CN110923147B - Method for inducing and culturing Phaeodactylum ovorans cells - Google Patents
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Abstract
The invention discloses an induction culture method of an egg-shaped phaeodactylum tricornutum cell. The method is characterized in that stress effect brought by extremely high cell density is utilized to promote most of phaeodactylum tricornutum cells to be converted into ovate in a short time, and the induction method is simple, efficient and feasible by combining with the optimization control of induction conditions. The method overcomes the defects that the conventional method for inducing the cells of the Phaeodactylum ovorans is overlong in period and high in cost, and the induced and cultured Phaeodactylum ovorans is low in density and not easy to collect, the period of the improved method for inducing and culturing the Phaeodactylum ovorans is greatly shortened, the Phaeodactylum ovorans can be stably induced and cultured within three months, and the cells of the Phaeodactylum ovorans induced and cultured are high in proportion and high in cell density and are very easy to collect.
Description
Technical Field
The invention belongs to the field of biotechnology. More particularly, relates to an induction culture method of an egg-shaped phaeodactylum tricornutum cell.
Background
Phaeodactylum tricornutum (Phaeodactylum tricornutum) is an important bait for aquatic animals and plants and an important research material for the physiology and ecology of algae. Phaeodactylum tricornutum is an atypical diatom that is generally believed to exist in three forms: oval, spindle, and radial with the most preferred being the spindle and the least oval. Research shows that only oval cells have true single silicified cell membranes, and silicon dioxide is embedded in an organic shell to form a middle gap, so that the cell membranes can slide; has special potential application value different from fusiform and three radial phaeodactylum tricornutum.
Some scholars have studied the morphological structure and variation of phaeodactylum tricornutum, and it is widely believed that the phaeodactylum tricornutum has three forms, namely fusiform form, ovoid form and three radial forms, and the three forms have the phenomenon of mutual transformation. However, the observation results of the respective scholars are inconsistent, and there is no consistent understanding about the laws and mechanisms of morphological variation. Since the Phaeodactylum tricornutum is separated and cultured in China, only a few scholars have simple description on the morphology and the mutation phenomenon, and no systematic research report exists.
At present, a relatively universal method for inducing transformation of Phaeodactylum ovorans is not available. In a few domestic related documents, scholars propose that low temperature (4-8 ℃) and low salinity (desalted seawater) are superposed to slowly induce the conversion of fusiform and three radial phaeodactylum tricornutum cells into ovoid phaeodactylum tricornutum cells; and with time, the culture broth aging and the number of cell passages increased also favoured the production of oval cells (half a year) (continental macros, 2001, proceedings of the university of Qingdao ocean). However, the method has the biggest defect that the required period is too long, and 1-2 years are required; and although high-proportion oval cells (more than 99%) are induced, the conditions of low temperature, low salt and culture solution aging are all stress conditions for phaeodactylum tricornutum, so that the cell density is low, the collection is not easy, and the comprehensive cost is high.
Disclosure of Invention
The invention aims to provide an induction culture method for the Phaeodactylum ovorans cells, which has the advantages of obviously shortened period, lower cost, high proportion of Phaeodactylum ovorans and high cell density.
The above purpose of the invention is realized by the following technical scheme:
an induction culture method of egg-shaped Phaeodactylum tricornutum cells comprises regulating Phaeodactylum tricornutum cell density with seawater to be not less than 5 × 107And (4) culturing per milliliter. Experimental studies have shown that the stress effects of very high cell densities are used to force the cells of phaeodactylum tricornutum to transform mostly into ovoid cells in a relatively short period of time.
Preferably, the method comprises the steps of firstly, carrying out activation culture on sterile phaeodactylum tricornutum algae seeds until the exponential growth phase, and then, transferring and carrying out amplification culture; then regulating the cell density of Phaeodactylum tricornutum to not less than 5 × 10 with seawater7Culturing the cells/ml under the conditions of 20-26 ℃, 3800-4200 lux illumination intensity and illumination dark ratio of 16-20h:4-8h for 2-3 months.
Wherein preferably the salinity of the seawater is 30-35 psu. Most preferably, the salinity of the seawater is 32 psu.
Preferably, the cell density of Phaeodactylum tricornutum is controlled at 5-25 × 107One/ml.
Preferably, the induction culture conditions are that the temperature is 25 +/-0.5 ℃, the illumination intensity is 4000 lux, and the illumination light-dark ratio is 20h:4 h.
In addition, preferably, the method for inducing and culturing the Phaeodactylum ovorans cells comprises the following steps:
s1, carrying out activation culture on sterile phaeodactylum tricornutum algae seeds until the exponential growth period, and transferring to an f/2 culture medium for amplification culture;
s2, step S1, expanding cultivation until the density of Phaeodactylum tricornutum reaches 5 × 106More than one per milliliter (about the tenth day after inoculation, more than 99 percent of phaeodactylum tricornutum is fusiform or radioactive, and the fusiform is slightly more), centrifuging and discarding the supernatant;
s3, adding seawater into the precipitate obtained in the step S2, and carrying out centrifugal cleaning (for 2-3 times);
s4, adding seawater into the sediment obtained in the step S3 until the cell density is not less than 5 multiplied by 107Slightly oscillating per milliliter to ensure that the phaeodactylum tricornutum cells are uniformly dispersed and not accumulated at the bottom;
s5, performing induction culture; the ratio of light to dark is 16-20h:4-8 h;
s6, after culturing for 15-25 days, adding seawater, centrifuging and discarding supernatant; adding seawater to cell density of not less than 5 × 107Slightly oscillating per milliliter to ensure that the phaeodactylum tricornutum cells are uniformly dispersed and not accumulated at the bottom;
s7, repeating the steps S5-S6 (repeating for 2-3 times).
Wherein preferably, seawater is added in steps S4 and S6 to a cell density of 5-25X 107One/ml.
Preferably, the light-to-dark ratio of the induction culture in the step S5 is 20h:4 h.
Preferably, the induction culture conditions of step S5: the culture temperature is 20-26 ℃, and the illumination intensity is 3800-.
More preferably, the induction culture conditions of step S5: the culture temperature is 25 +/-0.5 ℃, and the illumination intensity is 4000 lux.
Preferably, step S6 is performed for 20 days.
Preferably, the conditions for the expansion culture in step S1 are: the temperature is 20-26 ℃, and the illumination intensity is 3800-.
More preferably, the conditions for the expansion culture in step S1 are: the temperature is 25 +/-0.5 ℃, and the illumination intensity is 4000 lux.
More preferably, the light brightness ratio of the expanded culture in step S1 is 14h:10 h.
Preferably, in the expanding culture in step S1, the culture vessel is shaken slightly every morning and evening.
Preferably, the centrifugation conditions in step S2 are 2500-3500rpm, 20-26 ℃, 3-10 min.
More preferably, the centrifugation in step S2 is carried out at 3000rpm, 25 ℃ for 5 min.
Preferably, during the cultivation in step S5, the algae is shaken three times per day.
Preferably, in the culture in step S5, the cover of the culture vessel is slightly loosened.
The method adopts f/2 culture medium at the initial stage of Phaeodactylum tricornutum culture, so that the Phaeodactylum tricornutum can reach high cell density after 5 days of growth. The method adopts sterilized seawater without nutrition in the step of induced transformation, prolongs the illumination time, and has the key step of adding a considerable amount of algae cells (the cell density is more than 5 × 10)6one/mL, 50mL solution volume) in a minute volume of 5mL (i.e., cultured cells with a density greater than 5X 107One/ml) to create extreme oligotrophic conditions, thereby greatly improving the power of inducing and converting the cells of the Phaeodactylum tricornutum into the cells, shortening the induction period to three months while ensuring the cell density and the proportion of the cells of the Phaeodactylum tricornutum, and needing no other stress means such as low temperature and the like, and the method is simple, feasible and efficient.
The invention has the following beneficial effects:
the invention provides an induction culture method of an ovoid phaeodactylum tricornutum cell, which is characterized in that various stress effects, such as oligotrophy and the like, brought by extremely high cell density are utilized to promote the phaeodactylum tricornutum cell to be mostly converted into an ovoid shape in a short time, and a set of simple, efficient and feasible induction method is explored by combining with the optimization control of induction conditions. The method overcomes the defects of low proportion, low cell density, overlong required period and high cost of the ovoid phaeodactylum tricornutum induced by the existing culture method, the improved induction culture method has greatly shortened period, can stably induce and culture the ovoid phaeodactylum tricornutum within three months, and has high proportion of cells of the ovoid phaeodactylum tricornutum induced and cultured, basically stable at more than 80 percent, high cell density and easy collection.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Materials used in the following examples:
phaeodactylum tricornutum algae species: can be purchased from a specialized algae seed bank, such as the marine algae collection center of Xiamen university.
Chemical reagents were purchased from Sigma-Aldrich.
Natural seawater: collected from Shenzhen Dapeng Bay. Filtering with 0.22 micron filter membrane, autoclaving at 115 deg.C for 20 min, cooling, and adding deionized water to reduce seawater salinity to 32 psu.
f/2 medium preparation (Guillard 1962): to A: NaNO3 75g B:NaH2PO4.H2O 5g C:Na2SiO3.9H2O 20g D:Na2EDTA 4.16g，FeCl3.6H2O 3.16g，CuSO4.5H2O 0.01g，ZnSO4.7H2O 0.023g，CoCl2.6H2O 0.012g，MnCl.4H2O 0.18g，Na2MoO4.2H2O0.07 g E: adding a certain amount of deionized water into 10.1g of vitamin B, 120.5 g of vitamin B and 0.5g of biotin respectively, fixing the volume to 1L respectively, and carrying out autoclaving at 115 ℃ for 20 minutes. And then sucking 1mL of the prepared A-E by using a pipette gun respectively, adding the treated seawater, fixing the volume to 1L by using the seawater, and adjusting the pH to 8.0 to obtain the f/2 culture medium.
Example 1
1. The method for inducing and culturing the Phaeodactylum ovorans cells comprises the following steps:
s1, sucking 5mL of sterile phaeodactylum tricornutum algae seeds by using a liquid transfer gun when the seeds reach an exponential growth phase, transferring the seeds into a clean conical flask filled with 50mL of f/2 culture medium, and then placing the conical flask into a constant-temperature culture box. The culture temperature is 25 + -0.5 deg.C, the illumination intensity is 4000 lux, and the ratio of light time to dark time is 14h:10 h. Shake flasks in the morning and evening daily.
S2, on the tenth day after inoculation, the density of the phaeodactylum tricornutum reaches 5 multiplied by 106More than one/ml, at which time more than 99% of phaeodactylum tricornutum is fusiform or three-way radial (slightly more fusiform). The algal solution in the Erlenmeyer flask was poured into a 50mL centrifuge tube and collected by centrifugation (3000rpm, 25 ℃ C., 5min), and the supernatant was discarded.
S3, adding 50mL of seawater into the centrifuge tube, centrifuging under the same condition, and repeating twice.
S4, adding 5mL of seawater into the centrifuge tube again (after conversion, the density of the phaeodactylum tricornutum is more than 5 multiplied by 10)7One/ml), and slightly shaking to uniformly disperse the phaeodactylum tricornutum cells without accumulating at the bottom.
S5, placing the centrifuge tube into a constant-temperature incubator, and slightly unscrewing a bottle cap. The culture temperature and the illumination intensity are unchanged, and the ratio of the light time to the dark time is adjusted to 20h:4 h. Shake the algae three times a day.
S6, after 20 days of culture, adding 45mL of seawater into the centrifuge tube, centrifuging (the centrifugation condition is the same as that in the step S3), then discarding the supernatant, adding 5mL of seawater, and slightly shaking to uniformly disperse the phaeodactylum tricornutum cells without accumulating at the bottom.
S7, repeating the steps S5 and S6 three times.
2. Results of the experiment
According to the microscopic examination results of repeated experiments, the phaeodactylum tricornutum cells in the centrifuge tube are mostly converted into egg shape.
After the induction culture, the proportion of the cells of the Phaeodactylum tricornutum is more than 80%, the cell density is high, and the cell density of the cells of the egg is more than 3.5 multiplied by 107One/ml.
Example 2
1. The method for inducing and culturing the Phaeodactylum ovorans cells is the same as the method in example 1, except that: in step S1, the Phaeodactylum tricornutum species was cultured in 250mL f/2 medium.
Similarly, in step S4, 5mL of seawater is enriched, i.e., the converted density of Phaeodactylum tricornutum is greater than 25 × 107Is/areAnd (4) milliliters.
2. Results of the experiment
According to the microscopic examination results of repeated experiments, the phaeodactylum tricornutum cells in the centrifuge tube are mostly converted into egg shape. The proportion of the cells of Phaeodactylum tricornutum is above 80%, the cell density is high, the egg cell density is 14.4 × 107More than one/ml.
Comparative example 1 variation in culture Density
1. The experimental procedure is the same as in example 1, except that:
(1) in step S4, the 5mL seawater is replaced by 20mL seawater (the converted density of Phaeodactylum tricornutum is 1.25 × 10)7One/ml);
(2) after culturing for 20 days in step S6, 30mL of seawater was added to the centrifuge tube, the supernatant was discarded by centrifugation, and 20mL of seawater was added.
2. Results of the experiment
According to the microscopic examination results of repeated experiments, most of the phaeodactylum tricornutum cells in the centrifuge tube are still fusiform and radioactive.
The ratio of Phaeodactylum ovorans cells after the induction culture is below 22%.
Comparative example 2 variation in culture Density
1. The experimental procedure is the same as in example 1, except that:
(1) in step S4, 35mL of seawater is substituted for 5mL of seawater (converted to a density of about 0.7X 107One/ml);
(2) after culturing for 20 days in step S6, 15mL of seawater was added to the centrifuge tube, the supernatant was discarded by centrifugation, and 35mL of seawater was added.
2. Results of the experiment
According to the microscopic examination results of repeated experiments, most of the phaeodactylum tricornutum cells in the centrifuge tube are still fusiform and radioactive.
The ratio of Phaeodactylum ovorans cells after the induction culture is less than 9%.
Comparative example 3 variation in culture Density
1. The experimental procedure is the same as in example 1, except that:
(1) in step S4, 50mL of seawater is substituted for 5mL of seawater (converted to obtain Phaeodactylum tricornutum with a density of 0.5 × 107One/ml);
(2) after culturing for 20 days in step S6, the supernatant was discarded by centrifugation without adding any seawater, and 50mL of seawater was added.
2. Results of the experiment
According to the microscopic examination results of repeated experiments, most of the phaeodactylum tricornutum cells in the centrifuge tube are still fusiform and radioactive.
The ratio of Phaeodactylum ovorans cells after the induction culture is less than 6%.
Comparative example 4 variation in culture Density
1. The method for inducing and culturing the Phaeodactylum ovorans cells is the same as the method in example 1, except that: in step S1, the Phaeodactylum tricornutum species was cultured in 1000mL of f/2 medium.
Similarly, in step S4, the brown algae is enriched with 5mL of seawater, i.e., the converted density of the brown algae is 100X 107One/ml.
2. Results of the experiment
According to the microscopic examination results of repeated experiments, most of the phaeodactylum tricornutum cells in the centrifuge tube are still fusiform and radioactive, and the number of dead cells is increased.
The ratio of Phaeodactylum ovorans cells after the induction culture is 13% or less.
Comparative example 5 variation of nutrient level
1. The experimental procedure is the same as in example 1, except that:
(1) replacing 5mL of seawater with 5mL of f/2 culture medium in the step S4;
(2) after centrifugation in step S6, 5mL of seawater was replaced with 5mL of f/2 medium.
2. Results of the experiment
According to the microscopic examination results of repeated experiments, most of the phaeodactylum tricornutum cells in the centrifuge tube are still fusiform and radioactive.
The ratio of Phaeodactylum ovorans cells after the induction culture is less than 12%.
Comparative example 6 variation of nutrient level
1. The experimental procedure is the same as in example 1, except that:
(1) step S4 replacing 5mL of seawater with 5mL of f/2 medium diluted once (salinity is still 32 psu);
(2) after centrifugation in step S6, 5mL of seawater was replaced with 5mL of f/2 medium diluted once (salinity was still 32 psu).
2. Results of the experiment
According to the microscopic examination results of repeated experiments, most of the phaeodactylum tricornutum cells in the centrifuge tube are still fusiform and radioactive.
The ratio of Phaeodactylum ovorans cells after the induction culture is below 18%.
Comparative example 7 variation of nutrient level
1. The experimental procedure is the same as in example 1, except that:
(1) 5mL of seawater was replaced with 5mL of twice diluted f/2 medium (still 32psu salinity) in step S4;
(2) after centrifugation in step S6, 5mL of seawater was replaced with 5mL of twice diluted f/2 medium (salinity still 32 psu).
2. Results of the experiment
According to the microscopic examination results of repeated experiments, most of the phaeodactylum tricornutum cells in the centrifuge tube are still fusiform and radioactive.
The ratio of Phaeodactylum ovorans cells after the induction culture is below 21%.
The experimental results show that the initial cell density of the phaeodactylum tricornutum cells can be controlled under the high-density stress effect to remarkably promote the phaeodactylum tricornutum cells to be transformed into oval cells in a short time; but not limitless, experiments have shown that the density of Phaeodactylum tricornutum cells at the induction stage is higher than 25X 107At one/ml, the induced oval cell proportion is reduced. In addition, the research on the application level of the culture medium shows that the seawater with the salinity of 32psu is most suitable, the culture temperature is preferably controlled at 20-26 ℃, the illumination intensity is about 4000 lux, the illumination time is moderately prolonged, and the ratio of light to dark is controlled at 20h:4hPreferably.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which are made without departing from the spirit and principle of the present invention should be construed as equivalents and included in the protection scope of the present invention.
Claims (3)
1. An induction culture method of an egg-shaped phaeodactylum tricornutum cell is characterized by comprising the following steps:
s1, carrying out activation culture on sterile phaeodactylum tricornutum algae seeds until the exponential growth period, and transferring to an f/2 culture medium for amplification culture; the condition of the expanded culture is as follows: the temperature is 20-26 ℃, and the illumination intensity is 3800 and 4200 lux;
s2, step S1, expanding cultivation until the density of Phaeodactylum tricornutum reaches 5 × 106When the volume is more than one ml, centrifuging and discarding the supernatant;
s3, adding seawater into the precipitate obtained in the step S2 for centrifugal cleaning;
s4, adding seawater into the sediment obtained in the step S3 to enable the cell density to be (5-25) multiplied by 107Slightly oscillating per milliliter to ensure that the phaeodactylum tricornutum cells are uniformly dispersed and not accumulated at the bottom;
s5, performing induction culture; the ratio of light to dark is 16-20h:4-8 h; the culture temperature is 20-26 ℃, and the illumination intensity is 3800 and 4200 lux;
s6, after culturing for 15-25 days, adding seawater, centrifuging and discarding supernatant; adding seawater to cell density of 5-25 x 107Slightly oscillating per milliliter to ensure that the phaeodactylum tricornutum cells are uniformly dispersed and not accumulated at the bottom;
s7, repeating the steps S5-S6;
wherein the seawater has a salinity of 30-35 psu.
2. The method of claim 1, wherein the light-to-dark ratio of the induction culture of step S5 is 20h:4 h.
3. The method of claim 1, wherein the induction culture temperature of step S5 is 25 ± 0.5 ℃.
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