CN104004695B - Bacilli for environmental modification of stichopus japonicus aquaculture pond and application thereof - Google Patents
Bacilli for environmental modification of stichopus japonicus aquaculture pond and application thereof Download PDFInfo
- Publication number
- CN104004695B CN104004695B CN201410277400.7A CN201410277400A CN104004695B CN 104004695 B CN104004695 B CN 104004695B CN 201410277400 A CN201410277400 A CN 201410277400A CN 104004695 B CN104004695 B CN 104004695B
- Authority
- CN
- China
- Prior art keywords
- bacillus strain
- bacterial strain
- bacilli
- culture
- bacillus cereuss
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention provides the (bacilli sp.) A1 which is stored in a common microorganism center of the China Committee for Culture Collection of Microorganisms of Microbiology Research Institute of China Science Academy which is located at No.3 of yard 1 of the Beichen west road of Chaoyang District of Beijing, and the storage number is CGMCC No. 8686. The provided bacillus strain is original bacilli obtained by being directly separated and screened from aquatic water, and therefore the provided bacillus strain is low in potential fulminant perniciousness to stichopus japonicas and environment ecology when applied to a stichopus japonicus aquaculture pond. The bacillus strain is screened through low temperature and organic degradation capacity to obtain the target bacillus strain, and the target bacillus strain grows well under low temperature and is high in degradation capacity on ammonia nitrogen and COD in bait.
Description
Technical field
The invention belongs to Aquatic product beneficial microbe triage techniqueses field is and in particular to a kind of be used for apostichopus japonicus culture pond environment
The bacillus cereuss repaired.
Background technology
Radix Morinae Bulleyanae (apostichopus japonicus selenka) aquaculture is an emerging sea-farming industry, from 2002
Since year, apostichopus japonicus culture industry quickly grows, and national apostichopus japonicus culture area has reached 8.4 ten thousand hectares within 2006, and yield reaches 7.6 ten thousand
Ton, the output value is close to 10,000,000,000 yuan.However, the cultivation scale developing rapidly with it, relevant issues also gradually display.Due to
Most of apostichopus japonicus culture relies primarily on and feeds man-made feeds at present, feedstuff poor adherence, and at the bottom of pond is also specially provided with substantial amounts of people
Work attachment base, can produce the residual of a large amount of residual baits, feces therefore in breeding process.These organic pollutions are not only given birth to Radix Morinae Bulleyanae
Long environment causes severe contamination, and the healthy growth that can directly endanger Radix Morinae Bulleyanae leads to the generation of disease, seriously hinders Radix Morinae Bulleyanae
The sustainable development of the even whole aquatic products industry of aquaculture.
Research finds, residual bait and Excreta are piled up in breeding environment particularly in substrate, become water pollution
Important endogenous, they constantly can discharge n, p nutritive salt and solubility organic pollution in breeding environment, leads to water quality to be ruined,
Oxygen consumption increases, and phytoplankton, benthon multiformity reduce, so that the generation of wawter bloom or red tide.
And be directed to disease and frequently need medication unavoidably, various antibiotic such as sulfonamides, tetracycline, penicillin, furans etc.,
Result leads to the secondary pollution cultivating, and produces a lot of negative effects, and the medicine major part such as putting into all directly is lost to ring
In border, directly pollution environment is so that final constitute harm to closing on waters.
Degrading microorganism as one of decomposer most important in terrestrial ecosystem, in terms of the Degradation and Transformation of pollutant
Show huge application potential.From the eighties, research in terms of environmental pollution repairing and treating for the microorganism gets more and more,
People are own to be widely used in each pollution field, such as processes in heavy metal pollution, oil spill, pesticide residues, sanitary wastewater
Etc. a lot of aspects.Fast development with aquaculture and a series of problem of environmental pollutions of appearance, degrading microorganism here is led
The applied research in domain has caused the extensive attention of people.
Content of the invention
It is an object of the invention to provide a kind of bacillus cereuss, the spore bar that is, a plant can purify water under cryogenic
Bacteria strain, thus make up the deficiencies in the prior art.
The present invention provides bacillus (bacillus sp.) a1, is located at Beijing in preservation on the 06th in 01 month in 2014
The China Committee for Culture Collection of Microorganisms of city Chaoyang District North Star West Road No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute
Common micro-organisms center, preserving number is cgmccno.8686.
The bacillus cereuss being screened are used for the purification of culture-pool water quality；
Described cultivating pool is preferably the cultivating pool of Radix Morinae Bulleyanae；
The bacillus cereuss of present invention screening also act as immunostimulant or feed additive；
Described feedstuff is preferably apostichopus japonicus Selenka feed；
On the other hand, the present invention also provides the microbial inoculum made using above-mentioned fermentation of bacillus.
The bacterium of the present invention is used for repairing apostichopus japonicus culture water environment having the beneficial effect that compared with alternate manner
1st, compared to chemical means, using the bacterial strain existing original in nature, environment is repaired, decrease chemistry
Residual in water environment for the material, enables cultivation water environment to obtain sustainable use.
2nd, the bacterial strain of this offer is to be directly separated the indigenous bacterium that obtains of screening from breeding water body, therefore by its amplification culture
Reapply relatively low to Radix Morinae Bulleyanae itself and environmental ecology potential fulminant hazardness to apostichopus japonicus culture pond.
3rd, the bacterial strain that the present invention provides screens through low temperature, and organic degradation ability is screened, and finally gives aimed strain, gained
Aimed strain well-grown at low temperature is all stronger for the ammonia nitrogen in bait and cod degradation capability.
Specific embodiment
Although the research for microorganism remediation environment is relatively more, microorganism is used for purifying Stichopus japonicus pond water environment
Research report is few.On the other hand, Radix Morinae Bulleyanae growth temperature condition is low temperature, just progresses into aestivation, Radix Morinae Bulleyanae more than 24 DEG C of Radix Morinae Bulleyanaes
Suitable growth temperature is 12-20 DEG C, and optimum temperature is 17 DEG C -18 DEG C, therefore provides one plant of bacterium purifying water under cryogenic
Strain purifies for Apostichopus japonicus in Ponds pond water quality has highly important practice significance.
The proportioning of the materials such as culture medium used in the present invention is as follows:
1st, Nutrient agar culture plate:
The nutrient agar (peptone 10g/l, sodium chloride 5g/l, Carnis Bovis seu Bubali cream 3-5g/l, sea water 1l) that will prepare, in
Sterilize at 121 DEG C 15min.By sterilizing after culture medium, in sterilizing flat board, standby after cooling.
2nd, sterilize bait fluid medium:
Weigh 20g Stichopus japonicus Selenka bait, soaked overnight in 1000ml sterilizing sea water.Bait impregnation liquid is filtered, in filtrate
Add 2g Carnis Bovis seu Bubali cream, then be diluted to 1000ml with sea water, its subpackage is sterilized in conical flasks to 10, in each conical flask
150ml, remaining culture medium is loaded in conical flask, and sterilize at 121 DEG C 15min, and cooling is standby.
With reference to specific embodiment, the present invention is described in detail.
First, the screening of bacterial strain
1st, sample collecting
1.1 formulation acquisition scheme
Before gathering, lookup plan collection sample is existing provides about data such as geography, ecology and biological characteristicses
Material, formulates acquisition scheme according to the data grasped.
1.2 collections prepare
According to the acquisition scheme formulated, determine collection pond, acquisition time and collection sample.And perform collector, adopt
The arrangement of collection instrument, collection vehicle etc..Sampling instrument in advance prior to 121 DEG C at sterilize 15min.
1.3 collection in worksite
Pond have chosen the apostichopus japonicus culture pond of Jimo saltworks, and during sampling, in pond, four points are sampled.Adopt
Collection sample scope selects sediment of pond, cultivated animals, plant, water body, attachment base etc..Record sampling pond hydrologic condition such as temperature
Degree, ph value, salinity etc..Sterile working is noted during collection in worksite.Use disposable sterilized glove, by bed mud, cultivated animals with
Plant is loaded in aseptic sample sack, with sterile sampling bottle from the quantitative water body of setting position fill.
2nd, bacillus cereuss screening
2.1 sample pretreatment
Processed according to the physical state of sample.By the sample sediment of pond of collection, cultivated animals, plant, water body, attached
Base etc., take supernatant with grinding to beat after 10 times of dilutions of sterile saline, obtain stock sample solution.Liquid-like does not do pre- place
Reason.
2.2 bacillus cereuss separate
By stock sample solution after 80 DEG C of water-bath 10min, with 10 times of gradient dilutions of sterile saline to suitable concentration.
Concrete dilution process is by the regulation of gb4789.2-2010.Often two parts of inoculations will be made by a sample diluting liquid, using flat board coating
Method is inoculated on nutrient agar, cultivates 24h at 28 DEG C.The compound method of nutrient agar presses gb/
The regulation of t4789.28-2003.
2.3 bacillus cereuss purification
By well-grown on nutrient agar panel, the discrepant single bacterium colony of the bacterium colony that detects by an unaided eye respectively streak inoculation in
Nutrient agar is placed in culture 24h at 28 DEG C.Through multiple purification of ruling, obtain single strain.
3rd, bacillus cereuss postsearch screening and identification
Preparation before screening:
Sterilizing bait fluid medium
Weigh 20g Stichopus japonicus Selenka bait, soaked overnight in 1000ml sterilizing sea water.Bait impregnation liquid is filtered, in filtrate
Add 2g Carnis Bovis seu Bubali cream, then be diluted to 1000ml with sea water, its subpackage is sterilized in conical flasks to 5, in each conical flask
150ml, remaining culture medium is loaded in conical flask, and sterilize at 121 DEG C 15min, and cooling is standby.
The low temperature screening of 3.1 bacillus cereuss
The bacillus cereuss streak inoculation that primary screening is obtained, on nutrient agar plate medium, cultivates one at 17 DEG C
Zhou Hou, selects the bacterial strain having obvious growth to carry out follow-up experiment.
The organic matter degradation ability screening of 3.2 bacillus cereuss
The spore bacterial strain that primary screening is obtained, chooses a single bacterium colony with Inoculating needle, is inoculated in the taper of the sterilizing of labelling
In bottle, 17 DEG C, after 160r/min constant-temperature table shaken cultivation 5d, sample and take supernatant after 5000r/min centrifugation 10min, measure
The content of supernatant cod, nh4-n.Represented respectively with 100 × (1-cod5/cod0) %, 100 × (1-nh4-n5/nh4-n0) %
The degradation rate to organic pollution in Stichopus japonicus Selenka bait and the 5d of ammonia nitrogen for each bacterial strain.Utilize the ability of Stichopus japonicus Selenka bait according to antibacterial, that is,
Bacterial strain is weighed to the removal effect of cod in Stichopus japonicus Selenka bait, ammonia nitrogen, and the bacterial strain of screening good degrading effect is studied.Finally
Screening obtains one plant of bacterium, and after culture 5d, its cod and ammonia nitrogen degradation effect are optimum, and degradation rate is respectively 69.5% and 61.7%.
3.3 the potential pathogenic detection of bacterial strain a1
Had or not potentially pathogenic using blood plate (being purchased from Jinan Babio Biotech Co., Ltd.) detection bacterial strain,
To verify bacterial strain whether as pathogen.With sterile toothpick, picking bacterial strain a1 single bacterium colony dibbling, in blood plate, is subsequently placed in 17 respectively
DEG C, observe no haemolysis circle after constant temperature culture 24～72h.Bacterial strain a1 no pathogenicity is described, can be used for economic living cultivating pool
Throw in, and the feed additive as economic living.
The identification of 3.4 bacillus cereuss
The preparation of pcr template dna:
Genome dna adopts water-boiling method to extract.The single bacterium colony of the activated rear picking of bacterial strain in 50 μ l sterile distilled waters, in
Steaming and decocting 5-10min in 100 DEG C of boiling water baths, centrifuging and taking supernatant is as pcr template dna.
The mensure of the 16srdna sequence of bacterial strain:
16srdna amplimer is antibacterial 16srdna universal primer, forward primer: 5 '-agagtt tga tcc tgg
Ctc ag-3 ' (27f), reverse primer: 5 '-tac ggc tac ctt gtt acg act t-3 ' (1492r).According to pcr body
System (Niu Yufeng etc., 2009) carries out pcr reaction amplification to bacterial strain.Pcr product (1.5kb about) deliver to Beijing after electrophoresis detection
Three rich Radix Polygalae Bioisystech Co., Ltd carry out purification and sequencing.By sequencing result with blast software and genbank (http: //
Www.ncbi.nlm.nih.gov the related 16srrna sequence planted that belongs in) is compared, by identification of strains to genus or kind.
Bacterial strain is located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese science in January 6 preservation in 2014 now
The China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology of institute, preserving number is
cgmccno.8686.
2nd, the degraded application in a low temperature of bacterium
1st, the activation culture of bacillus cereuss
By the Flavobacterium a1 for cgmccno.8686 for the deposit number and other bacterial strain a2～a7 preserving and bacterium on the market
Totally 8 bacillus (numbering respectively is a1, a2, a3, a4, a5, a6, a7, a8) are inoculated in 28 DEG C on enrichment medium strain a8
After the activation of 160rpm condition, then streak inoculation, on nutrient agar panel, cultivates 24h at 28 DEG C.
2nd, degradation capability detection under bacterium low temperature
Embodiment 1:
A single bacterium colony will be chosen with Inoculating needle, be inoculated in accordingly going out of a1, a2, a3, a4, a5, a6, a7, a8 labelling respectively
In the conical flask of bacterium, two conical flasks are not inoculated as blank.All conical flasks are placed in 28 DEG C, 160r/min constant-temperature table shakes
After swinging culture 5d.Sample from corresponding conical flask respectively and take supernatant after 5000r/min centrifugation 10min, mensure supernatant cod,
The content of nh4-n.Represent each bacterial strain to thorn respectively with 100 × (1-cod5/cod0) %, 100 × (1-nh4-n5/nh4-n0) %
The degradation rate of the 5d of organic pollution and ammonia nitrogen in ginseng bait.Concrete numerical value is as shown in the table.
| Strain number | Cod degradation rate | Ammonia nitrogen degradation rate |
| a1 | 73.70% | 67.10% |
| a2 | 22.80% | - 82.60% |
| a3 | 9.80% | - 6.70% |
| a4 | 3.60% | 26.80% |
| a5 | 55.80% | 14.00% |
| a6 | 63.10% | - 10.60% |
| a7 | - 13.70% | - 30.20% |
| a8 | 67.90% | 23.40% |
From the point of view of experimental result, in 5 day time, 8 plants of bacteriums, all there is different degrees of degraded to ammonia nitrogen and cod
Effect, but exist bacterial strain when the 5th day itself degradation capability it is difficult to decompose itself generation ammonia nitrogen.
The degradation rate of cod is ordered as a1 > a8 > a6 > a5 > a2 > a3 > a4 > a7 from high to low, and the wherein degradation rate of bacterial strain a1 is high
Reach 73.70%, in all bacterial strains obvious to cod degradation effect, more than 20 times of about minimum bacterial strain, have good
Cod degradation capability.
The degradation rate of ammonia nitrogen is ordered as a1 > a4 > a8 > a5 > a3 > a6 > a7 > a2, the wherein degradation rate of bacterial strain a1 from high to low
Up to 67.10%, in all bacterial strains obvious to ammonia nitrogen degradation effect, five times of about minimum bacterial strain, have good
Ammonia nitrogen degradation ability.
Embodiment 2:
Prepare the 250ml conical flasks of four bait culture medium that sterilize equipped with 100ml, choose bacterial strain a1 and on the market with Inoculating needle
Two kinds of existing bacillus cereuss (a8, a9), are inoculated in the conical flask of three sterilizings, a conical flask is not inoculated as sky respectively
In vain.Conical flask is placed in 17 DEG C, after 160r/min constant-temperature table shaken cultivation 5d.Corresponding conical flask sample in 5000r/min from
Take supernatant after heart 10min, measure the content of supernatant cod, nh4-n.With 100 × (1-cod5/cod0) %, 100 × (1-nh4-
N5/nh4-n0) % represents the degradation rate to organic pollution in Stichopus japonicus Selenka bait and the 5d of ammonia nitrogen for each bacterial strain respectively.After 5d, bacterial strain
The cod of a1 and ammonia nitrogen degradation rate respectively 57.73%, 38.06%, the cod of market existing bacillus cereuss a8 and ammonia nitrogen degradation rate
Being respectively the 49.05%, cod of 18.31%, a9 and ammonia nitrogen degradation rate is 33.07%, 12.04%.As can be seen that being screened
The bacterial strain a1 arriving, compared with bacillus cereuss existing on the market, has more excellent decomposition efficiency at low temperature.
Embodiment 3:
With marking pen in starch plating medium (peptone 10g, nacl5g, Carnis Bovis seu Bubali cream 5g, soluble starch 2g, distillation
Water 1000ml, agar 15～20g, 121 DEG C of sterilizing 20min) bottom divides four parts into.Bacterial strain a1 is inoculated in the culture of starch
On base, four parallel, 18 DEG C of culture 2d, plus Lu's Ge Shi iodine solution is observed, and finds achromatic region around a1.Meanwhile, by its in
It is inoculated in the test kit of the moo1mr-vp being purchased from Beijing Luqiao Technology Co., Ltd. at 18 DEG C, after culture 48h, Deca first
Base red reagent cm1003 observes discovery and is changed into red.It is inoculated at 18 DEG C simultaneously and is purchased from Beijing overpass technology Limited Liability
The test kit of the m035 hydrogen sulfide of company, after culture 48h, finds to generate black precipitate.
Experimental result can be seen that the mr experiment of bacterial strain a1, h2S experiment, Starch Hydrolysis experiment all show as the positive, explanation
Bacterial strain a1 all has degradation capability for starch, sugar, aminoacid.This also partial interpretation bacterial strain a1 good organic matter degradation energy
Power.
Claims (7)
1. a kind of bacillus cereuss (bacillus sp.) a1 is it is characterised in that the deposit number of described bacillus cereuss is cgmcc
no.8686.
2. application in purifying water of culture pond for the bacillus cereuss described in claim 1.
3. application as claimed in claim 2 is it is characterised in that described cultivating pool is apostichopus japonicus culture pond.
4. application in preparing immunostimulant or feed additive for the bacillus cereuss described in claim 1.
5. a kind of immunostimulant is it is characterised in that described immunostimulant includes the bacillus cereuss described in claim 1
Viable bacteria.
6. a kind of feed additive is it is characterised in that described feed additive includes the bacillus cereuss described in claim 1
Viable bacteria.
7. feed additive as claimed in claim 6 is it is characterised in that described feedstuff is apostichopus japonicus Selenka feed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410277400.7A CN104004695B (en) | 2014-06-19 | 2014-06-19 | Bacilli for environmental modification of stichopus japonicus aquaculture pond and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410277400.7A CN104004695B (en) | 2014-06-19 | 2014-06-19 | Bacilli for environmental modification of stichopus japonicus aquaculture pond and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104004695A CN104004695A (en) | 2014-08-27 |
| CN104004695B true CN104004695B (en) | 2017-01-18 |
Family
ID=51365605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410277400.7A Active CN104004695B (en) | 2014-06-19 | 2014-06-19 | Bacilli for environmental modification of stichopus japonicus aquaculture pond and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104004695B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841747B (en) * | 2018-06-25 | 2020-08-14 | 青岛农业大学 | Protease-producing bacillus subtilis and method of use |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134558A (en) * | 2010-01-25 | 2011-07-27 | 辽宁省海洋水产科学研究院 | Bacillussubtilis and application thereof in raising Apostichopus japonicus |
| CN102559533A (en) * | 2010-12-23 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Bacillus atrophaeus, and preparation and application of bacillus atrophaeus |
| CN102559534A (en) * | 2010-12-23 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Bacillus cereus, and preparation and application of bacillus cereus |
-
2014
- 2014-06-19 CN CN201410277400.7A patent/CN104004695B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134558A (en) * | 2010-01-25 | 2011-07-27 | 辽宁省海洋水产科学研究院 | Bacillussubtilis and application thereof in raising Apostichopus japonicus |
| CN102559533A (en) * | 2010-12-23 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Bacillus atrophaeus, and preparation and application of bacillus atrophaeus |
| CN102559534A (en) * | 2010-12-23 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Bacillus cereus, and preparation and application of bacillus cereus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104004695A (en) | 2014-08-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105255782B (en) | There is fiber bacterium and the purposes of reducing power to Cr VI | |
| CN106399178B (en) | Bacillus amyloliquefaciens and its application with degradation Phos and bacteriostasis | |
| CN106399177B (en) | Bacillus amyloliquefaciens and its microbial inoculum with degradation Phos and bacteriostasis | |
| CN103008339B (en) | Microbial remediation method for basic chromium pollution soil | |
| CN103992963A (en) | Bacillus megaterium and application thereof | |
| CN105950495B (en) | One plant of Methylotrophic bacillus and its application in livestock breeding wastewater processing | |
| CN102703363B (en) | Bacillus methylotrophicus UTM401 and applications thereof | |
| CN106148232A (en) | The bacterial isolates of one strain antagonism plant pathogenetic bacteria and application thereof | |
| CN102703351A (en) | Bacillus sp. UTM03 and application thereof | |
| CN109234167A (en) | A kind of chlorella and its application in biogas slurry purification | |
| CN104004695B (en) | Bacilli for environmental modification of stichopus japonicus aquaculture pond and application thereof | |
| CN105886437A (en) | Composition or composite bacterium agent for controlling water body pollution | |
| CN103382450B (en) | Streptomyces sp. strain T115 with broad-spectrum antibacterial activity and its application | |
| CN105779367A (en) | Coral-associated marine bacillus amyloliquefaciens strain CoMb-9 and application thereof | |
| CN102382784A (en) | Salt-resistant Serratiasp. strain and application thereof in restoration of salinized petroleum-contaminated soil | |
| CN105349462B (en) | One plant of American aloe bacillus Hexi1 and its application in compost | |
| CN105060499B (en) | A kind of compound micro-ecological preparation for improving breeding water body transparency and its application | |
| CN104894026B (en) | The cultural method of bafillus natto and its application | |
| CN103820342B (en) | Selenium-enriched yeast and application thereof | |
| CN103695335B (en) | Mesorhizobium huakuii and use thereof | |
| CN106148218A (en) | Aquatic product photosynthetic bacteria Tiny ecosystem and application thereof | |
| CN105039218B (en) | One plant of simple bacillus and its cultural method and application | |
| CN115287212A (en) | Salt-tolerant growth-promoting bacterium ACP81 and application thereof | |
| CN103805527B (en) | StreptomycesYunnanensis var daliensis and fermentation broth and application thereof | |
| CN105925521A (en) | Method for supplementing salt in batches for promoting lactic acid bacteria salt resistance |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |