CN104152367B - Heterotrophic nitrification bacterial strain - Google Patents
Heterotrophic nitrification bacterial strain Download PDFInfo
- Publication number
- CN104152367B CN104152367B CN201310750912.6A CN201310750912A CN104152367B CN 104152367 B CN104152367 B CN 104152367B CN 201310750912 A CN201310750912 A CN 201310750912A CN 104152367 B CN104152367 B CN 104152367B
- Authority
- CN
- China
- Prior art keywords
- bacterial strain
- nitrogen
- heterotrophic nitrification
- bacterium
- hlnr02
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses a heterotrophic nitrification bacterial strain which is named as Klebsiella sp.HLNR02 and is collected in the China General Microbiological Culture Collection Center (CGMCC) in 3#, No.1 Courtyard, West Beichen Road, Chaoyang District, Beijin in October, 2013, and the collection number is CGMCC No.8397. The heterotrophic nitrification bacterial strain has the beneficial effects that the heterotrophic nitrification bacterial strain is relatively high in degradation effect on ammonia nitrogen and total nitrogen in a eutrophic water body, and a good microorganism material is provided for biological treatment on a water body polluted by ammonia nitrogen wastewater by further using the stain.
Description
Technical field:
The present invention relates to a kind of bacterial isolateses of microorganism field are and in particular to one plant of Heterotrophic nitrification bacterial strain.
Background technology:
Nitrogen transform is close with sewage sludge nitric efficiency, the improvement of denitrification process, solution of body eutrophication etc.
Cut is closed.The method solving the nitrogen eutrophication of water body at present has physics, chemistry and biological method.With nitrate pollution not
Disconnected aggravation and the pay attention to day by day to environmental problem for the people, denitrogenate technology, particularly bio-denitrification technology has become as control water body
The important directions of pollution and means.
Ammonia nitrogen is to cause one of pollutant of body eutrophication.At present it is believed that in biological denitrification process, waste water
In ammonia nitrogen first no is oxidized under aerobic condition by Autotrophic nitrification bacteriumx -.Then nox -Thin by denitrification under anoxic conditions
Bacterium is reduced to gaseous nitrogen such as n2Escape Deng from water.Traditional bio-denitrification technology such as biofilter, oxidation ditch, biological film process
The nitrifying bacteria community that system and fluid bed etc. adopt mostly, nitrifier mostly is autotrophic bacteria.Autotrophic bacteria growth rate is slow and is difficult to maintain
Higher biological concentration, needs first through Air Exposure to reduce organic concentration, bacterial strain could grow and show biologic activity, anti-impact
Hit ability weak；Ammonia nitrogen in high density and nitrite can suppress the growth of nitrifier again, make Nitrification not exclusively, lead to total nitrogen to go
Except rate is very low.
Nitrogen-containing compound can be nitrified while being grown using organic carbon source and generate azanol, nitrous by allotrophic nitrobacteria
The products such as hydrochlorate, nitrate, majority can also carry out aerobic denitrification simultaneously, directly nitration product is converted into containing nitrogen
Body.Therefore, this kind of antibacterial has become the important research object of New Biodenitrification Process in wastewater treatment.With autotrophic type nitrifier ratio
Relatively, the growth rate of nitrification bacteria is fast, and cell yield is high, and the dissolved oxygen concentration of needs is low, is resistant to sour environment and activity
Height, and it is capable of the nitrogen compound of the various form of metabolism.Due to the appearance of nitrification bacteria, technique can be implemented in one instead
Answer and complete nitration denitrification in device, operating cost not only can be reduced, reduce loaded down with trivial details operation in technique, autotrophy can also be expanded
The not treatable water quality scope of nitrifier institute.
Content of the invention:
The technical problem to be solved in the present invention is to separate one plant of Heterotrophic nitrification bacterial strain.
The technical solution of the present invention is to separate from eutrophication water and filter out heterotrophic nitrifying bacterium, this bacterium
Strain is named as Klebsiella bacteria (klebsiella sp.) hlnr02, and it is micro- that it was preserved in China on October 28th, 2013
Biological inoculum preservation administration committee common micro-organisms center (cgmcc), address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica, deposit number is: cgmcc no.8397.
The biological characteristicses of this bacterial strain are: Gram-negative, heterotrophic growth, optimum carbon source glucose sugar, and optimum ph value is
7.2,28 DEG C of optimum temperature.
Separation screening obtains Heterotrophic nitrification bacterial strain involved in the present invention by the following method:
(1) gather water sample in the general lake of Jiangxi Province Jian County, the water body 1l in water sampling with 0.22 μm of membrane filtration,
With 10ml aseptic water washing filter membrane, after algae precipitation, take supernatant 1ml in the enrichment medium containing ammonia nitrogen that 99ml sterilizes,
This culture medium is inserted 28 DEG C of temperature, frequency of vibration cultivates 4d in the shaken cultivation case for 200rpm/min.1ml bacterium solution is taken after 4d
In the enrichment medium containing ammonia nitrogen of 99ml sterilizing, cultivate 4d in above-mentioned condition of culture.It is repeated 3 times by same operation, enter
The enrichment culture in 4 cycles of row.
(2) take and in step (1), be enriched with bacterium solution 1ml obtaining to the 4th cycle, be added to the domestication training that ammonia nitrogen concentration improves 50%
In foster base, carry out domestication culture, inoculum concentration and the same enrichment culture of condition of culture in 4 cycles.Obtain domestication culture fluid.
(3) take 5 parts of bacterium solution in the domestication culture fluid obtaining from step (2), every part is 1ml, carries out gradient dilution respectively,
It is 10 from gradient-3、10-4、10-5、10-6、10-7；The dilution bacterium solution taking 0.1ml gained from every part respectively is respectively coated in containing
Have in the solid plate culture medium containing ammonia nitrogen, 37 DEG C of culture 2d.
(4) with sterilized liquid transfer gun head from the plating medium of step (3) the larger and independent bacterium colony of picking, adopt
It is inoculated on lb solid medium with method of scoring, cultivate in 28 DEG C.Single bacterium colony is obtained by 3 line.Single bacterium colony is in fresh lb
It is enlarged in culture medium cultivating, the strain after amplification culture, be partially disposed in conservation in -80 DEG C of refrigerators, be partially disposed in 4 DEG C of guarantors again
Deposit standby.
The beneficial effects of the present invention is: through multiple repetition test, this heterotrophic nitrifying bacterium is to ammonia in eutrophication water
Nitrogen and total nitrogen have stronger degradation effect.It is to be given birth to by the water body of ammonia nitrogen class contaminated wastewater to administering further with this bacterial strain
Thing method is administered and is provided good microbial material.
Brief description:
Fig. 1 is strain 16s rdna pcr-rflp restriction enzyme digestion and electrophoresis figure；
Fig. 2 is that bacterial strain hlnr02 adopts the systematic evolution tree of n-j method structure with similar sequences；
Fig. 3 is solution nitrogen effect in simulation eutrophic wastewater for the bacterial strain hlnr02.
Specific embodiment:
1. the separation of bacterial strain hlnr02
(1) gather water sample in the general lake of Jiangxi Province Ji'an City Jian County, the water body 1l in water sampling is with 0.22 μm of filter
Membrane filtration, with 10ml aseptic water washing filter membrane, takes the enrichment training containing ammonia nitrogen that supernatant 1ml sterilizes in 99ml after algae precipitation
In foster base, this culture medium is inserted 28 DEG C of temperature, frequency of vibration cultivates 4d in the shaken cultivation case for 200rpm/min.Take after 4d
1ml bacterium solution, in the enrichment medium containing ammonia nitrogen that 99ml sterilizes, cultivates 4d in above-mentioned condition of culture.Repeat by same operation
3 times, carry out the enrichment culture in 4 cycles.
(2) take and in step (1), be enriched with bacterium solution 1ml obtaining to the 4th cycle, be added to the domestication training that ammonia nitrogen concentration improves 50%
In foster base, carry out domestication culture, inoculum concentration and the same enrichment culture of condition of culture in 4 cycles.Obtain domestication culture fluid.
(3) take 5 parts of bacterium solution in the domestication culture fluid obtaining from step (2), every part is 1ml, carries out gradient dilution respectively,
It is 10 from gradient-3、10-4、10-5、10-6、10-7；The dilution bacterium solution taking 0.1ml gained from every part respectively is respectively coated in containing
Have in the solid plate culture medium containing ammonia nitrogen, 37 DEG C of culture 2d.
(4) with sterilized liquid transfer gun head from the plating medium of step (3) 40 plants of larger and independent bacterium of picking
Fall, be inoculated on lb solid medium using method of scoring, cultivate in 28 DEG C.Single bacterium colony is obtained by 3 line.Take this single bacterium colony
Fresh lb culture medium is enlarged again cultivate.Strain after amplification culture, is partially disposed in conservation in -80 DEG C of refrigerators, partly in 4
DEG C preserve, be that follow-up test is standby.
Above-mentioned enrichment medium, refers to ammonia nitrogen (nh4 +- n) for nitrogen source enrichment medium, this culture medium comprises following thing
Matter component and condition: glucose 5g, (nh4)2so41g, k2hpo40.5g, kcl63mg, mgso423mg, anhydrous cacl223mg、
nahco365mg、kh2po423mg, trace element solution 2ml, distilled water 1000ml, ph7.2.
Above-mentioned lb culture medium: include peptone 10g, yeast extract 10g, nacl5.0g, ph7.2.
Above-mentioned solid medium adds 1.5% agar powder for fluid medium.
Above-mentioned trace element solution, its formula (g l-1) it is: znso42.2, cacl25.5, mncl2·4h2O5.06,
feso4·7h2O5.0, cuso4·5h2O1.57, cocl2·6h2o1.61.
2. the pcr-rflp analysis of bacterial strain
(1) with nido pcr (nested-pcr), the 16s rdna of above-mentioned 40 plants of bacterium is expanded.Method is as follows:
It is template that first round reaction (volume 25 μ l) contains 1 μ l (about 1ng) dna, 0.5 μ l primer (10 μm), the dntp of 2 μ l
Mixture (2.5mm), 1.5 μ l, mgcl2(25mm), 2.5 μ l10 × pcr reaction buffer and 0.2 μ l taq dna polymerase
(5u/ μ l, takara company).The first round primer of reaction is: 27f (5 '-agagtttgatcctggctcag-3 ') and 1492r
(5′-taccttgttacgactt-3′).Pcr response procedures are: 94 DEG C of denaturations 5min, 94 DEG C of 30s, 55 DEG C of 40s, 72 DEG C
1min, totally 10 circulations, last 72 DEG C of extension 10min.
With the pcr product of first round reaction as template, primer is: 63f (5 '-caggcctaac for second wheel reaction
Acatgcaagtc-3 ') and 1389r (5 '-acgggcggtgtgtacaag-3 ').In the reaction of this wheel, 30 circulations of setting,
Reaction condition reacted with the first round.After pcr terminates, the agarose gel electrophoresiies containing 1% ethidium bromide for the product, detection are (about
The fragment of 1300bp length is target fragment).
(2) pcr product is used respectively restriction endonuclease rsai and hhai digest (37 DEG C, 1h).Enzyme action dna fragment
Agarose gel electrophoresiies with 2% separate, and are imaged (see accompanying drawing 1 strain 16s through ethidium bromide staining and gel imaging system
Rdna pcr-rflp restriction enzyme digestion and electrophoresis figure) after, gained dna banding pattern collection of illustrative plates carries out manpower comparing relatively under gis gel analysis software auxiliary
Analysis.Clustered based on the polymorphic collection of illustrative plates of genetic fragment, the clone with identical collection of illustrative plates being aggregated to together is considered as identical
Genotype.Each genotype as a sort operation unit (otu, operational taxonomic unit) or claims
For unique gene type, obtain 11 kinds of different strains, numbering is respectively 1,2,4,12,16,22,23,27,29,37,39.
3. the nitrification activity detection of bacterial strain
(1) from the step (2) of the pcr-rflp of above-mentioned bacterial strains analysis acquisition 11 plants of bacterium bacterium solution, respectively take 1ml, with aseptic
Water is inoculated in the fresh nitrification culture medium with ammonia nitrogen (nh4+-n) as nitrogen source of 100ml after cleaning 3 times respectively, is placed in 28 DEG C,
Frequency of vibration cultivates 1d in the shaken cultivation case for 200rpm/min, and ammonia nitrogen concentration (nh in 4 hours detection culture medium4 +-
N), nitrite nitrogen (no2 -- n), nitrate nitrogen (no3 -- n), the od value of total n concentration and bacterial growth；Arranged with this simultaneously
Blank control group.Wherein, nh4 +- n adopts Na Shi reagent spectrophotography；no2 -- n adopts n- (naphthalidine)-ethylenediamine luminosity
Method；no3 -- n adopts hydrochloric acid-naphthodiamide spectrophotography；Tn adopts potassium peroxydisulfate-ultraviolet spectrophotometry；Antibacterial od value is adopted
With using spectrophotometric determination bacteria suspension 600nm light absorption value.
Above-mentioned ammonia nitrogen (nh4 +- n) for nitrogen source nitrification culture medium: glucose 169mg, (nh4)2so437.71mg,
kcl63mg、mgso423mg, anhydrous cacl223mg、nahco365mg、kh2po4(formula is same for 23mg, trace element solution 2ml
On), distilled water 1000ml, ph7.2.
(2) testing result: 11 plants of bacterium with glucose as carbon source, cultivate in the Heterotrophic culture liquid of only nitrogen source by ammonium sulfate
During growth, ammonia nitrogen initial concentration is about 10.1mg/l, compares part folding after testing, No. 37 bacterium be the best bacterial strain of denitrification effect (see
Table 1 below, bacterium pearl nitrification activity, mg/l).
Table 1. bacterial strain nitrification activity
The Molecular Identification of 4.hlnr02 bacterial strain
(1) the 16s rdna pcr product of No. 37 bacterium is separated with 1% agarose gel electrophoresiies and uses agarose gel
Isolate and purify test kit and carry out purification.16s rdna fragment after purification is transformed into by e.coli dh5 α by ta clone technology
In, ampicillin and blue white macula screening picking positive colony Zi Song Sangon Biotech (Shanghai) Co., Ltd. are carried out
Sequencing.Sequencing result is compared with the 16s rdna sequence in genbank data base, obtains the most close bacterial strain
16s rdna sequence.By mega5.0 software, develop cladogram (see accompanying drawing 2 bacterial strain with neighbor-joining constructing system
Hlnr02 adopts the systematic evolution tree of n-j method structure with similar sequences).
(2) rdp cluster analyses show that bacterial strain hlnr02 belongs to β Bacillus proteuss subphylum (β-proteobacteria) Cray primary
Bordetella antibacterial (klebsiella sp.), similarity is 99%.Strain Designation is klebsiella sp.hlnr02 (see accompanying drawing
2 bacterial strain hlnr02 adopt the systematic evolution tree of n-j method structure with similar sequences).
Degradation effect in simulation eutrophic wastewater for the 5.hlnr02 bacterial strain
(1) experiment material
For examination waste water: simulation eutrophic wastewater: glucose 169mg, peptone 88.88mg, kcl63mg, anhydrous
cacl223mg, kh2po423mg, mgso423mg, nahco365mg, (nh4)2so437.71mg, trace element 2ml (formula and 1 bacterium
Formula described in the separation of strain hlnr02 is identical), distilled water 1l.
(2) experimental technique
Take 1ml bacterium solution, be inoculated in the fresh simulation eutrophic wastewater of 100ml with after sterile water wash 3 times, 28 DEG C,
1d is cultivated in the shaken cultivation case of 200r/min, and nh in 4 hours detection culture medium4 +- n and tn concentration.Wherein, nh4 +- n adopts Na Shi reagent spectrophotography；Tn adopts potassium peroxydisulfate-ultraviolet spectrophotometry.
(3) result of the test:
See solution nitrogen effect in simulation eutrophic wastewater for the accompanying drawing 3. bacterial strain hlnr02.
Hlnr02 is to nh in simulation eutrophic wastewater4 +- n and tn all has good degradation effect.Nh in 24 hours4 +-n
Clearance reach the clearance of 85.3%, tn and reach 77.3%.Plus bacterium cultivate 1 hour after record nh in culture medium4 +- n concentration is obvious
Increase, nh after 4 hours4 +- n concentration continuous decrease, illustrates that hlnr02 bacterium has and the organic nitrogen in simulated wastewater is converted into nh4 +-
The ability (see solution nitrogen effect in simulation eutrophic wastewater for the accompanying drawing 3 bacterial strain hlnr02) of n.
Claims (1)
1. one plant of Heterotrophic nitrification bacterial strain, this Strain Designation is Klebsiella bacteria klebsiella sp.hlnr02,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center cgmcc, address on October 28th, 2013
For Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, deposit number is: cgmcc
no.8397.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310750912.6A CN104152367B (en) | 2013-12-27 | 2013-12-27 | Heterotrophic nitrification bacterial strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310750912.6A CN104152367B (en) | 2013-12-27 | 2013-12-27 | Heterotrophic nitrification bacterial strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104152367A CN104152367A (en) | 2014-11-19 |
| CN104152367B true CN104152367B (en) | 2017-02-01 |
Family
ID=51877996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310750912.6A Expired - Fee Related CN104152367B (en) | 2013-12-27 | 2013-12-27 | Heterotrophic nitrification bacterial strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104152367B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726366B (en) * | 2015-01-29 | 2018-06-01 | 徐州工程学院 | The Denitrifying Phosphate Accumulating Organisms of one plant of high-efficient denitrification and dephosphorization and its application |
| CN110157637B (en) * | 2019-04-04 | 2021-03-16 | 华中农业大学 | Enterobacter Z1 and Klebsiella Z2 composite microbial inoculum for removing high-nitrogen polluted wastewater and application thereof |
| CN114958669B (en) * | 2022-05-31 | 2023-05-23 | 湖南农业大学 | Michigan klebsiella and product and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613156A (en) * | 2009-08-07 | 2009-12-30 | 合肥工业大学 | Adopt Klebsiella sp. DB-2 bacterium to repair the method that nitrate in groundwater is polluted |
| CN101033453B (en) * | 2006-03-07 | 2010-07-14 | 中国科学院过程工程研究所 | Klebsiella and application of the same for eliminating organic nitrogen in fossil fuels |
| CN101886053A (en) * | 2010-06-08 | 2010-11-17 | 东华大学 | Denitrification screening and purifying method for klebsiella ornithinolytica |
| CN101875526B (en) * | 2010-05-13 | 2011-09-07 | 宜兴蓝星化工环保研究院有限公司 | Method for treating coking waste water after ammonia removal |
| CN103013867A (en) * | 2012-12-04 | 2013-04-03 | 南京农业大学 | Acid-producing klebsiella pneumoniae DF-1 and application thereof in removing nitrous nitrogen in water body |
-
2013
- 2013-12-27 CN CN201310750912.6A patent/CN104152367B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033453B (en) * | 2006-03-07 | 2010-07-14 | 中国科学院过程工程研究所 | Klebsiella and application of the same for eliminating organic nitrogen in fossil fuels |
| CN101613156A (en) * | 2009-08-07 | 2009-12-30 | 合肥工业大学 | Adopt Klebsiella sp. DB-2 bacterium to repair the method that nitrate in groundwater is polluted |
| CN101875526B (en) * | 2010-05-13 | 2011-09-07 | 宜兴蓝星化工环保研究院有限公司 | Method for treating coking waste water after ammonia removal |
| CN101886053A (en) * | 2010-06-08 | 2010-11-17 | 东华大学 | Denitrification screening and purifying method for klebsiella ornithinolytica |
| CN103013867A (en) * | 2012-12-04 | 2013-04-03 | 南京农业大学 | Acid-producing klebsiella pneumoniae DF-1 and application thereof in removing nitrous nitrogen in water body |
Non-Patent Citations (1)
| Title |
|---|
| 异养硝化菌的筛选及其在污水脱氮中的应用;王鑫;《中国优秀博硕士学位论文全文数据库（硕士）工程科技I辑》;20070215(第2期);摘要，正文第2.3.2、2.3.3、3.2.4、3.3.1、4.3.4节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104152367A (en) | 2014-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107201325A (en) | Pseudomonad strain and its cultural method and application | |
| CN105861359A (en) | Heterotrophic nitrification-aerobic denitrification high temperature resisting strain for producing floc, and application thereof | |
| CN104845920B (en) | One plant of ocean Zhuo Beier Salmonella and its application | |
| CN108342339B (en) | Klebsiella strain and application thereof in river sewage and rural ammonia nitrogen-containing domestic sewage | |
| CN107460141B (en) | Heterotrophic nitrification aerobic denitrification citrobacter and application thereof | |
| CN113174345A (en) | Heterotrophic nitrification-aerobic denitrification strain for efficient denitrification and application thereof | |
| CN107177530B (en) | Novel efficient domestic sewage denitrifying bacterium and application thereof | |
| CN105420165A (en) | Aerobic denitrifying bacteria and applications therefor | |
| CN109401997A (en) | One plant of Stenotrophomonas and its application and microbial bacterial agent | |
| CN102373161A (en) | Bacterium for reducing mercury pollution in seawater and application thereof | |
| CN110656066B (en) | Acinetobacter strain for shortcut nitrification and denitrification and application thereof | |
| Feng et al. | Characterization of Pseudomonas mendocina LR capable of removing nitrogen from various nitrogen-contaminated water samples when cultivated with Cyperus alternifolius L. | |
| CN104152367B (en) | Heterotrophic nitrification bacterial strain | |
| CN103881947A (en) | Brevendimonas diminuta strain for forming biofilm and application thereof in cyanide-containing wastewater treatment | |
| CN113151063B (en) | Citrobacter freundii AS11 and application thereof in sewage treatment | |
| CN102167448B (en) | Application of aniline degradation strain NO.3 | |
| CN116622556B (en) | Low-temperature-resistant high-alkalinity high-efficiency denitrifying bacterium, screening method and application thereof | |
| CN115386520B (en) | Rhodococcus pyridine-philic RL-GZ01 strain and application thereof | |
| CN110157637A (en) | Enterobacteria Z1 and klebsiella Z2 composite bacteria agent removal high nitrogen pollutant effluents and application | |
| CN105670965B (en) | Strain with iron reduction capacity and application thereof | |
| CN104004674B (en) | Aerobic denitrifying bacterial strain | |
| CN103013867A (en) | Acid-producing klebsiella pneumoniae DF-1 and application thereof in removing nitrous nitrogen in water body | |
| CN113583918A (en) | River sediment degrading strain and application thereof | |
| CN102367423A (en) | Carbazole-degrading bacterium and its culture method | |
| CN114317382B (en) | Anaerobic strain applied to riverway water body COD degradation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| 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: 20170201 Termination date: 20191227 |