for culture, supplementing a culture medium to volatilize in the period, transferring the cells into a test tube for culture when the colors of the cells are visible to naked eyes, and abandoning the unchanged micropores to obtain 11 strains in total. And (3) gradually culturing and amplifying the 11 strains from a small volume, wherein 5 strains fail to be cultured, and finally obtaining 6 strains of purified monoclonal algae seeds, wherein the numbers of the 6 strains are as follows:
YZWFX029180805042101、YZWFX029180805042102、YZWFX029180805042103、YZWFX029180805042104、YZWFX029180805042105、YZWFX029180805042106。
s3, algae seed expanding culture: adding a fresh culture medium into 6 algae strains in S2 for propagation, wherein the volume of the medium propagation reaches 2.5L, the cell density is about 15x104cells/ml, the medium propagation is used as an algae strain series for screening, the algae strain propagation is carried out on an illumination culture frame of an independent breeding room, the culture medium adopts a sterile BBM culture medium, and after the culture medium formula is added, the system contains the following substances in per liter of algae solution: 1.5X 10-3g of sodium nitrate, 7.5X 10-3g of dipotassium phosphate, 1.75X 10-2g of monopotassium phosphate, 2.5X 10-2g of sodium chloride, 3.7X 10-2g of magnesium sulfate, 1.9X 10-2g of calcium chloride, 1.14X 10-2g of boric acid, 8.82X 10-3g of zinc sulfate, 1.44X 10-3g of manganese chloride, 1.2X 10-3g of sodium molybdate, 1.57X 10-3g of copper sulfate, 4.9X 10-4g of cobalt nitrate, 5.0X 10-2g of disodium ethylenediamine tetraacetic acid, 3.1X 10-2g of potassium hydroxide and 4.98X 10-3g of ferric sulfate. The light source is an LED white light lamp tube, the illumination intensity is adjusted according to the cell density, the photoperiod L is 12:12, the pH value is controlled to be 7.0-7.5, the temperature is controlled to be 24-26 ℃, the shake flask culture is carried out in the culture volume of 5L or more, the aeration culture is carried out in the volume of 5L or more, and the introduced gas is the mixed gas of compressed air and carbon dioxide which are subjected to sterilization and filtration;
s4, establishing an algae species series: expanding culture in S3 to reach algae liquid volume of 2.5L and cell density of 15x104cells/ml, using the algae liquid as screening algae seed series for later use, numbering the series and establishing archives;
s5, primary screening of algae: the method comprises the following steps of primarily screening the algae species subjected to propagation by using astaxanthin induction conditions of nitrogen deficiency, and specifically comprises the following steps: 2.5L of algae solution obtained by expanding and culturing algae seeds YZWFX 0291805042101, YZWFX 0291805042103, YZWFX 0291805042104 and YZWFX 0291805042106 is settled, filtered and washed to remove the interference of the original culture medium, then the cells are suspended by BBM culture medium without sodium nitrate and are subpackaged into 3 triangular flasks of 500mL, wherein each flask is 350mL, and each algae seed is divided into 3 parallel cells; all triangular flasks are placed on a culture rack, the conditions are kept consistent, except that the culture medium adopts a nitrogen-deficient culture medium, a light source is a special LED lamp tube for red-hastening haematococcus pluvialis and capable of emitting the following specific spectrum, the lamp tube is arranged above and below the culture rack, the illumination intensity is 15000Lux, the photoperiod L: D is 16:8, the pH value is controlled to be 8.5-9.0, and the temperature is controlled to be 27-29 ℃. Comparing to obtain the algae species series with fast astaxanthin accumulation, high astaxanthin accumulation amount, high cell dry weight, low cell death rate and clean culture system in the same time, entering a secondary sieve, and obtaining 2 series by primary sieve: yZWFX 0291805042102, YZWFX 0291805042106;
s6, secondary screening of algae: expanding and culturing 2 series of algae strains subjected to primary screening in S5 to reach the volume of 5L and about 30x104 cells/ml, settling, filtering, cleaning, re-suspending, then connecting into a simulation reactor, cleaning and disinfecting the reactor in advance, wherein the cell inoculation density is about 15x104cells/ml, turning on a built-in light source of the simulation reactor, adjusting the PH to 8.5-9.0, adjusting the light intensity to 15000Lux, adjusting the light period L: D to 16:8, controlling the temperature to 27-29 ℃, and controlling the gas flow to be 3m 3/h. Cells were examined daily and astaxanthin content and dry weight indices were analyzed daily beginning on day 7 to day 15. The selected alga strain YZWFX 0291805042106 has better performance, the astaxanthin content reaches 3.6 percent at the induction 10 day, the dry weight is 0.58g/L, the astaxanthin content reaches 4.75 percent at the culture 15 day, the dry weight is 0.68g/L, the cell death rate is lower in the whole induction process, and the culture system is clean;
s7, establishing and protecting the seeds in a quality library: and storing YZWFX 0291805042106 into a germplasm library and preserving the seeds. Three forms are manufactured: solid flat plate, inclined plane and liquid, wherein the number of the liquid is 3 in 100ml triangular bottles, and all the liquid is stored in a refrigerator;
s8, algal species production application test: expanding the strain YZWFX 02918050106 to about 30L and the cell density of 50x104cells/ml, inoculating the strain into a 1000L large-scale production photobioreactor, turning on a built-in light source, adjusting the pH to 8.5-9.0, the light intensity of 15000Lux, the light period L: D to 16:8, controlling the temperature to 27-29 ℃, and performing the erythropoiesis induction on the cells at the gas flow rate of 3m 3/h. Cell changes were examined daily, starting on day 10 and analyzing astaxanthin content and dry weight indices to day 15, with the following results:
TABLE 2 algal species YZWFX 0291805042106 production data
S9, determination of excellent algae species: according to the data in the table and by combining the whole cultivation process, the algae species YZWFX 0291805042106 can be used as a germplasm resource bank for storing and producing alternative algae species.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A haematococcus pluvialis strain breeding method suitable for industrial production and application is characterized by comprising the following steps: the method comprises the following steps: selecting original algae seeds → purifying original algae seeds → expanding culture of algae seeds → establishing algae seed series → primary screening of algae seeds → secondary screening of algae seeds → establishment and conservation of algae seed → production and application test of algae seeds → determination of excellent algae seeds, wherein:
s1, selection of original algae species: samples with outstanding performance are reserved in production application practice through field collection, communication or purchase and are used as original algae seeds of breeding seeds;
s2, purifying original algae species: purifying the original algae species according to the pollution degree of the algae species;
s3, algae seed expanding culture: performing cell culture on an illumination culture rack of an independent breeding room, wherein a sterile BBM culture medium is adopted as a culture medium;
s4, establishing an algae species series: carrying out propagation on the algae obtained after purification in the S2 to reach a certain volume and cell density, and establishing a series which can be used for carrying out selective breeding work under specific production conditions;
s5, primary screening of algae: carrying out primary screening on different series of algae seeds subjected to propagation by using astaxanthin induction conditions of nitrogen deficiency;
s6, secondary screening of algae: carrying out directional screening on algae seeds under the condition of industrial production;
s7, establishing and protecting the seeds in a quality library: preserving and establishing a library for the good algae screened by the simulation experiment;
s8, algal species production application test: testing the algae species in the germplasm library in S7, and detecting the astaxanthin content and dry weight indexes;
s9, determination of excellent algae species: determining the haematococcus pluvialis strain suitable for industrial production.
2. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: s1, selecting a sample which can realize artificial production and has formed dominant growth of algae in the original environment for collection in the field; when in communication and purchase, the selected algae species with the following characteristics are used as starting algae species of selective breeding, and the starting algae species comprise: the cell growth is rapid, the division is vigorous, the activity is strong, the chromoplast is full, the individual cell is generally large, the stress resistance is strong, and the dominant growth can be formed under the condition that other organisms exist.
3. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: in S2, pure stock seeds do not need to be specially purified, and rejuvenation can be carried out by replacing fresh sterile BBM culture medium by quantity; purifying algae species with general pollution, including microorganism and protozoan pollution but without other algae or other biological pollution by adopting a centrifugal cleaning and solid plate separation method; for samples with heavy pollution degree and impurities and various organisms in the samples, pure algae cells are obtained by adopting a capillary micro-operation method after pretreatment such as filtration and dilution; the method for purifying the stock with common pollution degree by adopting solid plate separation comprises the following specific steps:
a, performing low-speed centrifugation on original algae seeds, wherein the centrifugation speed is not more than 5000rpm, the time is 3.0min, removing supernatant, retaining bottom sediment, adding fresh sterile culture solution or buffer solution into a centrifuge tube, performing re-suspension dispersion on algae mud, performing centrifugation again after cleaning, collecting sediment, wherein the cleaning operation frequency can be different according to the pollution condition of the original seeds, and finally performing microscopic examination and rough counting on the obtained algae mud in the sterile culture solution;
b, diluting the algae liquid obtained in the step a to enable the final cell density to be about 500-1000cells/ml for later use;
c, adding agar powder into a conventional BBM liquid culture medium according to the proportion of 1.5%, adding antibiotics after sterilization when the culture medium is cooled, wherein the antibiotics consist of nystatin and chloramphenicol, the concentration is 80ppm, uniformly mixing, and immediately pouring the mixture into a flat plate;
d, taking 100 mu L of the cell diluent in the step b, inoculating the cell diluent into a flat plate, lightly and uniformly coating the cell diluent by using a coating rod, turning the culture dish upside down, and putting the culture dish into a constant-temperature illumination box for culture;
e, performing plate culture until macroscopic algae colonies grow out, selecting single algae colonies by using an inoculating loop, inoculating into a test tube, continuously culturing, gradually amplifying, and performing secondary streak inoculation and separation on the algae colonies in a solid culture medium according to the condition; for the original seeds with serious pollution, a capillary microscopy method is adopted for purification, and the specific steps are as follows:
a, filtering original algae seeds to remove impurities and partial pollutants, wherein a bolting silk or a filter membrane is adopted for filtering, the bottom sediment is reserved after the algae liquid is centrifuged at a low speed (5000rpm for 3.0min), fresh sterile culture solution or buffer solution is added into a centrifuge tube, algae mud is uniformly dispersed, cleaned and then centrifuged again, the sediment is collected, the cleaning times are different according to the pollution condition of the original seeds for 3-5 times, and finally the obtained algae mud is re-suspended by the sterile culture solution, and is subjected to microscopic examination and rough counting;
b, diluting the algae liquid obtained in the step a to enable the final cell density to be about 300-500cells/ml for later use;
c, drawing a capillary glass tube at high temperature by using an alcohol blast burner, wherein the aperture of the capillary tube is about 40-100 um;
d, dripping the diluted algae liquid obtained in the step b onto a glass slide sterilized in advance in a microdroplet mode, and quickly performing microscopic examination on the microdroplet size based on the fact that the visual field can be completely covered to determine target cells which are healthy, full in pigment and large in size;
e, sucking the target cells by using the glass capillary tube drawn by the step c under a microscope, transferring the target cells into a 24-hole cell culture plate filled with a sterilized culture medium, repeating the operation, and picking a plurality of cells into the culture plate, wherein one cell is placed into each hole;
f, culturing the cell plate in dark light, volatilizing the supplementary culture medium during the culturing, transferring the cell plate into a test tube for culturing when the color of the cell plate is visible to naked eyes, and gradually culturing and amplifying from a small volume to obtain the purified algae.
4. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: after the culture medium is added into S3, each L of algae solution contains nutrient salts with the following concentrations: 1.5 to 2.0 multiplied by 10 to 3g of sodium nitrate, 7.5 to 10.0 multiplied by 10 to 3g of dipotassium phosphate, 1.75 to 2.25 multiplied by 10 to 2g of monopotassium phosphate, 2.5 to 3.5 multiplied by 10 to 2g of sodium chloride, 3.7 to 4.7 multiplied by 10 to 2g of magnesium sulfate, 1.9 to 2.5 multiplied by 10 to 2g of calcium chloride, 1.10 to 1.50 multiplied by 10 to 2g of boric acid, 8.70 to 9.50 multiplied by 10 to 3g of zinc sulfate, 1.40 to 1.60 multiplied by 10 to 3g of manganese chloride, 1.2 to 1.4 multiplied by 10 to 3g of sodium molybdate, 1.50 to 1.80 multiplied by 10 to 3g of copper sulfate, 4.9 to 5.3 multiplied by 10 to 4g of cobalt nitrate, 5.0 to 6.0 multiplied by 10 to 2g of disodium ethylenediaminetetraacetate, 3.0 to 5.0 to 5.10 to 2g of potassium hydroxide, 4.9 to 5.5 multiplied by 10 to 3g of potassium phosphate, and a white light LED light source, the illumination intensity is adjusted according to the cell density, the photoperiod L: D is 12:12, the pH value is controlled to be 7.0-7.5, the temperature is controlled to be 24-26 ℃, the shake flask culture is carried out in a culture volume of 5L or more, the aeration culture is carried out in a volume of 5L or more, and the introduced gas is the mixed gas of compressed air and carbon dioxide which are subjected to sterilization and filtration.
5. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: the specific operation of S4 is: when the copied original algae species with pure sources reach 1L of volume and the cell density reaches 25x104cells/ml, diluting the algae liquid by 50 times with a fresh culture medium until the cell density is 5x103cells/ml, subpackaging the algae liquid into a plurality of triangular flasks for continuous culture, and when the volume is increased to 2.5L and the cell density is about 15x104cells/ml, using the algae liquid as a screening algae species series for later use, numbering the series and establishing a file.
6. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: the specific operation method of S5 is as follows: 2.5L of algae solution after each series of expanding culture is settled, filtered and cleaned, after the interference of the original culture medium is removed, the cells are suspended by nitrogen deficiency culture medium, and are subpackaged into 3 1L triangular flasks, wherein each flask is 750ml, and each algae seed is 3 parallel; all the triangular flasks were placed on a culture shelf and kept consistent under the following conditions: the culture medium adopts a nitrogen-deficient culture medium, namely nitrate components are removed from the conventional BBM culture medium, the light source is a special LED lamp tube capable of emitting the following specific spectrum for promoting red blood production by Haematococcus pluvialis, the specific spectrum comprises a first LED light-emitting chip with the wavelength of 370-: 32-50: 66-70, arranging the lamp tubes above and below the culture frame, controlling the illumination intensity of 15000Lux, the light period L: D to be 16:8, the pH value to be 8.5-9.0, controlling the temperature to be 27-29 ℃, and obtaining the algae series with quick astaxanthin accumulation, high astaxanthin accumulation amount in the same time, high cell dry weight, low cell death rate and clean culture system by primary screening.
7. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: in S6, because the industrial equipment is bulky and is not suitable for the algae species screening experiment, firstly, the simulation production conditions need to be established, and the specific operation method of S6 is as follows: according to the shape, accessories and related equipment of the vertical photobioreactor with built-in light sources adopted in industrial production, a small simulation photobioreactor is manufactured, experimental conditions which are consistent with the red-promoting induction conditions of haematococcus pluvialis adopted in production to the maximum extent are provided, and the simulation photobioreactor has the following characteristics: the capacity is reduced to 15L from 1000L of industrial equipment, the material is changed into acrylic tube from stainless steel, according to the light path of light source for production, 1 special red LED fluorescent tube that urges that is used for red ball algae is put to the simulation reactor, in addition, the monitoring regulation and control system of the aeration system, temperature, PH etc. of the simulation reactor is with industrial equipment, a plurality of simulation reactors are installed and fixed on the support in unison, each container package has the reflective membrane to carry out the isolation of light, guarantee that each container condition is unanimous.
8. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: the algae liquid adopted in S7 is experimental algae or corresponding algae liquid before experiment, and the specific operation method of S7 is as follows: the method comprises the following steps of numbering and registering algae seeds in a book, noting the original source, the primary screening result, the production simulation experiment result and other related characters of the algae seeds, dividing the algae seed preservation into three levels, adopting a solid form for primary seed preservation, comprising a solid flat plate and an inclined plane, adopting a liquid small-volume seed preservation for secondary seed preservation, comprising a test tube and a small triangular flask below 150ml, adopting a 250-plus-500-ml triangular flask for the tertiary seed preservation, wherein the primary seed preservation is stored in a refrigerator-20 ℃ after being sealed, the secondary seed preservation is stored in a refrigerator 4 ℃, the tertiary seed preservation is placed in a light incubator or an incubator, and the algae seed nursing period is as follows: the content of the inducer for the first-class seeds is 0.1-0.2g/L, the second-class seeds is 3 months, and the third-class seeds are 45 days.
9. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: the operation method in the S8 comprises the following steps: selecting algae seeds from a germplasm library established in S7, amplifying until the volume of algae liquid is 30L and the cell density is about 50x104cells/ml, inoculating the algae seeds into a 1000L large-scale production photobioreactor, turning on a built-in light source, adjusting the pH value to 8.5-9.0, adjusting the light intensity to 15000Lux, the light period L: D to 16:8, controlling the temperature to 27-29 ℃ and the gas flow to 3m3/h, performing red induction on cells, performing microscopic examination on cell changes every day, starting to analyze the astaxanthin content and dry weight indexes from the 10 th day to the 15 th day, and comparing experimental results.
10. The method for selectively breeding haematococcus pluvialis strain suitable for industrial production and application according to claim 1, wherein the method comprises the following steps: the haematococcus pluvialis strain suitable for industrial production in S9 has the following characteristics: (1) the cells are exuberant in division and fast in growth rate in the green amplification stage; (2) the size of the cells is generally larger, the proportion of green cells is more than 25 mu m is 80%, and the proportion of red cells is more than 4 mu m is 80%; (3) the cells are full of chromosomes, the color is heavy in the green stage, and the color is dark red in the red stage; (4) cells can rapidly turn red under the condition of erythroinduction, and the accumulation of astaxanthin is started earlier; (5) the death rate of the cells is lower under the condition of induced erythropoiesis, and the death rate is lower than 10 percent except for normal death; (6) the cells can continue to increase under the condition of erythropoiesis induction, and the final dry weight of the harvested cells exceeds 0.75 g/L; (7) the cells accumulate astaxanthin rapidly, and the time required for the astaxanthin accumulation amount to reach the same value is shorter under the same induction condition; (8) the astaxanthin content of the cell harvest is more than 4.5%; (9) the algae species are stable in heredity, and show stable performance in production and application, and the results of batch production experiment data are close; further comprising S10, follow-up work: the algae seed performance is monitored at any time in the subsequent production, the algae seed activation work is carried out regularly, and the aging and character degradation of the algae seeds are prevented.
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