WO2017169275A1 - Procédé de culture de micro-organisme et procédé de production d'un composé organique - Google Patents

Procédé de culture de micro-organisme et procédé de production d'un composé organique Download PDF

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Publication number
WO2017169275A1
WO2017169275A1 PCT/JP2017/006048 JP2017006048W WO2017169275A1 WO 2017169275 A1 WO2017169275 A1 WO 2017169275A1 JP 2017006048 W JP2017006048 W JP 2017006048W WO 2017169275 A1 WO2017169275 A1 WO 2017169275A1
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culture
microorganism
culture solution
organic compound
saccharide
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PCT/JP2017/006048
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English (en)
Japanese (ja)
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和典 澤田
秀一 湯村
川上　公徳
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三菱ケミカル株式会社
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Priority to JP2018508568A priority Critical patent/JP6911835B2/ja
Publication of WO2017169275A1 publication Critical patent/WO2017169275A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Patent Document 1 As a method of obtaining a sugar solution from a non-edible raw material, a method in which cellulose or hemicellulose in a non-edible raw material is hydrolyzed to a simple sugar such as hexose represented by glucose or pentose represented by xylose using concentrated sulfuric acid. (Patent Document 1) and a method (Patent Document 2) of hydrolyzing by an enzymatic reaction after performing a pretreatment for improving the reactivity of a non-edible raw material.
  • cellulose and hemicellulose in non-edible materials are hydrolyzed to obtain sugars such as glucose and xylose, and not only the decomposition reaction of these sugars proceeds, but also lignin is hydrolyzed. Disassembled.
  • a carbonyl compound other than a saccharide is produced by a sugar decomposition reaction or lignin hydrolysis.
  • water-soluble aldehyde compounds such as formaldehyde, acetaldehyde and glyoxal, and aldehyde compounds of furan derivatives such as furfural and hydroxymethylfurfural.
  • Carbonyl compounds such as aliphatic aldehyde compounds such as formic acid, aromatic aldehyde compounds such as vanillin, and ketone compounds such as benzoquinone are formed.
  • Non-Patent Documents 2 and 3 use sulfite gas or phosphoric acid that has the effect of suppressing the by-product of the inhibitor in the pretreatment step of the saccharification treatment, and the inhibitor concentration in the culture medium is low and sufficient for the inhibitor. It may not be adapted.
  • the concentration of hydroxymethylfurfural, which is an inhibitory substance is relatively low, and there is a possibility that microorganisms obtained by culturing are not sufficiently adapted to the inhibitory substance.
  • the culture solution used in the culture method (cultivation step) of the present invention can adapt the microorganism to the inhibitory substance by containing the microorganism growth inhibitory substance and / or fermentation inhibitory substance at a certain concentration or higher.
  • the inhibitor concentration in the culturing process is low, it cannot sufficiently adapt to the inhibitor, and the fermentation inhibition in the subsequent fermentation process cannot be significantly improved.
  • the value of the carbonyl value derived from the compound excluding saccharide is 100 ⁇ mol eq or more, preferably 175 ⁇ mol eq or more, more preferably 220 ⁇ mol eq or more, and more preferably 250 ⁇ mol eq or more per 1 g of the medium.
  • the concentration of furfural contained in the culture solution used in the culture method (cultivation step) of the present invention is not particularly limited as long as it contains an inhibitor necessary for sufficiently adapting the microorganism to the inhibitor. It is preferably 380 ⁇ g or more, more preferably 390 ⁇ g or more, and further preferably 400 ⁇ g or more.
  • Furfural is a typical inhibitor produced by the excessive decomposition of pentose, and if the furfural is within the above range, the microorganism can sufficiently adapt to the inhibitor.
  • the concentration of furfural is too high, the growth of microorganisms is remarkably deteriorated or stopped, and it is difficult to obtain a necessary amount of microorganisms or processed products thereof.
  • the growth rate is obtained from the following formula (1) using the bacterial cell concentration at the start of culture and the value of the bacterial cell concentration at an arbitrary time before reaching the stationary phase.
  • Growth rate ⁇ ln (Xt / X0) ⁇ / t (1) (Xt: Cell concentration at an arbitrary time point before reaching the stationary phase, X0: Cell concentration at the start of culture, t: Culture time until an arbitrary time point)
  • the bacterial cell concentration can be measured by a method known to those skilled in the art. For example, the absorbance at a wavelength of 660 nm can be used as the bacterial cell concentration.
  • coryneform bacteria that can be used in the present invention are not particularly limited as long as they are classified in this, but bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, bacteria belonging to the genus Arthrobacter Among them, preferred are those belonging to the genus Corynebacterium and Brevibacterium, and more preferred are Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes or Brevibacterium lactofermentum Bacteria classified as:
  • examples of the bacterium belonging to the genus Basfia that can be used in the present invention include Basfia succiniciproducens.
  • examples of the genus Zymomonas that can be used in the present invention include Zymomonas mobilis.
  • examples of the genus Zymobacter that can be used in the present invention include Zymobacter palmae.
  • filamentous fungi examples include Aspergillus, Penicillium, and Rhizopus. Among them, Aspergillus niger (Aspergillus niger), Aspergillus oryzae, etc. are mentioned in Aspergillus genus, and Penicillium chrysogenium (Penicillium chrysogenis) in Penicillium genus. . In the genus Rhizopus, Rhizopus oryzae and the like can be mentioned.
  • the microorganism is not only a wild strain, but also a mutant strain obtained by a normal mutation treatment such as UV irradiation or NTG treatment, a recombinant strain induced by a genetic technique such as cell fusion or gene recombination.
  • a stock of The microorganism used in the present invention may be a microorganism that inherently has an organic compound-producing ability, or may have been imparted with an organic compound-producing ability by breeding.
  • microorganism used in the present invention may be a microorganism originally having the ability to assimilate saccharides used in the present invention, or may be one imparted with the ability to assimilate saccharides by breeding.
  • the production method of the present invention is a method for producing an organic compound through a fermentation process in which a microorganism obtained in the culture process cultured by the culture method described above or a processed product thereof is prepared and allowed to act on a fermentation raw material containing saccharides. It is. Therefore, the production rate of the organic compound in the fermentation process can be improved by using a growth inhibitory substance and a microorganism adapted to the fermentation inhibitory substance in the culturing process.
  • the fermentation raw material used in the production method of the present invention contains saccharides.
  • a saccharide the same thing as the saccharide used with the culture solution of the above-mentioned culture
  • the fermentation raw material preferably contains a saccharide obtained by thermal decomposition or hydrolysis of the lignocellulose raw material, and it is particularly preferred to use the saccharified solution containing the saccharide in the fermentation raw material as it is.
  • the fermentation raw material used in the production method of the present invention preferably contains the same inhibitor as the culture solution in the culturing step in order to significantly exhibit the effects of the present invention.
  • the carbonyl value derived from the compound excluding saccharides in the fermentation raw material is preferably 40% or more of the carbonyl value derived from the compound excluding saccharides in the culture broth, more preferably 60% or more, and still more preferably 80%. % Or more, particularly preferably 100% or more, and usually 250% or less.
  • the fermentation raw material may be, for example, a medium suitable for culturing microorganisms or a buffer solution such as a phosphate buffer, but the reaction solution is an aqueous solution containing a nitrogen source, an inorganic salt, or the like. It is preferable.
  • the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated by the microorganism to produce an organic compound. Specifically, ammonium salt, nitrate, urea, soybean hydrolysate, casein degradation product And various organic and inorganic nitrogen compounds such as peptone, yeast extract, meat extract and corn steep liquor.
  • the organic compound produced by the microorganism is not particularly limited as long as the microorganism is an organic compound that can be produced and accumulated in the medium.
  • the microorganism is an organic compound that can be produced and accumulated in the medium.
  • ethanol propanol, butanol, 1, Alcohols such as 3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, glycerol, erythritol, xylitol, sorbitol Amines such as 1,5-pentamethylenediamine and 1,6-hexamethylenediamine; acetic acid, butyric acid, glycolic acid, lactic acid, 3-hydroxypropionic acid, pyruvic acid, succinic acid, fumaric acid, malic acid, oxalo Acetic acid, cis-acon
  • the amount of microbial cells used in the organic compound production reaction (fermentation process) is not particularly limited, but is usually 1 g / L or more, preferably 10 g / L or more, more preferably 20 g / L or more as the weight of wet cells. On the other hand, it is usually 400 g / L or less, preferably 300 g / L or less, more preferably 200 g / L or less.
  • the time for the organic compound production reaction is not particularly limited, but is usually 1 hour or longer, preferably 3 hours or longer, and is usually 168 hours or shorter, preferably 72 hours or shorter.
  • the organic compound production reaction (fermentation step) may be carried out at the same temperature as the optimum growth temperature of the microorganism to be used, but it is advantageous to carry out the reaction at a temperature higher than the optimum growth temperature. C., preferably 7 to 15 ° C. higher. Specifically, in the case of yeast, it is usually 33 ° C or higher, preferably 35 ° C or higher, and is usually 40 ° C or lower, preferably 38 ° C or lower.
  • an organic compound can be produced by the organic compound production reaction (fermentation step) and accumulated in the reaction solution.
  • the accumulated organic compound may further include a step of recovering from the aqueous medium according to a conventional method.
  • the accumulated organic compound is an alcohol such as ethanol, butanol, or butanediol
  • solid substances such as cells are removed by centrifugation, filtration, etc., and then concentrated by distillation or the like.
  • the alcohol can be recovered by dehydrating the solution.
  • the accumulated organic compound is a carboxylic acid such as succinic acid, fumaric acid, malic acid, etc.
  • carboxylic acid such as succinic acid, fumaric acid, malic acid, etc.
  • the carboxylic acid can be recovered from the solution by crystallization (crystallization) or purification by column chromatography.
  • LC analysis Glucose, xylose, fructose, sucrose, ethanol, furfural, and hydroxymethylfurfural abundance in the sugar solutions of the following production examples, examples and comparative examples are obtained by liquid phase chromatography (LC) analysis using an absolute calibration curve method. Asked.
  • the analysis conditions are as follows.
  • Carbonyl value derived from compounds other than sugars A method for analyzing a carbonyl value derived from a compound excluding saccharide is shown below.
  • the carbonyl value can be obtained by reacting a non-edible saccharified solution with hydroxylamine hydrochloride and measuring the amount of potassium hydroxide required for neutralization of the generated hydrochloric acid, thereby obtaining the total amount of carbonyl components in the sample.
  • this method not only neutralization titration containing the acid originally contained in the sample but also a carbonyl component derived from a reducing sugar is included.
  • Carbonyl number ⁇ (A1 ⁇ 500 / S1) ⁇ ((G1 + F1) ⁇ 7.6) ⁇ (X1 ⁇ 38) ⁇ ⁇ (A2 ⁇ 100 / S2) ( ⁇ moleq / g) (2)
  • A1 is a titration amount (0.5 mL) of 0.5 M potassium hydroxide required for neutralization of the sample reacted with hydroxylamine hydrochloride
  • A2 is 0.1 M water required for neutralization of the unreacted sample.
  • Bagasse was used as a non-edible material.
  • sulfuric acid and water were added to and mixed with bagasse to obtain a bagasse mixture.
  • the addition amount of sulfuric acid was 2% by weight with respect to the dry weight of bagasse, and the addition amount of water was adjusted so that the water content with respect to the total weight of the bagasse mixture was 60% by weight.
  • the bagasse mixture was mixed for 20 minutes with a drum mixer (manufactured by Sugiyama Heavy Industries, Ltd.) and stirred and stirred to obtain a diluted sulfuric acid treatment mixture.
  • Steam was added to the dilute sulfuric acid treatment mixture in a hydrolysis apparatus (manufactured by Yasima Co., Ltd.), and steamed at 180 ° C.
  • the water content of the obtained steamed product was 64.6% by weight.
  • the steamed product was charged into a saccharification apparatus so that the dry weight was 200 g / L, and a 10N-NaOH aqueous solution was added to adjust the pH to 6.0.
  • CTec2 for 15 FPU manufactured by Novozyme
  • hydrolysis was carried out with stirring at a temperature of 50 ° C. and a stirring speed of 200 rpm for 72 hours. Thereafter, centrifugation (8000 ⁇ g, 10 minutes) was performed to separate and remove undegraded cellulose and lignin to prepare a bagasse saccharified solution.
  • the composition of the obtained bagasse saccharified solution is shown in Table 1.
  • Example 2 The same procedure as in Example 1 was performed except that 177 mL of saccharified solution 1 and 1 mL of sterile distilled water were added to a 500 mL Erlenmeyer flask and main culture was performed.
  • Example 1 The carbonyl value derived from the compound excluding the saccharide in the culture broth at this time was 449 ⁇ mol eq / g.
  • a culture bacterial solution was prepared.
  • (B) as main culture in a 500 mL Erlenmeyer flask, 80 mL of 50 wt% glucose solution heated in advance at 121 ° C. for 20 minutes and 100 mL of sterilized distilled water were added, and 200 g / day heated in advance at 121 ° C. for 20 minutes. 20 mL of L yeast extract solution was added and mixed.
  • Example 2 The same procedure as in Example 1 was performed except that 35 mL of saccharified solution 1 and 143 mL of sterilized distilled water were added to a 500 mL Erlenmeyer flask and main culture was performed. The carbonyl value derived from the compound excluding saccharide in the culture broth at this time was 90 ⁇ mol eq / g.
  • O. D. The results of (660 nm) are shown in Table 3, and the ratio of each growth rate to the growth rate of Comparative Example 1, which is a condition that does not contain an inhibitor of non-edible sugar, and the growth rate calculated from this result. It is shown in Table 4.
  • the growth rate is calculated from the transition of the bacterial cell concentration during the combined period of the induction phase and the logarithmic growth phase.
  • Example 3 ⁇ Fermentation inhibition reduction evaluation using sugar solution> [Example 3] First, 66 mL of bagasse saccharified solution obtained in Production Example 1, 3 mL of distilled water, and 0.015 g of an antifoaming agent (LG-294, manufactured by ADEKA Corporation) were added to a 150 mL jar fermenter (manufactured by Biot Co., Ltd.). A fermentation raw material liquid was prepared.
  • LG-294 an antifoaming agent
  • the culture solution obtained in (B) main culture of Example 1 was separated into cells and culture supernatant by centrifugation at 1000 ⁇ g, 10 minutes, 20 ° C.
  • the culture supernatant was removed, sterilized water was added and resuspended so that the wet cell mass was 0.5 g / mL, and a bacterial solution for fermentation was prepared. 4.5 mL of this was inoculated into the fermentation raw material liquid. Aeration was not performed, and the temperature was 35 ° C. and stirring was performed at 200 rpm.
  • the pH was adjusted to 4.5 with 1M sulfuric acid or 5.4 wt% aqueous ammonia, and ethanol fermentation was performed.
  • Example 4 Ethanol fermentation was carried out in the same manner as in Example 3 except that the culture solution obtained in Example 2 was used for the preparation of the bacterial solution for fermentation.
  • Comparative Example 3 Ethanol fermentation was performed in the same manner as in Example 3 except that the culture solution obtained in Comparative Example 1 was used for the preparation of the bacterial solution for fermentation.
  • Example 4 Ethanol fermentation was performed in the same manner as in Example 3 except that the culture solution obtained in Comparative Example 2 was used for the preparation of the bacterial solution for fermentation. As a result of Examples 3 and 4 and Comparative Examples 3 and 4, transition of ethanol concentration obtained by LC analysis is shown in Table 5.
  • a microorganism capable of improving the production rate of the organic compound when used for the fermentation production of the organic compound using a sugar solution containing a growth inhibitory substance and / or a fermentation inhibitory substance can be obtained. it can.
  • the production rate of the organic compound can be improved in the fermentation production of the organic compound using a sugar solution containing a growth inhibitory substance and / or a fermentation inhibitory substance.

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Abstract

La présente invention concerne le problème de préparation d'un procédé de culture d'un micro-organisme, moyennant quoi le micro-organisme peut s'adapter à un inhibiteur et ainsi l'inhibition de la fermentation peut être suffisamment réduite, et un procédé de production d'un composé organique dans lequel le micro-organisme obtenu ci-dessus ou un produit transformé de ce dernier est utilisé. Un procédé de culture d'un micro-organisme dans un milieu de culture liquide contenant un saccharide. Dans le milieu de culture liquide, la valeur carbonyle basée sur le(s) composé(s) autre(s) que le saccharide est de 100 µmol éq. ou plus par gramme du milieu de culture liquide. Un procédé de production d'un composé organique dans lequel le micro-organisme ci-dessus mentionné ou un produit transformé de ce dernier est utilisé.
PCT/JP2017/006048 2016-03-29 2017-02-20 Procédé de culture de micro-organisme et procédé de production d'un composé organique WO2017169275A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN113980868A (zh) * 2021-12-02 2022-01-28 广西科学院 一株耐受五羟甲基糠醛的产琥珀酸放线杆菌及其选育方法和应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113980868A (zh) * 2021-12-02 2022-01-28 广西科学院 一株耐受五羟甲基糠醛的产琥珀酸放线杆菌及其选育方法和应用
CN113980868B (zh) * 2021-12-02 2023-02-03 广西科学院 一株耐受五羟甲基糠醛的产琥珀酸放线杆菌及其选育方法和应用

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