JP6620405B2 - Method for producing D-lactic acid - Google Patents

Description

  The present invention relates to a method for producing D-lactic acid using woody biomass as a raw material and using D-lactic acid-producing bacteria.

  A method of fermenting and producing a substance that can be used as a fuel such as ethanol and butanol by the action of microorganisms using woody biomass containing cellulose as a raw material has been implemented. Among those produced by fermentation, lactic acid is an organic acid that is produced by the decomposition of sugars such as glucose by the glycolytic system of organisms, and is an important compound that is induced by the TCA circuit and becomes the starting point for bioenergy production. . There is a possibility that methyl lactate or ethyl lactate esterified with lactic acid can be used as a biofuel. Lactic acid can be a solvent and a raw material for food. Furthermore, lactic acid with high optical purity becomes a raw material monomer for synthesizing poly-L-lactic acid or poly-D-lactic acid. Since the optical purity of the raw material monomer affects the degree of polymerization and the glass transition point, for the production of polylactic acid, the optical purity of the raw material lactic acid is required to be high, for example, 98% ee or higher.

  Various methods have been studied for efficient fermentation production of lactic acid. For example, Patent Document 1 proposes a method for producing lactic acid by lactic acid bacteria, wherein the lactic acid bacteria culture medium contains dry yeast as a nitrogen source as a method for producing lactic acid at a high yield and at low cost. Yes. Patent Document 2 discloses a method for efficiently producing a saccharified solution from lignocellulosic biomass, a step of pulverizing lignocellulosic biomass, and a step of hydrolyzing the obtained pulverized product using a hydrolase. A method for producing a saccharified solution is proposed. Furthermore, a method for producing a microbial metabolite such as lactic acid by culturing the microorganism in a medium containing the saccharified solution is proposed.

JP 2007-215428 A JP 2010-104361 A

  This invention made it the subject which should be solved to provide the method of manufacturing D-lactic acid of high optical purity by a high production amount using woody biomass as a raw material and using D-lactic acid producing microbe.

  The present inventors have made various studies on the production of D-lactic acid from biomass raw materials. Among them, we focused on magnesium, which has been a common sense to add in the culture of lactic acid bacteria. Conventionally, magnesium sulfate has usually been added to a medium for culturing lactic acid bacteria as a magnesium source. And when magnesium sulfate is not added, it has been thought that lactic acid bacteria do not grow and do not ferment (produce useful substances). However, the present inventors have found that magnesium derived from a biomass raw material is used, and that even when a medium containing a relatively low concentration of magnesium is used, it is possible to carry out a good culture of lactic acid bacteria. Not only that, but also unexpectedly, it has been found that high optical purity D-lactic acid can be produced in a high production amount using a medium containing a low concentration of magnesium, and the present invention has been completed.

That is, according to the present invention, the following inventions are provided.
[1] A method for producing D-lactic acid by fermentative production of D-lactic acid from woody biomass using a D-lactic acid-producing bacterium, wherein the fermentation by the D-lactic acid-producing bacterium has a magnesium concentration of 32 mg / L or more and 180 mg / L The manufacturing method of D-lactic acid performed in the culture medium which is the following.
[2] A method for producing D-lactic acid by fermentatively producing D-lactic acid from woody biomass using a D-lactic acid-producing bacterium, wherein fermentation by the D-lactic acid-producing bacterium has a magnesium concentration of 6500 mg / L or less, And the manufacturing method of D-lactic acid performed in the culture medium containing woody biomass.
[3] A method for producing D-lactic acid by fermentative production of D-lactic acid from woody biomass using a D-lactic acid-producing bacterium, wherein the fermentation by the D-lactic acid-producing bacterium has a magnesium concentration of 32 mg / L or more and 6500 mg / L A method for producing D-lactic acid, which is performed in a medium containing at least one selected from the group consisting of aluminum, manganese, iron, strontium, and barium.

[4] The D according to any one of [1] to [3], wherein the fermentation with D-lactic acid bacteria is performed by simultaneously acting an enzyme and a D-lactic acid-producing bacterium to perform saccharification and fermentation. -Method for producing lactic acid.
[5] The method for producing D-lactic acid according to any one of [1] to [4], wherein the woody biomass is pretreated before saccharification or before saccharification and fermentation are performed in parallel.
[6] The method for producing D-lactic acid according to any one of [1] to [5], wherein the D-lactic acid-producing bacterium is a lactic acid bacterium belonging to the genus Lactobacillus.
[7] The method for producing D-lactic acid according to any one of [1] to [6], wherein the D-lactic acid-producing bacterium is a lactic acid bacterium belonging to Lactobacillus delbruki.

[8] Fermentation by D-lactic acid bacteria is performed by simultaneously acting an enzyme and a D-lactic acid-producing bacterium to perform saccharification and fermentation, and the saccharification and fermentation are performed in the presence of a base. [1] The method for producing D-lactic acid according to any one of [7] to [7].
[9] The method for producing D-lactic acid according to [8], wherein the base is calcium carbonate.
[10] The method for producing D-lactic acid according to [7] or [8], wherein saccharification and fermentation are performed in parallel under anaerobic conditions.
[11] Any one of [1] to [10], wherein the fermentation by the D-lactic acid-producing bacterium is performed in a medium to which one or more selected from the group consisting of peptone, beef extract, and yeast extract is added. 2. The production method according to item 1.
[12] The production method according to any one of [1] to [11], wherein the yeast extract is a yeast extract derived from brewer's yeast.
[13] The production method according to [12], wherein the yeast extract derived from brewer's yeast is a degradation product decomposed by an enzyme.
[14] The production method according to [13], wherein the degradation product decomposed by an enzyme is a degradation product containing an amino acid, a peptide, and a nucleic acid.

  ADVANTAGE OF THE INVENTION According to this invention, D-lactic acid with high optical purity can be manufactured with a high production amount using D-lactic acid-producing bacteria using woody biomass as a raw material.

Hereinafter, the present invention will be described in more detail.
<Woody biomass>
Woody biomass used as a raw material in the method of the present invention includes paper-making trees, forest residue, chips such as thinned wood or bark, sprouting generated from stumps of woody plants, sawdust or sawdust generated from a sawmill, Pruning branches and leaves of street trees, building waste, etc. Furthermore, wood-derived paper, waste paper, pulp and the like can be used as raw materials. These woody biomass can be used alone or in combination. Further, the woody biomass can be used as a dry solid, a solid containing moisture, or a slurry.

  As a raw material of woody biomass, Eucalyptus genus plant, Salix genus plant, Poplar genus plant, Acacia genus plant, Cryptomeria genus plant and the like can be used. In particular, Eucalyptus plants, Acacia plants, and Willow plants are preferable because they can be easily collected in large quantities as raw materials. For example, bark of tree species such as Eucalyptus genus or Acacia genus commonly used for papermaking raw materials can be obtained in large quantities stably from lumber mills and chip factories for papermaking raw materials. Preferably used.

  In the present invention, the woody biomass as a raw material is not only a source of saccharides (carbon source) utilized by D-lactic acid-producing bacteria but also a magnesium source important for the growth and fermentation of D-lactic acid-producing bacteria. . Woody biomass is also important as one or more sources, optionally selected from the group consisting of aluminum, manganese, iron, strontium and barium.

(Preprocessing)
In the present invention, the woody biomass can be subjected to pretreatment suitable for saccharification and fermentation. A wood biomass that has been pretreated may be referred to as a “pretreated raw material”. As pre-processing, any of the following processes can be mentioned. By performing such pretreatment, the lignocellulose in the woody biomass is in a state capable of saccharification and fermentation. Mechanical treatment, chemical treatment, hydrothermal treatment, pressurized hot water treatment, carbon dioxide added hydrothermal treatment, steaming treatment, wet grinding treatment, dilute sulfuric acid treatment, steam explosion treatment, ammonia explosion treatment, carbon dioxide explosion treatment, ultrasonic irradiation Treatment, microwave irradiation treatment, electron beam irradiation treatment, gamma ray irradiation treatment, supercritical treatment, subcritical treatment, organic solvent treatment, phase separation treatment, wood decay fungus treatment, green solvent activation treatment, various catalyst treatments, radical reaction Treatment, ozone oxidation treatment.
These processes may be either single processes or a combination of a plurality of processes. Among these, it is preferable to perform one or more pretreatments selected from alkali treatment, pressurized hot water treatment, and mechanical treatment on woody biomass.

  Examples of the mechanical treatment include arbitrary mechanical means such as crushing, cutting, and grinding, and making lignocellulose in the woody biomass easy to be saccharified and fermented in the saccharification and fermentation treatment step. Although it does not specifically limit about the mechanical apparatus to be used, For example, a uniaxial crusher, a biaxial crusher, a hammer crusher, a refiner, a kneader etc. can be used.

  The chemical treatment is a treatment in which woody biomass is immersed in an aqueous solution of a chemical such as acid or alkali to make it suitable for enzymatic saccharification treatment. The chemicals used for the chemical treatment are not particularly limited. For example, at least one selected from alkali metal or alkaline earth metal hydroxides, sulfuric acid, dilute sulfuric acid sulfides, carbonates or sulfites. Is. As the chemical treatment, an alkali treatment obtained by immersing in an aqueous solution of one or more kinds of chemicals selected from sodium hydroxide, calcium hydroxide, sodium sulfide, sodium carbonate, calcium carbonate, sodium sulfite and the like is suitable. Further, chemical treatment with an oxidizing agent such as ozone or chlorine dioxide is also possible. The chemical treatment is preferably performed as a post-treatment of the pretreatment in combination with the mechanical treatment. The amount of chemicals used for chemical treatment can be arbitrarily adjusted according to the situation, but from the viewpoint of reducing chemical costs and from the viewpoint of preventing yield loss due to cellulose elution and overdegradation, It is desirable that it is 50 mass parts or less with respect to 100 mass parts of absolutely dry. The immersion time and the treatment temperature of the chemical in the chemical treatment can be arbitrarily set depending on the raw materials and chemicals to be used, but a treatment time of 30 minutes to 1 hour and a treatment temperature of 80 to 130 ° C. are preferable. By tightening the processing conditions, elution or excessive decomposition of cellulose in the raw material may occur, so that the processing time is preferably 1 hour or less and the processing temperature is 130 ° C. or less.

  Woody biomass that has been subjected to pretreatment suitable for saccharification and fermentation treatment may be sterilized before being used for preparing a suspension containing a lignocellulose raw material. When miscellaneous bacteria are mixed in the woody biomass raw material, a problem arises that when the saccharification is performed by the enzyme, the miscellaneous bacteria consume sugar and the yield of the product is reduced. The sterilization treatment may be a method in which the raw material is exposed to a pH at which bacteria are difficult to grow, such as acid or alkali, but may be a method in which treatment is performed at a high temperature, or a combination of both. About the raw material after acid and alkali treatment, it is preferable to use it as a raw material after adjusting it to neutral vicinity or pH suitable for a saccharification and fermentation process. In addition, even when pasteurized at high temperature, it is preferably used as a raw material after the temperature is lowered to room temperature or a temperature suitable for the saccharification / fermentation process. Thus, by feeding out the raw material after adjusting the temperature and pH, it is possible to prevent the enzyme from being exposed to the outside of the preferred pH and the preferred temperature and being deactivated.

(Hardwood kraft pulp)
In a particularly preferred embodiment of the present invention, hardwood kraft pulp can be used as woody biomass. The wood chip used as a raw material for producing the pulp is not particularly limited as long as it is a hardwood material such as eucalyptus, oak, acacia, beach, tan oak, and alder. Moreover, some softwoods may be included in the hardwood used.
Hardwood unbleached kraft pulp (LUKP) can be obtained by subjecting the above wood chips to kraft cooking. Subsequently, an oxygen delignified pulp can be obtained by an oxygen delignification step. Furthermore, a hardwood bleached kraft pulp (LBKP) can be obtained by subjecting the oxygen delignified pulp to a bleaching treatment.

Kraft cooking can be performed by a known method. For example, when kraft cooking wood, the sulfidity of the kraft cooking solution is 5 to 75%, preferably 20 to 35%, and the effective alkali addition rate is 5 to 30% by weight, preferably 10 to 10% by weight of the absolutely dry wood. The cooking temperature is 140-170 ° C. However, the conditions for kraft cooking are not limited to these. The kraft cooking method may be either a continuous cooking method or a batch cooking method, and when a continuous cooking kettle is used, it may be a cooking method in which cooking white liquor is added in portions, and the method is not particularly limited.
A cooking aid may be added to the cooking liquid used for cooking. Examples include known cyclic keto compounds, for example, benzoquinone, naphthoquinone, anthraquinone, anthrone, phenanthroquinone, and nuclear substitutes such as alkyl and amino of the quinone compounds. Alternatively, a hydroquinone compound such as anthrahydroquinone, which is a reduced form of the quinone compound, may be mentioned. Furthermore, the 9,10-diketohydroanthracene compound which is a stable compound obtained as an intermediate of the anthraquinone synthesis method by Diels Alder method, etc. are mentioned. One or more selected from these may be added. Although an addition rate is not specifically limited, Generally, it is 0.001-1.0 mass% per the absolute dry mass of a wood chip.

The unbleached chemical pulp obtained by the kraft cooking method can be delignified by a known oxygen delignification method through a washing step if desired. As the alkali used in the oxygen delignification method, caustic soda or oxidized kraft white liquor can be used. As the oxygen gas, oxygen from a cryogenic separation method, oxygen from PSA (PRESSURE Swing Adsorption), oxygen from VSA (Vacuum Swing Adsorption), or the like can be used.
In the oxygen delignification step, the oxygen gas and alkali are added to a medium concentration pulp slurry in a medium concentration mixer, and after sufficiently mixed, a reaction that can hold a mixture of pulp, oxygen and alkali for a certain period of time under pressure It is sent to the tower and delignified. Although the addition rate of oxygen gas is not specifically limited, it is 0.5-3 mass% with respect to the absolute dry pulp mass, and an alkali addition rate is 0.5-4 mass%. Moreover, although reaction temperature is 80-120 degreeC, reaction time is 15-100 minutes, and a pulp density | concentration is 8-15 mass%, these conditions are not specifically limited.

  Pulp that has been subjected to oxygen delignification can be sent to the washing step. The washing machine used in the washing process after oxygen delignification and the washing machine used for washing during the multi-stage bleaching process are not particularly limited. For example, a pressure diffuser, a diffusion washer, a pressure drum washer, a horizontal long washer, a press washer, and the like can be given.

  The pulp that has been delignified as described above can be subjected to a multistage bleaching step. The multi-stage bleaching step can be performed by combining known ECF bleaching methods such as chlorine dioxide (D), alkali (E), oxygen (O), hydrogen peroxide (P), and ozone (Z). Further, during the multi-stage bleaching step, a high-temperature acid treatment stage (A), an acid washing stage, an enzyme treatment stage, a high-temperature chlorine dioxide bleaching stage, a peracid bleaching stage using persulfuric acid or peracetic acid can be introduced. A chelating agent treatment stage with ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), or the like can be introduced into the multistage bleaching process.

  From the above, hardwood bleached kraft pulp (LBKP) that can be used as a raw material in a particularly preferred embodiment of the present invention can be obtained.

<Lactic acid bacteria>
In the present invention, D-lactic acid-producing bacteria are used. The D-lactic acid-producing bacterium used in the present invention is not particularly limited as long as it can produce D-lactic acid by fermenting saccharides (hexose, pentose). Examples of D-lactic acid producing bacteria include Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, and Leucostock. (Leuconostoc) or bacteria belonging to the genus Sporolactobacillus. However, it is not limited to these. Specific examples include Lactobacillus delbrueckii, Lactobacillus plantarum, Leuconostoc mesenteroides, and the like. However, it is not limited to these. Moreover, genetically modified microorganisms (bacteria etc.) produced using genetic recombination techniques can also be used. As the genetically modified microorganism, a microorganism capable of producing D-lactic acid by fermenting hexose or pentose can be used without particular limitation.

  Microorganisms may be immobilized and used. By immobilizing microorganisms, the process of separating and re-recovering microorganisms in the next process can be omitted, so that at least the burden required for the recovery process can be reduced and the loss of microorganisms can be reduced. is there. Moreover, the collection of microorganisms can be facilitated by selecting microorganisms having aggregating properties.

<Saccharification treatment and fermentation treatment>
In the embodiment of the present invention, enzymatic saccharification and fermentation using a D-lactic acid-producing bacterium may be sequentially performed, and saccharification and fermentation are performed simultaneously by causing an enzyme and a D-lactic acid-producing bacterium to act simultaneously on woody biomass. It may be done. In addition, in this specification, the fermentation process by a D-lactic acid production microbe may be demonstrated to the example which carries out saccharification and fermentation simultaneously among the embodiments of this invention. However, the explanation also applies to the fermentation process in the case where saccharification by an enzyme and fermentation using a D-lactic acid-producing bacterium are sequentially performed unless otherwise specified.

  D-lactic acid-producing bacteria are used, and saccharides produced by the action of enzymes from raw woody biomass are converted to D-lactic acid by the D-lactic acid-producing bacteria. The liquid used in the process in which such D-lactic acid-producing bacteria are used may be referred to as a culture medium. In the present invention, regarding the medium, when referring to the component concentration, it means the concentration at the start of culture (initial concentration) unless otherwise specified.

(Amount of raw material biomass)
The suspension concentration of the woody biomass used in the saccharification step or the concurrent saccharification and fermentation step can typically be 1.0% by mass or more, preferably 1.5% by mass or more. More preferably, it can be 3.0 mass% or more, More preferably, it can be 4.5 mass% or more, More preferably, it can be 5.0 mass% or more. If it is lower than this, it may not be sufficient as a magnesium source, and depending on the lactic acid bacteria used, growth and fermentation are not sufficiently performed. Moreover, it is because the problem that the density | concentration of a product will eventually become too low and the concentration cost of a product will become high will generate | occur | produce. The upper limit of the suspension concentration is, for example, 30% by mass or less, preferably 25% by mass or less, more preferably 20% by mass or less, and further preferably 15% by mass or less. More preferably, it can be 10 mass% or less. This is because as the concentration exceeds 30% by mass, it becomes difficult to stir the raw material, which may cause a problem that productivity is lowered.

(Magnesium sulfate, etc.)
In the present invention, a water-soluble magnesium salt such as magnesium sulfate can be added to the medium. According to the study by the present inventors, in culturing D-lactic acid-producing bacteria, the amount of magnesium salt added to the medium may be less than the amount normally considered necessary as described below. I found. Therefore, when adding a magnesium salt, it is preferable to add a relatively small amount such that the concentration in the medium is within the range described below. When it is considered that the magnesium concentration in the medium is sufficiently secured by the suspended woody biomass, it is not necessary to add a magnesium salt. Alternatively, if the concentration of one or more components selected from the group consisting of aluminum, manganese, iron, strontium and barium is considered to be sufficiently secured by the suspended woody biomass, the magnesium salt is added There is no need. In a preferred embodiment of the present invention, a magnesium salt such as magnesium sulfate is not added to the medium.

(Concentration of magnesium, etc. in the medium)
In the present invention, the concentration of magnesium in the medium is 6500 mg / L or less. In the present invention, the amount and concentration of magnesium in the medium are the total amount and concentration of magnesium in all forms contained in the medium, unless otherwise specified. The raw materials contained in the medium that can be the source of magnesium are mainly woody biomass, yeast extract, and added magnesium sulfate, so the magnesium concentration in the medium is calculated as the total contained in each of these raw materials. You can also The same applies to aluminum, manganese, iron, strontium and barium.

  According to the study by the present inventors, in the culture of D-lactic acid-producing bacteria, the magnesium concentration in the medium can be made lower than the concentration that is normally considered necessary. According to the study by the present inventors, in the present invention, the magnesium concentration in the medium can be set appropriately, but is typically 1000 mg / L or less, preferably 500 mg / L or less. . More preferably, it can be 180 mg / L or less, More preferably, it can be 100 mg / L or less, More preferably, it can be 75 mg / L or less. This is because, within this range, D-lactic acid with high optical purity is unexpectedly produced and the production amount of D-sulfuric acid is high. The lower limit value of magnesium in the medium can typically be 32 mg / L or more, preferably 35 mg / L or more. More preferably, it can be 40 mg / L or more, More preferably, it can be 45 mg / L or more, More preferably, it can be 47.5 mg / L or more. If it is lower than this, depending on the lactic acid bacteria used, growth and fermentation are not sufficiently performed.

The lower limit value of aluminum in the medium can typically be 0.41 mg / L or more, preferably 0.82 mg / L or more. The lower limit value of iron in the medium can be typically 0.33 mg / L or more, and preferably 0.66 mg / L or more. The lower limit of strontium in the medium can typically be 0.15 mg / L or more, and preferably 0.29 mg / L or more. Typically, the lower limit of manganese in the medium can be 0.13 mg / L or more, and preferably 0.26 mg / L or more. The lower limit of barium in the medium can typically be 0.09 mg / L or more, and preferably 0.18 mg / L or more.
In any case, the upper limit value in the medium can be appropriately determined as long as the culture and fermentation are not inhibited. Typically, the upper limit value is 50 mg / L or less, and preferably 25 mg / L or less. .
The amount and concentration of components such as magnesium and aluminum in the medium can be measured by methods well known to those skilled in the art, and can be determined as a calculated value from those amounts in the raw materials used.

(Peptone, beef extract, yeast extract)
In a preferred embodiment of the present invention, the medium for culturing the D-lactic acid-producing bacterium contains any one or more selected from the group consisting of yeast extract, peptone (including polypeptone), and beef extract. Also good. Examples of yeast extract include commercially available yeast extract (Difco Laboratories) yeast extract L (MC Food Specialties Co., Ltd.), yeast extract SL-W (MC Food Specialties Co., Ltd.), Ribonex B2-P (Sapporo Beer Co., Ltd.) Company), Ribonex N7-P (Sapporo Beer Co., Ltd.), Mist P1G (Asahi Food and Healthcare), Mist P2G (Asahi Food and Healthcare), Mist AP-1122 (Asahi Food and Healthcare), Eastoc S- Pd (Yeastock Co., Ltd.), fermented taste (fermented paste, fermented powder: MC Food Specialties Co., Ltd.), etc. When using a yeast extract, various types can be selected, and yeasts belonging to the genus Saccharomyces (for example, yeasts belonging to Saccharomyces cerevisiae, Saccharomyces rosei, Saccharomyces ubalum, Saccharomyces cavalieri) can be used. Alternatively, yeast belonging to the genus Candida (for example, yeast belonging to Candida utils) can be used. Extracts obtained from yeast classified as Saccharomyces cerevisiae, which are beer yeast or baker's yeast, can be used. Among the yeast extracts, it is preferable to use a yeast extract derived from brewer's yeast. Moreover, it is preferable to use the yeast extract containing the degradation product decomposed | disassembled with the enzyme in the yeast extract derived from beer yeast. Among yeast extracts derived from brewer's yeast, examples of yeast extracts containing degradation products (amino acids, peptides, nucleic acids) degraded by enzymes include fermentation taste (fermented paste, fermented powder). Examples of peptone include high-poly peptone (Nippon Pharmaceutical Co., Ltd.).

  When any one or more selected from the group consisting of peptone, beef extract, and yeast extract is used, the concentration in the medium (each concentration in the case of using a plurality thereof) can be appropriately determined. Typically, it can be 0.1 to 10% by mass, preferably 0.2 to 7.5% by mass, more preferably 0.4 to 5.0% by mass. Can do. This is because, within this range, the growth and fermentation of D-lactic acid-producing bacteria are sufficiently achieved and economical.

(Presence of base)
In a preferred embodiment of the present invention, saccharification and fermentation can be performed at pH 4.0 to 7.0 in the presence of a base. The pH is more preferably pH 4.5 to 5.8, further preferably pH 4.6 to 5.6, and still more preferably pH 4.9 to 5.5.

  The type of base used is not particularly limited as long as the pH of the culture solution can be adjusted to pH 4.0 to 7.0, and may be an inorganic base or an organic base. Examples of the inorganic base include calcium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide and the like. Examples of the organic base include monoethanolamine, triethanolamine, diisopropanolamine, triisopropanolamine, and lysine. The base is preferably an inorganic base among the above, and particularly preferably calcium carbonate.

  When performing saccharification and fermentation in parallel, the method of allowing an enzyme and a D-lactic acid-producing bacterium to simultaneously act on woody biomass is not particularly limited as long as the pH of the culture solution can be adjusted to pH 4.0 to 7.0. However, according to a preferred embodiment of the present invention, from the bottom of the culture vessel, the first layer containing the base, the second layer containing the woody biomass, and the third layer containing the enzyme and D-lactic acid-producing bacteria are in this order. It can be arranged to perform saccharification and fermentation in parallel. As above-mentioned, by arrange | positioning a 1st layer-a 3rd layer in this order, the effect | action of the base of adjusting the pH of a culture solution to pH 4.0-7.0 can be achieved well.

(Anaerobic / aerobic conditions)
Saccharification and fermentation may be performed under anaerobic conditions or aerobic conditions, but can be preferably performed under anaerobic conditions. By performing saccharification and fermentation under anaerobic conditions, it becomes possible to produce D-lactic acid with a higher optical purity than when it is performed under aerobic conditions. Examples of the anaerobic condition include a condition that the culture solution is not rotated and shaken during the saccharification and fermentation treatment. Even in this case, the culture solution may be stirred once to several times a day. Absent. On the other hand, aerobic conditions include performing saccharification and fermentation treatment while rotating the culture solution. Although the rotation speed at the time of carrying out rotation shaking of a culture solution is not specifically limited, For example, 10-200 rpm etc. can be mentioned.

(Saccharifying enzyme)
Enzymes used for saccharification are cellulolytic enzymes, which are enzymes collectively called cellulases having cellobiohydrolase activity, endoglucanase activity, and betaglucosidase activity. Each cellulolytic enzyme may be added with an appropriate amount of an enzyme having the respective activity. However, commercially available cellulase preparations have the above-mentioned various cellulase activities and also have hemicellulase activity. Since many products are available, a commercially available cellulase preparation may be used.

  Commercial cellulase preparations include the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Phanerochaete, the genus Trametes, and the genus Humiculas There are cellulase formulations derived from Examples of such commercially available cellulase preparations are all trade names, for example, cellulosin T2 (manufactured by HIPI), mecerase (manufactured by Meiji Seika Co., Ltd.), Novozyme 188 (manufactured by Novozyme), cellulase preparation manufactured by Genencor, etc. Is mentioned.

  0.5-100 mass parts is preferable and, as for the usage-amount of the cellulase formulation with respect to 100 mass parts of raw material solid content, 1-50 mass parts is especially preferable.

(Culture conditions)
The temperature for saccharification and fermentation treatment is not particularly limited as long as it is within the optimum temperature range for the enzyme and fermentation, and is usually preferably 25 ° C to 50 ° C, more preferably 37 ° C to 50 ° C. The culture is preferably a continuous type, but may be a batch type. The culture time varies depending on the enzyme concentration and the like, but in the case of a batch type, it is 10 to 240 hours, more preferably 15 to 160 hours. Also in the case of a continuous type, the average residence time is 10 to 150 hours, more preferably 15 to 100 hours.

<Other processes>
In the embodiment of the present invention, a step of recovering D-lactic acid may be included. D-lactic acid produced by fermentation as described above can be recovered from the fermentation broth by separation and purification by a known method. For example, after removing insoluble substances (such as bacterial cells) from the fermentation broth by centrifugation, filtration, etc., desalting with an ion exchange resin, etc., and then using the solution according to conventional methods such as crystallization and column chromatography Of lactic acid can be separated and purified.

<D-lactic acid obtained by the production method of the present invention>
According to the production method of the present invention, D-lactic acid can be produced with high optical purity. Specifically, according to the production method of the present invention, D-lactic acid can be produced with an optical purity of 96% ee or higher, preferably 97% ee or higher, more preferably 98% ee or higher. it can. Various means known in this technical field can be used for the optical purity of D-lactic acid. In the present invention, when referring to the optical purity of D-lactic acid, it means a value calculated by the formula in the section of Examples, unless otherwise indicated.

  According to the production method of the present invention, the production amount of D-lactic acid can be increased. Specifically, according to the production method of the present invention, on the third day of culture, the D-lactic acid concentration in the medium can be 84 g / L or more, preferably 85 g / L or more, More preferably, it can be 86 g / L or more. For the amount of D-lactic acid, various means known in the art can be used.

  D-lactic acid produced using the method of the present invention can be used as a raw material for producing, for example, poly-D-lactic acid or a stereocomplex of poly-L-lactic acid and poly-D-lactic acid. A stereocomplex of poly L-lactic acid and poly D-lactic acid can be a biodegradable plastic having high heat resistance. It is known that D-lactic acid also has uses as an agricultural intermediate.

  The effects of the present invention will be specifically described with reference to the following examples. However, the scope of the present invention is not limited by these examples.

[Example 1]
<Manufacture of hardwood kraft pulp (LBKP)>
Using hardwood mixed wood chips (eucalyptus 70%, acacia 30%), added to wood chips to cooking white liquor prepared to have a liquid ratio of 4, a sulfidity of 28%, and an effective alkali addition rate of 17% (as Na2O). Thereafter, kraft cooking was performed at a cooking temperature of 160 ° C. for 2 hours. After completion of the kraft cooking, the black liquor was separated, and the obtained chips were defibrated. Then, centrifugal dehydration and water washing were repeated three times, and then the uncooked material was removed with a screen to obtain a cooked unbleached pulp (LUKP). To this unbleached pulp absolutely dry mass, NaOH was added at 2.0%, oxygen gas was injected, and oxygen delignification treatment was performed at 100 ° C. for 60 minutes to obtain oxygen delignified pulp (LOKP). Subsequently, the oxygen delignified pulp was subjected to a four-stage bleaching process of D-E-P-D. The pulp concentration at the time of bleaching is adjusted to 10% by mass, and the first chlorine dioxide treatment (D) is performed by treating the dry dry pulp with chlorine dioxide at a rate of 1.0% by mass at 70 ° C. for 40 minutes. Washed with water and dehydrated. Next, an alkali extraction treatment (E) was performed at 70 ° C. for 90 minutes with an NaOH addition rate of 1% by mass with respect to the absolute dry mass of the pulp, washed with deionized water, and dehydrated. Next, the hydrogen peroxide addition rate is 0.2% and the NaOH addition rate is 0.5% with respect to the absolute dry mass of the pulp, and hydrogen peroxide treatment (P) is performed at 70 ° C. for 120 minutes to obtain ion-exchanged water. Washed and dehydrated. Next, the chlorine dioxide addition rate is 0.2% with respect to the absolute dry mass of the pulp, the chlorine dioxide treatment (D) is performed at 70 ° C. for 120 minutes, washed with ion-exchanged water, dehydrated, and the whiteness is 85%. Bleached pulp (LBKP) was obtained.

<Pre-culture of D-lactic acid producing bacteria>
Lactobacillus delbrueckii subsp. Delbrueckii NBRC3202 strain [available from National Institute for Product Evaluation and Technology (2-5-8 Kazusa-Kamazu, Kisarazu-shi, Chiba)] It was. After thawing, preculture medium sterilized by autoclaving (see Table 1 below for medium composition, pH adjusted to 5.5 with 70% sulfuric acid before autoclaving) 10 ml of platinum ear inoculation and stationary culture at 37 ° C for 48-72 hours The culture solution was prepared in advance. Furthermore, 0.9 ml of the previous culture solution was inoculated into 30 ml of the sterilized preculture medium. This was subjected to static culture at 37 ° C. for 48 hours to prepare a preculture liquid (hereinafter referred to as “lactic acid bacteria preculture liquid”).

<Fermentation paste>
Enzymatic decomposition yeast extract (Kirin Kyowa Foods Co., Ltd.) using 100% brewer's yeast. 61-67% solids, 5.5% or more total nitrogen per solid, pH 4.8-6.0.

<Concurrent saccharification and fermentation>
Next, 6 g of calcium carbonate and 7.5 g of LBKP were added as pH adjusters to a culture container (250 ml plastic conical flask with a sterilizing filter cap), and then sterilized by an autoclave.
Next, in the clean bench, the concentration of the fermented paste (diluted to 80 g / L solid content, adjusted to pH 5.5 with 70% sulfuric acid and sterilized by autoclave) as the nitrogen source is 1.4% by mass. Added. Thereafter, 2 ml of cellulase preparation manufactured by Genencor and 2 ml of lactic acid bacteria preculture solution were added, and a medium (hereinafter, “medium A”) was prepared with distilled water so that the total volume became 100 ml.

  A cut tape was put on a sterilizing filter attached to the cap of the culture vessel (flask), and carbon dioxide generated by the reaction of lactic acid and calcium carbonate produced by fermentation was released out of the flask. And it applied so that the air inflow from the outside might be suppressed as much as possible, and it tightened with the cap to the flask, and blocked | blocking ventilation | gas_flowing with the exterior.

  The culture vessel was cultured at 48 ° C. for 3 days. The culture was allowed to stand for the first day of the culture, and was subjected to rotary shaking for the remaining 2 days. The pH, glucose, and D-lactic acid of the culture solution were measured over time. Glucose and lactic acid were analyzed by the following method. The results are shown in Table 2. In addition, since the amount of magnesium in LBKP is 213 ppm and 214 ppm, respectively, and is 213.5 ppm on average in two measurements, the amount of magnesium in the medium of 7.5% LBKP is calculated to be 0.016 g / L. . In addition, the measurement of the amount of magnesium in LBKP etc. followed the following method.

LBKP and the like are incinerated (about 525 ° C., about 2 hours), added with 5 ml of concentrated nitric acid, and boiled for about 10 minutes.
Note 1) Concentrated nitric acid boiling point 121 ° C.
Note 2) Boiling time 10 minutes: From factory wastewater test method JIS K102. The boiling treatment with nitric acid is applied to samples with very little organic matter or suspension.
After boiling, the volume was increased to 50 ml, filtered through a membrane filter (0.45 μm), and then measured with ICP-OES (CIROS120 manufactured by Rigaku).

<Analysis of glucose and lactic acid>
Immediately after the start of the culture, 2 ml of the culture solution on the first day, the second day, and the third day was collected, centrifuged, and the supernatant was filtered through a 22 μm disk filter (DISMIC 13HP020AN, manufactured by ADVANTEC). Next, the sample was diluted 20 times with demineralized water to obtain a glucose and lactic acid quantification sample (hereinafter referred to as “quantification sample”).

(Quantification of glucose)
300 μL of the sample for quantification was collected in a dedicated cell, set in an autosampler of a biosensor (BF-5 / Oji Scientific Instruments) and automatically measured. As the electrode, a planar electrode replacement glucose electrode EDO05-0003 / manufactured by Oji Scientific Instruments) was used.

(Quantification of lactic acid)
1 ml of the sample for quantification was collected in a microvial, set in an autosampler, and measured using a high performance liquid chromatograph HPLC (alliance 2695 / Waters) under the following conditions.
Column: SUMICHIRAL OA-5000 manufactured by Sumitomo Analysis Center Co., Ltd. (inner diameter: 4.6 mm, column length: 25.0 cm) Temperature: 30 ° C.
Mobile phase: 2 mM CuSO4 · 7H2O in water-2-propanol (98: 2) Solution flow rate: 1.0 ml / min
Detection wavelength: 254 nm

<Measurement of optical purity>
The optical purity of D-lactic acid was calculated by the following formula.
Optical purity (% ee) = (D−L) / (D + L) × 100
Here, D represents the D-lactic acid concentration, and L represents the L-lactic acid concentration.

  The amount of D-lactic acid was 84.3 g / L on the third day of culture, and it was confirmed that the fermentation was good. The optical purity was 98.0% ee.

[Example 2]
<Preparation of raw material for pretreatment>
Chip-like eucalyptus and globula woodland residues (70% bark, 30% branches and leaves) were crushed with a uniaxial crusher (SC-15, manufactured by Saiho Kiko Co., Ltd.) equipped with a 20 mm round hole screen and used as a raw material.
After adding 200 g of 97% sodium sulfite and 10 g of sodium hydroxide to 1 kg (absolute dry weight) of the raw material, water was added to adjust the volume of the aqueous solution to 8 L. The raw material suspension was mixed and then heated at 170 ° C. for 1 hour. The raw material suspension after the heat treatment was ground by a refiner (manufactured by Kumagai Riki Kogyo Co., Ltd., KRK high concentration disc refiner) with a disc (plate) clearance of 1.0 mm. Next, the raw material suspension after the grinding treatment was subjected to solid-liquid separation (dehydration) using a 20 mesh (847 μm) screen, and washed with water until the electric conductivity of the solution reached 30 μS / cm. Simultaneous saccharification and fermentation was carried out using the solid content after solid-liquid separation as a raw material (hereinafter referred to as “pretreatment raw material”).

<Concurrent saccharification and fermentation>
In Example 1, it tested by the method similar to Example 1 except having used the pre-processing raw material prepared above instead of LBKP. The results are shown in Table 2.

  The amount of D-lactic acid was 79.8 g / L on the third day of culture, and it was confirmed that fermentation was good as in Example 1. The optical purity was 96.1% ee.

[Reference Example 1]
In Example 1, it tested by the method similar to Example 1 except having added so that the density | concentration of glucose might be 6 mass% instead of LBKP. The results are shown in Table 3.

  The amount of D-lactic acid produced was very low at 2.9 g / L compared with 79.8 g / L in Example 1, and almost no fermentation was possible.

[Reference Example 2]
In Example 1, the test was performed in the same manner as in Example 1 except that the final concentration of magnesium sulfate heptahydrate was 0.14% by mass to Medium A. The results are shown in Table 2.

  The production amount of D-lactic acid was 83.5 g / L on the third day of culture, and the optical purity was 97.7% ee. From the above results, it was confirmed that when LBKP was used as a raw material, D-lactic acid was efficiently produced without adding magnesium sulfate to the medium.

[Reference Example 3]
In Example 1, the test was conducted in the same manner as in Example 1 except that the final concentration of magnesium sulfate heptahydrate was 7.0% by mass in Medium A. The results are shown in Table 2.

The production amount of D-lactic acid was 83.9 g / L on the third day of culture, and the optical purity was 95.7% ee.

表中、「Mg濃度」として示した値は、マグネシウム分についての値
（硫酸マグネシウム・７水和物としての添加量は、参考例2は1,400mg/L、参考例3は70,000mg/L）

In the table, the value shown as “Mg concentration” is the value for magnesium content (magnesium sulfate heptahydrate added amount is 1,400 mg / L for Reference Example 2 and 70,000 mg / L for Reference Example 3)

[Example 3]
In Example 1 (<parallel saccharification and fermentation>), everything was tested in the same manner as in Example 1 except that the concentration of the fermented paste was 3.0% by mass. The results are shown in Table 3.

[Example 4]
In Example 1 (<concurrent saccharification and fermentation>), everything was tested in the same manner as in Example 1 except that the concentration of the fermentation paste was 2.0% by mass. The results are shown in Table 3.

[Example 5]
In Example 1 (<parallel saccharification and fermentation>), everything was tested in the same manner as in Example 1 except that the concentration of the fermented paste was 1.0% by mass. The results are shown in Table 3.

[Example 6]
In Example 1 (<parallel saccharification and fermentation>), everything was tested in the same manner as in Example 1 except that the concentration of the fermented paste was 0.75% by mass. The results are shown in Table 3.

[Example 7]
In Example 1 (<concurrent saccharification and fermentation>), everything was tested in the same manner as in Example 1 except that the concentration of the fermentation taste (powder) was 3.0% by mass. The results are shown in Table 4.

[Example 8]
In Example 1 (<parallel saccharification and fermentation>), everything was tested in the same manner as in Example 1 except that the concentration of the fermentation taste (powder) was 2.0% by mass. The results are shown in Table 4.

[Example 9]
In Example 1 (<concurrent saccharification and fermentation>), everything was tested in the same manner as in Example 1 except that the fermented taste (powder) concentration was 1.0% by mass. The results are shown in Table 4.

[Example 10]
In Example 1 (<parallel saccharification and fermentation>), everything was tested in the same manner as in Example 1 except that the concentration of the fermentation taste (powder) was 0.5% by mass. The results are shown in Table 4.

Claims (7)

A method for producing D-lactic acid by fermentative production of D-lactic acid from a hardwood bleached kraft pulp using a D-lactic acid-producing bacterium, or from a pretreatment material obtained by subjecting woody biomass to alkali treatment and mechanical treatment,
Fermentation with D-lactic acid producing bacteria is performed in a medium having a magnesium concentration of 32 mg / L or more and 180 mg / L or less and containing a fermented taste (registered trademark) paste,
The fermented taste (registered trademark) paste is an enzyme-degraded yeast extract using 100% brewer's yeast, containing amino acids, peptides, and nucleic acids, having a solid content of 61 to 67%, and a total nitrogen of 5 per solid content. More than 5%, pH 4.8-6.0 extract,
0.75-10 mass% of the fermented taste (registered trademark) paste is added to the medium,
Magnesium salt is not added to the medium,
Fermentation by D-lactic acid-producing bacteria is performed in parallel with saccharification and fermentation by simultaneously acting an enzyme and D-lactic acid-producing bacteria.
A method for producing D-lactic acid. The method for producing D-lactic acid according to claim 1, wherein the medium is a medium containing one or more selected from the group consisting of aluminum, manganese, iron, strontium and barium. The method for producing D-lactic acid according to claim 1 or 2, wherein the D-lactic acid-producing bacterium is a lactic acid bacterium belonging to the genus Lactobacillus. The method for producing D-lactic acid according to any one of claims 1 to 3, wherein the D-lactic acid-producing bacterium is a lactic acid bacterium belonging to Lactobacillus delbruki. The method for producing D-lactic acid according to any one of claims 1 to 4, wherein the saccharification and fermentation are performed in the presence of a base. The method for producing D-lactic acid according to claim 5, wherein the base is calcium carbonate. The method for producing D-lactic acid according to claim 5 or 6, wherein saccharification and fermentation are performed in parallel under anaerobic conditions.