CN106754559A - A kind of probiotics preparation method for effectively purifying water - Google Patents
A kind of probiotics preparation method for effectively purifying water Download PDFInfo
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- CN106754559A CN106754559A CN201710067235.6A CN201710067235A CN106754559A CN 106754559 A CN106754559 A CN 106754559A CN 201710067235 A CN201710067235 A CN 201710067235A CN 106754559 A CN106754559 A CN 106754559A
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- water
- bacillus
- ammonia nitrogen
- shrimp
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention discloses a kind of probiotics preparation method for effectively purifying water, its method is：Water sample, bed mud, fecal specimens and separation screening bacillus flora in gut of shrimp are gathered from shrimp culture pond, bacterial strain is obtained by 80 DEG C of water-baths, ammonia nitrogen Screening of Media, identified with morphological feature, physio-biochemical characteristics result and 16S rDNA again, Primary Study is carried out to it by safety testing, the indigenous seawater bacillus with good characteristic is filtered out；The bacillus that the present invention is filtered out can reduce the content of ammonia nitrogen and nitrite in water, and then influence is produced on prawn vivo immunization enzymatic activity, also can play a part of to promote prawn body immunity, it has broad application prospects in probiotics application.
Description
Technical field
The present invention relates to technical field of aquaculture, prepared by specifically a kind of probiotics for effectively purifying water
Method.
Background technology
With the fast development of culture fishery, intensive cultivation scale expands day by day, because the pattern cultivation density is high,
Feeding volume is big, unreasonable along with mode is fed, and causes substantial amounts of feed to remain, and makes body eutrophication, algae excessive propagation
And cause breeding water body severe depletion of oxygen, while in cultivated animals excrement and residuum connate water, be decomposed generation under anaerobic environment
The poisonous intermediate material such as hydrogen sulfide, ammonia nitrogen, nitrite, causes water quality deterioration, harmful microorganism to grow, and has a strong impact on China's water
Produce the long term growth of aquaculture.
The content of the invention
Defect and deficiency it is an object of the invention to be directed to prior art, there is provided it is a kind of effectively purify water it is prebiotic
Bacterium preparation method, the organic matter in the rapid degrading cultivation pond of bacillus energy, effectively improves water quality, and bacillus also has
Have no toxic side effect, noresidue, do not develop immunity to drugs, the advantage such as good stability.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of effectively probiotics preparation method for purifying water, its method is：Water sample, bottom are gathered from shrimp culture pond
Mud, fecal specimens and separation screening bacillus flora in gut of shrimp, obtain by 80 DEG C of water-baths, ammonia nitrogen Screening of Media
Bacterial strain, then identified with morphological feature, physio-biochemical characteristics result and 16S rDNA, it is carried out by safety testing
Primary Study, filters out the indigenous seawater bacillus with good characteristic.
Beneficial effects of the present invention are：The bacillus for filtering out can reduce the content of ammonia nitrogen and nitrite in water,
And then influence is produced on prawn vivo immunization enzymatic activity, it is also possible to play a part of to promote prawn body immunity, it is micro-
Had broad application prospects in ecological agent application.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is influence of the JNSY1 bacterial strains of present invention screening to breeding water body ammonia-nitrogen content；
Fig. 2 is influence of the JNSY1 bacterial strains of present invention screening to cultivation water nitrite content；
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with accompanying drawing and specific implementation
Mode, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain this
Invention, is not intended to limit the present invention.
First, strain isolation and screening：
(1) 5g samples, are taken, addition is filled in the triangular flask of 45ml SPSSs.Suspension is in holding at 80 DEG C
20min, kill can not form other thalline of gemma.
(2), take the treated suspension of 5ml to be linked into the triangular flask equipped with 45ml enriched mediums, 37 DEG C of enrichment trainings
Support 24h~48h.
(3) bacterium solution after 1ml enrichment cultures, is taken, with sterilized water dilution gradient to 10-1、10-2、10-3、10-4、10-5、10-6、10-7, dilution factor is 10-6、10-7Bacteria suspension respectively take 100 μ L and be coated on isolation medium, 37 DEG C of culture 24h.
(4), select well-grown bacterium colony line to separate, 4 DEG C of inclined-planes preservations of dominant colony are selected after repeating 2-3 times and is treated
With.
(5) each Bacillus strain for, obtaining, after being diluted with 1% SPSS, the bacterium solution coating that will be diluted
Rod flat board is applied on ammonia nitrogen degradation bacterium screening and culturing medium, 30 DEG C, 24h cultures, observes strain growing way, and picking colony is big, growth
Fireballing dominant strain conservation is standby.
(6), by enrichment, separate and purify, obtain 8 plants of bacteriums, respectively numbering be HDSY1, YKSY1, JNSY1,
YKCD1, HDCD1, JDCD1, HDFB1, JNFB1 and YKFB1.8 plants of bacterium after activated are inoculated on screening and culturing medium, 30 DEG C of trainings
After supporting 24h, observation discovery bacterial strain JNSY1 bacterium colonies are big, fast growth.Therefore selection JNSY1 is test strain.
2nd, identification of strains：
1st, picking single bacterium colony purifying, the dyeing microscopic examination on nutrient agar, JNSY1 bacterial strains single bacterium colony is Chinese wax shape table
The smooth milky circle projection in face, diameter about 2mm；Grain stain is the positive, in elongated rod shape.
2nd, take 1ml and be enriched with bacterium colony nutrient solution in centrifuge tube, 12000r/min is centrifuged 5min.Remove supernatant to be subsequently adding
100ul sterilized waters mix, 100 DEG C of heating water bath 10min, and ice bath 2min, 12000r/min centrifugation 5min, supernatant is the bacterium
DNA profiling.
PCR amplification system：Reaction volume is 11 μ L, including 5 10 × buffer of μ L, 1 μ L dNTPs, forward and reverse primer is each
1.5 μ L, 1 μ L template DNAs, 1 μ LTaqDNA polymerases.Amplification condition：95℃5min；95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C
1.5min, 35 circulations；72℃7min.(forward primer is 27F：5 '-AGAGTTTGATCCTGGCTCAG-3 ', reverse primer
1492R：5 '-CTACGGCTACCTTGTTACGA-3 ').
Amplified production is detected with 2% agarose gel electrophoresis, and amplified production is transferred into institute of microbiology of Chinese Academy of Sciences bacterium
Planting collection carries out sequencing identification, and gained sequence is committed to US National Bioinformatics Institute and carries out Blast sequence analysis,
16S rDNA sequence results are compared and understand that bacillus cereus similitude is 99.9%, category bacillus cereus category, with reference to its shape
State and physiological and biochemical property, strain JNSY1 are defined as bacillus cereus.
3rd, safety testing：
(1), JNSY1 strain cultured solutions are separately added into the breeding water body of Environment of Litopenaeus vannamei Low, make the final of water body bacterium
Concentration is adjusted to be 10 respectively8、107、106Cfu/mL, labeled as test group；And 2 control groups are set, in control group water body respectively
Deliver sterile saline and 108The bacteria suspension of cfu/mL Aeromonas hydrophilas, every group sets 2 parallel, guarantees in process of the test
Water normally is changed, 1 feed, and continuous charge are fed daily.
(2) after, observation finds 14d, SPSS is inoculated with test group and control group in Environment of Litopenaeus vannamei Low box for breeding
Prawn normal activity and can ingest, survival rate is 100%.The Environment of Litopenaeus vannamei Low dissected and randomly select is checked, bacterium is found
Strain JNSY1 does not cause the abnormal response of prawn；And prawn survival rate is only after Aeromonas hydrophila is vaccinated with control group
48%, therefore think that bacillus cereus JNSY1 is safe to prawn.
4th, effects of purification quality：
1st, add 1L distilled water to be made sludge leachate from the sludge of plant's sampling, be 10 concentration8Cfu/mL bacterial strains
The bacteria suspension of JNSY1 is accessed in sludge leachate, and the ultimate density for making bacterium solution in water is 107cfu/mL。
2nd, quiescent culture 10d under room temperature condition, if 1 parallel test, ammonia nitrogen is determined every 2d with reagent colorimetric method
Content, purification of water quality test ammonia nitrogen concentration measurement result (see Fig. 1).The concentration of test group ammonia nitrogen is dropped to from 2.26 μ g/mL
0.78 μ g/mL, decline obvious, and degradation rate is up to 65.5%；And ammonia nitrogen concentration is in slow downward trend in control group, from 2.19 μ g/
ML drops to 1.77 μ g/mL, declines 19.2%.Illustrating bacillus cereus JNSY1 can effectively degrade ammonia nitrogen in water body.
3 and every the 2d contents of alpha-naphthylamine colorimetric method for determining nitrite, purification of water quality tests the measure of ammonia nitrogen concentration
As a result (see Fig. 2).Test group nitrite concentration is decreased obviously, and degradation rate is up to 68.3%；Control group content of nitrite only under
23.5% is dropped, this experiment shows, after adding strain JNSY1 bacterial strains, ammonia nitrogen and content of nitrite in breeding water body are presented
Obvious downward trend, and effect is significant when using 2d, this shows that the strain being capable of effectively degradation of ammonia nitrogen and nitrite
Content, has good application prospect as microecological microbial agent.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires to be limited rather than described above, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.Any reference in claim should not be considered as the claim involved by limitation.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each implementation method is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should
Specification an as entirety, the technical scheme in each embodiment can also be formed into those skilled in the art through appropriately combined
May be appreciated other embodiment.
Claims (1)
1. a kind of effectively probiotics preparation method for purifying water, it is characterised in that：Its method is：Gathered from shrimp culture pond
Water sample, bed mud, fecal specimens and separation screening bacillus flora in gut of shrimp, by 80 DEG C of water-baths, ammonia nitrogen culture medium
Screening obtains bacterial strain, then is identified with morphological feature, physio-biochemical characteristics result and 16S rDNA, by safety testing
Primary Study is carried out to it, the indigenous seawater bacillus with good characteristic is filtered out.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048340A (en) * | 2017-10-10 | 2018-05-18 | 盐城裕达饲料有限公司 | A kind of bacillus cereus and its application |
| CN108060096A (en) * | 2017-12-12 | 2018-05-22 | 中国水产科学研究院黄海水产研究所 | A kind of probiotic combinations and its application in litopenaeus vannamei seed rearing |
| CN109456920A (en) * | 2018-11-30 | 2019-03-12 | 江苏大学 | The Halophilic Bacterium bacterial strain seawater bacillus of one plant of raising alec fermentation quality |
| LU102735A1 (en) * | 2021-04-06 | 2021-10-06 | Univ Qingdao Agricultural | A manufacturing process of probiotics to effectively purify water quality |
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| CN109456920A (en) * | 2018-11-30 | 2019-03-12 | 江苏大学 | The Halophilic Bacterium bacterial strain seawater bacillus of one plant of raising alec fermentation quality |
| CN109456920B (en) * | 2018-11-30 | 2021-09-10 | 江苏大学 | Moderately halophilic bacteria strain bacillus marinus for improving fermentation quality of fish paste |
| LU102735A1 (en) * | 2021-04-06 | 2021-10-06 | Univ Qingdao Agricultural | A manufacturing process of probiotics to effectively purify water quality |
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