CN102017913B - Energy-saving method for culturing haliotis diversicolor aquatilis - Google Patents
Energy-saving method for culturing haliotis diversicolor aquatilis Download PDFInfo
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Abstract
The invention discloses an energy-saving method for culturing haliotis diversicolor aquatilis. The method comprises the following steps of: (1) culturing algae; (2) adding nutrient salt; (3) collecting the spats and distributing eggs, namely, changing water in an algae culturing pool, adding bdellovibrio bacteriovorus plasmid bacterial liquid into the pool until the concentration of bdellovibrio bacteriovorus plasmid in the pool water is between 10 and 107 pfu/mL and distributing 40,000 to 50,000 haliotis diversicolor aquatilis fertilized eggs in each pool during spat distribution; (4) changing water, namely, changing the pool water once every 5 to 30 days and adding the bdellovibrio bacteriovorus plasmid bacterial liquid into the pool water after the water is changed each time until the concentration of the bdellovibrio bacteriovorus plasmid in the pool water is between 10 and 107 pfu/mL; and (5) stripping and collecting. The spat culturing method remarkably promotes the growth of the haliotis diversicolor aquatilis, greatly increases the survival rate of the haliotis diversicolor aquatilis and improves the water quality of a culturing environment. The bdellovibrio bacteriovorus plasmid does not have toxic or side effect on abalone, so that the method is suitable for large-scale culturing of the abalone and provides new culturing technology for promoting the spat culturing and growth of haliotis diversicolor aquatilis.
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
Haliotis diversicolor Reeve (having another name called Haliotis diversicolor) is one of economic species important in the Bao Ke of south China marine site, due to this kind of Bao, to have growth rate very fast, cultivating condition is required to the advantages such as relatively low, now developed into most important cultivation kind in Taiwan and continent southeastern coast Bao Ke.But since 2000, the coastal haliotis diversicolor Reeve artificial culture seedling mortality phenomenon that occurred from Fujian to Hainan, mass death can't be cultivated in the Bao Miao field more than 90%, forces many raisers to abandon cultivating haliotis diversicolor Reeve.Therefore, how research improves mass death is cultivated ecotope, reduces the generation of board falling phenomenon, improves its survival rate and growth rate, and in the production technology of haliotis diversicolor Reeve seed and even abalone culture industry, all tool is of great significance.
In the haliotis diversicolor Reeve seedling raising process, can usually prevent and treat Bao Miao by antibiosis disease occurs, improve survival rate.But use for a long time antibiotic, particularly abuse of antibiotics, also can cause the composition of gut flora imbalance of Bao, to the immunity of organisms generation harmful effect of Bao, produce the side effects such as drug resistance and medicament residue, give aquaculture and all brought potential harm as consumer's the mankind's health.In addition, some employing Chinese herbal medicines are prevented and treated, but exist again the slow shortcoming that takes effect.
Bdellovibrio is to parasitize other bacteriums, and can cause a bacterioid of host bacteria cracking; Less than general bacterium, can pass through bacterial filter, the effect of similar phage is arranged; Gram’s staining is negative, and can cracking comprise that vibrio parahaemolytious, vibrio alginolyticus etc. cause the potentially pathogenic organism of Bao disease.Can be divided into the leech plastid state that breaks away from host bacteria, there is the telotroch state of flagellum, free swimming and grow the history of life of Bdellovibrio in host bacteria.Lose flagellum in the time of periplasmic space that the telotroch Bdellovibrio is invaded the host, host cell, together with Bdellovibrio, becomes " leech plastid ".With telotroch, compare, the leech plastid not only has preparation, it is more convenient to preserve, and the environment tolerance is strong, though action time characteristics more of a specified duration slightly slowly, also there is potential inactivated vaccine function simultaneously.After telotroch Bdellovibrio invasion host cell, the respiration of host cell (respiration) is stopped very soon.Bdellovibrio also enters leech plastid state, starts 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, elongated.Finally, the leech plastid of strip is divided into a lot of short sections, grows flagellum, again becomes telotroch.Afterwards, they secrete essential enzyme, and 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 immunogenicity, but the stimulating organism body produces immune response.This potential function makes the application bdellovibrio bdelloplast bacterial in haliotis diversicolor Reeve is grown seedlings, and can effectively prevent the generation of Bao Miao bacteriosis, improves its survival rate.
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 is all safe.For example: abroad, Lenz and Hespell (1978) studies discovery, Bdellovibrio and animal and people's cell is not had to an infectivity [Lenz R.W., Hespell R.B.Attempts to grow bedellovibrios micurgically-injected int o 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 Bdellovibrio BDH2102 to fish cell and pathogen.The microbiology circular, 2006,33 (1): 7-11].
In addition, what in the present invention, seedling-cultivating method adopted is the cycle to change water, so not only reduces breeding water amount and discharge of wastewater, reduces especially energy consumption, has reduced aquaculture cost; With the closed circulating breeding method, compare, less investment, more easily promote.Therefore, the method be that a kind of facility is feasible, green, the seedling raising manners of low-carbon (LC), energy-saving and environmental protecting.
Summary of the invention
For overcoming the deficiency existed in 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.
Purpose of the present invention realizes by 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: collect seedling and start in first 30～45 days to be trained algae in training algae pond; In training algae process, controlled light intensity, at 2000～5000lux, is controlled water temperature 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 bdellovibrio bdelloplast bacterial bacterium liquid, and make the initial concentration of the bdellovibrio bdelloplast bacterial thalline of Chi Shuizhong reach 10～10
7pfu/mL; During lower seedling, every pond cloth haliotis diversicolor Reeve fertilized egg is 4～50,000, 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: changed water once every 5～30 days, add bdellovibrio bdelloplast bacterial bacterium liquid after changing water at every turn, make the bdellovibrio bdelloplast bacterial cell concentration of Chi Shuizhong reach 10～10
7pfu/mL;
(5) peel off and gather: lower seedling 30～50 days, young Bao shell reaches after 3.0～6.5mm to be peeled off gathers.
The described training algae of step (1) pond washes rear employing liquor potassic permanganate and carries out disinfection, and then injects the seawater through sand filtration and precipitation process; A filter bag, before injecting seawater, is wrapped up at the pond water inlet pipe mouth in training algae pond, prevents that fine sand from entering pond.
The described algae of step (1) is benthic diatom.Described benthic diatom is a month shape algae, rhombus algae, boat-shaped algae and avette algae.
The described training of step (1) algae be take the water white transparency polyethylene film as adherance.
It is 5: 1: 1 that the described nutritive salt of step (2) contains mass ratio: 1 N element, P element, Si element and Fe element.
Step (2) and (3) described Bdellovibrio are Bdellovibrio (Bdellovibrio sp.) BDFM05, and described Bdellovibrio is preserved in Chinese Typical Representative culture collection center on August 7th, 2009, and deposit number is CCTCCNO:M 209172.
Described Bdellovibrio BDFM05 carries out after negative staining carrying out morphologic observation: BDFM05 under electron microscope 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; Described Bdellovibrio BDFM05 cultivates the transparent circular plaque that can form diameter 2～3mm in three days with the double-layer plate method in 28 ℃;
Described bdellovibrio bdelloplast bacterial bacterium liquid prepares in accordance with the following methods: the host is in the nutrient broth liquid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, culture fluid is through 4 ℃, after the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium, access Bdellovibrio BDFM05, at 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain the culture fluid containing bdellovibrio bdelloplast bacterial and Bdellovibrio nectophore, culture fluid at 4 ℃ of temperature with 6000～8000rpm after centrifugal 15～20min, remove the supernatant containing Bdellovibrio nectophore, the precipitation obtained is bdellovibrio bdelloplast bacterial, the phosphate buffer that for bdellovibrio bdelloplast bacterial, DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value are 7.2～7.6 is suspended, and obtaining concentration is 10
6～10
9the bdellovibrio bdelloplast bacterial bacterium liquid of pfu/mL,
Described host is Aeromonas hydrophila (Aeromonas hydrophila); Described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolysate 0.5g and yeastex extract 0.1g are dissolved in 1000mL distilled water, regulates pH value to 7.2～7.6.
The present invention, with respect to prior art, has following advantage and effect:
(1) bdellovibrio bdelloplast bacterial of the present invention causes a disease to aquatic products or potentially pathogenic organism has strong cracking strength, and bdellovibrio bdelloplast bacterial itself can improve the aquaculture organism intestinal environment, promotes growth, improves Bao Miao immunity.Therefore, with other microorganism formulation, compare, the bdellovibrio bdelloplast bacterial in 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) with telotroch, compare, the leech plastid not only has preparation, it is more convenient to preserve, and the environment tolerance is strong, though action time characteristics more of a specified duration slightly slowly, also there is potential inactivated vaccine function simultaneously.During the leech development of plastid, 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.
(3) what existing existing haliotis diversicolor Reeve seedling growing process adopted is the mode of flowing water, thus, has not only expended huge human and material resources, financial resources, also can cause certain pollution to environment.And the employing cycle change water and carry out haliotis diversicolor Reeve and grow seedlings, can reduce the demand of output, saved production cost, and can not cause harmful effect to environment, the sustainable development of Bao Ye and other aquacultures is had great significance.
The accompanying drawing explanation
The testing result figure of the bacterium infection experiment that Fig. 1 is each group.
Embodiment
Below in conjunction with embodiment, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.For not dated especially technological parameter, can carry out with reference to routine techniques.
It is the invention process abalone culture field, Fujian.Culturing pool is built in cultivation greenhouse, and each pond specification is 2 * 2 * 0.75m, and the depth of water is 0.45m.Disinfecting solution of potassium permanganate is all used in each pond, repeatedly rinses well.The top, pond covers the gobo that obscurity is 60%, avoids illumination excessively strong.The intensity of illumination general control is at (200～5000) lux.
Embodiment 1
(1) preparation of the bdellovibrio bdelloplast bacterial bacterium liquid of growing seedlings for haliotis diversicolor Reeve (Haliotis diversicolor aquatilis)
Inoculation 0.5mL 10 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
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 are cultivated 12 hours, culture fluid, in 4 ℃, the centrifugal 15min of 5000rpm, is abandoned supernatant.Suspend and precipitate thalline with 5mL physiological saline, join afterwards one 100mL DNB liquid nutrient medium (nutrient broth 0.8g, caseinic acid hydrolysate 0.5g are housed, yeastex extract 0.1g, be dissolved in 1000mL distilled water, the pH value is 7.2～7.6) conical flask in, then access 1mL containing 10
3pfu/mL Bdellovibrio BDFM05 (CCTCC M209172).Constant-temperature table 250rpm, 28 ℃ of cultivation 36h.Culture fluid, in 4 ℃ of centrifugal 20min of 6000rpm, is removed the supernatant containing bdellovibrio bdelloplast bacterial, retains precipitation (being bdellovibrio bdelloplast bacterial), adds 10mL DNB liquid nutrient medium Eddy diffusion bdellovibrio bdelloplast bacterial, and obtaining concentration is 10
9the bdellovibrio bdelloplast bacterial bacterium liquid of pfu/mL.
(2) bdellovibrio bdelloplast bacterial bacterium liquid is in the haliotis diversicolor Reeve middle application of growing seedlings
The present invention can be divided into altogether two stages.First stage is the training algae, and the training algae time is 40 days, and this stage is not added bdellovibrio bdelloplast bacterial bacterium liquid; Second stage is the stage of growing seedlings, and content of the present invention is mainly launched in this stage.Seedling raise period is 50 days, and this one-phase experimental group is added immediately bdellovibrio bdelloplast bacterial bacterium liquid after the water of at every turn changing full pond.With different exchange water cycles, throw respectively the bdellovibrio bdelloplast bacterial bacterium liquid of variable concentrations 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, comprise 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 as follows:
The A group: positive perennial draingage, 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 bdellovibrio bdelloplast bacterial bacterium liquid, be the plant produced mode;
B group (B-1, B-2, B-3, B-4): water once within 5 days, to change (full pond);
C group (C-1, C-2, C-3, C-4): water once within 10 days, to change (full pond);
D group (D-1, D-2, D-3, D-4): water once within 15 days, to change (full pond);
E group (E-1, E-2, E-3, E-4): water once within 20 days, to change (full pond);
F group (F-1, F-2, F-3, F-4): water once within 25 days, to change (full pond);
G group (G-1, G-2, G-3, G-4): water once within 30 days, to change (full pond);
B-1, C-1, D-1, E-1, F-1, G-1 group, for change after water immediately toward the bdellovibrio bdelloplast bacterial bacterium liquid of splashing in pond at every turn, make bdellovibrio bdelloplast bacterial cell concentration in water body reach 10pfu/mL.
B-2, C-2, D-2, E-2, F-2, G-2 group, for change after water immediately toward the bdellovibrio bdelloplast bacterial bacterium liquid of splashing in pond at every turn, make bdellovibrio bdelloplast bacterial cell concentration in water body reach 10
3pfu/mL.
B-3, C-3, D-3, E-3, F-3, G-3 group, for change after water immediately toward the bdellovibrio bdelloplast bacterial bacterium liquid of splashing in pond at every turn, make bdellovibrio bdelloplast bacterial cell concentration in water body reach 10
5pfu/mL.
B-4, C-4, D-4, E-4, F-4, G-4 group, for change after water immediately toward the bdellovibrio bdelloplast bacterial bacterium liquid of splashing in pond at every turn, makes bdellovibrio bdelloplast bacterial cell concentration in water body reach 10
7pfu/mL.
In first stage training algae process, every pond cloth adheres to 20 of basement membranes, connects algae kind (naturally giving birth in local seawater), and controlled light intensity 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, it is N: P: Si: Fe=5 that nutritive salt applies ratio: 1: 1: 1 (ppm), after 10 days, can observe on whole film and be pale green and golden yellow edematus, it is for adhering to the skim algae, and microscopy under the microscope takes a morsel, 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 reasonable regulating illumination, the illumination general control, between 200-2000lux, for algae provides more modest condition, is crossed slow and is consumed by Bao Miao to prevent grow too fast accelerated ageing or growth rate of algae.
After algae is trained, the fertilized egg of haliotis diversicolor Reeve is splashed in pond.Every pond (also being the cloth ovum) 50,000 of splashing.Cloth ovum water temperature is controlled at 17-22 ℃ of left and right, in cloth ovum 3 days, can observe in water body a lot of trochophores that swim, and changes water this period and need to filter with 200 mesh filter screens, to prevent larva, runs off.After seven days, larva starts metamorphosis, can see on adherance and be attached with the small young that crawls, and microscopy under careful scraping microscope, observe its vigor.
From the training algae to receiving seedling, time is 90 days, young Bao body grows to 3.4～6.4mm, unicellular alga part on adherance is aging, algae on the minority adherance is eaten up, and blank or de-plate phenomenon are not found in all ponds, and the spatfall amount on every film is 500～800 and does not wait, can arrive 1300 left and right on maximum plates, the index of specifically growing seedlings and measuring method are as follows:
1) haliotis diversicolor Reeve Bao Miao growth and survival condition
A Bao Miao primary quantity, every pond 40,000 seedlings.
It is long that b receives the average shell of seedling, randomly draws 100 Bao Miao, measures shell long, averages.
The long average daily growth amount of c shell, receive the average shell of seedling long, gets the mean value of 50 days.
The average individual weight of d Bao Miao, get 100 Bao Miao at random, claims to average after its gross weight.
E receives the seedling amount, takes 10g Bao Miao, calculates its number, the Bao Miao of collection is claimed to the grain number of estimation Bao Miao after its gross weight.
The f survival rate, when certain stage finishes, Bao Miaoliang accounts for the percentage of this stage throwing seedling amount.
2) the Bao Miao immune indexes is measured
100 of every group of off-test samplings, 20/pipe is put in 5 Eppendoff pipes that fill respectively 1mL Hank ' s buffer solution (pH=7.8), stores 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 of supernatant for immune indexes.
With reference to the Bradford method (document Bradford M.A rapid and sensitive method for the quantification of microgram quantities of protein[J] .Analytical Biochemistry, 1976,72:248-254) working sample protein content, take Bovine serum albumin as standard protein, the Criterion curve, take Coomassie brilliant blue as developer, in 96 hole ELISA Plate, react 10min, by microplate reader, measure the OD in each reaction system
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 buffer solution, shake up rapidly, pour in the cuvette of optical path 1cm, under the 325nm wavelength, every 30s, survey the A value once, require autoxidation speed in the 0.070OD/min left and right.Enzyme assay, before adding 1,2,3,-thrihydroxy-benzene, add SOD sample to be measured, and data measured is calculated as follows enzymic activity:
Enzymic activity=(0.070-A
325nm)/min * 100% * reactant liquor cumulative volume * sample liquid extension rate/(0.070 * 50% * sample liquid is long-pending)
Enzyme unit definition alive: in every milliliter of reactant liquor, the enzyme amount of inhibition 1,2,3,-thrihydroxy-benzene autoxidation rate 50% per minute is defined as enzyme unit (U/mL) alive.
B, antalzyme activity are measured
Take the micrococcus lysodeikticus freeze-dried powder as 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, then add 50 μ L to treat test sample, mix, survey A
0value.Then test solution is moved into to 37 ℃ of temperature and bathe mid-30min, be placed in again ice bath 10min after taking-up, with cessation reaction, survey its A value.Bacteriolytic activity UL is by (A
0-A)/A formula is calculated.
3) bacterium infection experiment
Infection experiment is carried out in the pond that fills 5L sand filtration seawater of sterilization in advance.Randomly draw respectively 100 Bao Miao from control group (A group) and each experimental group (B-G group), the long 4.5～6.0mm of shell.Add Aeromonas hydrophila bacterium liquid in each group water body, make Aeromonas hydrophila cell concentration in water body reach 10
8cfu/mL.Experimental session is flowing water culture not, and normal bait throwing in, with 5L/h/m
3continuous aeration, water temperature is 20 ℃, pH is controlled at 7.2.After the artificial infection, Continuous Observation 5 days, finally be calculated to be motility rate, adopts 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 table 2.Table 1 refers to the impact of different seedling raising mannerses on the mass death growing state.Visible, compared with control group (A group), all experimental group improve the survival rate of Bao Miao more significantly, and promote the growth of Bao Miao.Under identical exchange water cycle, along with the rising of the bdellovibrio bdelloplast bacterial bacterial concentration added, the survival rate of Bao Miao and shell are long also in rising trend gradually.
Table 2 refers to the impact of different seedling raising mannerses on mass death immunity.Data show, compared with control group (A group), 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 bdelloplast bacterial bacterial concentration added, Bao Miao immunity also has certain enhancing.Bacterium infection experiment result as shown in Figure 1.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 bdelloplast bacterial concentration in water body, the relative survival rate of Bao Miao also can rise gradually.In addition, adopt the experimental group of adding bdellovibrio bdelloplast bacterial bacterium liquid, the relative survival rate of every group of Bao Miao all reaches more than 90%.This explanation bdellovibrio bdelloplast bacterial can be brought into play the effect of a potential vaccine, strengthens the immunity of mass death.
The water quality aspect, ammoniacal nitrogen is received oxidizing process with hypobromous acid and is measured (GB12763.4-91); Diazonium for nitrite nitrogen-azo spectrphotometric method for measuring (GB12763.4-91).Through measuring, the every water-quality determination value in A group pond is all higher than experimental group (B-G group).Wherein, the testing result of A group ammoniacal nitrogen and nitrite nitrogen is respectively 0.081mg/L and 0.012mg/L, and, in experimental group (B-G group), the testing result of ammoniacal nitrogen and nitrite nitrogen is respectively 0.076-0.080mg/L and 0.008-0.010mg/L.In addition, total number of bacteria and vibrios sum context of detection, adopt respectively coating nutrient broth flat band method and TCBS flat band method to detect.The result demonstration, total number of bacteria and vibrios sum in A group water body reach respectively 10
5with 10
3the order of magnitude (cfu/mL), the total number of bacteria and the vibrios sum that add the experimental group of bdellovibrio bdelloplast bacterial bacterium liquid are respectively 10
2～10
3with 10
1～10
2the order of magnitude (cfu/mL), lower than control group, illustrate that bdellovibrio bdelloplast bacterial can effectively control the number of bacteria of water body, improve water quality.
The synthesise various result, add bdellovibrio bdelloplast bacterial bacterium liquid, can significantly improve the growth rate of Bao Miao, improves survival rate, strengthens immunity, and can improve water quality, reaches the good result of saving water and energy.
The impact of the different seedling raising mannerses of table 1 on the mass death growing state
The impact of the different seedling raising mannerses of table 2 on mass death immunity
Above-described embodiment is preferably 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 principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (6)
1. an energy-saving method for culturing haliotis diversicolor aquatilis is characterized in that comprising following operating procedure:
(1) training algae: collect seedling and start in first 30～45 days to be trained algae in training algae pond; In training algae process, controlled light intensity, at 2000～5000lux, is controlled water temperature 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 bdellovibrio bdelloplast bacterial bacterium liquid, and make the initial concentration of the bdellovibrio bdelloplast bacterial thalline of Chi Shuizhong reach 10～10
7pfu/mL; During lower seedling, every pond cloth haliotis diversicolor Reeve fertilized egg is 4～50,000, 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: changed water once every 5～30 days, add bdellovibrio bdelloplast bacterial bacterium liquid after changing water at every turn, make the bdellovibrio bdelloplast bacterial cell concentration of Chi Shuizhong reach 10～10
7pfu/mL;
(5) peel off and gather: lower seedling 30～50 days, young Bao shell reaches after 3.0～6.5mm to be peeled off gathers;
The described algae of step (1) is benthic diatom;
Step (3) and (4) described Bdellovibrio are Bdellovibrio (Bdellovibrio sp.) BDFM05, and described Bdellovibrio is preserved in Chinese Typical Representative culture collection center on August 7th, 2009, and deposit number is CCTCCNO:M 209172.
2. a kind of energy-saving method for culturing haliotis diversicolor aquatilis according to claim 1, it is characterized in that: the described training algae of step (1) pond washes rear employing liquor potassic permanganate and carries out disinfection, and then injects the seawater through sand filtration and precipitation process; A filter bag, before injecting seawater, is wrapped up at the pond water inlet pipe mouth in training algae pond, prevents that fine sand from entering pond.
3. a kind of energy-saving method for culturing haliotis diversicolor aquatilis according to claim 1 is characterized in that: described benthic diatom is a month shape algae, rhombus algae, boat-shaped algae and avette algae.
4. a kind of energy-saving method for culturing haliotis diversicolor aquatilis according to claim 1, it is characterized in that: the described training of step (1) algae be take the water white transparency polyethylene film as adherance.
5. a kind of energy-saving method for culturing haliotis diversicolor aquatilis according to claim 1, it is characterized in that: the described nutritive salt of step (2) contains N element, P element, Si element and the Fe element that mass ratio is 5:1:1:1.
6. a kind of energy-saving method for culturing haliotis diversicolor aquatilis according to claim 1, it is characterized in that: described bdellovibrio bdelloplast bacterial bacterium liquid prepares in accordance with the following methods: the host is in the nutrient broth liquid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, culture fluid is through 4 ℃, after the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium, access Bdellovibrio BDFM05, at 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain the culture fluid containing bdellovibrio bdelloplast bacterial and Bdellovibrio nectophore, culture fluid at 4 ℃ of temperature with 6000～8000rpm after centrifugal 15～20min, remove the supernatant containing Bdellovibrio nectophore, the precipitation obtained is bdellovibrio bdelloplast bacterial, the phosphate buffer that for bdellovibrio bdelloplast bacterial, DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value are 7.2～7.6 is suspended, obtain bdellovibrio bdelloplast bacterial bacterium liquid, described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolysate 0.5g and yeastex extract 0.1g are dissolved in 1000mL distilled water, regulates pH value to 7.2～7.6.
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