CN101575566A - Two-step method for cultivating microalgae by using light conversion film - Google Patents
Two-step method for cultivating microalgae by using light conversion film Download PDFInfo
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Abstract
The invention provides a two-step method for cultivating microalgae by using a light conversion film, comprising the following steps of: step one, carrying out the cultivation by using a first light conversion film so as to quickly accumulate the biomass; and step two, cultivating the microalgae obtained in the first step by using a second light conversion film, thus realizing the conversion of useful products, wherein the first light conversion film converts the light from the light source into the light with the wavelength within 400-700nm; and the second light conversion film converts the light from the light source into the light with the wavelength within 400-500nm.
Description
Technical field
The invention belongs to little algae and cultivate the field.The present invention is the improvement of conventional microalgae culture method, particularly, the invention provides a kind of two-step approach of utilizing light-converting film to cultivate little algae.
Background technology
At present, the main mode of little algae cultivation employing is that open Da Chi cultivates (runway pond) and the closed bioreactor is cultivated.Open Da Chi cultivate be the earliest, the simplest microalgae culture system, little algae that suitable growth conditions is extensive relatively is as the cultivation of several little algaes such as spirulina, chlorella or Dunaliella salina, its simple structure, mainly utilize ambient lighting, temperature condition to cultivate, simple to operate, with low cost.But little algae is cultivated and is subjected to the envrionment conditions restriction effect obvious, and when intensity of illumination was hanged down, micro algae growth speed was slow; And intensity of illumination is too high or ultraviolet radiation will suppress micro algae growth even kill little algae, and simultaneously open Da Chi cultivation microalgae cell density is often very low.
The development of novel bioreactor and exploitation have remedied the deficiency of open Da Chi culture systems, some can be cultivated under the air tight condition that is fit to the relatively harsh little algae of growth conditions requirement, can prevent to pollute, and can be according to the micro algae growth needs, be convenient to adjust conditions such as intensity of illumination and temperature, can improve cultured microalgae density to a certain extent.But the general cost of bioreactor is higher, makes little algae cultivate the corresponding rising of cost.
Carry out the focus that both culturing microalgae is research always, difficult point especially with high-density culture.In order to realize high-density breeding, generally take the heterotrophism mode, heterotrophism need add organic carbon source, and cost is higher.Common autotrophy then cell concn is lower, and biomass can't effectively accumulate, and is difficult to realize large-scale farming.
The unit cell algae owing to be subjected to the restriction of factors such as illumination, nutritional condition, pollution, is difficult to reach highdensity cultivation in the process of propagating artificially, make the both culturing microalgae cost high always, is difficult to realize large-scale industrialized production.Illumination becomes the factor that seriously limits its growth in high-density breeding, most of green algas generally when the cell density absorbancy when 750nm is 1.0 left and right sides, be that 5cm place light has nearly attenuated 90% in the algae liquid degree of depth.Therefore, the utilising efficiency that effectively improves light becomes a great problem.The high-density culture general requirement can accumulate a large amount of biomasss in a short time, but intracellularly in a short time utilizes composition accumulation often less, therefore need carry out two-step approach and cultivate.
In state of the art, so-called two-step approach is cultivated, be optimal growth condition and the nutritional condition that guarantees little algae at the cultivation initial stage, make its growth accumulation biomass fast, and in the micro algae growth process, the accumulation of biomass is often just in time opposite with the accumulation of product, therefore after cultivating finite concentration little algae is inserted in the poor nutrition culture system, little algae accumulates the material that grease or pigment and so on have commercial application value in vivo in a large number in order to survive.
In theory, chlorophyllous absorption spectrum is the blood orange light district of royal purple light and the 600～680nm of 400～480nm, that is to say photosynthesis meaningfully royal purple light and blood orange light.Based on this, existing at present people is applied in light-converting film in little algae cultivation.Usually, photosynthesis curve according to institute's cultured microalgae, select to add the film of suitable light-converting agent, promptly be called light-converting film, light-converting agent mostly is the organic class material that contains πDian Zi, after being stimulated, can produce the transition of electron of energy level, inspire the fluorescence of different wave length, excite the wave band broad, between 400～600nm, and a plurality of obvious excitation peaks are arranged, emmission spectrum is consistent with the chlorophyll absorption spectrum just, convert effective light composition to making, increase the light utilising efficiency to the disadvantageous UV-light of micro algae growth and to the little green glow of little algae photosynthesis influence.
Light-converting film is widely used in agriculture production, can effectively improve the speed of growth and the output of farm crop.But the booth construction that light-converting film is used for both culturing microalgae does not then appear in the newspapers as yet, particularly utilizes light-converting film with the little algae of two-step approach high-density culture and induce the synthetic of useful products such as high added value pigment not appear in the newspapers as yet yet.
Summary of the invention
The invention provides a kind of two-step approach of utilizing light-converting film to cultivate little algae, particularly, described method comprises:
The first step utilizes first light-converting film to cultivate, and biomass is accumulated fast; With
Second step, utilize second light-converting film to cultivate and cultivate the little algae that obtains through the first step, realize the conversion of useful products;
Wherein, described first light-converting film will become the light of wavelength in the 400-700nm scope from the phototransformation of light source; And described second light-converting film will be the light of wavelength in the 400-500nm scope from the phototransformation of light source.
In the method for the invention, described light source comprises natural source and source of artificial light, for example, but is not limited to the sun, fluorescent lamp etc.Described first light-converting film will become to help the photosynthetic light of little algae from the phototransformation of light source, is mainly royal purple light or blood orange light, for example, and royal purple light or wavelength the blood orange light 550-700nm scope in of wavelength in the 400-480nm scope; Preferably, described first light-converting film will be blood orange light from the phototransformation of light source, for example, and the blood orange light of wavelength in the 550-700nm scope.Preferably, described second light-converting film will be blue light from the phototransformation of light source, and for example, the blue light of wavelength in the 400-500nm scope helps pigment accumulation such as carotenoid.
In addition, in the method for the invention, the first step utilizes first light-converting film to cultivate in the process of accumulation biomass, can use the nutritious substratum that helps micro algae growth; Utilize second light-converting film to cultivate in second step and cultivate in the process of the little algae that obtains, can combine, make quick, a large amount of accumulation of useful products with poor nutrition culture system through the first step.
In a preferred embodiment of the invention, used light-converting film is available from Jiangsu Mi Keduo company, and the light-converting film base-material is polyethylene SH150, and the used light-converting agent of first light-converting film and second light-converting film is the rare earth mineral compound, forms general formula and is respectively UV﹠amp; The mass content that GTRI-MS and UVTBI-MS, light-converting agent account for base-material is about 1.0%, and the thickness of film is 0.10-0.16mm.Wherein, UV﹠amp; GTRI-MS type light-converting agent excitation spectrum peak value is at 300nm, and 350nm and 550nm, emmission spectrum peak value be at 650nm, UV-light and green glow can be converted to be easy to the ruddiness that absorbed by little algae; UVTBI-MS type light-converting agent excitation spectrum peak value is at 276nm, and the emmission spectrum peak value is 447nm, can convert UV-light to blue light.Yet it should be appreciated by those skilled in the art that the needs of cultivating according to little algae, can use any suitable light-converting agent and light-converting film, be not limited to above-mentioned example.
In the present invention, described first or second light-converting film can directly overlay the surface of bioreactor, can directly be made into closed photoreactor, also can cover the ceiling of both culturing microalgae sunlight booth, or their arbitrary combination, preferably cover the ceiling of both culturing microalgae sunlight booth.
With reference to figure 1, in one embodiment of the invention, culturing process adopts two-step approach to carry out in being covered with the sunlight booth of different light-converting films, and the first and second used light-converting films have the blood orange light that UV-light is converted to 550-700nm respectively, and the ability of the blue light of 400-500nm.Compare with traditional sunlight booth, first light-converting film is cultivated to have increased in the sunlight booth in one-level and is helped photosynthetic blood orange light, reduce the light restriction, second light-converting film transforms in the sunlight booth at secondary, increased the blue light of being convenient to pigment accumulation, can realize fast and effectively that the later stage product transforms, effectively improve the product accumulation.
Fig. 1 shows a preferred embodiment of the present invention.Particularly, (available from Jiangsu Mi Keduo company, the light-converting film base-material is polyethylene SH150 to sunlight, and it is UV﹠amp that used light-converting agent is formed general formula by first light-converting film; GTRI-MS, the excitation spectrum peak value is at 300nm, 350nm and 550nm, the emmission spectrum peak value is at 650nm, the mass content that light-converting agent accounts for base-material is about 1.0%, the thickness of film is 0.10-0.16mm) enter one-level and cultivate the sunlight booth, in the one-level photoreactor, carry out the accumulation of biomass, because this light-converting film is converted to the required ruddiness of photosynthesis with sunlight, and the penetrativity height of ruddiness, can effectively reduce because cell density increases caused optical attenuation, reduce the inhibition to growing such as UV-light, thus the production that effectively improves biomass.Because a large amount of carbon sources of the quick growth needs of cell, make that the rising of pH value is very fast, at the same time can be with suitable flow velocity bubbling air and CO by a certain percentage
2Mixed gas, concrete ratio change according to the pH value to be regulated, to keep optimum growh pH environment and timely supplementary carbon source.Cultivate an about week, carry out tentatively concentrated, after removing former substratum, algae mud is squeezed into the secondary light reactor, the secondary light reactor places coated with second light-converting film that UV-light can be converted into blue light (available from Jiangsu Mi Keduo company, the light-converting film base-material is polyethylene SH150, it is UVTBI-MS that used light-converting agent is formed general formula, the excitation spectrum peak value is at 276nm, the emmission spectrum peak value is 447nm, the mass content that light-converting agent accounts for base-material is about 1.0%, and the thickness of film is 0.10-0.16mm) secondary transform in the sunlight booth, blue light helps the accumulation of pigment, and in the secondary light reactor zero nutritional medium (not containing any nutritive ingredient), be the environment of coercing of algal grown, can realize that quick product transforms, and then improve the economic worth of little algae.
The two-step approach that the present invention utilizes light-converting film to cultivate little algae is applicable to the cultivation of most of little algae, is specially adapted to the cultivation to the more green alga of light demand, for example, but is not limited to green alga, blue-green algae or variegated algae etc.
In the present invention, the advantage of the inventive method mainly is following aspect:
At first, adopt the light-converting film two-step approach to cultivate little algae, be characterized in carrying out the first step and the cultivation of second step with different light-converting films respectively according to actual needs, can optionally satisfy the demand of little algae different times to different wavelengths of light, accumulation and the product of effectively realizing biomass fast transform, and can not pollute the later stage product, and conversion rate is fast, and do not need other external source means, economically feasible;
Secondly, adopt the light-converting film two-step approach to cultivate little algae, do not need to add the mutagenic compound induced product and transform.Single nutritive deficiency, it is longer to coerce the growth needs time, and be in for a long time in the poor nutrient environment, have a large amount of frond death, and coercing under the environment, the frond growth is unfavorable, is very easily polluted, and add other mutagenic compound and all little algae had certain lethality rate, the part medicine also can to the later stage extract and product purity influential.Adopt light-converting film mutagenesis, can effectively improve the accumulation of conversion rate and product, the transformation period shortens, significantly reduce to cultivate the chance of polluting, and to the later stage product without any influence.
In addition, covering canopy with light-converting film compares with directly making bio-reactor and paste on the bio-reactor surface with light-converting film, cost is lower, be convenient to carry out large scale culturing, reduce and clean, improve work-ing life, and light-converting film can effectively be regulated and control the temperature of sunlight booth its damage that causes, can play good heat insulation effect in the winter time, reduce cost.Importantly be, because most of little algaes are cultivated with to transform desired growth conditions often different, therefore carry out in the sunlight booth of two different light-converting films of covering, realize its corresponding top condition respectively, contradiction not is convenient to form large-scale continuous production mutually.
It should be appreciated by those skilled in the art, cultivate in little algae process utilizing two-step approach of the present invention, cover sunlight booth ceiling and cover the surface of bioreactor and can arbitrary combination use with light-converting film with the closed photoreactor that light-converting film is directly made with light-converting film, for example, the first step is cultivated can carry out the biomass accumulative culture in the sunlight booth that covers the top with first light-converting film, second step cultivated in the closed photoreactor of can be in the bioreactor that pastes second light-converting film or being made by second light-converting film and cultivates, the second wherein used light-converting film can have the specific commentaries on classics luminous energy of specific product power, to realize the quick of different products, the a large amount of conversion.
Description of drawings
From the detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 shows the synoptic diagram of a preferred embodiment of the present invention;
Fig. 2 demonstration is being used light-converting film and is not being used chlorella growth graphic representation under the light-converting film condition according to embodiment one.
Fig. 3 demonstration is being used light-converting film and is not being used new green alga growth curve chart under the light-converting film condition according to embodiment two.
Embodiment
Come further to illustrate the present invention by the following examples.But should be appreciated that described embodiment is illustrational purpose, and be not intended to limit the scope of the invention and spirit.
Embodiment one: the light-converting film two-step approach is cultivated chlorella
With chlorella (Chlorella, a kind of green alga) is object, and employing covers the photoreactor outer wall with first and second light-converting films and carries out the two-step approach cultivation.
(available from Jiangsu Mi Keduo company, the light-converting film base-material is polyethylene SH150, and it is UV﹠amp that light-converting agent is formed general formula with first light-converting film; GTRI-MS, the excitation spectrum peak value is at 300nm, 350nm and 550nm, the emmission spectrum peak value is at 650nm, and the mass content that light-converting agent accounts for base-material is about 1.0%, and the thickness of film is 0.10-0.16mm) be covered in 10L bubbling style reactor outer wall, simultaneously do contrast with the identical reactor of no light-converting film, adopt improvement SM substratum (medium component and content thereof see the following form 1-6), with equal densities inoculation logarithmic phase algae kind (change the training back formed in 5-7 days), initial OD
750Be 0.102.Under natural lighting, cultivate this green alga, intensity of illumination by artificial adjustment at 10000-20000lux.The control room temperature is about 25 ℃.Pass through aeration head bubbling air and CO from reactor bottom
2Mixed gas, gas-liquid volume ratio are 1: 16.Culture cycle is 10 days.Fig. 2 shows that chlorella using light-converting film and do not using growth curve under the light-converting film condition, and the result shows that biomass has improved about 25% than control group biomass under this algae light-converting film condition.
To remove former substratum after centrifugal the concentrating of algae liquid of cultivating after 10 days, the tap water that adds no substratum, sunlight mainly can be converted into second light-converting film of blue light (available from Jiangsu Mi Keduo company, the light-converting film base-material is polyethylene SH150, it is UVTBI-MS that used light-converting agent is formed general formula, the excitation spectrum peak value is at 276nm, the emmission spectrum peak value is 447nm, the mass content that light-converting agent accounts for base-material is about 1.0%, the thickness of film is 0.10-0.16mm) cover the surface of another same reactor, do contrast with no light-converting film.The result shows that under the light-converting film condition, the comparison of the content of total carotinoid is according to having improved 1.5%.
The composition and the content thereof of table 1. improvement SM substratum
| | Composition | Amount | |
| 1 |
|
500mL/ |
|
| 2 |
|
500mL/L |
Annotate: underscore is represented to be mixed with mother liquor in advance, and the preparation of described mother liquor sees table 2-3.
The composition and the content thereof of table 2.Spir solution 1
| # | Composition | Amount | |
| 1 | NaHCO 3 | 6.805g/500mL dH 2O | 162mM |
| 2 | Na 2CO 3 | 2.00g/500mL dH 2O | 48mM |
| 3 | K 2HPO 4 | 0.5g/500mL dH 2O | 2.9mM |
The composition and the content thereof of table 3.Spir solution 2
| # | Composition | Amount | |
| 1 | NaNO 3 | 2.5g/500mL | 36.2mM |
| 2 | K 2SO 4 | 1g/500mL | 5.73 |
| 3 | NaCl | 1g/500mL | 17.1 |
| 4 | MgSO 4·7H 2O | 0.2g/500mL | 0.81 |
| 5 | CaCl 2·2H 2O | 0.04g/500mL | 0.36 |
| 6 | The P-IV metallic solution | 6mL/0.5 |
|
| 7 | Chu micronutrient solution | 1mL/0.5 |
|
| 8 | Vitamins B 12 | 1mL/0.5L |
Annotate: underscore is represented to be mixed with mother liquor in advance, and the preparation of described mother liquor sees table 4-6.
The composition and the content thereof of table 4.P-IV metallic solution
| # | Composition | Amount | |
|
| 1 | Na 2EDTA·2H 2O | 0.75g/ | 2mM | |
| 2 | FeCl 3·6H 2O | 0.097g/L | 0.36 |
|
| 3 | MnCl 2·4H 2O | 0.041g/L | 0.21 |
|
| 4 | ZnCl 2 | 0.005g/L | 0.037 |
|
| 5 | CoCl 2·6H 2O | 0.002g/L | 0.0084 |
|
| 6 | Na 2MoO 4·2H 2O | 0.004g/L | 0.017mM |
The composition and the content thereof of table 5.Chu micronutrient solution
| # | Composition | Amount | |
| 1 | CuSO 4·5H 2O | 0.02g/L | 0.78 |
| 2 | ZnSO 4·7H 2O | 0.044g/L | 0.15 |
| 3 | CoCl 2·6H 2O | 0.02g/L | 0.084 |
| 4 | MnCl 2·4H 2O | 0.012g/L | 0.064 |
| 5 | Na 2MoO 4·2H 2O | 0.012g/L | 0.052μM |
| 6 | H 3BO 3 | 0.62g/ | 10μM | |
| 7 | Na 2EDTA·2H 2O | 0.05g/L | 0.13μM |
Table 6. vitamins B
12The composition of liquid storage and content thereof
| | Composition | Amount | |
| 1 | HEPES pH of buffer 7.8 | 2.4g/200mL dH 2O | |
| 2 | Vitamins B 12(cyanocobalamin，Sigma V2876) | 0.027g/200mL dH 2O |
Annotate: it should be appreciated by those skilled in the art that except as otherwise noted, the used reagent of the present invention all can be bought from common biological reagent company or chemical reagents corporation, its compound method also is that those skilled in the art is known.
Embodiment two: the light-converting film two-step approach is cultivated new green alga
With embodiment one, cultivating the algae kind is new green alga (Neochloris oleoabundans, available from UTEX), adopt BBM substratum (reference: Stein, the 448th page of J. (ED.) Handbook ofPhycological methods.Culture methods and growth measurements.CambridgeUniversity Press.).The first used light-converting film is a ruddiness with the UV-light phototransformation mainly, and it is available from Jiangsu Mi Keduo company, and the light-converting film base-material is polyethylene SH150, and it is UV﹠amp that light-converting agent is formed general formula; GTRI-MS, excitation spectrum peak value be at 300nm, and 350nm and 550nm, emmission spectrum peak value be at 650nm, and the mass content that light-converting agent accounts for base-material is about 1.0%, and the thickness of film is 0.10-0.16mm; Second light-converting film mainly is converted into blue light with UV-light, producer is the same, the light-converting film base-material also is polyethylene SH150, it is UVTBI-MS that used light-converting agent is formed general formula, the excitation spectrum peak value is at 276nm, the emmission spectrum peak value is 447nm, and the mass content that light-converting agent accounts for base-material is about 1.0%, and the thickness of film is 0.10-0.16mm.Two-step approach is cultivated new green alga under the use light-converting film condition, and culture cycle is a week, and the cellular biomass comparison is according to improving 20%, and the total lipid content comparison is shone and improved 18% (Fig. 3).
Embodiment three: use is carried out two-step approach cultivation chlorella with the two-stage sunlight booth that first and second light-converting films cover the top
Object of Development is identical with the Object of Development of embodiment one, is chlorella.The one-level booth is UV﹠amp with the composition general formula of light-converting agent; GTRI-MS, the mass content that light-converting agent accounts for base-material is about 1.0%, thickness is that (the light-converting film base-material is polyethylene SH150 to the light-converting film produced of the Mi Keduo company of 0.10-0.16mm, light-converting agent excitation spectrum peak value is at 300nm, 350nm and 550nm, the emmission spectrum peak value is at 650nm) cover the top, there is the independently abat vent of folding outside, awning film top, inside have can free folding reflective curtain, to regulate strong excessively illumination at noon.There is the cascade cooling system booth inside, and booth is inner reactor used to be glass tubular type bubbling reactor, and culture system is 10m
3With SM culture medium culturing chlorella, inoculation initial OD 750 is 0.40, with abat vent and reflective curtain control light intensity, from initial incubation light intensity 8000lux, to the light intensity 30000lux in later stage, along with the increase of cultivating concentration, control between the light time and shorten gradually noon, cultivates after 10 days, and changing secondary over to after centrifugal the concentrating changes wide canopy.It is UVTBI-MS with light-converting agent composition general formula that secondary changes wide canopy, the mass content that light-converting agent accounts for base-material is about 1.0%, thickness is that (the light-converting film base-material is polyethylene SH150 to the light-converting film produced of the Mi Keduo company of 0.10-0.16mm, light-converting agent excitation spectrum peak value is at 276nm, and the emmission spectrum peak value is 447nm) cover the top, will add tap water in the algae liquid after concentrating, make that OD750 is about 1.00, no longer control light intensity, cultivate an about week, gained result and embodiment one basically identical.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and describe, but will be understood by those skilled in the art that, under the condition that does not deviate from by the defined the spirit and scope of the present invention of accompanying Claim, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
Claims (10)
1. utilize light-converting film to cultivate the two-step approach of little algae, described method comprises:
The first step utilizes first light-converting film to cultivate, and biomass is accumulated fast; With
Second step, utilize second light-converting film to cultivate and cultivate the little algae that obtains through the first step, realize the conversion of useful products;
Wherein said first light-converting film will become the light of wavelength in the 400-700nm scope from the phototransformation of light source; And described second light-converting film will be the light of wavelength in the 400-500nm scope from the phototransformation of light source.
2. method according to claim 1, it is characterized in that described first or second light-converting film directly overlays the surface of bioreactor, or directly is made into closed photoreactor, or cover the ceiling of both culturing microalgae sunlight booth or their arbitrary combination.
3. method according to claim 1 is characterized in that, the light-converting agent that described first light-converting film and second light-converting film use is the rare earth mineral compound.
4. according to each described method among the claim 1-3, it is characterized in that described first light-converting film will become royal purple light or wavelength the blood orange light 550-700nm scope in of wavelength in the 400-480nm scope from the phototransformation of light source.
5. method according to claim 4 is characterized in that, it is UV﹠amp that the used light-converting agent of described first light-converting film is formed general formula; The mass content that GTRI-MS, light-converting agent account for base-material is about 1.0%, and thickness is 0.10-0.16mm.
6. method according to claim 5 is characterized in that, the excitation spectrum peak value of the used light-converting agent of described first light-converting film is 300nm, and 350nm and 550nm, emmission spectrum peak value are 650nm.
7. according to each described method among the claim 1-3, it is characterized in that it is UVTBI-MS that the used light-converting agent of described second light-converting film is formed general formula, the mass content that light-converting agent accounts for base-material is about 1.0%, and thickness is 0.10-0.16mm.
8. method according to claim 7 is characterized in that, the excitation spectrum peak value of the used light-converting agent of described second light-converting film is 276nm, and the emmission spectrum peak value is 447nm.
9. method according to claim 1, wherein in described the first step culturing process, bubbling air and CO in micro algae culturing liquid
2Mixed gas.
10. method according to claim 1 is characterized in that, described little algae is green alga, blue-green algae or variegated algae etc.
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| CN104944769A (en) * | 2015-05-25 | 2015-09-30 | 长春理工大学 | Rare earth-doped light conversion glass for building agricultural greenhouse |
| CN106520852A (en) * | 2017-01-20 | 2017-03-22 | 中国科学院青岛生物能源与过程研究所 | Method for producing nervonic acid-containing microalgae oil by using freshwater microalgae |
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| CN104762212A (en) * | 2015-04-16 | 2015-07-08 | 青岛华盛绿能农业科技有限公司 | Method for culturing haematococcus pluvialis by photovoltaic greenhouse |
| CN104944769A (en) * | 2015-05-25 | 2015-09-30 | 长春理工大学 | Rare earth-doped light conversion glass for building agricultural greenhouse |
| CN106520852A (en) * | 2017-01-20 | 2017-03-22 | 中国科学院青岛生物能源与过程研究所 | Method for producing nervonic acid-containing microalgae oil by using freshwater microalgae |
| CN111172096A (en) * | 2020-04-11 | 2020-05-19 | 杭州富阳君晓农业科技有限公司 | Production process for high-density culture of heteroglena |
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| CN101575566B (en) | 2011-07-06 |
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