JP6331327B2 - Method for producing D-lactic acid - Google Patents

Description

  The present invention relates to a method for producing D-lactic acid using pulp as a raw material and using a D-lactic acid-producing bacterium.

  Many methods have been implemented in which woody biomass containing cellulose is used as a raw material, enzymes and microorganisms are added, and saccharification and saccharification and fermentation are performed to obtain a sugar solution and a fermentation broth. Lactic acid is an organic acid produced by the decomposition of sugars such as glucose by a biological glycolysis system, and is also an important compound that is induced by the TCA circuit and becomes the starting point of bioenergy production.

  Non-Patent Document 1 describes that lactic acid is produced by parallel saccharification and fermentation of cellulose. Non-Patent Documents 2 and 3 describe that lactic acid is produced by pulverizing and chemically treating a piece of wood, followed by parallel saccharification and fermentation using cellulase and lactic acid bacteria. Non-Patent Document 4 describes that L-lactic acid is produced by fermentation of a wood hydrolyzate by Entetococcus faecalism. Non-Patent Document 5 describes that D-lactic acid is produced by parallel saccharification and fermentation using cellulase and Lactobacillus coryniformis. Non-Patent Document 6 describes that lactic acid is produced by parallel saccharification and fermentation using paper sludge as a raw material.

  Patent Document 1 describes a method for producing lactic acid from cassava pulp at high yield and low cost. However, this method is a method for producing lactic acid, but is not a method for producing only D-lactic acid.

  Furthermore, Patent Document 2 describes that D-lactic acid is fermented using a lactic acid bacterium belonging to Lactobacillus delbrueckii. This document ferments D-lactic acid using glucose as a substrate, and there is no description about using a raw material other than glucose as a substrate.

Shin-ichiro Abe and Motoyoshi Takagi, Simultaneous Saccharification and Fermentation of Cellulose to Lactic Acid, Biotechnology and Bioengineering, Vol. 37, Pp. 93-96 (1991) Moldes, A. B., Alonso, J. L. and Parajo, J. C, Strategies to improve the bioconversion of processed wood into lactic acid by simultaneous saccharification and fermentation., J. Chem. Technol. Biotechnol., 76: 279-284 (2001) Moldes, A. B., Alonso, J. L. and Parajo, J. C., Multi-step feeding systems for lactic acid production by simultaneous saccharification and fermentation of processed wood, Bioprocess Engineering, Volume 22, Issue 2, pp 175-180 (2000) Young-Jung Wee, Jong-Sun Yun, Don-Hee Park, Hwa-Won Ryu, Biotechnological production of l (+)-lactic acid from wood hydrolyzate by batch fermentation of Enterococcus faecalis, Biotechnology Letters, Volume 26, Issue 1, pp 71-74 (2004) R Yanez, A Belen Moldes, JL Alonso, JC Parajo, Production of D (-)-lactic acid from cellulose by simultaneous saccharification and fermentation using Lactobacillus coryniformis subsp.torquens, Biotechnology letters, Volume 25, Issue 14, pp 1161-1164 ( 2003) S Marques, JAL Santos, FM Girio, JC Roseiro, Lactic acid production from recycled paper sludge by simultaneous saccharification and fermentation, Biochemical Engineering Journal, Volume 41, Issue 3, pp 210-216 (2008)

JP 2004-097136 A JP 2010-279332 A

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

  The present inventors diligently studied to solve the above problems. As a result, hardwood bleached kraft pulp (LBKP) is used as a raw material for woody biomass, and a saccharification step by an enzyme and a fermentation step by a D-lactic acid-producing bacterium are sequentially performed to produce a high production amount and high production of D-lactic acid. It was found that it can be manufactured at a speed. Thus, the present invention has been completed.

That is, according to the present invention, the following inventions are provided.
(1) (a) A step of obtaining an enzyme saccharified solution by saccharifying hardwood bleached kraft pulp with an enzyme: and (b) fermenting the enzyme saccharified solution obtained in the step (a) using a D-lactic acid-producing bacterium. To produce D-lactic acid by:
The manufacturing method of D-lactic acid containing this.

(2) The method for producing D-lactic acid according to (1), wherein the enzyme saccharified solution obtained in the step (a) is a solution containing glucose.
(3) The method for producing D-lactic acid according to (1) or (2), wherein the D-lactic acid-producing bacterium is a Lactobacillus lactic acid bacterium.

(4) The method for producing D-lactic acid according to any one of (1) to (3), wherein the D-lactic acid-producing bacterium is a lactic acid bacterium belonging to Lactobacillus delbruki.
(5) The method for producing D-lactic acid according to any one of (1) to (4), wherein the fermentation in the step (b) is performed with stirring at a pH of 5 to 7 and a temperature of 30 ° C to 50 ° C.

  According to the present invention, woody biomass pulp can be used as a raw material, and D-lactic acid can be produced with a high production amount and a high production rate using D-lactic acid-producing bacteria.

FIG. 1 shows changes in the amount of D-lactic acid produced and the amount of glucose when D-lactic acid was produced from an LBKP enzyme saccharified solution (present invention) and glucose (comparative example). Black circles show the case of LBKP enzyme saccharified solution (present invention), and white triangles show the case of glucose (comparative example).

Hereinafter, the present invention will be described in more detail.
<Raw material>
In the present invention, hardwood bleached kraft pulp (LBKP) is used as a raw material. The wood chip used as a raw material for producing LBKP 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 bleached kraft pulp (LBKP) can be obtained by subjecting the above wood chips to kraft cooking and then bleaching.
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 known cyclic keto compound as a cooking aid can be used in the cooking solution used for cooking. For example, benzoquinone, naphthoquinone, anthraquinone, anthrone, phenanthroquinone and the above-mentioned quinone compounds such as alkyl, amino and other nuclear substitutes, or hydroquinone compounds such as anthrahydroquinone, which is a reduced form of the quinone compounds, and Diels Alder One or more selected from 9,10-diketohydroanthracene compounds and the like, which are stable compounds obtained as intermediates in the method of synthesizing anthraquinones, 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.

  By the above, the hardwood bleached kraft pulp (LBKP) used as a raw material in this invention can be obtained.

  In addition, hardwood bleached kraft pulp (LBKP) can be sterilized before being used to prepare the LBKP suspension. When miscellaneous bacteria are mixed in the LBKP raw material, there is a problem that the miscellaneous bacteria consume sugar when the enzyme saccharifies and the yield of the product decreases. 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 an acid or an alkali. 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. Moreover, even when sterilized at high temperature and high pressure, it is preferable to use the raw material after lowering the temperature to room temperature or a temperature suitable for the saccharification 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.

<Saccharification treatment>
In the present invention, hardwood bleached kraft pulp (LBKP) is saccharified prior to fermentation.

  In the saccharification treatment, LBKP is mixed with an appropriate amount of water and an enzyme and supplied to the saccharification and fermentation process. The suspension concentration of LBKP is preferably 1 to 30% by mass. If it is less than 1% by mass, there is a problem in that the concentration of the product is ultimately too low and the cost for concentrating the product becomes high. Moreover, as the concentration exceeds 30% by mass, it becomes difficult to stir the raw materials, resulting in a problem that productivity is lowered.

  Cellulolytic enzymes used for saccharification 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, the genus Humicola, and the like. There are cellulase formulations derived from Commercially available products of such 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), GC220 (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.

  The pH in the saccharification treatment is preferably in the range of 3.5 to 10.0, and more preferably in the range of 4.0 to 7.5.

  The temperature of the saccharification treatment is not particularly limited as long as it is within the optimum temperature range of the enzyme, and is usually preferably 25 to 60 ° C and more preferably 45 to 55 ° C. The reaction is preferably continuous, but may be batch. The reaction time varies depending on the enzyme concentration, but in the case of a batch system, it is 10 to 240 hours, more preferably 15 to 120 hours. Also in the case of a continuous type, the average residence time is 10 to 150 hours, more preferably 15 to 100 hours.

<Fermentation treatment>
The enzyme saccharified solution obtained by the above saccharification step is subjected to fermentation treatment.
The microorganism used for the fermentation treatment is not particularly limited as long as it is a D-lactic acid-producing bacterium that can ferment sugars (hexose sugar, pentose sugar) to produce D-lactic acid. Examples of D-lactic acid-producing bacteria include Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, Leuconostoc (Leuconostoc) or bacteria belonging to the genus Spololactobacillus can be mentioned, but is not particularly limited. Specific examples include Lactobacillus delbrueckii, Lactobacillus plantarum, and Leuconostoc mesenteroides, but are not particularly limited thereto. 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. 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.

  The pH in the fermentation treatment is preferably in the range of 3.0 to 10.0, more preferably in the range of 3.5 to 7.5, and in the range of 4.0 to 7.0. More preferably.

  The temperature of the fermentation treatment is not particularly limited as long as it is within the optimum temperature range of the enzyme, and is usually preferably 25 to 60 ° C, more preferably 30 to 55 ° C. The reaction is preferably continuous, but may be batch. The reaction time varies depending on the enzyme concentration, but in the case of a batch system, 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.

<Recovery of D-lactic acid>
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.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1:
The saccharification and fermentation used in this example consists of (1) two steps of performing saccharification treatment with pulp to obtain an enzyme saccharified solution and then (2) lactic acid fermentation with D-lactic acid producing bacteria ( Lactobacillus delbrueckii ). Become.

(1) Preparation of Enzymatic Saccharification Solution In the present invention, LBKP was produced in order to saccharify hardwood bleached kraft pulp (LBKP) prior to fermentation.
<LBKP manufacturing method>
Using hardwood mixed wood chips (eucalyptus 70%, acacia 30%), the liquid ratio 4, sulfidity 28%, effective alkali addition rate 17% (as Na ₂ O) to cooked white liquor to wood chips After the addition, 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, and then centrifugal dehydration and water washing were repeated three times, and then the uncooked material was removed by a screen to obtain a cooked unbleached pulp. With respect to the absolute dry mass of unbleached pulp, 2.0% by mass of NaOH was added, oxygen gas was injected, and oxygen delignification treatment was performed at 100 ° C. for 60 minutes. 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 ion-exchanged water, and dehydrated. Next, the hydrogen peroxide addition rate is 0.2% by mass and the NaOH addition rate is 0.5% by mass with respect to the absolute dry mass of the pulp, and hydrogen peroxide treatment (P) is performed at 70 ° C. for 120 minutes to perform ion exchange. Washed with water and dehydrated. Next, the chlorine dioxide addition rate is 0.2% by mass with respect to the absolute dry mass of the pulp, and the chlorine dioxide treatment (D) is performed at 70 ° C. for 120 minutes, washed with ion-exchanged water, dehydrated, and the whiteness is 85%. Of bleached pulp (LBKP) was obtained.

<Saccharification treatment of LBKP>
Acetate buffer solution (pH 5.0) is added to a 300 mL culture container containing a predetermined amount of water so that the final concentration of LBKP is 10% and 10 mM, and then autoclaved (121 ° C., 20 minutes). did. Next, 10% by mass of cellulase preparation GC220 (manufactured by Genencor), which has been aseptically filtered through a 0.22 μm disk filter (DISMIC 13HP020AN, manufactured by ADVANTEC), is added to the reaction liquid to prepare a total volume of 100 mL of the reaction liquid. did. The reaction solution was subjected to a saccharification reaction at a treatment temperature of 50 ° C. for 24 hours at a rotation speed of 120 rpm in a rotary shaker.

  Glucose concentration of the resulting saccharification reaction solution is collected by collecting an appropriate amount of saccharification reaction solution, and after centrifugation, the supernatant is filtered through a 0.22 μm disk filter (DISMIC 13HP020AN, manufactured by ADVANTEC) and diluted with deionized water. A sample for measurement was obtained. This measurement sample was measured with a biosensor (BF-5 / Oji Scientific Instruments) using a glucose electrode (EDO05-0003 / Oji Scientific Instruments).

(2) D-lactic acid fermentation <pre-culture>
Lactobacillus delbrueckii subsp. Delbrueckii NBRC3202 strain [available from National Institute of Technology and Evaluation (2-5-8, Kazusa-Kamashita, Kisarazu-shi, Chiba)] thawed at −80 ° C. Pre-culture medium sterilized by autoclave (1 volume% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 1 volume% yeast extract (manufactured by BD), 1 volume% glucose (manufactured by Wako Pure Chemical Industries, Ltd.), 0.02 volume% magnesium sulfate seven Inoculated into 10 ml of hydrate (manufactured by Wako Pure Chemical Industries, Ltd., pH 6.6-7.0). Static culture was performed at 37 ° C. for 48-72 hours to prepare a culture solution in advance. Further, 0.9 ml of the culture solution is 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 solution.

<Main culture>
Main culture medium (1 volume% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 1 volume% yeast extract (manufactured by BD), 0.02 volume% magnesium sulfate heptahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) pH 6.6-7. 0) was placed in a 500 mL culture container and sterilized by autoclave. The saccharification reaction solution was added so that the final glucose concentration was 30 g / L, and the volume was adjusted to 200 mL. 4 ml of the above preculture was inoculated and cultured at 37 ° C. Under the present circumstances, it culture | cultivated anaerobically without performing control of pH for 24 hours after culture | cultivation start, and it stirred and culture | cultivated by controlling to pH 6.0 by adding 5M ammonia water after 24 hours. The glucose concentration of the culture solution was the same as the glucose concentration measurement of the saccharification reaction solution.

The concentration of D-lactic acid and L-lactic acid in the culture solution is determined by taking an appropriate amount of the culture solution, centrifuging, filtering the supernatant with a 0.22 μm disk filter, and diluting with deionized water under the following conditions: Was measured by HPLC method.
Column: Sumikal OA-5000 manufactured by Sumika Chemical Analysis Center (inner diameter: 4.6 mm, column length: 15.0 cm)
Solvent: 2 mM aqueous copper sulfate / 2-propanol 98/2
Flow rate: 1 mL / min Detector: UV (UV absorption, wavelength: 254 nm)
Column temperature: 30 ° C

The optical purity of D-lactic acid was calculated by the following formula.
Optical purity of D-lactic acid (% ee) = {(D-lactic acid concentration)-(L-lactic acid concentration)} / [(D-lactic acid concentration) + (L-lactic acid concentration)} × 100

Comparative Example 1:
Experiments were conducted in the same manner as in Example 1 except that a glucose (manufactured by Wako Pure Chemical Industries, Ltd.) solution (30 g / L) was used instead of the enzymatic saccharified solution of LBKP (prepared to a glucose concentration of 30 g / L after saccharification treatment) went. Thereby, the production of D-lactic acid was confirmed using the D-lactic acid-producing bacterium Lactobacillus delbrueckii subsp. Delbrueckii NBRC3202.

  The results of Example 1 and Comparative Example 1 are shown in FIG. From the LBKP enzyme saccharified solution (containing 30 g / L of glucose), the entire amount of glucose was consumed in 2 days, and D-lactic acid 48.5 g / L was produced. On the other hand, from the glucose solution (30 g / L), the entire amount of glucose was consumed in 5 days to produce 44.2 g / L of D-lactic acid. From the above results, it was shown that fermentation progresses faster in the LBKP enzyme saccharified solution than in glucose.

Claims (3)

(A) A step of obtaining an enzyme saccharified solution containing glucose by saccharifying a hardwood bleached kraft pulp having a suspension concentration of 1 to 30% by mass with cellulase at a pH of 3.5 to 7.5 and a temperature of 25 to 60 ° C . :
And (b) by anaerobically fermenting the enzyme saccharified solution obtained in the step (a) using lactic acid bacteria belonging to Lactobacillus delbruki at pH 3.0 to 7.0 and temperature 30 to 55 ° C. Process for producing D-lactic acid:
The manufacturing method of D-lactic acid containing this. The method for producing D-lactic acid according to claim 1, wherein the fermentation in the step (b) is performed with stirring at a pH of 4.0 to 7.0 and a temperature of 30C to 50C. The manufacturing method of D-lactic acid of Claim 1 or 2 whose pH of the saccharification process in the said process (a) is 3.5-5.0.

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