JP2009142255A - Method for producing pyruvic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce a useful substance from glycerol, especially a biodiesel waste liquid. <P>SOLUTION: Provided is a method for producing the pyruvic acid from glycerol, characterized by culturing a specific pyruvic acid-producing bacterium in a glycerol-containing culture medium, or contacting the cells of the bacterium, a treated cell product, or their immobilized products with glycerol. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing pyruvic acid from glycerol using a microorganism.

  Bio Diesel Fuel (BDF) is a carbon-neutral alternative to diesel oil, and has recently attracted attention as a fuel that contributes to solving environmental problems such as depletion of energy resources, global warming, and air pollution.

  Biodiesel fuel is produced from vegetable oil, animal oil, waste oil, and the like, and at that time, a waste liquid containing glycerol is produced as a by-product. This waste liquid has no particularly effective utilization method and is currently discarded.

  As a method for effectively using biodiesel waste liquid, Patent Document 1 describes a method for producing hydrogen and ethanol from glycerol in waste liquid using bacteria belonging to the genus Enterobactor.

  Regarding the production of organic acids, Patent Document 2 describes a method for producing organic acids using aerobic bacteria (particularly coryneform bacteria) using glucose as a carbon source. Patent Document 3 describes a bacterial strain (Mannheimia sp. 55E) that produces various organic acids from glucose.

  However, Arthrobacter genus, Bacillus genus, Microbacterium genus, Raoultella genus, Achromobacter genus, Brevibacterium genus, Corynebacterium genus, Curtobacterium genus, Devosia genus, Exiguobacterium genus, Frateuria genus, Hafnia genus, Mesorhizobium genus, Nocardioides genus, Paenibacillus Bacteria belonging to the bacterial genus selected from the genus (Paenibacillus), the genus Proteus, the genus Saccharopolyspora, the Spirillospora and the Streptomyces genus, and Citrobacter Bacteria belonging to the genus, or Pseudomonas azotoformans, Pseudomonas oryzihabitans, Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chloros phis ), Pseudomonas taetrolens and Nocardia uniformis, a bacterium belonging to a bacterial species selected from the bacterial species group, and Escherichia coli NBRC 12734 It has never been known that it can be generated.

Citrobacter freundii is known to produce 1,3-propanediol and the like from glycerol, but production of pyruvic acid is not suggested (Patent Document 4, Non-Patent Documents 1, 2 and 3). Citrobacter frundi is a gram-negative facultative anaerobic gonococci present in the intestines of animals. In particular, Non-Patent Document 3 describes that 1,3-propanediol, ethanol, acetic acid, and the like are produced when Citrobacter frundi is cultured with glycerol under anaerobic conditions. Production is not suggested.
JP 2006-180782 A Japanese Patent Laid-Open No. 2003-235592 JP-T-2004-501634 European Patent Application Publication 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

  In order to effectively use resources, it is required to produce useful substances from biodiesel waste liquid.

  As a result of intensive studies in view of the above problems, the present inventor has found that the genus Arthrobacter, the genus Bacillus, the genus Microbacterium, the genus Raoultella, the genus Achromobacter, the genus Archromobacter, Brevibacterium, Corynebacterium, Curtobacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium ), Nocardioides genus, Paenibacillus genus, Proteus genus, Saccharopolyspora genus, Spirillospora genus and Streptomyces genus Belonging to a bacterial genus selected from Bacteria and bacteria belonging to the genus Citrobacter, or Pseudomonas azotoformans, Pseudomonas oryzihabitans, Pseudomonas synxantha, Pseudomonas framon (Pseudomonas fraud)・ Bacteria belonging to bacterial species selected from the bacterial group consisting of Pseudomonas chlororaphis, Pseudomonas taetrolens and Nocardia uniformis, and Escherichia coli NBRC 12734 (Hereinafter, pyruvate-producing bacteria used in the present invention, or simply referred to as pyruvate-producing bacteria) is cultured in a glycerol-containing medium (aerobic conditions for bacteria belonging to the genus Citrobacter) In particular, pyruvic acid can be easily produced from glycerol by culturing under particularly aerobic conditions or by contacting glycerol with the cells, treated cells, or immobilized products thereof. Thus, the present invention has been completed.

That is, the present invention can provide:
[1] Arthrobacter genus, Bacillus genus, Microbacterium genus, Raoultella genus, Achromobacter genus, Brevibacterium genus, Corynebacterium genus , Genus Curtobacterium, genus Devosia, genus Exiguobacterium, genus Frateuria, genus Hafnia, genus Mesorhizobium, genus Nocardioides, It belongs to the genus of bacteria selected from the genus Paenibacillus, Proteus, Saccharopolyspora, Spirillospora and Streptomyces, glycerol to pyruvin Produce acid A method for producing pyruvic acid from glycerol, which comprises culturing a bacterium having an ability in a glycerol-containing medium, or bringing glycerol into contact with the cells, a treated product of the cells, or an immobilized product thereof;
[2] Arthrobacter genus, Bacillus genus, Microbacteria genus, Raoulutera genus, Achromobacter genus, Brevibacterium genus, Corynebacterium genus, Kurtobacteria genus, Devosia genus, Igujiobacterium genus, Fratheria genus, Hafnia genus, Bacteria belonging to the bacterial genus selected from the bacterial genus group consisting of the genus Mesozobium, Nocardioides, Paenibacillus, Proteus, Saccharopolyspora, Spirirospora and Streptomyces, Arthrobacter aurescens, Arthrobacter citreus, Arthrobacter histidinolovorans, Arthrobacter nicotianae, 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 ter rigena), Achromobacter denitrificans, Achromobacter lyticus, Brevibacterium butanicum, Brevibacterium ketoglutamicum, Brevibacterium fuscum ), Corynebacterium glutamicum, Curtobacterium albidum, Curtobacterium luteum, Curtobacterium citreum, Devosia riboflavina, Exiguobacterium acetylicum, Frateuria aurantia, Hafnia alvei, Mesolyso Bis roti (Mesorhizobium loti), Nocardioides simplex, Paenibacillus validus, Paenibacillus polymyxa, Paenibacillus proteus vulners, Paenibacillus prosus , Saccharopolyspora erythraea, Spirillospora albida, Streptomyces parvulus, Streptomyces virginiae, Streptomyces gals rep , Streptomyces canus, Streptomyces fradiae, Streptomyces alboflavus ), Streptomyces albovinaceus, Streptomyces cellulosae, Streptomyces fumanus or Streptomyces longispororuber [1] Method;
[3] Arthrobacter genus, Bacillus genus, Microbacteria genus, Raoulutera genus, Achromobacter genus, Brevibacterium genus, Corynebacterium genus, Kurtobacteria genus, Debosia genus, Hydrangea genus, Fratheria genus, Hafnia genus, Bacteria belonging to the bacterial genus selected from the bacterial genus group consisting of Mesozobium, Nocardioides, Paenibatilus, Proteus, Saccharopolispora, Spirirospora and Streptomyces are Arthrobacter aurescens NBRC 12136, Arthrobacter・ Siteureus NBRC 12957, Arthrobacter histidinorovorans JCM 2520, Arthrobacter Nicotianae JCM 1333, Arthrobacter protoformiae JCM 1973, Arthrobacter globiformis ATCC 4336 Slovacta Passense ATCC 13346, Arthrobacter paraffineus ATCC 21003, Bacillus Badius ATCC 14574, Bacillus sphaericus NBRC 3341, Bacillus lichenifomis NBRC 12197, Bacillus cereus bar juroy ATCC 21182, Micro Bacterium Barkeri JCM 1343, Microbacterium Testaceum JCM 1353, Microbacterium Liquefaciens JCM 3879, Microbacterium Estelaromaticum NBRC 3751, Lao Ulterra Planticocola JCM 7251, Lao Ulterra Terigena JCM 1687, Achromobacter denitrificans NBRC 12669, Achromobacter riticas ATCC 21456, Brevibacterium butanicam ATCC 21196, Brevibacterium ketoglutamicum ATCC 21004, Brevibacterium Huscum JCM 1488, Corynebacterium glutamicum ATCC 14020, Kurtbacterium albidam JCM 1344, Kurtbacterium rutheum JCM 1480, Kurtbacterium citreum JCM 1345, Devosia riboflavina NBRC 13584, Hydrangea acetilicum JCM 1968 strain, Fratheria Ourantia NBRC 3247 strain, Hafnia albey NBRC 3731 strain, Mesozobium roti NBRC 13336 strain, Nocardioides simplex NBRC 12069 strain, Paenibacillus varidais NBRC 13635 strain NBRC 13635 strain Polymyxia NBRC 3020, Paenibacillus macerans JCM 2500, Proteus vulgaris NBRC 3851, Saccharopolispora erythraea NBRC 13426, Spirirospora albida NBRC 12248, Streptomyces parvallus NBRC 13193, Streptomyces virginia NBRC 12827, Streptomyces galiraeus ATCC 31133, Streptomyces canus ATCC 12646, Streptomyces fradiae ATCC 10745, Streptomyces alboflavus NBRC 13196, Streptomyces The method according to [2] above, which is Myces arbobinaceus NBRC 12739 strain, Streptomyces cellulosae NBRC 3713 strain, Streptomyces fumanas NBRC 13042 strain or Streptomyces longis spolloba NBRC 13488 strain;
[4] Pseudomonas azotoformans, Pseudomonas oryzihabitans, Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chloros, Pudomonas chloros Culturing bacteria in a glycerol-containing medium, belonging to a bacterial species selected from the group of bacterial species consisting of Pseudomonas taetrolens and Nocardia uniformis and capable of producing pyruvate from glycerol, Or a method for producing pyruvic acid from glycerol, which comprises bringing glycerol into contact with the microbial cell, a treated microbial cell, or an immobilized product thereof;
[5] Bacteria belonging to a bacterial species selected from the bacterial species group consisting of Pseudomonas azotoformans, Pseudomonas oryzii habitans, Pseudomonas synxantha, Pseudomonas fragis, Pseudomonas chlorolaphis, Pseudomonas taetrurens and Nocardia uniformis Pseudomonas azotoformans JCM 2777, Pseudomonas oryzae habitans JCM 2952, Pseudomonas synxantha NBRC 3913, Pseudomonas fraj JCM 20552, Pseudomonas chlorolafis NBRC 3904, Pseudomonas taetrorens NBRC 3460 or The method according to [4] above, which is Nocardia Uniformis NBRC 13702 strain;
[6] Escherichia coli NBRC 12734 strain is cultured in a glycerol-containing medium, or glycerol is contacted with the cells, treated cells, or immobilized products thereof, A method for producing pyruvic acid from glycerol;
[7] Bacteria belonging to the genus Citrobacter and having the ability to produce pyruvic acid from glycerol are cultured under aerobic conditions in a glycerol-containing medium, or the microbial cells, processed microbial cells in glycerol Or a method for producing pyruvic acid from glycerol, characterized by contacting them with an immobilized product thereof;
[8] The method according to claim 7, wherein the bacterium belonging to the genus Citrobacter is Citrobacter freundii;
[9] The method according to claim 8, wherein the Citrobacter frundi is Citrobacter freundii JCM 1657 strain;
[10] The method according to any one of [1] to [6] above, wherein the culture is performed under aerobic conditions;
[11] The method according to any one of [1] to [10] above, which comprises a step of recovering pyruvic acid from the culture;
[12] The method according to any one of [1] to [11] above, wherein glycerol is derived from a biodiesel waste liquid.

  According to the method of the present invention, pyruvic acid can be easily produced from glycerol (biodiesel waste liquid) using microorganisms. Since pyruvic acid has high reactivity, it is used as an important intermediate in the synthesis of pharmaceuticals, agricultural chemicals and the like. Thus, the present invention provides a method for producing a useful substance from glycerol in biodiesel waste liquid that has been discarded.

Specific examples of pyruvic acid-producing bacteria used in the present invention include the bacterial genera, bacterial species, and bacterial strains described in [1] to [9] above. Among them, bacteria belonging to the genus Citrobacta are not particularly limited as long as they can assimilate glycerol to produce pyruvic acid. For example, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter braakii, Citrobacter brameli ( Citrobacter farmeri), Citrobacter koseri, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werobmanit, Citrobacter werobmanit, Citrobacter werobmanit It is done. Citrobacter frundi is preferable. A preferred example of Citrobacter frundi includes, but is not limited to, Citrobacter frundi JCM 1657 strain.
Among these pyruvic acid producing bacteria, bacteria capable of producing pyruvic acid from glycerol in a good yield are preferable. Examples of such bacteria include Arthrobacter aurescens NBRC 12136 strain, Arthrobacter histidinovololans JCM 2520 Stocks, Arthrobacter protoformiae JCM 1973, Bacillus sphaericus NBRC 3341, Arthrobacter globforis ATCC 4336, Arthrobacta Passense ATCC 13346, Arthrobacter paraffinus ATCC 21003, Bacillus Badius ATCC 14574 Nifomis NBRC 12197, Bacillus cereus bar juroy ATCC 21182, Corynebacterium glutamicum ATCC 14020, Kurtobacterium albidam JCM 1344, Kurtobacterium rutheum JCM 1480, Cartba Cterium citreum JCM 1345, Devosia riboflavina NBRC 13584, Mesorizobium roti NBRC 13336, Microbacterium barkeri JCM 1343, Microbacterium liquefaciens JCM 3879, Microbacterium estelalomaticum NBRC 3751 shares, Paenibacillus validas NBRC 13635 shares, Paenibacillus polymyxia NBRC 3020 shares, Paenibacillus macerans JCM 2500 shares, Proteus bulgaris NBRC 3851 shares, Lao Ulterra planticola JCM 7251 shares, Lao Ulterra terigenus JCM 1687 shares Azotoformance JCM 2777, Pseudomonas synxantha NBRC 3913, Pseudomonas fraj JCM 20552, Pseudomonas chlorolafis NBRC 3904, Pseudomonas taetrulens NBRC 3460, Achromobacter Nitrificans NBRC 12669, Achromobacter riticas ATCC 21456, Brevibacterium butanicum ATCC 21196, Brevibacterium ketoglutamicum ATCC 21004, Brevibacterium Huscum JCM 1488, Escherichia coli NBRC 12734, Citrobacter Freundi JCM 1657, Ikeguobacterium acetyricum JCM 1968, Frathelia aurantia NBRC 3247, Hafnia albey NBRC 3731, Microbacterium testaceum JCM 1353, Nocardia Uniformis NBRC 13702, Nocardio IDES SIMPLEX NBRC 12069, Pseudomonas oryzii habitans JCM 2952, Arthrobacter nicotianae JCM 1333, Saccharopolis spora erythraea NBRC 13426, Spirolos pora albida NBRC 12248, S Leptomyces parvallus NBRC 13193, Streptomyces virginiae NBRC 12827, Streptomyces gallillaeus ATCC 31133, Streptomyces canus ATCC 12646, Streptomyces fradiae ATCC 10745, Streptomyces alboflavus NBRC Examples include, but are not limited to, 13196 strains, Streptomyces albobinaceus NBRC 12739 strains, Streptomyces cellulosae NBRC 3713 strains, Streptomyces fumanus NBRC 13042 strains, and Streptomyces longispololaba NBRC 13488 strains. Among them, Achromobacter denitrificans NBRC 12669, Achromobacter riticas ATCC 21456, Ikugiobacterium acetyricum JCM 1968, Fratheria aurantia NBRC 3247, Microbacterium testaseum JCM 1353, Nocardia Uniformis NBRC 13702, Nocardioides simplex NBRC 12069, Pseudomonas origihabitans JCM 2952, Arthrobacter Nicotianae JCM 1333, Arthrobacter globiformis ATCC 4336, Streptomyces parvallus NBRC 13193, Streptomyces・ Virginia NBRC 12827, Streptomyces gallillaeus ATCC 31133, Streptomyces canus ATCC 12646, Hafnia albey NBRC 3731, Streptomyces fradiae ATCC 10745 , Streptomyces alboflavus NBRC 13196, Streptomyces albobinaceus NBRC 12739, Streptomyces cellulosae NBRC 3713, Streptomyces fumanus NBRC 13042, Streptomyces longispololaba NBRC 13488, Escherichia colii 12734, Citrobacter frundi JCM 1657 is more preferred, Arthrobacter Nicotianae JCM 1333, Arthrobacter globiformis ATCC 4336, Streptomyces parvallus NBRC 13193, Hafnia albey NBRC 3731, Streptomyces fradiae ATCC 10745 Strain, Streptomyces arboflavus NBRC 13196, Streptomyces arbobinaceus NBRC 12739, Streptomyces cerulosae NBRC 3713, Streptomyces fumanus NBRC 13 More preferred are the 042 strain, Streptomyces longispololaba NBRC 13488 strain, Escherichia colii NBRC 12734 strain, and Citrobacter Freundi JCM 1657 strain.

  Here, the generation of pyruvic acid with “good yield” means that pyruvic acid is accumulated from glycerol at a yield of about 0.1 g / L or more in the culture solution. preferable. For example, pyruvic acid is accumulated in the culture medium at a yield of about 0.5 g / L or more, preferably about 1 g / L or more.

  Arthrobacter histidinovobolans JCM 2520, Arthrobacter nicotianae JCM 1333, Arthrobacta protoformiae JCM 1973, Microbacterium Barkeri JCM 1343, Microbacterium testaceum JCM 1353, Microbacterium Riqueficiens JCM 3879, Pseudomonas azotoformans JCM 2777, Pseudomonas oryzii habitance JCM 2952, Brevibacterium Huscum JCM 1488, Citrobacter Freundi JCM 1657, Paenibacillus macerans JCM 2500, Lao Ultera Planticola JCM 7251, Raoulutera Terigena JCM 1687, Kurtobacterium albidam JCM 1344, Kurtobacterium rutheum JCM 1480, Kurtobacterium citreum JCM 1345, Ikujiguobacte Um Acetericum JCM 1968, Pseudomonas fraj JCM 20552 are from the Institute for Bioresources, RIKEN BioResource Center.・ Richenifomis NBRC 12197, Pseudomonas synxantha NBRC 3913, Pseudomonas chlorolafis NBRC 3904, Pseudomonas taetrulens NBRC 3460, Achromobacter denitrificans NBRC 12669, Debosia riboflavina NBRC 13584・ Ourrantia NBRC 3247, Hafnia albey NBRC 3731, Escherichia colii NBRC 12734, Microbacterium estellaromaticum NBRC 3751, Mesozobium roti NBRC 13336 , Nocardia Uniformis NBRC 13702, Nocardioides Simplex NBRC 12069, Paenibacillus validas NBRC 13635, Paenibacillus polymyxia NBRC 3020, Proteus Bulgaris NBRC 3851, Saccharopolispora erythraea NBRC 13426 , S. sp. Albida NBRC 12248, Streptomyces parvallus NBRC 13193, Streptomyces virginia NBRC 12827, Streptomyces albofurabus NBRC 13196, Streptomyces albobiaceus NBRC 12739, Streptomyces cerosa NBRC 3713 strain, Streptomyces fumanus NBRC 13042 strain, Streptomyces longis spollolaba NBRC 13488 strain is an independent administrative agency from National Institute of Product Evaluation Technology Biotechnology Division Kuta Globiformis ATCC 4336, Arthrobacta Passense ATCC 13346, Arthrobacta parafineus ATCC 21003, Bacillus Badius ATCC 14574, Bacillus cereus bar juroy ATCC 21182, Brevibacterium butanicum ATCC 21196, Brevibacterium Um ketoglutamicum ATCC 21004, Corynebacterium glutamicum ATCC 14020, Streptomyces gallillaeus ATCC 31133, Streptomyces canus ATCC 12646, Streptomyces fradiae ATCC 10745, Achromobacter riticas ATCC 21456 Available from the American Type Culture Collection.

  The pyruvate-producing bacterium used in the present invention is not limited to wild strains thereof, but any natural or artificial mutant such as X-ray irradiation, ultraviolet irradiation, N-methyl-N′-nitro-N— It may be a mutant obtained by treatment with a chemical mutagen such as nitrosoguanidine, or a recombinant derived by a genetic technique such as cell fusion or gene recombination. The host of the above gene recombinant may be any bacterial genus as long as it is a transformable microorganism, but it should be the same bacterial genus as the parent strain from which the gene of interest is derived. Is preferred. In that case, it is desirable to select a pyruvic acid-producing bacterium having improved ability to convert glycerol into pyruvic acid.

  The glycerol-containing medium may be a culture medium to which pure glycerol is added or a glycerol-containing mixture. The other components in the glycerol-containing mixture and their amounts are preferably those that do not adversely affect the pyruvic acid producing bacteria used in the present invention. The origin of the glycerol-containing mixture is not particularly limited, but it is preferable to use biodiesel waste liquid or the like from the viewpoint of effective use of resources.

  One method for producing biodiesel fuel is to produce fatty acid methyl ester (FAME) by alcoholysis of triglycerides using an alkali catalyst. In this method, a waste liquid containing glycerol as a by-product (referred to as biodiesel waste liquid) is generated. This waste liquid usually contains a catalyst, unconverted fatty acid (depending on the oil used), and the like. For example, the composition of the biodiesel waste liquid used in the examples described later is glycerol: 51%, methanol: 11%, potassium hydroxide: 8%, water: 4%, glycerides, etc .: 26%. Other than this, glycerol: 65%, potassium sodium salt: 4-5%, methanol: 1%, water: 28% described in S. Papanikolaou et al., Bioresource Technology (2002) 82: 43-49 Biodiesel wastewater having an arbitrary composition such as glycerol: 65%, sodium salt: 5% or less described in M. Gonzalez-Pajuelo et al., J IndMicrobiol Biotechnol (2004) 31: 442-446 is also used. be able to.

  When biodiesel waste liquid is added to the medium by the method of the present invention, pyruvic acid can be produced with a yield and conversion efficiency equivalent to or higher than when pure glycerol is added.

  The medium used in the method of the present invention is not limited to a specific medium as long as it is a medium containing components usually used for culturing bacteria. In the method of the present invention, pyruvic acid can be obtained even using a medium having a very simple composition containing a carbon source, a nitrogen source and an inorganic salt.

  The medium used in the method of the present invention contains glycerol as a carbon source. The concentration of glycerol contained in the medium can be appropriately selected within a range that does not adversely affect bacterial growth and pyruvic acid production, but is usually about 0.1 to about 500 g / L, preferably about 1 to about 300 g / L. L. When the biodiesel waste liquid is used as a glycerol source, it may be diluted or added with glycerol so that the amount of glycerol in the medium falls within the above range depending on the concentration of glycerol contained in the waste liquid.

  The medium may further contain substances other than glycerol as a carbon source, but other carbon sources should be added in an amount that does not interfere with the production of pyruvate from glycerol. Examples of other carbon sources used in the present invention include, but are not limited to, glucose, fructose, starch, lactose, arabinose, xylose, dextrin, molasses and the like. Preferably, the other carbon source is about 10 wt% or less, more preferably about 1 wt% or less based on glycerol. Most preferably, the medium contains glycerol as a single carbon source.

  As the nitrogen source, inorganic nitrogen compounds such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, urea, and the like can be used. Add additional ingredients such as organic nitrogen sources such as gluten powder, cottonseed powder, soybean powder, corn steep liquor, dry yeast, yeast extract, peptone, meat extract, casamino acid or various vitamins to the medium as needed. However, in the method of the present invention, pyruvic acid can be produced without including such relatively expensive nutrients.

  Carbon sources and nitrogen sources are advantageously used in combination, but low-purity substances containing trace amounts of growth factors and substantial amounts of mineral nutrients are also suitable for use, so they are used in pure form There is no need.

  If desired, inorganic salts such as primary potassium phosphate, dibasic sodium phosphate, sodium chloride, magnesium sulfate, manganese sulfate, calcium carbonate, calcium chloride, sodium iodide, potassium iodide, cobalt chloride and the like can be used. . In addition, when necessary, particularly when the medium is significantly foamed, an antifoaming agent such as liquid paraffin, higher alcohol, vegetable oil, mineral oil, or silicone may be added.

  Such nitrogen sources, inorganic salts and further components are known to those skilled in the art.

  The culture of various pyruvate-producing bacteria (excluding bacteria belonging to the genus Citrobacta) in the method of the present invention may be performed under anaerobic conditions, but is preferably performed under aerobic conditions. Bacteria belonging to the genus Citrobacter are cultured under aerobic conditions. Aerobic conditions refer to culturing in the presence of molecular oxygen. Aeration, stirring, shaking, etc. may be performed for supplying oxygen. As an apparatus used for culturing, various apparatuses usually used for culturing microorganisms can be used. When culturing under aerobic conditions in the method of the present invention, bacteria can be cultured and pyruvic acid production can be easily performed without using an apparatus or the like necessary to bring about anaerobic conditions.

  On the other hand, in the case of culturing under anaerobic conditions, for example, carbon dioxide gas, inert gas (nitrogen, argon, etc.) can be aerated or non-aerated.

  Liquid culture is preferred as a condition for mass production of pyruvic acid. When growing in a large tank, it is preferable to inoculate the production tank using bacteria in the growth phase for the purpose of avoiding growth delay in the pyruvic acid production step. That is, it is desirable to first inoculate a relatively small amount of the medium and incubate it to produce a growth phase inoculum, and then to aseptically transfer the inoculum to a large tank.

  Stirring and aeration of the culture solution can be performed by various methods. Stirring can be performed by using a propeller or similar mechanical stirring device, rotating or shaking a fermenter, using various pump devices, and the like. Further, aeration can be performed, for example, by passing sterilized air through the culture solution. In this case, it is possible to obtain an agitation effect by the aeration operation.

  When culturing in a liquid medium, culture methods such as batch culture, fed batch culture, and continuous culture can be appropriately selected and used.

  The culture conditions may be any suitable for culturing pyruvate-producing bacteria used in the present invention. For example, the culture temperature is about 4 ° C to about 40 ° C, preferably about 20 ° C to about 37 ° C. The pH of the medium is about 5 to about 9, preferably about 6 to about 8. Since the pH of the medium may decrease with the generation of pyruvic acid, an alkali such as aqueous ammonia, calcium carbonate, sodium hydroxide, or potassium hydroxide may be added to the medium as needed during the cultivation. Is adjusted within these ranges.

  The composition of the medium, the above and other culture conditions, and the like can be appropriately adjusted by those skilled in the art. In order to further improve the yield and / or yield of pyruvic acid, it may be considered to adjust them.

Bacteria used in the method of the present invention may be used in the form of cells, treated cells, or immobilized products thereof. Here, the treated microbial cell refers to a crushed cell or an enzyme extracted from a culture (including microbial cells and culture supernatant). Examples of treated cells include cells obtained by culturing cells treated with organic solvents (acetone, ethanol, etc.), freeze-dried or alkali-treated, or cells physically or enzymatically. Or a crude enzyme separated or extracted from these. Specifically, after collecting the cells from the culture by centrifugation or the like, the cells are subjected to physical disruption methods such as ultrasonic treatment, dynomill treatment, French press treatment, or chemical using a lytic enzyme such as a surfactant or lysozyme. Crush by crushing method. A cell-free extract is prepared by removing impurities from the obtained crushed liquid by centrifugation, membrane filter filtration, etc., and this is prepared by cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration chromatography. In addition, an enzyme or the like can be purified by fractionation using an appropriate separation and purification method such as metal chelate chromatography.
Examples of the carrier used for the chromatography include insoluble polymer carriers such as cellulose, dextrin, or agarose into which carboxymethyl (CM) group, diethylaminoethyl (DEAE) group, phenyl group or butyl group is introduced. A commercially available carrier-filled column can also be used. In addition to the methods described above, disruption of bacterial cells and extraction of enzymes can also be performed by methods known to those skilled in the art.

  The following is mentioned as an example of the method of making a microbial cell, a microbial cell processed material, or those fixed thing contact glycerol.

  Examples of a method for producing pyruvic acid from glycerol using bacterial cells or processed bacterial cells include a method in which bacterial cells are suspended in a substrate solution containing glycerol and reacted. Bacterial cells can be obtained by culturing pyruvic acid-producing bacteria, followed by centrifugation or the like. The concentration of glycerol in the substrate solution is preferably about 0.01 to 50% by weight. The reaction temperature is generally about 4 ° C to about 40 ° C, preferably about 20 ° C to about 37 ° C. The pH of the reaction solution is usually about 5 to about 9, preferably about 6 to about 8. Since the pH of the medium may decrease with the generation of pyruvic acid, an alkali such as aqueous ammonia, calcium carbonate, sodium hydroxide, or potassium hydroxide may be added to the medium as needed during the cultivation. Is adjusted within these ranges.

  As a method for producing pyruvic acid from glycerol using an immobilized product such as a microbial cell, a method in which an immobilized product such as a bacterial cell is packed in a column and a substrate solution containing glycerol is circulated can be exemplified. Bacterial cells and treated cells can be obtained by culturing pyruvic acid-producing bacteria and then performing centrifugation or the like. Examples of the method for immobilizing bacterial cells and the like include means for comprehensively immobilizing with a gel, means for immobilizing an ion exchanger, and the like. Examples of the gel used include carrageenan gel, agar gel, mannan gel, PVA gel, and polyacrylamide gel. The size of the gel sphere varies depending on the type of gel, but a diameter of about 1 to 10 mm is appropriate. Moreover, as an ion exchanger, ion exchangers, such as a cellulose type, a styrene divinylbenzene type, a phenol formalin type, can be mentioned, for example. The concentration of glycerol in the substrate solution is preferably about 0.01 to 50% by weight. In addition, SH compounds such as mercaptoethanol, cysteine, and glutathione, reducing agents such as sulfites, and enzyme activators such as magnesium ions and manganese ions can be added to the solution. The speed of the solution to be circulated varies depending on the size of the column and the amount of the immobilized substance, but the space velocity (ml / ml resin · hr), which is an index of the speed for treating the solution, is suitably 0.05 to 10. .

  Separation and purification of pyruvic acid can be performed according to a conventionally known method. For example, a culture supernatant can be obtained by filtering or centrifuging the culture solution after completion of the culture, and pyruvic acid can be separated from the culture supernatant by, for example, concentrated crystallization. A method for separating and purifying pyruvic acid Is not limited to them. Specifically, pyruvic acid is separated from the culture supernatant by a solvent extraction method, or adsorbed onto an ion exchange resin, and then eluted and separated. It is separated and purified by a method such as a separation method, a fractional precipitation method by insolubilization treatment, a fractional crystallization method by crystallization, a membrane separation method by a reverse osmosis membrane, and a concentrated crystallization method. Specifically, for example, pyruvic acid may be separated and purified according to the method described in JP-A-6-91.

  The present invention is illustrated by the following examples, but it should be understood that the present invention is not limited thereto and that various modifications can be made within the scope of the present invention.

Example 1
Acromobacter denitrificans NBRC 12669 strain was prepared from A culture medium for plate culture (composition: primary potassium phosphate 3 g, dibasic sodium phosphate 6 g, sodium chloride 0.5 g, ammonium chloride 1 g, magnesium sulfate It was applied to 492 mg of 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 same strain grown on the above plate was platinum in 3 ml of B medium (composition: the same composition as the above-mentioned A agar medium except that it does not contain calcium chloride, yeast extract and agar). The cells were inoculated with ears and cultured with shaking (preculture) at 30 ° C. for 24 hours at 200 rpm. 30 μl of the culture solution of the same strain grown in this manner was added to C medium (composition: glycerol 10 g instead of glycerol 10 g instead of 10 g of glycerol (glycerol: 51%, methanol: 11%). , Potassium hydroxide: 8%, water: 4%, glycerides, etc .: 26%) The composition is the same as that of the above B tube culture medium except that 19.6 g is contained) And shaking culture (main culture) at 200 rpm. Four days after the start of the reaction, 3.9 g of glycerol was consumed per liter, and 0.3 g of pyruvic acid was accumulated.

(Example 2)
Acromobacter denitrificans NBRC 12669 strain was prepared from A culture medium for plate culture (composition: primary potassium phosphate 3 g, dibasic sodium phosphate 6 g, sodium chloride 0.5 g, ammonium chloride 1 g, magnesium sulfate It was applied to 492 mg of 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 same strain grown on the above plate was inoculated with platinum ears in 3 ml of D medium (composition: the same composition as that of A agar medium except that it does not contain agar). , And shaking culture (preculture) at 200 rpm for 24 hours at 30 ° C. 30 μl of the same culture broth thus grown was added to E medium (composition: glycerol 10 g instead of glycerol 10 g instead of 10 g of glycerol (glycerol: 51%, methanol: 11%). , Potassium hydroxide: 8%, water: 4%, glycerides, etc .: 26%) Except for containing 19.6 g, the composition is the same as that of the above E-tube culture medium. And shaking culture (main culture) at 200 rpm. Four days after the start of the reaction, 9.4 g of glycerol was consumed per liter 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 was consumed per litter and 0.4 g of pyruvic acid was accumulated.

Example 4
Pseudomonas synxanta NBRC 3913 strain was reacted in the same manner as in Example 1. As a result, 4.5 g of glycerol was consumed and 0.1 g of pyruvic acid was accumulated per liter.

(Example 5)
Raoulutera terigena JCM 1687 strain was reacted in the same manner as in Example 1. As a result, 1.9 g of glycerol was consumed and 0.1 g of pyruvic acid was accumulated per liter.

(Example 6)
A. agar medium (composition: 3 g primary potassium phosphate, 6 g dibasic sodium phosphate, 0.5 g sodium chloride, 1 g ammonium chloride, magnesium sulfate. It was applied to 492 mg of 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 same strain grown on the above plate was inoculated with platinum ears in 3 ml of D medium (composition: the same composition as that of A agar medium except that it does not contain agar). And shaking culture (preculture) at 200 rpm for 6 days at 30 ° C. 30 μl of the same strain culture solution grown in this way was transferred to 3 ml of D medium, which is a test tube culture medium, and subjected to shaking culture (main culture) at 30 ° C. and 200 rpm. Six days after the start of the reaction, 1.4 g of glycerol was consumed per liter and 0.5 g of pyruvic acid was accumulated.

(Example 7)
When Devosia riboflavina NBRC 13584 strain was reacted in the same manner as in Example 6, 0.9 g of glycerol was consumed per litter and 0.3 g of pyruvic acid was accumulated.

(Example 8)
Mesozobium roti NBRC 13336 strain was reacted in the same manner as in Example 6. As a result, 0.4 g of glycerol was consumed per liter 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 that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 7.1 g of glycerol was consumed per liter, and 1.7 g of pyruvic acid was accumulated.

(Example 10)
Arthrobacter silleus NBRC 12957 was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 2.4 g of glycerol was consumed per liter, and 0.3 g of pyruvic acid was accumulated.

(Example 11)
Arthrobacter aurescens NBRC 12136 was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 2.5 g of glycerol was consumed per liter, and 0.2 g of pyruvic acid was accumulated.

Example 12
Arthrobacter histidinovololans JCM 2520 was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. As a result, 3.8 g of glycerol was consumed per liter, and 0.1 g of pyruvic acid was accumulated.

(Example 13)
Arthrobacter protoformiae JCM 1973 was reacted in the same manner as in Example 6 except that the culture time for preculture was 2 days instead of 6 days and the culture time for main culture was 4 days instead of 6 days. However, 3.7 g of glycerol was consumed per liter, and 0.1 g of pyruvic acid was accumulated.

(Example 14)
Streptomyces parvallus NBRC 13193 was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 9.4 g of glycerol was consumed per liter, and 2.2 g of pyruvic acid was accumulated.

(Example 15)
Frateuria / Ourantia NBRC 3247 strain was prepared from A culture medium for plate culture (composition: primary potassium phosphate 3 g, dibasic sodium phosphate 6 g, sodium chloride 0.5 g, ammonium chloride 1 g, magnesium sulfate-7 water The mixture was applied to Japanese 492 mg, calcium chloride dihydrate 147 mg, yeast extract 100 mg, glycerol 10 g, agar 20 g and distilled water 1 L (final pH 7.4)), and allowed to stand at 30 ° C. for 4 days. The same strain grown on the above plate was platinum in 3 ml of B medium (composition: the same composition as the above-mentioned A agar medium except that it does not contain calcium chloride, yeast extract and agar). The cells were inoculated with ears and cultured with shaking (preculture) at 30 ° C. for 24 hours at 200 rpm. 30 μl of the culture solution of the same strain thus grown was transferred to 3 ml of B medium, which is a test tube culture medium, and subjected to shaking culture (main culture) at 30 ° C. and 200 rpm. Four days after the start of the reaction, 0.7 g of glycerol was consumed per liter and 0.1 g of pyruvic acid was accumulated.

(Example 16)
As a result of reacting Fratheria aurantia NBRC 3247 strain in the same manner as in Example 6, 2.4 g of glycerol was consumed per litter and 0.5 g of pyruvic acid was accumulated.

(Example 17)
Microbacterium testaceum JCM 1353 was reacted in the same manner as in Example 6 except that the culture time for preculture was 2 days instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 3.1 g of glycerol was consumed per liter, and 0.6 g of pyruvic acid was accumulated.

(Example 18)
Microbacterium Barkeri JCM 1343 was reacted in the same manner as in Example 6 except that the culture time for preculture was 2 days instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 4.2 g of glycerol was consumed per liter, and 0.1 g of pyruvic acid was accumulated.

Example 19
When Nocardioides simplex NBRC 12069 strain was reacted in the same manner as in Example 6, 2.2 g of glycerol was consumed per litter 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 was consumed per litter and 0.2 g of pyruvic acid was accumulated.

(Example 21)
The Bacillus sphaericus NBRC 3341 strain was reacted in the same manner as in Example 6 except that the culture time was 24 hours instead of 6 days for preculture and 4 days instead of 6 days for main culture. 1.9 g of glycerol was consumed per liter, and 0.3 g of pyruvic acid was accumulated.

(Example 22)
The Bacillus badius ATCC 14574 strain was reacted in the same manner as in Example 6 except that the culture time was 24 hours instead of 6 days for preculture and 4 days instead of 6 days for main culture. 2.7 g of glycerol per liter was consumed and 0.3 g of pyruvic acid was accumulated.

(Example 23)
Nocardia Uniformis NBRC 13702 was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 3.7 g of glycerol was consumed per liter, 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 was consumed per litter and 0.4 g of pyruvic acid was accumulated.

(Example 25)
When the Kurtobacterium albidum JCM 1344 strain was reacted in the same manner as in Example 6, 1.4 g of glycerol was consumed and 0.1 g of pyruvic acid was accumulated per liter.

(Example 26)
Corynebacterium glutamicum ATCC 14020 strain was reacted in the same manner as in Example 6. As a result, 1.9 g of glycerol was consumed and 0.1 g of pyruvic acid was accumulated per liter.

(Example 27)
Pseudomonas oridihabitans JCM 2952 was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 9.5 g of glycerol was consumed per liter, 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 was consumed per litter and 0.1 g of pyruvic acid was accumulated.

(Example 29)
Lao Ulterra 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 polymyxia NBRC 3020 strain was reacted in the same manner as in Example 6. As a result, 0.4 g of glycerol was consumed per litter 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 was consumed per litter and 0.8 g of pyruvic acid was accumulated.

(Example 32)
Streptomyces galiraeuus ATCC 31133 strain was reacted in the same manner as in Example 6. As a result, 4.3 g of glycerol was consumed per litter 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 was consumed per litter 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)
As a result of reacting Spirorospora albida NBRC 12248 strain in the same manner as in Example 6, 0.3 g of glycerol was consumed per litter 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 was consumed per liter 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 was consumed per liter 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)
When Hafnia albei NBRC 3731 strain was reacted in the same manner as in Example 1, 8.5 g of glycerol was consumed per litter and 2.4 g of pyruvic acid was accumulated.

(Example 41)
When Hafnia albei NBRC 3731 strain was reacted in the same manner as in Example 6, 8.1 g of glycerol was consumed per liter and 0.1 g of pyruvic acid was accumulated.

(Example 42)
When the Kurtobacterium ruthenium JCM 1480 strain was reacted in the same manner as in Example 6, 2.1 g of glycerol was consumed and 0.1 g of pyruvic acid was accumulated per liter.

(Example 43)
Pseudomonas fraj 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 fraj JCM 20552 strain was reacted in the same manner as in Example 2. As a result, 4.5 g of glycerol was consumed and 0.2 g of pyruvic acid was accumulated per liter.

(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 was consumed and 0.1 g of pyruvic acid was accumulated per liter.

(Example 46)
Achromobacter riticas ATCC 21456 strain was reacted in the same manner as in Example 6 except that the culture time was 24 hours instead of 6 days for preculture and 4 days instead of 6 days for main culture. 2.2 g of glycerol was consumed per liter and 0.7 g of pyruvic acid was accumulated.

(Example 47)
Bacillus licheniformis NBRC 12197 was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. However, 1.8 g of glycerol was consumed per liter, and 0.3 g of pyruvic acid was accumulated.

(Example 48)
Microbacterial liquifaciens JCM 3879 strain was reacted in the same manner as in Example 6. As a result, 2.3 g of glycerol was consumed per litter 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 was consumed per litter 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 strain ATCC 4336 was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 5.3 g of glycerol was consumed per liter, 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 was consumed per liter and 0.1 g of pyruvic acid was accumulated.

(Example 53)
Arthrobacta passense ATCC 13346 was reacted in the same manner as in Example 6 except that the culture time was 24 hours instead of 6 days for preculture and 4 days instead of 6 days for main culture. 2.9 g of glycerol per liter was consumed and 0.1 g of pyruvic acid was accumulated.

(Example 54)
Bacillus cereus bar juroy ATCC 21182 strain was reacted in the same manner as in Example 6 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. As a result, 0.7 g of glycerol was consumed per liter, and 0.2 g of pyruvic acid was accumulated.

(Example 55)
Pseudomonas taetzulorens NBRC 3460 was reacted in the same manner as in Example 15 except that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 3.2 g of glycerol was consumed per liter, and 0.3 g of pyruvic acid was accumulated.

(Example 56)
Pseudomonas chlorolafis NBRC 3904 strain was reacted in the same manner as in Example 1. As a result, 5.4 g of glycerol was consumed and 0.2 g of pyruvic acid was accumulated per liter.

(Example 57)
Streptomyces arboflavus 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 arbobinaceus NBRC 12739 strain was reacted in the same manner as in Example 6. As a result, 10.3 g of glycerol was consumed and 3.0 g of pyruvic acid was accumulated per liter.

(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 that the culture time for preculture was 24 hours instead of 6 days and the culture time for main culture was 4 days instead of 6 days. 7.5 g of glycerol was consumed per liter, and 3.3 g of pyruvic acid was accumulated.

(Example 61)
Streptomyces longispololava NBRC 13488 was reacted in the same manner as in Example 2 except that the culture time was 6 days instead of 24 hours for the preculture and 6 days instead of 4 days for the main culture. 11 g of glycerol was consumed per liter, 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 that the culture time for main culture was 13 days instead of 6 days. As a result, 1.7 g of glycerol was consumed per liter and pyruvic acid was consumed. Accumulated 0.2 g.

(Example 63)
Microbacterial Estellaromaticum NBRC 3751 strain was reacted in the same manner as in Example 6 except that the culture time for main culture was 8 days instead of 6 days, and 1.3 g of glycerol was consumed per liter. 0.6 g of pyruvic acid was accumulated.

(Example 64)
Kurtobacterium citreum JCM 1345 strain was reacted in the same manner as in Example 6 except that the culture time for main culture was 13 days instead of 6 days. As a result, 1.2 g of glycerol was consumed per liter, and pyruvin was consumed. Accumulated 0.2 g of acid.

(Example 65)
Brevibacterium huscum JCM 1488 strain was reacted in the same manner as in Example 6 except that the culture time for main culture was 7 days instead of 6 days. As a result, 0.9 g of glycerol was consumed per liter, and pyruvin was consumed. Accumulated 0.3 g of acid.

Example 66
Citrobacter Freundi JCM 1657 strain was cultured on A culture medium for plate culture (composition: primary potassium phosphate 3g, dibasic sodium phosphate 6g, sodium chloride 0.5g, ammonium chloride 1g, magnesium sulfate heptahydrate 250 mg, 20 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 same strain as the above-mentioned A agar medium except that the same strain grown on the above plate is B medium (composition: not containing agar and containing 10 g instead of 20 g of glycerol). ) 3 ml was inoculated with platinum ears and cultured with shaking at 200 rpm at 30 ° C. for 24 hours. 30 μl of the same strain culture solution grown in this way was transferred to 3 ml of B medium, which is a test tube culture medium, and cultured with shaking at 30 ° C. for 24 hours at 200 rpm. Four days after the start of the reaction, 3.1 g / L of pyruvic acid was accumulated.

(Example 67)
Citrobacter Freundi JCM 1657 strain was cultured on A culture medium for plate culture (composition: primary potassium phosphate 3g, dibasic sodium phosphate 6g, sodium chloride 0.5g, ammonium chloride 1g, magnesium sulfate heptahydrate 250 mg, 20 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 same strain as the above-mentioned A agar medium except that the same strain grown on the above plate is B medium (composition: not containing agar and containing 10 g instead of 20 g of glycerol). ) 3 ml was inoculated with platinum ears and cultured with shaking at 200 rpm at 30 ° C. for 24 hours. 30 μl of the culture solution of the same strain grown in this manner was added to C medium (composition: glycerol 10 g instead of glycerol 10 g instead of 10 g of glycerol (glycerol: 51%, methanol: 11%). Potassium hydroxide: 8%, water: 4%, glycerides, etc .: 26%) Except for containing 19.6 g, the composition is the same as that of the B agar medium. And cultured at 200 rpm with shaking. Four days after the start of the reaction, 4.2 g / L of pyruvic acid was accumulated.

  According to the method of the present invention, pyruvic acid can be easily produced from glycerol (biodiesel waste liquid) using microorganisms. Since pyruvic acid has high reactivity, it is used as an important intermediate in the synthesis of pharmaceuticals, agricultural chemicals and the like. Thus, the present invention is useful for producing useful substances from waste.

Claims (12)

  Arthrobacter, Bacillus, Microbacterium, Raoultella, Archromobacter, Brevibacterium, Corynebacterium, Cartobacteria The genus Curtobacterium, Devosia, Exiguobacterium, Frateuria, Hafnia, Mesorhizobium, Nocardioides, Paenibacillus (Paenibacillus) ) Belongs to the bacterial genus selected from the genus Bacteria, Proteus, Saccharopolyspora, Spirillospora and Streptomyces, and produces pyruvic acid from glycerol The ability to How the bacteria, or cultured in glycerol containing medium, or glycerol to the cells, which comprises contacting the treated bacterial cells, or their immobilized products, the production of pyruvate from glycerol.   Arthrobacter, Bacillus, Microbacteria, Raoulutera, Achromobacter, Brevibacterium, Corynebacterium, Kurtobacterium, Devosia, Iquigobacterium, Fratheria, Hafnia, Mesolyzobium, Bacteria belonging to the genus Bacteria selected from the genus Nocardioides, Paenibacillus, Proteus, Saccharopolispora, Spirirospora and Streptomyces are Arthrobacter aurescens, Arthrobacter aurescens, Arthrobacter citrus (Arthrobacter citreus), Arthrobacter histidinolovorans, Arthrobacter nicotianae, Arthrobacter protophormae (Arthrobacter protophorm) iae), Arthrobacter globiformis, Arthrobacter pascens, Arthrobacter paraffineus, Bacillus badius, Bacillus sphaericus (Bacillus sphaericus) Bacillus licheniformis, Bacillus cereus var. Juroi, Microbacterium barkeri, Microbacterium testaceum, Microbacterium liquefaciens , Microbacterium esteraromaticum, Raoultella planticola, Raoultella terrigena Achromobacter denitrificans, Achromobacter lyticus, Brevibacterium butanicum, Brevibacterium ketoglutamicum, Brevibacterium fuscum, Brevibacterium fuscum Corynebacterium glutamicum, Curtobacterium albidum, Curtobacterium luteum, Curtobacterium citreum, Devosia riboflavina, Ijiguobacterium Exiguobacterium acetylicum, Frateuria aurantia, Hafnia alvei, mesolizobium Loti (Mesorhizobium loti), Nocardioides simplex, Paenibacillus validus, Paenibacillus polymyxa, Paenibacillus macerte vulgaris, Prote vulgaris Lopolispora erythraea (Saccharopolyspora erythraea), Spirillospora albida, Streptomyces parvulus, Streptomyces virginiae, Streptomyces ilagines gal Streptomyces galtomy Streptomyces canus, Streptomyces fradiae, Streptomyces alboflavus, Reputomaisesu-Arubobinaseusu (Streptomyces albovinaceus), Streptomyces Serurosae (Streptomyces cellulosae), is Streptomyces Fumanasu (Streptomyces fumanus) or Streptomyces Rongisupororaba (Streptomyces longispororuber), The method of claim 1, wherein.   Arthrobacter, Bacillus, Microbacteria, Raoulutera, Achromobacter, Brevibacterium, Corynebacterium, Kurtobacterium, Devosia, Iquigobacterium, Fratheria, Hafnia, Mesolyzobium, The bacteria belonging to the bacterial genus selected from the genus Nocardioides, Paenibacillus, Proteus, Saccharopolisporia, Spirorospora, and Streptomyces are Arthrobacter aurescens NBRC 12136, Arthrobacter silleus NBRC 12957 shares, Arthrobacter histidinorovorans JCM 2520 shares, Arthrobacta Nicotianae JCM 1333 shares, Arthrobacta protoformiae JCM 1973 shares, Arthrobacta globiformis ATCC 4336 shares, Arturo Kuta Passens ATCC 13346, Arthrobacta paraffineus ATCC 21003, Bacillus Badius ATCC 14574, Bacillus sphaericus NBRC 3341, Bacillus lichenifomis NBRC 12197, Bacillus cereus bar Juroy ATCC 21182, Bacterium Barkeri JCM 1343, Microbacterium Testaceum JCM 1353, Microbacterium Liquefaciens JCM 3879, Microbacterium Estelaromaticum NBRC 3751, Lao Ulterra Planticocola JCM 7251, Lao Ulterra Terigena JCM 1687, Achromobacter denitrificans NBRC 12669, Achromobacter riticas ATCC 21456, Brevibacterium butanicam ATCC 21196, Brevibacterium ketoglutamicum ATCC 21004, Brevibacterium fu Scum JCM 1488, Corynebacterium glutamicum ATCC 14020, Kurtbacterium Albidam JCM 1344, Kurtbacterium rutheum JCM 1480, Kurtbacterium citreum JCM 1345, Devosia riboflavina NBRC 13584, Ijiguo Bacterium acetyricum JCM 1968, Fratheria aurantia NBRC 3247, Hafnia albey NBRC 3731, Mesozobium roti NBRC 13336, Nocardioides simplex NBRC 12069, Paenibacilas validas NBRC 13635 3020 shares, Paenibacillus macerans JCM 2500 shares, Proteus Bulgaris NBRC 3851 shares, Saccharopolis pora erythraea NBRC 13426 shares, Spirorospola albida NBRC 12248 shares, Streptomyces parvallus NBRC 13193 shares Streptomyces virginiae NBRC 12827, Streptomyces gallillaeus ATCC 31133, Streptomyces canus ATCC 12646, Streptomyces fradiae ATCC 10745, Streptomyces alboflavus NBRC 13196, Streptomyces The method according to claim 2, which is Arbobinaceus NBRC 12739 strain, Streptomyces cellulosae NBRC 3713 strain, Streptomyces fumanas NBRC 13042 strain or Streptomyces longis sporola NBRC 13488 strain.   Pseudomonas azotoformans, Pseudomonas oryzihabitans, Pseudomonas synxantha, Pseudomonas fragi, Pseudomonas chlororaphis, Pseudomonas chlororafis Pseudomonas taetrolens) and Nocardia uniformis belonging to a bacterial species selected from the bacterial species group and capable of producing pyruvic acid from glycerol are cultured in glycerol-containing medium or in glycerol A method for producing pyruvic acid from glycerol, which comprises contacting the cells, treated cells of the cells, or immobilized products thereof.   Bacteria belonging to the bacterial species selected from the bacterial species group consisting of Pseudomonas azotoformans, Pseudomonas oryzii habitans, Pseudomonas synxantha, Pseudomonas fragrance, Pseudomonas chlorolaphis, Pseudomonas taetulorens and Nocardia uniformis・ Azotoformance JCM 2777, Pseudomonas oryzii habitans JCM 2952, Pseudomonas synxantha NBRC 3913, Pseudomonas fraj JCM 20552, Pseudomonas chlorolafis NBRC 3904, Pseudomonas taetrurens NBRC 3460 or Nocardia The method according to claim 4, which is Uniformis NBRC 13702 strain.   Escherichia coli NBRC 12734 is cultured in a glycerol-containing medium, or glycerol to pyrubin, characterized in that the cells, treated cells or their immobilizates are contacted with glycerol. A method for producing an acid.   Bacteria belonging to the genus Citrobacter and capable of producing pyruvic acid from glycerol are cultured under aerobic conditions in a glycerol-containing medium, or their cells, treated cells, or those in glycerol A method for producing pyruvic acid from glycerol, which comprises contacting an immobilized product of glycerol.   The method according to claim 7, wherein the bacterium belonging to the genus Citrobacter is Citrobacter freundii.   9. The method of claim 8, wherein the Citrobacter frundi is Citrobacter frundi JCM 1657 strain.   The method in any one of Claims 1-6 by which culture | cultivation is performed on aerobic conditions.   The method in any one of Claims 1-10 including the process of collect | recovering pyruvic acids from a culture.   The method according to any one of claims 1 to 11, wherein glycerol is derived from biodiesel waste liquor.