WO2010095975A2 - Method for regulation of ratio of organic solvents during biosynthesis - Google Patents
Method for regulation of ratio of organic solvents during biosynthesis Download PDFInfo
- Publication number
- WO2010095975A2 WO2010095975A2 PCT/RU2009/000640 RU2009000640W WO2010095975A2 WO 2010095975 A2 WO2010095975 A2 WO 2010095975A2 RU 2009000640 W RU2009000640 W RU 2009000640W WO 2010095975 A2 WO2010095975 A2 WO 2010095975A2
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- WO
- WIPO (PCT)
- Prior art keywords
- fermentation
- ratio
- pressure reduction
- butanol
- culture
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
- C12P7/28—Acetone-containing products
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to a process for production of organic solvents, particularly acetone, butanol, ethanol using the anaerobic fermentation.
- Clostridium acetobutylicum is usually used in the production of the solvents.
- Clostridium acetobutylicum is a particularly interesting culture since it possesses a rather complicated and branched sugar metabolising pathway leading to the production of the solvents butanol, acetone and ethanol as its end products.
- the typical ratio of acetone to butanol to ethanol in the final product is usually 30:60:10 when using traditional strains and traditional batch fermentation processes. The classical ratio can be changed slightly to increase the yield of one or another solvent.
- substrate for fermentation has the significant effect on the solvent ratio.
- butanol/acetone ratios obtained after fermentation of whey by Clostridium acetobutylicum have been shown to be different from those obtained from glucose.
- CN 101250496 discloses the strain Clostridium acetobutylicum SB-I CGMCC No. 2287. The strain is used to produce butanol through fermentation, and the ratio of butanol in the final product reaches 68-75%.
- CN 101250496 discloses the strain Clostridium acetobutylicum SB-I CGMCC No. 2287. The strain is used to produce butanol through fermentation, and the ratio of butanol in the final product reaches 68-75%.
- some workers have believed that
- Clostridium are not ideal because of the relative lack of genetic tools to manipulate their metabolism, their slow growth.
- a method for regulation of ratio of organic solvents produced by a culture of Clostridium acetobutylicum during the fermentation on carbohydrate contained raw material by subjecting the culture to pressure reduction.
- all culture is subjected to pressure reduction during the fermentation.
- a portion of said culture is subjected to pressure reduction out of the fermentation with return of the subjected culture into the fermentation.
- the pressure reduction is carried out periodically.
- solvent removal is carried out during the pressure reduction.
- the pressure reduction is set up when butanol concentration in the fermentor is approaching to toxic for producing culture, it means when butanol concentration in the fermentor is 8-9 g/1, the pressure reduction is kept on until butanol concentration in the fermentor is 5-2 g/1.
- all culture of Clostridium acetobutylicum is subjected to pressure reduction during the fermentation, but it is possible that a portion of said fermentation culture is subjected to pressure reduction out of the fermentation in a separate tank and then the said culture returns into the fermentation.
- the pressure reduction is performed periodically because the ratio fixed for the pressure reduction retains for 20 hours.
- the solvents produced in the fermentation are removed from the fermentor.
- the pressure reduction is set up when butanol concentration in the fermentor is approaching to toxic for producing bacterium, it means when butanol concentration in the fermentor is 8-9 g/1.
- the pressure reduction is kept on until butanol concentration in the fermentor is 5-2 g/1.
- the present invention is carried out as follows:
- the standard mixture of carbohydrate and minerals (mineral salts, vitamin and one of the following carbon sources: glucose, mannose, xylose, galactose, fermentolysate of non-food plant polysaccharides, containing glucose, mannose, xylose, arabinose separated from the lignin residues in concentration of 2-4%) was added into a fermentor. Then the medium was inoculated with cells of Clostridium acetobutylicum VKM B-2531D with density of 1-2 milliard/ml. 30 min after inoculation the intensive fermentation gas formation began, in 5-6 hours the organic acids production took place, in 10-12 hours the intensive production of organic solvents, the rate of solvent production was maximal to 28-36 hours.
- a four-liter fermentor containing 2.5 1 of 4% glucose solution, 120 g of flour was inoculated with 300 ml of inoculum of Clostridium acetobutylicum VKPM B-4786 with density of 1-2 milliard/ml, water and flour (40 g/1).
- the fermentation was allowed to proceed in the batch mode for 72 h.
- the temperature was maintained at 37 0 C.
- the pressure in the fermentor was reduced to - 0.94 kg/cm 2 for 3 hours and the vapors were taken off.
- the ABE vapors were cooled in a condenser and in result 250 ml of solution, containing 5% butanol, 1.5% acetone and 0.5% ethanol was obtained with ratio 60:30:10.
- a four-liter fermentor containing 2.5 1 of 4% glucose solution, 120 g of flour was inoculated with 300 ml of inoculum of Clostridium acetobutylicum VKM B-2531D with density of 1-2 milliard/ml, water and flour (40 g/1).
- the fermentation was allowed to proceed at 37 0 C.
- butanol concentration approached 9 g/1
- the pressure in the fermentor was reduced to - 0.92 kg/cm 2 for 0,5 hour, it results in changing of ABE ratio to 15:80:5.
- the procedure was repeated in 8 hours and ABE ratio was 9:90:1.
- the ABE vapors were cooled in a condenser and in result 250 ml of solution (organic solvents 19.9 ml), containing 6.5% butanol, 1.2% acetone and 0.25% ethanol was obtained, ABE ratio 16:81:3.
- solution organic solvents 19.9 ml
- acetone 1.2% acetone
- 0.25% ethanol was obtained, ABE ratio 16:81:3.
- the ABE vapors were cooled in a condenser and in result 50 ml of solution (organic solvents 4,2 ml), containing 3.75 ml of butanol, 0.35 ml of acetone and 0.125 ml of ethanol was obtained, ABE ratio 8:89:3.
- the fermentation broth in the tank containing reduced butanol content (2 g/1) was recycled to the fermentor.
- the nutrient medium containing 50 g/1 of glucose was added into the fermentor and the fermentation was allowed to proceed. When butanol concentration approached 9 g/1, the procedure was repeated.
- the ABE vapors were cooled in a condenser and in result of the fermentation 250 ml of solution (organic solvents 19.9 ml), containing 6.5% butanol, 1.2% acetone and 0.25% ethanol was obtained, ABE ratio 16:81:3. Productivity was 10 g/l/day.
- the advantage of the present invention is the focused change of ratio of organic solvents produced by the fermentation of carbohydrate contained raw material during the process of fermentation in situ.
- the method allows increasing the ratio of the solvent depending on the current needs at the moment.
Abstract
The present invention relates to a process for production of organic solvents, particularly acetone, butanol, ethanol using the anaerobic fermentation by butanol, acetone, and ethanol- producing bacteria. A method for regulation of ratio of organic solvents produced by Clostridium acetobutylicum for the fermentation on carbohydrate contained raw material is provided.
Description
Method for regulation of ratio of organic solvents during biosynthesis
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a process for production of organic solvents, particularly acetone, butanol, ethanol using the anaerobic fermentation.
BACKGROUND OF THE INVENTION
Production of butanol and acetone by fermentation processes is one of the oldest industrial fermentations. A strain of the anaerobic bacterium Clostridium acetobutylicum is usually used in the production of the solvents. Clostridium acetobutylicum is a particularly interesting culture since it possesses a rather complicated and branched sugar metabolising pathway leading to the production of the solvents butanol, acetone and ethanol as its end products. The typical ratio of acetone to butanol to ethanol in the final product is usually 30:60:10 when using traditional strains and traditional batch fermentation processes. The classical ratio can be changed slightly to increase the yield of one or another solvent.
It is attractive to have the possibility to affect the solvent ratio for preferred synthesis of butanol or acetone. Ethanol ratio in the process is quite low moreover there are other processes of ethanol production.
The different factors can affect the solvent ratio. For example, substrate for fermentation has the significant effect on the solvent ratio. For example, the butanol/acetone ratios obtained after fermentation of whey by Clostridium acetobutylicum have been shown to be different from those obtained from glucose. Butanol/acetone ratios as high as 12:1 to 20:1 from whey as compared with 3:1 from glucose, but a whey permeate has proved to be a relatively poor substrate when overall reactor productivities are considered (Jones D.T. et al., Acetone-Butanol Fermentation Revisited, MICROBIOLOGICAL REVIEWS, Dec. 1986, Vol. 50, No. 4, p. 484- 524).
Further it is known about the effect of temperature on the solvent ratio. For example, Carnarius (U.S. Patent No. 2,198,104) reports that an increase in the butanol ratio (it was 77.6%) is obtained in the acetone, butanol, ethanol (ABE) process by decreasing the temperature of the fermentation from 30 0C to 24 0C after 16 hours. However it should to be noted that temperature decreasing retards substantially the fermentation, results in by-products formation and decreases the total yield of solvents.
There have been efforts to increase the butanol ratio through metabolic engineering of Clostridium. For example, CN 101250496 discloses the strain Clostridium acetobutylicum SB-I CGMCC No. 2287. The strain is used to produce butanol through fermentation, and the ratio of
butanol in the final product reaches 68-75%. However some workers have believed that
(Eric J. Steen et al., Metabolic engineering of Saccharomyces cerevisiae for the production of n- butanol, Microbial Cell Factories 2008, 7:36) Clostridium are not ideal because of the relative lack of genetic tools to manipulate their metabolism, their slow growth.
Therefore inventors could not find out the technical decisions which give the possibility to regulate significantly the ratio of organic solvents in the fermentation of carbohydrate contained raw materials moreover to change the ratio during the fermentation.
Summarizing the aforesaid, a need exists for an improved process to change the ratio of organic solvents during the fermentation of carbohydrate contained raw materials for preferred production of one or another organic solvent.
BRIEF SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a method for regulation of ratio of organic solvents produced by a culture of Clostridium acetobutylicum during the fermentation on carbohydrate contained raw material, by subjecting the culture to pressure reduction. In one preferred embodiment all culture is subjected to pressure reduction during the fermentation. In another preferred embodiment a portion of said culture is subjected to pressure reduction out of the fermentation with return of the subjected culture into the fermentation. In another preferred embodiment the pressure reduction is carried out periodically. In another preferred embodiment solvent removal is carried out during the pressure reduction. In one preferred embodiment the pressure reduction is set up when butanol concentration in the fermentor is approaching to toxic for producing culture, it means when butanol concentration in the fermentor is 8-9 g/1, the pressure reduction is kept on until butanol concentration in the fermentor is 5-2 g/1.
DETAILED DESCRIPTION OF THE INVENTION
We have discovered surprisingly that when the overhead pressure in fermenter for ABE fermentation is negative (subatmospheric), the classical ABE ratio 30:60:10 has changed. Without wishing to be bound by theory of operation, the reason of this effect is the action of periodic pressure into biological systems, probably it causes the irregularity (disturbance) of mass transfer processes through membranes, change in fermentative processes in cell, initiation and development of repair reactions accompanied by novel synthesis (Akopyan V.B., Korzhevenko G.N., Shangin-Berezovskiy G.N., The buried reserve of growth and development of live system, Vestnik selskohozyastvennoy nauki, 1988, No 4 (380), p 69-105).
In one preferred embodiment all culture of Clostridium acetobutylicum is subjected to pressure reduction during the fermentation, but it is possible that a portion of said fermentation
culture is subjected to pressure reduction out of the fermentation in a separate tank and then the said culture returns into the fermentation.
Preferably the pressure reduction is performed periodically because the ratio fixed for the pressure reduction retains for 20 hours.
In one preferred embodiment during the period of pressure reduction the solvents produced in the fermentation are removed from the fermentor. The pressure reduction is set up when butanol concentration in the fermentor is approaching to toxic for producing bacterium, it means when butanol concentration in the fermentor is 8-9 g/1. The pressure reduction is kept on until butanol concentration in the fermentor is 5-2 g/1.
On practical grounds this technique gives option to carry out the process providing the preferred production of butanol, a high-price solvent and chemical feedstock. The fact of changing solvent ratio during the fermentation is very important.
The present invention is carried out as follows:
The standard mixture of carbohydrate and minerals (mineral salts, vitamin and one of the following carbon sources: glucose, mannose, xylose, galactose, fermentolysate of non-food plant polysaccharides, containing glucose, mannose, xylose, arabinose separated from the lignin residues in concentration of 2-4%) was added into a fermentor. Then the medium was inoculated with cells of Clostridium acetobutylicum VKM B-2531D with density of 1-2 milliard/ml. 30 min after inoculation the intensive fermentation gas formation began, in 5-6 hours the organic acids production took place, in 10-12 hours the intensive production of organic solvents, the rate of solvent production was maximal to 28-36 hours. At this moment the cell concentration was also peak - 3 109 cells/ml of suspension. When butanol concentration in the fermentor was 8-9 g/1, the pressure in the fermentor was reduced for 0,3-1 hour to -0,80 - 0,95 kg/cm2, the ABE ratio 30:60:10 has changed to 21:70:9. The procedure was repeated in 6-8 hours and ABE ratio was 9:90:1. After the fermentation gas removal feeding of the carbohydrate and mineral solution was carried out.
The invention may be further understood by the following non-limiting examples.
Example 1
A four-liter fermentor containing 2.5 1 of 4% glucose solution, 120 g of flour was inoculated with 300 ml of inoculum of Clostridium acetobutylicum VKPM B-4786 with density of 1-2 milliard/ml, water and flour (40 g/1). The fermentation was allowed to proceed in the batch mode for 72 h. The temperature was maintained at 37 0C. In 72 hours the pressure in the fermentor was reduced to - 0.94 kg/cm2 for 3 hours and the vapors were taken off. The ABE
vapors were cooled in a condenser and in result 250 ml of solution, containing 5% butanol, 1.5% acetone and 0.5% ethanol was obtained with ratio 60:30:10.
Example 2
A four-liter fermentor containing 2.5 1 of 4% glucose solution, 120 g of flour was inoculated with 300 ml of inoculum of Clostridium acetobutylicum VKM B-2531D with density of 1-2 milliard/ml, water and flour (40 g/1). The fermentation was allowed to proceed at 37 0C. When butanol concentration approached 9 g/1, the pressure in the fermentor was reduced to - 0.92 kg/cm2 for 0,5 hour, it results in changing of ABE ratio to 15:80:5. The procedure was repeated in 8 hours and ABE ratio was 9:90:1.
The ABE vapors were cooled in a condenser and in result 250 ml of solution (organic solvents 19.9 ml), containing 6.5% butanol, 1.2% acetone and 0.25% ethanol was obtained, ABE ratio 16:81:3. After the ABE vapors and fermentation gases removal the nutrient medium containing 50 g/1 of glucose was added into the fermentor. One time in three days 28 g of yeast autolyzate was added into the fermentor.
Example 3
A four-liter fermentor containing 2.5 1 of 4% fermentolyzate of non-food plant polysaccharides, 120 g of flour was inoculated with 300 ml of inoculum of Clostridium acetobutylicum VKM B-2531D with density of 1-2 milliard/ml, water and flour (40 g/1). The fermentation was allowed to proceed at 37 0C. Total time of process is one week. Productivity was 4 g/l/day. When butanol concentration approached 8 g/1, 0.5 1 of the fermentation broth was charged to the separate tank operated at a reduced pressure of about -0,94 KΓ/CM2. The ABE vapors were cooled in a condenser and in result 50 ml of solution (organic solvents 4,2 ml), containing 3.75 ml of butanol, 0.35 ml of acetone and 0.125 ml of ethanol was obtained, ABE ratio 8:89:3. The fermentation broth in the tank containing reduced butanol content (2 g/1) was recycled to the fermentor. The nutrient medium containing 50 g/1 of glucose was added into the fermentor and the fermentation was allowed to proceed. When butanol concentration approached 9 g/1, the procedure was repeated. The ABE vapors were cooled in a condenser and in result of the fermentation 250 ml of solution (organic solvents 19.9 ml), containing 6.5% butanol, 1.2% acetone and 0.25% ethanol was obtained, ABE ratio 16:81:3. Productivity was 10 g/l/day.
The advantage of the present invention is the focused change of ratio of organic solvents produced by the fermentation of carbohydrate contained raw material during the process of fermentation in situ. The method allows increasing the ratio of the solvent depending on the current needs at the moment.
Claims
1. A method for regulation of ratio of organic solvents produced by a culture Clostridium acetobutylicum during fermentation on carbohydrate contained raw material, by subjecting the culture to pressure reduction.
2. The method of claim 1 wherein all culture is subjected to pressure reduction during the fermentation.
3. The method of claim 1 wherein a portion of said culture is subjected to pressure reduction out of the fermentation with return of the subjected culture into the fermentation.
4. The method of claim 1 wherein the pressure reduction is carried out periodically.
5. The method of claim 1 wherein the solvent removal is carried out during the pressure reduction.
6. The process of claim 1 wherein the pressure reduction is set up when butanol concentration in the fermentor is approaching to toxic for producing culture.
7. The process of claim 1 wherein the pressure reduction is set up when butanol concentration in the fermentation broth is 8-9 g/1.
8. The process of claim 1 wherein the pressure reduction is kept on until butanol concentration in the fermentation broth is 5-2 g/1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2009105442 | 2009-02-18 | ||
| RU2009105442/10A RU2405829C2 (en) | 2009-02-18 | 2009-02-18 | Method of preparing organic solvents |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2010095975A2 true WO2010095975A2 (en) | 2010-08-26 |
| WO2010095975A3 WO2010095975A3 (en) | 2011-01-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/RU2009/000640 WO2010095975A2 (en) | 2009-02-18 | 2009-11-24 | Method for regulation of ratio of organic solvents during biosynthesis |
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| RU (1) | RU2405829C2 (en) |
| WO (1) | WO2010095975A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010095976A3 (en) * | 2009-02-18 | 2011-03-03 | Limited Liability Company "Prof Business" | Abe fermentation, wherein the fermentation broth is subjected to pressure reduction in a seperate vessel |
| CN102653777A (en) * | 2012-05-08 | 2012-09-05 | 上海中科高等研究院 | Method for producing acetone-butanol-ethanol (ABE) by virtue of fermentation of wheat bran |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2198104A (en) | 1938-12-15 | 1940-04-23 | Commercial Solvents Corp | Butyl alcohol fermentation process |
| CN101250496A (en) | 2008-03-25 | 2008-08-27 | 中国科学院微生物研究所 | Acetone-butanol clostridium strain and uses thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2375454C1 (en) * | 2008-08-28 | 2009-12-10 | Открытое акционерное общество "Корпорация Биотехнологии" | Method of producing organic solvents, mainly butanol |
| RU2405826C2 (en) * | 2008-12-29 | 2010-12-10 | Дэвон Инвестмент Лимитед | Method of preparing organic solvents, apparatus for realising said method, product obtained using said method |
| RU2405830C2 (en) * | 2009-02-18 | 2010-12-10 | Дэвон Инвестмент Лимитед | Method of preparing organic solvents |
-
2009
- 2009-02-18 RU RU2009105442/10A patent/RU2405829C2/en not_active IP Right Cessation
- 2009-11-24 WO PCT/RU2009/000640 patent/WO2010095975A2/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2198104A (en) | 1938-12-15 | 1940-04-23 | Commercial Solvents Corp | Butyl alcohol fermentation process |
| CN101250496A (en) | 2008-03-25 | 2008-08-27 | 中国科学院微生物研究所 | Acetone-butanol clostridium strain and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| AKOPYAN V.B.; KORZHEVENKO G.N.; SHANGIN-BEREZOVSKIY G.N.: "The buried reserve of growth and development of live system", VESTNIK SELSKOHOZYASTVENNOY NAUKI, 1988, pages 69 - 105 |
| ERIC J. STEEN ET AL.: "Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol", MICROBIAL CELL FACTORIES, vol. 7, 2008, pages 36 |
| JONES D.T. ET AL.: "Acetone-Butanol Fermentation Revisited", MICROBIOLOGICAL REVIEWS, vol. 50, no. 4, December 1986 (1986-12-01), pages 484 - 524 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010095976A3 (en) * | 2009-02-18 | 2011-03-03 | Limited Liability Company "Prof Business" | Abe fermentation, wherein the fermentation broth is subjected to pressure reduction in a seperate vessel |
| CN102653777A (en) * | 2012-05-08 | 2012-09-05 | 上海中科高等研究院 | Method for producing acetone-butanol-ethanol (ABE) by virtue of fermentation of wheat bran |
Also Published As
| Publication number | Publication date |
|---|---|
| RU2405829C2 (en) | 2010-12-10 |
| WO2010095975A3 (en) | 2011-01-13 |
| RU2009105442A (en) | 2010-08-27 |
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