CN102027883B - Energy-saving type sea cucumber culturing method - Google Patents
Energy-saving type sea cucumber culturing method Download PDFInfo
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Abstract
The invention discloses an energy-saving type sea cucumber culturing method which comprises the steps of: selecting a culturing water source, processing pool water, processing feed, controlling a water exchanging period, and controlling the culture environment by adopting a bioecology method according to the culturing water body ecological principle, the growth environment requirements on sea organisms, correlations among the organisms and non-polluted culture standard of the organisms. The energy-saving type sea cucumber culturing method has the key technology that bdellovibrio bacteriovorus nectophore and bdelloplast mixed bacteria liquid is added in the water body, the feed is soaked with the bdellovibrio bacteriovorus nectophore and bdelloplast mixed bacteria liquid, and the pool water can be replaced in 40 days once during the culture. The energy-saving type sea cucumber culturing method is a biological culturing method, is green and safe without pollution, has remarkable effect, can improve the water quality, promotes the sea cucumber ingestion, improves the immunity, promotes the growth, and increases the success rate, thereby having better development prospect. The invention has important significance on improving the economic benefit and the eating safety and guaranteeing the human health.
Description
Technical field
The invention belongs to holothurian cultural technique field, particularly a kind of raising sea cucumber immunity promotes the sea cucumber Fast Growth, improves water quality, saves water and energy the sea cucumber non-nuisance method of green low-carbon.
Background technology
Whole world sea cucumber has kind more than 1200 approximately, is the ocean kind, can be edible have more than 40 to plant.There are 140 kinds of sea cucumbers in China, and about more than 20 kinds are edible.The protein content of sea cucumber is high, and does not contain carbohydrate and cholesterol, and nutritive value is high.In addition, the sweet temperature of the sea cucumber property of medicine has the effect of " dissolving phlegm saliva, impotence is treated in establishing-Yang for kidney tonifying warp, beneficial marrow ", and the good reputation of " marine ginseng " is arranged, and has unique medical value.Along with the raising of people's living standard and the enhancing of health care consciousness, sea cucumber consumption heats up gradually, its price also rises steadily, the propagating artificially at the coastal again new mariculture tide that formed after sea-tangle, prawn, scallop, fish of northern China of sea cucumber.
Continuous intensification along with the culture fishery intensive degree, add the shortage science management and normalized operation, cultural method falls behind, cause fulminant disease frequent occurrence, multiple obvious illness and the extensive phenomena of mortality occur, seriously restricted continuing, stablize, developing in a healthy way of holothruian cultures industries.
For many years, people mainly adopt antibiotic that its illness is prevented and treat, but antibiotic a large amount of use can cause chemical sproof generation, make the healing difficulty of disease increasing.Simultaneously, also may produce medicament residue and affect product quality and human health.The use of other chemical classes disinfectant also can severe contamination water quality, welding.
Although Chinese herbal medicine can have drug residue free, advantage that economic benefit is high, it is slower to take effect, and can't resolve in a short time epidemic disease.
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 saline Land, has caused harm to ecotope.According to method commonly used waste water is carried out cycle for the treatment of and use, then need higher investment, increased aquaculture cost.
Thereby seek survival rate and the immunity that a kind of more efficient, more economical way improves sea cucumber, seem very urgent." green cultivation " not only overcome the abuse of antibiotics band side effect, can also improve water quality, strengthen seed immunity, improve survival rate and the quality of sea cucumber, become the in recent years great demand of country, cause the extensive concern of aquatic products circle.The mode of at present, carrying out holothruian cultures with the mixed bacteria liquid of telotroch and Bdellovibrio nectophore and leech plastid there is not yet report at home and abroad.
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) studies discovery, 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.Bdellovibrio BDH2102 is to the effect of fish cell 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 energy-saving sea cucumber (Apostichopus japonicus) cultural method; The method realizes by the mixed bacteria liquid that adds Bdellovibrio nectophore and leech plastid in feed and water body, specifically comprises the cultivation choosing source, the processing of Chi Shui, the processing of feed, the control of exchange water cycle, its feature is according to the cultural ecology principle, halobiontic growing environment requirement, correlation between the biology and non-nuisance standard are manually created breeding environment, reduce input cost, improve water quality, improve sea cucumber immunity, promote the sea cucumber Fast Growth, increase economic efficiency.
Purpose of the present invention realizes by following technical proposals: a kind of energy-saving type sea cucumber culturing comprises following operating procedure:
(1) gets free of contamination deep-sea, marine site water as breeding water; Breeding water injects culturing pool after sand filtration, precipitation and sterilization; Place the corrugated plating (apposition growth that is used for sea cucumber) through sterilization in the culturing pool;
(2) in culturing pool, add the mixture bacterium liquid of Bdellovibrio nectophore and leech plastid, and strengthen aeration, stir, make the concentration of the mixture bacterium liquid of the Bdellovibrio nectophore of Chi Shuizhong and leech plastid reach 10～10
7Pfu/mL;
(3) in culturing pool, throw in sea cucumbers;
(4) normal aeration during the cultivation, the control water temperature is at 15～30 ℃, and the salinity of Chi Shui remains on 23 ‰～30 ‰, and daily ration of feeding is the mass percent 3～5% of sea cucumber body weight, and bait is 10～10 with concentration before input
7The mixture bacterium liquid of the Bdellovibrio nectophore of pfu/mL and leech plastid soaks 15～30min; Changed the mixture bacterium liquid that adds Bdellovibrio nectophore and leech plastid after Chi Shui (full pond) once, changes (full pond) water at every turn every 5～40 days, make the Bdellovibrio nectophore of Chi Shuizhong and the mixture concentration of leech plastid reach 10～10
7Pfu/mL.
The described culturing pool of step (1) cleaning and sterilizing before injecting breeding water is clean; 3m in the described culturing pool
2～4m
2Arrange 1 gas exhausting stone (being used for inflation).
The method of the mixture bacterium liquid of the described adding Bdellovibrio nectophore in step (2) and (4) and leech plastid is: behind the dilution of the Chi Shui in culturing pool bacterium liquid, 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 sea cucumbers of step (3) when 15～30 ℃ of water temperatures according to following standard: when the sea cucumbers specification was 400/kg, throwing in density was 600/m
2When the sea cucumbers specification was 200/kg, input density was 400 tails/m
2When the sea cucumbers specification was 100/kg, input density was 300 tails/m
2
The described bait of step (4) is mixed bait, and every day, bait throwing in was twice; Every 5～7 days, adopt the soil pick-up at the bottom of siphonage carries out culturing pool.
Step (2) and (4) described Bdellovibrio are Bdellovibrio (Bdellovibrio sp.Strain) BDM01, described Bdellovibrio is preserved in Chinese Typical Representative culture collection center in the Wuhan University of Luojia Mountain, Wuhan, Hubei Province on April 28th, 2008, and deposit number is CCTCC NO:M 208066.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 ℃.
Step (2) and (4) described mixture bacterium liquid are that Bdellovibrio nectophore bacterium liquid and bdellovibrio bdelloplast bacterial bacterium liquid mix by the bacterium number at 1: 1.
Described bdellovibrio bdelloplast bacterial bacterium liquid prepares in accordance with the following methods: inoculation 0.5mL concentration is 10
7Cfu/mL Aeromonas hydrophila (Aeromonas hydrophila, be purchased from 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, accesses 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 nectophore, 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
9The bdellovibrio bdelloplast bacterial bacterium liquid of pfu/mL;
Described Bdellovibrio nectophore bacterium liquid prepares in accordance with the following methods: with the supernatant that contains Bdellovibrio nectophore among the bdellovibrio bdelloplast bacterial preparation method behind the centrifugal 15～20min of 16000～18000rpm, 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
9The Bdellovibrio nectophore 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:
(1) 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 Bdellovibrio nectophore is invaded the host, produce a hole at the host cell wall, invade host's periplasmic space when Bdellovibrio after hole again seal.Telotroch loses flagellum, and host cell is called as " leech plastid " together.Next, the hydrolysis of host's periplasmic space, the Bdellovibrio cell is elongated, and splits into a plurality of short segments, grows flagellum, spins off from host cell by discharging the dissolubility enzyme, again forms telotroch.
Compare with telotroch, the leech plastid not only has preparation, it is more convenient to preserve, and the environment tolerance is strong, though action time slightly slow characteristics more of a specified duration, also have simultaneously potential inactivated vaccine function.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 large 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, again becomes telotroch.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.During this leech development of plastid, 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 improves cultivation survival rate etc. so that application leech plastid Bdellovibrio in holothruian cultures, can effectively prevent the generation of seedling bacteriosis.
The mixed bacteria liquid of Bdellovibrio nectophore of the present invention and leech plastid Bdellovibrio can play the effect of the vaccine of deactivation with the common advantage of telotroch and leech plastid, and effect is better.
(2) the present invention adopts the scheme that nutrition preparation, the prevention of Bdellovibrio mixed bacteria liquid and adjusting, environment and feed regulation and control combine, reach the survival rate and the immunity that improve sea cucumber, promoted ingesting and Fast Growth of sea cucumber, improved water quality, reduce production costs the effect of increasing economic efficiency.
(3) mixed bacteria liquid of Bdellovibrio nectophore of the present invention and the leech plastid energy most aquatic pathogenic bacteria of cracking or potential pathogenic bacteria, soak feed with the Bdellovibrio mixed bacteria liquid, and the certain density Bdellovibrio mixed bacteria liquid of splashing in the pool water within, can promote the growth of sea cucumber, improve immunity, can purify water again, the longlyest reach 40 days and do not change water.Thereby overcome side effect that abuse of antibiotics brings and to breeding wastewater process expensive, for the mankind provide safety, green, non-harmful aquatic products, improve raiser's income.
(4) Bdellovibrio nectophore of the present invention and the safety in the application of sea cucumber of leech plastid mixed bacteria liquid are good; The method that Bdellovibrio is eliminated pathogenic bacteria by cracking is biological method, and is green, safe, pollution-free.Bdellovibrio can be infected, the characteristic of cracking host bacteria makes it to be suitable as the biological cleaning factor of restraining or removing pathogenic bacteria in organism and the environment thereof, and it is behind the complete Host Strains of cracking, can automatically wither away because of hungry, existingly studies show that Bdellovibrio is to echinoderm class free of toxic effects, to human no pathogenicity.
(5) Bdellovibrio nectophore of the present invention and leech plastid mixture action effect in the application of sea cucumber are good, and are widely used, and extend to other industries; Promote that with Bdellovibrio the growth of sea cucumber is a kind of promising cultivation new method.
Embodiment
Below in conjunction with embodiment the present invention is done further detailed description, but embodiments of the present invention are not limited to this.
Implementation 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 culturing pool after the sand filtration of seawater process, the precipitation process, meet (NY5052-2001) requirement of Ministry of Agriculture's " pollution-free food mariculture water water quality ".The culturing pool of sea cucumber is built in the cultivation greenhouse, selects uniformly children's ginseng of physical health, Individual Size, and the long 4～6cm of average body adopts the randomized block experiment design, according to 55～70/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.The processing of test is as follows:
A group: normal flowing water, the manufactured feed feed of throwing something and feeding (breeding way of plant produced);
B group: the mixed bacteria liquid of splash in water Bdellovibrio nectophore and leech plastid makes that Bdellovibrio nectophore and leech plastid mixed bacteria liquid concentration reach respectively 10,10 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 nectophore of pfu/mL and leech plastid mixed bacteria liquid soak;
D group: in water, add Bdellovibrio nectophore and leech plastid mixed bacteria liquid, make cell concentration reach respectively 10,10
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 nectophore of pfu/mL and leech plastid mixed bacteria liquid soak;
B1, C1, D1 organize per 5 days and change (full pond) water once, again add bacterium after changing water;
B2, C2, D2 organize per 10 days and change (full pond) water once, again add bacterium after changing water;
B3, C3, D3 organize per 15 days and change (full pond) water once, again add bacterium after changing water;
B4, C4, D4 organize per 20 days and change (full pond) water once, again add bacterium after changing water;
B5, C5, D5 organize per 25 days and change (full pond) water once, again add bacterium after changing water;
B6, C6, D6 organize per 30 days and change (full pond) water once, again add bacterium after changing water;
B7, C7, D7 organize per 35 days and change (full pond) water once, again add bacterium after changing water;
B8, C8, D8 organize per 40 days and change (full pond) water once, again add bacterium 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;
B 1-3, C1-3, D1-3 ... the bacterial concentration that D8-3 throws in is 10
5Pfu/mL;
B 1-4, 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, early, the evening each once.Growth indexes, immune indexes and the microorganism situation of sea cucumber measured in off-test.
1, the assay method of sea cucumber growth indexes is as follows:
After going through 4 months, add up the sea cucumber number of surviving in final every mouthful of pond, randomly draw respectively 100 sea cucumbers 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 is carried out in the pond that fills 1 ton of sand filtration seawater (salinity 29～30 ‰) of in advance sterilization.From experiment A group, B1-1, B1-2 ... D8-3, D8-4 in totally 97 groups each group get at random 100 of sea cucumbers, body length is 10 ± 1cm.Be 10 with asepsis injector through sea cucumber lumbar injection 300uL concentration
8Cfu/mL Aeromonas hydrophila (Aeromonas hydrophila is purchased from Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.172) infects sea cucumber.The test water temperature remains on 15～30 ℃, flowing water culture, and inflation is cultivated.Behind the artificial infection, the record sea cucumber motion, ingest and death condition.Continuous Observation 10 days finally is calculated to be motility rate, adopts 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 water quality situation 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 83.25%, 843.78%, 282.89%, 0.033U/mL, 0.037U/mL, and vibrios adds up to 5.90 * 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 affect 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 mixed bacteria liquid 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 bdelloplast bacterial is described is higher, and the immunity of sea cucumber is stronger, and survival rate is higher, and Expectancy is less.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,
Concentration is added in explanation in water or in feed be 10～10
7Pfu/mL Bdellovibrio nectophore and leech plastid mixed bacteria 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 impact of vibrios sum on 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 mixed bacteria liquid 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 mixed bacteria liquid is described, can obviously improve the relative survival rate of sea cucumber, namely improve the Immunoresistance 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 cultivation method that do not change water in 5-40 days, bdellovibrio sp. mixture bacterium solution can both strengthen the immunocompetence of sea cucumber.With regard to B, C, three groups of Different treatments of D, all can improve the relative survival rate of sea cucumber, and in water body and feed, add simultaneously best results.
Therefore, no matter add certain density bdellovibrio bdelloplast bacterial bacterium liquid in water or in the feed, reach 40 days and do not change water, all can improve the Immunoresistance of sea cucumber, add simultaneously in water with in the feed, effect is best.Thereby show that further bdellovibrio mixture also 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 0.288mg/L, is higher than the 0.093～0.197mg/L of 0.128～0.231mg/L, D group of 0.123～0.212mg/L, the C group of B group; The testing result of the nitrite nitrogen of A group is 0.079mg/L, is higher than the 0.015～0.031mg/L of 0.029～0.060mg/L, D group of 0.023～0.053mg/L, the C group of B group.By above interpretation of result as can be known, using concentration is 10-10
7Pfu/mL Bdellovibrio nectophore and leech plastid mixed bacteria liquid can effectively improve the breeding water body environment, regulate the content of ammoniacal nitrogen, nitrite nitrogen in the water, thereby provide the water body environment that is beneficial to sea cucumber existence, and in water body and feed, add simultaneously best results.
This shows no matter adding concentration in water or in feed is 10～10
7The mixture bacterium liquid of the Bdellovibrio Bdellovibrio nectophore of pfu/mL and leech plastid changed (full pond) water and once can both reach the immunity and the survival rate that improve sea cucumber in 40 days, promote growth, and can control bacterial number in the water, regulate the content of ammoniacal nitrogen, nitrite nitrogen in the water, improve water quality.Add simultaneously in water and in the feed, effect is better.Namely can descend frugally water resource, protection of the environment can reduce energy resource consumption again, saves cost, increases economic efficiency, and promotes the fast development of aquaculture.
The mensuration of the growth indexes of table one sea cucumber, immune indexes and water quality
The mensuration of the growth indexes of table two sea cucumber, immune indexes and water quality
The mensuration of the growth indexes of table three sea cucumber, immune indexes and water quality
The mensuration of growth indexes, immune indexes and the water quality of table four seas ginseng
Table five is attacked malicious infection experiment relative survival rate
Above-described embodiment is the better embodiment 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 Spirit 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 (2)
1. energy-saving type sea cucumber culturing is characterized in that comprising following operating procedure:
(1) gets free of contamination deep-sea, marine site water as breeding water; Breeding water injects culturing pool after sand filtration, precipitation and sterilization; Place the corrugated plating through sterilization in the culturing pool;
(2) in culturing pool, add the mixture bacterium liquid of Bdellovibrio nectophore and leech plastid, and strengthen aeration, stir, make the concentration of the mixture of the Bdellovibrio nectophore of Chi Shuizhong and leech plastid reach 10～10
7Pfu/mL;
(3) in culturing pool, throw in sea cucumbers;
(4) normal aeration during the cultivation, the control water temperature is at 15～30 ℃, and the salinity of Chi Shui remains on 23 ‰～30 ‰, and daily ration of feeding is the mass percent 3～5% of sea cucumber body weight, and bait is 10～10 with concentration before input
7The mixture bacterium liquid of the Bdellovibrio nectophore of pfu/mL and leech plastid soaks 15～30min; Changed Chi Shui once every 5～40 days, change the mixture bacterium liquid that adds Bdellovibrio nectophore and leech plastid behind the water at every turn, make the Bdellovibrio nectophore of Chi Shuizhong and the mixture concentration of leech plastid reach 10～10
7Pfu/mL;
The described input sea cucumbers of step (3) when 15～30 ℃ of water temperatures according to following standard: when the sea cucumbers specification was 400/kg, throwing in density was 600/m
2When the sea cucumbers specification was 200/kg, input density was 400 tails/m
2When the sea cucumbers specification was 100/kg, input density was 300 tails/m
2
Step (2) and (4) described Bdellovibrio be Bdellovibrio (
BdellovibrioSp.) BDM01, described Bdellovibrio is preserved in Chinese Typical Representative culture collection center on April 28th, 2008, and deposit number is CCTCC NO:M208066;
Described mixture bacterium liquid is that Bdellovibrio nectophore bacterium liquid and bdellovibrio bdelloplast bacterial bacterium liquid mix by bacterium number 1:1;
Described bdellovibrio bdelloplast bacterial bacterium liquid prepares in accordance with the following methods: inoculation 0.5mL concentration is 10
7The Aeromonas hydrophila of cfu/mL (
Aeromonas hydrophila) in the nutrient broth liquid nutrient medium, cultivate 20h for 26～30 ℃, 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, accesses 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 nectophore, 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
9The bdellovibrio bdelloplast bacterial bacterium liquid of pfu/mL;
Described Bdellovibrio nectophore bacterium liquid prepares in accordance with the following methods: with the supernatant that contains Bdellovibrio nectophore among the bdellovibrio bdelloplast bacterial preparation method behind the centrifugal 15～20min of 16000～18000rpm, 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
9The Bdellovibrio nectophore 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.
2. a kind of energy-saving type sea cucumber culturing according to claim 1 is characterized in that: the described culturing pool of step (1) cleaning and sterilizing before injecting breeding water is clean; 3m in the described culturing pool
2～4m
2Arrange 1 gas exhausting stone.
3. a kind of energy-saving type sea cucumber culturing according to claim 1, it is characterized in that: the method for the mixture bacterium liquid of the described adding Bdellovibrio nectophore in step (2) and (4) and leech plastid is: behind the dilution of the Chi Shui in culturing pool bacterium liquid, and the full pond of evenly splashing.
4. a kind of energy-saving type sea cucumber culturing 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. a kind of energy-saving type sea cucumber culturing according to claim 1, it is characterized in that: the described bait of step (4) is mixed bait, and every day, bait throwing in was twice; Soil pick-up at the bottom of 5～7 days employing siphonages carry out culturing pool.
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| 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 |
| CN101440362A (en) * | 2008-12-26 | 2009-05-27 | 华南理工大学 | Use of UV induction mutation for reinforcing cracking performance of Bdellovibrio |
| CN101691550A (en) * | 2009-08-28 | 2010-04-07 | 华南理工大学 | Microbial inoculum for improving water body and structure of biological intestinal colony, and preparation method and application thereof |
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| CN101440362A (en) * | 2008-12-26 | 2009-05-27 | 华南理工大学 | Use of UV induction mutation for reinforcing cracking performance of Bdellovibrio |
| CN101691550A (en) * | 2009-08-28 | 2010-04-07 | 华南理工大学 | Microbial inoculum for improving water body and structure of biological intestinal colony, and preparation method and application thereof |
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| 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03期),1-4. * |
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