CN102017906B - Energy-saving emission-reducing haliotis diversicolor supertexta culturing method - Google Patents
Energy-saving emission-reducing haliotis diversicolor supertexta culturing method Download PDFInfo
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Abstract
The invention discloses an energy-saving emission-reducing haliotis diversicolor supertexta culturing method, which comprises the steps of: (1) culturing aquatic plants, (2) adding nutritive salt, (3) picking seedlings and distributing eggs by exchanging the water in an aquatic plants culturing pool, adding the mixed preparation of leech and vibrio till the initial concentration of the mixed preparation in the pool water reaches to 10 to 107pfu/ml, and arraying forty thousand to fifty thousand germ cells of haliotis diversicolor supertexta in each pool, (4) exchanging the water: exchanging the water in pool every 5 to 30 days and adding the mixed preparation of leech and vibrio after exchanging the water every time till the concentration of the mixed preparation in the pool water reaches to 10 to 107pfu/ml, and (5) peeling off and harvesting. The culturing method of the invention has obvious facilitation to the growth of haliotis diversicolor supertexta, greatly increases the livability of the haliotis diversicolor supertexta and improves the water quality of the culturing environment. The leech and the vibrio are non-toxic to abalone, therefore being fit for the large-scale industrial culturing of abalone and providing a new culturing technique for boosting the culturing and growth of the haliotis diversicolor supertexta.
Description
Technical field
The invention belongs to aquaculture shellfish hatchery method field, particularly a kind of can promote haliotis diversicolor Reeve (Haliotis diversicolor aquatilis) Bao Miao growth novel, save water and energy, the seedling-cultivating method of environmental protection.
Background technology
Bao is one of eight sea treasure of China, has very high economy and medical value.The abalone culture of China begins starting the seventies in 20th century, has formed large-scale production to the mid-80, and after this development is swift and violent especially, and the foster Bao Ye of coastal various places has formed bigger scale.Yet along with the raising of the aggravation of environmental pollution, the expansion of culturing scale and intensification degree, the disease of Bao Miao also takes place in succession, and the trend that increases the weight of is year by year arranged, and the development to the foster Bao Ye of China constitutes a serious threat.Especially haliotis diversicolor Reeve Bao Miao, since 2000, the phenomenon of the extensive board falling of haliotis diversicolor Reeve seedling appearred in each plant in succession, and the disease problem is frequent, has had a strong impact on abalone culture production, causes many raisers to abandon culturing haliotis diversicolor Reeve.
Pathogenic infection is the main cause that causes the haliotis diversicolor Reeve board falling, and propagating artificially under the condition mainly is bacteriosis and viral disease.Common bacterial pathogen has vibrio parahaemolytious, vibrio fluvialis, secondary vibrio alginolyticus etc.Vibrio parahaemolytious mainly causes the abalone muscular dystrophy, and lethality can reach about 50%, and the infection that vibrio fluvialis and secondary vibrio alginolyticus cause can cause 60～70% lethality.The method of conventional control abalone board falling mainly is regular administration of antibiotics at present, and antibiotic advantage is that the direct entry of medicine, concentration are high, absorption is fast, good effect; Shortcoming is may produce excitant to abalone, cause new illness.If long-term a large amount of antibiotic that uses not only can make the pathogenic bacteria pesticide resistance strengthen, some Bao cases are become be difficult to cure.Antibiotic residue can influence the abalone quality in the Bao body in addition, is unfavorable for selling, and then causes culturist's economic loss.The board falling of herbal control abalone also is a kind of common way, though have drug residue free, advantage that economic benefit is high, the slow shortcoming that takes effect is arranged also, thereby seeks a kind of more efficient, economic seedling-cultivating method and seem very urgent.
Bdellovibrio is to parasitize other bacteriums, and can cause a bacterioid of host bacteria cracking; Littler than general bacterium, can pass through bacterial filter, the effect of similar phage is arranged; Gram is negative, and can cracking comprise that vibrio parahaemolytious, vibrio alginolyticus etc. cause the potentially pathogenic organism of Bao disease.With the microorganism formulation that the mix preparation of Bdellovibrio telotroch and leech plastid is grown seedlings as haliotis diversicolor Reeve, it is rapid-action to have telotroch concurrently, and energetic and leech plastid prepares, preserves easily, the more of a specified duration and stronger advantage of environment tolerance of bactericidal action time.These characteristics make the Bdellovibrio mix preparation be suitable as a kind of microorganism formulation, in the seedling raising process that is applied to haliotis diversicolor Reeve.In addition, what seedling growing process adopted among the present invention is the cycle to change water, so not only reduces breeding water amount and discharge of wastewater, reduces energy consumption especially, has reduced aquaculture cost.Therefore, this method be that a kind of facility is feasible, green, the seedling-cultivating method of low-carbon (LC), energy-saving and environmental protecting.
Existing research shows, as a kind of effective microorganism preparation, no matter 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 02 pair of fish cell of Bdellovibrio BDH21 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 seedling growing process, the object of the present invention is to provide a kind of haliotis diversicolor Reeve seedling-cultivating method of energy-saving and emission-reduction.
The object of the invention is realized through following technical proposals: a kind of haliotis diversicolor Reeve seedling-cultivating method of energy-saving and emission-reduction comprises following operating procedure:
(1) training algae: the beginning in preceding 30～45 days of collecting seedling is trained algae in training algae pond; The control intensity of illumination is at 2000～5000lux in the training algae process, and the control water temperature is at 15～28 ℃;
(2) Ensure Liquid salt: during the training algae, according to 3～5g/m
3Breeding water body, the mode of adding once in 2～4 days is added nutritive salt;
(3) the cloth ovum of collecting seedling: will train the algae pond and change Chi Shui, and add the Bdellovibrio mix preparation, and make the initial concentration of the Bdellovibrio mix preparation of Chi Shuizhong reach 10～10
7Pfu/mL; Every pond cloth haliotis diversicolor Reeve fertilized egg is 4～50,000 during following seedling, and according to aeration rate 10～60L/h/m
3Carry out aeration, illumination is controlled at 200～3000lux, and water temperature is controlled at 15～28 ℃;
(4) change water: whenever changed water once, change at every turn and add the Bdellovibrio mix preparation behind the water, make the Bdellovibrio mix preparation concentration of Chi Shuizhong reach 10～10 at a distance from 5～30 days
7Pfu/mL;
(5) peel off and gather: following seedling 30～50 days, young Bao shell reach to peel off behind 3.0～7.0mm gathers.
The said training of step (1) algae pond washes the back and adopts liquor potassic permanganate to carry out disinfection, and injects the seawater through sand filtration and precipitation process then; Training algae pond is before injecting seawater, and water inlet pipe mouth is wrapped up a filter bag in the pond, prevents that fine sand from going into the pond.
The said algae of step (1) is a benthic diatom; Said benthic diatom is preferably a moon shape algae, rhombus algae, boat-shaped algae and avette algae.
The said training of step (1) algae is an adherance with the water white transparency polyethylene film.
It is 5: 1: 1 that the said nutritive salt of step (2) contains mass ratio: 1 N element, P element, Si element and Fe element.
Step (3) and (4) said Bdellovibrio are Bdellovibrio (Bdellovibrio sp.) BDFM05; Said Bdellovibrio is preserved in the Chinese typical culture collection center in the Wuhan University of Luojia Mountain, Wuhan, Hubei Province on August 7th, 2009, and deposit number is CCTCC NO:M 209172.
Said Bdellovibrio BDFM05 carries out under electron microscope, carrying out morphologic observation: BDFM05 after the negative staining and is unicellular, ellipse, and size is 1.43 * 0.53um, and end is given birth to flagellum, and flagellum length is 2um at least; Said Bdellovibrio BDFM05 cultivates the transparent circular plaque that can form diameter 2～3mm in three days with the double-layer plate method in 28 ℃;
The said Bdellovibrio mix preparation in step (3) and (4) is that Bdellovibrio telotroch bacterium liquid and Bdellovibrio leech plastid bacterium liquid mix by the bacterium number at 1: 1.
Said Bdellovibrio leech plastid bacterium liquid prepares according to following method: the host is in the nutrient broth liquid nutrient medium in inoculation, cultivates 12h at 35 ℃ with the 200rpm shaking table, and culture fluid is through 4 ℃; Behind the centrifugal 10～20min of 5000～8000rpm, deposition is added in the DNB liquid nutrient medium, inserts Bdellovibrio BDFM05; Under 25～35 ℃ of temperature; Cultivate 36～48h with 150～300rpm shaking table, obtain containing the culture fluid of Bdellovibrio leech plastid and Bdellovibrio telotroch, culture fluid under 4 ℃ of temperature with 6000～8000rpm behind centrifugal 15～20min; Removal contains the supernatant of Bdellovibrio telotroch, and the deposition that obtains is Bdellovibrio leech plastid; It is 7.2～7.6 phosphate buffer suspension that Bdellovibrio leech plastid is used DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
6～10
9The Bdellovibrio leech plastid bacterium liquid of pfu/mL;
Said Bdellovibrio telotroch bacterium liquid prepares according to following method: with the supernatant that contains the Bdellovibrio telotroch among the Bdellovibrio leech plastid preparation method behind the centrifugal 15～20min of 16000～18000rpm; Deposition use DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value are 7.2～7.6 phosphate buffer suspension, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL.
Said host is Aeromonas hydrophila (Aeromonas hydrophila); Said 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.
The present invention has following advantage and effect with respect to prior art:
(1) Bdellovibrio mix preparation of the present invention causes a disease to aquatic products or potentially pathogenic organism has strong cracking strength, and Bdellovibrio mix preparation itself can improve the aquaculture organism intestinal environment, promotes growth, improves Bao Miao immunity.Therefore, compare with other microorganism formulation, the Bdellovibrio mix preparation among the present invention has the immunity that improves Bao Miao self, improves its survival rate, accelerates the characteristics such as growth rate of Bao Miao.
(2) the Bdellovibrio mix preparation comprises Bdellovibrio leech plastid, and Bdellovibrio leech plastid also has potential inactivated vaccine function.Between the leech plastid puberty, Bdellovibrio does not change the surface texture of host cell, makes former host still have immunogenicity, but the stimulating organism body produces immune response.Therefore, can effectively prevent the generation of the bacteriosis of haliotis diversicolor Reeve Bao Miao, improve survival rate of seedling.
What (3) existing haliotis diversicolor Reeve seedling growing process adopted is the mode of flowing water, has not only expended great amount of manpower and material resources, financial resources, also can cause certain pollution to environment.And the haliotis diversicolor Reeve seedling-cultivating method that utilizes this Bdellovibrio mix preparation can reduce the demand of output; Practiced thrift production cost to a great extent; And can not cause harmful effect to environment, the sustainable development of Bao Ye and other aquacultures is had great significance.
Description of drawings
Fig. 1 is the testing result figure of the bacterial infection experiment of each group.
Embodiment
Below in conjunction with embodiment the present invention is done further detailed description, but embodiment of the present invention is not limited thereto.Technological parameter for not indicating especially can carry out with reference to routine techniques.
The present invention is abalone culture field, Fujian with implementing.Culture the pond and build in the breed booth, each pond specification is 2 * 2 * 0.75m, and the depth of water is 0.40m.Disinfecting solution of potassium permanganate is all used in each pond, rinses well repeatedly.It is 80% gobo that pond top covers obscurity, avoids illumination strong excessively.Intensity of illumination generally is controlled at (200～5000) lux.
Embodiment 1
(1) is used for the preparation of the Bdellovibrio mix preparation that haliotis diversicolor Reeve (Haliotis diversicolor aquatilis) grows seedlings
The preparation of Bdellovibrio leech plastid bacterium liquid: in a conical flask that 100mL nutrient broth liquid nutrient medium (sodium chloride 5g, pH 7.4 ± 0.2 for peptone 10g, beef extract powder 3g) are housed, inoculate 0.5mL10
7Cfu/mL Aeromonas hydrophila (Aeromonas hydrophila, bacterium numbering: GIM 1.172 derive from microorganism fungus kind preservation center, Guangdong Province), 200rpm, 35 ℃ of shaking tables were cultivated 12 hours, and culture fluid is abandoned supernatant in 4 ℃, the centrifugal 15min of 5000rpm.With 5mL physiological saline suspension deposition thalline, join one afterwards 100mL DNB liquid nutrient medium (nutrient broth 0.8g, caseinic acid hydrolysate 0.5g are housed; Yeast extract 0.1g; Be dissolved in the 1000mL distilled water, the pH value is 7.2～7.6) conical flask in, insert 1mL again and contain 10
3Pfu/mL Bdellovibrio BDFM05 (CCTCC M209172).Constant temperature shaking table 250rpm, 28 ℃ of cultivation 36h.Culture fluid is in 4 ℃ of centrifugal 20min of 6000rpm, removes the supernatant that contains the Bdellovibrio telotroch, keeps deposition (being Bdellovibrio leech plastid), adds the 10mL DNB liquid nutrient medium Bdellovibrio leech plastid that suspends again, and promptly obtaining concentration is 10
9The Bdellovibrio BDFM05 leech plastid bacterium liquid of pfu/mL;
The preparation of Bdellovibrio telotroch bacterium liquid: with the supernatant that contains the Bdellovibrio telotroch in the above-mentioned Bdellovibrio leech plastid bacterium liquid and preparation method thereof behind the centrifugal 20min of 16000rpm; The deposition that obtains promptly is the Bdellovibrio telotroch; Add 10mL DNB liquid nutrient medium suspension Bdellovibrio telotroch promptly, obtaining concentration is 10
9The Bdellovibrio BDFM05 telotroch bacterium liquid of pfu/mL.
The Bdellovibrio mix preparation is mixed by bacterial population by Bdellovibrio BDFM05 telotroch bacterium liquid and Bdellovibrio BDFM05 leech plastid bacterium liquid and forms at 1: 1;
(2) application of Bdellovibrio mix preparation in haliotis diversicolor Reeve is grown seedlings
The present invention can be divided into two stages altogether.Phase I is the training algae, and the training algae time is 35 days, and this stage is not added the Bdellovibrio mix preparation; Second stage is the stage of growing seedlings, and content of the present invention is mainly launched in this stage.Seedling raise period is 40 days, and this stage experimental group is added the Bdellovibrio mix preparation immediately behind the water of at every turn changing full pond.With different exchange water cycles, throw the Bdellovibrio mix preparation of variable concentrations respectively to breeding water body.Exchange water cycle is respectively: 5,10,15,20,25,30 days, each quantity of exchanged water was a range.All experiment pool environmental condition of living in comprises that temperature, illumination, pH value, salinity etc. are identical.All test tanks are divided into 25 groups, are A, B-1, B-2, B-3, B-4, C-1, C-2, C-3, C-4, and D-1, D-2, D-3, D-4, E-1, E-2, E-3, E-4, F-1, F-2, F-3, F-4, G-1, G-2, G-3, G-4,2 every group are parallel.The exchange water cycle of each group and to add bacteria concentration following:
Following mask body is described this implementation step:
A group: normal flowing water, day quantity of exchanged water is 3 times of the pond water yield, every morning, 7:00 used the high pressure water washing pond, during do not add the Bdellovibrio mix preparation, be the plant produced mode;
B organizes (B-1, B-2, B-3, B-4): water once to change (full pond) in 5 days;
C organizes (C-1, C-2, C-3, C-4): water once to change (full pond) in 10 days;
D organizes (D-1, D-2, D-3, D-4): water once to change (full pond) in 15 days;
E organizes (E-1, E-2, E-3, E-4): water once to change (full pond) in 20 days;
F organizes (F-1, F-2, F-3, F-4): water once to change (full pond) in 25 days;
G organizes (G-1, G-2, G-3, G-4): water once to change (full pond) in 30 days;
B-1, C-1, D-1, E-1, F-1, G-1 group be for change behind the water immediately the Bdellovibrio mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10pfu/mL in the water body.
B-2, C-2, D-2, E-2, F-2, G-2 group be for change behind the water immediately the Bdellovibrio mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
3Pfu/mL.
B-3, C-3, D-3, E-3, F-3, G-3 group be for change behind the water immediately the Bdellovibrio mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
5Pfu/mL.
B-4, C-4, D-4, E-4, F-4, G-4 group be for change behind the water immediately the Bdellovibrio mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
7Pfu/mL.
In phase I training algae process, every pond cloth adheres to 20 of basement membranes, connects algae kind (giving birth to naturally in the local seawater), and the control intensity of illumination is at 2000～5000lux, and water temperature is at 15～25 ℃; According to the algae upgrowth situation, according to 5g/m
3Water body, the mode of adding once in 4 days is added nutritive salt, and the ratio of applying is N: P: Si: Fe=5: 1: 1: 1 (ppm); After 10 days; Can observe and be pale green and golden yellow edematus on whole the film, it takes a morsel at the microscopically microscopy for adhering to the skim algae; The finding algae mainly contains large-scale boat-shaped algae, small-sized boat-shaped algae, rhombus algae and avette algae etc., and ratio differs.
In the second stage seedling raising process, also need rationally to regulate illumination, illumination generally is controlled between the 200-2000lux, for algae provides more modest condition, crosses slow and is consumed by Bao Miao to prevent grow too fast accelerated ageing or growth rate of algae.
After algae is trained, splash the fertilized egg of haliotis diversicolor Reeve in the pond.Every pond (also being the cloth ovum) 50,000 of splashing.Cloth ovum water temperature is controlled at about 17-22 ℃, in the cloth ovum 3 days, can observe a lot of trochophores that swim in the water body, changes water this period and need use 200 mesh filter screens to filter, and runs off to prevent larva.After seven days, larva begins metamorphosis, on adherance, can see being attached with the small young that crawls, and carefully scrapes and gets the microscopically microscopy, observes its vigor.
To receiving seedling, the time is 75 days from the training algae, and young Bao body grows to 3.0～7.0mm; Unicellular alga on the adherance is partly aging; Algae on the minority adherance is eaten up, and all ponds do not find blank or take off the plate phenomenon, and the spatfall amount on every film is 300～700 and does not wait; Can arrive about 1100 on the maximum plates, the index of specifically growing seedlings and measuring method are following:
1) haliotis diversicolor Reeve Bao Miao growth and survival
A Bao Miao primary quantity, every pond 50,000 seedlings.
It is long that b receives the average shell of seedling, randomly draws 100 Bao Miao, and it is long to measure shell, averages.
The long average daily growth amount of c shell, it is long to receive the average shell of seedling, gets 40 days mean value.
The average individual weight of d Bao Miao is got 100 Bao Miao at random, claims to average after its gross weight.
E receives the seedling amount, takes by weighing 10g Bao Miao, calculates its number, the Bao Miao that collects is claimed the grain number of estimation Bao Miao after its gross weight.
The f survival rate, Bao Miaoliang accounted for the percentage of this stage throwing seedling amount when certain stage finished.
2) the Bao Miao immune indexes is measured
100 of every group of off-test samplings, 20/pipe are put in 5 Eppendoff pipes that fill 1mL Hank ' s buffer solution (pH=7.8) respectively, store in-80 ℃ of mensuration in order to anti-immune indexes.Take out sample during mensuration, it is being melted on ice.Carry out homogenate, 6000r/min then, 4 ℃ of centrifugal 5min get the mensuration that supernatant is used for immune indexes.
With reference to Bradford method (document Bradford M.A rapid and sensitive method for thequantification of microgram quantities of protein [J] .Analytical Biochemistry; 1976,72:248-254) working sample protein content is a standard protein with calf serum albumen; Set up calibration curve; With the Coomassie brilliant blue is developer, in 96 hole ELISA Plates, reacts 10min, measures the OD in each reaction system with ELIASA
595nmValue.
The mensuration of a, superoxide dismutase (SOD) vigor
The mensuration of 1,2,3,-thrihydroxy-benzene autoxidation speed, under 25 ℃, in 4.5mL 50mmol/L, the K of Ph=8.30
2HPO
4-KH
2PO
4Add 10 μ L 50mmol/L 1,2,3,-thrihydroxy-benzenes in the buffer solution, shake up rapidly, pour in the cuvette of optical path 1cm, every separated 30s surveys the A value once under the 325nm wavelength, requires autoxidation speed about 0.070OD/min.Enzyme assay before adding 1,2,3,-thrihydroxy-benzene, adds SOD appearance to be measured, records data, is calculated as follows enzymic activity:
Enzymic activity=(0.070-A
325nm)/min * 100% * reactant liquor cumulative volume * appearance liquid extension rate/(0.070 * 50% * appearance liquid is long-pending)
The enzyme unit definition of living: in every milliliter of reactant liquor, per minute suppresses 1,2,3,-thrihydroxy-benzene autoxidation speed and reaches 50% enzyme amount and be defined as the enzyme unit (U/mL) that lives.
B, antalzyme activity are measured
With the micrococcus lysodeikticus freeze-dried powder is substrate.Use 0.1mol/L, the phosphate buffer of pH=6.4 is made into substrate suspension (OD
570nm≈ 0.3), get this suspension of 3.0mL in the built-in ice bath of test tube, add 50 μ L again and treat test sample, mix, survey A
0Value.Then test solution is moved into 37 ℃ of temperature and bathe mid-30min, place ice bath 10min after the taking-up again,, survey its A value with cessation reaction.Bacteriolyze vigor U
LBy (A
0-A)/calculating of A formula.
3) bacterial infection experiment
Infection experiment is carried out in the pond that fills 5L sand filtration seawater of sterilization in advance.Randomly draw 100 Bao Miao respectively from control group (A group) and each experimental group (B-G group), the long 4.5～6.0mm of shell.In each group water body, add Aeromonas hydrophila bacterium liquid, make that the Aeromonas hydrophila cell concentration reaches 10 in the water body
8Cfu/mL.Experimental session not flowing water is cultured, and normal bait throwing in is with 5L/h/m
3Continuous aeration, water temperature is 20 ℃, pH is controlled at 7.2.Behind the artificial infection, observed continuously 5 days, finally be calculated to be motility rate, the employing relative survival rate (Relative Percent Survival, RPS) calculating formula:
RPS (%)=(1-immune group lethality/control group lethality) * 100%
Experimental result is by shown in table 1 and the table 2.Table 1 refers to the influence of different seedling-cultivating method to haliotis diversicolor Reeve seedling growing state.It is thus clear that compared with control group (A group), all experimental group improve the survival rate of Bao Miao more significantly, and the growth of promotion Bao Miao.Under identical exchange water cycle, along with the rising of the Bdellovibrio mix preparation concentration of adding, the survival rate of Bao Miao and shell are long also in rising trend gradually.
Table 2 refers to the influence of different seedling raising mannerses to haliotis diversicolor Reeve seedling immunity.Data show that compared with control group (A group), the SOD vigor and the antalzyme activity of all experimental group all are significantly improved; Under identical exchange water cycle, along with the rising of the Bdellovibrio mix preparation concentration of adding, Bao Miao immunity also has certain enhancing.
The bacterial infection experimental result is by shown in Figure 1.The result shows, adopts the control group (A group) of factory's water-flowing type aquaculture, and the relative survival rate of its Bao Miao is 0; In other experimental group, no matter adopt which kind of exchange water cycle, along with the rising of Bdellovibrio mix preparation concentration in water body, the relative survival rate of Bao Miao also can rise gradually.In addition, adopt the experimental group of adding the Bdellovibrio mix preparation, the relative survival rate of every group of Bao Miao all reaches more than 90%.Bdellovibrio leech plastid in this explanation Bdellovibrio mix preparation can be brought into play the effect of a potential vaccine, strengthens the immunity of haliotis diversicolor Reeve seedling.
The water quality aspect, ammoniacal nitrogen is received oxidizing process with hypobromous acid and is measured (GB12763.4-91); Nitrite nitrogen is with diazonium-azo spectrphotometric method for measuring (GB12763.4-91).Through measuring, each item water-quality determination value in the A group pond all is higher than experimental group (B-G group).Wherein, the testing result of A group ammoniacal nitrogen and nitrite nitrogen is respectively 0.084mg/L and 0.011mg/L, and in the experimental group (B-G group), the testing result of ammoniacal nitrogen and nitrite nitrogen is respectively 0.075-0.081mg/L and 0.006-0.009mg/L.In addition, total number of bacteria and vibrios sum context of detection adopt coating nutrient broth flat band method and TCBS flat band method to detect respectively.The result shows that total number of bacteria and vibrios sum in the A group water body reach 10 respectively
5With 10
3The order of magnitude (cfu/mL), the total number of bacteria and the vibrios sum that add the experimental group of Bdellovibrio mix preparation are respectively 10
2～10
3With 10
1～10
2The order of magnitude (cfu/mL), lower than control group.Explain that the Bdellovibrio mix preparation can effectively control the bacterial number of water body, improve water quality.
Comprehensive each side result adds the Bdellovibrio mix preparation, can significantly improve the growth rate of Bao Miao, improves survival rate, enhancing immunity, and can improve water quality, reach the good result of saving water and energy.
The different seedling-cultivating method of table 1 is to the influence of haliotis diversicolor Reeve seedling growing state
The different seedling-cultivating method of table 2 is to the influence of haliotis diversicolor Reeve seedling immunity
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit 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 (8)
1. the haliotis diversicolor Reeve seedling-cultivating method of energy-saving and emission-reduction is characterized in that comprising following operating procedure:
(1) training algae: the beginning in preceding 30～45 days of collecting seedling is trained algae in training algae pond; The control intensity of illumination is at 2000～5000lux in the training algae process, and the control water temperature is at 15～28 ℃;
(2) Ensure Liquid salt: during the training algae, according to 3～5g/m
3Breeding water body, the mode of adding once in 2～4 days is added nutritive salt;
(3) the cloth ovum of collecting seedling: will train the algae pond and change Chi Shui, and add the Bdellovibrio mix preparation, and make the initial concentration of the Bdellovibrio mix preparation of Chi Shuizhong reach 10～10
7Pfu/mL; Every pond cloth haliotis diversicolor Reeve fertilized egg is 4～50,000 during following seedling, and according to aeration rate 10～60L/h/m
3Carry out aeration, illumination is controlled at 200～3000lux, and water temperature is controlled at 15～28 ℃;
(4) change water: whenever changed water once, change at every turn and add the Bdellovibrio mix preparation behind the water, make the Bdellovibrio mix preparation concentration of Chi Shuizhong reach 10～10 at a distance from 5～30 days
7Pfu/mL;
(5) peel off and gather: following seedling 30～50 days, young Bao shell reach to peel off behind 3.0～7.0mm gathers;
The said algae of step (1) is a benthic diatom;
Step (3) and (4) said Bdellovibrio are Bdellovibrio (Bdellovibrio sp.) BDFM05, and said Bdellovibrio is preserved in Chinese typical culture collection center on August 7th, 2009, and deposit number is CCTCCNO:M 209172.
2. the haliotis diversicolor Reeve seedling-cultivating method of a kind of energy-saving and emission-reduction according to claim 1 is characterized in that: adopt liquor potassic permanganate to carry out disinfection after the said training of step (1) algae pond washes, inject the seawater through sand filtration and precipitation process then; Training algae pond is before injecting seawater, and water inlet pipe mouth is wrapped up a filter bag in the pond, prevents that fine sand from going into the pond.
3. the haliotis diversicolor Reeve seedling-cultivating method of a kind of energy-saving and emission-reduction according to claim 1 is characterized in that: said benthic diatom is a month shape algae, rhombus algae, boat-shaped algae and avette algae.
4. the haliotis diversicolor Reeve seedling-cultivating method of a kind of energy-saving and emission-reduction according to claim 1, it is characterized in that: the said training of step (1) algae is an adherance with the water white transparency polyethylene film.
5. the haliotis diversicolor Reeve seedling-cultivating method of a kind of energy-saving and emission-reduction according to claim 1, it is characterized in that: it is 5: 1: 1 that the said nutritive salt of step (2) contains mass ratio: 1 N element, P element, Si element and Fe element.
6. the haliotis diversicolor Reeve seedling-cultivating method of a kind of energy-saving and emission-reduction according to claim 1, it is characterized in that: the said Bdellovibrio mix preparation in step (3) and (4) is that Bdellovibrio telotroch bacterium liquid and Bdellovibrio leech plastid bacterium liquid mix by the bacterium number at 1: 1.
7. the haliotis diversicolor Reeve seedling-cultivating method of a kind of energy-saving and emission-reduction according to claim 6, it is characterized in that: said Bdellovibrio leech plastid bacterium liquid prepares according to following method: the host is in the nutrient broth liquid nutrient medium in inoculation, cultivates 12h at 35 ℃ with the 200rpm shaking table; Culture fluid is through 4 ℃; Behind the centrifugal 10～20min of 5000～8000rpm, deposition is added in the DNB liquid nutrient medium, inserts Bdellovibrio BDFM05; Under 25～35 ℃ of temperature; Cultivate 36～48h with 150～300rpm shaking table, obtain containing the culture fluid of Bdellovibrio leech plastid and Bdellovibrio telotroch, culture fluid under 4 ℃ of temperature with 6000～8000rpm behind centrifugal 15～20min; Removal contains the supernatant of Bdellovibrio telotroch, and the deposition that obtains is Bdellovibrio leech plastid; It is 7.2～7.6 phosphate buffer suspension that Bdellovibrio leech plastid is used DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
6～10
9The Bdellovibrio leech plastid bacterium liquid of pfu/mL;
Said Bdellovibrio telotroch bacterium liquid prepares according to following method: with the supernatant that contains the Bdellovibrio telotroch among the Bdellovibrio leech plastid preparation method behind the centrifugal 15～20min of 16000～18000rpm; Deposition use DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value are 7.2～7.6 phosphate buffer suspension, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL.
8. the haliotis diversicolor Reeve seedling-cultivating method of a kind of energy-saving and emission-reduction according to claim 7 is characterized in that: said host is Aeromonas hydrophila (Aeromonas hydrophila); Said 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.
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| CN101638629A (en) * | 2009-08-28 | 2010-02-03 | 华南理工大学 | Bdellovibrio bacteriovorus for preventing and curing rice bacterial diseases and application thereof |
| CN101647406A (en) * | 2009-09-04 | 2010-02-17 | 福建蓝鲸水产有限公司 | Benthic diatom culture method for growing seedlings and abalone fry culture method |
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| CN101638629A (en) * | 2009-08-28 | 2010-02-03 | 华南理工大学 | Bdellovibrio bacteriovorus for preventing and curing rice bacterial diseases and application thereof |
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| 宋志萍等.一种消除九孔鲍苗细菌性病原的无公害绿色生物方法的研究.《海洋科学》.2006,第30卷(第01期), * |
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