JP5968733B2 - Method for producing butanol using microorganisms - Google Patents

Description

  The present invention relates to a method for producing butanol using microorganisms. More specifically, the present invention relates to a method for producing butanol suitable for producing butanol using a butanol-producing microorganism that produces butanol-containing metabolites in a growth-independent manner.

  Butanol is attracting attention as a next-generation biofuel because it has a higher energy content than ethanol. It is also useful as a raw material for various chemically synthesized substances such as isoprene, isobutene and butene. Furthermore, it is useful as an additive for improving the combustion efficiency of biodiesel fuel. For these reasons, establishment of an efficient butanol production process is desired (see Non-Patent Documents 1 and 2).

  In recent years, as one of the methods for producing butanol, a method using acetone / butanol / ethanol fermentation (ABE fermentation) of Clostridium bacteria represented by C. acetobutylicum and the like has attracted attention. In ABE fermentation, a substrate such as glucose is consumed, and organic acids such as acetic acid and butyric acid are mainly produced in the logarithmic growth phase (acid production phase), and butanol, acetone and ethanol are mainly 6: 3 in the stationary phase. It is produced in a growth-independent manner at a ratio of 1 (alcohol production phase). That is, butanol and ethanol can be simultaneously produced as alcohol from a substrate such as saccharide, and butanol can be produced predominantly (see, for example, Non-Patent Document 3).

Pfromma PH, Amanor-Boadub V, Nelson R, Vadlanid P, Madl R., Bio-butanol vs. bio-ethanol: A technical and economic assessment for corn and switchgrass fermented by yeast or Clostridium acetobutylicum. Biomass and Bioenergy, 2010. 34 (4): p. 515-524. Vert A, Qureshi N, Yukawa H, Blaschek HP., Biomass to Biofuels: Strategies for Global Industries, 2010. D.T.JONES.1981.Solvent Production and Morphological changes in Clostridium acetobutylicum.Applied and Environmental Microbiology.

  However, as shown in FIG. 32, the standard butanol production pathway using glucose as a substrate in ABE fermentation requires 4 molecules of NADH as a reducing power for synthesizing butanol in a downstream reaction from pyruvic acid. . In contrast, the upstream glucose consumption reaction (glucose to pyruvate) only produces two molecules of NADH. Therefore, in order to synthesize one molecule of butanol, at least two molecules of glucose are consumed. Actually, since the reducing power is dispersed also in other metabolites such as hydrogen and ethanol, the yield of butanol to consumed glucose is low. That is, in the production of butanol using ABE fermentation, there is a problem that the yield of butanol is low with respect to the substrate such as saccharide that has been added, and the improvement has been desired.

  In addition, this problem is not limited to the production of butanol using ABE fermentation of Clostridium bacteria, but can also be applied to the overall production of butanol using butanol-producing microorganisms that produce butanol-containing metabolites in a growth-independent manner. Therefore, the improvement was desired.

  Therefore, the present invention significantly improves the production amount (yield) of butanol with respect to the input substrate when producing butanol using butanol-producing microorganisms that produce butanol-containing metabolites in a growth-independent manner. It is an object to provide a method that can be used.

  In order to solve such problems, the present inventor considers that there is a possibility that the productivity of butanol can be greatly improved by supplying the reducing power necessary for butanol production from the outside of the butanol-producing microorganism. went.

  As a result, the electrode is immersed in a culture solution containing C. acetobutylicum, glucose and methyl viologen, and C. acetobutylicum consumes glucose while producing metabolites containing butanol in a growth-independent manner, By starting energization (application of reduction potential) to the electrode, and continuing energization for 10 minutes to reduce methylviologen from the oxidized form to the reduced form, and then stopping the energization, the amount of butanol produced (yield relative to the input glucose) ) Has been found to be greatly improved. And it came to discover that this yield improvement effect was notably show | played when the timing of an energization start was made into the consumption middle stage of glucose especially.

  Based on the above findings, the inventors of the present invention are not limited to C. acetobutylicum, but also about Clostridium bacteria that perform ABE fermentation, and also about all butanol-producing microorganisms that produce butanol-containing metabolites in a growth-independent manner. The amount of butanol produced relative to the input substrate by temporarily supplying electrons through an electron medium material that can take both oxidant and reductant forms while producing butanol while consuming The present inventors have found that the possibility that the (yield) can be greatly improved is derived, and have further studied variously to complete the present invention.

  That is, the butanol production method of the present invention provides an electronic medium substance that can take both forms of oxidized form and reduced form to a butanol-producing microorganism that produces butanol-containing metabolites while consuming a substrate. Including a step of temporarily supplying electrons via.

Here, in the butanol production method of the present invention, the supply of electrons is performed by immersing an electrode in a culture solution containing a substrate, a butanol-producing microorganism and an electron medium substance, and applying a reduction potential to the electrode for 5 to 15 minutes. This is preferably done by reducing the electronic media material.

  Moreover, in the butanol production method of the present invention, it is preferable to carry out the above process in the middle of the substrate consumption period of the butanol-producing microorganism.

  Furthermore, in the butanol production method of the present invention, a bacterium belonging to the genus Clostridium that performs acetone / butanol / ethanol fermentation (ABE fermentation) is preferable.

  According to the present invention, when butanol is produced using a butanol-producing microorganism that produces butanol-containing metabolites in a non-growth-linked manner, the production amount (yield) of butanol relative to the input substrate is greatly improved. Is possible. Therefore, it is possible to increase the butanol production amount for the input substrate as compared with the conventional method, and to achieve both the reduction of butanol production cost and the improvement of butanol production efficiency.

It is a figure which shows the concept of the butanol production method of this invention. It is the structure schematic of the electroculture apparatus used in the Example. It is a cyclic voltammogram of methyl viologen. It is a redox reaction formula of methyl viologen. It is a figure which shows the time-dependent change of the glucose concentration in a culture solution in the electroculture test by continuous electricity supply. It is a figure which shows the measurement result of the butyric acid and acetic acid density | concentration in a culture solution in the electroculture test by continuous electricity supply. It is a figure which shows the measurement result of the ethanol and butanol density | concentration in a culture solution in the electroculture test by continuous electricity supply. It is a figure which shows the natural potential change of the culture solution at the time of non-energization. It is a figure which shows the electric current value change at the time of the electricity supply by continuous electricity supply. It is a figure which shows the flow of the metabolism estimated at the time of -0.7V application. It is a figure which shows a time-dependent change of the electric current value at the time of reduce | restoring a methyl viologen by electricity supply. It is a figure which shows the time-dependent change of the glucose concentration in a culture solution in the electroculture test by temporary electricity supply. It is a figure which shows the measurement result of the butyric acid and acetic acid density | concentration in a culture solution in the electroculture test by temporary electricity supply. It is a figure which shows the measurement result of the ethanol and butanol density | concentration in a culture solution in the electroculture test by temporary electricity supply. It is a figure which shows the time-dependent change of the glucose concentration in a culture solution in the electric culture test by temporary or intermittent electricity supply. It is a figure which shows the measurement result of the butyric acid and acetic acid density | concentration in a culture solution in the electroculture test by temporary or intermittent electricity supply. It is a figure which shows the measurement result of the ethanol and butanol density | concentration in a culture solution in the electroculture test by temporary or intermittent electricity supply. It is a figure which shows the flow of metabolism estimated in the culture | cultivation which applied -0.7V for 10 minutes in the glucose consumption middle stage. It is a figure which shows the culture result (glucose concentration) of the sample used for the comprehensive analysis of a metabolite. It is a figure which shows the culture result (acetic acid concentration) of the sample used for the comprehensive analysis of a metabolite. It is a figure which shows the culture result (butyric acid concentration) of the sample used for the comprehensive analysis of a metabolite. It is a figure which shows the culture result (ethanol concentration) of the sample used for the comprehensive analysis of a metabolite. It is a figure which shows the culture result (butanol concentration) of the sample used for the comprehensive analysis of a metabolite. It is a figure which shows the relative value (central metabolic system, amino acid metabolism) of a main metabolite. It is a figure which shows the relative value (amino acid metabolism, purine pyrimidine metabolism) of the main metabolites. It is a figure which shows the relative value (nicotinic acid and nicotinamide metabolism, others) of the main metabolites. It is a figure which shows the relative area of a butyric acid. It is a figure which shows the relative area of NAD ⁺ . It is a figure which shows the relative area of NADH. It is a figure which shows the flow of metabolism at the time of -0.7V application (glucose consumption middle period) estimated from the comprehensive metabolic analysis. It is a figure which shows a time-dependent change of a butanol density | concentration when electricity supply is performed 8 times intermittently. It is a figure which shows the metabolic pathway of C.acetobutylicum.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings.

  In FIG. 1, an example of embodiment of the butanol production method of this invention is shown notionally. The butanol production method of the present invention is an electronic medium substance that can take both forms of an oxidant and a reductant on a butanol-producing microorganism 2 that is producing a metabolite 7 containing butanol in a non-growth manner while consuming the substrate 3. 5 includes a step of temporarily supplying electrons via 5.

  In this embodiment, the supply of electrons is performed by, for example, immersing the electrode 9 in a culture solution 4 containing the substrate 3, butanol-producing microorganism 2, and the electron medium material 5, and applying a reduction potential to the electrode 9 to apply the electron medium material 5. This is done by reducing.

  The butanol-producing microorganism 2 is not particularly limited as long as it is a butanol-producing microorganism that produces butanol-containing metabolites in a growth-independent manner, but a Clostridium bacterium that performs ABE fermentation is suitable. Examples of the genus Clostridium performing ABE fermentation include C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, and the use of C. acetobutylicum is not particularly limited thereto. .

  The cell density in the culture environment of the butanol-producing microorganism 2 is preferably set to a cell density corresponding to the stationary phase of growth or close to it. For example, when a Clostridium bacterium that carries out ABE fermentation is used as the butanol-producing microorganism 2, since the solvent (acetone, butanol, ethanol) is produced in a growth-independent manner in the stationary growth phase, the cells in the culture environment By setting the density to the cell density corresponding to the stationary phase of growth, the metabolite containing butanol can be produced in the microorganism 2 immediately after the start of the culture.

  The substrate 3 is a substance that becomes a metabolite generation source of the butanol-producing microorganism 2, and is appropriately selected according to the butanol-producing microorganism 2 to be used. Here, when using a Clostridium bacterium that performs ABE fermentation, it is possible to use monosaccharides such as glucose and polysaccharides such as starch, cellulose, and xylan as substrates, paper, wheat straw, rice straw, etc. Various types of biomass waste such as vegetable fiber, kitchen waste, agricultural waste, shochu distillation waste, surplus sludge (activated sludge) waste mixture, starch waste, waste palm fiber, etc. can be widely used as a substrate . By using such biomass waste, it is possible to promote effective utilization of biomass waste while further reducing the cost of butanol production.

  The added amount of the substrate 3 may be an amount suitable for butanol production not linked to the growth of the butanol-producing microorganism 2. However, an extremely high concentration of the substrate 3 is an inhibition factor for fermentation (butanol production). Further, the concentration of butanol generated by the conversion of the substrate 3 becomes 2% or more is also an inhibiting factor for fermentation (butanol production). Therefore, for example, when a clostridium bacterium performing ABE fermentation is used and the substrate 3 is glucose, glucose is added in an amount of 10 to 500 mM, preferably 10 to 300 mM, more preferably 10 to 100 mM, and even more preferably about 50 mM. However, the amount is not necessarily limited.

  As the electronic medium material 5, a material that can take both forms of an oxidized form and a reduced form and that does not deactivate the butanol-producing microorganism 2 is appropriately selected. For example, when a Clostridium bacterium that performs ABE fermentation is used, the electron medium material 5 is preferably a viologen or a viologen derivative. As the viologen derivative, methyl viologen, ethyl viologen, benzyl viologen and the like can be used, and it is preferable to use methyl viologen. However, the electronic medium material 5 is not limited to these.

  The optimum amount of the electronic medium substance 5 varies depending on the type of the electronic medium substance 5, the type of butanol-producing microorganism 2 to be used, the cell density, etc., but is generally 0.5 to 10 mM, preferably May be 0.5 to 5 mM, more preferably 0.5 to 3 mM, and even more preferably about 2 mM. If the addition amount of the electronic medium material 5 is too small, it is difficult to obtain an effect of improving butanol productivity. Moreover, when there is too much addition amount of the electronic medium substance 5, depending on the kind of the electronic medium substance 5, the activity of the butanol producing microorganism 2 may be inhibited. For example, when the electron medium substance 5 is a viologen derivative and the concentration thereof is 0.5 to 3 mM, particularly 1 to 2 mM, the Clostridium bacterium performing ABE fermentation can exert a remarkable remarkable butanol productivity improvement effect.

  The composition of the culture solution 4 is appropriately selected according to the butanol-producing microorganism 2 to be used. For example, when a Clostridium bacterium performing ABE fermentation is used, it is preferable to use a TYA medium, more preferably a TYA medium containing acetic acid. By using a TYA medium containing acetic acid, it becomes possible to cause the Clostridium bacterium performing ABE fermentation to produce butanol early. However, the composition of the culture solution 4 is not limited to these. Examples of the culture method include continuous culture methods such as fed-batch culture, but are not limited to this method.

  Examples of the electrode 9 include a carbon electrode such as a carbon plate and glassy carbon, a platinum electrode, and the like, but are not limited thereto.

  By applying a reduction potential to the electrode 9, the electron medium substance 5 in the culture solution 4 is reduced from an oxidant to a reductant. Then, supply of electrons from the reduced electron medium material 5 to the butanol-producing microorganism 2 occurs, and at that time, the reduced electron medium material 5 is oxidized to an oxidant. That is, supply of electrons to the butanol-producing microorganism 2 through the electronic medium substance 5 occurs.

The value of the reduction potential applied to the electrode 9 can be obtained based on the cyclic voltammogram of the electronic medium material 5. The case where the electron medium material 5 is methyl viologen (MV) will be described as an example. From the cyclic voltammogram, the MV oxidation-reduction reaction is -0.61 V, -0.93 V is an oxidation peak, -0.68 V This is a two-electron reaction with a reduction peak at −1.0 V, and the two-electron reductant (MV ⁰ ) of MV is insolubilized, so that the reduction potential for generating a one-electron reductant is about −0.7 V. It can be judged that it is sufficient. Even when other electronic medium materials are used, the reduction potential can be set in the same way.

  Here, in the present invention, a reduction potential is temporarily applied to the electrode 9 to temporarily supply electrons via the electron medium material 5 to the butanol-producing microorganism 2. . The energization start timing is set during the substrate consumption period of the butanol-producing microorganism 2. By doing in this way, the substrate consumption suppression effect which arises when a reduction potential is continuously applied to the electrode 9 can be avoided. Furthermore, while an increase in intracellular reducing power and an increase in NADH occur, synthesis of metabolic byproducts other than butanol and ethanol is inhibited, and synthesis of bacterial cell components (amino acids and nucleic acids) is inhibited. That is, waste of unnecessary carbon and reducing power can be suppressed, and butanol (and ethanol) can be easily produced. Thereby, butanol productivity with respect to the substrate 3 is improved.

  In other words, the temporary supply of electrons via the electron medium material 5 to the butanol-producing microorganism 2 according to the present invention plays a role of switching the metabolic pathway of the butanol-producing microorganism 2 to a metabolic pathway that easily produces butanol, or butanol. It can be said that it plays a role as a trigger for inducing the metabolic pathway of the production microorganism 2 to a state where butanol can be easily produced.

  Here, the energization start timing may be during the substrate consumption period of the butanol-producing microorganism 2, but when the entire substrate consumption period is 1, it is within 0.1 to 0.9 of the substrate consumption period. It is preferable to start energization, more preferably to start energization within a period of 0.3 to 0.7, and further to start energization within a period of 0.4 to 0.6. Is preferred.

  Further, if the energization time is too short, it is difficult to obtain the effect of improving the butanol production amount with respect to the input substrate, and if it is too long, the consumption of the substrate may be suppressed and the production amount of butanol may be reduced. Accordingly, a time during which the entire amount of the electronic medium substance 5 in the culture environment can be reduced, for example, 5 minutes to 15 minutes, preferably 7 minutes to 13 minutes, more preferably about 9 minutes to 11 minutes, and more preferably. However, it is not necessarily limited to these times, and these times may be deviated as long as the effects of the present invention can be achieved.

  In the present invention, the temporary supply of electrons to the butanol-producing microorganism 2 via the electronic medium substance 5 is sufficient if it is performed once, but may be performed a plurality of times within the above period. Good. Thereby, the productivity of butanol with respect to the input substrate can be further improved. However, if the interval between the first energization and the next energization is too short, the consumption of the substrate may be suppressed and the butanol production may be reduced. Therefore, the interval of about 2 to 4 hours, preferably about 3 hours is preferred. It is preferable to provide it. Further, since the effect of improving butanol productivity is saturated even if the number of temporary electron supply times is excessively increased, it is preferable to set the number of times to about 8 times at the maximum. It is suitable, and it is more suitable to set it as about 3 times.

  The above-described embodiment is an example of a preferred embodiment of the present invention, but is not limited thereto, and various modifications can be made without departing from the gist of the present invention. For example, in the above-described embodiment, the electron medium material 5 is reduced by applying a reduction potential to the electrode 9 in the culture solution in which the butanol-producing microorganism 2 is inhabited. It may be electrochemically reduced in the inside to form a reductant, which may be added to the culture solution in which the butanol-producing microorganism 2 lives. Also in this case, supply of electrons via the electron medium substance 5 to the butanol-producing microorganism 2 occurs, and the effect of improving the amount of butanol produced with respect to the input substrate can be achieved.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

1. Experimental method (1-1) Bacterial strain used Among the bacteria belonging to the genus Clostridium that fermentate and produce butanol, C. acetobutylicum NBCR13948 ^T was purchased from National Institute for Product Evaluation Technology and used in this example. did.

(1-2) Culture method
C. acetobutylicum is cultured using shaking culture at 30 ° C under anaerobic conditions (the gas phase in the glass vial as a culture vessel is replaced with nitrogen) using a thioglycolic acid medium having the composition shown below. From this, it was subjected to the culture test described later.

[Thioglycolic acid medium composition (g / L)]
L-cysteine: 0.5
・ Sodium chloride: 2.5
・ Glucose: 5.0
・ Yeast extract: 5.0
Casein peptone: 15.0
・ Thioglycolic acid: 0.5
pH 7.0

(1-3) Electroculture apparatus and culture test method The configuration of the electroculture apparatus 1 used in this example is shown in FIG. A 250 mL glass vial made of Duran (hereinafter referred to as container 20) provided with two openings on the side surface was used as a culture tank. A lid 30 was attached to the container 20. A silicone rubber stopper was provided on the upper surface 30a of the lid 30 to ensure the hermeticity of the container 20 when wires, electrodes, and tubes were passed.

A small container 21 was used as a counter electrode tank. Specifically, a 15 mL plastic tube is cut vertically, and an anion exchange membrane 6 (manufactured by Astom Co., Ltd., model number: Neoceptor AMX) is bonded to the cut surface and processed into a semi-cylindrical shape with a small container 21. did. The area of the anion exchange membrane 6 was 12 cm ² . The small container 21 contained the electrolyte solution 4a and the counter electrode 10 and was immersed in the electrolyte solution 4a. The upper part of the small container 21 was filled with a silicon adhesive and sealed.

  The culture solution 4 was accommodated in the container 20, and the small container 21 and the working electrode 9 were immersed in the culture solution 4. The wiring between the working electrode 9 and the counter electrode 10 was drawn to the outside of the container 20 through a silicone rubber stopper provided on the lid 30. The silver / silver chloride reference electrode 11 (RE-1B, BAS Co., Ltd.) was inserted from below the two openings provided on the side surface of the container 20 and brought into contact with the culture solution 4.

  The gas generated in the container 20 during the culture period was collected in an aluminum sampling bag 40 (trade name: aluminum bag, 1 L, manufactured by GL Science) installed at the upper part of the container 20.

  Both the working electrode 9 and the counter electrode 10 were carbon plates (7.5 cm × 2.5 cm).

  The culture solution 4 contained in the container 20 was a TYA medium. The composition of TYA medium is shown below. In addition, oxidized methylviologen (MV) was added to the culture solution 4 as an electronic medium substance so as to have a final concentration of 2 mM.

[Composition of TYA medium (g / L)]
・ Glucose: 10.0
・ Tryptone: 6.0
-Yeast extract: 2.0
-Ammonium acetate: 3.0
-Potassium dihydrogen phosphate: 0.5
Magnesium sulfate heptahydrate: 0.3
・ Iron sulfate heptahydrate: 0.01
pH 7.0

  The electrolyte solution 4a accommodated in the small container 21 was a TYA medium (MV not added).

  Incidentally, 40 mM acetic acid was added to the TYA medium as the culture solution 4 to suppress the inactivation of C. acetobutylicum butanol-producing ability that had reached the stationary phase due to long-term culture (Paredes CJ, Jones SW, Senger RS). Borden JR, Sillers R, Papoutsakis ET., Molecular Aspects of Butanol Fermentation. Bioenergy, 2008).

  Moreover, since C. acetobutylicum has been reported to induce alcohol production under conditions of pH 4.7 or lower, the pH of the culture solution 4 was adjusted to 4.5 with 1 M KOH.

  After sealing the container 20, nitrogen gas was passed through the upper opening of the two openings provided on the side surface of the container 20 for 1 hour. After the electroculture device is installed in the thermostat, the working electrode 9, the counter electrode 10 and the reference electrode 11 are connected to a three-electrode constant potential setting device (potentiostat) 12 so that the potential of the working electrode 9 is strictly controlled. Can be controlled.

As a source of inoculation, preculture using thioglycolic acid medium (1.2 L) was performed, and the cells that shifted to the stationary phase were collected, washed with a new medium, and then initially placed in an electric culture apparatus containing 190 mL of culture solution 4 The cells were inoculated so as to have a cell density of 1 × 10 ⁹ cells / mL (corresponding to the cell density in the stationary phase), and electroculture tests were performed under various conditions. During the test, the temperature of the culture solution 4 was controlled at 30 ° C. In addition, a stirring bar was placed in the culture solution 4, and the culture solution 4 was continuously stirred during the test.

  During the electrical culture test period, the natural potential (non-energization experiment) and current value (energization experiment) are measured over time, and the culture solution 4 is periodically extracted from the opening on the side surface of the container 20 to determine the cell density. The components of the culture solution 4 were analyzed.

  Further, as a control test, a similar electroculture test was carried out in the presence of oxidized MV and without energization.

(1-4) Analysis of culture solution The culture solution 4 was fractionated in the culture process, and the cell density measurement and component analysis of the culture solution 4 were performed. The cell density was measured by observation with a microscope (Eclipse E-40, Nicon). Among the components of the culture solution 4, glucose is glucose CII test Wako (Wako), acetic acid, butyric acid, ethanol and butanol are liquid chromatography (Lachrome Elite, Hitachi), Gelpack column (GL-C610H-S), as a detector Qualitative and quantitative analysis was performed using an RI detector.

(1-5) Calculation of energy efficiency and conversion efficiency of electrons into butanol Energy efficiency (ΔW _butanol / W _in ) was calculated from the following equation.

W _in = _{E ap} · C
ΔW _butanol = (Butanol combustion calorie) x (Butanol production when not energized-Butanol production when energized)

In the above formula, E _ap is an interelectrode voltage (unit: V), and C is an electric quantity (unit: C). The butanol combustion heat amount is 2675.9 kJ / mol, and the unit of butanol production is mol.

The conversion efficiency of electrons to butanol is based on the following formula, assuming that all currents are used for conversion of NADH and two molecules of NADH are used for the production of one molecule of butanol. The calculated number of moles was calculated.
NAD ⁺ + H ⁺ + 2e ⁻ → NADH

2. Examination of applied potential (2-1) Determination of set potential The applied potential was determined based on a cyclic voltammogram of methyl viologen (MV). A cyclic voltammogram of MV is shown in FIG. Moreover, the oxidation-reduction reaction formula of MV is shown in FIG. The MV oxidation-reduction reaction is a two-electron reaction in which −0.61 V and −0.93 V are oxidation peaks, and −0.68 V and −1.0 V are reduction peaks. Since it has been reported that the two-electron reduced form of MV (MV ⁰ ) is insolubilized (Paul MS Monk., The Viologens: Physicochemical Properties, Synthesis and Applications of the Salts of 4,4'-Bipyridine, 1999.) In order to generate a one-electron reductant, -0.7 V was set as the lower limit as the setting of the reduction potential.

(2-2) Electroculture test The energization potential of the working electrode 9 is set to −0.7 V which is the lower limit of the reduction peak or the oxidation potential (+0.9 V, +0.6 V, −0.5 V), and the current value is stabilized. The cells recovered by the preculture were inoculated at a high cell density (1.0 × 10 ⁹ cells / mL), and an electroculture test was performed. The glucose concentration was 55.6 mM (10 g / L).

  The effects of energization and applied potential were examined by comparing the current value, natural potential, glucose concentration, and metabolite concentration after the end of culture under energized and non-energized conditions. The time course of the glucose concentration of the culture solution 4 is shown in FIG. 5, the acetic acid and butyric acid concentrations of the culture solution 4 after the end of the test are shown in FIG. 6, and the ethanol and butanol concentrations of the culture solution 4 after the end of the test are shown in FIG. .

    From the results shown in FIG. 5, the consumption rate and consumption of glucose as a substrate in the condition where −0.7 V was applied were compared with those in other energized conditions (+0.9 V to −0.5 V) and non-energized conditions. There was a tendency to be suppressed. When the accumulation of each metabolite was compared between non-energized and energized, there was no significant difference between the non-energized condition and the oxidation potential applied condition, but in the case where -0.7 V, which is the MV reduction potential, was applied. Only showed a different trend. In other words, the concentration of acetic acid, butyric acid, and ethanol in the culture broth was small in correlation with the reduced glucose consumption when -0.7 V was applied, but the butanol production was comparable to other potential conditions. (FIGS. 6 and 7).

  Next, Table 1 shows the results of calculating the carbon yield with respect to glucose consumed for the metabolites under each condition. For acetic acid, the increase relative to the initial value of 40 mM was calculated as the sugar yield.

  The carbon yield of butanol was comparable to 1.6% to 4.1% under the application of oxidation potential, compared with the non-energized condition (3.2% (mol-C / mol-C)). On the other hand, when -0.7V was applied, it was shown that it increased with 28.8%. It was estimated that this increase in yield was due to a small amount of by-products generated compared to a small total glucose consumption, and the carbon derived from glucose was concentrated in butanol production.

  Next, FIG. 8 shows a change in natural potential during non-energization, and FIG. 9 shows a change in current value during energization. When the culture was performed while applying a potential of −0.7 V, an increase in the reduction current was observed so that the current value during the culture period was synchronized with the decrease in glucose (maximum: −3.0 mA). This means that the electrode re-reduced the regenerated oxidized MV as a result of C. acetobutylicum receiving electrons from the reduced MV. That is, it can be said that electrons were supplied from the electrode into the cell as a reducing power. In addition, under the condition where -0.7 V was applied, the medium was blue throughout the culture period. From this, it was suggested that reduced MV (blue) is always present under the condition where −0.7 V is applied. It was presumed that the supply of 1-electron reduced MVs or electrons improved butanol yield, but suppressed glucose consumption (see FIG. 10: the thickness of the arrow indicates the flow of metabolism). Thick arrows indicate increased metabolism, and the gray and dotted lines indicate pathways with decreased metabolism).

3. Examination of an efficient energization method to increase butanol production The applied voltage was fixed at -0.7 V, which was effective for butanol production increase, and the electrocultivation conditions that had been energized continuously from the start to the end of the culture were changed, The glucose consumption and butanol productivity when the energization start timing and energization time were changed were examined.

(3-1) Examination of energization time The energization time required for the generation of the reduced MV was examined. When a culture solution 4 containing bacterial cells was prepared in an electric culture apparatus and a potential of −0.7 V was applied, the medium color changed to blue indicating a reduced form of MV within 10 minutes after the start of energization, The current used for reduction was stable and showed a constant value (FIG. 11). From the above results, it was judged that 10 minutes was sufficient for the energization time.

(3-2) Examination of energization timing As the timing of energization, the following four conditions were set using glucose consumption as an index after the start of the growth non-interlocking test using stationary bacteria.
(1) Before starting culture (2) Early glucose consumption (3) Middle glucose consumption (4) Late glucose consumption

  First, (1) before the start of culture, (2) the initial stage of glucose consumption and (3) the middle stage of glucose consumption were tested. In addition, the same test was carried out in the case of non-energization. The glucose concentration of the culture solution 4 was 55.6 mM (10 g / L). The time course of the glucose concentration of the culture solution 4 is shown in FIG. In the figure, (1) indicates the energization start position in condition (1), (2) indicates the energization start position in condition (2), and (3) indicates the energization start position in condition (3). Yes.

  Under any condition, when the energization was stopped after the MV was reduced by energization for 10 minutes, the reduced color of the culture medium faded with time, the reduced color disappeared in about 30 minutes, and returned to the pre-energized media color. . From this, it was shown that the electrons of reduced MVs migrated into C. acetobutylicum cells.

  When the influence of energization on metabolism was examined, glucose degradation was confirmed in all energization conditions as in the case of non-energization, and no difference was observed in the consumption (FIG. 12). Therefore, it was estimated that the effect of suppressing the glucose decomposition activity could be avoided by extremely reducing the energization time and reducing the supply of reducing power.

  Next, in order to investigate the influence of electricity on metabolism, quantitative analysis of metabolites under each culture condition was performed. FIG. 13 shows the acetic acid and butyric acid concentrations in the culture solution 4 after the test is completed, and FIG. 14 shows the ethanol and butanol concentrations in the culture solution 4 after the test is completed. Regarding the condition (1) in which the energization was performed before the start of the culture, no significant difference was observed in the accumulation of metabolites including butanol as compared to the non-energized condition. On the other hand, in the condition (2) in which electricity was supplied at the initial stage of glucose consumption (after 19 hours of culture) and the condition (3) in which electricity was supplied in the middle stage of glucose consumption (after 28 hours of culture), Reduced butyric acid accumulation and increased ethanol / butanol production compared to the non-energized state. Especially, the butyric acid accumulation and butanol increased production effect was achieved under the condition (3) in which energization was conducted for a short time in the middle of glucose consumption. It was conspicuous (the butanol production-increasing effect was 1.5 times that at the time of non-energization).

Next, in addition to the effect on the butanol production of intermittent energization in the late stage of glucose consumption, the conditions for energizing in the middle period were examined in detail. Specifically, the following conditions were examined. The glucose concentration of the culture solution 4 was 55.6 mM. Moreover, in this examination, the middle stage of glucose consumption means 13 hours after culture, and the late stage of glucose consumption means 19 hours after culture.
(A) Energization for 10 minutes in the middle glucose consumption period (B) Energization for 10 minutes in the middle glucose consumption period 3 times every 3 hours (C) Energization for 10 minutes in the later glucose consumption period (D) Energization for 10 minutes in the later glucose consumption period 3 Repeated 3 times every hour (E) Continuous energization from the late stage of glucose consumption

  The change with time of the glucose concentration of the culture solution 4 is shown in FIG. In the figure, (A) and (B) indicate the energization start positions in the conditions (A) and (B), and (C), (D) and (E) indicate the conditions (C), (D) and ( The energization start position in E) is shown.

  From the results shown in FIG. 15, in this experiment, it was confirmed that glucose consumption started immediately after the start of culture under any condition, and showed the same consumption rate as in the non-energized case in the three conditions energized late. It was done. Although no significant decrease in glucose consumption rate was observed for those energized in the middle period, a slight delay was observed in the time until the glucose was completely consumed (non-energized: 20 hours → energized / mid-term: 24 time).

  Moreover, the acetic acid and butyric acid concentration of the culture solution 4 after the end of the test is shown in FIG. 16, and the ethanol and butanol concentrations of the culture solution 4 after the end of the test are shown in FIG. From the results shown in FIGS. 16 and 17, the condition (E) among the three conditions energized in the latter period shows no change in the production of organic acids and alcohols compared to non-energization, and the effect of energization is It became clear that there was no. On the other hand, in the conditions (A) to (D), the production suppression of organic acid and the production increase effect of ethanol and butanol were recognized as compared with the case of non-energization, and the effect was particularly in the conditions (A) and (B). It was conspicuous (an effect of increasing ethanol and butanol by about twice as much as that in the non-energized case was observed). In any condition, it was confirmed that the cell density did not change from the time of inoculation.

  From the above results, it became clear that the suppression of glucose consumption can be avoided by energization (application of the reduction potential) for a short time. Moreover, it became clear that the butanol production-increasing effect can be easily obtained by energizing in the middle period when glucose is actively consumed.

4). Consideration on Butanol Production Increase Effect by Supply of Reducing Power and Its Mechanism In the test of 3 above, according to the increase in the number of times of energization (reducing power input) in 10 minutes × 1 time and 10 minutes × 3 times as the condition of power supply. Butanol production increased, but the increased production was not directly proportional to the increase in the number of energizations. Table 2 shows the yield of each metabolite under both conditions. In Table 2, for acetic acid, the increase relative to the initial value of 40 mM was calculated as the yield to sugar.

  In both conditions, the yield to sugar was improved about twice as much as that in the non-energized state, and it became clear that metabolic control to improve butanol production was possible with only 10 minutes of energization.

  Next, Table 3 shows the result of calculating the energy recovery efficiency with respect to the input energy.

  The amount of electricity calculated from the integration of the current values that flowed during electroculture and the voltage between the electrodes (about 2.3 V) is 11.3 J (10 minutes × 1) and 44.2 J (10 minutes × 3) at each potential. there were.

The amount of energy calculated from the increased production of butanol was 2358.1 J (10 minutes × 1) and 3173.4 J (10 minutes × 3). Therefore, the energy efficiency of both conditions is calculated as ΔW _butanol / ΔW _in = 209.3 (10 minutes × 1) and 71.2 (10 minutes × 3), and the method of the present invention is more dramatic than the amount of electricity charged by energization. It can be said that it can be said to be a metabolic control technology that can achieve a significant increase in energy substance production.

  From the above, it is possible to dramatically reduce the amount of input electricity by temporarily or intermittently energizing while narrowing the energization time, rather than energizing the entire culture period as in the conventional electroculture method. It was revealed that butanol productivity can be improved efficiently.

The method of the present invention is based on the idea of increasing butanol by supplying reducing power as electrons to the butanol production pathway. Therefore, in this test, the amount of MV reduced was calculated based on the current value. Assuming that all of the current was used for production of MV ^{· +} , the MVs reduced in the current culture test (mid-term) were 0.048 mmol (10 minutes × 1) and 0.18 mmol (10 minutes × 3), respectively. It was. Further, when it is assumed that all the electrons of the one-electron reduced MV are directly NADH converted and all of the NADH is used for butanol production, the increased production of butanol is 0.024 mmol (10 minutes × 1), 0.09 mmol. It is estimated that (10 minutes × 3). This value is 1 / 10-1 / 40 of the butanol production increase actually obtained by energization, and the difference in the production increase cannot be explained by the concept of direct butanol production by supplying reducing power. In addition, the enormous increase in butanol produced by energization was particularly noticeable when energized in the middle of glucose consumption, which means that energization not only supplies simple reducing power but also unexpected metabolic fluctuations that contribute to increased butanol production. (See FIG. 18: the thickness of the arrow indicates the magnitude of the metabolic flow. The thick arrow indicates an increase in metabolic rate, and the pathway indicated in gray and dotted lines is the metabolism.) Indicates a reduced path).

5. Comprehensive analysis of metabolites Since metabolites are direct and chemical expressions of changes in the state of the organism (transcribed and translated), analyzing all metabolites is very important for understanding changes in the state of the organism It is thought that it will provide useful knowledge. Therefore, in order to understand the mechanism by which butanol productivity was improved by applying a reduction potential and to establish it as a metabolic control method, we conducted a comprehensive analysis of intracellular metabolites. Of the primary metabolites involved in the basic function of cells, 92% are water-soluble, and 96% of them are ionic metabolites that are charged in aqueous solution. Therefore, in this study, CE-MS was selected which is effective for the analysis of these water-soluble and ionic metabolites and at the same time capable of measuring more than 900 metabolites.

(5-1) Experimental Method Culture was carried out in the same manner as in the above 3 for 3 samples of non-energized conditions and 2 samples of energized conditions (−0.7 V). After the start of culture, energization is performed every 3 hours for 10 minutes from the middle stage of glucose consumption (after 19 hours), and 1 mL of sample is taken during 10 minutes at the timing when the residual glucose concentration becomes 2 mM. did. The control sample was similarly cultured under non-energized conditions, and 1 mL of the sample was collected when the residual glucose concentration reached 2 mM. The collected sample was collected on a filter (Millipore Isopore Membrane Filter HTTP 0.4 μm pore 47 mm diameter, Millipore) using a suction filtration device. The cells collected on the filter were washed twice with 10 mL of MilliQ water, and the filter bacteria were placed in a sealed petri dish containing 2 mL of methanol (LC / MS, Wako) containing internal standard (H3304-1002, HMT). The filter was immersed in methanol with the body adhesion surface down. A filter immersed in methanol was subjected to ultrasonic treatment for 30 seconds to obtain a metabolite extract. The extract was stirred by adding 1600 μL of chloroform and 640 μL of MilliQ water, followed by centrifugation. After centrifugation, 325 μL × 4 aqueous layers were transferred to an ultrafiltration tube (ultra free MC PLHCC centrifugal filter unit). This was centrifuged and subjected to ultrafiltration treatment. The filtrate was dried and dissolved again in 50 μL of MilliQ water and used for measurement of metabolites. The analysis of metabolites was carried out using CE-TOF MS system (Agilent), and cationic substances and anionic substances were measured in cation mode and anion mode, respectively (Soga T, Heiger DN., Amino acid analysis by capillary electrophoresis). Electrospray ionization mass spectrometry. Anal Chem, 2000. 72 (6): p. 1236-1241., Soga T, Ohashi Y, Ueno Y, Naraoka H, Tomita M, Nishioka T., Quantitative metabolome analysis using capillary electrophoresis mass spectrometry. J Proteome Res, 2003. 2 (5): p. 488-494., Soga T, Ueno Y, Naraoka H, Ohashi Y, Tomita M, Nishioka T., Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry. Anal Chem, 2002. 74 (10): p. 2233-2239.).

Peaks detected by CE-TOF MS were automatically integrated with MasterHands ver. 2.9.0.9 was automatically extracted to obtain mass-to-charge ratio (m / z), migration time and peak area value as peak information. The obtained peak area value was converted into a relative area value using the following [Equation 1], and correction was performed based on the number of collected cells of each sample. Next, based on the values of m / z and MT, the peaks between the samples were collated and aligned. At this time, noise other than the peak was deleted. Moreover, since these data include adduct ions such as Na ⁺ and K ⁺ and fragment ions such as dehydration and deammonium, these molecular weight related ions were deleted.

  Relative area value = Area value of target peak / (Area value of internal standard substance × Number of bacterial cells) [Equation 1]

  The detected peak was collated and searched with the HMT metabolite database based on m / z (mass) and MT value (electrophoresis time). The tolerance for searching was ± 0.5 minutes for MT and ± 10 ppm for m / z. Relative values indicate average values in independent triple (control test) or double (energization test) experiments, and error bars indicate standard deviations or average value ranges.

(5-2) Experimental results The number of cells of the collected samples was (1.4 ± 0.1) × 10 ⁹ cells in all samples. After collecting the sample, the metabolite was immediately extracted from the cell and subjected to intracellular metabolite analysis. In addition, changes in metabolites in the culture were also performed by liquid chromatography. The results for glucose are shown in FIG. 19, the results for acetic acid in FIG. 20, the results for butyric acid in FIG. 21, the results for ethanol in FIG. 22, and the results for butanol in FIG. Metabolite fluctuations showed the same trend as before, with a slight decrease in glucose reduction rate due to energization, a decrease in organic acid accumulation, and an increase in ethanol and butanol concentrations.

  When metabolome analysis of intracellular metabolites was performed on 5 samples (3 samples, 2 samples), 179 peaks were detected as candidate compounds (cation 62, anion 117). Relative quantification was performed for each of these detected compounds, and hierarchical clustering analysis (HCA) was performed on the obtained data. As a result of heat map notation, a clear difference was confirmed between the non-energized sample and the energized sample. It was revealed macroscopically that energization gave large fluctuations in intracellular metabolism.

  Further, the relative area ratio obtained for each metabolite was compared between the non-energized sample and the energized sample, and the relative value (energized sample / non-energized sample) was calculated. These values were classified for each metabolic pathway, arranged in descending order of the rate of increase due to energization, and compiled as a graph. The results are shown in FIGS.

In addition, 69 types of compounds not classified as major metabolic pathways were detected. Of these, only butyric acid, NAD ⁺ , and NADH involved in butanol production are shown in FIGS. FIG. 27 shows butyric acid, FIG. 28 shows NAD ⁺ , and FIG. 29 shows NADH.

  24 to 29 are relative values of energization / non-energization, a value greater than 1 indicates a product increased by energization, and a value less than 1 indicates a decrease due to energization.

From FIG. 27, it was confirmed that there was a decrease in the amount of intracellular butyric acid accumulated under energization conditions, and the results were consistent with the analysis of the culture solution. Furthermore, under energization conditions, an increase in the amount of NADH, which is an important reducing power in metabolic reactions, and a decrease in NAD ⁺ linked thereto are observed (FIGS. 28 and 29). By applying a reducing potential, the reducing power is reduced in the cells. This result also suggested that it was supplied.

  The overall trend is that most of the metabolites grouped in amino acid metabolism, nucleic acid (purine / pyrimidine) metabolism, and other sugar metabolisms involved in cell wall biosynthesis, which are the main anabolic reactions in cells, tend to be reduced by energization. It was seen. Among them, putrescine, asparagine, N-carbamoylaspartic acid, tyramine (FIGS. 24 and 25), purine, and pyrimidine metabolism showed that inosine, hypoxanthine, adenosine, AMP, and guanine were increased by energization. (FIG. 25). In other sugar metabolism groups, the accumulation of activated saccharides was also reduced by energization (FIG. 26).

  In this way, while the anabolic reaction is suppressed by energization, in the central metabolic system (glycolysis system, pentose phosphate pathway, TCA cycle) that supports the catabolism reaction, an increase was observed for a relatively large number of metabolites by energization. The largest increase was in ribulose 5-phosphate on the pentose phosphate pathway, which increased 2.5-fold due to energization (FIG. 24).

  In the glycolytic system, the amount of metabolite decreased by energization in the upstream portion starting from glyceraldehyde 3-phosphate, and increased in the downstream. In addition, dihydroxyacetone phosphate, glycerol 3-phosphate, and lactic acid increased as a branched product from the glycolysis system due to energization.

  C. acetobutylicum has been inferred from its genome sequence that it does not have a complete TCA cycle. From the results of recent metabolite analysis, it may actually have a complete TCA cycle reaction. (Amador-Noguez D, Feng XJ, Fan J, Roquet N, Rabitz H, Rabinowitz JD., Systems-level metabolic flux profiling elucidates a complete, bifurcated tricarboxylic acid cycle in Clostridium acetobutylicum. J Bacteriol, 2010. 192 (17): p. 4452-4461.). Also in this example, a plurality of metabolites positioned within the TCA cycle were detected. While the accumulated amount of citric acid slightly increased by energization, a decrease in the accumulated amount was observed for succinic acid and malic acid in the latter half of the TCA cycle.

(5-3) Interpretation of metabolic fluctuation and estimation of metabolic control mechanism As a result of supplying reducing power into the cell via MV reduced by extracellular electrode reaction, the increased NADH / NAD ⁺ ratio is as follows It was estimated that it caused metabolic changes. The cell components (amino acids, nucleic acids, cell walls) showed a decrease in intracellular amount as a whole. Furthermore, as components increased by energization, degradation products of amino acids such as putrescine and tyramine, N-carbamoylaspartic acid, which is an intermediate of pyrimidine (UMP) synthesis, and low-energy intermediates of nucleic acids were detected. It was inferred that the anabolic reaction that synthesizes cells as a whole was suppressed by energization, or that there was a significant change in the direction of degradation. Since the reaction for synthesizing the cell constituents uses a large amount of carbon and energy resources (ATP and NAD (P) H), it is considered that their suppression contributed to increased butanol production.

  On the other hand, metabolic changes were also observed in the central metabolic pathway, which is a catabolic pathway. In the preparation period of glycolysis upstream of glucose metabolism (glucose → glyceraldehyde triphosphate), a decrease in the overall product amount is seen due to energization, and the metabolite ribulose 5- on the pentose phosphate pathway, which is a bypass pathway. Since phosphoric acid was increased by energization, it was estimated that the carbon distribution ratio between the glycolytic system and the pentose phosphate pathway was varied by energization. While ATP (adenosine triphosphate) is consumed in the preparation period of glycolysis, NADPH is produced without consuming ATP in the bypass pentose phosphate pathway. C.acetobutylicum ATCC824 has three butanol-producing enzymes, one of which is NADPH-dependent (Saint-Amans S, Girbal L, Andrade J, Ahrens K, Soucaille P., Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixture. J Bacteriol, 2001. 183 (5): p. 1748-1754. Thus, it was estimated that metabolic fluctuations in the tendency to produce were suppressed, constraining the consumption of limited energy resources, similar to the fluctuations observed in the assimilation reaction. In the reward period downstream of the glycolysis (glyceraldehyde 3-phosphate → pyruvic acid), the amount of metabolites tended to increase due to energization, and an active reaction was estimated.

It has been reported so far that an increase in the NADH / NAD ⁺ ratio in C. acetobutylicum cells inhibits the activity of glyceraldehyde 3-phosphate dehydrogenase (Girbal L, Soucaille P., Regulation of Clostridium acetobutylicum metabolism). as revealed by mixed-substrate steady-state continuous cultures: role of NADH / NAD ratio and ATP pool. J Bacteriol, 1994. 176 (21): p. 6433-6438). As a result of the balance of metabolism and consumption, the intracellular NADH / NAD ⁺ ratio was maintained at a low level (FIGS. 28 and 29), and it was estimated that glucose consumption was not suppressed.

  Interestingly, lactic acid, dihydroxyacetone phosphate and glycerol triphosphate increased in metabolites branched from the central metabolic pathway. Since NADH is required for the pathway for producing lactic acid from pyruvic acid and the branching pathway for producing glycerol triphosphate via dihydroxyacetone phosphate from fructose 1,6-bisphosphate, it can be used in cells by energization. With the increase in NADH, the production of metabolites existing in the branching pathway as with butanol is considered to be activated.

  As a branch point to the TCA cycle, which is the latter stage of glucose catabolism, there are two types of pathways: pyruvate directly flows into oxaloacetate on the TCA cycle and pyruvate → acetyl CoA → citrate. In the latter route, acetyl CoA branches to the butanol production route. In the former route, pyruvic acid is converted to amino acids such as aspartic acid and pyrimidine via oxaloacetic acid. It is presumed that the amount of carbon flowing into the TCA cycle is reduced because the accumulated amount of amino acids such as aspartic acid, pyrimidine, and succinic acid and malic acid in the TCA cycle is reduced by energization. On the other hand, since the concentration of acetyl-CoA itself was decreased by energization, it was suggested that the amount of carbon flowing into the TCA cycle was reduced as a whole, and the distribution to the butanol production pathway was increased. .

As described above, increasing the NADH / NAD ⁺ ratio in a non-proliferating cell by energization induces the following two large metabolic fluctuations, thereby conserving limited carbon resources and energy resources in the cell, It was suggested that a metabolic switch to concentrate on butanol production occurred (see FIG. 30: the thickness of the arrow indicates the magnitude of the metabolic flow. The thick arrow indicates an increase in metabolic rate, The pathways shown in gray and dotted lines indicate pathways with reduced metabolism).
(1) The assimilation reaction is suppressed, and further, excess amino acids and nucleic acid substances are degraded. (2) The TCA cycle is also suppressed in the central metabolic system.

6). Examination of Butanol Production Increase Effect by Multiple Energization In addition to the experimental results of 3 and 4 above, the case where the energization was intermittently conducted 8 times was examined.

  From the middle stage of glucose consumption (19 hours after the start of culture), electricity was applied 8 times for 10 minutes every 3 hours.

  The results are shown in FIG. It has been clarified that a significant increase in butanol production can be obtained compared with the case of non-energization.

  Next, Table 4 shows the results of calculating the energy recovery efficiency with respect to the input energy.

There is more energy derived from butanol that is increased by energization than the input electric energy, and the increased production of butanol by this method is a beneficial method from the viewpoint of energy. Increasing the number of energizations increases the absolute value of increased butanol production, but energy efficiency tends to decrease because the reducing power supplied is excessive. Therefore, it is possible to take the option of suppressing the number of energizations if importance is placed on energy efficiency and increasing the number if importance is placed on butanol production. When energized 8 times, the absolute amount is large and the energy efficiency exceeds 1, so it can be said that it is a sufficiently efficient condition.

2 Butanol-producing microorganism 3 Substrate 4 Culture solution 5 Electronic medium substance 7 Metabolite

Claims (2)

Temporary supply of electrons via butterfly-producing microorganisms that produce butanol-containing metabolites in a non-growth-coupled manner while consuming substrates through an electron medium that can take both oxidized and reduced forms the process only contains,
The supply of the electrons is performed by immersing an electrode in a culture solution containing the substrate, the butanol-producing microorganism, and the electron medium substance, and applying a reduction potential to the electrode for 5 to 15 minutes to reduce the electron medium substance. By doing
Performing the process in the middle of the substrate consumption period of the butanol-producing microorganism,
The butanol-producing microorganism is a Clostridium bacterium that performs acetone-butanol-ethanol fermentation (ABE fermentation),
The substrate is a substance that becomes a metabolite generation source of the butanol-producing microorganism,
The butanol production method , wherein the substrate consumption period is a period until the butanol-producing microorganism consumes the introduced substrate . The butanol production method according to claim 1, wherein the step is performed 2 to 8 times every 2 to 4 hours.