JP2012213405A - Method for producing lactic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a useful material from glycerol, biodiesel effluent in particular.SOLUTION: A method for producing lactic acid from glycerol is provided, which includes culturing a specific bacterium 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

  The present invention relates to a method for producing lactic 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 (including lactic acid) from glucose.

  Several bacteria are known so far that can produce lactic acid from glycerol. For example, Patent Document 4 and Non-Patent Documents 1, 2 and 3 describe bacteria capable of producing lactic acid from glycerol, but the bacterial genus group consisting of the genus Nocardioides and the genus Proteus Bacteria belonging to the genus Bacterium selected from Arthrobacter aurescens, Arthrobacter citreus, Bacillus badius, Bacillus sphaericus, Nocardia uniforis ), Streptomyces carnosus and Streptomyces cellulosae, there is no description suggesting that such a bacterium belongs to a bacterial species selected from the bacterial species group. In any literature, the amount of lactic acid produced from glycerol is still insufficient, and a microorganism having a better ability to produce lactic acid has been desired.

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, as a result, a bacterium belonging to a genus of bacteria selected from the genus Nocardioides and Proteus, or Arthrobacter aurescens, Arthrobacter citreus, Bacillus badius, Bacillus sphaericus, Nocardia uniformis, Streptomyces carnosus and Streptomyces celestes A bacterium belonging to a bacterial species selected from the group of bacterial species consisting of Streptomyces cellulosae (hereinafter also referred to as a lactic acid-producing bacterium used in the present invention, or simply referred to as a lactic acid-producing bacterium), or cultured under aerobic conditions, or Glycerol and its fungus By contacting the body-treated product or the immobilized product thereof, lactic acid was successfully produced from glycerol at a high conversion rate, and the present invention was completed.

That is, the present invention can provide:
[1] Bacteria belonging to the genus Bacteria selected from the genus Nocardioides and Proteus and capable of producing lactic acid from glycerol are cultured in a glycerol-containing medium, Or a method for producing lactic acid from glycerol, which comprises bringing glycerol into contact with the microbial cell, a treated microbial cell, or an immobilized product thereof;
[2] The above-mentioned [1], wherein the bacterium belonging to the bacterial genus selected from the genus Nocardioides and Proteus is Nocardioides simplex or Proteus vulgaris Described method;
[3] The bacterium described in [2] above, wherein the bacterium belonging to the genus of bacteria selected from the genus Nocardioides and Proteus is Nocardioides simplex NBRC 12069 or Proteus bulgaris NBRC 3851. Method;
[4] Arthrobacter aurescens, Arthrobacter citreus, Bacillus badius, Bacillus sphaericus, Nocardia uniformis, Nocardia uniformis (Streptomyces carnosus) and Streptomyces cellulosae belonging to the bacterial species group selected from the bacterial species group and having the ability to produce lactic acid from glycerol is cultured in glycerol-containing medium or in glycerol A method for producing lactic acid from glycerol, which comprises contacting the cells, treated cells of the cells, or immobilized products thereof; [5] Arthrobacter aurescens, Arthrobacter silleus, Bacillus Bacteria belonging to the bacterial species selected from the group consisting of Dius, Bacillus sphaericus, Nocardia Uniformis, Streptomyces carnosus and Streptomyces cerrozae are the Arthrobacter aurescens NBRC 12136 and Arthrobacter citrus NBRC 12957 strains The above-mentioned [4], which is Bacillus badius ATCC 14574, Bacillus sphaericus NBRC 3341, Nocardia Uniformis NBRC 13702, Streptomyces carnosus NBRC 13025 or Streptomyces cerrosae NBRC 3713 Method;
[6] The method according to any one of the above [1] to [5], wherein the culture is performed under aerobic conditions; [7] The above [1] to [[including the step of recovering lactic acid from the culture] 6];
[8] The method according to any one of [1] to [7] above, wherein glycerol is derived from a biodiesel waste liquid.

  According to the method of the present invention, lactic acid can be easily produced from glycerol (biodiesel waste liquid) with a high conversion rate using microorganisms.

  Lactic acid is used in various applications such as food additives for foods, pH adjustment and bioplastic raw materials for industrial applications, and infusions and intestinal disinfection for pharmaceuticals.

  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 the lactic acid-producing bacterium used in the present invention include the genus bacteria, bacterial species and bacterial strains described in [1] to [5] above. Among these lactic acid-producing bacteria, bacteria that can produce lactic acid from glycerol with a good conversion rate are preferable. Here, lactic acid is produced with a “good conversion rate” means that lactic acid is produced from glycerol at a conversion rate of about 10% (mol / mol) or more. preferable. For example, lactic acid is produced at a conversion rate of about 20% (mol / mol) or higher, preferably about 40% (mol / mol) or higher.

  Examples of such bacteria include Proteus vulgaris NBRC 3851 strain, Nocardioides simplex NBRC 12069 strain, Nocardia uniformis NBRC 13702 strain, Bacillus sphaericus NBRC 3341 strain, Bacillus badius ATCC 14574 strain, Arthrobacter -Oursense NBRC 12136, Arthrobacter silleus NBRC 12957, Streptomyces carnosus NBRC 13025, Streptomyces cerrosae NBRC 3713, but are not limited thereto. Among them, Arthrobacter aurescens NBRC 12136, Arthrobacta citrus NBRC 12957, Bacillus Badius ATCC 14574, Nocardia Uniformis NBRC 13702, Proteus Bulgaris NBRC 3851, Streptomyces cerrosae NBRC 3713 Streptomyces cellulosae NBRC 3713 strain is more preferable.

  Arthrobacter aurescens NBRC 12136, Arthrobacta citrus NBRC 12957, Bacillus sphaericus NBRC 3341, Nocardia Uniformis NBRC 13702, Nocardioides Simplex NBRC 12069, Proteus Bulgaris NBRC 3851, Streptomyces・ Carnosus NBRC 13025 and Streptomyces cerrosae NBRC 3713 are available from the Biotechnology Division, Biotechnology Division, National Institute of Technology and Evaluation, and Bacillus Badius ATCC 14574 is available from the American Type Culture Collection. is there.

  In addition, the lactic acid-producing bacteria used in the present invention are not limited to wild strains thereof, and any natural or artificial mutant such as X-ray irradiation, ultraviolet irradiation, N-methyl-N′-nitro-N-nitroso It may be a mutant obtained by treatment with a chemical mutagen such as guanidine, 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 lactic acid-producing bacterium that has improved conversion ability from glycerol to lactic 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 lactic acid-producing bacteria used in the present invention. The origin of the glycerol-containing mixture is not particularly limited, but biodiesel waste liquid is preferably used from the viewpoint of effective utilization 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 of the present invention 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 Ind Microbiol Biotechnol (2004) 31: 442-446 can do.

  When biodiesel waste liquid is added to a culture medium by the method of the present invention, lactic 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, lactic acid can be obtained at a high conversion rate even when a medium having a very simple composition containing a carbon source, a nitrogen source and an inorganic salt is used.

  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 lactic acid production, but is usually about 0.1 to about 500 g / L, preferably about 1 to about 300 g / L. It is. 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 lactic acid 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, malt extract and the like. Preferably, the other carbon source is about 10% by weight or less, more preferably about 1% by weight 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. Alternatively, organic nitrogen sources such as gluten powder, cottonseed powder, soybean powder, corn steep liquor, dry yeast, yeast extract, peptone, meat extract, and casamino acid may be used.

  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.

  If necessary, additional components such as various vitamins may be added to the medium.

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

  The culture of various lactic acid-producing bacteria in the method of the present invention may be performed under anaerobic conditions, but is preferably performed 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 lactic acid can be easily produced 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 lactic 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 lactic acid production process. 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 lactic acid-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. As the lactic acid is produced, the pH of the medium may decrease. If necessary, an alkali such as aqueous ammonia, calcium carbonate, sodium hydroxide, or potassium hydroxide may be added during cultivation to adjust the pH of the medium. Adjust 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 of lactic acid, it may be considered to adjust these.

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 lactic 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 lactic acid-producing bacteria and then performing 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. As the lactic acid is produced, the pH of the medium may decrease. If necessary, an alkali such as aqueous ammonia, calcium carbonate, sodium hydroxide, or potassium hydroxide may be added during cultivation to adjust the pH of the medium. Adjust within these ranges.

  Examples of a method for producing lactic acid from glycerol using an immobilized product such as bacterial cells include a method in which a column is filled with an immobilized product such as bacterial cells and a substrate solution containing glycerol is circulated. Bacterial cells and treated cells can be obtained by culturing lactic 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 lactic acid can be performed according to a conventionally known method. For example, the method of directly distilling the culture after acidification, the method of distilling by forming lactide of lactic acid, the method of distilling after adding lactic acid by adding an alcohol and a catalyst, the method of extracting lactic acid in an organic solvent, By a method such as separation of lactic acid with an ion exchange column that is adsorbed to an ion exchange resin, and then eluted and separated, a method of producing and isolating a metal salt with calcium ions, a method of concentrating and separating lactic acid by electrodialysis, etc. Separation and purification. Specifically, for example, lactic acid may be separated and purified according to the examples described in JP-A-2003-88392.

  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
Arthrobacter schileuus NBRC 12957 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 hydrate) 492 mg, 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 B 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 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, 2.4 g of glycerol was consumed per liter, 0.5 g of lactic acid was accumulated, and the conversion rate was 21% (mol / mol).

(Example 2)
Arthrobacter aurescens NBRC 12136 strain was reacted in the same manner as in Example 1. As a result, 2.5 g of glycerol was consumed per liter, 0.5 g of lactic acid was accumulated, and the conversion rate was 20% (mol / mol). It was.

(Example 3)
When Bacillus badius ATCC 14574 strain was reacted in the same manner as in Example 1, 2.7 g of glycerol was consumed per liter, 0.5 g of lactic acid was accumulated, and the conversion rate was 19% (mol / mol). It was.

Example 4
When Bacillus sphaericus NBRC 3341 was reacted in the same manner as in Example 1, 1.9 g of glycerol was consumed per liter, 0.3 g of lactic acid was accumulated, and the conversion rate was 16% (mol / mol). It was.

(Example 5)
When Nocardia Uniformis NBRC 13702 strain was reacted in the same manner as in Example 1, 3.7 g of glycerol was consumed per liter, 0.5 g of lactic acid was accumulated, and the conversion rate was 14% (mol / mol). Met.

(Example 6)
Nocardioides simplex NBRC 12069 was reacted in the same manner as in Example 12 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. 2.2 g of glycerol was consumed per liter, 0.3 g of lactic acid was accumulated, and the conversion rate was 14% (mol / mol).

(Example 7)
Proteus bulgaris NBRC 3851 strain was reacted in the same manner as in Example 1 except that the culture time was 6 days instead of 24 hours for preculture and 6 days instead of 4 days for main culture. 5.3 g of glycerol was consumed per liter, 0.6 g of lactic acid was accumulated, and the conversion rate was 12% (mol / mol).

(Example 8)
Streptomyces carnosus NBRC 13025 strain was reacted in the same manner as in Example 1 except that the culture time for preculture was 6 days instead of 24 hours and the culture time for main culture was 6 days instead of 4 days. 1.8 g of glycerol was consumed per liter, 0.2 g of lactic acid was accumulated, and the conversion rate was 11% (mol / mol).

Example 9
Streptomyces cellulosae NBRC 3713 strain was reacted in the same manner as in Example 1. As a result, 12.4 g of glycerol was consumed per liter, 2.1 g of lactic acid was accumulated, and the conversion rate was 17% (mol / mol). Met.

(Example 10)
Streptomyces cellulosae NBRC 3713 strain was added to 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 It was applied to 492 mg of hydrate, 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 plated with 3 ml of D medium (composition: the same composition as that of 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 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. Four days after the start of the reaction, 7.5 g of glycerol was consumed per liter, 1.0 g of lactic acid was accumulated, and the conversion rate was 14% (mol / mol).

  According to the method of the present invention, lactic acid can be easily produced from glycerol (biodiesel waste liquid) with a high conversion rate using microorganisms. Lactic acid is used in various applications such as food additives for food, pH adjustment and bioplastic raw materials for industrial use, and infusion and intestinal disinfection for pharmaceutical use. Thus, the present invention is useful for producing useful substances from waste.

Claims (6)

  Culturing a bacterium belonging to the genus Proteus and capable of producing lactic acid from glycerol in a medium containing glycerol, or contacting the glycerol with the cells, treated cells thereof, or immobilization products thereof A process for producing lactic acid from glycerol.   The method according to claim 1, wherein the bacterium belonging to the genus Proteus is Proteus vulgaris.   The method according to claim 2, wherein the bacterium belonging to the genus Proteus is Proteus vulgaris NBRC 3851 strain.   The method according to claim 1, wherein the culture is performed under aerobic conditions.   The method in any one of Claims 1-4 including the process of collect | recovering lactic acid from a culture.   The method according to any one of claims 1 to 5, wherein glycerol is derived from biodiesel waste liquor.