CN101353631A - Cultivation method of Rhodopseudomonas sphaeroides for sea cucumber - Google Patents
Cultivation method of Rhodopseudomonas sphaeroides for sea cucumber Download PDFInfo
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Abstract
The invention relates to a marine microorganism, in particular to a method for culturing rhodopseudomonas spheroids used for sea cucumber; the method comprises the following steps: the rhodopseudomonas spheroids is cultured by liquid one-grade seeds, cultured by liquid two-grade reproduction and cultured by three-grade mass production, and the rhodopseudomonas spheroids products used for sea cucumber with high concentration are produced by centrifugation and concentration; the culture solution contains the rhodopseudomonas spheroids which are more than or equal to 2 billion viable bacteria/ml; the product obtained by concentration contains rhodopseudomonas spheroids which are more than or equal to 10 billion viable bacteria/ml; the invention adopts the common rhodopseudomonas spheroids used for sea cucumber as strain, and takes old seawater, chelatase ferrum and sodium acetate, etc. as main materials and carries out liquid culture under the conditions of being anaerobic, the illumination of 1000 to10000Lux and the temperature of 15 DEG C to 32 DEG C; the production technique has simple technique and operation, short period and high product quality; when being applied in the culture of seedling and the cultivation of sea cucumber, the invention can improve the metamorphosis ratio and the survival rate of each stage of sea cucumber larvae, can enhance the growth of the sea cucumber, can simultaneously improve the ecological environment of water body in a culture pond and purify the water quality.
Description
Technical field
The present invention relates to marine microorganism, specifically be meant a kind of cultural method of Rhodopseudomonas sphaeroides for sea cucumber.
Background technology
Rhodopseudomonas sphaeroides for sea cucumber (Rhodpseudomonas sphaeroides), be a kind of of photosynthetic bacterium, photosynthetic bacterium (Photosynthetic Bacteria is called for short PSB) is the prokaryotic organism with original luminous energy synthetic system, is a class luminous energy nutrition, amphimicrobian aquatic, hydrosphere microorganism.Photosynthetic bacterium distributes very extensive at occurring in nature, mainly be distributed in the habitats such as ocean, rivers, lakes and marhshes, active sludge, soil and haline water body.Photosynthetic bacterium is the energy with luminous energy, is carbon source with multiple organic acids such as sodium acetate, Sodium Propionate, Sodium.alpha.-hydroxypropionate, N.F,USP MANNITOL, oxysuccinic acid, citric acid, glycerine.Have growth and breeding speed fast, characteristics such as be easy to cultivate, meta-bolites is nutritious.According to surveying and determination, photosynthetic bacterium dry mycelium protein content is up to more than 60%, wherein amino acid is formed completely, also contains multivitamin (vitamin B group is very abundant), folic acid, pantothenic acid, vitamin H, ubiquinone and a large amount of carotenoid, tomatine, astaxanthin, exocellular polysaccharide etc.Therefore, photosynthetic bacterium has very high using value, and its product is widely used in all conglomeraties such as agricultural, feed, medicines and health protection, environment protection.(document: a kind of photosynthetic bacteria culture medium additive, the patent No.: ZL 02133074.3, contriver: Xue Delin, Hu Jiangchun, Ma Chengxin, Wang Wei, Liu Jun, Wang Shujin)
In recent years, a large amount of test-results show, the application of Rhodopseudomonas spheroides in aquaculture is the most successful in the photosynthetic bacterium: 1. Rhodopseudomonas spheroides is the fine biological feed, can directly and indirectly provide nutritive substance, improve distortion ratio, surviving rate, enhancing disease resistance, promote growth, improve quality, improve output for culturing the sea cucumber and the young.No matter be that the surviving rate or the raising of output all can reach 10～40%.2. Rhodopseudomonas spheroides has purifying aquaculture pond water quality function, can utilize small molecules nitrogenous compound and heterotrophic microorganism are decomposed to form in the water organic acid and amino acid, organic circulation at the bottom of the promotion breed pond, can under various condition of water quality, purify or assist to purify water, ammonia nitrogen, hydrogen sulfide in the aquaculture pond water body are descended, and dissolved oxygen improves, and item indexs such as COD, BOD value and pH value reach normal, improve the ecotope of culturing pool body, purify water.
In the natural, ecological scavenging process, because the participation of Rhodopseudomonas spheroides, microorganism species is formed and is tending towards reasonable in the water body, that is: the growth and breeding of the growth and breeding of the growth and breeding → Rhodopseudomonas spheroides of organotrophy type microflora → promotion algae grows breeding → promotion aquatic animal is kept the water ecology balance
The subject matter that exists in the existing Rhodopseudomonas spheroides culture technique: 1. the cultural method strain growth reproduction speed of routine is slow, to contain bacteria concentration low, and incubation time is long, easy pollution microbes.2. strain properties is not obvious, and carotenoid in the thalline and lysine content are not high, and salt tolerance is poor, and bacterial classification is easily degenerated.3. Rhodopseudomonas spheroides is that fresh water is cultivated in production in the past, and survival rate is low, and the precious marine product and the sea cucumber young are bad to its utilization, can not satisfy demands of applications.
The applicant adopts from the bed mud and salt flat of coastal waters holothruian cultures circle, and the Rhodopseudomonas sphaeroides for sea cucumber that separation and purification obtains carries out application test in Dalian five island choice seafood aquiculture company sea cucumbers are grown seedlings and form, obtained very ideal effect.The result shows: Rhodopseudomonas sphaeroides for sea cucumber can improve distortion ratio and the surviving rate of the sea cucumber young, promotes the sea cucumber growth and can strengthen immunizing power, disease resistance and the sea cucumber body colour pool of sea cucumber; Organism in water quality that can the purifying aquaculture pond, the water of decomposition descends indexs such as hydrogen sulfide, ammonia nitrogen, and dissolved oxygen improves, COD, BOD and pH value reach normal, nontoxic, harmless, without any side effects, be a kind of biological feed and water quality cleansing agent of green, have vast market prospect.
Summary of the invention,
The object of the invention is to provide a kind of cultural method of Rhodopseudomonas sphaeroides for sea cucumber.The present invention is easy and simple to handle, and the cycle is short, quality product is high, and product is grown seedlings and formed middle application sea cucumber, can improve each stage distortion ratio of the sea cucumber young, surviving rate, can promote growing of sea cucumber, can improve the ecotope of culturing pool body, purifies water.
For achieving the above object, the technical solution used in the present invention is:
With Chen Haishui, chelated iron, sodium acetate etc. is main raw material, with the Rhodopseudomonas spheroides of routine in the anaerobic jar plate, separation, purifying, the cultivation production of hybrid seeds, expand numerous cultivation and the cultivation of three grades of property produced in enormous quantities through the cultivation of liquid first order seed, liquid secondary, centrifugal concentrating made high density Rhodopseudomonas sphaeroides for sea cucumber product.
A kind of cultural method of Rhodopseudomonas sphaeroides for sea cucumber comprises the steps:
1) actication of culture: will be inoculated in the liquid nutrient medium through activatory Rhodopseudomonas spheroides slant strains, and, obtain Rhodopseudomonas spheroides seed mother liquor through cultivating;
2) first order seed is cultivated: the seed mother liquor that obtains in the step 1) is inoculated in the first order seed substratum, and the weight percent of seed mother liquor and first order seed nutrient solution is 20-25: 75-80, obtains primary seed solution through cultivation;
3) secondary expands numerous cultivation: with step 2) in the primary seed solution that obtains be inoculated into secondary and expand in the breeding culture medium, primary seed solution is 20-25: 75-80 with the weight percent that expands numerous liquid medium, obtains secondary seed solution through cultivation;
4) three grades of property produced in enormous quantities cultivation: the secondary seed solution that obtains in the step 3) is inoculated in the three grades of property produced in enormous quantities substratum, and the weight percent of secondary seed solution and the three grades of property produced in enormous quantities nutrient solutions is 30-35: 65-70; Through cultivating, promptly obtain liquidly, be pink colour, redness and red-purple, the thalline size is 1.0～1.5 μ m or 1.0～1.1 μ m, contains the bacterium liquid of the Rhodopseudomonas sphaeroides for sea cucumber of viable count 〉=2,000,000,000/ml;
The substratum composition that above-mentioned Rhodopseudomonas spheroides culturing process is adopted is: ammonium chloride 1.0-3.0g, sodium acetate 2.0-5.0g, sal epsom 0.2-0.3g, sodium bicarbonate 1.0-3.0g, dipotassium hydrogen phosphate 0.2-0.5g, yeast extract paste 1.0-3.0g, micro-mother liquor 1-3ml, Chen Haishui 1000ml, wherein contain chelated iron 5-10ppm, pH value nature;
Culture condition is: anaerobism, illumination 1000-10000Lux, cultivated 6-8 days;
Described old seawater is that natural sea-water is placed into shady and cool place and stores 1-2 week, treats that the contamination precipitation after-filtration forms, and salinity is 2.0-3.0%.
Described substratum composition specifically can be, ammonium chloride 1.0g, sodium acetate 3.0g, sal epsom 0.2g, sodium bicarbonate 2.0g, dipotassium hydrogen phosphate 0.2g, yeast extract paste 1.0g, micro-mother liquor 1ml, Chen Haishui 1000ml wherein contain chelated iron 5ppm, pH value nature.
The process for preparation of described chelated iron mother liquor is, in the 500ml deionized water, add disodium ethylene diamine tetraacetate 13.1g, ferrous sulfate 12.5g, 1 normal potassium hydroxide 134ml, under alkaline condition, carry out ventilatory response, the back liquid that reacts completely is red-brown, pH value is transferred to 5.5, be the chelated iron mother liquor; If add chelated iron mother liquor 0.2ml in every liter of solution, the chelated iron concentration in the solution is 1ppm.
Described micro-mother liquor consists of, boric acid 1.40g, manganous sulfate 0.14g, ammonium molybdate 0.26g, zinc sulfate 0.06g, copper sulfate 0.20g, cobalt chloride 0.01g, deionized water 1000ml.
Described step 1) and 2) substratum that is adopted and nutrient solution are through autoclave sterilization, and the inoculation back belongs to pure culture; Step 3) and 4) nutrient solution that is adopted is chemosterilization, and Chen Haishui adopts clorox and Sulfothiorine neutralisation to handle; The capacity of Chen Haishui, clorox and Sulfothiorine and part by weight are: 1t: 100ml: 8-10g; Be specially: it is 8% clorox 100ml that 1 ton of Chen Haishui adds content, fully react 6-8 hour after, with the neutralization of 8-10g Sulfothiorine, the potassiumiodide indicator reaction is colourless, adding other nutrition again is exactly secondary or three grades of liquid nutrient mediums.
The high-quality biological feed that the production in enormous quantities bacterium liquid of described Rhodopseudomonas sphaeroides for sea cucumber can be used as breeding sea valued product or forms also can be used as the seawater quality scavenging agent.
The present invention has following advantage:
1. the bacterial classification wide material sources are bred fast.The bacterial classification that the present invention adopts be the Rhodopseudomonas sphaeroides for sea cucumber of routine, and is main raw material with Chen Haishui, chelated iron, sodium acetate etc., cultivates under anaerobism, illumination, 15 ℃～32 ℃ conditions.Under the isometric growth condition, use cultural method of the present invention, can make the Rhodopseudomonas sphaeroides for sea cucumber reproduction speed general the fast 2-3 of collective media days, bacteria concentration improves more than 50%.
2. culture medium raw material is formed simple, wide material sources, cost is low.It is simply general that the Rhodopseudomonas sphaeroides for sea cucumber that the present invention adopts is cultivated raw material, during production in enormous quantities, can adopt urban industry product tankage, agricultural byproducts waste,, can be made into the Rhodopseudomonas sphaeroides for sea cucumber product of high density after the cultivation through comprehensive treating process.
3. the quality product height of Rhodopseudomonas sphaeroides for sea cucumber.With the Rhodopseudomonas sphaeroides for sea cucumber is provenance, thalline carotenoid and lysine content height in the product of making, and good salt tolerance.The liquid first order seed is cultivated, the liquid secondary expands numerous cultivation, the property produced in enormous quantities cultivation and centrifugal production technique such as concentrate are easy and simple to handle, and the cycle is short, quality product is high.
4. the present invention has the low temperature modification sea cucumber and characteristics such as uses, Rhodopseudomonas sphaeroides for sea cucumber, adaptability is extensive, nutrition is complete, security is good, to eco-friendly, application can improve the young each stage distortion ratio and surviving rate in sea cucumber is grown seedlings, and can accelerate the sea cucumber young and grow, and improves the ecotope of culturing pool body, purify water, improve the quality and yield of sea cucumber.Welcome by the holothruian cultures user, be easy to apply.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1:
A kind of cultural method of Rhodopseudomonas sphaeroides for sea cucumber can follow these steps to operation:
(1) the female activation of inclined-plane kind
With the kind mother of Rhodopseudomonas spheroides, the female activation of kind of receiving new preparation is put into anaerobic jar with on plate, test tube or the Kolle flask inclined-plane of solid medium, under 25 ℃ of conditions of temperature, in indoor natural light 1000-3000Lux scope, anaerobism was cultivated 6--8 days, carried out kind of a female activation.
(2) plant female activation and obtain the seed mother liquor
The inclined-plane kind mother that activation is good chooses the female lawn of 1-2 ring inclined-plane kind with transfering loop, and the volume that is inoculated into the bacterium of going out is the vial interior (culture condition is the same) of the transparent interior dress liquid nutrient medium of 500ml, obtains the seed mother liquor.
The substratum that the female reactivation process of Rhodopseudomonas spheroides kind is adopted consists of: ammonium chloride 1.0g, sodium acetate 3.0g, sal epsom 0.2g, sodium bicarbonate 1.0g, dipotassium hydrogen phosphate 0.2g, yeast extract paste 1.0g, micro-mother liquor 1ml, chelated iron mother liquor 1ml, Chen Haishui 1000ml, agar 22-25g, PH=nature; 1 normal atmosphere, the 20min sterilization.
The liquid nutrient medium composition of planting female activation usefulness is the same, but does not add agar.
(3) the liquid seeds one-level is cultivated inoculation and culture condition
Liquid seeds one-level culture medium preparation and sterilization.The liquid seeds one-level is cultivated, and its substratum is formed and prepared with above-mentioned (2) liquid nutrient medium.With the liquid nutrient medium that the configures volume of packing into is (the substratum loading amount is 75-80%, and surplus is a grain weight) in 500 milliliters of transparent vials, 1 normal atmosphere, and the 20min sterilization, stand-by.The Rhodopseudomonas sphaeroides for sea cucumber liquid seeds mother liquor that above-mentioned (2) have been activated, the volume of pouring the bacterium of having gone out in sterilisable chamber into is in the transparent vial of 500 milliliters of liquid seed one-level substratum, be placed under the nature sunlight and cultivate, culture temperature is 15 ℃～32 ℃, and the time is 6-8 days, the seed culture maturation, contain viable count 〉=500,000,000/ml, nutrient solution is scarlet or scarlet, and the motion of test under microscope thalline is active, neatly, can use for expanding numerous cultivation;
(4) the liquid seeds secondary expands numerous cultivation sterilization and culture condition
The liquid seeds secondary expands the breeding culture medium composition, substratum is prepared with above-mentioned (2) liquid nutrient medium.Nutrient solution adopts chemosterilization, and Chen Haishui adopts clorox and Sulfothiorine neutralisation to handle, and the capacity of Chen Haishui, clorox and Sulfothiorine and part by weight are: 1t: 100ml: 8-10g; Be specially: it is 8% clorox 100ml that 1 ton of Chen Haishui adds content, fully react 6-8 hour after, with the neutralization of 8-10g Sulfothiorine, detect with potassiumiodide-Starch Indicator, get final product when presenting water white transparency.This method can be killed hydrobiont, ocean animalcule and bacterium (gemma kills not dead).
The cultured secondary of Rhodopseudomonas sphaeroides for sea cucumber is expanded numerous liquid seeds with the inoculation of 20-25% inoculum size, receive splendid attire (the cultivation base unit weight is 75-80%, the liquid seeds amount of surplus for inserting) in 5000 milliliters of transparent vials.Under natural sunlight, temperature is 15 ℃～32 ℃, and incubation time is 6～8 days, cultivate maturation after, contain viable count 〉=1,000,000,000/ml, nutrient solution is scarlet or scarlet, the motion of test under microscope thalline is active, stalwartness, pollution-free, free from extraneous odour, no abnormal phenomenon.Can use for producing in enormous quantities to cultivate.
(5) liquid is produced inoculation and culture condition in enormous quantities
The substratum that liquid is produced in enormous quantities is formed, substratum is prepared with above-mentioned (2) liquid nutrient medium.Clorox-Sulfothiorine neutralisation is adopted in sterilization, and method is with above-mentioned (4).It mainly is the sterilising treatment of container containing and seawater that liquid is produced cultivation in enormous quantities, selecting container containing is 50 kilograms of transparent plastics bags, the nutrient solution loading amount is 65-70%, again secondary seed solution is linked in the plastics bag with the 30-35% inoculum size, with sealing machine mouth is sealed, be placed into the cultivation that is hung in the plastic greenhouse, culture condition is with above-mentioned (4), expand numerous cultivation maturation and contain viable count 〉=2,000,000,000/ml, nutrient solution is scarlet or scarlet, and test under microscope is active with the Rhodopseudomonas sphaeroides for sea cucumber motion, the thalline stalwartness, pollution-free, free from extraneous odour, no abnormal phenomenon can be used aborning.
(6) centrifugal concentrates and products thereof standard
It is liquid that cultivation obtains, be pink colour, orange, red, brown and red-purple, the thalline size is 1.0～1.5 μ m or 1.0～1.1 μ m, contains the bacterium liquid in enormous quantities of the Rhodopseudomonas sphaeroides for sea cucumber of viable count 〉=2,000,000,000/ml, can be used as bait and the water quality cleansing agent of sea cucumber.
Centrifugal concentration process is: the bacterium liquid in enormous quantities of Rhodopseudomonas sphaeroides for sea cucumber is carried out 5000rpm on whizzer, 20-25min is centrifugal, and centrifugal back is diluted to desired standard with the Chen Haishui that has handled with the Rhodopseudomonas sphaeroides for sea cucumber thalline.Product standard is decided to be respectively: contain viable count and be 10,000,000,000/ml, 15,000,000,000/ml, 20,000,000,000/ml.
The Rhodopseudomonas sphaeroides for sea cucumber product can be preserved at normal temperatures.Quality guaranteed period is 12 months.Can in seawater, quantitatively dilute during use, in container, shake up, disperse to splash and use to culturing in the pond according to the consumption that calculates.
The Rhodopseudomonas sphaeroides for sea cucumber production technique is: slant strains → liquid seeds mother liquor → liquid seeds one-level cultivation → liquid seeds secondary expands numerous cultivation → liquid production in enormous quantities cultivation → centrifugal and concentrates → the rhodopseudomonas product.
Embodiment 2:
The cultivation factor optimization test of Rhodopseudomonas sphaeroides for sea cucumber follows these steps to operation: plant female activation: with embodiment 1 (1), (2).
Under laboratory condition, cultivate the optimization test of the factor, the cultivation factor is: sodium acetate, yeast extract paste, micro-mother liquor, chelated iron mother liquor.Test design adopts 4 factors, 5 levels; 5 processing are numbered 1,2,3,4,5.In the 500ml reagent bottle, seawater and other nutritive ingredients that dress 350ml handles through clorox, again according to test design, add other and cultivate the factor, every then bottle graft kind Rhodopseudomonas spheroides seed liquor 150ml, be placed under following 15 ℃～32 ℃ of the nature solar irradiation, anaerobism was cultivated more than 120 hours, measured the Rhodopseudomonas spheroides number.It is as shown in table 1 that test design, bacterium are counted measurement result, and test is organized in contrast not add the substratum of cultivating the factor.
Other nutritive ingredient substratum content prescription is composed as follows: the aseptic water containing chlorine ammonium of 1000ml 1.0g, sal epsom 0.2g, sodium bicarbonate 1.0g,, dipotassium hydrogen phosphate 0.2g.
Table 1. Rhodopseudomonas sphaeroides for sea cucumber culture medium prescription composite test
| Numbering | 1 | 2 | 3 | 4 | 5 | Control group |
| Sodium acetate (g/mL) | 1.0 | 2.0 | 3.0 | 4.0 | 5.0 | 0 |
| Yeast extract paste (g/mL) | 0.5 | 1.0 | 1.5 | 2.0 | 2.5 | 0 |
| Trace element mother liquor (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 |
| The bacterium number (hundred million/mL) | 10 | 13 | 12 | 10 | 8 | 3 |
Test-results shows that it is best of breed (being to contain sodium acetate 2.0g, yeast extract paste 1g, micro-mother liquor 1ml, chelated iron mother liquor 1ml in the 1000ml nutrient solution) that the add-on of cultivating the factor in 2 is handled in test.
When the present invention used, the composition substratum was chosen the order of cultivating the factor and is: Chen Haishui, sodium acetate, yeast extract paste, chelated iron, micro-mother liquor.
Embodiment 3
A kind of implementation result of Rhodopseudomonas sphaeroides for sea cucumber can follow these steps to operation:
(1) plants female activation: with embodiment 1 (1), (2);
(2) the liquid seeds one-level is cultivated inoculation and culture condition: with embodiment 1 (3);
(3) the liquid seeds secondary expands numerous cultivation inoculation and culture condition: with embodiment 1 (4);
(3) liquid is produced inoculation and culture condition in enormous quantities: with embodiment 1 (5);
With Rhodopseudomonas sphaeroides for sea cucumber, by above-mentioned steps expand numerous after, 500ml/666.7m is pressed in viable count 〉=2,000,000,000/ml
2Consumption, finely dispersed splashing to 50 * 666.7m
2In (depth of water 2-2.5m) holothruian cultures pond, through breeding in 20 days, it is as shown in table 2 below that its technical indicator can reach:
The implementation result of table 2. Rhodopseudomonas sphaeroides for sea cucumber
| Test is handled | Photosynthetic bacteria hundred million/ml | Water temperature ℃ | The PH value | Salinity ‰ | Dissolved oxygen mg/L | COD mg/L | BOD mg/L | Ammonium salt mg/L |
| Before the use | 0 | 19.6 | 8.2 | 34 | 2.50 | 6.84 | 7.12 | 0.079 |
| After the use | 0.2 | 21.6 | 8.4 | 35 | 5.50 | 2.10 | 3.20 | 0.039 |
Duration of service: on May 8th, 2006; Analysis time: on May 28th, 2006
From Measurement results as can be seen: after using Rhodopseudomonas spheroides, in the holothruian cultures pond, dissolved oxygen improves, and ammonium salt descends to some extent, and item indexs such as COD, BOD value and pH value reach normal.
The all commercially available acquisition of the Rhodopseudomonas spheroides kind mother who is adopted in the embodiment of the invention there is no particular requirement to it.
The applicant has adopted the Rhodopseudomonas sphaeroides for sea cucumber (Rhodpseudomonas sphaeroides RSY96) of Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences's separation, purifying, evaluation, in the production practice of Dalian Wu Dao precious marine product company cultivation base, tame, purify, obtain the Rhodopseudomonas sphaeroides for sea cucumber of high density.Its effect is very good, the coastland widespread uses such as geographic Lvshun, Changxing Island, interchange island in Dalian, and can realize the commercialization production of product.
High density Rhodopseudomonas sphaeroides for sea cucumber gramstaining through domestication, purification is negative, and no gemma can be than good utilisation H
2S examines under a microscope, and cellular form is: ovoid or sphere, ossphere shape diameter are 1.0 * 15 or 10 * 1.1 μ m.Extremely give birth to single flagellum or two flagellum, the photosynthetic membrane system is arranged.The anaerobism illumination cultivation has photosynthetic membrane to generate, and is aerobic, and unglazed photograph is cultivated and do not generated photosynthetic membrane.Observe on the plate substratum, colonial morphology is: circular protrusions or flat, and colony diameter is 2.0-5.0mm, and neat in edge is regular, and surface wettability is smooth, opaque.Color is pink or red.Rhodopseudomonas sphaeroides for sea cucumber can make the sea cucumber surviving rate improve 20% in the sea cucumber middle application of growing seedlings; Join in the breed the children and to use, can accelerate the raise immunity of growing, disease resistance enhancemen, promoting digestion absorbs, can obviously increase sea cucumber body colour pool, as can be seen from the test results, Rhodopseudomonas sphaeroides for sea cucumber is to sea cucumber metamorphosis, children ginseng and become to join to culture all very ideal effect of tool, is subjected to the welcome at local holothruian cultures family.
Claims (5)
1. the cultural method of a Rhodopseudomonas sphaeroides for sea cucumber is characterized in that, comprises the steps:
1) actication of culture: will be inoculated in the liquid nutrient medium through activatory Rhodopseudomonas spheroides slant strains, and, obtain Rhodopseudomonas spheroides seed mother liquor through cultivating;
2) first order seed is cultivated: the seed mother liquor that obtains in the step 1) is inoculated in the first order seed substratum, and the weight percent of seed mother liquor and first order seed nutrient solution is 20-25: 75-80, obtains primary seed solution through cultivation;
3) secondary expands numerous cultivation: with step 2) in the primary seed solution that obtains be inoculated into secondary and expand in the breeding culture medium, primary seed solution is 20-25: 75-80 with the weight percent that expands numerous liquid medium, obtains secondary seed solution through cultivation;
4) three grades of property produced in enormous quantities cultivation: the secondary seed solution that obtains in the step 3) is inoculated in the three grades of property produced in enormous quantities substratum, and the weight percent of secondary seed solution and the three grades of property produced in enormous quantities nutrient solutions is 30-35: 65-70; Through cultivating, promptly obtain liquidly, be pink colour, redness and red-purple, the thalline size is 1.0～1.5 μ m or 1.0～1.1 μ m, contains the bacterium liquid of the Rhodopseudomonas sphaeroides for sea cucumber of viable count 〉=2,000,000,000/ml;
The substratum composition that above-mentioned Rhodopseudomonas spheroides culturing process is adopted is: ammonium chloride 1.0-3.0g, sodium acetate 2.0-5.0g, sal epsom 0.2-0.3g, sodium bicarbonate 1.0-3.0g, dipotassium hydrogen phosphate 0.2-0.5g, yeast extract paste 1.0-3.0g, micro-mother liquor 1-3ml, Chen Haishui 1000ml, wherein contain chelated iron 5-10ppm, pH value nature;
Culture condition is: anaerobism, illumination 1000-10000 Lux, cultivated 6-8 days;
Described old seawater is that the shady and cool place of natural sea-water placement stores 1-2 week, treats that the contamination precipitation after-filtration forms, and salinity is 2.0-3.0%.
2. according to the described cultural method of claim 1, it is characterized in that: described substratum composition is, ammonium chloride 1.0g, sodium acetate 3.0g, sal epsom 0.2g, sodium bicarbonate 2.0g, dipotassium hydrogen phosphate 0.2g, yeast extract paste 1.0g, micro-mother liquor 1ml, Chen Haishui 1000ml wherein contain chelated iron 5ppm, pH value nature.
3. according to the described cultural method of claim 1, it is characterized in that: the process for preparation of described chelated iron mother liquor is, in the 500ml deionized water, add disodium ethylene diamine tetraacetate 13.1g, ferrous sulfate 12.5g, 1 normal potassium hydroxide 134ml, under alkaline condition, carry out ventilatory response, the back liquid that reacts completely is red-brown, and pH value is transferred to 5.5, is the chelated iron mother liquor; If add chelated iron mother liquor 0.2ml in every liter of solution, the chelated iron concentration in the solution is 1ppm.
4. according to the described cultural method of claim 1, it is characterized in that: described micro-mother liquor consists of, boric acid 1.40g, manganous sulfate 0.14g, ammonium molybdate 0.26g, zinc sulfate 0.06g, copper sulfate 0.20g, cobalt chloride 0.01g, deionized water 1000ml.
5. according to the described cultural method of claim 1, it is characterized in that: described step 1) and 2) substratum that is adopted and nutrient solution are through autoclave sterilization, and the inoculation back belongs to pure culture; Step 3) and 4) nutrient solution that is adopted is chemosterilization, and Chen Haishui adopts clorox and Sulfothiorine neutralisation to handle; The capacity of Chen Haishui, clorox and Sulfothiorine and part by weight are: 1t: 100ml: 8-10g; Be specially: it is 8% clorox 100ml that 1 ton of Chen Haishui adds content, fully react 6-8 hour after, with the neutralization of 8-10g Sulfothiorine, the potassiumiodide indicator reaction is colourless, adding other nutrition again is exactly secondary or three grades of liquid nutrient mediums.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100122884A CN101353631A (en) | 2007-07-27 | 2007-07-27 | Cultivation method of Rhodopseudomonas sphaeroides for sea cucumber |
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| CN101935632A (en) * | 2010-06-29 | 2011-01-05 | 东华大学 | Strain for degrading wool scale layer |
| CN101735968B (en) * | 2009-12-29 | 2011-12-28 | 中国科学院南海海洋研究所 | Marine bacteria chromogenic isolation medium and application thereof |
| CN103004740A (en) * | 2012-11-29 | 2013-04-03 | 大连金砣水产食品有限公司 | Method for removing harmful dinoflagellates in chrysophyte bait cultivating pool |
| CN103289913A (en) * | 2013-01-27 | 2013-09-11 | 大连海宝渔业有限公司 | Apostichopus japonicus Pseudomonas elyacovii HS1 strain and its use |
| CN103284029A (en) * | 2013-06-14 | 2013-09-11 | 广州格拉姆生物科技有限公司 | Selenium enriched rhodopseudomonas palustris preparation and preparation method thereof |
| CN103609910A (en) * | 2013-12-04 | 2014-03-05 | 领绿生物镇江有限公司 | Ecological preparation used for sea cucumber breeding and preparation method thereof |
| CN105075948A (en) * | 2015-08-27 | 2015-11-25 | 张家港鸿蚓生物科技有限公司 | Cultivation method of benthonic animals |
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| CN101735968B (en) * | 2009-12-29 | 2011-12-28 | 中国科学院南海海洋研究所 | Marine bacteria chromogenic isolation medium and application thereof |
| CN101935632A (en) * | 2010-06-29 | 2011-01-05 | 东华大学 | Strain for degrading wool scale layer |
| CN103004740A (en) * | 2012-11-29 | 2013-04-03 | 大连金砣水产食品有限公司 | Method for removing harmful dinoflagellates in chrysophyte bait cultivating pool |
| CN103289913A (en) * | 2013-01-27 | 2013-09-11 | 大连海宝渔业有限公司 | Apostichopus japonicus Pseudomonas elyacovii HS1 strain and its use |
| CN103289913B (en) * | 2013-01-27 | 2015-03-11 | 大连海宝渔业有限公司 | Apostichopus japonicus Pseudomonas elyacovii HS1 strain and its use |
| CN103284029A (en) * | 2013-06-14 | 2013-09-11 | 广州格拉姆生物科技有限公司 | Selenium enriched rhodopseudomonas palustris preparation and preparation method thereof |
| CN103609910A (en) * | 2013-12-04 | 2014-03-05 | 领绿生物镇江有限公司 | Ecological preparation used for sea cucumber breeding and preparation method thereof |
| CN105075948A (en) * | 2015-08-27 | 2015-11-25 | 张家港鸿蚓生物科技有限公司 | Cultivation method of benthonic animals |
| CN105075948B (en) * | 2015-08-27 | 2017-12-15 | 张家港鸿蚓生物科技有限公司 | A kind of cultural method of zoobenthos |
| CN109207388A (en) * | 2017-07-03 | 2019-01-15 | 浙江工业大学 | A kind of compound immobilization microbe stuffing preparation of glass fibre/high polymer and its application |
| CN109207388B (en) * | 2017-07-03 | 2020-11-13 | 浙江工业大学 | Preparation and application of glass fiber/high polymer composite immobilized microbial filler |
| CN109619269A (en) * | 2018-11-21 | 2019-04-16 | 浙江海洋大学 | Special feed for sea cucumber cultured in net cage |
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