CN103146621B - Pseudomonas aeruginosa and application thereof - Google Patents
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- CN103146621B CN103146621B CN201310095235.9A CN201310095235A CN103146621B CN 103146621 B CN103146621 B CN 103146621B CN 201310095235 A CN201310095235 A CN 201310095235A CN 103146621 B CN103146621 B CN 103146621B
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- 241000589517 Pseudomonas aeruginosa Species 0.000 title claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 230000000813 microbial effect Effects 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 238000000746 purification Methods 0.000 claims abstract description 3
- 241000894006 Bacteria Species 0.000 claims description 22
- 239000000843 powder Substances 0.000 claims description 13
- 239000002351 wastewater Substances 0.000 claims description 13
- 238000009395 breeding Methods 0.000 claims description 6
- 230000001488 breeding effect Effects 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 229920002472 Starch Polymers 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 238000000855 fermentation Methods 0.000 claims description 4
- 230000004151 fermentation Effects 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- 235000019698 starch Nutrition 0.000 claims description 4
- 239000010865 sewage Substances 0.000 claims description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 abstract description 33
- 241000238553 Litopenaeus vannamei Species 0.000 abstract description 5
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 230000000593 degrading effect Effects 0.000 abstract 1
- 230000035558 fertility Effects 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 238000004065 wastewater treatment Methods 0.000 abstract 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 230000015556 catabolic process Effects 0.000 description 12
- 238000006731 degradation reaction Methods 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 8
- 238000009360 aquaculture Methods 0.000 description 7
- 244000144974 aquaculture Species 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 238000005273 aeration Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 101100406487 Drosophila melanogaster Or47a gene Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012851 eutrophication Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000251511 Holothuroidea Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
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- 231100000719 pollutant Toxicity 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention aims at providing a pseudomonas aeruginosa and an application thereof. Pseudomonas aeruginosa AN5 has a preservation number of CCTCC NO:M2013068. Pseudomonas aeruginosa can be applied to the field of water body purification. Pseudomonas aeruginosa AN5 is screened from Litopenaeus vannamei culture pond bottom mud. The strain can be used for efficiently degrading ammonium nitrogen in a water body and has strong fecundity, so that the strain can be developed for an aquatic microbial preparation and a waste water treatment agent, and therefore, the application prospect is wide.
Description
Technical field
The invention belongs to technical field of microbe application, be specifically related to strain Pseudomonas aeruginosa and an application thereof.
Background technology
In recent decades, China's culture fishery fast development, intensive, high-density breeding scale expanding day, but because causing, kind of bait throwing in aquaculture water, accumulates a large amount of residual baits, and the reason such as the ight soil of cultivated animals and animals and plants corpse causes water body nitrogen element content severe overweight, eutrophication aggravation, is destroyed breeding ecological environment, fish and shrimp diseases takes place frequently, and finally causes huge financial loss.
Research shows, ammonia nitrogen is the Main Morphology of nitrogen in aqueous environment, also be the principal pollutant that cause body eutrophication, wherein maximum with ammonium nitrogen, nonionic form in ammonium nitrogen has stronger toxicity to aquatic animal, and along with pH and temperature and change, ammonia nitrogen is too high very easily easily causes aquatic animal death.
At present, in aquaculture production process, the way that people control ammonia and nitrogen pollution normally reduces ammonia nitrogen concentration by changing the new water of note, adopts aeration mode to make the part ammonia nitrogen effusion water surface, for example, adopt chlorine disinfectant to remove ammonia nitrogen, or carrys out regulating water quality with oxygenator etc.Can be reduced to water quality requirement standard completely because aforesaid method can not make ammonia nitrogen, for example, limited by water source, or fish bubbling disease, bacillary gill disease etc., therefore unsatisfactory to the control effect of ammonia and nitrogen pollution.And in recent years, the microbial technique that Microbial denitrogenation technology is removed starts to develop rapidly, the stabilised efficiency of microbiological deterioration ammonia nitrogen, the little advantage of secondary pollution is subject to showing great attention to of aquaculture and environmental protection industry, and this just requires to screen the microorganism with high-efficiency ammonia nitrogen degradation function.
Summary of the invention
The object of this invention is to provide strain Pseudomonas aeruginosa and an application thereof, described Pseudomonas aeruginosa screening is from the bed mud on the culture of Penaeus vannamei pool, and the ammonia-state nitrogen in energy efficient degradation water body, can be widely used in the processing to aquaculture and sanitary wastewater.
One aspect of the present invention provides a strain Pseudomonas aeruginosa AN5(Pseudomonas aeruginosa AN5), be preserved on March 6th, 2013 the Chinese Typical Representative culture collection center that is positioned at Wuhan, China Wuhan University, its deposit number is CCTCC NO:M2013068.
Microorganism of the present invention can be applicable to water body purification field.
Another aspect of the present invention provides the microbial preparation and the application thereof that contain above-mentioned Pseudomonas aeruginosa.
Above-mentioned Pseudomonas aeruginosa can bacterium powder form make microbe additive, preparation process is: by Pseudomonas aeruginosa AN5 activation, enlarged culturing, through liquid state fermentation, centrifugal collection bacterium mud, adds starch carrier, dries and makes the bacterium powder that bacterium amount is 100～1,000 hundred million/g.
Above-mentioned Pseudomonas aeruginosa AN5 bacterium powder is for the purifying treatment of breeding wastewater or sanitary sewage.
The present invention screens a strain Pseudomonas aeruginosa AN5 from the bed mud of the culture of Penaeus vannamei pool.Ammonium nitrogen in this bacterial strain energy efficient degradation water body, and this bacterial strain nutritional need is simple, and reproductivity is strong, can be developed as aquatic microorganisms preparation and waste water conditioner, has a extensive future.
Embodiment
Embodiment 1: Pseudomonas aeruginosa AN5 separation screening and qualification
1, the mud of screening is taken from the shining sun culture of Penaeus vannamei pool, Jiangsu.
2, substratum:
1) enrichment medium: glucose 5g, ammonium sulfate 2g, sodium-chlor 2g, ferrous sulfate 0.4g, dipotassium hydrogen phosphate 1g, pH7.2, magnesium sulfate 0.5g, water 1000mL;
2) isolation medium: the agar powder that adds 20g on enrichment medium basis;
3) screening culture medium: glucose 5g, ammonium sulfate 0.225g, sodium-chlor 1g, dipotassium hydrogen phosphate 0.5g, pH7.2, magnesium sulfate 0.25g, deionized water are settled to 1000mL, wherein ammonia-nitrogen content is 45mg/L, be sub-packed in the little triangular flask of 250mL, every bottle of 100mL, 121 DEG C of sterilizing 30min.
4) glucose peptone liquid nutrient medium: peptone 5g, glucose 5g, dipotassium hydrogen phosphate 2g, sodium-chlor 5g, water 1000mL, pH7.0, filters packing, 121 DEG C of sterilizing 30min.
3, screening:
Get culture of Penaeus vannamei pool mud, add half water and stir evenly, after the centrifugal 5min of 1000r/min, get supernatant and be inoculated in enrichment medium, in 30 DEG C of cultivations, enrichment 7d, and add wherein ammoniumsulphate soln 1ml every 1d, can not utilize NH to eliminate
4 +microorganism.Fermented liquid is coated on inverted isolation medium flat board after gradient dilution, in 30 DEG C of cultivations, the single bacterium colony growing on picking flat board carries out repeated isolation purifying, until obtain the pure culture of single strain, this experiment obtains 10 strain list bacterium colonies altogether, and difference called after AN1, AN2, AN3 to AN10 successively.The inoculation that purifying is obtained, in screening culture medium, is tested each bacterial strain above-mentioned 10 strain bacterium degraded situation to ammonia nitrogen in the time that 30 DEG C, 150r/min are cultivated 24h, and wherein strains A N5 is the highest to the degradation rate of ammonia nitrogen, reaches 71.93%(table 1).
Table 1:10 strain bacterium is in the experimental result of 24h degradation of ammonia nitrogen
| Bacterial strain | Initial ammonia nitrogen (mg/mL) | Finish ammonia nitrogen (mg/mL) | Degradation rate (%) |
| Control group | 45 | 43.47±2.11 | 3.40 |
| AN1 | 45 | 26.43±4.28 | 41.27 |
| AN2 | 45 | 35.20±3.82 | 32.89 |
| AN3 | 45 | 27.24±5.73 | 39.47 |
| AN4 | 45 | 24.46±2.71 | 45.64 |
| AN5 | 45 | 12.63±3.44 | 71.93 |
| AN6 | 45 | 23.36±1.82 | 48.09 |
| AN7 | 45 | 20.44±2.34 | 54.58 |
| AN8 | 45 | 21.28±1.32 | 52.71 |
| AN9 | 45 | 18.69±2.05 | 58.47 |
| AN10 | 45 | 24.36±1.15 | 45.87 |
4, Pseudomonas aeruginosa qualification
1) colony morphology characteristic: AN5 bacterial strain is cultivated to 18-24 hour on nutrient agar, be rendered as lark, opaque, surface wettability, non-wrinkled, diameter 2-3mm bacterium colony, its cell of microscopy is rod-short.
2) utilize test kit to extract the genomic dna of AN5 bacterial strain, utilize round pcr amplification 16S rRNA sequence, after order-checking, obtain the fragment that length is 1771bp.By BLAST compare of analysis, it is 99% with the 16S rRNA sequence similarity degree of many strains Pseudomonas aeruginosa of announcing, identifies and confirms that this bacterial strain is Pseudomonas aeruginosa, consistent with biochemical identification result.
By AN5 bacterial strain called after Pseudomonas aeruginosa AN5(Pseudomonas aeruginosa AN5), be preserved on March 6th, 2013 the Chinese Typical Representative culture collection center that is positioned at Wuhan, China Wuhan University, its deposit number is CCTCC NO:M2013068.
Embodiment 2: Pseudomonas aeruginosa AN5 microbial preparation degradation water aquaculture waste experiment
By Pseudomonas aeruginosa AN5 activation, enlarged culturing, through liquid state fermentation, centrifugal collection bacterium mud, adds starch carrier, dries the bacterium powder of making 10,000,000,000/g.Breeding wastewater is taken from the horizontal sea cucumber in field, the Qingdao pond of keeping a full stand of seedings, its ammonia-nitrogen content is 3.14 ± 0.65mg/L after measured, experiment container is plastic tank, experiment water body is 10L, and experimental group is added above-mentioned bacterium powder according to mass ratio 100ppm, and control group does not add bacterium powder, experimental group and control group all arrange 3 parallel, the uninterrupted aeration of experimental session, each container aeration rate is adjusted to unanimously, and experimental temperature is 28 DEG C of constant temperature.Every 24h measures ammonia-nitrogen content in each experimental group and control group water body to 96h(table 2).
The degradation rate of table 2 Pseudomonas aeruginosa AN5 to ammonia nitrogen in breeding wastewater
| Time | Control group (mg/L) | Test group (mg/L) | Ammonia nitrogen removal frank (%) |
| 24h | 3.01±0.45 | 2.33±0.72 | 22.59 |
| 48h | 2.86±0.27 | 1.87±0.48 | 34.62 |
| 72h | 2.92±0.61 | 1.24±0.63 | 57.53 |
| 96h | 2.58±0.29 | 0.97±0.45 | 62.40 |
From this experimental result, AN5 bacterium powder all has stronger Degradation (p < 0.05) to the ammonia nitrogen in aquaculture rich water, and along with ability of its degraded ammonium nitrogen of prolongation of bacterium powder duration of service is significantly strengthened (p < 0.05)., the microorganism that in this experiment, AN5 bacterial classification is made can reduce the too high problem of ammonia nitrogen in aquaculture process, for raiser reduces cultivation risk.
Embodiment 3: Pseudomonas aeruginosa AN5 degraded sanitary wastewater experiment
By Pseudomonas aeruginosa AN5 activation, enlarged culturing, through liquid state fermentation, centrifugal collection bacterium mud, adds starch carrier, dries the bacterium powder of making 10,000,000,000/g.Sanitary wastewater is taken from waterways, Li Cun fish market, Licang District, Qingdao City, after staticly settling, get its supernatant liquor, measuring its ammonia-nitrogen content is 118.27 ± 6.17mg/L, and experiment container is plastic tank, and experiment water body is 10L, experimental group is added Pseudomonas aeruginosa AN5 microbial preparation according to mass ratio 100ppm, control group does not add microorganism, experimental group and control group all arrange 3 parallel, the uninterrupted aeration of experimental session, each container aeration rate is adjusted to unanimously, and experimental temperature is 28 DEG C of constant temperature.Every 24h measures ammonia-nitrogen content in each experimental group and control group water body to 96h(table 3).
The degradation rate of table 3:AN5 microbial preparation to ammonia nitrogen in sanitary wastewater
| Time | Control group (mg/L) | Test group (mg/L) | Ammonia nitrogen removal frank (%) |
| 24h | 121.21±8.22 | 103.63±6.42 | 14.50 |
| 48h | 110.06±9.38 | 91.77±5.49 | 16.62 |
| 72h | 116.42±9.21 | 65.24±4.63 | 43.96 |
| 96h | 108.58±7.31 | 31.62±4.40 | 70.88 |
Result shows, the bacterium powder microbial preparation that strains A N5 makes has remarkable Degradation (p < 0.05) to the ammonia nitrogen in sanitary wastewater, and along with the ability of its degradation of ammonia nitrogen of prolongation of microorganism duration of service is significantly strengthened (p < 0.05).Due to the ammonia nitrogen severe overweight in sanitary wastewater, breath malodor, the sanitary wastewater of high ammonia nitrogen can not directly discharge, must be through denitrogenation processing, and the strains A N5 of the present invention's screening can reduce by 70% by ammonia nitrogen in 4d, the microbial preparation that this bacterial classification is made as seen can be used for sanitary wastewater processing.
Claims (4)
1. a Pseudomonas aeruginosa, is characterized in that, the deposit number of described Pseudomonas aeruginosa is CCTCC NO:M2013068.
2. the application of Pseudomonas aeruginosa claimed in claim 1 in water body purification field.
3. a microbial preparation, described microbial preparation is microbial germ powder, is by Pseudomonas aeruginosa activation claimed in claim 1, enlarged culturing, through liquid state fermentation, centrifugal collection bacterium mud, adds starch carrier, dries and makes the bacterium powder that bacterium amount is 100～1,000 hundred million/g.
4. the application of microbial preparation claimed in claim 3 in breeding wastewater or purifying domestic sewage processing.
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| CN103496793A (en) * | 2013-10-21 | 2014-01-08 | 中国科学院城市环境研究所 | Method for fast removing azo dyes from sewage in aerobic conditions |
| CN103966121B (en) * | 2014-04-18 | 2016-10-12 | 中国科学院南海海洋研究所 | One Pseudomonas aeruginosa strain and the application in preparation antibacterials thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102167448A (en) * | 2010-12-03 | 2011-08-31 | 中国地质大学（武汉） | Application of aniline degradation strain NO.3 |
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| CN102167448A (en) * | 2010-12-03 | 2011-08-31 | 中国地质大学（武汉） | Application of aniline degradation strain NO.3 |
Non-Patent Citations (6)
| Title |
|---|
| ByJ.R.POTTS等.TheEffectofNitrogenLimitationonCataboliteRepressionofAmidase Histidase and Urocanase in Pseudomonas aeruginosa.《Journal af General Microbiology》.1976 |
| CATHERINE N. MULLIGAN 等.Correlation of Nitrogen Metabolism with Biosurfactant Production by Pseudomonas aeruginosa.《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》.1989,第55卷(第11期),3016－3019. |
| Correlation of Nitrogen Metabolism with Biosurfactant Production by Pseudomonas aeruginosa;CATHERINE N. MULLIGAN 等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19891130;第55卷(第11期);3016-3019 * |
| The Effect of Nitrogen Limitation on Catabolite Repression of Amidase, Histidase and Urocanase in Pseudomonas aeruginosa;By J. R. POTTS 等;《Journal af General Microbiology》;19760430;第93卷(第2期);377-387 * |
| 两株异养硝化菌的筛选鉴定及其脱氮性能研究;张献旭;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20090215(第2期);论文摘要、正文第24页图3－1，第25页第2段等 * |
| 张献旭.两株异养硝化菌的筛选鉴定及其脱氮性能研究.《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》.2009,(第2期),B027-260. |
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