CN103911332B - A kind of method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria - Google Patents
A kind of method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria Download PDFInfo
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- CN103911332B CN103911332B CN201410136717.9A CN201410136717A CN103911332B CN 103911332 B CN103911332 B CN 103911332B CN 201410136717 A CN201410136717 A CN 201410136717A CN 103911332 B CN103911332 B CN 103911332B
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- black
- odorous water
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- composite bacteria
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 241000894006 Bacteria Species 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000002131 composite material Substances 0.000 title claims abstract description 15
- 230000001580 bacterial effect Effects 0.000 claims abstract description 23
- 239000010865 sewage Substances 0.000 claims abstract description 15
- 239000010802 sludge Substances 0.000 claims abstract description 5
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 238000012216 screening Methods 0.000 claims description 9
- 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 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000002953 phosphate buffered saline Substances 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 235000019156 vitamin B Nutrition 0.000 claims description 3
- 239000011720 vitamin B Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 14
- 239000000126 substance Substances 0.000 abstract description 8
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract description 7
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 7
- 229910052698 phosphorus Inorganic materials 0.000 abstract description 7
- 239000011574 phosphorus Substances 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 5
- 239000003795 chemical substances by application Substances 0.000 abstract description 4
- 230000000050 nutritive effect Effects 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 5
- 241000193755 Bacillus cereus Species 0.000 description 4
- 241000589597 Paracoccus denitrificans Species 0.000 description 4
- 241000589614 Pseudomonas stutzeri Species 0.000 description 4
- 238000003794 Gram staining Methods 0.000 description 3
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 description 3
- 238000012851 eutrophication Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 239000010866 blackwater Substances 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 230000002542 deteriorative effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000002352 surface water 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)
- Treatment Of Sludge (AREA)
Abstract
The invention discloses a kind of method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria, comprise (1) bacterial classification sample collecting, (2) amplification cultivation, (3) four steps such as domesticated strain and (4) purifying bacterial classification are screened, first the present invention filters out the indigenous microorganism with denitrogenation dephosphorizing in the active sludge of municipal sewage plant and black and odorous water bed mud, with the sewage of black and odorous water as main carbon source and energy, add the nutritive element needed for other growth again, obtained from the composite bacteria with denitrogenation dephosphorizing in native environment by domestication.It is fast that the bacterial classification that the present invention turns out has propagation, to the feature such as the Nitrogen/Phosphorus Removal of black and odorous water is obvious, the composite bacteria process black and odorous water using the present invention to obtain both had avoided the secondary pollution using chemical water purification agent, also do not need the security worrying bacterial classification, processing efficiency is also far above planting aquatic plants.
Description
Technical field
The present invention relates to microbial strains culture technique field, be specifically related to a kind of method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria.
Background technology.
At the city inland river of China, close water body, all there is serious body eutrophication and pollute in small watershed, black smelly, peculiar smell, even blue-green algae red tide, cause huge infringement in water body safety and HUMAN HEALTH.The nutritive substance such as nitrogen, phosphorus surplus causes the basic reason of black and odorous water eutrophication, when just there is the possibility that eutrophication occurs in water body during N concentration >0.2mg/L, P concentration >0.02mg/L.Comprehensive due to the planning of China urban pipe network, the impact causing many city inland rivers to be all subject to sanitary sewage discharge becomes sewage ditch, loses self-purification capacity.And sanitary sewage is the maximum emission source of the nutritive substance such as nitrogen, phosphorus, the nitrogen phosphorus therefore how solving black and odorous water is restoration of the ecosystem city inland river, closes water body, the key problem of small watershed.The more of current use adds chemical water purification agent, the methods such as additional bacterial classification and planting aquatic plants.Add chemical water purification agent and can reduce phosphorus content in water body fast, but produce inorganic sediment, cause bed mud state deteriorating, produce secondary pollution, and nitrogen is not acted on; Additional bacterial classification, can increase the quantity of microorganisms of nitrogen and phosphors removal in water body, but there is the risk of external bacterial classification to the adaptation of water body and alien species long-term safety, and cost is higher, and the expense of the additional bacterial classification of every square meter water body is in 1-2 unit; Plantation has the waterplant separated nitrogen and gather phosphorus, can absorb the nitrogen phosphorus in water body, but growth cycle is longer, and needs harvesting in time, and labour cost is higher, limited use.
In view of the above problems, be necessary that cultivating one solves the problems referred to above for black and odorous water denitrogenation dephosphorizing composite bacteria method of cultivation.
Summary of the invention
The object of the present invention is to provide a kind of for black and odorous water denitrogenation dephosphorizing composite bacteria method of cultivation, to effectively solving the problem of nitrogen phosphorus surplus in black and odorous water.
Technical scheme of the present invention is:
For a method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria, comprise the following steps:
(1) bacterial classification sample collecting: gather the active sludge of municipal sewage plant and the bed mud of black and odorous water respectively, put into thermostat container stand-by, temperature is 28 DEG C ~ 35 DEG C;
(2) amplification cultivation: under aerobic condition, in two kinds of mud samples, add liquid nutrient medium respectively carry out perseverance surely cultivation, temperature is 28 DEG C ~ 35 DEG C, and incubation time is 2 ~ 3 days;
(3) domesticated strain is screened: added in screening domestication substratum according to 1 ~ 3:1 mixing by two kinds of mud mixed liquids, aerobic vibration at 28 DEG C ~ 35 DEG C temperature 2 ~ 3 days is an acclimation period, needs 5-8 acclimation period; After each acclimation period terminates, get 5-10mL nutrient solution and be inoculated in fresh screening domestication substratum;
(4) purifying bacterial classification: by the strain inoculation of having screened in activation enrichment medium, 28 DEG C ~ 35 DEG C temperature, cultivate 2-3 days, use supercentrifuge centrifugal treating, then with phosphate buffered saline buffer cleaning, more centrifugally make bacterial classification suspension.
The allocation ratio of the liquid nutrient medium described in step (2) is: add K in 1000mL substratum
2hPO
40.0-1.5g, MgSO
47H
2o1-3g, extractum carnis 1-3g, (NH
4)
2sO
41-2g, NaH
2pO43H
2o1-2g, urea 0.2-0.3g, FeSO
47H
2o0.002-0.003g, MnSO
47H
2o0.003-0.004g, ZnSO
47H
2o0.003-0.004g, CoSO
47H
2o0.001-0.002g, glucose 15-16g, adjust pH value to be 7.0, and autoclaving.
The allocation ratio of the screening domestication substratum described in step (3) is: sewage 50-100g, K of adding black and odorous water in 1000mL substratum
2hPO
41.5-2.5g, NaH
2pO
43H
2o1-2g, MgSO
47H
2o3-4g, NH
4cl2-3g, vitamins B
10.1-0.2g, adjusts pH value to be 7.0, and autoclaving.
The allocation ratio of the activation enrichment medium described in step (4) is: add NH in 1000mL substratum
4cl2-3g, extractum carnis 5-6g, glucose 15-16g, agar 15-16g, adjust pH value to be 7.0, and autoclaving.
Through qualification, gained bacterial classification comprises following four kinds of microorganisms: bacillus cereus, Paracoccus denitrificans, Pseudomonas stutzeri, subtilis.
First the present invention filters out the indigenous microorganism with denitrogenation dephosphorizing in the active sludge of municipal sewage plant and black and odorous water bed mud, with the sewage of black and odorous water as main carbon source and energy, add the nutritive element needed for other growth again, obtained from the composite bacteria with denitrogenation dephosphorizing in native environment by domestication.It is fast that the bacterial classification that the present invention turns out has propagation, to the feature such as the Nitrogen/Phosphorus Removal of black and odorous water is obvious, the composite bacteria process black and odorous water using the present invention to obtain both had avoided the secondary pollution using chemical water purification agent, also do not need the security worrying bacterial classification, processing efficiency is also far above planting aquatic plants.
Accompanying drawing explanation
Fig. 1 is the schema for black and odorous water denitrogenation dephosphorizing composite bacteria method of cultivation of the present invention.
Embodiment
Embodiment:
Schema for black and odorous water denitrogenation dephosphorizing composite bacteria method of cultivation of the present invention refers to Fig. 1, and concrete implementation step is as follows:
(1) bacterial classification sample collecting: each 1000mL of bed mud gathering the black smelly inland river of active sludge and city one of Nanjing one municipal sewage plant respectively with sterile sampling bottle, put into thermostat container stand-by, temperature is 28 DEG C ~ 35 DEG C.
(2) amplification cultivation: add liquid nutrient medium according to the ratio of 10g/L respectively in two kinds of mud samples, and carry out aeration constant temperature culture, temperature is 28 DEG C ~ 35 DEG C, and incubation time is 2-3 days.Wherein the composition of liquid nutrient medium is: add K in 1000mL substratum
2hPO
41.5g, MgSO
47H
2o1g, extractum carnis 1g, (NH
4)
2sO
41g, NaH
2pO
43H
2o1g, urea 0.2g, FeSO
47H
2o0.002g, MnSO
47H
2o0.003g, ZnSO
47H
2o0.003g, CoSO
47H
2o0.001g, glucose 15g, adjust pH value to be 7.0, and autoclaving.
(3) screen domesticated strain: two kinds of mud mixed liquids are got 5mL mixing altogether according to 1:1 ratio, add 1000mL screening domestication substratum, aerobic vibration at 28 DEG C ~ 35 DEG C temperature, 2 days is an acclimation period, carries out 6 acclimation periods altogether.After each acclimation period terminates, get 5mL nutrient solution and be inoculated in fresh 1000mL screening domestication substratum.Wherein screening domestication medium component is: sewage 100g, K of adding black and odorous water in 1000mL substratum
2hPO
41.5g, NaH
2pO
43H
2o1.0g, MgSO
47H
2o3.0g, NH
4cl2g, vitamins B
10.1g, adjusts pH value to be 7.0, and autoclaving.
(4) purifying bacterial classification: the bacterial classification 5mL screened is inoculated into 1000mL and activates in enrichment medium, 28 DEG C ~ 35 DEG C temperature, cultivate 2 days, with supercentrifuge (8000r/min) centrifugal treating 10min, again with phosphate buffered saline buffer cleaning, more centrifugal 10min makes bacterial classification suspension.Wherein activating enrichment culture based component is add NH in 1000mL substratum
4cl2g, extractum carnis 5g, glucose 15g, agar 15g, adjust pH value to be 7.0, and autoclaving.
(5) strain identification: the bacterium colony that the extraction provided with MIDI lipid acid identification systems, authentication method purification Identification go out.The bacterial classification identified comprises following four kinds of microorganisms, and number consecutively is: A bacillus cereus B Paracoccus denitrificans C Pseudomonas stutzeri D subtilis.
A bacillus cereus falls for oyster white, irregularly shaped, and gramstaining is positive; B Paracoccus denitrificans, milk yellow, the individual ovalize of bacterial strain, gramstaining is negative; C Pseudomonas stutzeri falls for yellow, straight or micro-curved bacilliform, and gramstaining is negative; D bacillus subtilis bacterium colony is micro-yellow, oval to column, Gram-positive.The ratio that four kinds of bacterium survive in the medium is: A bacillus cereus 38%, B Paracoccus denitrificans 35%, C Pseudomonas stutzeri 18%, D subtilis 9%.By the composite bacteria suspension obtained, liquid nutrient medium is added at 28-35 DEG C of temperature, cultivate 2 days, aerobic treatment takes from the sewage in malodorous black water, experiment finds, according to the dosage of 1%, the aerobic treatment of 8-16 hour efficiently can remove the suspended substance (SS) in sewage, CODcr, ammonia nitrogen and total phosphorus (TP).As table 1, by the process of 16 hours, the requirement of surface water four class standard can be reached.
Table 1
Above-described embodiment is interpreted as only being not used in for illustration of the present invention limiting the scope of the invention.After the content of having read the present invention's record, those skilled in the art can make various changes or modifications the present invention, and these equivalence changes and modification fall into the scope of the claims in the present invention equally.
Claims (1)
1. for a method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria, it is characterized in that, comprise the following steps:
(1) bacterial classification sample collecting: gather the active sludge of municipal sewage plant and the bed mud of black and odorous water respectively, put into thermostat container stand-by, temperature is 28 DEG C ~ 35 DEG C;
(2) amplification cultivation: under aerobic condition, adds liquid nutrient medium respectively and carries out constant temperature culture in two kinds of mud samples, and temperature is 28 DEG C ~ 35 DEG C, and incubation time is 2 ~ 3 days; The allocation ratio of described liquid nutrient medium is: add K in 1000mL substratum
2hPO
40.0-1.5g, MgSO
47H
2o1-3g, extractum carnis 1-3g, (NH
4)
2sO
41-2g, NaH
2pO
43H
2o1-2g, urea 0.2-0.3g, FeSO
47H
2o0.002-0.003g, MnSO
47H
2o0.003-0.004g, ZnSO
47H
2o0.003-0.004g, CoSO
47H
2o0.001-0.002g, glucose 15-16g, adjust pH is 7.0, and autoclaving;
(3) domesticated strain is screened: added in screening domestication substratum according to 1 ~ 3:1 mixing by two kinds of mud mixed liquids, aerobic vibration at 28 DEG C ~ 35 DEG C temperature 2 ~ 3 days is an acclimation period, needs 5-8 acclimation period; After each acclimation period terminates, get 5-10mL nutrient solution and be inoculated in fresh screening domestication substratum; The allocation ratio of described screening domestication substratum is: sewage 50-100g, K of adding black and odorous water in 1000mL substratum
2hPO
41.5-2.5g, NaH
2pO
43H
2o1-2g, MgSO
47H
2o3-4g, NH
4cl2-3g, vitamins B
10.1-0.2g, adjust pH is 7.0, and autoclaving;
(4) purifying bacterial classification: by the strain inoculation of having screened in activation enrichment medium, 28 DEG C ~ 35 DEG C temperature, cultivate 2-3 days, centrifugal treating, then with phosphate buffered saline buffer cleaning, more centrifugally make bacterial classification suspension; The allocation ratio of described activation enrichment medium is: add NH in 1000mL substratum
4cl2-3g, extractum carnis 5-6g, glucose 15-16g, agar 15-16g, adjust pH is 7.0, and autoclaving.
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| CN201410136717.9A CN103911332B (en) | 2014-04-04 | 2014-04-04 | A kind of method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria |
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| CN201410136717.9A CN103911332B (en) | 2014-04-04 | 2014-04-04 | A kind of method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria |
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| CN103911332A CN103911332A (en) | 2014-07-09 |
| CN103911332B true CN103911332B (en) | 2016-01-20 |
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| CN104877916B (en) * | 2015-06-01 | 2017-11-17 | 云南省烟草农业科学研究院 | A kind of secondary coccus and its microbial inoculum and preparation method and application |
| CN106337028A (en) * | 2015-07-17 | 2017-01-18 | 上海海洋大学 | Cleaning agent, and preparation method and application thereof |
| CN106676022A (en) * | 2015-11-06 | 2017-05-17 | 丹阳市尚德生物科技有限公司 | Method for preparing microbe bacteria liquid for treating black-odor riverway |
| CN106148246B (en) * | 2016-08-30 | 2019-11-12 | 蓝德环保科技集团股份有限公司 | Purify the compounding microbial inoculum and preparation method thereof of black and odorous water |
| CN106396133B (en) * | 2016-12-01 | 2019-03-26 | 东莞理工学院 | A method of black and odorous water is handled with ethyl alcohol stalk waste residue immobilization polyP bacteria processed |
| CN106745804B (en) * | 2016-12-16 | 2020-12-04 | 北京桑德环境工程有限公司 | Microbial quick clarifying agent for black and odorous water body and preparation method thereof |
| CN108178327A (en) * | 2017-12-26 | 2018-06-19 | 中冶京诚工程技术有限公司 | Microorganism screening and enriching method for treating black and odorous water river and application thereof |
| CN109052624B (en) * | 2018-08-08 | 2021-08-03 | 中国科学院水生生物研究所 | Mobile method and device for purifying sewage and black and odorous water |
| CN110484458A (en) * | 2018-08-28 | 2019-11-22 | 华中科技大学 | It is a kind of to handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage |
| CN109231711B (en) * | 2018-11-06 | 2021-05-18 | 江苏澳洋生态园林股份有限公司 | Black and odorous river sewage treatment system and method |
| CN109576189B (en) * | 2018-12-29 | 2022-04-19 | 合肥市东方美捷分子材料技术有限公司 | Composite microbial inoculum for black and odorous water body and preparation method and application thereof |
| CN109576201A (en) * | 2019-01-31 | 2019-04-05 | 上海同瑞环保科技有限公司 | A method of culture indigenous microorganism efficiently removes water body ammonia nitrogen |
| CN109678257A (en) * | 2019-03-01 | 2019-04-26 | 北京高能时代环境技术股份有限公司 | A method of being suitable for the functional flora construction method and application flora improvement black and odorous water of black and odorous water improvement |
| CN111268857B (en) * | 2020-01-21 | 2023-04-18 | 武汉工程大学 | Method for removing high-concentration ammonia nitrogen in residual ammonium salt leachate of rare earth leaching site by chemical-biological method |
| CN111268807B (en) * | 2020-01-21 | 2023-04-07 | 武汉工程大学 | Method for removing ammonia nitrogen in residual ammonium salt leachate of rare earth leaching site by using indigenous denitrification microbial flora |
| CN112520854A (en) * | 2020-12-30 | 2021-03-19 | 魏一明 | Method for treating sewage by using biological membrane |
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| CN103539251A (en) * | 2013-10-22 | 2014-01-29 | 江西金达莱环保股份有限公司 | Method for synchronously degrading carbon, nitrogen and phosphorus in wastewater without discharging sludge |
| CN103626302B (en) * | 2013-11-26 | 2015-12-02 | 广州市地下铁道总公司 | The sewage water treatment method that a kind of denitrogenation dephosphorizing bacterial classification On-line testing is cultivated and tamed |
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Effective date of registration: 20190515 Address after: Room D7, Room 205, 2nd Floor, Lane 13, 16299, Puwei Highway, Shanyang Town, Jinshan District, Shanghai, 201500 Patentee after: Shanghai Yousilk Environmental Technology Co., Ltd. Address before: 2001-135 Yushan Road 100 Lane 4-301, Pudong New Area, Shanghai Patentee before: Fan Min |
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| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160120 Termination date: 20200404 |