WO2006103531A1 - A process for production of succinic acid from glucose - Google Patents
A process for production of succinic acid from glucose Download PDFInfo
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- WO2006103531A1 WO2006103531A1 PCT/IB2006/000716 IB2006000716W WO2006103531A1 WO 2006103531 A1 WO2006103531 A1 WO 2006103531A1 IB 2006000716 W IB2006000716 W IB 2006000716W WO 2006103531 A1 WO2006103531 A1 WO 2006103531A1
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- glucose
- succinic acid
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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
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
Definitions
- the present invention relates a new process for production of succinic acid from glucose.
- Succinic acid is a dicarboxylic acid produced as an intermediate of tricarboxylic acid cycle and also as one of the fermentation products of anaerobic metabolism [Gottschalk 1986; Zeikus 1980]. It has been considered as an important chemical because it can be used as the precursor of 1,4- butanediol, tetrahydrofuran and gamma- butyrolactone and in polymers, foods, pharmaceuticals and cosmetics.
- succinic acid is produced commercially through chemical synthesis.
- the fermentative production of succinic acid can be regarded as a "Green Technology" because renewable substrates are used for its production [Biotechnol.Bioprocess Eng. 2000, 5:379-381].
- Succinic acid is produced from glucose by various fermentative methods. Escherichia coli strain NZNl I l gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AF Pi l l, ferment glucose more slowly than did its wild type ancestor, strain W 1485, and generated a very different spectrum of products. AFP 111 produced succinic acid, acetic acid, and ethanol in proportions of approx. 2:1:1. 1 mole of glucose was converted to 1 mole of succinic acid and 0.5 mol each of acetic acid and ethanol [Appl. Biochem. Biotechnol. 1998 Spring, 70-72: 187-98]
- Anaerobic bacterium Anaerobiospirillum succiniciproducens has most often been employed because it can produce a significant amount of succinic acid from glucose [P.C.Lee et al 1999].
- the main objective of the present invention is to provide one step transformation of glucose to succinic acid.
- Yet another object is to provide a new process for production of succinic acid in which yeast cells require less restricted conditions and cheaper carbon sources.
- the present invention provides a new process for production of succinic acid from glucose, fructose or any other hexoses using Candida bombicola which comprises growing Candida bombicola in a conventional growth medium consisting of a carbon, nitrogen sources and carbohydrate source and other micro-ingredients for a period of 24 to 96 hrs. at a temperature ranging between 26 to 30 0 C under aseptic conditions, harvesting the medium, separating the biomass by conventional methods, extracting the separated broth with an organic solvent, separating the product extracted in solvent by conventional methods, such as extraction with polar solvents, washing the product by solvent, drying the product by conventional method and purifying by column chromatography.
- Candida bombicola has been isolated from bumble bee honey and it's culture was previously known as Torulopsis bombicola. This strain is available in ATCC 22214. First reference of this organism is by J.F.T. Spencer., Gorin P.A.J, and AJ. Tulloch Journal of Microbiology and serology (Antonie van Leeuwenhock) Volume 36 (1970) 1 pp. p 129-133 and is publicly available.
- yeast cells are round to oval and is of 1.7 - 2.6 x 1.0 - 2.0 micron. No pellicle is formed. No true or psudomycelium is formed and Ascospore formation was not observed.
- the present invention provides a process for the preparation of succinic acid from glucose by using Candida bombicola (ATCC 22214), which comprises growing Candida bombicola in a conventional growth medium consisting of a carbon, nitrogen sources and other micro-ingredients, having pH in the range of pH 4-7, for a period of 24 - 100 hrs., at a temperature ranging between 26 to 30 0 C under aseptic conditions, harvesting the above said medium, separating the biomass by known method and extracting the separated broth with an organic solvent, separating the resultant product from the above said extract by known method, washing the product by a solvent followed by drying and purifying it by known method to obtain the desired product in pure form.
- Candida bombicola ATCC 22214
- the organic solvent used for extraction is a polar solvent.
- the organic solvent used for extraction is selected from the group consisting of ethyl acetate, methanol and chloroform.
- MGYP media Malt Extract (0.3 g), Glucose (5 g), Yeast Extract (0.3 g), and Peptone was dissolved in 100 ml of distilled water. The resulting mixture was stirred for 10 min. on magnetic stirrer at room temperature and autoclaved for 15-20 min. Then Candida bombicola culture 10% inoculation kept at 30 0 C for 24 hrs. was inoculated into sterilized flasks and kept for a period of 96 hrs. at 30 0 C. The pH of media was pH 6.8. The broth was harvested at 96 hrs and same was separated from solid and liquid broth was extracted with ethyl acetate.
- the extract was concentrated on Rota vapor at 40 0 C and solid form was purified by column chromatography.
- 60-120 mesh particle size silica was used as the stationary phase and organic solvents like petroleum ether, acetone, chloroform, methanol, ethyl acetate may be used as the mobile phase for column chromatography.
- Crystalline from of succinic acid was identified by various methods like infra-red spectroscopy (IR), NMR, melting point, and X-ray crystallography. Melting point of succinic acid was 182-186 degree C. The yield succinic acid was approximately 120 mg/g-glucose by Candida bombicola.
- Another advantage of the invention is there are no other bye products from glucose.
- yeast can be grown on cheaper carbon source such as molasses keeping the end product value minimum. 5.
- the present invention provides an eco friendly (Green Technology) as most of the enzymatic reactions are carried out in aqueous medium.
- Another advantage of the present invention is use of immobilized system for continuous conversion of glucose into succinic acid.
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Abstract
The present invention provides an environment friendly new process for production of succinic acid from glucose or fructose, using Candida bombicola (ATCC 22214) by fermentation in conventional MGYP medium and its purification by column chromatography.
Description
A PROCESS FOR PRODUCTION OF SUCCINIC ACID FROM GLUCOSE
FIELD OF THE INVENTION
The present invention relates a new process for production of succinic acid from glucose.
BACKGROUND OF THE INVENTION Succinic acid is a dicarboxylic acid produced as an intermediate of tricarboxylic acid cycle and also as one of the fermentation products of anaerobic metabolism [Gottschalk 1986; Zeikus 1980]. It has been considered as an important chemical because it can be used as the precursor of 1,4- butanediol, tetrahydrofuran and gamma- butyrolactone and in polymers, foods, pharmaceuticals and cosmetics. Currently most succinic acid is produced commercially through chemical synthesis. However, much attention has recently been focused on the biological production of succinic acid using microorganisms as an alternative to chemical synthesis. The fermentative production of succinic acid can be regarded as a "Green Technology" because renewable substrates are used for its production [Biotechnol.Bioprocess Eng. 2000, 5:379-381].
Succinic acid is produced from glucose by various fermentative methods. Escherichia coli strain NZNl I l gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AF Pi l l, ferment glucose more slowly than did its wild type ancestor, strain W 1485, and generated a very different spectrum of products. AFP 111 produced succinic acid, acetic acid, and ethanol in proportions of approx. 2:1:1. 1 mole of glucose was converted to 1 mole of succinic acid and 0.5 mol each of acetic acid and ethanol [Appl. Biochem. Biotechnol. 1998 Spring, 70-72: 187-98]
There is no report on succinic acid production by Candida bombicola where glucose is source of carbohydrate. Further there is no report of purification of crude succinic acid by column chromatography where it is obtained in crystalline form.
A PfI ldha double mutant Escherichia coli strain NZNl I l was used to produced succinic acid by over expressing the E.coli malic enzyme Escherichia coli strain NZNl Il harboring pTrcML produced 6 and 8 g/L of succinic acid from 20 g/L of glucose in flask culture at 37 degree C and 30 degree C, respectively. When NZNl I l (pTrcML ) was cultured at 30 degree with intermittent glucose feeding the final succinic acid concentration obtained was 9.5 g/L and the ratio of succinic acid to acetic acid was 13:1 [ Biotechnol. Bioeng. 2001 JuI 20; 74(2): 89-95]
Several anaerobic and facultative anaerobic bacteria produce succinic acid from carbohydrates e.g. Ruminococcus albus [Leatherwood 1965], cellulolytic Prevolella ruminicola [Howlett et al. 1976], Bacteroides amylophilus [Caldwell et al. 1973], Bacteroides fragilis [Caspari and Macy 1983], and Actinobacillus succiniciproducens MBEL55E [P.C.Lee et al. 2002]
The anaerobic bacterium Anaerobiospirillum succiniciproducens has most often been employed because it can produce a significant amount of succinic acid from glucose [P.C.Lee et al 1999].
The process of production of succinic acid from glucose, fructose or hexoses by the present invention is illustrated using Candida bombicola. It is a new aerobic method for production of succinic acid. The process of present invention is described herein below with reference to examples.
Most of the reported reactions require perfect pH condition, accurate temperature and typical media composition i.e reactions require certain conditions. In certain reactions side products are formed e.g. acetic acids, Ethanol, CO2 gas etc. antifoaming agents are required for some reactions for control of foaming. Also some reactions are two step reactions.
We are reporting here a biocon version process for succinic acid production from glucose by yeast Candida bombicola (ATCC 22214) which obviates the drawbacks as detailed above.
OBJECTIVES OF THE INVENTION
The main objective of the present invention is to provide one step transformation of glucose to succinic acid.
Yet another object is to provide a new process for production of succinic acid in which yeast cells require less restricted conditions and cheaper carbon sources.
SUMMARY OF THE INVENTION
The present invention provides a new process for production of succinic acid from glucose, fructose or any other hexoses using Candida bombicola which comprises growing Candida bombicola in a conventional growth medium consisting of a carbon, nitrogen sources and carbohydrate source and other micro-ingredients for a period of 24 to 96 hrs. at a temperature ranging between 26 to 30 0C under aseptic conditions, harvesting the medium, separating the biomass by conventional methods, extracting the separated broth with an organic solvent, separating the product extracted in solvent by conventional methods, such as extraction with polar solvents, washing the product by solvent, drying the product by conventional method and purifying by column chromatography.
Candida bombicola has been isolated from bumble bee honey and it's culture was previously known as Torulopsis bombicola. This strain is available in ATCC 22214. First reference of this organism is by J.F.T. Spencer., Gorin P.A.J, and AJ. Tulloch Journal of Microbiology and serology (Antonie van Leeuwenhock) Volume 36 (1970) 1 pp. p 129-133 and is publicly available.
Typical characteristic of this organism are:
In alt agar the yeast cells are round to oval and is of 1.7 - 2.6 x 1.0 - 2.0 micron. No pellicle is formed. No true or psudomycelium is formed and Ascospore formation was not observed.
DETAIL DESCRIPTION OF THE INVENTION
Accordingly the present invention provides a process for the preparation of succinic acid from glucose by using Candida bombicola (ATCC 22214), which comprises growing Candida bombicola in a conventional growth medium consisting of a carbon,
nitrogen sources and other micro-ingredients, having pH in the range of pH 4-7, for a period of 24 - 100 hrs., at a temperature ranging between 26 to 30 0C under aseptic conditions, harvesting the above said medium, separating the biomass by known method and extracting the separated broth with an organic solvent, separating the resultant product from the above said extract by known method, washing the product by a solvent followed by drying and purifying it by known method to obtain the desired product in pure form.
In an embodiment of the present invention the MGYP growth medium used is consisting of:
Glucose 10 - 100
Yeast Extract 0.2 - 1.0
Ammonium sulfate 1.0 - 0.8 MgSO4.7H2O in the range of 0.1 - 0.3
Na2HPO4 in the range of 0.2 - 2.0
KH2PO4 in the range of 0.7 - 7.0
In yet another embodiment the organic solvent used for extraction is a polar solvent.
In still another embodiment the organic solvent used for extraction is selected from the group consisting of ethyl acetate, methanol and chloroform.
The process of the present invention is described here in below with reference to examples, which are illustrative in nature and should not be construed to limit the scope of the present invention in any manner.
Example 1
MGYP media Malt Extract (0.3 g), Glucose (5 g), Yeast Extract (0.3 g), and Peptone was dissolved in 100 ml of distilled water. The resulting mixture was stirred for 10 min. on magnetic stirrer at room temperature and autoclaved for 15-20 min. Then Candida bombicola culture 10% inoculation kept at 30 0C for 24 hrs. was inoculated into sterilized flasks and kept for a period of 96 hrs. at 30 0C. The pH of media was pH 6.8. The broth was harvested at 96 hrs and same was separated from solid and liquid broth
was extracted with ethyl acetate. The extract was concentrated on Rota vapor at 40 0C and solid form was purified by column chromatography. 60-120 mesh particle size silica was used as the stationary phase and organic solvents like petroleum ether, acetone, chloroform, methanol, ethyl acetate may be used as the mobile phase for column chromatography. Crystalline from of succinic acid was identified by various methods like infra-red spectroscopy (IR), NMR, melting point, and X-ray crystallography. Melting point of succinic acid was 182-186 degree C. The yield succinic acid was approximately 120 mg/g-glucose by Candida bombicola.
The main advantage of the present invention are
1. There is no reported method by which succinic acid can be produced using Candida bombicola ATCC 22214 under condition which are exemplified in the text.
2. In the present method provides a process for succinic acid by biotransformation of glucose using Candida bombicola.
3. Another advantage of the invention is there are no other bye products from glucose.
4. Another advantage is yeast can be grown on cheaper carbon source such as molasses keeping the end product value minimum. 5. The present invention provides an eco friendly (Green Technology) as most of the enzymatic reactions are carried out in aqueous medium.
6. Another advantage of the present invention is use of immobilized system for continuous conversion of glucose into succinic acid.
Claims
1. A novel process for production of succinic acid from glucose by Candida bombicola (ATCC 22214), which comprises growing C.bombicola in a conventional growth medium consisting of a carbon, nitrogen sources and other micro-ingredients having pH in the range of pH 4 - 7, for a for a period of 24 to
100 hrs., at a temperature ranging between 26 to 30 0C under aseptic conditions, harvesting the medium, separating the biomass by known methods and extracting the separated broth with an organic solvent, separating the product extracted in solvent by conventional methods, washing the product by solvent followed by drying and purifying it by known method to obtain the desired product in pure form.
2. A process as claimed in claim 1, wherein the solvent used for extraction is any polar solvent like ethyl acetate, methanol, chloroform or pet ether.
Glucose 10 - 100
Yeast Extract 0.2 - 1.0
Ammonium sulfate 1.0 - 0.8
MgSO4JH2O in the range of 0.1 - 0.3 Na2HPO4 in the range of 0.2 - 2.0
KH2PO4 in the range of 0.7 - 7.0
3. A process as claimed in claim 1 and 2, wherein the organic solvent used for extraction is a polar solvent.
4. A process as claimed in claim 1-3, wherein the organic solvent used for extraction is selected from the group consisting of ethyl acetate, methanol and chloroform.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN757/DEL/2005 | 2005-03-31 | ||
| IN757DE2005 | 2005-03-31 |
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| Publication Number | Publication Date |
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| WO2006103531A1 true WO2006103531A1 (en) | 2006-10-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2006/000716 WO2006103531A1 (en) | 2005-03-31 | 2006-03-29 | A process for production of succinic acid from glucose |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210090870A (en) * | 2020-01-13 | 2021-07-21 | 주식회사 고운세상코스메틱 | Cosmetic composition with excellent skin barrier using skin microbiome |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3352759A (en) * | 1959-01-14 | 1967-11-14 | Kaken Kagaku Kk | Process for producing cis-succinic acid of gauche form |
| JPS5617077B2 (en) * | 1971-11-19 | 1981-04-20 | ||
| WO2000020620A2 (en) * | 1998-10-05 | 2000-04-13 | Cognis Corporation | Process for recovering carboxylic acides from a fermentation broth |
-
2006
- 2006-03-29 WO PCT/IB2006/000716 patent/WO2006103531A1/en not_active Application Discontinuation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3352759A (en) * | 1959-01-14 | 1967-11-14 | Kaken Kagaku Kk | Process for producing cis-succinic acid of gauche form |
| JPS5617077B2 (en) * | 1971-11-19 | 1981-04-20 | ||
| WO2000020620A2 (en) * | 1998-10-05 | 2000-04-13 | Cognis Corporation | Process for recovering carboxylic acides from a fermentation broth |
Non-Patent Citations (2)
| Title |
|---|
| COLLINGS A ET AL: "SECRETION OF END-PRODUCTS OF METABOLISM BY CANDIDA-SPP", BIOMEDICAL LETTERS, vol. 46, no. 184, 1991, pages 285 - 296, XP008066991, ISSN: 0961-088X * |
| DATABASE WPI Section Ch Week 198120, Derwent World Patents Index; Class D16, AN 1973-72963U, XP002391767 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210090870A (en) * | 2020-01-13 | 2021-07-21 | 주식회사 고운세상코스메틱 | Cosmetic composition with excellent skin barrier using skin microbiome |
| KR102443283B1 (en) | 2020-01-13 | 2022-09-15 | 주식회사 고운세상코스메틱 | Cosmetic composition with excellent skin barrier using skin microbiome |
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