WO2008126669A2 - Method for producing pyruvic acid - Google Patents

Method for producing pyruvic acid Download PDF

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WO2008126669A2
WO2008126669A2 PCT/JP2008/055618 JP2008055618W WO2008126669A2 WO 2008126669 A2 WO2008126669 A2 WO 2008126669A2 JP 2008055618 W JP2008055618 W JP 2008055618W WO 2008126669 A2 WO2008126669 A2 WO 2008126669A2
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strain
nbrc
streptomyces
glycerol
jcm
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PCT/JP2008/055618
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French (fr)
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WO2008126669A3 (en
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Kazuhisa Kishimoto
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Sumitomo Chemical Company, Limited
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Priority claimed from JP2008010896A external-priority patent/JP2009142255A/en
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Publication of WO2008126669A2 publication Critical patent/WO2008126669A2/en
Publication of WO2008126669A3 publication Critical patent/WO2008126669A3/en

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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

Definitions

  • the present invention relates to a method for producing pyruvic acid from glycerol using a microorganism.
  • BDF BACKGROUND ART Bio Diesel Fuel
  • Bio Diesel Fuel is produced from vegetable oils , animal fats , waste oil, and the like. In this time, a waste liquid containing glycerol is produced as a by-product . There is no specific use for the waste liquid, so that it is currently just discarded.
  • Japanese Unexamined Patent Publication (Kokai) No. 2006-180782 discloses a method for producing hydrogen and ethanol from glycerol in the waste using a bacterium of the genus Enterobactor.
  • Japanese Unexamined Patent Publication (Kokai) No. 2003-235592 discloses a method for producing organic acid from glucose as a carbon source, using aerobacter (particularly, coryneform bacteria) . Japanese
  • Unexamined Patent Publication (Kokai) No. 2004-501634 discloses a bacterial strain (Mannheimia sp. 55E) which produces various organic acids from glucose.
  • Citrobacter freundii is a Gram-negative facultative anaerobic rod existing in places such as animal intestine. Citrobacter freundii is known to produce 1, 3-propanediol etc. from glycerol, but it is not suggested that Citrobacter freundii produces pyruvic acid (European Patent Application EP 0 361 082 A2; Ke-Ke Cheng et al.. Biotechnology Letters (2004) 26: 911-915; F. Barbirato et al., Appl Microbiol Biotechnol (1995) 43: 786-793; T. Homann et al. , Appl Microbiol biotechnol (1990) 33: 121-126). Particularly, T.
  • An object of the present invention is to provide a method for manufacturing pyruvic acid from glycerol by a simple manner, by culturing a bacterium of a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobatcer , Brevibacterium, Corynebacterium, Curtobacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides , aenibacillus , Proteus, Saccharopolyspora, Spirillospora and Streptomyces ; a bacterium of the genus Citrobacter; a bacterium of a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans
  • a method for producing pyruvic acid from glycerol which comprises : culturing in a culture medium containing glycerol, a bacterium which belongs to a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobacter, Brevibacterium, Corynebacterium, Curtbacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides , Paenibacillus , Proteus, Saccharopolyspora, Spirillospora and Streptomyces , and has an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol;
  • Achromobacter lyticus Brevibacterium butanicum, Brevibacterium ketoglutamicum, Brevibacterium fuscum, Corynebacterium glutamicum, Curtobacterium albidum, Curtobacterium luteum.
  • Curtbacterium citreum Devosia riboflavina, Exiguobacterium acetylicum, Frateuria aurantia, Hafnia alvei, Mesorhizobium loti, Nocardioid.es simplex, Paenibacillus validus , Paenibacillus polymyxa, Paenibacillus macerans , Proteus vulgaris, Saccharopolyspora erythraea, Spirillospora albida, Streptomyces parvulus , Streptomyces virginiae , Streptomyces galilaeus , Streptomyces canus , Streptomyces fradiae, Streptomyces albofla
  • Spirillospora and Streptomyces is a bacterium of Arthrobacter aurescens NBRC 12136 strain, Arthrobacter citreus NBRC 12957 strain, Arthrobacter histidinolovorans JCM 2520 strain, Arthrobacter nicotianae JCM 1333 strain, Arthrobacter protophormiae JCM 1973 strain, Arthrobacter globiformis ATCC 4336 strain, Arthrobacter pas ⁇ ens ATCC 13346 strain, Arthrobacter paraffineus ATCC 21003 strain. Bacillus badius ATCC 14574 strain. Bacillus sphaericus NBRC 3341 strain. Bacillus licheniformis NBRC 12197 strain. Bacillus cereus var. juroi ATCC 21182 strain, Microbacterium barker! JCM 1343 strain, Microbacterium testaceum JCM 1353 strain,
  • a method for producing pyruvic acid from glycerol which comprises : culturing in a culture medium containing glycerol, a bacterium which belongs to a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans , Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis , Pseudomonas taetrolens and Nocardia uniformis , and has an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol; 5.
  • the bacterium belonging to a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans , Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis , Pseudomonas taetrolens and Nocardia uniformis is a bacterium of Pseudomonas azotoformans JCM 2777 strain, Pseudomonas oryzihabitans JCM 2952 strain, Pseudomonas synxantha NBRC 3913 strain, Pseudomonas fragi JCM 20552 strain, Pseudomonas chlororaphis NBRC 3904 strain, Pseudomonas taetrolens NBRC 3460 strain or Nocardia uniformis NBRC
  • a method for producing pyruvic acid from glycerol which comprises : culturing a bacterium of Escherichia coli NBRC 12734 strain in culture medium containing glycerol, or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol;
  • a method for producing pyruvic acid from glycerol which comprises culturing in a culture medium containing glycerol under an aerobic condition a bacterium belonging to the genus Citrobacter and having an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol;
  • the method according to any of the above 1 to 10, further comprises recovering pyruvic acid from a culture obtained by culturing;
  • the pyruvic acid-producing bacteria used for the present invention are specifically exemplified by the bacterial genera, bacterial species and bacterial strains described in the above 1 to 9.
  • bacteria of the genus Citrobacter are not specially limited as long as they can produce pyruvic acid by utilizing glycerol, and exemplified by a bacterium of Citrobacter amalonaticus , Citrobacter braakii, Citrobacter farmer!, Citrobacter freundii, Citrobacter koseri, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii and Citrobacter youngae.
  • a preferable bacterium is a bacterium of the species Citrobacter freundii.
  • Citrobacter freundii include Citrobacter freundii JCM 1657 strain, but it is not limited thereto .
  • preferred bacteria are those being able to produce pyruvic acid from glycerol in higher yield.
  • preferred bacteria include a Arthrobacter aurescens NBRC 12136 strain, Arthrobacter histidinolovorans JCM 2520 strain, Arthrobacter protophormiae JCM 1973 strain.
  • JCM 1343 strain Microbacterium liquefaciens JCM 3879 strain, Microbacterium esteraromaticum NBRC 3751 strain, Paenibacillus validus NBRC 13635 strain, Paenibacillus polymyxa NBRC 3020 strain, Paenibacillus macerans JCM 2500 strain, Proteus vulgaris NBRC 3851 strain, Raoultella planticola JCM 7251 strain, Raoultella terrigena JCM 1687 strain, Pseudomonas azotoformans JCM 2777 strain, Pseudomonas synxantha NBRC 3913 strain, Pseudomonas fragi JCM 20552 strain, Pseudomonas chlororaphis NBRC 3904 strain, Pseudomonas taetrolens NBRC 3460 strain, Archromobacter denitrificans NBRC 12669 strain, Achromoba ⁇ ter lyticus ATCC 214
  • Archromobacter denitrificans NBRC 12669 strain Achromobacter lyticus ATCC 21456 strain, Exiguobacterium acetylicum JCM 1968 strain, Frateuria aurantia NBRC 3247 strain, Microbacterium testaceum JCM 1353 strain, Nocardia uniformis NBRC 13702 strain, Nocardioides simplex NBRC 12069 strain, Pseudomonas oryzihabitans JCM 2952 strain, Arthrobacter nicotianae JCM 1333 strain, Arthrobacter globiformis ATCC 4336 strain, Streptomyces parvulus NBRC 13193 strain, Streptomyces virginiae NBRC 12827 strain, Streptomyces galilaeus ATCC 31133 strain, Streptomyces canus ATCC 12646 strain, Hafnia alvei NBRC 3731 strain, Streptomyces fradiae ATCC 10745 strain, Streptomyces alboflavus NB
  • pyruvic acid being produced in "higher yield” denotes that pyruvic acid is accumulated from glycerol in a culture medium in a yield of about 0.1 g/L or more.
  • pyruvic acid is accumulated in a culture medium in a yield of about 0.5 g/L or more, and preferably about 1 g/L or more .
  • Bacillus sphaericus NBRC 3341 strain Bacillus licheniformis NBRC 12197 strain, Pseudomonas synxantha NBRC 3913 strain, Pseudomonas chlororaphis NBRC 3904 strain, Pseudomonas taetrolens NBRC 3460 strain, Archromobacter denitrificans NBRC 12669 strain, Devosia riboflavina NBRC 13584 strain, Frateuria aurantia NBRC 3247 strain, Hafnia alvei NBRC 3731 strain, Escherichia coli NBRC 12734 strain, Microbacterium esteraromaticum NBRC 3751 strain, Mesorhizobium loti NBRC 13336 strain, Nocardia uniformis NBRC 13702 strain, Nocardioides simplex NBRC 12069 strain, Paenibacillus validus NBRC 13635 strain, Paenibacillus polymy
  • Arthrobacter globiformis ATCC 4336 strain Arthrobacter pascens ATCC 13346 strain, Arthrobacter paraffineus ATCC 21003 strain.
  • Bacillus badius ATCC 14574 strain Bacillus cereus var. juroi ATCC 21182 strain, Brevibacterium butanicum ATCC 21196 strain, Brevibacterium ketoglutamicum ATCC 21004 strain, Corynebacterium glutamicum ATCC 14020 strain, Streptomyces galilaeus ATCC 31133 strain.
  • Streptomyces canus ATCC 12646 strain, Streptomyces fradiae ATCC 10745 strain and Achromobacter lyticus ATCC 21456 strain are available from American Type Culture Collection, P.O.Box 1549 Manassas, VA 20108 USA.
  • the pyruvic acid-producing bacteria used for the present invention may not only be their wild-type strains, but also be any given naturally-occurring or artificial mutants, including those obtained by treatment with X-ray irradiation, ultraviolet-ray irradiation or a chemical mutagen such as
  • a host of the recombinant can belong to any bacterial genera as long as it is a transformable microorganism. However, it is preferred that the host belongs to the same bacterial genus as a parent strain that a targeted gene is originated from. It is desirable to select a pyruvic acid-producing bacterium with improved conversion capacity from glycerol into pyruvic acid.
  • a culture medium containing glycerol may be a medium obtained by adding pure glycerol or a glycerol-containing mixture.
  • Components other than glycerol in the glycerol-containing mixture or their amount are preferably those which do not have harmful effects on the pyruvic acid-producing bacteria used for the present invention.
  • Origin of the glycerol-containing mixture is not particularly limited, but using Bio Diesel waste is preferable for effective use of resources .
  • One of a method for producing Bio Diesel Fuel is to produce Fatty Acid Methyl Ester (FAME) by alcoholysis of triglyceride using an alkali catalyst. In this method, waste fluid containing glycerol is produced as a by-product (called Bio Diesel waste) .
  • This waste fluid is usually contaminated with a catalyst, unconverted fatty acids (they differ depending on used oil) and the like.
  • a Bio Diesel waste used after-mentioned Examples is glycerol: 51%, methanol: 11%, potassium hydroxide: 8%, water: 4%, others such as glyceride: 26%.
  • the medium used for the method of the present invention may be any kind as long as it contains common components necessary for culture of bacteria, and is not limited to a particular medium. In the present invention, it is possible to obtain pyruvic acid even in a medium with plain composition containing carbon sources, nitrogen sources and inorganic salts .
  • the medium used for the method of the present invention contains glycerol as a carbon source .
  • the concentration of glycerol contained in the medium can be properly selected within a range which exerts no harmful effect on growth of bacteria, and production of pyruvic acid. However, it is usually from about 0.1 to 500 g/L, and preferably from about 1 to 300 g/L.
  • Bio Diesel waste When used as a glycerol source, it is allowed to dilute the waste liquid, or to add glycerol thereto, until the amount of glycerol in the medium falls within the above range, depending on the concentration of glycerol contained in the waste.
  • the medium may contain substances other than glycerol as carbon sources , but the amount thereof should be limited to the extent which does not interfere production of pyruvic acid from glycerol.
  • the carbon sources used for the present invention are exemplified by glucose, fructose, starch, lactose, arabinose, xylose, dextrin, molasses and malt extract, but are not limited thereto.
  • the amount of other carbon sources is preferably about 10% by weight or less of glycerol, and more preferably about 1% by weight or less. It is most preferable that the medium contains glycerol as a single carbon source.
  • Examples of a nitrogen source include inorganic nitrogen compounds such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate, urea, and the like. It is also allowed to add to a culture medium organic nitrogen sources such as gluten flour, cottonseed flour, soybean flour, corn steep liquor, dried yeast, yeast extract, peptone, meat extract and casamino acid, or various vitamin groups, as needed, but a method of the present invention can produce pyruvic acid without these relatively expensive nutrients .
  • inorganic nitrogen compounds such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate, urea, and the like. It is also allowed to add to a culture medium organic nitrogen sources such as gluten flour, cottonseed flour, soybean flour, corn steep liquor, dried yeast, yeast extract, peptone, meat extract and casamino acid, or various vitamin groups, as needed, but a method of the present invention can produce pyruvic acid without these relatively expensive nutrients .
  • inorganic salts such as potassium phosphate monobasic, potassium phosphate dibasic, sodium chloride, magnesium sulfate , manganese sulfate, calcium carbonate, calcium chloride, sodium iodide, potassium iodide, and cobalt chloride. It is also allowed to add defoaming agents, such as liquid paraffin, higher alcohol, vegetable oil, mineral oil and silicon, as needed, particularly when the medium foams markedly.
  • the nitrogen sources, inorganic salts and other components are known by those skilled in the art.
  • pyruvic acid-producing bacteria excluding bacteria of the genus Citrobacter
  • Citrobacter are cultured under aerobic condition.
  • the aerobic condition denotes culture in the presence of molecular oxygen. Ventilation, stirring and shaking can be performed for supplying oxygen. It is available to use any common devices for culture of microorganisms .
  • the method of the present invention allows to culture bacteria, and to produce pyruvic acid, by a simple manner without using any devices necessary for bringing about an anaerobic condition.
  • Culture of bacteria under an anaerobic condition can be performed by introducing carbon dioxide or inert gas (nitrogen argon, etc.), or without ventilation.
  • pyruvic acid It is preferred for mass production of pyruvic acid to be performed under a submerged culture condition.
  • bacteria When bacteria are propagated in a large tank, it is preferred to inoculate bacteria in a vegetative period into a production tank, so as to avoid delay in propagation in a pyruvic acid producing process. That is, it is preferred that bacteria are first inoculated to a relatively small amount of medium, and cultured to produce seed bacteria in a vegetative period, and then the seed bacteria is transferred into the large tank in a sterile manner.
  • Stirring and ventilation of the culture solution can be performed in various manners.
  • Stirring can be performed using a propeller or a mechanical stirring device similar to a propeller, rotation or shake of a fermenter, or a pumping device.
  • Ventilation can be performed by allowing sterilized air to pass through in the culture solution. In doing so, the ventilation operation may provide stirring effect as well.
  • a culture method such as batch culture, fed batch culture and continuous culture can be properly selected and used.
  • the culture conditions are discretional as long as they are suitable for culture of pyruvic acid-producing bacteria used for the present invention.
  • the culture temperature is from about 4 to 40 0 C, preferably from about 20 to 37 0 C.
  • the pH of the medium is from about 5 to 9 , and preferably from about 6 to 8.
  • alkali such as an aqueous ammonia solution, calcium carbonate, sodium hydroxide and potassium hydroxide
  • composition of the medium and other culture conditions are appropriately adjustable by those skilled in the art. It will also be considered to adjust the conditions , in order to further enhance yield of pyruvic acid.
  • the bacteria used for the method of the present invention may take a bacterial cell, processed bacterial cell or immobilized product thereof.
  • the processed bacterial cell denotes a disrupted bacterial cell or an enzyme extracted from cultured substances (include a bacterial cell and culture supernatant) .
  • Examples of the processed bacterial cell include that obtained by treating a cultured bacterial cell with an organic acid (such as acetone and ethanol), freeze dry treatment or alkali treatment, that obtained by physically or enzymatically disrupting a bacterial cell, or a crude enzyme separated or extracted therefrom.
  • cultured bacteria are subjected to a centrifugal treatment, and the cells to be collected are disrupted by a physical milling method such as an ultrasonic, Dyno-mill and French press treatment, or a chemical disrupting method using a surfactant or a lytic enzyme such as lyzozyme.
  • the resultant solution is subjected to centrifuge or membrane filtration to remove insoluble materials, and the resultant cell-free extract is subjected to a separation/purification method, such as cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and metal chelate chromatography, to fractionate and purify the enzyme.
  • Examples of a carrier used for the chromatography include insoluble polymer carriers such as cellulose, dextrin and agarose introduced with a carboxymethyl (CM) group, diethylaminoethyl (DEAE) group, phenyl group or butyl group. It is also allowed to use a commercially available carrier-packed column. Disruption of the bacterial cell and extraction of the enzyme can be performed by a known method by those skilled in the art, as well as the above method.
  • the method for producing pyruvic acid from glycerol using a bacterial cell or processed bacterial cell is exemplified by a method that the bacterial cell is suspended and reacted in a 18
  • the bacterial cell can be prepared by culturing pyruvic acid-producing bacteria, followed by centrifuge thereof . It is preferred that the concentration of glycerol in the substrate solution is approx. from 0.01 to 50% by weight.
  • the reaction temperature is usually from about 4 to 40° C, and preferably from about 20 to 37° C.
  • the pH of the reaction solution is usually from about 5 to 9 , and preferably from 6 to 8. When the pH of the medium declines along with production of pyruvic acid, it is adjusted to be fallen within the above range, by adding alkali such as an aqueous ammonia solution, calcium carbonate, sodium hydroxide and potassium hydroxide to the culture system, as needed.
  • the method for producing pyruvic acid from glycerol using an immobilized bacterial cell or processed bacterial cell is exemplified by a method that the immobilized bacterial cell or processed bacterial cell is filled in a column, and a glycerol-containing substrate solution is allowed to pass it through.
  • the bacterial cell or processed bacterial cell is obtained by culturing the pyruvic acid-producing bacteria, followed by centrifuge thereof .
  • the method for immobilizing the bacterial cell is exemplified by a comprehensive immobilization means using a gel, and immobilization means by supporting an ion exchange material. Examples of the gel to be used include carrageenan, agar, mannan, PVA and polyacrylamide gels.
  • the proper particle size of the gel is from about 1 to 10 mm in diameter, although the size varies depending on a kind of gel.
  • the ion exchange material include a cellulose-based material. styrenedivinylbenzene-based material and phenolformalin-based ion exchange material. It is preferred that the concentration of glycerol in the substrate solution is from about 0.01 to 50% by weight. It is also allowed to add a SH compound such as mercaptoethanol, cysteine and glutathione, reducing agent such as sulfite, and enzyme activator such as a magnesium ion and manganese ion.
  • the velocity of the solution passing through varies depending on the column size and amount of the immobilized substance. It is proper that the space velocity (ml/ml resin'hr) is from 0.05 to 10, as an index of velocity for treating a solution.
  • Separation and purification of pyruvic acid are performed in accordance with a conventional known method. For example, filtration or centrifugation is performed to a culture solution after completion of culture, to obtain a supernatant. From the supernatant, for example, pyruvic acid can be separated such as by concentrated crystallization, but separation and purification of pyruvic acid are not limited thereto.
  • pyruvic acid can be separated by such methods as solvent extraction from the supernatant, or separated and purified by methods such as ion exchange chromatography in which pyruvic acid is eluted and separated after absorbed in an ion exchange resin, isolation by forming metal salts such as a calcium ion, fractional precipitation by an insolubilizing treatment, fractional crystallization by crystallization, membrane separation by a reverse osmosis membrane, and concentrated crystallization method.
  • pyruvic acid can be separated and purified according to a method described in Japanese Unexamined Patent Publication 5618
  • Achromobacter denitrifleans NBRC 12669 strain was spread on an agar medium A, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic , 6 g of sodium phosphate dibasic , 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and 1 L of distilled water (final pH 7.4)), and allowed to stand at 30° C for 4 days .
  • composition 3 g of potassium phosphate monobasic , 6 g of sodium phosphate dibasic , 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of
  • the bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium B, being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no calcium chloride, yeast extract and agar) , and subjected to shaking culture (pre-culture) at 30° C at 200 rpm for 24 hours .
  • a medium B being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no calcium chloride, yeast extract and agar)
  • the bacterial strain culture solution of 30 ⁇ L grown above was transferred to 3 mL of a medium C, being a medium for test tube culture (composition: the same composition as the above medium B for test tube culture, except for containing 19.6 g of a glycerol fraction (glycerol: 51%, methanol: 11%, potassium hydroxide: 8%, water: 4%, and others including glyceride: 26%) which was by-produced upon production of the Bio Diesel Fuel, instead of 10 g of glycerol) , and subjected to shaking culture (main culture) at 3O 0 C at 200 rpm.
  • shaking culture main culture
  • Achromobacter denitrifleans NBRC 12669 strain was spread on an agar medium A, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic , 6 g of sodium phosphate dibasic , 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate , 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4)), and allowed to stand at 30° C for 4 days.
  • agar medium A being a medium for plate culture
  • the bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium D, being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no agar) , and subjected to shaking culture (pre-culture) at 30° C at 200 rpm for 24 hours.
  • the bacterial strain culture solution of 30 ⁇ L grown above was transferred to 3 mL of a medium E, being a medium for test tube culture, (composition: the same composition as the above medium D for test tube culture, except for containing 19.6 g of a glycerol fraction (glycerol: 51%, methanol: 11%, potassium: hydroxide: 8%, water: 4%, and others including glyceride: 26%) which was by-produced upon production of the Bio Diesel Fuel, instead of 10 g of glycerol) , and subjected to shaking culture (main culture) at 30° C at 200 rpm. Four (4) days after initiation of the reaction, 9.4 g of glycerol was consumed per litter, and 0.7 g of pyruvic acid was accumulated.
  • a medium E being a medium for test tube culture
  • Pseudomonas azotoformans JCM 2777 strain was reacted in the same manner as in Example 1. As a result , 3.3 g of glycerol per one liter was consumed and 0.4 g of pyruvic acid was accumulated.
  • Pseudomonas synxantha NBRC 3913 strain was reacted in the same manner as in Example 1. As a result, 4.5 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Raoultella terrigena JCM 1687 strain was reacted in the same manner as in Example 1. As a result, 1.9 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Exiguobacterium a ⁇ etylicum JCM 1968 strain was spread on an agar medium A, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4)), and allowed to stand at 3O 0 C for 4 days.
  • composition 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar
  • the bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium D, being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no agar) , and subjected to shaking culture (pre-culture) at 30° C at 200 rpm for 24 hours.
  • the bacterial strain culture solution of 30 ⁇ L grown above was transferred to 3 mL of a medium D, being a medium for test tube culture, and subjected to shaking culture (main culture) at 30° C at 200 rpm.
  • Six (6) days after initiation of the reaction 1.4 g of glycerol was consumed per litter, and 0.5 gof pyruvic acid was accumulated.
  • Arthrobacter nicotianae JCM 1333 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days .
  • 7.1 g of glycerol per one liter was consumed and 1.7 g of pyruvic acid was accumulated.
  • Arthrobacter citreus NBRC 12957 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 2.4 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
  • Arthrobacter aurescens NBRC 12136 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days. As a result, 2.5 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Arthrobacter histidinolovorans JCM 2520 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 3.8 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Arthrobacter protophorminae JCM 1973 strain was reacted in the same manner as in Example 6 , except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 3.7 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Streptomyces parvulus NBRC 13193 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days .
  • 9.4 g of glycerol per one liter was consumed and 2.2 g of pyruvic acid was accumulated.
  • Frateuria aurantia NBRC 3247 strain was spread on an agar medium A, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and 1 L of distilled water (final pH 7.4)), and allowed to stand at 30° C for 4 days.
  • composition 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and 1 L of distilled
  • the bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium B, being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no calcium chloride, yeast extract and agar) , and subjected to shaking culture (pre-culture) at 30° C at 200 rpm for 24 hours.
  • the bacterial strain culture solution of 30 ⁇ L grown above was transferred to 3 mL of a medium B, being a medium for test tube culture, and subjected to shaking culture (main culture) at 30° C at 200 rpm.
  • Four (4) days after initiation of the reaction 0.7 g of glycerol was consumed per litter, and 0.1 g of pyruvic acid was accumulated.
  • Frateuria aurantia NBRC 3247 strain was reacted in the same manner as in Example 6. As a result , 2.4 g of glycerol per one liter was consumed and 0.5 g of pyruvic acid was accumulated.
  • Microbacterium testaceum JCM 1353 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 2 days , and the main culture period from 6 days to 4 days . As a result , 3.1 g of glycerol per one liter was consumed and 0.6 g of pyruvic acid was accumulated.
  • Microbacterium barkeri JCM 1343 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 2 days , and the main culture period from 6 days to 4 days . As a result , 4.2 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Nocardioides simplex NBRC 12069 strain was reacted in the same manner as in Example 6. As a result, 2.2 g of glycerol per one liter was consumed and 0.5 g of pyruvic acid was accumulated.
  • Paenibacillus validus NBRC 13635 strain was reacted in the same manner as in Example 6. As a result , 1.1 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Bacillus sphaericus NBRC 3341 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 1.9 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
  • Bacillus badius ATCC 14574 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result, 2.7 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
  • Nocardia uniformis NBRC 13702 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 3.7 g of glycerol per one liter was consumed and 0.6 g of pyruvic acid was accumulated.
  • Proteus vulgaris NBRC 3851 strain was reacted in the same manner as in Example 6. As a result , 5.3 g of glycerol per one liter was consumed and 0.4 g of pyruvic acid was accumulated.
  • Cortnebacterium glutamicum ATCC 14020 strain was reacted in the same manner as in Example 6. As a result, 1.9 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Pseudomonas oryzihabitans JCM 2952 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from
  • Example 28 Pseudomonas azotoformans JCM 2777 strain was reacted in the same manner as in Example 15. As a result, 5.6 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Example 29 Raoultella planticola JCM 7251 strain was reacted in the same manner as in Example 15. As a result, 1.3 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Example 30 Paenibacillus polymyxa NBRC 3020 strain was reacted in the same manner as in Example 6. As a result , 0.4 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
  • Example 31
  • Streptomyces virginiae NBRC 12827 strain was reacted in the same manner as in Example 6. As a result, 4.0 g of glycerol per one liter was consumed and 0.8 g of pyruvic acid was accumulated.
  • Streptomyces galilaeus ATCC 31133 strain was reacted in the same manner as in Example 6. As a result , 4.3 g of glycerol per one liter was consumed and 0.7 g of pyruvic acid was accumulated.
  • Streptomyces canus ATCC 12646 strain was reacted in the same manner as in Example 6. As a result , 5.7 g of glycerol per one liter was consumed and 0.8 g of pyruvic acid was accumulated.
  • Streptomyces fradiae ATCC 10745 strain was reacted in the same manner as in Example 6. As a result, 4.0 g of glycerol per one liter was consumed and 2.4 g of pyruvic acid was accumulated.
  • Spirillospora albida NBRC 12248 strain was reacted in the same manner as in Example 6. As a result, 0.3 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Escherichia coli NBRC 12734 strain was reacted in the same manner as in Example 15. As a result, 8.3 g of glycerol per one liter was consumed and 2.0 g of pyruvic acid was accumulated.
  • Escherichia coli NBRC 12734 strain was reacted in the same manner as in Example 1. As a result, 8.9 g of glycerol per one liter was consumed and 2.9 g of pyruvic acid was accumulated.
  • Escherichia coli NBRC 12734 strain was reacted in the same manner as in Example 6. As a result, 8.1 g of glycerol per one liter was consumed and 1.9 g of pyruvic acid was accumulated.
  • Escherichia coli NBRC 12734 strain was reacted in the same manner as in Example 2. As a result, 10.2 g of glycerol per one liter was consumed and 3.3 g of pyruvic acid was accumulated.
  • Hafnia alvei NBRC 3731 strain was reacted in the same manner as in Example 1. As a result, 8.5 g of glycerol per one liter was consumed and 2.4 g of pyruvic acid was accumulated.
  • Hafnia alvei NBRC 3731 strain was reacted in the same manner as in Example 6. As a result, 8.1 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Example 42 Curtbacterium luteum JCM 1480 strain was reacted in the same manner as in Example 6. As a result , 2.1 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Pseudomonas fragi JCM 20552 strain was reacted in the same manner as in Example 1. As a result , 4.4 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Pseudomonas fragi JCM 20552 strain was reacted in the same manner as in Example 2. As a result , 4.5 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Saccharopolyspora erythraea NBRC 13426 strain was reacted in the same manner as in Example 6. As a result , 3.2 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Achromobacter lyticus ATCC 21456 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days .
  • 2.2 g of glycerol per one liter was consumed and 0.7 g of pyruvic acid was accumulated.
  • Bacillus licheniformis NBRC 12197 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 1.8 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
  • Microbacterium liquefaciens JCM 3879 strain was reacted in the same manner as in Example 6. As a result , 2.3 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Brevibacterium butanicum ATCC 21196 strain was reacted in the same manner as in Example 2. As a result, 2.8 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Arthrobacter globiformis ATCC 4336 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days. As a result, 5.3 g of glycerol per one liter was consumed and 1.3 g of pyruvic acid was accumulated.
  • Example 52 Arthrobacter paraffineus ATCC 21003 strain was reacted in the same manner as in Example 6. As a result, 10 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
  • Arthrobacter pascens ATCC 13346 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from
  • Bacillus cereus var. juroi ATCC 21182 strain was reacted in the same manner as in Example 6 , except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from
  • Example 55 Pseudomonas taetrolens NBRC 3460 strain was reacted in the same manner as in Example 15, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 3.2 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
  • Pseudomonas chlororaphis NBRC 3904 strain was reacted in the same manner as in Example 1. As a result , 5.4 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Streptomyces alboflavus NBRC 13196 strain was reacted in the same manner as in Example 6. As a result, 10.4 g of glycerol per one liter was consumed and 2.0 g of pyruvic acid was accumulated.
  • Streptomyces albovinaceus NBRC 12739 strain was reacted in the same manner as in Example 6. As a result, 10.3 g of glycerol per one liter was consumed and 3.0 g of pyruvic acid was accumulated.
  • Streptomyces fumanus NBRC 13042 strain was reacted in the same manner as in Example 6. As a result, 8.7 g of glycerol per one liter was consumed and 3.2 g of pyruvic acid was accumulated.
  • Streptomyces cellulosae NBRC 3713 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from
  • Streptomyces longispororuber NBRC 13488 strain was reacted in the same manner as in Example 2 , except for changing the pre-culture period from 24 hours to 6 days, and the main culture period from 4 days to 6 days. As a result, 11 g of glycerol per one liter was consumed and 2.9 g of pyruvic acid was accumulated.
  • Example 62 Paenibacillus macerans JCM 2500 strain was reacted in the same manner as in Example 6 , except for changing the main culture period from 6 days to 13 days. As a result, 1.7 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Microbacterium esteraromaticum NBRC 3751 strain was reacted in the same manner as in Example 6 , except for changing the main culture period from 6 days to 8 days . As a result , 1.3 g of glycerol per one liter was consumed and 0.6 g of pyruvic acid was accumulated.
  • Curtobacterium citreum JCM 1345 strain was reacted in the same manner as in Example 6 , except for changing the main culture period from 6 days to 13 days. As a result, 1.2 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
  • Brevibacterium fuscum JCM 1488 strain was reacted in the same manner as in Example 6 , except for changing the main culture period from 6 days to 7 days. As a result, 0.9 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
  • Example 66 Citrobacter freundii JCM 1657 strain was spread on an agar medium F, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 250 mg of magnesium sulfate heptahydrate , 20 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4) ), and allowed to stand at 30° C for 4 days .
  • composition 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 250 mg of magnesium sulfate heptahydrate , 20 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4)
  • composition 3 g of potassium phosphate monobasic, 6 g of sodium
  • the bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium G, being a medium for test tube culture, (composition: the same composition as the agar medium F, except for containing no agar and containing 10 g or glycerol instead of 20 g) , and subjected to shaking culture at 30° C at 200 rpm for 24 hours .
  • the bacterial strain culture solution of 30 ⁇ L grown above was transferred to 3 mL of the medium G, being a medium for test tube culture, and subjected to shaking culture at 3O 0 C at 200 rpm for 24 hours.
  • Four (4) days after initiation of the reaction 3.1 g/L of pyruvic acid was accumulated.
  • Citrobacter freundii JCM 1657 strain was spread on an agar medium F, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g of sodium chloride, 1 g of ammonium chloride, 250 mg of magnesium sulfate heptahydrate, 20 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4) ) , and allowed to stand at 30° C for 4 days.
  • composition 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g of sodium chloride, 1 g of ammonium chloride, 250 mg of magnesium sulfate heptahydrate, 20 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4)
  • the bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium G, being a medium for test tube culture (composition: the same composition as the agar medium F, except for containing no agar and containing 10 g or glycerol instead of 20 g) , and subjected to shaking culture at 30° C at 200 rpm for 24 hours .
  • a medium G being a medium for test tube culture
  • the bacterial strain culture solution of 30 ⁇ L grown above was transferred to 3 mL of the medium H, being a medium for test tube culture (composition: the same composition as the above medium G for test tube culture, except for containing 19.6 g of a glycerol fraction (glycerol: 51%, methanol: 11%, potassium: hydroxide: 8%, water: 4%, and others including glyceride: 26%) which was by-produced upon production of the Bio Diesel Fuel, instead of 10 g of glycerol) , and subjected to shaking culture at 30° C at 200 rpm for 24 hours. Four (4) days after initiation of the reaction, 4.2 g/L of pyruvic acid was accumulated.
  • pyruvic acid is produced from glycerol (Bio Diesel waste) using a microorganism by a simple manner. Pyruvic acid has high reactivity, so it is used as an important intermediate in synthesis of medicine, pesticide, and the like. Thus, the present invention serves for producing useful substances from a waste material.

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Abstract

There is provided is a method for producing pyruvic acid from glycerol, which comprises culturing a specific bacterium capable of producing pyruvic acid in a culture medium containing glycerol, or making cells of the bacterium, processed cells of the bacterium or immobilized product thereof contact with glycerol.

Description

DESCRIPTION
METHOD FOR PRODUCING PYRUVIC ACID
TECHNICAL FIELD
The present invention relates to a method for producing pyruvic acid from glycerol using a microorganism.
BACKGROUND ART Bio Diesel Fuel (BDF) is a carbon-neutral alternative fuel for light oil, and has recently been receiving attention as a fuel contributing to resolving environmental problems such as depletion of energy resources, global warming, and air pollution.
Bio Diesel Fuel is produced from vegetable oils , animal fats , waste oil, and the like. In this time, a waste liquid containing glycerol is produced as a by-product . There is no specific use for the waste liquid, so that it is currently just discarded.
As a method for utilizing Bio Diesel waste, Japanese Unexamined Patent Publication (Kokai) No. 2006-180782 discloses a method for producing hydrogen and ethanol from glycerol in the waste using a bacterium of the genus Enterobactor.
Regarding production of organic acids , Japanese Unexamined Patent Publication (Kokai) No. 2003-235592 discloses a method for producing organic acid from glucose as a carbon source, using aerobacter (particularly, coryneform bacteria) . Japanese
Unexamined Patent Publication (Kokai) No. 2004-501634 discloses a bacterial strain (Mannheimia sp. 55E) which produces various organic acids from glucose. However , it has not been known yet that the following bacteria can produce pyruvic acid from glycerol: a bacterium of a genus selected from the bacterial genus group consisting of genus Arthrobacter, genus Bacillus, genus Microbacterium, genus Raoultella, genus Archromobacter, genus Brevibacterium, genus Corynebacterium, genus Curtobacterium, genus Devosia, genus Exiguobacterium, genus Frateuria, genus Hafnia, genus Mesorhizobium, genus Nocardioides , genus Paenibacillus , genus Proteus, genus Saccharopolyspora, genus Spirillospora and genus Streptomyces ; a bacterium of the genus Citrobacter; a bacterium of a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans, Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis , Pseudomonas taetrolens and Nocardia uniformis; and Escherichia σoli NB RC 12734 strain.
Citrobacter freundii is a Gram-negative facultative anaerobic rod existing in places such as animal intestine. Citrobacter freundii is known to produce 1, 3-propanediol etc. from glycerol, but it is not suggested that Citrobacter freundii produces pyruvic acid (European Patent Application EP 0 361 082 A2; Ke-Ke Cheng et al.. Biotechnology Letters (2004) 26: 911-915; F. Barbirato et al., Appl Microbiol Biotechnol (1995) 43: 786-793; T. Homann et al. , Appl Microbiol biotechnol (1990) 33: 121-126). Particularly, T. Homann et al., Appl Microbiol biotechnol (1990) 33: 121-126 discloses that 1, 3-propandiol, ethanol, acetic acid, and the like are produced when Citrobacter frendu is cultured with glycerol under an anaerobic condition, but does not suggest production of pyruvic acid. DISCLOSURE OF INVENTION
It is desired to produce useful substances from the Bio Diesel waste for effective utilization of resources . An object of the present invention is to provide a method for manufacturing pyruvic acid from glycerol by a simple manner, by culturing a bacterium of a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobatcer , Brevibacterium, Corynebacterium, Curtobacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides , aenibacillus , Proteus, Saccharopolyspora, Spirillospora and Streptomyces ; a bacterium of the genus Citrobacter; a bacterium of a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans , Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis , Pseudomonas teatrolens and Nocardia uniformis; or a bacterium of Escherichia coli NBRC 12734 strain (hereinafter, these bacteria are collectively referred to as pyruvic acid-producing bacteria used for the present invention, or simply as pyruvic acid-producing bacteria) in a culture medium containing glycerol (under an aerobic condition for a bacterium of Citrobacter) , particularly by culturing the bacterium under an aerobic condition, or by contacting to glycerol cells of the bacterium, processed cells of the bacterium, or immobilized product thereof.
Namely, the present invention can provide the followings : 1. A method for producing pyruvic acid from glycerol, which comprises : culturing in a culture medium containing glycerol, a bacterium which belongs to a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobacter, Brevibacterium, Corynebacterium, Curtbacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides , Paenibacillus , Proteus, Saccharopolyspora, Spirillospora and Streptomyces , and has an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol;
2. The method according to the above 1 , wherein the bacterium belonging to a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobacter, Brevibacterium, Corynebacterium, Curtbacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides, Paenibacillus, Proteus, Saccharopolyspora, Spirillospora and Streptomyces is a bacterium of Arthrobacter aurescens , Arthrobacter citreus , Arthrobacter histidinolovorans , Arthrobacter nicotianae, Arthrobacter protophormiae , Arthrobacter globiformis , Arthrobacter pascens, Arthrobacter paraffineus , Bacillus badius , Bacillus sphaericus , Bacillus licheniformis , Bacillus cereus var. juroi, Microbacterium barkeri, Microbacterium testaceum, Microbacterium liquefaciens , Microbacterium esteraromaticum, Raoultella planticola, Raoultella terrigena, Archromobacter denitrifleans ,
Achromobacter lyticus, Brevibacterium butanicum, Brevibacterium ketoglutamicum, Brevibacterium fuscum, Corynebacterium glutamicum, Curtobacterium albidum, Curtobacterium luteum. Curtbacterium citreum, Devosia riboflavina, Exiguobacterium acetylicum, Frateuria aurantia, Hafnia alvei, Mesorhizobium loti, Nocardioid.es simplex, Paenibacillus validus , Paenibacillus polymyxa, Paenibacillus macerans , Proteus vulgaris, Saccharopolyspora erythraea, Spirillospora albida, Streptomyces parvulus , Streptomyces virginiae , Streptomyces galilaeus , Streptomyces canus , Streptomyces fradiae, Streptomyces alboflavus, Streptomyces albovinaceus , Streptomyces cellulosae, Streptomyces fumanus , or Streptomyces longispororuber; 3. The method according to the above 2 , wherein the bacterium belonging to a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobacter, Brevibacterium, Corynebacterium, Curtbacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides , Paenibacillus, Proteus, Saccharopolyspora,
Spirillospora and Streptomyces is a bacterium of Arthrobacter aurescens NBRC 12136 strain, Arthrobacter citreus NBRC 12957 strain, Arthrobacter histidinolovorans JCM 2520 strain, Arthrobacter nicotianae JCM 1333 strain, Arthrobacter protophormiae JCM 1973 strain, Arthrobacter globiformis ATCC 4336 strain, Arthrobacter pasσens ATCC 13346 strain, Arthrobacter paraffineus ATCC 21003 strain. Bacillus badius ATCC 14574 strain. Bacillus sphaericus NBRC 3341 strain. Bacillus licheniformis NBRC 12197 strain. Bacillus cereus var. juroi ATCC 21182 strain, Microbacterium barker! JCM 1343 strain, Microbacterium testaceum JCM 1353 strain,
Microbacterium liquefaciens JCM 3879 strain, Microbacterium esteraromaticum NBRC 3751 strain, Raoultella planticola JCM 7251 strain, Raoultella terrigena JCM 1687 strain, Archromobacter denitrificans NBRC 12669 strain, Achromobacter lyticus ATCC 21456 strain, Brevibacteriumbutanicum ATCC 21196 strain, Brevibacterium ketoglutamicum ATCC 21004 strain, Brevibacterium fuscum JCM 1488 strain, Corynebacterium glutamicum ATCC 14020 strain, Curtobacterium albidum JCM 1344 strain, Curtobacterium luteum JCM 1480 strain, Curtbacterium citreum JCM 1345 strain, Devosia riboflavina NBRC 13584 strain, Exiguobacterium acetylicum JCM 1968 strain, Frateuria aurantia NBRC 3247 strain, Hafnia alvei NBRC 3731 strain, Mesorhizobium loti NBRC 13336 strain, Nocardioid.es simplex NBRC 12069 strain, Paenibacillus validus NBRC 13635 strain,
Paenibacillus polymyxa NBRC 3020 strain, Paenibacillus macerans JCM 2500 strain, Proteus vulgaris NBRC 3851 strain, Saccharopolyspora erythraea NBRC 13426 strain, Spirillospora albida NBRC 12248 strain, Streptomyces parvulus NBRC 13193 strain, Streptomyces virginiae NBRC 12827 strain, Streptomyces galilaeus ATCC 31133 strain, Streptomyces canus ATCC 12646 strain, Streptomyces fradiae ATCC 10745 strain, Streptomyces alboflavus NBRC 13196 strain, Streptomyces albovinaceus NBRC 12739 strain, Streptomyces cellulosae NBRC 3713 strain, Streptomyces fumanus NBRC 13042 strain or Streptomyces longispororuber NBRC 13488 strain;
4. A method for producing pyruvic acid from glycerol, which comprises : culturing in a culture medium containing glycerol, a bacterium which belongs to a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans , Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis , Pseudomonas taetrolens and Nocardia uniformis , and has an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol; 5. The method according to the above 4 , wherein the bacterium belonging to a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans , Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis , Pseudomonas taetrolens and Nocardia uniformis is a bacterium of Pseudomonas azotoformans JCM 2777 strain, Pseudomonas oryzihabitans JCM 2952 strain, Pseudomonas synxantha NBRC 3913 strain, Pseudomonas fragi JCM 20552 strain, Pseudomonas chlororaphis NBRC 3904 strain, Pseudomonas taetrolens NBRC 3460 strain or Nocardia uniformis NBRC 13702 strain; 6. A method for producing pyruvic acid from glycerol, which comprises : culturing a bacterium of Escherichia coli NBRC 12734 strain in culture medium containing glycerol, or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol;
7. A method for producing pyruvic acid from glycerol, which comprises culturing in a culture medium containing glycerol under an aerobic condition a bacterium belonging to the genus Citrobacter and having an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol;
8. The method according to the above 7 , wherein the bacterium belonging to genus Citrobacter is a bacterium of Citrobacter freundii ;
9. The method according to the above 8 , wherein the bacterium of Citrobacter freundii is a bacterium of Citrobacter freundii JCM 1657 strain;
10. The method according to any of the above 1 to 6 , wherein the bacterium is cultured under an aerobic condition;
11. The method according to any of the above 1 to 10, further comprises recovering pyruvic acid from a culture obtained by culturing; and
12. The method according to any of the above 1 to 11, wherein glycerol is derived from Bio Diesel waste.
MODE FOR CARRYING OUT THE INVENTION The pyruvic acid-producing bacteria used for the present invention are specifically exemplified by the bacterial genera, bacterial species and bacterial strains described in the above 1 to 9. Among them, bacteria of the genus Citrobacter are not specially limited as long as they can produce pyruvic acid by utilizing glycerol, and exemplified by a bacterium of Citrobacter amalonaticus , Citrobacter braakii, Citrobacter farmer!, Citrobacter freundii, Citrobacter koseri, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii and Citrobacter youngae. A preferable bacterium is a bacterium of the species Citrobacter freundii. Preferable examples of Citrobacter freundii include Citrobacter freundii JCM 1657 strain, but it is not limited thereto .
Among these pyruvic acid-producing bacteria, preferred bacteria are those being able to produce pyruvic acid from glycerol in higher yield. Examples of such bacteria include a Arthrobacter aurescens NBRC 12136 strain, Arthrobacter histidinolovorans JCM 2520 strain, Arthrobacter protophormiae JCM 1973 strain. Bacillus sphaericus NBRC 3341 strain, Arthrobacter globiformis ATCC 4336 strain, Arthrobacter pascens ATCC 13346 strain, Arthrobacter paraffineus ATCC 21003 strain. Bacillus badius ATCC 14574 strain. Bacillus licheniformis NBRC 12197 strain. Bacillus cereus var. juroi ATCC 21182 strain, Corynebacterium glutamicum ATCC 14020 strain, Curtobacterium albidum JCM 1344 strain, Curtobacterium luteum JCM 1480 strain, Curtbacterium citreum JCM 1345 strain, Devosia riboflavina NBRC 13584 strain, Mesorhizobium loti NBRC 13336 strain, Microbacterium barker! JCM 1343 strain, Microbacterium liquefaciens JCM 3879 strain, Microbacterium esteraromaticum NBRC 3751 strain, Paenibacillus validus NBRC 13635 strain, Paenibacillus polymyxa NBRC 3020 strain, Paenibacillus macerans JCM 2500 strain, Proteus vulgaris NBRC 3851 strain, Raoultella planticola JCM 7251 strain, Raoultella terrigena JCM 1687 strain, Pseudomonas azotoformans JCM 2777 strain, Pseudomonas synxantha NBRC 3913 strain, Pseudomonas fragi JCM 20552 strain, Pseudomonas chlororaphis NBRC 3904 strain, Pseudomonas taetrolens NBRC 3460 strain, Archromobacter denitrificans NBRC 12669 strain, Achromobaσter lyticus ATCC 21456 strain, Brevibacterium butanicum ATCC 21196 strain, Brevibacterium ketoglutamicum ATCC 21004 strain, Brevibacterium fuscum JCM 1488 strain, Escherichia coli NBRC 12734 strain, Citrobacter freundii JCM 1657 strain, Exiguobacterium acetylicum JCM 1968 strain, Frateuria aurantia NBRC 3247 strain, Hafnia alvei NBRC 3731 strain, Microbacterium testaceum JCM 1353 strain, Nocardia uniformis NBRC 13702 strain, Nocardioides simplex NBRC 12069 strain, Pseudomonas oryzihabitans JCM 2952 strain, Arthrobacter niσotianae JCM 1333 strain, Saccharopolyspora erythraea NBRC 13426 strain, Spirillospora albida NBRC 12248 strain, Streptomyces parvulus NBRC 13193 strain, Streptomyces virginiae NBRC 12827 strain, Streptomyces galilaeus ATCC 31133 strain, Streptomyces canus ATCC 12646 strain, Streptomyces fradiae ATCC 10745 strain, Streptomyces alboflavus NBRC 13196 strain, Streptomyces albovinaceus NBRC 12739 strain, Streptomyces cellulosae NBRC 3713 strain, Streptomyces fumanus NBRC 13042 strain, and Streptomyces longispororuber NBRC 13488 strain, , but they are not limited thereto. Archromobacter denitrificans NBRC 12669 strain, Achromobacter lyticus ATCC 21456 strain, Exiguobacterium acetylicum JCM 1968 strain, Frateuria aurantia NBRC 3247 strain, Microbacterium testaceum JCM 1353 strain, Nocardia uniformis NBRC 13702 strain, Nocardioides simplex NBRC 12069 strain, Pseudomonas oryzihabitans JCM 2952 strain, Arthrobacter nicotianae JCM 1333 strain, Arthrobacter globiformis ATCC 4336 strain, Streptomyces parvulus NBRC 13193 strain, Streptomyces virginiae NBRC 12827 strain, Streptomyces galilaeus ATCC 31133 strain, Streptomyces canus ATCC 12646 strain, Hafnia alvei NBRC 3731 strain, Streptomyces fradiae ATCC 10745 strain, Streptomyces alboflavus NBRC 13196 strain, Streptomyces albovinaceus NBRC 12739 strain, Streptomyces cellulosae NBRC 3713 strain, Streptomyces fumanus NBRC 13042 strain, Streptomyces longispororuber NBRC 13488 strain, Escherichia coli NBRC 12734 strain and Citrobaσter freundii JCM 1657 strain are preferred, and Arthrobacter niσotianae JCM 1333 strain, Arthrobacter globiformis ATCC 4336 strain, Streptomyces parvulus NBRC 13193 strain, Hafnia alvei NBRC 3731 strain, Streptomyces fradiae ATCC 10745 strain, Streptomyces alboflavus NBRC 13196 strain, Streptomyces albovinaceus NBRC 12739 strain, Streptomyces cellulosae NBRC 3713 strain, Streptomyces fumanus NBRC 13042 strain, Streptomyces longispororuber NBRC 13488 strain, Escherichia coli NBRC 12734 strain and Citrobacter freundii JCM 1657 strain are more preferred.
Herein, pyruvic acid being produced in "higher yield" denotes that pyruvic acid is accumulated from glycerol in a culture medium in a yield of about 0.1 g/L or more. In general, the higher the yield, the better it is. For example, pyruvic acid is accumulated in a culture medium in a yield of about 0.5 g/L or more, and preferably about 1 g/L or more .
Arthrobacter histidinolovorans JCM 2520 strain, Arthrobacter nicotianae JCM 1333 strain, Arthrobacter protophormiae JCM 1973 strain, Microbacterium barker! JCM 1343 strain, Microbacterium testaceum JCM 1353 strain, Microbacterium liquefaciens JCM 3879 strain, Pseudomonas azotoformans JCM 2777 strain, Pseudomonas oryzihabitans JCM 2952 strain, Brevibacterium fuscum JCM 1488 strain, Citrobacter freundii JCM 1657 strain, Paenibacillus macerans JCM 2500 strain, Raoultella planticola JCM 7251 strain, Raoultella terrigena JCM 1687 strain, Curtobacterium albidum JCM 1344 strain, Curtobacterium luteum JCM 1480 strain, Curtbacterium citreum JCM 1345 strain, Exiguobacterium acetylicum JCM 1968 strain, and Pseudomonas fragi JCM 20552 strain are available from Microbe Division, RIKEN BioResource Center, and Arthrobacter aurescens NBRC 12136 strain, Arthrobacter citreus NBRC 12957 strain. Bacillus sphaericus NBRC 3341 strain. Bacillus licheniformis NBRC 12197 strain, Pseudomonas synxantha NBRC 3913 strain, Pseudomonas chlororaphis NBRC 3904 strain, Pseudomonas taetrolens NBRC 3460 strain, Archromobacter denitrificans NBRC 12669 strain, Devosia riboflavina NBRC 13584 strain, Frateuria aurantia NBRC 3247 strain, Hafnia alvei NBRC 3731 strain, Escherichia coli NBRC 12734 strain, Microbacterium esteraromaticum NBRC 3751 strain, Mesorhizobium loti NBRC 13336 strain, Nocardia uniformis NBRC 13702 strain, Nocardioides simplex NBRC 12069 strain, Paenibacillus validus NBRC 13635 strain, Paenibacillus polymyxa NBRC 3020 strain, Proteus vulgaris NBRC 3851 strain, Saccharopolyspora erythraea NBRC 13426 strain, Spirillospora albida NBRC 12248 strain, Streptomyces parvulus NBRC 13193 strain, Streptomyces virginiae NBRC 12827 strain, Streptomyces alboflavus NBRC 13196 strain, Streptomyces albovinaceus NBRC 12739 strain, Streptomyces cellulosae NBRC 3713 strain, Streptomyces fumanus NBRC 13042 strain and Streptomyces longispororuber NBRC 13488 strain are available from Biological Resource Center, Department of Biotechnology, Incorporated Administrative Agency National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818 Japan. Arthrobacter globiformis ATCC 4336 strain, Arthrobacter pascens ATCC 13346 strain, Arthrobacter paraffineus ATCC 21003 strain. Bacillus badius ATCC 14574 strain. Bacillus cereus var. juroi ATCC 21182 strain, Brevibacterium butanicum ATCC 21196 strain, Brevibacterium ketoglutamicum ATCC 21004 strain, Corynebacterium glutamicum ATCC 14020 strain, Streptomyces galilaeus ATCC 31133 strain. Streptomyces canus ATCC 12646 strain, Streptomyces fradiae ATCC 10745 strain and Achromobacter lyticus ATCC 21456 strain are available from American Type Culture Collection, P.O.Box 1549 Manassas, VA 20108 USA.
The pyruvic acid-producing bacteria used for the present invention may not only be their wild-type strains, but also be any given naturally-occurring or artificial mutants, including those obtained by treatment with X-ray irradiation, ultraviolet-ray irradiation or a chemical mutagen such as
N-methyl-N' -nitro-N-nitrosoguanidine, or recombinants obtained through genetic engineering techniques such as cell fusion and gene recombination. A host of the recombinant can belong to any bacterial genera as long as it is a transformable microorganism. However, it is preferred that the host belongs to the same bacterial genus as a parent strain that a targeted gene is originated from. It is desirable to select a pyruvic acid-producing bacterium with improved conversion capacity from glycerol into pyruvic acid.
A culture medium containing glycerol may be a medium obtained by adding pure glycerol or a glycerol-containing mixture. Components other than glycerol in the glycerol-containing mixture or their amount are preferably those which do not have harmful effects on the pyruvic acid-producing bacteria used for the present invention. Origin of the glycerol-containing mixture is not particularly limited, but using Bio Diesel waste is preferable for effective use of resources . One of a method for producing Bio Diesel Fuel is to produce Fatty Acid Methyl Ester (FAME) by alcoholysis of triglyceride using an alkali catalyst. In this method, waste fluid containing glycerol is produced as a by-product (called Bio Diesel waste) . This waste fluid is usually contaminated with a catalyst, unconverted fatty acids (they differ depending on used oil) and the like. For example, the composition of a Bio Diesel waste used after-mentioned Examples is glycerol: 51%, methanol: 11%, potassium hydroxide: 8%, water: 4%, others such as glyceride: 26%. Bio Diesel waste fluids having arbitrary composition, such as glycerol: 65%, potassium/sodium salt : 4 to 5%, methanol: 1%, water: 28% described in S. Papanikolaou et al., Bioresource Technology (2002) 82: 43-49, and glycerol: 65%, sodium salt: 5% or less described in M. Gonzalez-Pajuelo et al. , J IndMicrobiol Biotechnol (2004) 31: 442-446, can also be used.
When a Bio Diesel waste is added to a medium in a method of the present invention, it is possible to produce pyruvic acid in the same or higher level of yield and conversion efficiency as in the case of adding pure glycerol.
The medium used for the method of the present invention may be any kind as long as it contains common components necessary for culture of bacteria, and is not limited to a particular medium. In the present invention, it is possible to obtain pyruvic acid even in a medium with plain composition containing carbon sources, nitrogen sources and inorganic salts . The medium used for the method of the present invention contains glycerol as a carbon source . The concentration of glycerol contained in the medium can be properly selected within a range which exerts no harmful effect on growth of bacteria, and production of pyruvic acid. However, it is usually from about 0.1 to 500 g/L, and preferably from about 1 to 300 g/L. When the Bio Diesel waste is used as a glycerol source, it is allowed to dilute the waste liquid, or to add glycerol thereto, until the amount of glycerol in the medium falls within the above range, depending on the concentration of glycerol contained in the waste.
The medium may contain substances other than glycerol as carbon sources , but the amount thereof should be limited to the extent which does not interfere production of pyruvic acid from glycerol. The carbon sources used for the present invention are exemplified by glucose, fructose, starch, lactose, arabinose, xylose, dextrin, molasses and malt extract, but are not limited thereto. The amount of other carbon sources is preferably about 10% by weight or less of glycerol, and more preferably about 1% by weight or less. It is most preferable that the medium contains glycerol as a single carbon source.
Examples of a nitrogen source include inorganic nitrogen compounds such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate, urea, and the like. It is also allowed to add to a culture medium organic nitrogen sources such as gluten flour, cottonseed flour, soybean flour, corn steep liquor, dried yeast, yeast extract, peptone, meat extract and casamino acid, or various vitamin groups, as needed, but a method of the present invention can produce pyruvic acid without these relatively expensive nutrients .
It is advantageous to use carbon sources and nitrogen sources in combination. Since the sources of low purity, containing a trace amount of growth factors and a great deal of inorganic nutrients,are also suitable for the use, there is no need to use them in a pure form.
Upon request, it is allowed to use inorganic salts, such as potassium phosphate monobasic, potassium phosphate dibasic, sodium chloride, magnesium sulfate , manganese sulfate, calcium carbonate, calcium chloride, sodium iodide, potassium iodide, and cobalt chloride. It is also allowed to add defoaming agents, such as liquid paraffin, higher alcohol, vegetable oil, mineral oil and silicon, as needed, particularly when the medium foams markedly.
The nitrogen sources, inorganic salts and other components are known by those skilled in the art.
In the present invention, it is allowed to culture pyruvic acid-producing bacteria (excluding bacteria of the genus Citrobacter) under an anaerobic condition, but preferably it is performed under an aerobic condition. Bacteria of the genus
Citrobacter are cultured under aerobic condition. The aerobic condition denotes culture in the presence of molecular oxygen. Ventilation, stirring and shaking can be performed for supplying oxygen. It is available to use any common devices for culture of microorganisms . In the case of culturing under an aerobic condition, the method of the present invention allows to culture bacteria, and to produce pyruvic acid, by a simple manner without using any devices necessary for bringing about an anaerobic condition.
Culture of bacteria under an anaerobic condition can be performed by introducing carbon dioxide or inert gas (nitrogen argon, etc.), or without ventilation.
It is preferred for mass production of pyruvic acid to be performed under a submerged culture condition. When bacteria are propagated in a large tank, it is preferred to inoculate bacteria in a vegetative period into a production tank, so as to avoid delay in propagation in a pyruvic acid producing process. That is, it is preferred that bacteria are first inoculated to a relatively small amount of medium, and cultured to produce seed bacteria in a vegetative period, and then the seed bacteria is transferred into the large tank in a sterile manner.
Stirring and ventilation of the culture solution can be performed in various manners. Stirring can be performed using a propeller or a mechanical stirring device similar to a propeller, rotation or shake of a fermenter, or a pumping device. Ventilation can be performed by allowing sterilized air to pass through in the culture solution. In doing so, the ventilation operation may provide stirring effect as well. In the case of culture in a submerged medium, a culture method such as batch culture, fed batch culture and continuous culture can be properly selected and used.
The culture conditions are discretional as long as they are suitable for culture of pyruvic acid-producing bacteria used for the present invention. For example, the culture temperature is from about 4 to 400C, preferably from about 20 to 370C. The pH of the medium is from about 5 to 9 , and preferably from about 6 to 8. When the pH of the medium declines along with production of pyruvic acid, it is adjusted to be fallen within the above range, by adding alkali such as an aqueous ammonia solution, calcium carbonate, sodium hydroxide and potassium hydroxide to the culture system, as needed.
The composition of the medium and other culture conditions are appropriately adjustable by those skilled in the art. It will also be considered to adjust the conditions , in order to further enhance yield of pyruvic acid.
The bacteria used for the method of the present invention may take a bacterial cell, processed bacterial cell or immobilized product thereof. Herein, the processed bacterial cell denotes a disrupted bacterial cell or an enzyme extracted from cultured substances (include a bacterial cell and culture supernatant) . Examples of the processed bacterial cell include that obtained by treating a cultured bacterial cell with an organic acid (such as acetone and ethanol), freeze dry treatment or alkali treatment, that obtained by physically or enzymatically disrupting a bacterial cell, or a crude enzyme separated or extracted therefrom. Specifically, cultured bacteria are subjected to a centrifugal treatment, and the cells to be collected are disrupted by a physical milling method such as an ultrasonic, Dyno-mill and French press treatment, or a chemical disrupting method using a surfactant or a lytic enzyme such as lyzozyme. The resultant solution is subjected to centrifuge or membrane filtration to remove insoluble materials, and the resultant cell-free extract is subjected to a separation/purification method, such as cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and metal chelate chromatography, to fractionate and purify the enzyme. Examples of a carrier used for the chromatography include insoluble polymer carriers such as cellulose, dextrin and agarose introduced with a carboxymethyl (CM) group, diethylaminoethyl (DEAE) group, phenyl group or butyl group. It is also allowed to use a commercially available carrier-packed column. Disruption of the bacterial cell and extraction of the enzyme can be performed by a known method by those skilled in the art, as well as the above method.
Examples of contacting the bacterial cell, processed bacteria cell or immobilized thereof to glycerol are given below.
The method for producing pyruvic acid from glycerol using a bacterial cell or processed bacterial cell is exemplified by a method that the bacterial cell is suspended and reacted in a 18
20
glycerol-containing substrate solution. The bacterial cell can be prepared by culturing pyruvic acid-producing bacteria, followed by centrifuge thereof . It is preferred that the concentration of glycerol in the substrate solution is approx. from 0.01 to 50% by weight. The reaction temperature is usually from about 4 to 40° C, and preferably from about 20 to 37° C. The pH of the reaction solution is usually from about 5 to 9 , and preferably from 6 to 8. When the pH of the medium declines along with production of pyruvic acid, it is adjusted to be fallen within the above range, by adding alkali such as an aqueous ammonia solution, calcium carbonate, sodium hydroxide and potassium hydroxide to the culture system, as needed.
The method for producing pyruvic acid from glycerol using an immobilized bacterial cell or processed bacterial cell is exemplified by a method that the immobilized bacterial cell or processed bacterial cell is filled in a column, and a glycerol-containing substrate solution is allowed to pass it through. The bacterial cell or processed bacterial cell is obtained by culturing the pyruvic acid-producing bacteria, followed by centrifuge thereof . The method for immobilizing the bacterial cell is exemplified by a comprehensive immobilization means using a gel, and immobilization means by supporting an ion exchange material. Examples of the gel to be used include carrageenan, agar, mannan, PVA and polyacrylamide gels. The proper particle size of the gel is from about 1 to 10 mm in diameter, although the size varies depending on a kind of gel. Examples of the ion exchange material include a cellulose-based material. styrenedivinylbenzene-based material and phenolformalin-based ion exchange material. It is preferred that the concentration of glycerol in the substrate solution is from about 0.01 to 50% by weight. It is also allowed to add a SH compound such as mercaptoethanol, cysteine and glutathione, reducing agent such as sulfite, and enzyme activator such as a magnesium ion and manganese ion. The velocity of the solution passing through varies depending on the column size and amount of the immobilized substance. It is proper that the space velocity (ml/ml resin'hr) is from 0.05 to 10, as an index of velocity for treating a solution.
Separation and purification of pyruvic acid are performed in accordance with a conventional known method. For example, filtration or centrifugation is performed to a culture solution after completion of culture, to obtain a supernatant. From the supernatant, for example, pyruvic acid can be separated such as by concentrated crystallization, but separation and purification of pyruvic acid are not limited thereto. Specifically, pyruvic acid can be separated by such methods as solvent extraction from the supernatant, or separated and purified by methods such as ion exchange chromatography in which pyruvic acid is eluted and separated after absorbed in an ion exchange resin, isolation by forming metal salts such as a calcium ion, fractional precipitation by an insolubilizing treatment, fractional crystallization by crystallization, membrane separation by a reverse osmosis membrane, and concentrated crystallization method. Specifically, for example, pyruvic acid can be separated and purified according to a method described in Japanese Unexamined Patent Publication 5618
22
( Kokai ) No . 6 - 91 .
The present invention is further illustrated by the following examples. It is to be understood that the present invention is not limited to the examples , and various variations can be made within a range of the present invention.
EXAMPLES Example 1 Achromobacter denitrifleans NBRC 12669 strain was spread on an agar medium A, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic , 6 g of sodium phosphate dibasic , 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and 1 L of distilled water (final pH 7.4)), and allowed to stand at 30° C for 4 days . The bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium B, being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no calcium chloride, yeast extract and agar) , and subjected to shaking culture (pre-culture) at 30° C at 200 rpm for 24 hours . The bacterial strain culture solution of 30 μL grown above was transferred to 3 mL of a medium C, being a medium for test tube culture (composition: the same composition as the above medium B for test tube culture, except for containing 19.6 g of a glycerol fraction (glycerol: 51%, methanol: 11%, potassium hydroxide: 8%, water: 4%, and others including glyceride: 26%) which was by-produced upon production of the Bio Diesel Fuel, instead of 10 g of glycerol) , and subjected to shaking culture (main culture) at 3O0C at 200 rpm. Four (4) days after initiation of the reaction, 3.9 g of glycerol was consumed per litter, and 0.3 g of pyruvic acid was accumulated.
Example 2
Achromobacter denitrifleans NBRC 12669 strain was spread on an agar medium A, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic , 6 g of sodium phosphate dibasic , 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate , 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4)), and allowed to stand at 30° C for 4 days. The bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium D, being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no agar) , and subjected to shaking culture (pre-culture) at 30° C at 200 rpm for 24 hours. The bacterial strain culture solution of 30 μL grown above was transferred to 3 mL of a medium E, being a medium for test tube culture, (composition: the same composition as the above medium D for test tube culture, except for containing 19.6 g of a glycerol fraction (glycerol: 51%, methanol: 11%, potassium: hydroxide: 8%, water: 4%, and others including glyceride: 26%) which was by-produced upon production of the Bio Diesel Fuel, instead of 10 g of glycerol) , and subjected to shaking culture (main culture) at 30° C at 200 rpm. Four (4) days after initiation of the reaction, 9.4 g of glycerol was consumed per litter, and 0.7 g of pyruvic acid was accumulated.
Example 3
Pseudomonas azotoformans JCM 2777 strain was reacted in the same manner as in Example 1. As a result , 3.3 g of glycerol per one liter was consumed and 0.4 g of pyruvic acid was accumulated.
Example 4
Pseudomonas synxantha NBRC 3913 strain was reacted in the same manner as in Example 1. As a result, 4.5 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 5
Raoultella terrigena JCM 1687 strain was reacted in the same manner as in Example 1. As a result, 1.9 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 6
Exiguobacterium aσetylicum JCM 1968 strain was spread on an agar medium A, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4)), and allowed to stand at 3O0C for 4 days. The bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium D, being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no agar) , and subjected to shaking culture (pre-culture) at 30° C at 200 rpm for 24 hours. The bacterial strain culture solution of 30 μL grown above was transferred to 3 mL of a medium D, being a medium for test tube culture, and subjected to shaking culture (main culture) at 30° C at 200 rpm. Six (6) days after initiation of the reaction, 1.4 g of glycerol was consumed per litter, and 0.5 gof pyruvic acid was accumulated.
Example 7
Devosia riboflavina NBRC 13584 strain was reacted in the same manner as in Example 6. As a result , 0.9 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
Example 8
Mesorhizobium loti NBRC 13336 strain was reacted in the same manner as in Example 6. As a result , 0.4 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 9
Arthrobacter nicotianae JCM 1333 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 7.1 g of glycerol per one liter was consumed and 1.7 g of pyruvic acid was accumulated.
Example 10
Arthrobacter citreus NBRC 12957 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 2.4 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
Example 11
Arthrobacter aurescens NBRC 12136 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days. As a result, 2.5 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 12
Arthrobacter histidinolovorans JCM 2520 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 3.8 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 13
Arthrobacter protophorminae JCM 1973 strain was reacted in the same manner as in Example 6 , except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 3.7 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 14
Streptomyces parvulus NBRC 13193 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 9.4 g of glycerol per one liter was consumed and 2.2 g of pyruvic acid was accumulated.
Example 15
Frateuria aurantia NBRC 3247 strain was spread on an agar medium A, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium chloride dihydrate, 100 mg of yeast extract, 10 g of glycerol, 20 g of agar and 1 L of distilled water (final pH 7.4)), and allowed to stand at 30° C for 4 days. The bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium B, being a medium for test tube culture (composition: the same composition as the agar medium A, except for containing no calcium chloride, yeast extract and agar) , and subjected to shaking culture (pre-culture) at 30° C at 200 rpm for 24 hours. The bacterial strain culture solution of 30 μL grown above was transferred to 3 mL of a medium B, being a medium for test tube culture, and subjected to shaking culture (main culture) at 30° C at 200 rpm. Four (4) days after initiation of the reaction, 0.7 g of glycerol was consumed per litter, and 0.1 g of pyruvic acid was accumulated.
Example 16
Frateuria aurantia NBRC 3247 strain was reacted in the same manner as in Example 6. As a result , 2.4 g of glycerol per one liter was consumed and 0.5 g of pyruvic acid was accumulated.
Example 17
Microbacterium testaceum JCM 1353 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 2 days , and the main culture period from 6 days to 4 days . As a result , 3.1 g of glycerol per one liter was consumed and 0.6 g of pyruvic acid was accumulated.
Example 18
Microbacterium barkeri JCM 1343 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 2 days , and the main culture period from 6 days to 4 days . As a result , 4.2 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 19
Nocardioides simplex NBRC 12069 strain was reacted in the same manner as in Example 6. As a result, 2.2 g of glycerol per one liter was consumed and 0.5 g of pyruvic acid was accumulated.
Example 20
Paenibacillus validus NBRC 13635 strain was reacted in the same manner as in Example 6. As a result , 1.1 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 21
Bacillus sphaericus NBRC 3341 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 1.9 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
Example 22
Bacillus badius ATCC 14574 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result, 2.7 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
Example 23
Nocardia uniformis NBRC 13702 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 3.7 g of glycerol per one liter was consumed and 0.6 g of pyruvic acid was accumulated.
Example 24
Proteus vulgaris NBRC 3851 strain was reacted in the same manner as in Example 6. As a result , 5.3 g of glycerol per one liter was consumed and 0.4 g of pyruvic acid was accumulated.
Example 25
Curtobacterium albidum JCM 1344 strain was reacted in the same manner as in Example 6. As a result, 1.4 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated. Example 26
Cortnebacterium glutamicum ATCC 14020 strain was reacted in the same manner as in Example 6. As a result, 1.9 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 27
Pseudomonas oryzihabitans JCM 2952 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from
6 days to 4 days . As a result , 9.5 g of glycerol per one liter was consumed and 0.7 g of pyruvic acid was accumulated.
Example 28 Pseudomonas azotoformans JCM 2777 strain was reacted in the same manner as in Example 15. As a result, 5.6 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 29 Raoultella planticola JCM 7251 strain was reacted in the same manner as in Example 15. As a result, 1.3 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 30 Paenibacillus polymyxa NBRC 3020 strain was reacted in the same manner as in Example 6. As a result , 0.4 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated. Example 31
Streptomyces virginiae NBRC 12827 strain was reacted in the same manner as in Example 6. As a result, 4.0 g of glycerol per one liter was consumed and 0.8 g of pyruvic acid was accumulated.
Example 32
Streptomyces galilaeus ATCC 31133 strain was reacted in the same manner as in Example 6. As a result , 4.3 g of glycerol per one liter was consumed and 0.7 g of pyruvic acid was accumulated.
Example 33
Streptomyces canus ATCC 12646 strain was reacted in the same manner as in Example 6. As a result , 5.7 g of glycerol per one liter was consumed and 0.8 g of pyruvic acid was accumulated.
Example 34
Streptomyces fradiae ATCC 10745 strain was reacted in the same manner as in Example 6. As a result, 4.0 g of glycerol per one liter was consumed and 2.4 g of pyruvic acid was accumulated.
Example 35
Spirillospora albida NBRC 12248 strain was reacted in the same manner as in Example 6. As a result, 0.3 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 36
Escherichia coli NBRC 12734 strain was reacted in the same manner as in Example 15. As a result, 8.3 g of glycerol per one liter was consumed and 2.0 g of pyruvic acid was accumulated.
Example 37
Escherichia coli NBRC 12734 strain was reacted in the same manner as in Example 1. As a result, 8.9 g of glycerol per one liter was consumed and 2.9 g of pyruvic acid was accumulated.
Example 38
Escherichia coli NBRC 12734 strain was reacted in the same manner as in Example 6. As a result, 8.1 g of glycerol per one liter was consumed and 1.9 g of pyruvic acid was accumulated.
Example 39
Escherichia coli NBRC 12734 strain was reacted in the same manner as in Example 2. As a result, 10.2 g of glycerol per one liter was consumed and 3.3 g of pyruvic acid was accumulated.
Example 40
Hafnia alvei NBRC 3731 strain was reacted in the same manner as in Example 1. As a result, 8.5 g of glycerol per one liter was consumed and 2.4 g of pyruvic acid was accumulated.
Example 41
Hafnia alvei NBRC 3731 strain was reacted in the same manner as in Example 6. As a result, 8.1 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 42 Curtbacterium luteum JCM 1480 strain was reacted in the same manner as in Example 6. As a result , 2.1 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 43
Pseudomonas fragi JCM 20552 strain was reacted in the same manner as in Example 1. As a result , 4.4 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 44
Pseudomonas fragi JCM 20552 strain was reacted in the same manner as in Example 2. As a result , 4.5 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 45
Saccharopolyspora erythraea NBRC 13426 strain was reacted in the same manner as in Example 6. As a result , 3.2 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 46
Achromobacter lyticus ATCC 21456 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 2.2 g of glycerol per one liter was consumed and 0.7 g of pyruvic acid was accumulated.
Example 47
Bacillus licheniformis NBRC 12197 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 1.8 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
Example 48
Microbacterium liquefaciens JCM 3879 strain was reacted in the same manner as in Example 6. As a result , 2.3 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 49
Brevibacterium butanicum ATCC 21196 strain was reacted in the same manner as in Example 2. As a result, 2.8 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 50
Brevibacterium ketoglutamicum ATCC 21004 strain was reacted in the same manner as in Example 6. As a result , 1.4 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
Example 51
Arthrobacter globiformis ATCC 4336 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days. As a result, 5.3 g of glycerol per one liter was consumed and 1.3 g of pyruvic acid was accumulated.
Example 52 Arthrobacter paraffineus ATCC 21003 strain was reacted in the same manner as in Example 6. As a result, 10 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 53
Arthrobacter pascens ATCC 13346 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from
6 days to 4 days . As a result , 2.9 g of glycerol per one liter was consumed and 0.1 g of pyruvic acid was accumulated.
Example 54
Bacillus cereus var. juroi ATCC 21182 strain was reacted in the same manner as in Example 6 , except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from
6 days to 4 days. As a result, 0.7 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 55 Pseudomonas taetrolens NBRC 3460 strain was reacted in the same manner as in Example 15, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from 6 days to 4 days . As a result , 3.2 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
Example 56
Pseudomonas chlororaphis NBRC 3904 strain was reacted in the same manner as in Example 1. As a result , 5.4 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 57
Streptomyces alboflavus NBRC 13196 strain was reacted in the same manner as in Example 6. As a result, 10.4 g of glycerol per one liter was consumed and 2.0 g of pyruvic acid was accumulated.
Example 58
Streptomyces albovinaceus NBRC 12739 strain was reacted in the same manner as in Example 6. As a result, 10.3 g of glycerol per one liter was consumed and 3.0 g of pyruvic acid was accumulated.
Example 59
Streptomyces fumanus NBRC 13042 strain was reacted in the same manner as in Example 6. As a result, 8.7 g of glycerol per one liter was consumed and 3.2 g of pyruvic acid was accumulated.
Example 60
Streptomyces cellulosae NBRC 3713 strain was reacted in the same manner as in Example 6, except for changing the pre-culture period from 6 days to 24 hours, and the main culture period from
6 days to 4 days . As a result , 7.5 g of glycerol per one liter was consumed and 3.3 g of pyruvic acid was accumulated.
Example 61
Streptomyces longispororuber NBRC 13488 strain was reacted in the same manner as in Example 2 , except for changing the pre-culture period from 24 hours to 6 days, and the main culture period from 4 days to 6 days. As a result, 11 g of glycerol per one liter was consumed and 2.9 g of pyruvic acid was accumulated.
Example 62 Paenibacillus macerans JCM 2500 strain was reacted in the same manner as in Example 6 , except for changing the main culture period from 6 days to 13 days. As a result, 1.7 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 63
Microbacterium esteraromaticum NBRC 3751 strain was reacted in the same manner as in Example 6 , except for changing the main culture period from 6 days to 8 days . As a result , 1.3 g of glycerol per one liter was consumed and 0.6 g of pyruvic acid was accumulated.
Example 64
Curtobacterium citreum JCM 1345 strain was reacted in the same manner as in Example 6 , except for changing the main culture period from 6 days to 13 days. As a result, 1.2 g of glycerol per one liter was consumed and 0.2 g of pyruvic acid was accumulated.
Example 65
Brevibacterium fuscum JCM 1488 strain was reacted in the same manner as in Example 6 , except for changing the main culture period from 6 days to 7 days. As a result, 0.9 g of glycerol per one liter was consumed and 0.3 g of pyruvic acid was accumulated.
Example 66 Citrobacter freundii JCM 1657 strain was spread on an agar medium F, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g or sodium chloride, 1 g of ammonium chloride, 250 mg of magnesium sulfate heptahydrate , 20 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4) ), and allowed to stand at 30° C for 4 days . The bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium G, being a medium for test tube culture, (composition: the same composition as the agar medium F, except for containing no agar and containing 10 g or glycerol instead of 20 g) , and subjected to shaking culture at 30° C at 200 rpm for 24 hours . The bacterial strain culture solution of 30 μL grown above was transferred to 3 mL of the medium G, being a medium for test tube culture, and subjected to shaking culture at 3O0C at 200 rpm for 24 hours. Four (4) days after initiation of the reaction, 3.1 g/L of pyruvic acid was accumulated.
Example 67
Citrobacter freundii JCM 1657 strain was spread on an agar medium F, being a medium for plate culture, (composition: 3 g of potassium phosphate monobasic, 6 g of sodium phosphate dibasic, 0.5 g of sodium chloride, 1 g of ammonium chloride, 250 mg of magnesium sulfate heptahydrate, 20 g of glycerol, 20 g of agar and IL of distilled water (final pH 7.4) ) , and allowed to stand at 30° C for 4 days. The bacterial strain grown on the above plate was inoculated with a platinum loop in 3 mL of a medium G, being a medium for test tube culture (composition: the same composition as the agar medium F, except for containing no agar and containing 10 g or glycerol instead of 20 g) , and subjected to shaking culture at 30° C at 200 rpm for 24 hours . The bacterial strain culture solution of 30 μL grown above was transferred to 3 mL of the medium H, being a medium for test tube culture (composition: the same composition as the above medium G for test tube culture, except for containing 19.6 g of a glycerol fraction (glycerol: 51%, methanol: 11%, potassium: hydroxide: 8%, water: 4%, and others including glyceride: 26%) which was by-produced upon production of the Bio Diesel Fuel, instead of 10 g of glycerol) , and subjected to shaking culture at 30° C at 200 rpm for 24 hours. Four (4) days after initiation of the reaction, 4.2 g/L of pyruvic acid was accumulated.
INDUSTRIAL APPLICABILITY
According to the present invention, pyruvic acid is produced from glycerol (Bio Diesel waste) using a microorganism by a simple manner. Pyruvic acid has high reactivity, so it is used as an important intermediate in synthesis of medicine, pesticide, and the like. Thus, the present invention serves for producing useful substances from a waste material.

Claims

1. A method for producing pyruvic acid from glycerol, which comprises : culturing in a culture medium containing glycerol, a bacterium which belongs to a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobacter, Brevibacterium, Corynebacterium, Curtbacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides , Paenibacillus , Proteus,
Saccharopolyspora, Spirillospora and Streptomyces , and has an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol.
2. The method according to claim 1 , wherein the bacterium belonging to a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobacter, Brevibacterium, Corynebacterium, Curtbacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides, Paenibacillus, Proteus, Saccharopolyspora, Spirillospora and Streptomyces is a bacterium of Arthrobacter aurescens , Arthrobacter citreus , Arthrobacter histidinolovorans , Arthrobacter nicotianae, Arthrobacter protophormiae, Arthrobacter globiformis , Arthrobacter pascens , Arthrobacter paraffineus , Bacillus badius , Bacillus sphaericus , Bacillus licheniformis , Bacillus cereus var. juroi, Microbacterium barker! , Microbacterium testaceum, Microbacterium liquefaciens , Microbacterium esteraromaticum, Raoultella planticola, Raoultella terrigena, Archromobacter denitrifleans , Achromobacter lyticus, Brevibacterium butanicum, Brevibacterium ketoglutamicum, Brevibacterium fuscum, Corynebacterium glutamicum, Curtobacterium albidum, Curtobacterium luteum, Curtbacterium citreum, Devosia riboflavina, Exiguobacterium acetylicum, Frateuria aurantia, Hafnia alvei, Mesorhizobium loti, Nocardioides simplex, Paenibacillus validus, Paenibacillus polymyxa, Paenibacillus macerans, Proteus vulgaris, Saccharopolyspora erythraea, Spirillospora albida, Streptomyces parvulus , Streptomyces virginiae , Streptomyces galilaeus , Streptomyces canus, Streptomyces fradiae, Streptomyces alboflavus, Streptomyces albovinaceus , Streptomyces cellulosae, Streptomyces fumanus , or Streptomyces longispororuber .
3. The method according to claim 2 , wherein the bacterium belonging to a genus selected from the bacterial genus group consisting of Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobacter, Brevibacterium, Corynebacterium, Curtbacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides, Paenibacillus, Proteus, Saccharopolyspora, Spirillospora and Streptomyces is a bacterium of Arthrobacter aurescens NBRC 12136 strain, Arthrobacter citreus NBRC 12957 strain, Arthrobacter histidinolovorans JCM 2520 strain, Arthrobacter nicotianae JCM 1333 strain, Arthrobacter protophormiae JCM 1973 strain, Arthrobacter globiformis ATCC 4336 strain, Arthrobacter pascens ATCC 13346 strain, Arthrobacter paraffineus ATCC 21003 strain. Bacillus badius ATCC 14574 strain. Bacillus sphaeriσus NBRC 3341 strain. Bacillus licheniformis NBRC 12197 strain. Bacillus cereus var. juroi ATCC 21182 strain, Microbacterium barker! JCM 1343 strain, Microbacterium testaceum JCM 1353 strain, Microbacterium liquefaciens JCM 3879 strain, Microbacterium esteraromaticum NBRC 3751 strain, Raoultella planticola JCM 7251 strain, Raoultella terrigena JCM 1687 strain, Archromobacter denitrificans NBRC 12669 strain, Achromobacter lyticus ATCC 21456 strain, Brevibacterium butanicum ATCC 21196 strain, Brevibacterium ketoglutamicum ATCC 21004 strain, Brevibacterium fuscum JCM 1488 strain, Corynebacterium glutamicum ATCC 14020 strain,
Curtobacterium albidum JCM 1344 strain, Curtobacterium luteum JCM 1480 strain, Curtbacterium citreum JCM 1345 strain, Devosia riboflavina NBRC 13584 strain, Exiguobacterium acetylicum JCM 1968 strain, Frateuria aurantia NBRC 3247 strain, Hafnia alvei NBRC 3731 strain, Mesorhizobium loti NBRC 13336 strain, Nocardioides simplex NBRC 12069 strain, Paenibacillus validus NBRC 13635 strain, Paenibacillus polymyxa NBRC 3020 strain, Paenibacillus macerans JCM 2500 strain, Proteus vulgaris NBRC 3851 strain, Saccharopolyspora erythraea NBRC 13426 strain, Spirillospora albida NBRC 12248 strain, Streptomyces parvulus NBRC 13193 strain, Streptomyces virginiae NBRC 12827 strain, Streptomyces galilaeus ATCC 31133 strain, Streptomyces canus ATCC 12646 strain, Streptomyces fradiae ATCC 10745 strain, Streptomyces alboflavus NBRC 13196 strain, Streptomyces albovinaceus NBRC 12739 strain, Streptomyces cellulosae NBRC 3713 strain, Streptomyces fumanus NBRC 13042 strain or Streptomyces longispororuber NBRC 13488 strain.
4. A method for producing pyruvic acid from glycerol , which comprises : culturing in a culture medium containing glycerol, a bacterium which belongs to a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans , Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis , Pseudomonas taetrolens and Nocardia uniformis , and has an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol.
5. The method according to claim 4 , wherein the bacterium belonging to a species selected from the bacterial species group consisting of Pseudomonas azotoformans , Pseudomonas oryzihabitans, Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis , Pseudomonas taetrolens and Nocardia uniformis is a bacterium of Pseudomonas azotoformans JCM 2777 strain, Pseudomonas oryzihabitans JCM 2952 strain, Pseudomonas synxantha NBRC 3913 strain, Pseudomonas fragi JCM 20552 strain, Pseudomonas chlororaphis NBRC 3904 strain, Pseudomonas taetrolens NBRC 3460 strain or Nocardia uniformis NBRC 13702 strain.
6. A method for producing pyruvic acid from glycerol , which comprises: culturing a bacterium of Escherichia coli NBRC 12734 strain in culture medium containing glycerol, or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol.
7. A method for producing pyruvic acid from glycerol , which comprises culturing in a culture medium containing glycerol under an aerobic condition a bacterium belonging to the genus Citrobacter and having an ability to produce pyruvic acid from glycerol; or contacting cells of the bacterium, processed cells of the bacterium or immobilized product thereof with glycerol.
8. The method according to claim 7 , wherein the bacterium belonging to genus Citrobacter is a bacterium of Citrobacter freundii .
9. The method according to claim 8, wherein the bacterium of Citrobacter freundii is a bacterium of Citrobacter freundii JCM 1657 strain.
10. The method according to any of claims 1 to 6 , wherein the bacterium is cultured under an aerobic condition.
11. The method according to any of claims 1 to 10, further comprises recovering pyruvic acid from a culture obtained by culturing .
12. The method according to any of claims 1 to 11, wherein glycerol is derived from Bio Diesel waste.
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WO2011099023A1 (en) * 2010-02-09 2011-08-18 Patel, Babubhai C. Composition and method of preparation of potash mobilizing bacterial based product that mobilizes potash and makes it available to plant
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CN110066757A (en) * 2019-04-18 2019-07-30 江南大学 One plant of pseudomonad for producing feruloyl esterase and its application
CN110066757B (en) * 2019-04-18 2020-08-04 江南大学 Pseudomonas capable of producing feruloyl esterase and application thereof
CN110951656A (en) * 2020-01-02 2020-04-03 山东农业大学 Acetobacter and application thereof in preventing and treating plant parasitic nematode disease

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