CN102115719A - Aerobic denitrifying bacterium, and screening method and application thereof - Google Patents
Aerobic denitrifying bacterium, and screening method and application thereof Download PDFInfo
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- CN102115719A CN102115719A CN2010105069310A CN201010506931A CN102115719A CN 102115719 A CN102115719 A CN 102115719A CN 2010105069310 A CN2010105069310 A CN 2010105069310A CN 201010506931 A CN201010506931 A CN 201010506931A CN 102115719 A CN102115719 A CN 102115719A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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- Y02W10/10—Biological treatment of water, waste water, or sewage
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Abstract
The invention discloses an aerobic denitrifying bacterium, and a screening method and application thereof, belonging to the fields of biological engineering technology and environmental protection. The aerobic denitrifying bacterium (Pseudomonas sp.) disclosed by the invention is obtained by screening active sludge in a sewage treatment plant, and a screening scheme is determined according to the alkali-producing performance of the aerobic denitrifying bacterium in the culturing process. The screening method comprises the following specific steps: carrying out enrichment culture; carrying out preliminary screening; carrying out secondary screening; and testing the denitrification performance, thus finally obtaining a strain with better performance. The strain can effectively remove nitrate nitrogen and nitrite nitrogen in the sewage, and the capabilities of removing nitrate nitrogen and nitrite nitrogen are respectively 1.954gNO3-N/d/g and 0.978gNO2-N/d/g. When the initial nitrate nitrogen concentration is 40mg/L, the removal rate in 8 hours is up to 99% or above. Thus, the aerobic denitrifying bacterium can be used for environmental sewage treatment, and has fine application prospects.
Description
Technical field:
The present invention relates to a strain aerobic denitrifying bacteria and screening method and application, belong to biotechnology and field of environment protection.
Background technology:
Serious day by day along with body eutrophication removed the hot issue that nitrate pollution in the water has become current water prevention and cure of pollution field.For a long time, removing of ammonia nitrogen in the sewage mainly emphasized in denitrogenation, nitric nitrogen do not handled, and causes that nitrate content constantly raises in the water body.The traditional biological denitrogenation comprises two processes of nitrification and denitrification, and promptly ammonia nitrogen is converted into nitric nitrogen by nitrobacteria, and then is reduced to nitrogen by denitrifying bacterium, discharges water body.Traditional denitrification mainly is to carry out under anaerobism or anoxia condition, and in this process, nitrate is reduced to nitrite, NO, N successively
2O and nitrogen.Yet up-to-date studies show that, some microorganisms also can show certain denitrifying capacity under different dissolved oxygen conditions.Such as the Thiosphaera pantotropha that is reported on the document, Alcaligenes faecalis, Pseudomonas aeruginosa, Thaurea mechernichensis, Acinetobacter calcoaceticus, Providencia rettgeri, Pseudomonas stutzeri and Bacillus strains etc. all can carry out denitrification under aerobic condition.The biological denitrificaion that appears as of aerobic denitrifying bacteria provides a brand-brand-new way, makes that traditional biological denitrificaion approach is more perfect, also provides certain theoretical basis for denitrification process research.Traditional biological denitrificaion is divided into nitration processes and denitrification process because nitrifier is different to the demand of oxygen with denitrifying bacteria.Now is along with the appearance of aerobic denitrifying bacteria, short-cut nitrification and denitrification, and nitration denitrification all becomes possibility simultaneously.
The screening method of aerobic denitrifying bacteria is a lot, mainly comprises: 1. enrichment culture method: utilize selective medium to screen, add restricted carbon source and nitrogenous source in substratum, or be that the process that alkali increases is added the pH indicator in substratum according to denitrification.2. dilution method will be cultivated after the diluted sample certain multiple, then with diluting again that the pH value changes, and repetitive operation, not dominant aerobic denitrifying bacteria was separated originally.3. intermittent aeration method, aerobic denitrifying bacteria can utilize oxygen molecule and nitric nitrogen simultaneously, and aerobic, anoxybiotic frequent transitions helps its status that gets the mastery in competition.
Summary of the invention:
Technical problem to be solved of the present invention provides a strain aerobic denitrifying bacteria, nitric nitrogen and nitrite nitrogen in the water body of can effectively degrading under aerobic condition.
Described aerobic denitrifying bacteria 16s rDNA Molecular Identification is Rhodopseudomonas (Pseudomonas sp.), on September 7th, 2010, is preserved in Chinese typical culture collection center, and culture presevation is numbered CCTCC M 2010209.
Another technical problem to be solved by this invention provides a kind of method of screening aerobic denitrifying bacteria.
Bacterial screening of the present invention adopts multiple screening method to combine, and at first by adding restricted nitrogenous source, intermittent aeration comes the enrichment aerobic denitrifying bacteria, and then fast and effeciently screens aerobic denitrifying bacteria by add the pH indicator in substratum.
For achieving the above object, the technical scheme taked of the present invention is:
Active sludge to sewage work carries out the aeration enrichment culture, filters out aerobic bacteria; Produce the performance of alkali then according to denitrifying bacteria,, screen aerobic denitrifying bacteria effectively by in substratum, adding the method for brooethyl Finland indicator; Use at last and simulated the well foster denitrifying bacteria that sewage sieves high denitrifying capacity again.
Its steps in sequence is sampling, the aeration enrichment culture, and primary dcreening operation, multiple sieve obtains 1 strain under aerobic condition, nitric nitrogen and nitrite nitrogen in the simulated sewage of degrading simultaneously, the bacterial strain that denitrifying capacity is higher at last.
Nitric nitrogen detection method: ultraviolet spectrophotometry (HJ/T 346-2007)
Nitrite nitrogen detection method: N-(1-naphthyl)-quadrol spectrophotometry (GB 7493-87)
Another technical problem to be solved by this invention provides a kind of method of application sewage disposal of aerobic denitrifying bacteria, be specially the thalline enlarged culturing after, be inoculated in and contain the water body that nitric nitrogen and nitrite nitrogen pollute.
Bacterial strain provided by the invention can effectively be removed nitric nitrogen and the nitrite nitrogen in the sewage, and the ability of removing nitric nitrogen and removal nitrite nitrogen is respectively 1.954gNO
3-N/d/g and 0.978gNO
2-N/d/g, when initial nitric nitrogen concentration was 40mg/L, the 8h clearance reached more than 99%, can be used for environmental wastewater and handled, and had good application prospects.
Embodiment:
The screening of embodiment 1 aerobic denitrifying bacteria
1. enrichment culture: will carry out the aeration enrichment culture from the active sludge that sewage work obtains, and change fresh culture every day.Liquid amount 2L.Leave standstill 1h earlier when changing fresh culture, remove the 600mL volume, and then add the 600mL fresh culture, enrichment is about 40 days altogether.
2. primary dcreening operation: the active sludge that enrichment is good dilutes, and is coated with on the dibromothymolsulfonphthalein substratum, places 30 ℃ of incubators to cultivate then, cultivates 2-3 days.The blue single bacterium colony that occurs on the dibromothymolsulfonphthalein substratum is classified, and gramstaining is selected the bacterium colony of each form 3-4 the separation of ruling then, places 30 ℃ of incubators to cultivate, and cultivates 2-3 days.Line is separated the blue single bacterium colony that obtains classify, select characteristic bacterium colony and be saved on the inclined-plane, use for multiple sieve.
3. multiple sieve: the bacterial strain that primary dcreening operation is preserved inserts thalline enlarged culturing base, and 30 ℃, 150rpm cultivates 24h, inserts 100mL respectively in 5% ratio then and detects substratum, cultivates 48h, and 30 ℃, 150rpm measures the variation of nitric nitrogen and nitrite nitrogen before and after the inoculation.The bacterial strain that selection nitric nitrogen degradation rate reaches more than 40% and the nitrite nitrogen degradation rate reaches more than 25% carries out the denitrification capability test, obtains the best bacterial strain of a strain performance.
4. the mensuration of nitric nitrogen and nitrite nitrogen
Nitric nitrogen: ultraviolet spectrophotometry (HJ/T 346-2007)
Nitrite nitrogen: N-(1-naphthyl)-quadrol spectrophotometry (GB 7493-87)
5. relevant substratum
Enrichment medium (g/L): (NH
4)
2SO
40.5; Soduxin 6H
2O 2.17; Vickers salts solution 50ml; PH 7.2.
Vickers salts solution (g/L): K
2HPO
45.0; MgSO
47H
2O 2.5; NaCl 2.5; FeSO
47H
2O 0.05; MnSO
44H
2O 0.05.
Primary dcreening operation substratum (g/L): agar 20; KNO
31; KH
2PO41; FeSO
47H
2O 0.5; CaCl
20.2; MgSO
47H
2O1; Soduxin 6H
2O 8.5; Dibromothymolsulfonphthalein (get the 0.1g dibromothymolsulfonphthalein and be dissolved in 10mL alcohol) 1mL, pH 7.0～7.3.
Thalline enlarged culturing base (g/L): KNO
31; KH
2PO
41; FeSO
47H
2O 0.05; CaCl
20.02; MgSO
47H
2O1; Soduxin 6H
2O 8.5.
Detect substratum (g/L): KH
2PO
41; MgSO
47H
2O 1; Soduxin 6H
2O 2.8; Be nitrogenous source with SODIUMNITRATE and Sodium Nitrite respectively, nitrogen concentration 100mg/L.
Embodiment 2. aerobic denitrifying bacteria treatment of simulated sewage
1. relevant substratum:
Thalline enlarged culturing base (/L): KNO
31g; KH
2PO
41g; FeSO
47H
2O 0.05g; CaCl
20.02g; MgSO
47H
2O 1g; Soduxin 8.5g.
Detect substratum (g/L): KH
2PO
41g; MgSO
47H
2O 1g; Soduxin 2.8g; Be nitrogenous source with SODIUMNITRATE and Sodium Nitrite respectively, nitrogen concentration is decided according to experiment.
2. processing condition:
Temperature: 30 ℃; Shaking speed: 150rpm; Liquid amount: 100/250mL does not have stream and adds
3. the application of aerobic denitrifying bacteria
Denitrifying bacteria picking one articulating on the inclined-plane is gone into the enlarged culturing base, at 30 ℃, cultivate 24h under the condition of 150rpm, insert respectively in the simulated sewage that is rich in nitric nitrogen and nitrite nitrogen in 5% ratio then, 30 ℃, 150rpm cultivates after 24 hours, centrifugal 5min under the condition of 8000rpm detects nitric nitrogen and nitrite nitrogen content in the supernatant liquor.The ability that described bacterial strain is removed nitric nitrogen and removal nitrite nitrogen is respectively 1.954gNO
3-N/d/g and 0.978gNO
2-N/d/g, when initial nitric nitrogen concentration was 40mg/L, the 8h clearance reached more than 99%.
The denitrifying capacity test of table one bacterial strain
Annotate: initial nitrogen concentration is 100mg/L, cultivates 24h
The removal nitric nitrogen ability of table two bacterial strain
Annotate: initial nitrogen concentration 40mg/L, cultivate 8h
Embodiment 3. aerobic denitrifying bacterias are disposed of sewage
Denitrifying bacteria picking one articulating on the inclined-plane is gone into the enlarged culturing base, at 30 ℃, cultivate 24h under the condition of 150rpm, be rich in the sewage of nitric nitrogen and nitrite nitrogen in 5% ratio access then, 30 ℃, 150rpm cultivates after 24 hours, centrifugal 5min under the condition of 8000rpm, nitrogen removal rate are 86.59%.
The denitrification effect of table three actual sewage
| Numbering | DCW(g/L) | Initial total nitrogen (mg/L) | Residue total nitrogen (mg/L) | Clearance (%) |
| 4 | 1.00 | 36.01 | 4.83 | 86.59 |
Annotate: cultivate 24h
Embodiment 4: denitrifying bacteria is identified
Bacterial strain is Pseudomonas (pseudomonas sp.) through 16S rDNA Molecular Identification, and its 16S rDNA sequence has now been submitted NCBI to, is numbered HM218829.This bacterial strain is preserved in Chinese typical culture collection center on September 7th, 2010, and culture presevation is numbered CCTCC M 2010209.
Claims (10)
1. a strain aerobic denitrifying bacteria belongs to Rhodopseudomonas (Pseudomonas sp.), on September 7th, 2010, is preserved in Chinese typical culture collection center, and culture presevation is numbered CCTCC M 2010209.
2. the described screening method of supporting denitrifying bacteria well of claim 1 is characterized in that the active sludge that will obtain from sewage work carries out the aeration enrichment culture; Then that enrichment is good active sludge dilutes coating dibromothymolsulfonphthalein flat board, selects the blue colonies line and obtains pure growth; Utilize the screening of thalline denitrifying capacity to obtain the high-efficiency aerobic denitrifying bacteria at last.
3. screening method according to claim 2 is characterized in that described enrichment medium is (g/L): (NH
4)
2SO
40.5; Soduxin 2.17; Vickers salts solution 50ml; PH 7.2.
4. screening method according to claim 3 is characterized in that the Vickers salts solution is (g/L): K
2HPO
45.0; MgSO
47H
2O 2.5; NaCl 2.5; FeSO
47H
2O 0.05; MnSO
44H
2O 0.05.
5. screening method according to claim 2 is characterized in that the dibromothymolsulfonphthalein flat board is (g/L): agar 20; KNO
31; KH
2PO41; FeSO
47H
2O 0.5; CaCl
20.2; MgSO
47H
2O 1; Soduxin 8.5; Dibromothymolsulfonphthalein (get the 0.1g dibromothymolsulfonphthalein and be dissolved in 10mL alcohol) 1mL, pH 7.0～7.3.
6. screening method according to claim 2 is characterized in that the screening of thalline denitrifying capacity by detecting substratum, detects substratum and consists of (g/L): KH
2PO
41; MgSO
47H
2O 1; Soduxin 2.8; Be nitrogenous source with SODIUMNITRATE and Sodium Nitrite respectively, nitrogen concentration 100mg/L.
7. the application of the described aerobic denitrifying bacteria of claim 1 in degraded nitric nitrogen and nitrite nitrogen.
8. application according to claim 7 is characterized in that the ratio in 5% after the denitrifying bacteria enlarged culturing is inserted respectively in the water body that is rich in nitric nitrogen.
9. application according to claim 7 is characterized in that the ratio in 5% after the denitrifying bacteria enlarged culturing is inserted respectively in the water body that is rich in nitrite nitrogen.
10. according to Claim 8 or 9 described application, the condition that it is characterized in that described enlarged culturing is 30 ℃, cultivates 24h under the condition of 150rpm.
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| CN103525730A (en) * | 2013-10-14 | 2014-01-22 | 青岛蔚蓝生物集团有限公司 | Pseudomonas otitidis strain and application thereof |
| CN103849588A (en) * | 2014-03-05 | 2014-06-11 | 北京市环境保护科学研究院 | Aerobic denitrifying bacterium and application thereof in sewage denitrification |
| CN104250625A (en) * | 2014-05-28 | 2014-12-31 | 北京市环境保护科学研究院 | Aerobic denitrifying bacterium strain and application thereof to water denitrification |
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| CN107686820A (en) * | 2017-09-11 | 2018-02-13 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of aerobic denitrifying bacteria and its application in water body denitrification |
| CN107686820B (en) * | 2017-09-11 | 2020-04-03 | 广东省微生物研究所（广东省微生物分析检测中心） | Aerobic denitrifying bacteria and application thereof in water denitrification |
| CN109486699A (en) * | 2018-08-21 | 2019-03-19 | 四川大学 | One plant of salt tolerant aerobic denitrifying bacteria and application |
| CN109182192A (en) * | 2018-09-26 | 2019-01-11 | 北京化工大学 | One plant of aerobic denitrifying bacteria HY3-2 and its application in sewage water denitrification |
| CN109810923A (en) * | 2019-03-14 | 2019-05-28 | 北京化工大学 | One plant of aerobic denitrifying bacteria SLY2-21 and its application for sewage water denitrification |
| CN109810923B (en) * | 2019-03-14 | 2021-02-19 | 北京化工大学 | Aerobic denitrifying bacterium SLY2-21 for sewage denitrification and application thereof |
| CN111454859A (en) * | 2020-04-07 | 2020-07-28 | 山东海景天环保科技股份公司 | Method for cultivating low-temperature aerobic denitrifying bacteria |
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