CN104004695A - 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
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- CN104004695A CN104004695A CN201410277400.7A CN201410277400A CN104004695A CN 104004695 A CN104004695 A CN 104004695A CN 201410277400 A CN201410277400 A CN 201410277400A CN 104004695 A CN104004695 A CN 104004695A
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- 230000007613 environmental effect Effects 0.000 title description 2
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- 238000012986 modification Methods 0.000 title 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
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 products beneficial microorganism triage techniques field, be specifically related to a kind of genus bacillus for apostichopus japonicus culture pond environment remediation.
Background technology
Stichopus japonicus (Apostichopus japonicus Selenka) aquaculture is an emerging sea farming industry, since two thousand two, rapidly, within 2006, national apostichopus japonicus culture area has reached 8.4 ten thousand hectares to the development of apostichopus japonicus culture industry, output reaches 7.6 ten thousand tons, and the output value approaches 10,000,000,000 yuan.Yet along with its cultivation scale developing rapidly, associated problem also display gradually.Because current most of apostichopus japonicus culture mainly relies on the artificial diet of throwing something and feeding, feed poor adherence, is also specially provided with a large amount of artificial adherances at the bottom of pond, therefore in breeding process, can produce the residual of a large amount of residual baits, ight soil.These organic pollutants not only cause severe contamination to stichopus japonicus growing environment, and understand the generation that the direct healthy growth that endangers stichopus japonicus causes disease, have seriously hindered the Sustainable development of the even whole water industry of apostichopus japonicus culture industry.
Research is found, residual bait and movement are particularly piled up in breeding environment in substrate, become the most important endogenous of water pollution, they can constantly discharge N, P nutritive salt and solubility organic pollutant in breeding environment, cause water quality to be ruined, oxygen-consumption increases, and plant plankton, bottom-dwelling diversity reduce, to such an extent as to the generation of wawter bloom or red tide.
And frequently need unavoidably medication for disease, various microbiotic are as sulfamido, tsiklomitsin, penicillin, furans etc., result causes the secondary pollution in cultivation, produce a lot of negative effects, as the medicine major part dropping into is all directly lost in environment, directly contaminate environment consequently finally forms harm to closing on waters.
Degrading microorganism, as one of most important decomposer in terrestrial ecosystem, is showing huge application potential aspect the Degradation and Transformation of pollutent.Since the eighties, the research of microorganism aspect environmental pollution repairing and treating is more and more, and oneself is widely used in each pollution field people, as heavy metal contamination, oil spill, pesticide residue, sanitary wastewater processing etc. a lot of aspect.Along with the fast development of aquaculture and a series of problem of environmental pollutions that occur, the applied research of degrading microorganism in this field caused people's extensive attention.
Summary of the invention
The object of this invention is to provide a kind of genus bacillus, i.e. the Bacillus strain that a strain can purify water under cold condition, thus make up the deficiencies in the prior art.
The invention provides a bacillus (Bacillus sp.) A1, in preservation on the 06th in 01 month in 2014, be positioned at China Committee for Culture Collection of Microorganisms's common micro-organisms center of No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCCNo.8686.
The genus bacillus of screening is for the purification of culture-pool water quality;
Described cultivating pool is preferably the cultivating pool of stichopus japonicus;
The genus bacillus of the present invention's screening also can be used as immunostimulant or fodder additives;
Described feed is preferably apostichopus japonicus Selenka feed;
Another aspect, the present invention also provides the microbial inoculum that utilizes above-mentioned fermentation of bacillus to make.
Bacterium of the present invention is compared with the usefulness of alternate manner for repairing apostichopus japonicus culture water surrounding:
1, compared to chemical means, utilize the original bacterial strain existing of occurring in nature to repair environment, reduced residual in water surrounding of chemical substance, make cultivation water environment can obtain sustainable use.
2, this bacterial strain providing is the indigenous bacterium that obtains from the direct separation screening of aquaculture water, therefore it is lower to stichopus japonicus itself and the potential fulminant hazardness of environmental ecology that its enlarged culturing is applied to apostichopus japonicus culture pond again.
3, bacterial strain provided by the invention is through low temperature screening, and the screening of organic degradation ability, finally obtains aimed strain, and gained aimed strain is well-grown at low temperatures, all stronger for the ammonia nitrogen in bait and COD degradation capability.
Embodiment
Although the research for microorganism repairing environment is many, microorganism is reported few for purifying the research of Stichopus japonicus pond water environment.On the other hand, stichopus japonicus growth temperature condition is low temperature, surpass 24 ℃ of stichopus japonicus and just progress into aestivation, stichopus japonicus suitable growth temperature is 12-20 ℃, optimum temperuture is 17 ℃-18 ℃, therefore provide the bacterial strain that a strain purifies water under cold condition to purify and have very important practice significance for Apostichopus japonicus in Ponds pond water quality.
The proportioning of the materials such as substratum used in the present invention is as follows:
1, nutrient agar medium culture plate:
By the nutrient agar (peptone 10g/L, sodium-chlor 5g/L, extractum carnis 3-5g/L, seawater 1L) of preparation, sterilizing 15min at 121 ℃.By the substratum after sterilizing, fall in the flat board of sterilizing, cooling rear standby.
2, sterilizing bait liquid nutrient medium:
Take 20g Stichopus japonicus Selenka bait, soaked overnight in 1000mL sterilizing seawater.Bait steeping fluid is filtered, in filtrate, add 2g extractum carnis, then be diluted to 1000mL with seawater, its minute is filled in 10 sterilizing Erlenmeyer flasks, 150mL in each Erlenmeyer flask, residue substratum is loaded in Erlenmeyer flask, and sterilizing 15min at 121 ℃ is cooling standby.
Below in conjunction with specific embodiment, the present invention is described in detail.
One, the screening of bacterial strain
1, sample collecting
1.1 formulate acquisition scheme
Before gathering, search plan collected specimens existing about data informations such as geography, ecology and biological characteristicses, according to the data of grasping, formulate acquisition scheme.
1.2 gather preparation
According to the acquisition scheme of formulating, determine and gather pond, acquisition time and collected specimens.And perform the arrangement of collector, sampling instrument, collection vehicle etc.Sampling instrument is in advance prior to sterilizing 15min at 121 ℃.
1.3 collection in worksite
The apostichopus japonicus culture pond of Jimo saltworks has been chosen in pond, during sampling, respectively at four points in pond, samples.Collected specimens scope is selected sediment of pond, cultivated animals, plant, water body, adherance etc.Record is sampled pond hydrologic condition as temperature, pH value, salinity etc.In collection in worksite process, note aseptic technique.Use disposable sterilized gloves, bed mud, cultivated animals and plant are loaded in aseptic sample sack, use sterile sampling bottle from the filling quantitative water body of desired location.
2, genus bacillus screening
2.1 sample pretreatment
Physical condition is per sample processed.By the sample sediment of pond gathering, cultivated animals, plant, water body, adherance etc., with grinding to beat after 10 times of dilutions of sterile saline, get supernatant liquor, obtain sample storing solution.Liquid-like is not done pre-treatment.
2.2 genus bacillus are separated
After 80 ℃ of water-bath 10min, use 10 times of gradient dilutions of sterile saline to suitable concentration sample storing solution.The regulation that concrete dilution process is pressed GB4789.2-2010.Each duplicate samples diluent is done to two parts of inoculations, adopt the method for dull and stereotyped coating to be inoculated on nutrient agar, cultivate 24h at 28 ℃.The compound method of nutrient agar is pressed the regulation of GB/T4789.28-2003.
2.3 genus bacillus purifying
By well-grown on nutrient agar plate, the discrepant single bacterium colony of the bacterium colony that detects by an unaided eye respectively streak inoculation in nutrient agar, be placed at 28 ℃ and cultivate 24h.Through the purifying of repeatedly ruling, obtain single strain.
3, genus bacillus postsearch screening and evaluation
Preparation work before screening:
Sterilizing bait liquid nutrient medium
Take 20g Stichopus japonicus Selenka bait, soaked overnight in 1000mL sterilizing seawater.Bait steeping fluid is filtered, in filtrate, add 2g extractum carnis, then be diluted to 1000mL with seawater, its minute is filled in 5 sterilizing Erlenmeyer flasks, 150mL in each Erlenmeyer flask, residue substratum is loaded in Erlenmeyer flask, and sterilizing 15min at 121 ℃ is cooling standby.
The low temperature screening of 3.1 genus bacillus
The genus bacillus streak inoculation that primary screening is obtained, on nutrient agar plate medium, is cultivated after one week at 17 ℃, and selecting has the bacterial strain of obvious growth to carry out follow-up experiment.
The organic matter degradation ability screening of 3.2 genus bacillus
The gemma bacterial strain that primary screening is obtained, with inoculating needle, choose a single bacterium colony, be inoculated in the Erlenmeyer flask of sterilizing of mark, after 17 ℃, 160r/min constant-temperature table shaking culture 5d, sample after the centrifugal 10min of 5000r/min and get supernatant, measure the content of supernatant C OD, NH4-N.With 100 * (1-COD5/COD0) %, 100 * (1-NH4-N5/NH4-N0) % represents respectively the degradation rate of each bacterial strain to the 5d of organic pollutant in Stichopus japonicus Selenka bait and ammonia nitrogen.According to bacterium, utilize the ability of Stichopus japonicus Selenka bait, bacterial strain is weighed the removal effect of COD, ammonia nitrogen in Stichopus japonicus Selenka bait, and the bacterial strain of screening good degrading effect is studied.Final screening obtains a strain bacterium, and after cultivation 5d, its COD and ammonia nitrogen degradation effect are optimum, and degradation rate is respectively 69.5% and 61.7%.
The potential pathogenic detection of 3.3 strains A 1
Utilize blood agar (being purchased from Jinan Babio Biotech Co., Ltd.) to detect bacterial strain and have or not potential pathogenicly, take and verify whether bacterial strain is pathogenic bacteria.With aseptic toothpick respectively the 1 single bacterium colony dibbling of picking strains A in blood agar, be then placed in 17 ℃, constant temperature culture 24～72h after observation without haemolysis circle.Strains A 1 no pathogenicity is described, can throws in for economic living cultivating pool, and as the fodder additives of economic living.
The evaluation of 3.4 genus bacillus
The preparation of pcr template DNA:
Genomic 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, boiling 5-10min in 100 ℃ of boiling water baths, centrifuging and taking supernatant liquor is as pcr template DNA.
The mensuration of the 16SrDNA sequence of bacterial strain:
16SrDNA amplimer is bacterial 16 S rDNA 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 system (Niu Yufeng etc., 2009), bacterial strain is carried out to PCR reaction amplification.PCR product (1.5kb left and right) is delivered to Beijing San Bo polygala root Bioisystech Co., Ltd and is carried out purifying and order-checking after electrophoresis detection.Sequencing result is compared to the 16SrRNA sequence of relevant genus kind in GenBank (http://www.ncbi.nlm.nih.gov) with Blast software, by identification of strains to belonging to or planting.
Bacterial strain has been positioned at China Committee for Culture Collection of Microorganisms's common micro-organisms center of No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City in preservation on January 6 in 2014 now, and preserving number is CGMCCNo.8686.
Two, the application of the degraded under the low temperature of bacterium
1, the activation culture of genus bacillus
Strains A 2～the A7 of the Flavobacterium A1 that is CGMCCNo.8686 by deposit number and other preservation and strains A on the market 8 totally 8 bacillus (are numbered respectively A1, A2, A3, A4, A5, A6, A7, A8) be inoculated on enrichment medium after 28 ℃ of 160rpm conditions activation, then streak inoculation is on nutrient agar plate, at 28 ℃, cultivates 24h.
2, under bacterium low temperature, degradation capability detects
Embodiment 1:
To choose a single bacterium colony with inoculating needle, and be inoculated in respectively in the Erlenmeyer flask of corresponding sterilizing of A1, A2, A3, A4, A5, A6, A7, A8 mark, two Erlenmeyer flasks are not inoculated as blank.All Erlenmeyer flasks are placed in after 28 ℃, 160r/min constant-temperature table shaking culture 5d.From corresponding Erlenmeyer flask, sample in the centrifugal 10min of 5000r/min and get supernatant respectively, measure the content of supernatant C OD, NH4-N.With 100 * (1-COD5/COD0) %, 100 * (1-NH4-N5/NH4-N0) % represents respectively the degradation rate of each bacterial strain to the 5d of organic pollutant in Stichopus japonicus Selenka bait and ammonia nitrogen.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 experimental result, through 5 day time, in 8 strain bacteriums, ammonia nitrogen and COD are all had to degradation effect in various degree, but have bacterial strain self degradation capability in the time of the 5th day, hard degradation falls the ammonia nitrogen that self produces.
The degradation rate of COD sorts from high to low as A1>A8>A6>A5Gre atT.GreaT.GTA2>A3>A4GreatT.G reaT.GTA7, wherein the degradation rate of strains A 1 is up to 73.70%, all to the obvious bacterial strain of COD degradation effect in, be approximately the more than 20 times of minimum bacterial strain, there is good COD degradation capability.
The degradation rate of ammonia nitrogen sorts from high to low as A1>A4>A8>A5Gre atT.GreaT.GTA3>A6>A7GreatT.G reaT.GTA2, wherein the degradation rate of strains A 1 is up to 67.10%, all to the bacterial strain of ammonia nitrogen degradation successful in, be approximately five times of minimum bacterial strain, there is good ammonia nitrogen degradation ability.
Embodiment 2:
Prepare four 250mL Erlenmeyer flasks that 100mL sterilizing bait substratum is housed, with inoculating needle, choose strains A 1 and existing two kinds of genus bacillus (A8, A9) on the market, be inoculated in respectively in the Erlenmeyer flask of three sterilizings, an Erlenmeyer flask is not inoculated as blank.Erlenmeyer flask is placed in after 17 ℃, 160r/min constant-temperature table shaking culture 5d.Corresponding Erlenmeyer flask samples after the centrifugal 10min of 5000r/min and gets supernatant, measures the content of supernatant C OD, NH4-N.With 100 * (1-COD5/COD0) %, 100 * (1-NH4-N5/NH4-N0) % represents respectively the degradation rate of each bacterial strain to the 5d of organic pollutant in Stichopus japonicus Selenka bait and ammonia nitrogen.After 5d, the COD of strains A 1 and ammonia nitrogen degradation rate are respectively 57.73%, 38.06%, and COD and the ammonia nitrogen degradation rate of the existing genus bacillus A8 in market are respectively 49.05%, 18.31%, and the COD of A9 and ammonia nitrogen degradation rate are 33.07%, 12.04%.Can find out, screen the strains A 1 that obtains compared with existing on the market genus bacillus, have at low temperatures more excellent decomposition efficiency.
Embodiment 3:
With marking pen, in starch plate culture medium (peptone 10g, NaCl5g, extractum carnis 5g, Zulkovsky starch 2g, distilled water 1000mL, agar 15～20g, 121 ℃ of sterilizing 20min) bottom, divide four parts into.Strains A 1 is inoculated on the substratum of starch, four parallel, cultivates 2d for 18 ℃, adds Lu Ge Shi iodine liquid and observe, and finds that achromatic region appears around in A1.Meanwhile, it is inoculated at 18 ℃ to the test kit of the MOO1MR-VP that is purchased from Beijing Luqiao Technology Co., Ltd., cultivates after 48h, drip methyl red reagent C M1003 observation and find to become redness.It is inoculated at 18 ℃ to the test kit of the M035 hydrogen sulfide that is purchased from Beijing Luqiao Technology Co., Ltd. simultaneously, cultivates after 48h, find to generate black precipitate.
Experimental result can be found out, the MR experiment of strains A 1, H
2s experiment, Starch Hydrolysis experiment all show as the positive, illustrate that strains A 1 all has degradation capability for starch, sugar, amino acid.This also partial interpretation the good organic matter degradation ability of strains A 1.
Claims (8)
1. a genus bacillus, is characterized in that, described genus bacillus volume deposit number is CGMCC No.8686.
2. genus bacillus claimed in claim 1, is characterized in that, described genus bacillus is what from the direct separation screening of aquaculture water, obtain.
3. the application of genus bacillus claimed in claim 1 in purifying water of culture pond.
4. application as claimed in claim 3, is characterized in that described cultivating pool is apostichopus japonicus culture pond.
5. the application of genus bacillus claimed in claim 1 in preparing immunostimulant or fodder additives.
6. an immunostimulant, is characterized in that, described immunostimulant includes the viable bacteria of genus bacillus claimed in claim 1.
7. a fodder additives, is characterized in that, described fodder additives includes the viable bacteria of genus bacillus claimed in claim 1.
8. fodder additives as claimed in claim 7, is characterized in that, described feed 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 |
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| CN201410277400.7A CN104004695B (en) | 2014-06-19 | 2014-06-19 | Bacilli for environmental modification of stichopus japonicus aquaculture pond and application thereof |
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| CN108841747A (en) * | 2018-06-25 | 2018-11-20 | 青岛农业大学 | Produce bacillus subtilis and the application method of protease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
| 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 |
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| 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 |
| 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 |
| 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108841747A (en) * | 2018-06-25 | 2018-11-20 | 青岛农业大学 | Produce bacillus subtilis and the application method of protease |
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