CN102017914B - Energy-saving emission-reducing sea cucumber factory culture method - Google Patents
Energy-saving emission-reducing sea cucumber factory culture method Download PDFInfo
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Abstract
The invention discloses an energy-saving emission-reducing sea cucumber culture method. The method comprises culture water source selection, pool water treatment, feed treatment and water change cycle control. According to the culture water body ecological principle, the growth environment requirements of marine organisms, relationship among organisms, and a harmless culture criterion, a bioecological method is used for controlling culture environment. A key technology is to add Bdellovibrio leech plasmid bacterial liquid to water, meanwhile, the feed is soaked with Bdellovibrio leech plasmidbacterial liquid, and the pool water can be changed every 40 days during the culture period. The organism culture method which is green, safe and unpolluted and has obvious effects; the method can improve water quality, promote sea cucumbers to ingest food, increase immunity, promote growth and increase survival rate; the method has excellent development prospect, and has very important meaning for increasing economic benefit and edible safety and guaranteeing human health.
Description
Technical field
The invention belongs to holothurian cultural technique field, particularly a kind of raising sea cucumber survival rate and immunity promote that sea cucumber grows, improves water quality fast, saves water and energy, the nuisanceless breed new method of sea cucumber of green low-carbon.
Background technology
Sea cucumber (Apostichopus japonicus) belongs to Holothuroidea (Holothurioidea), is widely distributed in each ocean, the world.The same genseng of sea cucumber, bird's nest, shark's fin are equally celebrated for their achievements, are one of the world's eight big treasures.Sea cucumber is not only precious food, also is famous and precious medicinal material.In recent years, the increase year by year of sea cucumber consumption demand amount causes the excessive exploitation of sea cucumber natural resources and the rapid decline of population quantity.Therefore, propagating artificially thereupon of sea cucumber risen.
Along with the cumulative year after year of holothruian cultures, cultivation density is excessive, the deterioration of carrying capacity overload and breeding environment, and disease frequently breaks out, the putrid skin disease of mature stage commonly, swollen mouth disease etc.At present, the sea cucumber prevention and treatment of diseases is mainly handled by the antibiolics bath method.Antibiotic use repeatedly, the pesticide resistance of pathogenic microorganism increases, and can not tackle the problem at its root, and the state of an illness is shown effect repeatedly, has caused new difficulty to the raiser again.And excessive antibiotic and the use of chemicals cause the antibiotic residue in the sea cucumber body to exceed standard, and not only influence the growth of sea cucumber, also influence the marketing quality of sea cucumber simultaneously, and people's use is threatened safely.The use of other class chemical classes disinfectant is severe contamination water quality also, destroys environment.
Though Chinese herbal medicine has drug residue free, advantage that economic benefit is high, it is slower to take effect, and can't resolve epidemic disease in a short time
On the other hand, at present the aquaculture model of sea cucumber needs a large amount of water and ponds frequently changed, and has not only wasted human and material resources, and a large amount of waters causes the littoral zone seawater invasion easily, causes the soil salinization of soil, has caused harm to ecotope.According to method commonly used waste water is handled, recycled waste water, then need higher investment, increased energy resource consumption and aquaculture cost.
Thereby seek survival rate and the immunity that a kind of more efficient, more economical way improves sea cucumber, and promote the quick growth of sea cucumber, seem very urgent." green cultivation " not only overcome the antibiotic abuse with side effect, can also improve water quality, strengthen seed immunity, improve the survival rate of sea cucumber, promote the growth of sea cucumber.Become the great demand of country in recent years, cause the extensive concern of aquatic products circle.At present, the mode of carrying out holothruian cultures with leech plastid Bdellovibrio is not at home and abroad appeared in the newspapers as yet.
No matter existing studies show that as a kind of effective microorganism preparation, is in food industry or in fields such as (ocean) aquacultures, the application of Bdellovibrio all is safe.For example: abroad, Lenz and Hespell (1978) discovers, Bdellovibrio and animal and people's cell do not had an infectivity [Lenz R.W., Hespell R.B.Attempts to grow bedellovibrios micurgically-injected into animal cells.Archives of Microbiology, 1978,119 (3): 245-248].At home, Lin Mao etc. (2006) have studied the effect of Bdellovibrio to fish cell, find that it does not have dissemination [Lin Mao, Yang Xianle, Xue Hui, Cao Haipeng, Qiu Junqiang to the fish bacterium.The effect of the fish cell of Bdellovibrio BDH2102 and pathogen.The microbiology circular, 2006,33 (1): 7-11].
Summary of the invention
For overcoming the deficiency that exists in the above-mentioned existing cultural technique, the object of the present invention is to provide a kind of holothurian culture method of energy-saving and emission-reduction; This method realizes by add Bdellovibrio leech plastid in feed and water body, specifically comprises the breed choosing source, the processing of Chi Shui, the processing of feed, the control of exchange water cycle, its feature is learned principle according to breeding ecological, halobiontic growing environment requirement, correlation between the biology and nuisanceless breed standard are manually created breeding environment, reduce input cost, improve water quality, improve sea cucumber immunity, promote that sea cucumber grows fast, increase economic efficiency.
Purpose of the present invention realizes by following technical proposals: a kind of holothurian culture method of energy-saving and emission-reduction comprises following operating procedure:
(1) gets the free of contamination seawater in marine site as breeding water; Breeding water injects and cultures the pond through sand filtration, post precipitation; Culture the corrugated plating of placing in the pond through sterilization (apposition growth that is used for sea cucumber);
(2) in culturing the pond, add Bdellovibrio leech plastid bacterium liquid, and strengthen aeration, stir, make the concentration of the Bdellovibrio leech plastid of Chi Shuizhong reach 10～10
7Pfu/mL;
(3) throw in the sea cucumber seedling in the pond to culturing;
(4) culture during normal aeration, the control water temperature is at 15～30 ℃, the salinity of Chi Shui remains on 23 ‰～30 ‰, daily ration of feeding be the sea cucumber body weight mass percent 3～5%, bait is 10～10 with concentration before input
7The Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks 15～30min; Adding Bdellovibrio leech plastid bacterium liquid makes the Bdellovibrio leech plastid bacterial concentration of Chi Shuizhong reach 10～10 at every turn after changing Chi Shui (full pond) once, to change (full pond) water in 5～40 days
7Pfu/mL.
The described breed of step (1) pond cleaning and sterilizing before injecting breeding water is clean; 3m in the described breed pond
2～4m
2Arrange 1 gas exhausting stone (being used for inflation).
The method of the described adding in step (2) and (4) Bdellovibrio leech plastid bacterium liquid is: behind the Chi Shui dilution bacterium liquid of culturing in the pond, and the full pond of evenly splashing.
The aeration rate of the described increasing aeration of step (2) is 90～100L/h/m
3The aeration rate of the described normal aeration of step (4) is 70～80L/h/m
3
The described input of step (3) sea cucumber seedling when 15～30 ℃ of water temperatures according to following standard: when sea cucumber seedling specification was 400/kg, throwing in density was 600/m
2When sea cucumber seedling specification was 200/kg, input density was 400 tails/m
2When sea cucumber seedling specification was 100/kg, input density was 300 tails/m
2
The described bait of step (4) is manufactured feed, and every day, bait throwing in was twice; Soil pick-up at the bottom of 5～7 days employing siphonages are cultured the pond.
Step (2) and (4) described Bdellovibrio are Bdellovibrio (Bdellovibrio sp.Strain) BDM01, described Bdellovibrio is preserved in Chinese typical culture collection center in the Wuhan University of Luojia Mountain, Wuhan, Hubei Province on April 28th, 2008, and deposit number is CCTCC NO:M208066.BDM01 carries out ultraviolet mutagenesis by Bdellovibrio BDJ01 (deposit number is CCTCC M 208011) and obtains, it is carried out morphologic observation under electron microscope after the negative staining: Bdellovibrio BDM01 is unicellular, arc, and size is 1.7 * 1.0 μ m, end is given birth to flagellum, and flagellum length is 3.5 μ m; It is cultivated the transparent circular plaque that can form diameter 3-4mm in three days with the double-layer plate method in 28 ℃.
Described Bdellovibrio leech plastid bacterium liquid prepares in accordance with the following methods: inoculation 0.5mL concentration is 10
7Cfu/mL Aeromonas hydrophila (Aeromonas hydrophila, purchase in Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.172) in the nutrient broth liquid nutrient medium, cultivate 20h for 26～30 ℃, culture fluid is behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium after suspending with 1mL physiological saline, inserts 1mL again and contains 10
3The bacterium liquid of pfu/mL Bdellovibrio BDM01, cultivate 36～48h in 25～35 ℃, 150～300rpm, culture fluid under 4 ℃ behind the centrifugal 15～20min of 6000～8000rpm, removal contains the supernatant of Bdellovibrio telotroch, precipitation is the suspension of 7.2～7.6 phosphate buffer with DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
8The Bdellovibrio leech plastid bacterium liquid of pfu/ml;
Described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolysate 0.5g and yeast extract 0.1g are dissolved in the 1000ml distilled water, regulates pH value to 7.2～7.6.
Compared with prior art, the present invention has following beneficial effect:
Developing nuisanceless breed, improve survival rate and the immunity of sea cucumber, promote the quick growth of sea cucumber, reduce production costs, increase economic efficiency, is the road that must have of China's mariculture industry development.Bdellovibrio can be improved the gastrointestinal tract environment of sea cucumber, improves immunity, promotes growth.Existingly studies show that Bdellovibrio is to echinoderm class class free of toxic effects, to human no pathogenicity.
Can be divided into the leech plastid state that breaks away from host bacteria, has the telotroch state of flagellum, free swimming and in host bacteria, grow the history of life of Bdellovibrio.When the Bdellovibrio telotroch is invaded the host, produce a hole at the host cell wall, invade host's periplasmic space when Bdellovibrio after hole seal again.Telotroch loses flagellum and host cell is called as " leech plastid " together.
Compare with telotroch, the leech plastid not only has preparation, preservation is more convenient, and the environment tolerance is strong, though omit characteristics more of a specified duration slowly action time, also has potential inactivated vaccine function simultaneously.Behind telotroch Bdellovibrio invasion host cell, the respiration of host cell (respiration) is stopped very soon.Bdellovibrio also enters leech plastid state, begins to secrete plurality of enzymes, and the big molecule of clearing up host cell is small-molecule substance, and then is integrated into the nutriment of self, makes self growth, and is elongated.Finally, the leech plastid of strip is divided into the sections of a lot of weak points, grows flagellum, becomes telotroch again.Afterwards, the essential enzyme of they secretions, broken wall and going out becomes the telotroch that breaks away from host bacteria, has flagellum, free swimming again.Between this leech plastid puberty, Bdellovibrio does not change the surface texture of host cell, makes former host still have antigenic action, but the stimulating organism body produces immune response.This potential function makes uses leech plastid Bdellovibrio in holothruian cultures, can effectively prevent the generation of seedling bacteriosis, improves and cultures survival rate etc.
These advantages make the leech plastid be suitable as the biological cleaning factor of restraining or removing pathogenic bacteria in organism and the environment thereof equally.The leech plastid in the water body cause a disease or potentially pathogenic organism has stronger cracking strength, water quality is also had certain catharsis, soak feed with the leech plastid, and the certain density leech plastid bacterium liquid of splashing in the pool water within, the longlyest reach 40 days and do not change water.Can promote the growth of sea cucumber, improve immunity, can purify water again, reduce quantity of exchanged water and energy resource consumption, thereby overcome side effect that the antibiotic abuse brings and to breeding wastewater handle expensive, for the mankind provide safety, green, non-harmful aquatic products, improve raiser's income.
Embodiment
Do further detailed description below in conjunction with the present invention of embodiment, but embodiments of the present invention are not limited thereto.
Concrete enforcement of the present invention place is holothruian cultures field, Shandong, and the sea cucumber kind is imitative stichopus japonicus (Apostichopus japonicus).The enforcement time is 4 months.Experimental field the source quality in marine site meets State Standard of the People's Republic of China's water quality standard for fishery (GB11607-89) requirement, enter after the sand filtration of seawater process, the precipitation process and culture the pond, meet (NY5052-2001) requirement of Ministry of Agriculture's " pollution-free food mariculture water water quality ".The breed pond of sea cucumber is built in and is cultured in the booth, selects physical health, individual children's ginseng of uniform size, and the long 4～6cm of average body adopts the randomized block experiment design, according to 50～70/m
2Cultivation density drop in the pond.
Be equally divided into 4 experimental group of A-D, B, C, each experimental group of D have 8 groups, are respectively B1, C1, D1; B2, C2, D2; , B3, C3, D3; B4, C4, D4; B5, C5, D5; B6, C6, D6; B7, C7, D7; B8, C8, B8; The every group of B1-D8 is further divided into 4 groups, i.e. B1-1～B1-4 ... D8-1～D8-4, every group of 2 repetitions.Test processing is as follows:
A group: normal flowing water, the normal manufactured feed of throwing something and feeding (breeding way of plant produced)
B group: the Bdellovibrio leech plastid bacterium liquid of splashing in water makes that Bdellovibrio leech plastid concentration reaches 10,10 respectively in the water body
3, 10
5, 10
7Pfu/mL, the manufactured feed of throwing something and feeding;
The C group: the concentration of throwing something and feeding is 10,10
3, 10
5, 10
7Manufactured feed after the Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks;
D group: in water, add Bdellovibrio leech plastid bacterium liquid, make cell concentration reach 10,10 respectively
3, 10
5, 10
7Pfu/mL, the concentration of throwing something and feeding respectively simultaneously is 10,10
3, 10
5, 10
7Manufactured feed after the Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks;
B1, C1, D1 organize per 5 days and change (full pond) water once, add bacterium again after changing water;
B2, C2, D2 organize per 10 days and change (full pond) water once, add bacterium again after changing water;
B3, C3, D3 organize per 15 days and change (full pond) water once, add bacterium again after changing water;
B4, C4, D4 organize per 20 days and change (full pond) water once, add bacterium again after changing water;
B5, C5, D5 organize per 25 days and change (full pond) water once, add bacterium again after changing water;
B6, C6, D6 organize per 30 days and change (full pond) water once, add bacterium again after changing water;
B7, C7, D7 organize per 35 days and change (full pond) water once, add bacterium again after changing water;
B8, C8, D8 organize per 40 days and change (full pond) water once, add bacterium again after changing water;
B1-1, C1-1, D1-1 ... the bacterial concentration that D8-1 throws in is 10pfu/mL;
B1-2, C1-2, D1-2 ... the bacterial concentration that D8-2 throws in is 10
3Pfu/mL;
B1-3, C1-3, D1-3 ... the bacterial concentration that D8-3 throws in is 10
5Pfu/mL;
B1-4, C1-4, D1-4 ... the bacterial concentration that D8-4 throws in is 10
7Pfu/mL;
Feeds utilized People's Republic of China's agricultural industry criteria " pollution-free food fishing manufactured feed safety limit " regulation (NY5072-2002) that meets.Duration of test is thrown something and fed 2 every day, and morning, evening are respectively once.Growth indexes, immune indexes, water quality and the microorganism situation of sea cucumber measured in off-test.
1, the assay method of sea cucumber growth indexes is as follows:
Feed after 4 months, add up the sea cucumber number of surviving in every mouthful of pond, randomly draw 120 sea cucumbers respectively from every mouthful of pond, with its body of vernier caliper measurement long (mm), electronic balance is surveyed its weight (g), calculates following parameter:
(1) survival rate (%)=N
t/ N
o* 100%;
(2) rate of body weight gain (%)=(W
t-W
o) W
o* 100%;
(3) the long growth rate (%) of body=(L
t-L
o)/L
o* 100%;
N in the formula
oTotal number of sea cucumber during for on-test; N
tTotal number of sea cucumber during for off-test; W
oSea cucumber body weight during for on-test; W
tSea cucumber body weight during for off-test; L
oThe sea cucumber body is long during for on-test; L
tThe sea cucumber body is long during for off-test;
2, the mensuration of the immune indexes of sea cucumber
(1) alkaline phosphatase (AKP) and acid phosphatase (ACP) activity all adopt the disodium phenyl phosphate method of Kruzel (1982)
Blood is extracted in 1/3 puncture behind the outside of belly mouth of sea cucumber, places centrifuge tube, and in 4 ℃, the centrifugal 10min of 3000r/min gets supernatant, is the serum of sea cucumber.Place ice chest to preserve.
The mensuration of acid phosphatase (ACP) adopts the disodium phenyl phosphate method., produce 1mg phenol person and be defined as an enzyme activity unit at 37 ℃ and substrate disodium phenyl phosphate effect 60min with every 100mL serum.
The mensuration of alkaline phosphatase (AKP) adopts the disodium phenyl phosphate method., produce 1mg phenol person and be defined as an enzyme activity unit at 37 ℃ and substrate disodium phenyl phosphate effect 15min with every 100mL serum.
(2) infection experiment
Infection experiment is carried out in the pond that fills 1 ton of sand filtration seawater (salinity 29～30 ‰) of sterilization in advance.From experiment A group, B1-1, B1-2 ... D8-3, D8-4 in totally 97 groups each group get 100 of sea cucumbers at random, body length is 10 ± 1cm.Be 10 with asepsis injector through sea cucumber lumbar injection 300uL concentration
8Cfu/mL Aeromonas hydrophila (Aeromonas hydrophila purchases in Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.172) infects sea cucumber.The test water temperature remains on 15～30 ℃, and flowing water is cultured, and inflation is cultivated.Behind the artificial infection, the record sea cucumber motion, ingest and death condition.Observed 10 days continuously, finally be calculated to be motility rate, the employing relative survival rate (Relative Percentage Survival, RPS) calculating formula:
RPS (%)=(1-immune group lethality/control group lethality) * 100%
3, the mensuration of water quality
Ammoniacal nitrogen is received oxidizing process with hypobromous acid and is measured (GB12763.4-91);
Nitrite nitrogen diazonium-azo spectrphotometric method for measuring (GB12763.4-91)
4, the mensuration of vibrios sum
Detect the vibrios sum with the TCBS medium
Concrete steps are: with phosphate-buffered salt (PBS) institute's water sampling sample is carried out 10 dilutions, to 10
-4Get the dilution (10 of stoste and each concentration
-1, 10
-2, 10
-3, 10
-4) 0.5mL, to coat on the TCBS solid culture medium, the vibrios sum in the water is calculated in colony counting.
The measurement result of the growth indexes of sea cucumber, immunocompetence index and total bacteria count sees Table one～table four: as can be seen from the table, A forms that motility rate, rate of body weight gain, the long growth rate of body, acid phosphatase (ACP), alkaline phosphatase (AKP) are respectively 82.76%, 838.88%, 281.29%, 0.030U/mL, 0.033U/mL, and vibrios adds up to 6.89 * 10
2CFU/mL.Compare with the A group, C, D, the survival rate of E group, rate of body weight gain, the long growth rate of body, acid phosphatase (ACP), alkaline phosphatase (AKP) all obviously raise, and the vibrios sum obviously reduces.Explanation is under the breeding way of routine, and not changing water can influence the survival rate of sea cucumber, rate of body weight gain, body long growth rate, acid phosphatase and alkaline phosphatase activity.Under identical exchange water cycle, along with the concentration of Bdellovibrio leech plastid raises, survival rate, rate of body weight gain, the long growth rate of body, acid phosphatase and alkaline phosphatase activity are in gradually rising, and the vibrios sum obviously reduces; The concentration that Bdellovibrio leech plastid is described is more high, and the immunity of sea cucumber is more strong, and survival rate is more high, and Expectancy is more little.B1-1～B8-1 ... survival rate between D1-4～D8-4, rate of body weight gain, the long growth rate of body, acid phosphatase (ACP), alkaline phosphatase (AKP), vibrios sum be no significant difference almost, and illustrating in water or add concentration in feed is 10～10
7Pfu/mL leech plastid Bdellovibrio bacterium liquid, water once to change (full pond) in 5-40 days, the long growth rate of survival rate of seedling, rate of body weight gain, body, acid phosphatase (ACP), alkaline phosphatase (AKP), the influence of vibrios sum to sea cucumber are little, all can reach and improve survival rate and immunity, promote the purpose of growth.
The result of infection experiment shows, relative survival rate (the Relative Percentage Survival of A group, RPS) be 0, compare with the A group, the relative survival rate of B, C, D group all is significantly improved, as can be seen from Table V, under identical exchange water cycle, rising along with Bdellovibrio leech plastid bacterial concentration, relative survival rate raises gradually, and all apparently higher than the A group of plant produced, the cultural method that adopts novel interpolation Bdellovibrio leech plastid is described, can obviously improve the relative survival rate of sea cucumber, namely improve the immune resistance of sea cucumber; From different exchange water cycles, the sea cucumber relative survival rate of respectively organizing under the same concentrations does not change significantly.Illustrate in the novel cultural method that do not change water in 5-40 days, Bdellovibrio leech plastid bacterium liquid can both strengthen the immunocompetence of sea cucumber.With regard to B, C, three groups of different disposal modes of D, all can improve the relative survival rate of sea cucumber, and in water body and feed, add best results simultaneously.
Therefore, no matter still add certain density Bdellovibrio leech plastid bacterium liquid in the feed in water, reach 40 days and do not change water, all can improve the immune resistance of sea cucumber, add simultaneously in water with in the feed, effect is best.Thereby show that further Bdellovibrio leech plastid has potential vaccine effect, can stimulate sea cucumber to produce immune response, resist the infringement of Aeromonas, help to improve the survival rate of sea cucumber.
Through measuring, the every water-quality determination value in the A group pond all is higher than experiment group B, C, D organize.Wherein, the testing result of A group ammoniacal nitrogen is 0.286mg/L, is higher than the 0.095～0.198mg/L of 0.127～0.232mg/L, D group of 0.124～0.211mg/L, the C group of B group; The testing result of the nitrite nitrogen of A group is 0.078mg/L, is higher than the 0.019～0.036mg/L of 0.031～0.062mg/L, D group of 0.024～0.055mg/L, the C group of B group.By above interpretation of result as can be known, using concentration is 10-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid can effectively improve the breeding water body environment, regulates the content of ammoniacal nitrogen, nitrite nitrogen in the water, thereby provides the water body environment that is beneficial to sea cucumber existence, and adds best results in water body and feed simultaneously.
This shows no matter adding concentration in water or in feed is 10～10
7The Bdellovibrio leech plastid bacterium liquid of pfu/mL changed (full pond) water in 40 days and once can both reach the immunity and the survival rate that improve sea cucumber, promoted growth, and can control amount of vibrio in the water, reduced the content of ammoniacal nitrogen, nitrite nitrogen in the water, improved water quality.Add simultaneously in water and in the feed, effect is better.Can either save groundwater resources, the protection environment can be saved cost again, increases economic efficiency, and promotes the fast development of aquaculture.
The mensuration of the growth indexes of table one sea cucumber, immune indexes and vibrios sum
The mensuration of the growth indexes of table two sea cucumber, immune indexes and vibrios sum
The mensuration of the growth indexes of table three sea cucumber, immune indexes and vibrios sum
The mensuration of growth indexes, immune indexes and the vibrios sum of table four seas ginseng
Table five is attacked malicious infection experiment relative survival rate
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spiritual essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. the holothurian culture method of energy-saving and emission-reduction is characterized in that comprising following operating procedure:
(1) gets the free of contamination seawater in marine site as breeding water; Breeding water injects and cultures the pond through sand filtration, post precipitation; Culture the corrugated plating of placing in the pond through sterilization;
(2) in culturing the pond, add Bdellovibrio leech plastid bacterium liquid, and strengthen aeration, stir, make the concentration of the Bdellovibrio leech plastid of Chi Shuizhong reach 10～10
7Pfu/mL;
(3) throw in the sea cucumber seedling in the pond to culturing;
(4) culture during normal aeration, the control water temperature is at 15～30 ℃, the salinity of Chi Shui remains on 23 ‰～30 ‰, daily ration of feeding be the sea cucumber body weight mass percent 3～5%, bait is 10～10 with concentration before input
7The Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks 15～30min; Changed Chi Shui once every 5～40 days, change at every turn and add Bdellovibrio leech plastid bacterium liquid behind the water, make the Bdellovibrio leech plastid concentration of Chi Shuizhong reach 10～10
7Pfu/mL;
The described input of step (3) sea cucumber seedling when 15～30 ℃ of water temperatures according to following standard: when sea cucumber seedling specification was 400/kg, throwing in density was 600/m
2When sea cucumber seedling specification was 200/kg, input density was 400 tails/m
2When sea cucumber seedling specification was 100/kg, input density was 300 tails/m
2
Step (2) and (4) described Bdellovibrio leech plastid bacterium liquid prepare in accordance with the following methods: inoculation 0.5mL concentration is 10
7The cfu/mL Aeromonas hydrophila is cultivated 20h for 26～30 ℃ in the nutrient broth liquid nutrient medium, culture fluid is behind the centrifugal 10～20min of 5000～8000rpm, and precipitation is added in the DNB liquid nutrient medium after suspending with 1mL physiological saline, inserts 1mL again and contains 10
3The bacterium liquid of pfu/mL Bdellovibrio BDM01, cultivate 36～48 h in 25～35 ℃, 150～300 rpm, culture fluid under 4 ℃ behind the centrifugal 15～20min of 6000～8000rpm, removal contains the supernatant of Bdellovibrio telotroch, precipitation is the suspension of 7.2～7.6 phosphate buffer with DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
8The Bdellovibrio leech plastid bacterium liquid of cfu/mL; Described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolysate 0.5g and yeast extract 0.1g are dissolved in the 1000ml distilled water, regulates pH value to 7.2～7.6;
Described Bdellovibrio BDM01 is preserved in Chinese typical culture collection center on April 28th, 2008, and deposit number is CCTCC NO:M208066.
2. the holothurian culture method of a kind of energy-saving and emission-reduction according to claim 1 is characterized in that: the described breed of step (1) pond cleaning and sterilizing before injecting breeding water is clean; 3m in the described breed pond
2～4m
2Arrange 1 gas exhausting stone.
3. the holothurian culture method of a kind of energy-saving and emission-reduction according to claim 1 is characterized in that: the method for the described adding in step (2) and (4) Bdellovibrio leech plastid bacterium liquid is: behind the Chi Shui dilution bacterium liquid of culturing in the pond, and the full pond of evenly splashing.
4. the holothurian culture method of a kind of energy-saving and emission-reduction according to claim 1, it is characterized in that: the aeration rate of the described increasing aeration of step (2) is 90～100L/h/m
3The aeration rate of the described normal aeration of step (4) is 70～80L/h/m
3
5. the holothurian culture method of a kind of energy-saving and emission-reduction according to claim 1, it is characterized in that: the described bait of step (4) is manufactured feed, and every day, bait throwing in was twice; Soil pick-up at the bottom of 5～7 days employing siphonages are cultured the pond.
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| CN101082038A (en) * | 2006-05-31 | 2007-12-05 | 上海泓宝绿色水产科技发展有限公司 | Preparation of holothurian-benefit bacterium and method for restoring sea cucumber cultivation pool environment |
| CN101275119A (en) * | 2008-01-24 | 2008-10-01 | 华南理工大学 | Application of bdellovibrio to eliminating pathogen in freshwater product and culture water thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101082038A (en) * | 2006-05-31 | 2007-12-05 | 上海泓宝绿色水产科技发展有限公司 | Preparation of holothurian-benefit bacterium and method for restoring sea cucumber cultivation pool environment |
| CN101275119A (en) * | 2008-01-24 | 2008-10-01 | 华南理工大学 | Application of bdellovibrio to eliminating pathogen in freshwater product and culture water thereof |
Non-Patent Citations (3)
| Title |
|---|
| 吴湛辉 等.海参健康苗种培育和生态养殖关键技术.《河北渔业》.2010,(第5期),第23-26页. * |
| 黄华伟 等.噬菌蛭弧菌——养殖刺参疾病生物防治的新工具.《河北渔业》.2007,(第1期),第37-38页. * |
| 黄华伟 等.蛭弧菌的生物学特性及其在海参养殖中的应用前景.《齐鲁渔业》.2007,第24卷(第3期),第1-3页. * |
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