CN103421701A - Bacillus subtilis strain and application thereof to degradation of 17 beta-estradiol - Google Patents
Bacillus subtilis strain and application thereof to degradation of 17 beta-estradiol Download PDFInfo
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- CN103421701A CN103421701A CN2012101646731A CN201210164673A CN103421701A CN 103421701 A CN103421701 A CN 103421701A CN 2012101646731 A CN2012101646731 A CN 2012101646731A CN 201210164673 A CN201210164673 A CN 201210164673A CN 103421701 A CN103421701 A CN 103421701A
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Abstract
The invention discloses a bacillus subtilis strain and application thereof to degradation of 17 beta-estradiol. The bacillus subtilis CHS provided by the invention has preservation number of CGMCC No.6128, and can be used for degradation of 17 beta-estradiol. The invention also patents a preparation method of a product. The method comprises the following steps: inoculating bacillus subtilis CHS to a LB medium containing ammonia nitrogen; oscillating and culturing for 12-36 h at 37 DEG C; and freeze-drying the culture system to obtain a degraded 17 beta-estradiol product. The bacillus subtilis CHS or the product can be applied to degradation of livestock waste water. The bacillus subtilis strain provided by the invention aims at the characteristic of high pH value of the livestock and poultry culture waste water, has the advantages of low cost, simple operation, high degradation efficiency; besides, the bacillus subtilis strain has good environment compatibility, reduces usage of chemical reagents, avoids secondary pollution, and shows good application prospects in biological restoration of environment polluted by natural estrogens.
Description
Technical field
The present invention relates to a bacillus subtilis and the application in degraded 17 beta estradiols thereof.
Background technology
Oestrogenic hormon (English name: estrogen; Estrin; Oestrogen), be the 18 carbon steroid hormones that produced by vertebrate ovary, testis, placenta or adrenal cortex.The mammiferous main oestrogenic hormon of the overwhelming majority is 17 beta estradiols (E2), and other important oestrogenic hormon have trihydroxy-oestrin and oestrone.
Natural estrogen extensively is distributed in waste water, animal excrements, surface water and underground water.Natural estrogen demonstrates very strong endocrine disrupting under very low concentrations, as E2, will make some wild male fish the feminisation phenomenon occur when the 1-10ng/L.With other sewage, compare, in livestock and poultry breeding industry waste water, the content of estrogenic chemicals is very high, and TO can reach 1000-21000ng/L, and wherein the concentration of E2 in the waste water of raising pigs can reach 1000-16000ng/L, is more than 10 times of municipal effluent content.
Summary of the invention
The purpose of this invention is to provide a bacillus subtilis and the application in degraded 17 beta estradiols thereof.
Subtilis provided by the invention (Bacillus subtilis) CHS, in on 05 22nd, 2012, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.6128.Subtilis (Bacillus subtilis) CHS CGMCC No.6128 is called for short subtilis CHS.
Described subtilis CHS 17 beta estradiols that can be used for degrading.When applying described subtilis CHS and degrading 17 beta estradiol, the pH value of reaction system can be 6.25-8.75(as 6.25-7.40 or 7.40-8.75).
When applying described subtilis CHS and degrading 17 beta estradiol, the ammonia nitrogen concentration of reaction system can be 0.6-10mg/L(as 0.6-2mg/L or 2-10mg/L).Described ammonia nitrogen is specifically by (NH
4)
2CO
3Or NH
4HCO
3Provide.
The present invention also protects a kind of method for preparing the product of 17 beta estradiols of degrading; comprise the steps: described subtilis CHS is seeded in the LB substratum that contains ammonia nitrogen; 37 ℃ of shaking culture 12-36 hour, then by the culture system freeze-drying, the product of 17 beta estradiols that obtain degrading.
The described LB substratum that contains ammonia nitrogen can be and contains (NH
4)
2CO
3The LB substratum or contain NH
4HCO
3The LB substratum.The pH value of described LB substratum specifically can be 8.25.After described inoculation, the starting point concentration of described subtilis CHS specifically can be 10
4Cfu/mL.After completing described shaking culture, described in described culture system, the concentration of subtilis CHS specifically can be 10
8-10
9Cfu/mL.Described (the NH that contains
4)
2CO
3The LB substratum specifically can be and contain 10mg/L (NH
4)
2CO
3The LB substratum.The described NH that contains
4HCO
3The LB substratum specifically can be and contain 10mg/LNH
4HCO
3The LB substratum.Described vibration specifically can adopt the rotating speed of 200r/min.
The product of degraded 17 beta estradiols that above arbitrary described method prepares also belongs to protection scope of the present invention.
Described product 17 beta estradiols that can be used for degrading.
Described subtilis CHS or described product can be used for the fowl poultry waste water of degrading.
The present inventor adopts the biotechnology means, is separated to the bacterial strain that a strain can efficient degradation 17 beta estradiols from the active sludge of livestock breeding wastewater biological treatment sbr reactor device.Observe through form, cultural characteristic and Physiology and biochemistry experimental result, identify this bacterium for being subtilis.This bacterial strain has very high degradation effect to 17 beta estradiols.This bacterium, under the condition of the high-concentration ammonia-nitrogen solution of livestock breeding wastewater and high pH solution, still shows good degradation capability.In practical application, this bacterial strain enlarged culturing can be made to finished product bacteria suspension or pulvis, be invested in actual waste water system.The present invention has that cost is low, simple to operate, the advantages such as degradation efficiency is high, strong adaptability.
Advantage of the present invention and effect: the characteristic that is applicable to the high pH of the high ammonia nitrogen of livestock breeding wastewater; (2) have advantages of that cost is low, simple to operate, degradation efficiency is high; (3) Environmental compatibility is good, reduces and uses chemical reagent, avoids secondary pollution; (4) aspect the biological restoration of natural estrogen class material contaminate environment, having a good application prospect.
The accompanying drawing explanation
Fig. 1 is the concentration of 17 beta estradiols in each sample in embodiment 2.
Fig. 2 is the concentration of 17 beta estradiols in each sample in embodiment 3.
Fig. 3 is part PCR-DGGE result in embodiment 4.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method.Test materials used in following embodiment, if no special instructions, be and purchase available from routine biochemistry reagent shop.Quantitative test in following examples, all arrange repeated experiments three times, results averaged.
17 beta estradiol (E2; 17 beta estradiol standard substance): Sigma company, article No. is E8875-5G.
The preparation method of inorganic salt damping fluid: by 4.0g K
2HPO
4, 4.0g NaH
2PO
4, 2.0g (NH
4)
2SO
4, 0.5gMgSO
47H
2O, 0.01g CaCl
22H
2O, 0.01g FeSO
47H
2O and 0.01g MnSO
42H
2O is by water dissolution and be settled to 1L.
The preparation method of LB substratum: by 10g tryptone, 5g yeast extract and 10g NaCl by water dissolution and be settled to 1L.
In embodiment, PBS damping fluid used is pH7.0, the PBS damping fluid of 1M.
The acquisition of embodiment 1, subtilis CHS and evaluation
One, the acquisition of subtilis CHS
The mud sample is the aerobic activated sludge of taking from the sbr reactor device of Tongzhou District, Beijing City boar factory biological treatment livestock breeding wastewater in September, 2011.
In container, add the mud sample of the standing 24h of aeration, then add the inorganic salt damping fluid containing 10mg/L 17 beta estradiols, 25 ℃ of constant temperature oscillations in shaking table, repeatable operation one month, inducible strain produces the enzyme of degraded 17 beta estradiols.Then adopt the liquid diluting method to obtain the bacterial strain of pure culture, more therefrom screen anti-pollution concentration high and to contaminant removal capacity strong bacterial strain.
Final obtain a strain and take the bacterial strain that 17 beta estradiols are sole carbon source, by its called after CHS.
Two, the evaluation of subtilis CHS
1, identification of morphology
Colonial morphology: oval bacterium colony, neat in edge, translucent, canescence, be combined with substratum and loosely easily provoke; 0.7～0.8 * 2～3 microns of individual cells, without pod membrane, peritrichous, can move.
2, Physiology and biochemistry is identified
Can utilize the several kinds of carbon source such as dextrin, D-Fructose, maltose, glucose, uridine.
3, Molecular Identification
Bacterial strain is carried out to 16S rRNA evaluation, and its encoding sequence is as shown in the sequence 1 of sequence table.The encoding sequence of 16S rRNA is carried out to the BLAST analysis, find that the bacterial strain best with its homology is Bacillus subtilis (GENBANK ACCESSION NO.NR 027552.1), homology is 98%.
Three, the preservation of subtilis CHS
The result of identifying according to cellular form, Physiology and biochemistry, 16S rRNA, bacterial strain belongs to subtilis (Bacillus subtilis).
Subtilis (Bacillus subtilis) CHS, in on 05 22nd, 2012, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.6128.Subtilis (Bacillus subtilis) CHS CGMCC No.6128 is called for short subtilis CHS.
The pH value of waste water of raising pigs is generally 8.5-9.0, and different pH can have a certain impact to subtilis CHS 17 beta estradiols of degrading, so the present embodiment is for detection of subtilis CHS degradation capability to 17 beta estradiols in different pH environment.
1, subtilis CHS being mixed with to bacteria concentration with the PBS damping fluid is 10
8The bacteria suspension of cfu/mL.
2, packet transaction (adopt parallel processing, every group arranges three re-treatments)
First group: 1mL PBS damping fluid is seeded to 20mL containing in the inorganic salt damping fluid (the pH value is 7.40) of 1mg/L 17 beta estradiols, 37 ℃, 200r/min shaking culture;
Second group: by the bacterial suspension inoculation of 1mL step 1, to 20mL, containing in the inorganic salt damping fluid (the pH value is 6.25) of 1mg/L 17 beta estradiols, (starting point concentration that is subtilis CHS is 4.76 * 10
6Cfu/mL), 37 ℃, 200r/min shaking culture;
The 3rd group: the bacterial suspension inoculation of 1mL step 1 is contained in the inorganic salt damping fluid (the pH value is 7.40) of 1mg/L 17 beta estradiols to 37 ℃, 200r/min shaking culture to 20mL;
The 4th group: the bacterial suspension inoculation of 1mL step 1 is contained in the inorganic salt damping fluid (the pH value is 8.75) of 1mg/L 17 beta estradiols to 37 ℃, 200r/min shaking culture to 20mL;
From having inoculated the beginning timing, after 0 hour, 12 hours, 24 hours, 48 hours, 72 hours and 96 hours, sample.
3, the sample of each time being obtained is handled as follows respectively: at first use Ultrasonic Cleaners (KQ5200DE type numerical control supersonic, Kunshan Ultrasonic Instruments Co., Ltd.) carry out supersound process 20min, to guarantee absorption and to enter undegradable 17 beta estradiols in cell, enter supernatant liquor; Then centrifugal (4000r/min, 10min) gets supernatant liquor, adopts UPLC to detect the concentration of 17 beta estradiols, and adopts mass spectrum further to identify 17 beta estradiols that UPLC collects.
The UPLC parameter: chromatographic column is ACQUITY UPLC BEH C18 post (2.1mm * 50mm 1.7 μ m), 40 ℃ of column temperatures, sample size 10 μ L, flow velocity 0.2mL/min;
UPLC elution process: the mixture that elutriant is A liquid (water), B liquid (acetonitrile) or A liquid and B liquid;
At the 0th minute, A liquid/elutriant=65%, B liquid/elutriant=35%; The 0th minute to the 4th minute, A liquid/elutriant dropped to 50%, B liquid/elutriant by 65% linearity and rises to 50% by 35% linearity; The 4th to 4.5 minutes, A liquid/elutriant dropped to 0%, B liquid/elutriant by 50% linearity and rises to 100% by 50% linearity; The 4.5th to 7 minutes, elutriant was B liquid; The 7th to 7.5 minutes, A liquid/elutriant rose to 65%, B liquid/elutriant by 0% linearity and drops to 35% by 100%; The ratio of this section is volume ratio;
Adopt 17 beta estradiol standard substance to carry out above-mentioned UPLC, its retention time is 2.11, so the peak that retention time is 2.11 is the purpose peak.
Concentration and peak area production standard curvilinear equation by 17 beta estradiol standard substance are as follows: y=58.761x+0.3935(x represents concentration, and unit is mg/L; Y represents peak area).
Can obtain 17 beta estradiol concentration in supernatant liquor according to above-mentioned typical curve equation.
In each sample, the concentration of 17 beta estradiols is shown in Fig. 1 (mean values of three re-treatments).In the environment of pH6.25-8.75, subtilis CHS all has good degradation effect to 17 beta estradiols.The degradation efficiency of 17 beta estradiols that subtilis CHS is 1mg/L to starting point concentration after 96 hours is about 95%.
Collect the purpose peak, and adopt the material at this purpose peak of Mass Spectrometric Identification to form.The mass spectrum condition is in Table 1 and table 2.
Table 1 mass spectrum condition-1
| Ionization mode | The ESI negatively charged ion |
| Capillary voltage | 3.3kV |
| Desolvation air pressure | 600L/h |
| Taper hole air pressure | 50L/h |
| Ion source temperature | 100℃ |
| The desolvation othermohygrometer | 400℃ |
| Ion energy | 1.0 |
Table 2 mass spectrum condition-2
| Compound | Parent ion (m/z) | Daughter ion (m/z) a | Collision voltage (ev) | Taper hole voltage (V) |
| 17 β estradiol standard substance | 271.4 | 183.1，145.2 | 40,45 | 45 |
The mass spectrum result at purpose peak is consistent with the mass spectrum result of 17 beta estradiol standard substance, and the purpose peak is 17 beta estradiols really.
The ammonia nitrogen concentration of raising pigs in influent waste water, up to 996 ± 10mg/L, is 30mg/L by sbr reactor device diluted ammonia nitrogen concentration, so the present embodiment is for detection of subtilis CHS degradation capability to 17 beta estradiols in different ammonia nitrogen concentrations.
1, with the step 1 of embodiment 2.
2, packet transaction (adopt parallel processing, every group arranges three re-treatments)
First group (blank group): 1mL PBS damping fluid is seeded to 20mL containing in the inorganic salt damping fluid (the pH value is 8.75) of 1mg/L 17 beta estradiols, 37 ℃, 200r/min shaking culture;
Second group: the bacterial suspension inoculation of 1mL step 1 is contained in the inorganic salt damping fluid (the pH value is 8.75) of 1mg/L 17 beta estradiols to 37 ℃, 200r/min shaking culture to 20mL;
The 3rd group: by the bacterial suspension inoculation of 1mL step 1 to 20mL containing 1mg/L 17 beta estradiols and 0.6mg/L (NH
4)
2CO
3Inorganic salt damping fluid (the pH value is 8.75) in, 37 ℃, 200r/min shaking culture;
The 4th group: by the bacterial suspension inoculation of 1mL step to 20mL containing 1mg/L 17 beta estradiols and 2mg/L (NH
4)
2CO
3Inorganic salt damping fluid (the pH value is 8.75) in, 37 ℃, 200r/min shaking culture;
The 5th group: by the bacterial suspension inoculation of 1mL step to 20mL containing 1mg/L 17 beta estradiols and 10mg/L (NH
4)
2CO
3Inorganic salt damping fluid (the pH value is 8.75) in, 37 ℃, 200r/min shaking culture;
From having inoculated the beginning timing, after 0 hour, 12 hours, 24 hours, 48 hours, 72 hours and 96 hours, sample.
3, with the step 3 of embodiment 2.
In each sample, the concentration of 17 beta estradiols is shown in Fig. 2 (mean values of three re-treatments).In each ammonia nitrogen concentration, subtilis CHS all has good degradation effect to 17 beta estradiols.As (NH
4)
2CO
3Concentration while being 10mg/L, subtilis CHS 17 beta estradiols of degrading are had to promoter action.
One, the preparation of subtilis CHS pulvis
1, subtilis CHS is seeded to and contains 10mg/L (NH
4)
2CO
3LB substratum (the pH value is 8.25) in, making its starting point concentration is 10
4Cfu/mL, 37 ℃, 200r/min shaking culture 12 hours, completing after cultivating the concentration of subtilis CHS in culture system is 10
8Cfu/mL, by the culture system freeze-drying, obtain pulvis, is the product I.
2, subtilis CHS is seeded to and contains 10mg/L (NH
4)
2CO
3LB substratum (the pH value is 8.75) in, making its starting point concentration is 10
4Cfu/mL, 37 ℃, 200r/min shaking culture 24 hours, completing after cultivating the concentration of subtilis CHS in culture system is 10
9Cfu/mL, by the culture system freeze-drying, obtain pulvis, is the product II.
3, subtilis CHS is seeded to and contains 10mg/L NH
4HCO
3LB substratum (the pH value is 8.25) in, making its starting point concentration is 10
4Cfu/mL, 37 ℃, 200r/min shaking culture 12 hours, completing after cultivating the concentration of subtilis CHS in culture system is 10
8Cfu/mL, by the culture system freeze-drying, obtain pulvis, is the product III.
4, subtilis CHS is seeded to and contains 10mg/L NH
4HCO
3LB substratum (the pH value is 8.75) in, making its starting point concentration is 10
4Cfu/mL, 37 ℃, 200r/min shaking culture 24 hours, completing after cultivating the concentration of subtilis CHS in culture system is 10
9Cfu/mL, by the culture system freeze-drying, obtain pulvis, is the product IV.
Two, the Degradation of subtilis CHS pulvis to 17 beta estradiols
1, the product I respectively prepared by step 1, product II, product III and product IV are tested as follows:
Product release prepared by the 1g step 1 to 20mL containing in the inorganic salt damping fluid (the pH value is 8.75) of 1mg/L 17 beta estradiols, 37 ℃, 200r/min shaking culture 96 hours.Then (method is with the step 3) of embodiment 2 to detect 17 beta estradiol concentration in culture system.
Throw in product I shaking culture after 96 hours, in culture system, the concentration of 17 beta estradiols is 0.12mg/L.
Throw in product II shaking culture after 96 hours, in culture system, the concentration of 17 beta estradiols is 0.08mg/L.
Throw in product III shaking culture after 96 hours, in culture system, the concentration of 17 beta estradiols is 0.10mg/L.
Throw in product IV shaking culture after 96 hours, in culture system, the concentration of 17 beta estradiols is 0.07mg/L.
2, the product I respectively prepared by step 1, product II, product III and product IV are tested as follows:
Livestock culture waste water is taken near the sewage draining exit certain pig farm, Tongzhou District, Beijing City, and parameter is as follows: COD mean value is that 7250mg/L, total nitrogen (TN) mean concns are 930mg/L, ammonia nitrogen (NH
3-N) mean concns is that 620mg/L, total phosphorus (TP) mean concns are that 470mg/L, 17 beta estradiol mean concnss are 4230ng/L, and pH mean value is 8.68.COD, NH
3-N, TN, TP adopt respectively method 8000, method 10031, method 10072, the method 10127 of U.S. Hash company and use spectrophotometer (DR5000 HACH) to measure.The measuring method of 17 beta estradiol concentration is with the step 3 of embodiment 2.
Product release prepared by the 1g step 1 is to 20mL livestock culture waste water, 37 ℃, 200r/min shaking culture, after 96 hours, detect 17 beta estradiol concentration in culture system (method is with the step 3) of embodiment 2, respectively with 48 hours after and 96 hours after extract total DNA and carry out PCR-DGGE.
Throw in product I shaking culture after 96 hours, in culture system, the concentration of 17 beta estradiols is 62ng/L.
Throw in product II shaking culture after 96 hours, in culture system, the concentration of 17 beta estradiols is 54ng/L.
Throw in product III shaking culture after 96 hours, in culture system, the concentration of 17 beta estradiols is 58ng/L.
Throw in product IV shaking culture after 96 hours, in culture system, the concentration of 17 beta estradiols is 43ng/L.
The primer pair following (the about 240bp of the target sequence of subtilis CHS) that PCR-DGGE is used:
5’-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGCCTACGGGAGGCAGCAG -3’；
5’-ATTACCGCGGCTGCTGG-3’。
Part PCR-DGGE the results are shown in Figure 3(arrow mark purpose band), wherein swimming lane 1 is the product I, swimming lane 2 is livestock culture waste water, swimming lane 3 is for throwing in product I the livestock culture waste water of shaking culture after 48 hours, swimming lane, 4 for throwing in product I the livestock culture waste water of shaking culture after 96 hours.The PCR-DGGE result of product II, product III and product IV is consistent with Fig. 3.Result shows, throws in product shaking culture after 96 hours, and subtilis CHS still preponderates in active sludge, and can effectively remove 17 beta estradiols, and degradation rate can reach more than 90%.
Claims (10)
1. subtilis (Bacillus subtilis) CHS, its deposit number is CGMCC No.6128.
2. the application of the described subtilis CHS of claim 1 in degraded 17 beta estradiols.
3. application as claimed in claim 2 is characterized in that: when applying described subtilis CHS and degrading 17 beta estradiol, the pH value of reaction system is 6.25-8.75.
4. application as claimed in claim 2 or claim 3, it is characterized in that: when applying described subtilis CHS and degrading 17 beta estradiol, the ammonia nitrogen concentration of reaction system is 0.6-10mg/L.
5. application as claimed in claim 4 is characterized in that: described ammonia nitrogen is by (NH
4)
2CO
3Or NH
4HCO
3Provide.
6. a method for preparing the product of 17 beta estradiols of degrading, comprise the steps: the described subtilis CHS of claim 1 is seeded in the LB substratum that contains ammonia nitrogen, 37 ℃ of shaking culture 12-36 hour, then by the culture system freeze-drying, the product of 17 beta estradiols that obtain degrading.
7. method as claimed in claim 6, it is characterized in that: the described LB substratum that contains ammonia nitrogen is for containing (NH
4)
2CO
3The LB substratum or contain NH
4HCO
3The LB substratum.
8. the product of degraded 17 beta estradiols that the described method of claim 6 or 7 prepares.
9. the application of the described product of claim 8 in degraded 17 beta estradiols.
10. the described subtilis CHS of claim 1 or the described product of claim 9 application in degraded fowl poultry waste water.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104774798A (en) * | 2015-05-04 | 2015-07-15 | 四川农业大学 | Bacillus subtillis capable of effectively degrading diethylstilbestrol |
| CN105779322A (en) * | 2014-12-23 | 2016-07-20 | 北京大北农科技集团股份有限公司 | Bacillus subtilis strain, microecological preparation, and applications thereof |
| CN110129226A (en) * | 2019-05-14 | 2019-08-16 | 东北师范大学 | A kind of lysine bacillus and its acquisition methods and application |
| CN110818206A (en) * | 2019-12-04 | 2020-02-21 | 北京金城泰尔制药有限公司 | Method for treating promestrene synthetic wastewater |
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| CN102443558A (en) * | 2011-12-21 | 2012-05-09 | 江苏江达生态科技有限公司 | Composite heterotrophic nitrifying bacterial agent and application of same in nitrogen removal treatment of waste water containing ammonia and nitrogen |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779322A (en) * | 2014-12-23 | 2016-07-20 | 北京大北农科技集团股份有限公司 | Bacillus subtilis strain, microecological preparation, and applications thereof |
| CN105779322B (en) * | 2014-12-23 | 2021-07-27 | 北京大北农科技集团股份有限公司 | Bacillus subtilis strain, microecological preparation and application |
| CN104774798A (en) * | 2015-05-04 | 2015-07-15 | 四川农业大学 | Bacillus subtillis capable of effectively degrading diethylstilbestrol |
| CN110129226A (en) * | 2019-05-14 | 2019-08-16 | 东北师范大学 | A kind of lysine bacillus and its acquisition methods and application |
| CN110129226B (en) * | 2019-05-14 | 2023-01-24 | 东北师范大学 | Lysine bacillus and obtaining method and application thereof |
| CN110818206A (en) * | 2019-12-04 | 2020-02-21 | 北京金城泰尔制药有限公司 | Method for treating promestrene synthetic wastewater |
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|---|---|
| CN103421701B (en) | 2015-01-14 |
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