CN102485884B - Arthrobacter CW9 and its purpose - Google Patents
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- CN102485884B CN102485884B CN2010105724266A CN201010572426A CN102485884B CN 102485884 B CN102485884 B CN 102485884B CN 2010105724266 A CN2010105724266 A CN 2010105724266A CN 201010572426 A CN201010572426 A CN 201010572426A CN 102485884 B CN102485884 B CN 102485884B
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Abstract
The invention relates to Arthrobacter CW9 (Arthrobacter sp. CW9) with CGMCC of No.4197. The bacterial strain is Gram positive bacteria without spore, capsule and motion. The colony on a 2216E flat possesses the characteristics of yellow, circle, intermediate protrusion, smoothness and moisture; The aerobic strain possesses optimum growth temperature of 26 DEG C and the optimum pH value of 7.6; the concentration for NaCl resistance is 10%, the optimum salinity is 1%, the growth can not generated without NaCl. The Arthrobacter CW9 can be used for raising the metamorphosis rate and survival rateof seawater crustaceans larvae. The bacterial strain screening method of the present invention is concise and high efficient, the Arthrobacter possesses probiotic function on the crustaceans larvae, the production method as well as the storage and transportation are simple, the application method is also simple; the Arthrobacter is employed only in crustaceans animal zoea one-stage, so that the metamorphosis rate of the post-larvae can be enhanced and stabilized, and the sprout seed output can be greatly increased.
Description
Technical field
The present invention relates to a strain from the mitten crab water for larval nursing, separate the aquatic products probiotic bacterium Arthrobacter CW9 that obtains (
Arthrobacter Sp.CW9) CGMCC N0. 4197(is deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on October 08th, 2010, and deposit number is CGMCC N0. 4197); The invention still further relates to the purposes of this bacterial strain.
Background technology
In recent years, because being on the rise of the aggravation of culture environment of aquatic products self-contamination and physical environment pollution causes the cultivated animals living environment constantly to worsen, pathogenic micro-organism increases, and velocity of propagation is accelerated, and has caused great loss for the aquatic products aquaculture.Simultaneously in animal body residual of employed some the violated pharmaceutical chemicals of disease preventing and treating, greatly had influence on the quality of product.But because in animal body residual of forbidden drugs such as the chloromycetin that in the sea farming production process, uses in order to prevent and treat disease, malachite green, caused the crustacean quality of breeding seawater to descend, price glides, export volume falls sharply, even is listed in " Black List " import prohibition in most traditional export State and area; Thereby whole seawater crustaceans aquaculture has been caused huge impact, grievous injury health and the Sustainable development of seawater crustaceans aquaculture.Therefore in the long run, must change traditional breed and disease control pattern, the screening beneficial microorganism replaces anti-microbial type pharmaceutical chemicalss such as microbiotic from natural water, solve fundamentally that resistance that microbiotic causes increases and the superinfection and the autogenous infection that cause and destroy problems such as microecological balance, avoid negative impacts such as drug residue that microbiotic brings, secondary pollution, realize cleaning and eco-friendly seawater crustaceans ecologic breeding pattern.
In recent years, the method for several selectable biological control diseases has been successfully used in the crustacean seed production of seawater.The marine bacteria PM-4 that Noganmi K etc. screens can occupy advantage in seawater bacteria group, and make vibrios (
VibrioSpp) quantity descends rapidly, even reaches non-detectable low-level, with this bacterial strain make an addition to Portunus trituberculatus Miers (
Portuns trituberculatus) can increase substantially the surviving rate of the young in the seedling water.Discovery PM-4 such as Maeda M have prebiotic effect to the tigar prawn young equally.Garriques etc. have improved the output of Wan Shi prawn seedling with potential pathogenic bacteria in the vibrio alginolyticus competitive exclusion seedling water.Usefulness such as Rengpipat be added with bud pole bacterium Bacillus S11 the bait feeding tigar prawn (
Penaeus monodon) promoted the prawn growth, and improved the prawn survival rate.Domestic correlative study started to walk from the nineties, many scientific payoffss have been obtained, wherein screening obtains from Chinese prawn probiotic bacterium Arthrobacter XE-7 such as Li Ji autumn can reach the effect of the inhibition morbid vibrio similar to paraxin, the effect that simultaneously can also serve as nitrobacteria, harmful NH3-N is converted into harmless NO3-N, thereby reached the double effects of bacteriosis control and water correction, do not added the surviving rate nearly 50% that has improved young shrimp (post-larvae) under any antibiotic condition.These probioticss are all from the normal bacteria fauna in aquaculture water or cultivated animals body surface or the body, can not work the mischief and become a kind of pattern of biological control day by day cultivated animals.
The feasibility of above-mentioned description of test research and development marine crustacean probiotic bacterium.But the domestic product that yet there are no relevant seawater crustaceans young probiotic bacterium emerges at present.Microniological proudcts such as photosynthetic bacterium, genus bacillus are difficult to that aquaculture water is carried out the master that act as of water quality regulation seawater crustaceans pathogenetic bacteria is had specific inhibition.
Summary of the invention
Technical problem to be solved by this invention is at the deficiencies in the prior art, selects a kind of new aquatic products probiotic bacterium Arthrobacter CW9.
Another technical problem to be solved by this invention has provided the purposes of above-mentioned Arthrobacter CW9, and it carries out biological control to the aquaculture disease.
Feature of the present invention comprise Arthrobacter CW9 (
Arthrobacter Sp.CW9) bacterial strain (following or title bacterial strain CW9), and the method (purposes) of utilizing this bacterial strain itself.
Bacterial strain CW9 involved in the present invention be the Arthrobacter CW9 that from the mitten crab water for larval nursing, is separated to (
Arthrobacter Sp.CW9), this bacterial strain CW92010 was deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms October 08, and deposit number is CGMCC N0. 4197.Depositary institution address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Below technical solution of the present invention is further set forth.
One, the separation of bacterial strain CW9 of the present invention and screening.
(1) bacterial strain screening and cultivation.
Gather the mitten crab water for larval nursing, behind this water gradient dilution, be applied on the 2216E marine bacteria solid medium 20 ℃ of constant temperature culture 72~96h; The inoculation of selecting different colonial morphologies is stand-by to the preservation of 2216E marine bacteria slant medium; Then the inclined-plane inoculation is arrived the 2216E liquid nutrient medium, 20 ℃, 160r/min, behind the cultivation 48h, centrifugal back obtains the bacterium thalline; Get each centrifugal back and obtain the bacterium thalline, infect the method for the mitten crab flea shape first phase young of firm membrane by individual plant bacterium thalline, relatively flea shape second phase larval metamorphosis rate causes the highest bacterium of flea shape second phase larval metamorphosis rate to be aquaculture probiotic strain CW9.
(2) morphological specificity of bacterial strain CW9 and growth characteristics.
1, morphological specificity
Gram-positive microorganism, no gemma, no pod membrane does not move.Bacterium colony yellow on the 2216E flat board, circle, center protrusion, smooth, moistening.Stationary phase, cell was most spherical, φ 0.5~1 μ m, a small amount of rod-short φ 0.2~0.4 m * 1.4~2.0 m; Vigorous period is all shaft-like, the end circle, and arrangement often is in the shape of the letter V.The rod bacterium gram is weak positive even negative.
2, the kind of bacterial strain CW9 is identified.According to the 16S rDNA sequence of bacterial strain CW9 and the comparison result of BLAST, this bacterium is genus arthrobacter
Arthrobacter
3, bacterial strain CW9 growth characteristics.
Bacterial strain CW9 is aerobic bacteria, and is aerobic, and growth temperature range is 0 ℃~28 ℃, and optimum growth temperature is 26 ℃, and growth pH scope is 5.5~11.0, and optimal pH is 7.6; The concentration of anti-NaCl is 10%, and optimal salt concn is 1%, and no NaCl does not grow.Bacterial strain CW9 can utilize glucose, lactose, semi-lactosi and sucrose, can not utilize starch, rhamnosyl, fructose, cellobiose and seminose; N.F,USP MANNITOL can be utilized, Xylitol, arabitol and inositol can not be utilized; Citrate, urea, arginine decarboxylase, catalase reacting positive, malonate, salicin, L-Ala desaminase, polychrom, lysine decarboxylase, hydrogen sulfide, indole reaction feminine gender; Can not liquefy gelatin.Below make a more detailed description.
Culture medium prescription progressively is optimized from basic fermention medium.The basis fermentative medium formula is: glucose 5 g/L, ammonium sulfate 4g/L, extractum carnis 1g/L, NaCl 2 g/L, PH7.6.
(1) NaCl concentration is to the influence of bacterial strain CW9 growth.
Other components unchanged of basis fermention medium change NaCl concentration, and the salt tolerance of bacterial strain CW9 is investigated.Fig. 1 has shown that NaCl concentration is respectively under 0~12g/L condition, and bacterial strain CW9 shaking table is measured absorbancy (diluting 4 times) after cultivating 21h under the 620nm.As can be seen, bacterial strain CW9 does not have NaCl and does not grow among Fig. 1; When NaCl concentration was 1g/L, bacterial strain CW9 growing state was best; When NaCl concentration reached 10g/L, bacterial strain CW9 growth was suppressed fully.
(2) influence that substratum is formed and culture condition is grown to bacterial strain CW9.
1. different carbon sources are to the influence of bacterial strain CW9 growth.
Other components unchanged of basis fermention medium, carbon source is selected following 4 kinds for use: glucose, lactose, semi-lactosi and sucrose, concentration is constant, still is 5g/L.Other compositions of substratum are: peptone 5g, and yeast extract paste 1g, NaCl 2g, distilled water 1000ml, pH 7.6.
Preparation 300ml nutrient solution, 5 250ml triangular flasks of packing.Add 5 kinds of carbon source compositions respectively by the 6g/L amount.Insert bacterial strain CW9 slant strains behind the autoclaving.28 ℃, behind the 180r/min shake-flask culture 21h, nutrient solution is measured the OD value with 4 times of distilled water dilutings, 620nm.The results are shown in Figure 2.Can be found out intuitively that by Fig. 2 glucose is the optimum carbon source of bacterial strain CW9 growth.
2. glucose concn is to the influence of bacterial strain CW9 growth.
Other components unchanged of basis fermention medium, glucose concn is respectively 2 g/L, 4 g/L, 6 g/L, 8 g/L and 10g/L.Behind the shake-flask culture 21h, 4 times of dilutions of nutrient solution, 620nm measures the OD value.The results are shown in Figure 3.Graphic representation by Fig. 3 can obtain, and the optimum concn of glucose is 6g/L in the substratum.
3. different nitrogen sources is to the influence of bacterial strain CW9 growth.
Other conditions are constant, change nitrogenous source into peptone, urea, ammonium nitrate and ammonium sulfate, concentration 6g/l.Other compositions of substratum: glucose 6g, extractum carnis 1g(is as somatomedin), NaCl 2g, distilled water 1000ml, PH7.6-7.8.28 ℃, behind the 180r/min shake-flask culture 21h, nutrient solution is measured the OD value and be the results are shown in Figure 4 with 4 times of distilled water dilutings, 620nm.As seen from Figure 4, the bacterial strain CW9 of ammonium sulfate correspondence compares with other nitrogenous source compositions, and biomass is the highest, is optimum nitrogen source.
4. ammonium sulfate concentrations is to the influence of bacterial strain CW9 growth.
Ammonium sulfate concentrations is respectively 2g/L in the experiment, 4 g/L, 6 g/L, 8 g/L, 10g/L.Knot is looked into and is seen Fig. 5, and as shown in Figure 5, the ammonium sulfate optimal concentration is 6g/L.
5. somatomedin is to the influence of bacterial strain CW9 growth.
With the corn starch, extractum carnis and yeast extract paste are as somatomedin respectively, and 28 ℃, behind the 180r/min shake-flask culture 21h, nutrient solution is measured the OD value with 4 times of distilled water dilutings, 620nm.The results are shown in Figure 6.Fig. 6 shows that 2 kinds of somatomedins of yeast extract paste and other compare, and absorbancy is the highest.Therefore choose yeast extract paste as the optimum growh factor.
6. the optimization of orthogonal test substratum is formed.
Select glucose, yeast extract and ammonium sulfate to design the orthogonal test of 3 levels as 3 factors, the fermented liquid of preparation utilizes L15(3
3) the orthogonal table contrived experiment carries out the optimization of fermention medium.Behind the shake-flask culture 21h, nutrient solution is measured with 4 times of distilled water dilutings, 620nm.Level of factor is seen Figure 11, the results are shown in Figure 12.
As seen from Figure 12: A1B2C3 is best collocation, i.e. glucose 7g/L, ammonium sulfate 6g/L, yeast extract paste 1.5g/L.
Can find out by the R value: R
CR
BR
A, namely secondly somatomedin kind and ratio are nitrogenous sources to the bacterial strain CW9 growth maximum that exerts an influence, carbon source influence minimum.
7. the influence of the bacterial strain CW9 growth of initial pH.
Initial pH value is respectively 6.8,7.2 in the experiment, and 7.6,7.8,8.0,8.4.Behind the shake-flask culture 21h, nutrient solution is measured with 4 times of distilled water dilutings, 620nm, the results are shown in Figure 7.Absorbancy maximum when initial pH is 7.6 is chosen 7.6 and is best pH as seen from Figure 7.
8. inoculum size is to the influence of bacterial strain CW9 growth.
Inoculum size in the experiment is made as 1%, 2%, 3%, 4%, 5%, 6%, and behind the shake-flask culture 21h, nutrient solution is measured with 8 times of distilled water dilutings, 620nm, the results are shown in Figure 8.Fig. 8 shows: the nutrient solution absorbancy reaches the highest when inoculum size is 5%.
9. shaking speed is to the influence of bacterial strain CW9 growth.
The shaking speed of setting in the experiment is respectively 125 r/min150r/min, 175r/min, and 200r/min, behind the shake-flask culture 21h, nutrient solution is measured with 8 times of distilled water dilutings, 620nm, the results are shown in Figure 9.Obtaining best shaking speed by Fig. 9 is 175r/min.
10. temperature is to the influence of bacterial strain CW9 growth.
Temperature in this experimentation is set at 20 ℃ respectively, and 23 ℃, 26 ℃, 29 ℃, behind the shake-flask culture 21h, nutrient solution is measured with 8 times of distilled water dilutings, 620nm, the results are shown in Figure 10.Obtaining best temperature condition by Figure 10 is 26 ℃.
Based on the above results, Optimal compositions of fermentation medium component and the optimal culture condition of the bacterial strain CW9 that obtains are: glucose 7g/L, ammonium sulfate 4g/L, yeast extract paste 1.5g/L, NaCl 2g/L, initial pH7.6, shaking speed 175r/min, inoculum size 5%, 26 ℃ of temperature, incubation time 21h.
(3) purposes of bacterial strain CW9 of the present invention and using method.
The resulting bacterial strain CW9 of isolation cultivation method of the present invention can improve distortion ratio and the survival rate of the seawater crustaceans young.The described seawater crustaceans young comprises the seawater crustaceans young such as mitten crab or Penaeus vannamei.The using method that is used for the bacterial strain of this purposes is: after this bacterial strain was carried out large scale culturing according to the optimization culture condition, the centrifugal thalline that gets added the nature seawater of sterilizing and is made into 1~10 * 10
8The dense bacterium liquid of cfu/ml, flea shape first phase stage of the seawater crustaceans young according to 1~10 * 10
5The concentration of cfu/ml is thrown in to aquaculture water, throws in 1-2 time in every 1-2 days, and bait adopts 75 ℃ of pasteurizations to throw something and feed after 30 minutes, and every day, quantity of exchanged water control was at 10%-30%; After metamorphosis is the flea shape second phase young, by the normal method management.
Compared with prior art, technical solution of the present invention has the following advantages:
1, the method for screening bacterial strain CW9 involved in the present invention is succinctly efficient;
2, this bacterial strain CW9 has prebiotic effect to the multiple Crustacean young;
3, this probiotic bacterium production and conveying method are simple, and using method is easy;
4, this probiotic bacterium is only thrown at Crustacean flea shape first phase, can improve and the distortion ratio in stable young later stage, thereby increase substantially seed output.
Description of drawings
Fig. 1 is that NaCl concentration is to the figure that influences of thalli growth.
Fig. 2 is that different carbon sources are to the figure that influences of thalli growth.
Fig. 3 is that glucose concn is to the figure that influences of thalli growth.
Fig. 4 is that different nitrogen sources is to the figure that influences of thalli growth.
Fig. 5 is that ammonium sulfate concentrations is to the figure that influences of thalli growth.
Fig. 6 is the figure that influences of growth factor pair thalli growth.
Fig. 7 is the figure that influences of the thalli growth of initial pH.
Fig. 8 is that inoculum size is to the figure that influences of thalli growth.
Fig. 9 is that shaking speed is to the figure that influences of thalli growth.
Figure 10 is that temperature is to the figure that influences of thalli growth.
Figure 11 is the level of factor chart.
Figure 12 takes into account the interpretation of result chart for the substratum orthogonal experiment plan.
Figure 13 is the abnormal situation parallel tables of the mitten crab flea shape I phase young.
Figure 14 is the distortion ratio growing state parallel tables of the Penaeus vannamei flea shape I phase young.
The bacterial strain Arthrobacter CW9(that the present invention relates to
Arthrobacter Sp.CW9) oneself is deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on October 08th, 2010, and deposit number is CGMCC N0. 4197.Depositary institution address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment
Below further describe concrete technical scheme of the present invention, so that those skilled in the art further understands the present invention, and do not constitute the restriction to its right.
Embodiment 1.A kind of Arthrobacter CW9(
Arthrobacter Sp.CW9) CGMCC N0. 4197.
Embodiment 2.Embodiment 1 described Arthrobacter CW9(
Arthrobacter Sp.CW9) among the CGMCC N0. 4197, the strain characteristics of described Arthrobacter CW9 is: this bacterial strain is gram-positive microorganism, no gemma, and no pod membrane does not move; Bacterium colony yellow on the 2216E flat board, circle, center protrusion, smooth, moistening; Stationary phase, the cell majority was spherical in shape, and φ 0.5~1 μ m is rod-short φ 0.2~0.4 m * 1.4~2.0 m on a small quantity; It is shaft-like that vigorous period all is, the end circle, and arrangement often is in the shape of the letter V; Characteristic feature with Arthrobacter, namely spherical bacterium Gram-positive in period is positive even negative a little less than the rod bacterium gram in period; Aerobic, growth temperature range is 0 ℃~28 ℃, and optimum growth temperature is 26 ℃, and growth pH scope is 5.5~11.0, and optimal pH is 7.6; The concentration of anti-NaCl is 10%, and optimal salt concn is 1%, and no NaCl does not grow.
Embodiment 3. Embodiment 1 or 2 described Arthrobacter CW9(
Arthrobacter Sp.CW9) among the CGMCC N0. 4197, the fermention medium of described Arthrobacter CW9 is: glucose 7g/L, ammonium sulfate 6g/L, yeast extract paste 1.5g/L, NaCl 1g/L, pH7.6; Fermentation condition is: 5%, 20 ℃ of inoculum size, and 175r/min, culture cycle is 21h.
Embodiment 4.With embodiment 1 described Arthrobacter CW9(
Arthrobacter Sp.CW9) CGMCC N0. 4197 improves distortion ratio and the survival rate of the seawater crustaceans young; The described seawater crustaceans young is mitten crab or Penaeus vannamei.The using method of Arthrobacter CW9 is: after Arthrobacter CW9 enlarged culturing, the centrifugal thalline that gets adds the nature seawater of sterilizing and is made into 1~10 * 10
8The dense bacterium liquid of cfu/ml, flea shape first phase stage of the seawater crustaceans young according to 1~10 * 10
5The concentration of cfu/ml is thrown in to aquaculture water, throws in 1-2 time in every 1-2 days.
Embodiment 5.With reference to Figure 13-14.The infectation of bacteria of the seawater crustaceans first phase young that carries out with embodiment 1 described bacterial strain CW9 and the benefit fruit that comes into force is tested.
Bacterial strain used in this experiment is: after bacterial strain CW9 enlarged culturing, the centrifugal thalline that gets adds the nature seawater of sterilizing and is made into bacterium liquid.
The mitten crab of incubation of membrane or rapid the copying with 150 order nets of the flea shape first phase young of Penaeus vannamei nauplius larva metamorphosis are fished for, in the beaker of the aseptic seawater of 1000ml that after aseptic seawater cleans, is placed in, 30 in each beaker, 3 every group are parallel; One group is contrast, and all the other each group groups are added bacterial strain CW9 bacterium liquid (10 respectively
6CFU/ml), continuous charge, 20 ℃ of water temperatures are changed water (aseptic seawater) every day and once and are again added bacterium liquid (10 of the same race
6CFU/ml); The sterilized bait of throwing something and feeding was in right amount cultivated 3-5 days, recorded the survival number of the young every day, and statistics flea 2 phase of shape young number is also calculated distortion ratio, and experiment repeats twice, gets the mean value of respectively organizing data.
The influence of the seawater crustaceans of bacterial strain CW9 survival rate of seedling is as follows:
1, Figure 13 is seen in the influence of the distortion ratio of the mitten crab flea of the bacterial strain CW9 shape I phase young.
Compare with blank, the distortion ratio (50%) of the mitten crab flea shape first phase of interpolation bacterial strain of the present invention is higher by 51.5% than contrast (33%).
2, Figure 14 is seen in the influence of the distortion ratio of the Penaeus vannamei flea of the bacterial strain CW9 shape I phase young.
Compare with blank, the distortion ratio (70%) of the Penaeus vannamei flea shape first phase of interpolation bacterial strain of the present invention is higher by 40% than contrast (50.3%).
Embodiment 6.The culture experiment that Application Example 1 described bacterial strain CW9 carries out.
Experiment one.
Choose healthy and strong mitten crab parent crab to be produced, placing 3 volumes respectively is that 30 cubic metres cement pit is concentrated hatching, make in the water body about flea shape I phase young density 150,000 tail/cubic meters, continuous charge, water temperature 20-21 ℃, the yolk that 200 meshes of throwing something and feeding are in right amount filtered once adds bacterial strain (bacterial density 10
5-10
6CFU/ml), change water (the sterilization seawater after the processing) 30% every day, and add bacterial strain again and (throw in bacterial density 10
5-10
6CFU/ml), after 90% individuality becomes the flea shape II phase young, operate according to Routine Management.Test-results is, 3 nursery ponds obtain 9 kilograms of mitten crab megalopa larvas, 10 kilograms and 12 kilograms respectively, average 10.3 kilograms; And the mean yield that this other nursery pond of not throwing in bacterial strain of the present invention the same period obtains the mitten crab megalopa larva is 7.5 kilograms, on average increases production 37.3%.
Experiment two.
The Penaeus vannamei nauplius larva that the vigor of choosing is good, placing 3 volumes respectively is the concentrated hatching of cement pit of 30 cubic metres (floorage is 20 square metres), make that the density of nauplius larva is about 120,000 tail/cubic meters in the water body, continuous charge, water temperature 22-23 ℃, treat that nauplius larva metamorphosis is throw something and feed in right amount behind the flea shape I phase young yolk and the yeast of the filtration of 250 meshes, once adds bacterial strain (bacterial density 10
5-10
6CFU/ml), change water (the sterilization seawater after the processing) 30% every day, embodying strain (is thrown in bacterial density 10
5-10
6CFU/ml), after 90% individuality becomes the flea shape II phase young, operate according to Routine Management.Test-results is that 3 nursery ponds obtain young shrimp 2,480,000 tails of Penaeus vannamei, 2,800,000 tails and 3,100,000 tails, average 2,790,000 tails respectively; And the mean yield of not throwing in the young shrimp of this other nursery pond acquisition Penaeus vannamei of bacterial strain of the present invention the same period is 2,340,000 tails, on average increases production 19.2%.
Claims (3)
1. an Arthrobacter (Arthrobacter sp.) CW9 is characterized in that its biological preserving number is CGMCC No. 4197.
2. an Arthrobacter as claimed in claim 1 (Arthrobacter sp.) CW9 is for the preparation of the purposes of the additive of the distortion ratio that improves the seawater crustaceans young and survival rate; The described seawater crustaceans young is Eriocheir or Penaeus vannamei.
3. purposes according to claim 2 is characterized in that: the using method that is used for the Arthrobacter CW9 of this purposes is: after Arthrobacter CW9 enlarged culturing, centrifugal thalline adds the nature seawater of sterilizing and is made into 1~10 * 10
8The dense bacterium liquid of cfu/ml, flea shape first phase stage of the seawater crustaceans young according to 1~10 * 10
5The concentration of cfu/ml is thrown in to aquaculture water, throws in 1-2 time in every 1-2 days.
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