CN102776140B - Cold-tolerant pseudomonas strain Den-05, and screening method and application thereof - Google Patents
Cold-tolerant pseudomonas strain Den-05, and screening method and application thereof Download PDFInfo
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- CN102776140B CN102776140B CN2012101529210A CN201210152921A CN102776140B CN 102776140 B CN102776140 B CN 102776140B CN 2012101529210 A CN2012101529210 A CN 2012101529210A CN 201210152921 A CN201210152921 A CN 201210152921A CN 102776140 B CN102776140 B CN 102776140B
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- 241000589516 Pseudomonas Species 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title abstract description 13
- 238000012216 screening Methods 0.000 title description 5
- 230000001580 bacterial effect Effects 0.000 claims abstract description 9
- 239000010865 sewage Substances 0.000 claims abstract description 8
- 229910002651 NO3 Inorganic materials 0.000 claims description 7
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 6
- 230000015556 catabolic process Effects 0.000 claims description 5
- 238000006731 degradation reaction Methods 0.000 claims description 5
- 241000589774 Pseudomonas sp. Species 0.000 claims 1
- 241000084283 Pseudomonas sp. Den-05 Species 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 5
- 238000005273 aeration Methods 0.000 abstract description 4
- 238000004904 shortening Methods 0.000 abstract description 4
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 36
- 239000007788 liquid Substances 0.000 description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000010802 sludge Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000002609 medium Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- 241001057811 Paracoccus <mealybug> Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000010841 municipal wastewater Substances 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 235000019263 trisodium citrate Nutrition 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- 238000004065 wastewater treatment Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108090000913 Nitrate Reductases Proteins 0.000 description 1
- 108010025915 Nitrite Reductases Proteins 0.000 description 1
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 1
- 241000190932 Rhodopseudomonas Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 208000035844 biological anomaly Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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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
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A cold-tolerant pseudomonas strain Den-05 belongs to the technical field of the biological engineering and environmental engineering, and is Pseudomonas psychrophilia Den-05, which is preserved in China General Microbiological Culture Collection Center (CGMCC) on March 27, 2012 with the preservation number of CGMCC NO.5942. Low-temperature aerobic denitrifying bacteria Pseudomonas psychrophilia Den-05 is screened from the natural environment by a microbial pure culture technique, so that the preparation of a biological denitrifying bacterial agent is realized and a high-efficiency denitrifying bacterial agent is provided for a biological enhancement system; besides, the winter denitrification effect of an urban sewage plant is improved in the control and regulation way of shortening the period of aeration by shortening the anaerobic period and the removal of nitrate nitrogen under the aerobic condition is realized; as a result, the water quality discharged is capable of meeting the requirements.
Description
Technical field
The invention belongs to biotechnology, field of environment engineering technology.Relate to the cold-resistant pseudomonas Den-05 of strain Pseudomonas psychrophilia Den-05() and screening method and the application in municipal sewage treatment under cold condition.
Background technology
In water body, appropriate nitrogen is the fundamental element that synthesizes the microorganisms such as algae, be to form the important material conditions of aquatic ecological environment, and be also the eucyclic basis of water surrounding.But the total amount of some Waters of China has surpassed the water body capacity at present, and excessive nitrogen element causes the biological anomalies propagation such as algae and water, causes water quality deterioration.Simultaneously, ammonia nitrogen and nitrite nitrogen are toxic to biologies such as fish.Due to the conversion that can circulate under certain condition of each existing way of Water element, how to control the pollution of Water, just become at present and topic that whole " 12 " reduction of discharging process all must be faced.China has begun to carry out " municipal sewage plant's integrated wastewater discharge standard " (GB18918-2002) one-level A standard (ammonia nitrogen＜5mg/L, TN＜15mg/L), the highest priority of municipal sewage treatment has turned to the removal of the organic nutrient substances such as nitrogen phosphorus.Therefore, the efficient biological denitrification method of exploiting economy and technology have become one of water pollution control field focus.
It is generally acknowledged, the denitrifying bacterium that carries out nitrate respiration is all amphimicrobe, because only under oxygen free condition, just can induce nitrate reductase A and nitrite reductase that denitrification needs.Yet people constantly found the loss of total nitrogen under aerobic condition in recent years, at present Aerobic Denitrification Phenomenon is formed reason and there is no final conclusion, but because aerobic denitrification exists obvious advantage than the traditional biological denitride technology, the aerobic denitrification technology becomes the new developing direction of domestic and international wastewater biological denitrificaion in recent years.The main body that aerobic denitrification is used is the aerobic denitrification flora, the aerobic denitrifying bacteria of having found at present mainly concentrates on Rhodopseudomonas (Pseudomonas), Alkaligenes (Alcaligenes), paracoccus (Paracoccus) and bacillus (Bacillus), bacterial classification is used at most, secondly be mechanism research, the utilization of aerobic denitrification microbial inoculum report less.
Summary of the invention
Total N is difficult for up to standard for the sewage disposal in winter, long processing period, the problem that expense is high, this patent filters out the strain Pseudomonas psychrophilia Den-05 that can significantly improve biological denitrificaion efficient under the low-temperature aerobic condition from nature, and be made into biological bacteria preparation, provide new realization approach to municipal effluent qualified discharge under cold condition, to improving the Sewage Plant operational efficiency, having reduced costs obvious actual application value.
Bacterial strain provided by the invention was Pseudomonas psychrophilia Den-05, was preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC NO. 5942 on 03 27th, 2012.
The cold-resistant pseudomonas Den-05(Pseudomonas of bacterial strain psychrophilia Den-05) be to separate to obtain from Harbin City's good faith ditch municipal wastewater treatment plant Aerobic Pond active sludge, the domestication process of aerobic denitrification flora is all carried out under 10 ℃ of conditions.At first get 15mL active sludge and some granulated glass spherees to the aerobic denitrification liquid nutrient medium of having sterilized, shake-flask culture 4 ~ 5d.Sterilising conditions is 121 ℃, high pressure steam sterilization 20 minutes.After first culture cycle finishes, the domestication bacterium liquid that obtains is joined by 15% inoculum size continue shake-flask culture 4 ~ 5d in fresh aerobic denitrification liquid nutrient medium, repeat aforesaid operations 3 ~ 4 times.This mixed bacteria liquid is coated on the aerobic denitrification solid medium, repeatedly carries out plate streaking, the bacterial strain that carries out aerobic denitrification under a plant height effect low temperature is isolated in solid, liquid alternated cultivation
Pseudomonas psychrophiliaDen-05.
Concrete screening step is as follows:
(1) the fresh active sludge of getting 15mL Harbin City good faith ditch municipal effluent factory adds in 100mL aerobic denitrification substratum triangular flask, and being placed in temperature is 10
oC, rotating speed cultivate 4 ~ 5d in the shaking table of 150r/min, obtain enrichment bacterium liquid;
(2) get 15mL enrichment bacterium liquid and add in 100mL aerobic denitrification substratum triangular flask, the culture condition of repeating step (1) 3 ~ 4 times obtains domestication bacterium liquid;
(3) gradient dilution domestication bacterium liquid, the dilution gradient is respectively 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, to get respectively 100 μ L domestication bacterium liquid and add in solid aerobic denitrification culture medium flat plate, coating method smoothens, and is placed in temperature and is the incubator of 10 ℃, cultivates 3 ~ 4d, obtains single bacterium colony;
(4) the stroke line of utilization, three rides continuation purifying obtain single bacterium colony, and aerobic denitrification substratum (liquid), the domestication of solid aerobic denitrification substratum alternate culture are accelerated sieve bacterium process, repeatedly the pure bacterium colony of operation acquisition;
Wherein the composition of aerobic denitrification substratum is: 7.9 g Na
2HPO
47H
2O, 0.3 g NH
4Cl, 4.7 g Trisodium Citrates, 1.5 g KH
2PO
4, 0.1 g MgSO
47H
2O, 1.5 g KNO
3, the 1mL liquid microelement is dissolved in 1000 mL distilled water, regulates pH 7.2; Solid aerobic denitrification substratum separately is added with 18g agar in the aerobic denitrification substratum;
The composition of trace element is: 0.1 g CuSO
45H
2O, 0.3 g MnSO
44H
2O, 0.2 g ZnSO
47H
2O, 2.0 g NaHCO
310H
2O, 0.5 g CoCl
26H
2O, 0.05g CaCl
22H
2O, 0.5g FeSO
47H
2O, 0.02g (NH
4)
6Mo
7O
24H
2O is dissolved in the distilled water of 1000mL.
The domestication bacterium liquid OD that obtains in the step of screening method of the present invention (2)
660Be 1.0, substratum is all using after sterilization 20 min under 121 ℃.
Strain Pseudomonas psychrophilia Den-05 degradation capability to nitrate under cold condition is extremely strong, is applied in the municipal sewage treatment under winter low temperature, aerobic condition.
The present invention adopts the microorganism pure culture technigne to filter out low temperature, aerobic denitrifying bacteria from natural habitat
Pseudomonas psychrophiliaDen-05, realized the preparation of biological denitrogenation microbial agent, for biological fortification system provides the efficient denitrification bacterial classification, and by shortening the control methods of anaerobism cycle and then shortening aeration period, improve municipal wastewater treatment plant denitrification effect in winter, realize the removal of nitrate nitrogen under aerobic condition, reach the effluent quality requirement.
Description of drawings
Fig. 1 is that the aerobic denitrifying bacteria of the present invention thalli growth situation of 108 hours and contaminant degradation rate change, and wherein ■ represents that the bacterium of aerobic denitrifying bacteria in 96 hours growth cycles is turbid.
Fig. 2 is the clearance trend of different nitrates in aerobic denitrifying bacteria low temperature denitrification process of the present invention, wherein ● the Degradation and Transformation rate of expression aerobic denitrifying bacteria when nitrate starting point concentration 120mg/L, the wherein ▲ Degradation and Transformation rate of expression aerobic denitrifying bacteria when nitrate starting point concentration 60mg/L.
Embodiment
The invention will be further described below in conjunction with accompanying drawing.
Embodiment one:
(1) the fresh active sludge of getting 15mL Harbin City good faith ditch municipal effluent factory adds in 100mL aerobic denitrification substratum triangular flask, and being placed in temperature is 10
oC, rotating speed cultivate 4 ~ 5d in the shaking table of 150r/min, obtain enrichment bacterium liquid;
(2) get 15mL enrichment bacterium liquid and add in 100mL aerobic denitrification substratum triangular flask, the culture condition of repeating step (1) 3 ~ 4 times obtains domestication bacterium liquid, and carries out the drafting of growth curve;
(3) gradient dilution domestication bacterium liquid, the dilution gradient is respectively 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, to get respectively 100 μ L domestication bacterium liquid and add in solid aerobic denitrification culture medium flat plate, coating method smoothens, and is placed in temperature and is the incubator of 10 ℃, cultivates 3 ~ 4d, obtains single bacterium colony;
(4) the stroke line of utilization, three rides continuation purifying obtain single bacterium colony, and aerobic denitrification substratum (liquid), the domestication of solid aerobic denitrification substratum alternate culture are accelerated sieve bacterium process, repeatedly the pure bacterium colony of operation acquisition;
Wherein the composition of aerobic denitrification substratum is: 7.9 g Na
2HPO
47H
2O, 0.3 g NH
4Cl, 4.7 g Trisodium Citrates, 1.5 g KH
2PO
4, 0.1 g MgSO
47H
2O, 1.5 g KNO
3, the 1mL liquid microelement is dissolved in 1000 mL distilled water, regulates pH 7.2; Solid aerobic denitrification substratum separately is added with 18g agar in the aerobic denitrification substratum;
The composition of trace element is: 0.1 g CuSO
45H
2O, 0.3 g MnSO
44H
2O, 0.2 g ZnSO
47H
2O, 2.0 g NaHCO
310H
2O, 0.5 g CoCl
26H
2O, 0.05g CaCl
22H
2O, 0.5g FeSO
47H
2O, 0.02g (NH
4)
6Mo
7O
24H
2O is dissolved in the distilled water of 1000mL.
The every 12h of domestication bacterium liquid that obtains in step (2) gets bacterium liquid and measures OD
660, draw growth curve as shown in Figure 1 after cultivation 120h.The bacterial strain absorbancy OD of 60h place
660Can reach maximum value 1.0 left and right, after this enter decline phase.
Embodiment two: will add after strain expanded culture in the sbr reactor device.Method is 10
oC, under 3000 * g condition, bacterium liquid is centrifugal 5 minutes, and thalline adds in reactor, and it is 200mLOD that per cycle adds biomass
6601.0 the centrifugal gained thalline of bacterium liquid, add continuously 4 cycles of thalline, NO in the assaying reaction device
3 -Content.Sbr reactor device volume 12L, the cycle is 8h, sedimentation time 2h, aeration time, churning time are respectively 4h, 2h, and operation scheme is to connect after aeration 2h to stir 1h.Water distribution composition: contain 25.58g sodium acetate, 10g NH in every 20L water distribution
4Cl, 2g MgSO
47H
2O, 2g CaCl
22H
2O, 2g KH
2PO
4, 2g K
2HPO
4Add a certain amount of KNO
3, record continuous 20 days NO
3 -Concentration, data such as Fig. 2.
Nitrate obviously raise at front 4 days, this and add the thalline time consistency, should be residual substratum, after this NO in thalline
3 -Concentration begins to reduce, starting point concentration 120mg/L lowering speed is slightly slower than starting point concentration 60mg/L, but in two reactors, nitrate has all reached the 10mg/L left and right after the 7th day, and steady running 13 days, show that the aerobic denitrifying bacteria that screens has the effect that promotes that nitric nitrogen transforms, and can long-term existence in active sludge, shorten the anaerobism time and then shorten the cycle in reactor, can realize good denitrification effect in actual applications.
Claims (2)
1. a cold-resistant pseudomonas Den-05 of strain, is characterized in that bacterial strain is Pseudomonas sp. Den-05, was preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC NO. 5942 on 03 27th, 2012.
2. cold-resistant pseudomonas Den-05 according to claim 1, is characterized in that strain Pseudomonas sp.Den-05 has degradation capability to nitrate under cold condition, be applied in the municipal sewage treatment under winter low temperature, aerobic condition.
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| CN103232954B (en) * | 2013-03-27 | 2015-01-07 | 南开大学 | Aerobic denitrification bacteria and applications thereof |
| CN103275908B (en) * | 2013-06-21 | 2014-10-08 | 黑龙江省科学院微生物研究所 | Low-temperature denitrification pseudomonas fluorescens |
| CN113801824B (en) * | 2021-10-14 | 2024-06-14 | 中交和美环境生态建设有限公司 | Pseudomonas Y1 with efficient heterotrophic nitrification and aerobic denitrification function and embedded pellets and application thereof |
| CN114250173B (en) * | 2021-12-10 | 2022-06-14 | 青岛蔚蓝赛德生物科技有限公司 | Zhaodong pseudomonas strain and application thereof |
| CN114804373A (en) * | 2022-04-30 | 2022-07-29 | 北京赛富威环境工程技术有限公司 | Microbial treatment method for ultralow-temperature wastewater |
| CN116162561B (en) * | 2022-07-08 | 2024-12-17 | 碧沃丰生物科技(广东)股份有限公司 | Strain with low temperature resistance and aerobic denitrification functions and application thereof |
| CN115558625B (en) * | 2022-11-10 | 2023-05-12 | 哈尔滨工业大学 | A kind of low-temperature denitrification Pseudomonas urzii strain and its application |
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| US7345217B2 (en) * | 1998-09-22 | 2008-03-18 | Mendel Biotechnology, Inc. | Polynucleotides and polypeptides in plants |
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| CN101200699A (en) * | 2007-11-21 | 2008-06-18 | 淮海工学院 | Low temperature amylase from marine microorganisms and its producing strain Pseudomonas alternata GS230 |
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