CN101353631A - A kind of cultivation method of sea cucumber with rhodopseudomonas sphaericus - Google Patents

Abstract

The invention relates to marine microorganisms, and relates to a method for cultivating rhodopseudomonas spheroidus for sea cucumbers. Rhodopseudomonas sphaericus, after liquid first-level seed culture, liquid second-level multiplication culture and third-level mass production culture, and then centrifuged and concentrated to make high-concentration Rhodopseudomonas spheroidus products for sea cucumbers. The culture solution contains Rhodopseudomonas spheroids ≥ 2 billion live bacteria/ml; the concentrated product contains Rhodopseudomonas spheroids ≥ 10 billion live bacteria/ml. The present invention adopts conventional Rhodopseudomonas sphaeroides for sea cucumbers, and uses aged seawater, chelated iron, sodium acetate, etc. Liquid culture, the production process is easy to operate, the cycle is short, and the product quality is high. When used in sea cucumber seedling and cultivation, it can increase the metamorphosis rate and survival rate of sea cucumber larvae at each stage, promote the growth and development of sea cucumber, improve the ecological environment of the water body in the breeding pond, and purify the water quality.

Description

A kind of cultivation method of sea cucumber with rhodopseudomonas sphaericus

technical field

The invention relates to marine microorganisms, in particular to a method for cultivating Rhodopseudomonas spheroidus for sea cucumbers.

Background technique

Rhodopseudomonas sphaeroides (Rhodpseudomonas sphaeroides) for sea cucumbers is a kind of photosynthetic bacteria. aquatic and hydrosphere microorganisms. Photosynthetic bacteria are widely distributed in nature, mainly in habitats such as oceans, rivers, lakes, activated sludge, soil and high-salinity water. Photosynthetic bacteria use light energy as energy source and various organic acids such as sodium acetate, sodium propionate, sodium lactate, mannitol, malic acid, citric acid, and glycerin as carbon source. It has the characteristics of fast growth and reproduction, easy cultivation, and rich metabolites. According to the measurement, the protein content of photosynthetic bacteria is as high as 60%, among which the amino acid composition is complete, and it also contains a variety of vitamins (very rich in B vitamins), folic acid, pantothenic acid, biotin, coenzyme Q and a large amount of carotenoids, tomato Astaxanthin, exopolysaccharide, etc. Therefore, photosynthetic bacteria have high application value, and their products are widely used in many industries such as agriculture, feed, medicine and health care, and environmental protection. (Document: An additive for photosynthetic bacteria culture medium, patent number: ZL 02133074.3, inventors: Xue Delin, Hu Jiangchun, Ma Chengxin, Wang Wei, Liu Jun, Wang Shujin)

In recent years, a large number of test results have shown that among the photosynthetic bacteria, Rhodopseudomonas sphaericus is the most successful application in aquaculture: 1. Rhodopseudomonas sphaericus is a high-quality biological bait, which can directly and indirectly provide sea cucumbers and larvae. Nutrients, improve metamorphosis rate, survival rate, enhance disease resistance, promote growth, improve quality, and increase output. Both the survival rate and the yield increase can reach 10-40%. 2. Rhodopseudomonas spheroides has the function of purifying the water quality of the breeding pond. It can use the organic acids and amino acids formed by the decomposition of small molecule nitrogen-containing compounds and heterotrophic microorganisms in the water to promote the circulation of organic matter at the bottom of the breeding pond. It can be used under various water quality conditions. Purifying or assisting in purifying water quality can reduce the ammonia nitrogen and hydrogen sulfide in the water body of the aquaculture pond, increase the dissolved oxygen, and bring the COD, BOD value and pH value and other indicators to normal, improve the ecological environment of the aquaculture pond water body, and purify the water quality.

In the process of natural ecological purification, due to the participation of Rhodopseudomonas spheroides, the composition of microbial flora in water tends to be reasonable, that is: the growth and reproduction of organic nutritional microbial communities → the growth and reproduction of Rhodopseudomonas spheroids → the promotion of algae growth and reproduction → promote the growth and reproduction of aquaculture animals and maintain the ecological balance of water bodies

The main problems existing in the existing culture technology of Rhodopseudomonas spheroides: 1. The growth and reproduction speed of strains in conventional culture methods is slow, the concentration of bacteria is low, the culture time is long, and it is easy to contaminate miscellaneous bacteria. 2. The characteristics of the strain are not obvious, the content of carotenoids and lysine in the strain is not high, the salt tolerance is poor, and the strain is easy to degenerate. 3. In the previous production, Rhodopseudomonas sphaericus was cultured in fresh water, and the survival rate was low. Sea treasures and sea cucumber larvae were not well utilized for it, which could not meet the application requirements.

The applicant used Rhodopseudomonas sphaeroides isolated and purified from the bottom mud and salt flats of offshore sea cucumber breeding circles to carry out application tests in sea cucumber seedling cultivation and cultivation in Liaoning Dalian Wudao Haizhenpin Breeding Company, and obtained Very ideal effect. The results showed that Rhodopseudomonas sphaeroides used for sea cucumbers can improve the metamorphosis rate and survival rate of sea cucumber larvae, promote the growth of sea cucumbers, and enhance the immunity, disease resistance and color of sea cucumbers; Organic matter in water can reduce the indicators such as hydrogen sulfide and ammonia nitrogen, increase dissolved oxygen, and achieve normal COD, BOD and pH values. It is non-toxic, harmless, and has no toxic or side effects. market prospects.

content of the invention,

The purpose of the present invention is to provide a method for cultivating Rhodopseudomonas sphaericus for sea cucumbers. The invention has the advantages of simple operation, short cycle and high product quality. The product can be used in sea cucumber seedling cultivation and cultivation, which can increase the metamorphosis rate and survival rate of sea cucumber larvae at each stage, can promote the growth and development of sea cucumber, and can improve the ecological environment of the water body in the culture pond. Purify water quality.

To achieve the above object, the technical solution adopted in the present invention is:

Using old seawater, chelated iron, sodium acetate, etc. as the main raw materials, the conventional Rhodopseudomonas spheroides is separated, purified, cultured and produced in an anaerobic tank plate. After liquid primary seed cultivation, liquid secondary expansion Propagation culture and three-level mass production culture, centrifugal concentration to make high-concentration Rhodopseudomonas spheroidus products for sea cucumbers.

A method for cultivating sea cucumbers with Rhodopseudomonas sphaericus, comprising the steps of:

1) Strain activation: Inoculate the activated Rhodopseudomonas sphaeroides slant strains in the liquid medium, and culture to obtain the Rhodopseudomonas sphaeroids seed mother liquor;

2) Primary seed culture: Inoculate the seed mother liquor obtained in step 1) into the primary seed culture medium, the weight percentage of the seed mother liquor and the primary seed culture solution is 20-25: 75-80, and obtain the primary seed liquor through cultivation. seed liquid;

3) Secondary propagation and cultivation: Inoculate the primary seed liquid obtained in step 2) into the secondary propagation medium, the weight percentage of the primary seed liquid and the propagation liquid culture medium is 20-25: 75-80 , to obtain secondary seed liquid through cultivation;

4) Three-level large-scale productive culture: the second-level seed liquid obtained in step 3) is inoculated into a third-level large-scale productive medium, and the weight percentage of the second-level seed liquid and the third-level large-scale productive culture medium is 30-35∶65-70; after cultivation, sea cucumbers can be obtained in liquid form, pink, red and purple in color, with a cell size of 1.0-1.5 μm or 1.0-1.1 μm, and the number of viable bacteria ≥ 2 billion/ml Use the bacterial solution of Rhodopseudomonas sphaericus;

The culture medium composition that above-mentioned rhodopseudomonas spheroides adopts is: ammonium chloride 1.0-3.0g, sodium acetate 2.0-5.0g, magnesium sulfate 0.2-0.3g, sodium bicarbonate 1.0-3.0g, hydrogen phosphate Dipotassium 0.2-0.5g, yeast extract 1.0-3.0g, trace element mother solution 1-3ml, aged sea water 1000ml, which contains chelated iron 5-10ppm, pH value is natural;

The culture conditions are: anaerobic, light 1000-10000Lux, culture 6-8 days;

The aged seawater is natural seawater stored in a cool place for 1-2 weeks, filtered after impurities are precipitated, and the salinity is 2.0-3.0%.

The composition of the medium can specifically be: 1.0g of ammonium chloride, 3.0g of sodium acetate, 0.2g of magnesium sulfate, 2.0g of sodium bicarbonate, 0.2g of dipotassium hydrogenphosphate, 1.0g of yeast extract, 1ml of trace element mother solution, aged seawater 1000ml, which contains chelated iron 5ppm, PH value is natural.

The preparation process of the chelated iron mother liquor is, in 500ml deionized water, add disodium edetate 13.1g, ferrous sulfate 12.5g, potassium hydroxide 134ml of 1 equivalent, carry out ventilation under alkaline condition Reaction, after the reaction is complete, the liquid is brownish red, and the pH value is adjusted to 5.5, which is the chelated iron mother solution; if 0.2ml of the chelated iron mother solution is added in every liter of the solution, the concentration of the chelated iron in the solution is 1ppm.

The trace element mother liquor is composed of 1.40 g of boric acid, 0.14 g of manganese sulfate, 0.26 g of ammonium molybdate, 0.06 g of zinc sulfate, 0.20 g of copper sulfate, 0.01 g of cobalt chloride, and 1000 ml of deionized water.

The medium and nutrient solution used in the steps 1) and 2) are sterilized by high temperature and high pressure, and belong to pure culture after inoculation; the nutrient solution used in steps 3) and 4) is chemically sterilized, and the old seawater is sterilized by sodium hypochlorite and sulfur Sodium sulfite neutralization process; the capacity and weight ratio of old seawater, sodium hypochlorite and sodium thiosulfate are: 1t: 100ml: 8-10g; specifically: add 100ml of 8% sodium hypochlorite to 1 ton of old seawater, and fully react After 6-8 hours, neutralize with 8-10g of sodium thiosulfate, the reaction of potassium iodide indicator is colorless, then add other nutrients to form secondary or tertiary liquid medium.

The mass-produced bacterial liquid of Rhodopseudomonas sphaeroides for sea cucumbers can be used as high-quality biological bait for sea treasure seedling cultivation or cultivation, and can also be used as a seawater water quality purifier.

The present invention has the following advantages:

1. The strains have a wide range of sources and reproduce rapidly. The bacterial species used in the present invention is conventional rhodopseudomonas sphaeroides for sea cucumbers, and the main raw materials are aged seawater, chelated iron, sodium acetate, etc., and are cultivated under the conditions of anaerobic, light, and 15°C to 32°C. Under the same growth condition, applying the cultivation method of the present invention can make the propagation speed of the rhodopseudomonas spheroidus for sea cucumbers be 2-3 days faster than that of general general-purpose culture medium, and the bacteria concentration can be increased by more than 50%.

2. The raw material of the culture medium has simple composition, wide sources and low cost. The raw materials for cultivating Rhodopseudomonas spheroidus used in the present invention are simple and common. When mass-produced, wastes from urban industrial products and agricultural and sideline products can be used. After comprehensive treatment, they can be made into high-concentration spherical bacterium for sea cucumbers. Rhodopseudomonas products.

3. The products of Rhodopseudomonas sphaericus used in sea cucumbers are of high quality. Using Rhodopseudomonas sphaeroides for sea cucumbers as the provenance, the products produced have high content of carotenoids and lysine, and have good salt tolerance. Production processes such as liquid primary seed cultivation, liquid secondary multiplication cultivation, large-scale productive cultivation and centrifugal concentration are easy to operate, with short cycle times and high product quality.

4. The present invention has the characteristics of being used for low-temperature type sea cucumbers. Sea cucumbers use Rhodopseudomonas spheroides, which has wide adaptability, complete nutritional components, good safety, and is friendly to the ecological environment. The application in sea cucumber seedlings can increase the metamorphosis rate of larvae at each stage and survival rate, and can accelerate the growth and development of sea cucumber larvae, improve the ecological environment of the water body in the breeding pond, purify the water quality, and improve the quality and output of sea cucumbers. It is welcomed by sea cucumber farming users and is easy to promote and apply.

Detailed ways

The present invention is described in further detail below in conjunction with embodiment.

Example 1:

A kind of cultivation method of rhodopseudomonas sphaericus for sea cucumber can be operated according to the following steps:

(1) Activation of slanted seed mother

The seed stock of Rhodopseudomonas spheroides is connected to the plate, test tube or the slant of the Kjeller flask of the newly prepared solid medium for seed stock activation, and placed in an anaerobic tank. In the range of natural light 1000-3000Lux, anaerobic culture for 6--8 days, the seed mother is activated.

(2) Seed mother activation to obtain seed mother liquor

With the slant seed mother that is activated, use inoculation ring to pick 1-2 rings of slant seed mother bacterial lawn, inoculate into the sterilized volume that is 500ml in the transparent glass bottle of liquid culture medium (cultivation condition is the same as above), obtain seed mother liquor.

The composition of the medium used in the activation process of Rhodopseudomonas sphaeroides is: 1.0 g ammonium chloride, 3.0 g sodium acetate, 0.2 g magnesium sulfate, 1.0 g sodium bicarbonate, 0.2 g dipotassium hydrogen phosphate, and 1.0 g yeast extract g. 1ml of trace element mother solution, 1ml of chelated iron mother solution, 1000ml of aged sea water, 22-25g of agar, PH = natural; 1 atmospheric pressure, 20min sterilization.

The composition of the liquid medium used for seed activation is the same as above, but without adding agar.

(3) Liquid seed primary culture inoculation and culture conditions

Preparation and sterilization of liquid seed primary medium. For liquid seed primary culture, its medium composition and preparation are the same as the above (2) liquid medium. Put the configured liquid culture medium into a transparent glass bottle with a volume of 500 ml (75-80% of the culture medium content, and the balance is the amount of seeds), sterilize at 1 atmospheric pressure for 20 minutes, and set aside. The above-mentioned (2) activated sea cucumber rhodopseudomonas sphaeroides liquid seed mother liquor is poured into a sterilized transparent glass bottle with a volume of 500 milliliters of liquid seed primary medium in a sterile room, and placed Cultivate under natural sunlight at a temperature of 15°C to 32°C for 6-8 days. The seeds are mature and contain more than 500 million live bacteria/ml. The culture medium is bright red or dark red. Check the bacteria under a microscope. The body movement is lively and neat, which can be used for multiplication and cultivation;

(4) Sterilization and culture conditions of liquid seed secondary multiplication culture

The composition and preparation of the liquid seed secondary propagation medium are the same as the above-mentioned (2) liquid medium. The culture medium is chemically sterilized, and the aged seawater is treated by the neutralization method of sodium hypochlorite and sodium thiosulfate. The capacity and weight ratio of aged seawater, sodium hypochlorite and sodium thiosulfate are: 1t: 100ml: 8-10g; specifically: 1 ton Add 100ml of sodium hypochlorite with a content of 8% to the old seawater, and after fully reacting for 6-8 hours, neutralize with 8-10g of sodium thiosulfate, and detect with potassium iodide-starch indicator until it becomes colorless and transparent. This method can kill aquatic organisms, small marine animals and bacteria (spores cannot be killed).

Sea cucumber is inoculated with 20-25% inoculum amount with the good secondary expansion liquid seed of Rhodopseudomonas sphaeroides cultivation, receives and dresses in the transparent glass bottle of 5000 milliliters (the culture medium amount is 75-80%, surplus is the amount of liquid seed inserted). Under natural sunlight, the temperature is 15°C-32°C, and the culture time is 6-8 days. After the culture is mature, the number of viable bacteria is ≥1 billion/ml, and the culture medium is bright red or deep red. Check the bacteria under a microscope. The movement is lively, robust, free from pollution, odor and abnormality. It can be used for mass production and cultivation.

(5) Liquid mass production inoculation and culture conditions

The medium composition and medium preparation of liquid mass production are the same as the above (2) liquid medium. Sterilization adopts sodium hypochlorite-sodium thiosulfate neutralization method, the method is the same as above (4). The mass production and cultivation of liquid is mainly to sterilize the container and seawater, choose a transparent plastic bag of 50 kg for the container, the amount of culture liquid is 65-70%, and then inoculate the secondary seed liquid with 30-35% Put it into a plastic bag, seal the mouth with a sealing machine, place it in a plastic greenhouse for hanging culture, the culture conditions are the same as the above (4), the number of viable bacteria is more than 2 billion/ml when the expansion culture is mature, and the culture solution is bright red Or deep red, check under the microscope that the sea cucumber with Rhodopseudomonas sphaericus is active, the bacteria are strong, no pollution, no peculiar smell, no abnormal phenomenon, and it can be used in production.

(6) Liquid centrifugal concentration and its product standards

The cultured liquid is pink, orange, red, brown and purple, the cell size is 1.0-1.5 μm or 1.0-1.1 μm, and the number of viable bacteria is ≥ 2 billion/ml spherical rhodopseudomonas for sea cucumbers Bacteria in large quantities can be used as bait and water purifier for sea cucumbers.

The process of centrifugal concentration is as follows: the sea cucumber with Rhodopseudomonas sphaeroides is centrifuged in a centrifuge at 5000rpm for 20-25min, and after centrifugation, the sea cucumber is diluted with Rhodopseudomonas sphaericus cells with the treated old seawater to the required standard. The product standards are set as follows: the number of viable bacteria is 10 billion/ml, 15 billion/ml, and 20 billion/ml.

Sea cucumber products with Rhodopseudomonas sphaericus can be stored at room temperature. The shelf life is 12 months. When in use, it can be quantitatively diluted in seawater according to the calculated dosage, shake well in the container, disperse and splash it into the culture pond for application.

The production process of Rhodopseudomonas spheroidus for sea cucumbers is as follows: slant strain → liquid seed mother liquor → liquid seed primary cultivation → liquid seed secondary expansion cultivation → liquid mass production cultivation → liquid centrifugal concentration → Rhodopseudomonas products .

Example 2:

Sea cucumber culture factor optimization test of rhodopseudomonas sphaericus, according to the following steps: seed mother activation: the same as embodiment 1 (1), (2).

Under laboratory conditions, the optimal test of culture factors is carried out, and the culture factors are: sodium acetate, yeast extract, trace element mother solution, and chelated iron mother solution. The experimental design adopts 4 factors and 5 levels; the 5 treatment numbers are 1, 2, 3, 4, 5. In a 500ml reagent bottle, fill 350ml of seawater treated with sodium hypochlorite and other nutrients, and then add other culture factors according to the experimental design, and then inoculate 150ml of Rhodopseudomonas sphaeroides seed solution in each bottle, and place it under natural sunlight at 15°C At ~32°C, culture anaerobically for more than 120 hours, and measure the number of Rhodopseudomonas spheroides. The design of the experiment and the results of the determination of the number of bacteria are shown in Table 1. The medium without adding culture factors was used as the control group.

Other nutrient components The composition of the medium content formula is as follows: 1000ml of sterile water contains 1.0g of ammonium chloride, 0.2g of magnesium sulfate, 1.0g of sodium bicarbonate, and 0.2g of dipotassium hydrogenphosphate.

Table 1. Combination test of rhodopseudomonas sphaeroides culture medium formula for sea cucumbers

serial number 1 2 3 4 5 control group Sodium acetate (g/mL) 1.0 2.0 3.0 4.0 5.0 0 Yeast extract (g/mL) 0.5 1.0 1.5 2.0 2.5 0 Trace element mother solution (mL/L) 0.5 1.0 1.5 2.0 2.5 0 Chelated iron mother liquor (mL/L) 0.5 1.0 1.5 2.0 2.5 0 Bacteria count (100 million/mL) 10 13 12 10 8 3

The test results showed that the addition of culture factors in test treatment 2 was the best combination (that is, 1000ml of culture solution contained 2.0g of sodium acetate, 1g of yeast extract, 1ml of trace element mother solution, and 1ml of chelated iron mother solution).

When the present invention is applied, the order of selecting culture factors to form the culture medium is: old sea water, sodium acetate, yeast extract, chelated iron, and trace element mother liquor.

Example 3

A kind of implementation effect of rhodopseudomonas sphaericus for sea cucumber can be operated according to the following steps:

(1) seed mother activation: with embodiment 1 (1), (2);

(2) liquid seed primary culture inoculation and culture conditions: with embodiment 1 (3);

(3) liquid seed two-stage multiplication culture inoculation and culture conditions: with embodiment 1 (4);

(3) Liquid mass production inoculation and culture conditions: the same as Example 1 (5);

After sea cucumbers are propagated with Rhodopseudomonas sphaericus according to the above steps, the number of viable bacteria is more than 2 billion/ml, and the amount of 500ml/666.7m ² is dispersed and evenly sprinkled to 50×666.7m ² (water depth 2- 2.5m) in the sea cucumber breeding pond, after 20 days of breeding, its technical index can reach as shown in the following table 2:

Table 2. Implementation effect of sea cucumber with Rhodopseudomonas sphaericus

Test treatment 100 million photosynthetic bacteria/ml Water temperature ℃ PH value Salinity‰ Dissolved oxygen mg/L CODmg/L BODmg/L Ammonium salt mg/L before use 0 19.6 8.2 34 2.50 6.84 7.12 0.079 After use 0.2 21.6 8.4 35 5.50 2.10 3.20 0.039

Time of use: May 8, 2006; time of analysis: May 28, 2006

From the test and analysis results, it can be seen that after using Rhodopseudomonas sphaericus, in the sea cucumber culture pond, the dissolved oxygen increased, the ammonium salt decreased, and the COD, BOD value and pH value and other indicators reached normal.

The seed stocks of Rhodopseudomonas sphaericus used in the examples of the present invention can be obtained commercially, and there is no special requirement for them.

The applicant used Rhodopseudomonas sphaeroides RSY96 for sea cucumbers (Rhodpseudomonas sphaeroides RSY96) isolated, purified and identified by the Shenyang Institute of Applied Ecology, Chinese Academy of Sciences, domesticated and purified in the production practice of the breeding base of Dalian Wudao Haizhenpin Company, and obtained High concentrations of Rhodopseudomonas sphaericus in sea cucumbers. Its application effect is very good, and it has been widely used in coastal areas such as Lushun, Changxing Island, and Exchange Island in Dalian, and can realize commercial production of products.

The domesticated and purified high-concentration sea cucumbers are negative for Gram staining with Rhodopseudomonas sphaericus, without spores, and can better utilize H ₂ S. Observed under a microscope, the cell shape is: ovoid or spherical, ovoid diameter The size is 1.0×15 or 10×1.1 μm. Polar single or double flagella, with photosynthetic membrane system. Photosynthetic membranes were formed in anaerobic light culture, but in aerobic culture without light, photosynthetic membranes were not formed. Observed on the plate culture medium, the colony shape is: round convex or flat, the diameter of the colony is 2.0-5.0mm, the edges are neat and regular, and the surface is moist, smooth and opaque. The color is pink or red. The application of Rhodopseudomonas sphaeroides for sea cucumbers in sea cucumber seedlings can increase the survival rate of sea cucumbers by 20%; it can accelerate growth and development, enhance immune function, improve disease resistance, promote digestion and absorption, and significantly increase the survival rate of sea cucumbers. The body color of sea cucumbers. From the test results, it can be seen that Rhodopseudomonas sphaericus for sea cucumbers has very ideal effects on sea cucumber metamorphosis, young ginseng and adult ginseng cultivation, and is welcomed by local sea cucumber farmers.

Claims (5)

1. a culture method for sea cucumber rhodopseudomonas sphaericus, is characterized in that, comprises the steps: 1) Strain activation: Inoculate the activated Rhodopseudomonas sphaeroides slant strains in the liquid medium, and culture to obtain the Rhodopseudomonas sphaeroids seed mother liquor; 2) Primary seed culture: Inoculate the seed mother liquor obtained in step 1) into the primary seed culture medium, the weight percentage of the seed mother liquor and the primary seed culture solution is 20-25: 75-80, and obtain the primary seed liquor through cultivation. seed liquid; 3) Secondary propagation and cultivation: Inoculate the primary seed liquid obtained in step 2) into the secondary propagation medium, the weight percentage of the primary seed liquid and the propagation liquid culture medium is 20-25: 75-80 , to obtain secondary seed liquid through cultivation; 4) Three-level large-scale productive culture: the second-level seed liquid obtained in step 3) is inoculated into a third-level large-scale productive medium, and the weight percentage of the second-level seed liquid and the third-level large-scale productive culture medium is 30-35∶65-70; after cultivation, sea cucumbers can be obtained in liquid form, pink, red and purple in color, with a cell size of 1.0-1.5 μm or 1.0-1.1 μm, and the number of viable bacteria ≥ 2 billion/ml Use the bacterial solution of Rhodopseudomonas sphaericus; The culture medium composition that above-mentioned rhodopseudomonas spheroides adopts is: ammonium chloride 1.0-3.0g, sodium acetate 2.0-5.0g, magnesium sulfate 0.2-0.3g, sodium bicarbonate 1.0-3.0g, hydrogen phosphate Dipotassium 0.2-0.5g, yeast extract 1.0-3.0g, trace element mother solution 1-3ml, aged sea water 1000ml, which contains chelated iron 5-10ppm, pH value is natural; The culture conditions are: anaerobic, light 1000-10000 Lux, culture for 6-8 days; The old sea water is natural sea water stored in a cool place for 1-2 weeks, filtered after impurities are precipitated, and the salinity is 2.0-3.0%. 2. According to the described cultivation method of claim 1, it is characterized in that: the composition of the medium is: ammonium chloride 1.0g, sodium acetate 3.0g, magnesium sulfate 0.2g, sodium bicarbonate 2.0g, dipotassium hydrogen phosphate 0.2 g. Yeast extract 1.0g, trace element mother solution 1ml, aged seawater 1000ml, which contains chelated iron 5ppm, pH value is natural. 3. according to the described culture method of claim 1, it is characterized in that: the preparation process of described chelated iron mother liquor is, in 500ml deionized water, add disodium edetate 13.1g, ferrous sulfate 12.5g, 1 One equivalent of potassium hydroxide 134ml is carried out under alkaline conditions for ventilating reaction. After the reaction is complete, the liquid is brownish red. Adjust the pH value to 5.5, which is the mother liquor of chelated iron; if 0.2 ml, the concentration of chelated iron in the solution is 1ppm. 4. According to the described cultivation method of claim 1, it is characterized in that: the trace element mother liquor consists of boric acid 1.40g, manganese sulfate 0.14g, ammonium molybdate 0.26g, zinc sulfate 0.06g, copper sulfate 0.20g, chloride Cobalt 0.01g, deionized water 1000ml. 5. according to the described culture method of claim 1, it is characterized in that: described step 1) and 2) the culture medium and culture fluid that adopt are through high temperature and high pressure sterilization, belong to pure culture after the inoculation; Step 3) and 4) The culture solution used is chemically sterilized, and the old seawater is treated by the neutralization method of sodium hypochlorite and sodium thiosulfate; the capacity and weight ratio of the old seawater, sodium hypochlorite and sodium thiosulfate are: 1t: 100ml: 8-10g; specifically: Add 100ml of sodium hypochlorite with a content of 8% to 1 ton of old seawater. After fully reacting for 6-8 hours, neutralize with 8-10g of sodium thiosulfate. grade liquid medium.