CN106701622A - Bacillus and chlorella mixture, and preparation method and application of bacillus and chlorella mixture - Google Patents
Bacillus and chlorella mixture, and preparation method and application of bacillus and chlorella mixture Download PDFInfo
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Abstract
The invention provides a bacillus and chlorella mixture. A volume ratio of bacillus to chlorella in the mixture is (1-9):3; the bacillus and the chlorella are compatible reasonably according to water quality of different cultivation periods; due to a synergistic effect of the bacillus and the chlorella, concentrations of ammonia nitrogen and nitrite in a water body can be reduced obviously; a dissolved oxygen content is increased; the mixture can effectively inhibit growth of enteric pathogenic bacteria of aquatic products, improve resistance and increase a survival rate when the mixture is applied to aquaculture of fishes, shrimps, crabs and the like.
Description
Technical field
The present invention relates to aquaculture alimentary field, more particularly to a kind of bacillus and chlorella intermixture and its system
Preparation Method and application.
Background technology
Aquatic products industry high density, intensive culture also bring many new problems while yield is greatly improved, research
Show, bait utilization is relatively low in breeding process, in addition, the organic matter such as biological waste, corpse largely accumulates on bottom of pond, has
Evil microbe species and increasing number, the noxious material that organic matter is produced under its decomposition are consequently increased.In addition, pond
Originally it is closing, a single ecosystem, its self-cleaning itself and regulating power are weaker, and aquaculture organism is chronically at biochemical oxygen demand
In the water bodys of index severe overweight such as amount (COD), BOD (BOD), ammonia nitrogen, nitrate, nitrite, sulfide, disease
Evil outburst probability is greatly improved, and aquatic products quality is also heavily affected.According to incompletely statistics, the whole nation occurs moderate every year
The farming disease harms area above accounts for more than the 20% of the cultivation gross area, and year, lost units was ten thousand tons more than 146.
Bacillus is a kind of aerobic gram-positive bacteria, is the bacterium nontoxic to people and animals, and bacillus is in water
The many ectoenzymes secreted after propagation can decompose the starch in water body and bed mud, the organic matter such as protein and fat；And it is many with ammonia
The end product of metabolisms such as gas, nitrogen and carbon dioxide are permeated into air, greatly reduce the concentration of poisonous and harmful substance in water, surely
Determine water body items physical and chemical index, reached improvement water quality and maintained the purpose of the breeding water body ecological balance.But, bacillus is more
Number is aerobic bacteria, and during its quick breeding, dissolved oxygen is reduced rapidly in water body, is easily caused cultivated animals death by suffocation
Die.
Chlorella is the producer in aquaculture pond, is the excellent bait of cultivated animals containing abundant nutriment
Material.But its photosynthesis can only utilize the inorganic salts ingredients in water body, the organic matters such as excrement, residual bait not acted on, especially
In the cultivation later stage, water body organism accumulation is more, and water body light transmittance is more low to be all unfavorable for that chlorella growth is bred, therefore bead
Algae is used alone, and its proliferative speed does not reach ideal effect.
The content of the invention
At present, bacillus is used alone in aquaculture or chlorella is very universal, bacterium algae is used in mixed way and has no
Report.For problem present in existing aquaculture, the present invention mixes bacillus and chlorella and for aquaculture,
The environment of breeding water body and bed mud can effectively be improved, larger molecular organicses can be converted into chlorella and directly utilized by bacillus
Small-molecule substance, while produce growth factor promote chlorella growth；Chlorella passes through photosynthesis using big in water simultaneously
The elements such as N, P of amount, so as to reduce the content of nutrient salt in water, and discharge a large amount of oxygen, improve content of oxygen dissolved in water, more
Mend the defect that bacillus is used alone, for bacillus and aquaculture organism provide abundant oxygen, give full play to chlorella and
The synergy of bacillus, gets good clean-up effect.
The present invention provides a kind of bacillus for being particularly suitable for aquaculture and chlorella intermixture, gemma in the intermixture
Bacillus is (1-9) with the consumption volume ratio of chlorella:3.
Bacillus of the present invention and the preparation method of chlorella intermixture, the method are comprised the following steps：
(1) bacillus kind is activated, using LB culture mediums, pH7-7.5,121 DEG C of autoclaving 20min, in 30-35 DEG C of training
Support 16-20 hours；
(2) one-level of bacillus, secondary liquid Amplification Culture, using 50L fermentation tank cultures, by 10-20% inoculum concentrations
Inoculation；
(3) by two grades of bacillus 4000rpm of culture, centrifugation, collects thalline is diluted cell concentration with LB culture mediums
To 5.0 × 109CFU/mL；
(4) chlorella algae kind is activated, using BG11 nutrient solutions, pH6-8,121 DEG C of high-temperature sterilization 20min, algae kind presses 1:2 or
1:3 inoculations, in 25-30 DEG C of temperature, cultivate 5-7 days under the conditions of illumination 5000-10000lx；
(5) one-level of chlorella, two grades of Amplification Cultures, using 5 tons of glass pipe cultures, by 1:5 or 1:10 inoculations, in temperature
25-30 DEG C of degree, cultivates 5-7 days under the conditions of illumination 5000-10000lx；
(6) algae mud is collected in the bead algae solution 4000rpm for two grades spreading cultivation, centrifugation, with BG11 nutrient solutions that algae mud concentration is dilute
Release to 2.0 × 109cell/mL；
(7) bacillus, the chlorella for obtaining above-mentioned culture, respectively by volumes below than mixing：Bacillus with it is small
Ball algae 1:3；Bacillus and chlorella 1:1；Bacillus and chlorella 3:1, it is standby in 0-5 DEG C of preservation.
The LB culture mediums, its formula is included：Dusty yeast 5-7g/L, tryptone 10-12g/L；
The BG11 nutrient solutions, its formula is included：NaNO30.15-0.3g/L, K2HPO40.04-0.06g/L,
MgSO4·7H2O 0.075-0.1g/L, CaCl2·2H2O 0.036-0.05g/L, Citric acid 0.006-0.01g/L,
Ferric ammonium citrate 0.01-0.02g/L, EDTANa20.001-0.005g/L, Na2CO3 0.02-0.1g/
L, micro- A5Mother liquor 1-2ml/L；
The micro- A5Mother liquor formula is H3BO32.86g/L, MnCl2·4H2O 1.86g/L, ZnSO4·7H2O
0.22g/L, Na2MoO4·2H2O 0.021g/L, CuSO4·5H2O 0.08g/L, Co (NO)3·6H2O 0.05g/L。
The present invention also provides the application of bacillus and chlorella intermixture, is applied to aquaculture：
(1) in plantlet stage, i.e., 1-15 days, spice fed：The intermixture and feed are fully mixed thoroughly, per kilogram feed
Stirring 0.1L intermixtures, feed 3 times daily；
If (a) seedling ingest it is bad, digest it is bad, from bacillus and chlorella 3:1 intermixture, specific aim changes
Good seedling intestinal environment.
If b () seedling individuality is modest, slow-growing, from bacillus and chlorella 1:1 intermixture, for seedling provides
Sufficient protein and unrighted acid.
(2) big pool cultivation stages, i.e., 16-90 days：
A () cultivates 16-25 days, it is 1 to take volume ratio:3 intermixtures, by intermixture 5L/ mus of consumption full pool spilling head, sprinkle for every 2 days
Spill 1 time；Using the quick breeding of chlorella, stabilization algae phase forms cultivation early stage preferably water colour.
B () cultivates 26-60 days, it is 1 to take volume ratio:1 intermixture, by intermixture 5L/ mus of consumption full pool spilling head, splashes daily
1 time；Using residual bait and excrement in bacillus fast decoupled water body, ammonia in water body is quickly reduced with reference to chlorella photosynthesis
Nitrogen and content of nitrite, increase dissolved oxygen.
C () cultivates 61-90 days, it is 3 to take volume ratio:1 intermixture, by intermixture 5L/ mus of consumption full pool spilling head, splashes daily
1 time；Effect is that after cultivation 61 days, the poisonous and harmful substance accumulated in water body is more, and water environment is more severe, and pathogen invasion is several
Rate is larger, using the quick breeding of bacillus, can play and suppress pathogen well, adjusts the effect of Bacterial community, then tie
Chlorella photosynthesis is closed, the recovery of water quality can be accelerated, reduce the incidence of disease.
Beneficial effects of the present invention：(1) it is for the water quality situation in different breeding period, bacillus is reasonable with chlorella
Compatibility, using both synergies, can substantially reduce ammonia nitrogen, the nitrite concentration in water body, increase dissolved oxygen content；
(2) it is applied to the aquacultures such as fish shrimp crab, can effectively suppresses the growth of aquatic products enteric pathogenic bacteria, build up resistance, carries
High viability.
Specific embodiment
Technical scheme is described further below：
A kind of bacillus and the preparation method of chlorella intermixture, the method are comprised the following steps：
(1) bacillus kind is activated, using LB culture mediums, pH7-7.5,121 DEG C of autoclaving 20min, in 30 DEG C of cultures
18 hours；
(2) one-level of bacillus, secondary liquid Amplification Culture, using 50L fermentation tank cultures, are connect by 20% inoculum concentration
Kind；
(3) by two grades of bacillus 4000rpm of culture, centrifugation, collects thalline is diluted cell concentration with LB culture mediums
To 5.0 × 109CFU/mL；
(4) chlorella algae kind is activated, using BG11 nutrient solutions, pH6-8,121 DEG C of high-temperature sterilization 20min, algae kind presses 1:2 connect
Kind, in 25 DEG C of temperature, cultivated 5 days under the conditions of illumination 10000lx；
(5) one-level of chlorella, two grades of Amplification Cultures, using 5 tons of glass pipe cultures, by 1:10 inoculations, in temperature 25
DEG C, cultivated 5 days under the conditions of illumination 10000lx；
(6) algae mud is collected in the bead algae solution 4000rpm for two grades spreading cultivation, centrifugation, with BG11 nutrient solutions that algae mud concentration is dilute
Release to 2.0 × 109cell/mL。
(7) bacillus, the chlorella for obtaining above-mentioned culture, respectively by volumes below than mixing：Bacillus with it is small
Ball algae 1:3；Bacillus and chlorella 1:1；Bacillus and chlorella 3:1, it is standby in 0-5 DEG C of preservation.
The LB culture mediums, its formula is included：Dusty yeast 5g/L, tryptone 10g/L；
The BG11 nutrient solutions, its formula is included：NaNO30.15g/L, K2HPO40.04g/L, MgSO4·7H2O
0.075g/L, CaCl2·2H2O 0.036g/L, Citric acid 0.006g/L, Ferric ammonium citrate
0.01g/L, EDTANa20.001g/L, Na2CO30.02g/L, micro- A5Mother liquor 1ml/L；
The micro- A5Mother liquor formula is H3BO32.86g/L, MnCl2·4H2O 1.86g/L, ZnSO4·7H2O
0.22g/L, Na2MoO4·2H2O 0.021g/L, CuSO4·5H2O 0.08g/L, Co (NO)3·6H2O 0.05g/L。
Experimental group
By bacillus obtained above and chlorella intermixture, for Environment of Litopenaeus vannamei Low cultivation：
(1) in plantlet stage, i.e., 1-15 days, spice fed：Take bacillus described in 5L and chlorella 1：1 intermixture sprays
In 50 kg diet surfaces, fully mix thoroughly, feed daily 3 times；
(2) big pool cultivation stages, i.e., 16-90 days：
A () cultivates 16-25 days, it is 1 to take volume ratio:3 intermixtures, by intermixture 5L/ mus of consumption full pool spilling head, sprinkle for every 2 days
Spill 1 time；
B () cultivates 26-60 days, it is 1 to take volume ratio:1 intermixture, by intermixture 5L/ mus of consumption full pool spilling head, splashes daily
1 time；
C () cultivates 61-90 days, it is 3 to take volume ratio:1 intermixture, by intermixture 5L/ mus of consumption full pool spilling head, splashes daily
1 time.
Control group 1
By bacillus obtained above, for Environment of Litopenaeus vannamei Low cultivation：
(1) in plantlet stage, i.e., 1-15 days, spice fed：Take 5L bacillus bodies and be sprayed on 50 kg diet surfaces, fill
Divide and mix thoroughly, feed daily 3 times；
(2) big pool cultivation stages, i.e., 16-90 days：
A () cultivates 16-25 days, by 5L/ mus of consumption full pool spilling head of bacillus body, splash 1 time within every 2 days；
B () cultivates 26-90 days, by 5L/ mus of consumption full pool spilling head of bacillus body, splash daily 1 time.
Control group 2
By chlorella obtained above, for Environment of Litopenaeus vannamei Low cultivation：
(1) in plantlet stage, i.e., 1-15 days, spice fed：Take 5L bead algae solutions and be sprayed on 50 kg diet surfaces, fully
Mix thoroughly, feed daily 3 times；
(2) big pool cultivation stages, i.e., 16-90 days：
A () cultivates 16-25 days, by 5L/ mus of consumption full pool spilling head of bead algae solution, splash 1 time within every 2 days；
B () cultivates 26-90 days, by 5L/ mus of consumption full pool spilling head of bead algae solution, splash daily 1 time.
Control other cultivating conditions consistent, periodically sampling detects COD, ammonia nitrogen, nitrous acid in breeding water body (every 9 days)
Salt, vibrios, total bacterial content, as a result as shown in following table 1-5, after cultivation terminates, determine Growth of Litopenaeus vannamei index, as a result as follows
Shown in table 6.
Result shows that the present invention can not only be effectively reduced COD, ammonia nitrogen, the content of nitrite in breeding water body, reach
To remarkable effects of purification quality, suppress the breeding of pathogenic bacteria, reduce the incidence of disease of cultivated animals, and can also promote well
Growth of Litopenaeus vannamei, improves survival rate.
The breeding water body COD contents of table 1
The breeding water body ammonia-nitrogen content of table 2
The cultivation water nitrite content of table 3
The culture water vibrios content of table 4
The total bacterial content of the breeding water body of table 5
The Environment of Litopenaeus vannamei Low of table 6 items growth indexes
| Project | Experimental group (numerical value ± SE) | Control group 1 (numerical value ± SE) | Control group 2 (numerical value ± SE) |
| Body (cm)/tail long | 6.35±0.38a | 5.86±0.29b | 5.77±0.24b |
| Body weight (g)/tail | 24.72±0.21a | 21.28±0.15b | 21.89±0.27b |
| Survival rate (%) | 69.3±0.04a | 62.1±0.01b | 57.3±0.04b |
Remarks：Ab represents the otherness (P of experimental group, control group growth indexes in lattice<0.01)；SE represents standard error.
Claims (7)
1. a kind of bacillus and chlorella intermixture, it is characterised in that the use of bacillus and chlorella in the intermixture
Amount volume ratio is (1-9):3.
2. the preparation method of a kind of bacillus according to claim 1 and chlorella intermixture, it is characterised in that including
Following steps：
(1) bacillus kind is activated, using LB culture mediums, pH7-7.5,121 DEG C of autoclaving 20min, in 30-35 DEG C of culture
16-20 hours；
(2) one-level of bacillus, secondary liquid Amplification Culture, using 50L fermentation tank cultures, are inoculated with by 10-20% inoculum concentrations；
(3) by two grades of bacillus 4000rpm of culture, be diluted to for cell concentration with LB culture mediums by centrifugation, collects thalline
5.0×109CFU/mL；
(4) chlorella algae kind is activated, using BG11 nutrient solutions, pH6-8,121 DEG C of high-temperature sterilization 20min, algae kind presses 1:2 or 1:3
Inoculation, in 25-30 DEG C of temperature, cultivates 5-7 days under the conditions of illumination 5000-10000lx；
(5) one-level of chlorella, two grades of Amplification Cultures, using 5 tons of glass pipe cultures, by 1:5 or 1:10 inoculations, in temperature
25-30 DEG C, cultivated 5-7 days under the conditions of illumination 5000-10000lx；
(6) the bead algae solution 4000rpm for two grades spreading cultivation, centrifugation, collect algae mud, with BG11 nutrient solutions by algae mud concentration dilution extremely
2.0×109cell/mL；
(7) bacillus, the chlorella for obtaining above-mentioned culture, respectively by volumes below than mixing：Bacillus and chlorella
1:3；Bacillus and chlorella 1:1；Bacillus and chlorella 3:1, it is standby in 0-5 DEG C of preservation.
3. the preparation method of bacillus according to claim 2 and chlorella intermixture, it is characterised in that the LB trainings
Support based formulas and include dusty yeast 5-7g/L, tryptone 10-12g/L.
4. the preparation method of bacillus according to claim 2 and chlorella intermixture, it is characterised in that the BG11
Culture formula of liquid is included：NaNO30.15-0.3g/L, K2HPO40.04-0.06g/L, MgSO4·7H2O 0.075-0.1g/L,
CaCl2·2H2O 0.036-0.05g/L, Citric acid 0.006-0.01g/L, Ferric ammonium citrate
0.01-0.02g/L, EDTANa20.001-0.005g/L, Na2CO30.02-0.1g/L, micro- A5Mother liquor 1-2ml/L.
5. the preparation method of bacillus according to claim 4 and chlorella intermixture, it is characterised in that described micro
Elements A5Mother liquor formula is H3BO32.86g/L, MnCl2·4H2O 1.86g/L, ZnSO4·7H2O 0.22g/L, Na2MoO4·
2H2O 0.021g/L, CuSO4·5H2O 0.08g/L, Co (NO)3·6H2O 0.05g/L。
6. the application of a kind of bacillus according to claim 1 and chlorella intermixture, it is characterised in that for aquatic products
Product are cultivated.
7. the application of bacillus according to claim 6 and chlorella intermixture, it is characterised in that comprising following step
Suddenly：
(1) in plantlet stage, i.e., 1-15 days, spice fed：The intermixture and feed are fully mixed thoroughly, the stirring of per kilogram feed
0.1L intermixtures, feed 3 times daily；
If (a) seedling ingest it is bad, digest it is bad, from bacillus and chlorella 3:1 intermixture；
If b () seedling individuality is modest, slow-growing, from bacillus and chlorella 1:1 intermixture；
(2) big pool cultivation stages, i.e., 16-90 days：
A () cultivates 16-25 days, it is 1 to take volume ratio:3 intermixtures, by intermixture 5L/ mus of consumption full pool spilling head, splash 1 time for every 2 days；
B () cultivates 26-60 days, it is 1 to take volume ratio:1 intermixture, by intermixture 5L/ mus of consumption full pool spilling head, splashes 1 time daily；
C () cultivates 61-90 days, it is 3 to take volume ratio:1 intermixture, by intermixture 5L/ mus of consumption full pool spilling head, splashes 1 time daily.
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