JP5985331B2 - Metabolism control method of hydrogen bacteria - Google Patents

Description

  The present invention relates to a method for controlling metabolism of hydrogen bacteria. More specifically, the present invention relates to a metabolic control method suitable for application particularly to Hydrogenobacter themophilus, a useful substance production method using this method, and the like.

  An increase in greenhouse gases such as carbon dioxide in the atmosphere can be a factor causing global warming, climate change, and the like, and is therefore regarded as an important environmental problem on a global scale. For example, in the electric power business, a large amount of carbon dioxide is emitted from a thermal power plant. As such a method for recovering and disposing of discharged carbon dioxide, a carbon dioxide fixing method using hydrogen bacteria is known (see, for example, Non-Patent Document 1).

  Hydrogen bacteria are autotrophic organisms that can grow using hydrogen (free hydrogen) as an energy source and carbon dioxide as the only carbon source. Since hydrogen bacteria have a high growth rate (carbonic acid assimilation rate), they are considered to be very useful for fixing and recycling a large amount of carbon dioxide.

Central Research Institute of Electric Power Industry report U92058

  By the way, in order to further improve the usefulness of hydrogen bacteria, it is effective to control the metabolism of hydrogen bacteria and increase the amount of useful substances in the metabolite produced by hydrogen bacteria as a carbon dioxide fixed product. Conceivable. However, a method for controlling the metabolism of hydrogen bacteria has not yet been established.

  Then, an object of this invention is to provide the method of controlling the metabolism of hydrogen bacteria.

  Another object of the present invention is to provide a method for producing useful substances by controlling the metabolism of hydrogen bacteria.

Another object of the present invention is to provide a hydrogen bacterium in which useful substances are accumulated more than usual by controlling the metabolism of the hydrogen bacterium, and a method for producing a microorganism-containing composition containing the hydrogen bacterium. And

In order to solve this problem, the present inventor has conducted extensive research, and as a result, from the cell of Hydrogenobacter themophilus TK-6 via an electronic medium substance that can take both oxidant and reductant forms. completed by performing the cultivation while pulling electrons led to the findings that can control the metabolism in a direction to increase the production of amino acids and fatty acids than when performing normal growth is, the present invention further extensive studies Te It came to do.

  That is, in the method for controlling metabolism of hydrogen bacteria of the present invention, hydrogen and carbon dioxide are extracted from the hydrogen bacteria in the medium while extracting electrons through an electron medium substance that can take both oxidant and reductant forms. Cultivation is performed by providing a substance that functions as the final electron acceptor of hydrogen bacteria.

Here, in the method for controlling metabolism of hydrogen bacteria according to the present invention, extraction of electrons is performed by one or more of the following (1) to (4) .
(1) Adsorb the electron medium substance on the cell surface of hydrogen bacteria and bring the electrode into contact with the medium, and apply an oxidation potential to the electrode. (2) Add the electron medium substance to the medium and bring the electrode into contact with the medium. Apply an oxidation potential to the electrode
(3) Adsorbing an electron medium substance on the cell surface of hydrogen bacteria and supplying oxygen continuously to the medium
(4) Adding an electron medium substance to the medium and supplying oxygen continuously to the medium

Further, in the metabolism control method of the hydrogen bacterium of the present invention, the hydrogen bacterium is hydro Genoa compactors thermophilus (Hydrogenobacter themophilus).

Next , the method for producing a useful substance of the present invention includes a step of performing the method for controlling metabolism of hydrogen bacteria of the present invention using Hydrogenobacter themophilus, thereby comprising an amino acid and a fatty acid. One or more useful substances selected from the group can be produced.

Next, the production method of the present invention includes a step of performing metabolic control method of the present invention, method der Hydro Genova compactors thermophilus are useful substance is accumulated than normal (Hydrogenobacter themophilus) is, from the usual Hydrogenobacter thermophilus (Hydrogenobacter themophilus) in which amino acids and fatty acids are accumulated is also obtained. And in the manufacturing method of the microorganism-containing composition of this invention, it is made to manufacture the microorganism-containing composition containing this Hydrogenobacter thermophilus (Hydrogenobacter themophilus).

According to the metabolism control method of the present invention, the metabolism of Hydrogenobacter thermophilus (Hydrogenobacter themophilus) is controlled, and the amount of useful substances in the metabolite produced by Hydrogenobacter thermophilus (Hydrogenobacter themophilus) as a carbon dioxide fixed product is reduced. It can be increased. Therefore, it is possible to produce useful substances more efficiently by using the metabolic control method of the present invention. Moreover, it becomes possible to provide Hydrogenobacter themophilus in which useful substances are accumulated more than before .

It is a figure which shows an example of embodiment of the metabolic control method of hydrogen bacteria of this invention. It is a figure which shows the other example of embodiment of the metabolic control method of the hydrogen bacteria of this invention. It is a figure which shows the result of having examined the influence which the electronic medium substance has on the growth of TK-6 (Example 1). It is the figure which examined whether TK-6 is utilizing the electronic medium substance as a final electron acceptor (respiration substrate) (Example 2). It is the structure schematic of the electroculture apparatus used in the Example. It is a figure which shows the time-dependent change of the electric current value in a culture test (Example 4). (Example 4) which is a figure which shows the measurement result of NAD ⁺ and NADH. (Example 4) which is a figure which shows the conversion result of NAD ^<+ > / NADH with energization when NAD ^<+ > / NADH is set to 1 about the result without energization of FIG. It is a figure which shows the microbial cell density after completion | finish of culture | cultivation (Example 6). It is a figure which shows the nitric acid concentration after completion | finish of culture | cultivation (Example 6). It is a figure which shows the time-dependent change of the electric current value in a culture test (Example 6). It is a chemical structural formula of various fatty acids increased by energization (Example 7). This is an estimated metabolic mechanism of hydrogen bacteria by electron extraction estimated from the results of this example.

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

In FIG. 1A, an example of embodiment of the metabolic control method of hydrogen bacteria of this invention is shown notionally. The method for controlling the metabolism of hydrogen bacteria according to the present invention allows hydrogen (H _{2) to be} extracted from the hydrogen bacteria 2 in the medium 4 through the electron medium substance 5 that can take both oxidant and reductant forms. ), Carbon dioxide (CO ₂ ), and a substance (for example, O ₂ or NO ₃ ⁻ ) that functions as a final electron acceptor of hydrogen bacteria, and culture is performed.

  In the embodiment shown in FIG. 1A, the extraction of electrons from the hydrogen bacterium 2 is performed by contacting (immersing) the electrode 9 with the culture medium (culture medium) 4 and applying an oxidation potential to the electrode 9. . By applying an oxidation potential to the electrode 9, the electron medium material 5 reduced to the hydrogen bacterium 2 is oxidized, and the electrochemical electrons can be continuously extracted from the hydrogen bacterium 2.

  Here, as shown in FIG. 1B, the electronic medium substance 5 may be adsorbed on the cell surface of the hydrogen bacterium 2. In this case, extraction of electrons from the hydrogen bacterium 2 through the electron medium material 5 can be performed more efficiently.

  The hydrogen bacterium 2 to which the metabolic control method of the present invention is applied is not particularly limited as long as it is an autotrophic organism that can grow using hydrogen (free hydrogen) as an energy source and carbon dioxide as a sole carbon source. . For example, Alcaligenes eutrophus (Cupriavidus necator), Alcaligenes hydrogenophilus, Alcaligenes ruhlandii, Alcaligenes latus, Alcaligenes paradoxus, Aquaspirillum autotrophicum, Azospirillum lipoferus, Calderobacterium hydrogenophilus, Derxia gummoser, Flavobacterium Hydrogentheractrum facilis, Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas hydrogenovora, Pseudomonas hydrogenothermophila, Pseudomonas palleronii, Pseudomonas thermophila, Pseudomonas saccharophila, Renobacter vacuolatum, Rhizobium ja ponicuni, Xanthobactertro Examples include autotrophica, Nocardia opaca, Hydrogenovibrio marinus and the like, and particularly, Hydrogenogenacter themophilus is preferable. Among the hydrogenobacter themophilus, the hydrogenobacter themophilus TK-6 strain is particularly suitable. The Hydrogenobacter themophilus TK-6 strain has a high growth rate (double growth in 1.5 hours), and has the highest growth rate among microorganisms capable of fixing carbon dioxide. Further, the hydrogen bacterium 2 may have a new substance-producing ability or the like incorporated by gene recombination.

  Hydrogen bacteria are microorganisms that can be easily isolated from all over the natural world, and have the advantage of being easily available.

  The culture medium 4 for culturing the hydrogen bacterium 2 may be appropriately selected from a general medium for the hydrogen bacterium 2 according to the type of the hydrogen bacterium 2. Moreover, what is necessary is just to select suitably according to the kind of hydrogen bacteria 2 to be used also about the supply amount of hydrogen and carbon dioxide, and culture | cultivation temperature.

  The substance that functions as the final electron accepting pair of the hydrogen bacterium 2 is appropriately selected according to the conditions of a preferable culture environment for the hydrogen bacterium 2 (whether it is aerobic or anaerobic). That is, when the culture environment is aerobic, oxygen supplied to the culture medium 4 by bubbling or the like may be used. When the culture environment is anaerobic, for example, nitrate respiration may be performed by hydrogen bacteria 2 as nitrate ions or the like. Hydrogenobacter themophilus can grow and proliferate regardless of whether the culture environment is an aerobic condition or an anaerobic condition.

The electronic medium substance 5 is a substance that can take both forms of an oxidant and a reductant, and a substance that can be reduced by the hydrogen bacterium 2 without deactivating the hydrogen bacterium 2 may be appropriately selected. Illustratively, anthraquinone derivatives (sodium anthraquinone-1-sulfonate, disodium anthraquinone-1,5-disulfonate, dipotassium anthraquinone-1,8-disulfonate, sodium anthraquinone-2-sulfonate, 1-amino-4-bromo Anthraquinone-2-sulfonic acid sodium and anthraquinone-2,6-disulfonic acid disodium, etc.) and quinone compounds such as 2-methyl-1,4-naphthoquinone, agricultural chemicals such as methylviologen, indigo carmine, neutral red, rema Zole brilliant blue, crystal violet, 2,6-dichlorophenol-indophenol, alcian blue, safranine, thionine and other pigment materials, or potassium hexacyanoferrate, iron ion complex (iron (II )-EDTA, _{etc.), Mn (II) Cl 2} , cited metallic materials such as potassium iodide and selenate.

  Here, in the case where the electronic medium material 5 is adsorbed on the cell surface of the hydrogen bacterium 2, it is preferable to use a pigment-based material as the electronic medium material 5. For example, when the hydrogen bacterium 2 is a hydrogenobacter themophilus, the use of neutral red is preferable. However, the effect of the present invention can be obtained without adsorbing the electronic medium substance 5 on the cell surface of the hydrogen bacterium 2. For example, when the hydrogen bacterium 2 is Hydrogenobacter themophilus, a quinone compound such as an anthraquinone derivative can also be preferably used.

  The optimum amount of the electronic medium substance 5 varies depending on the type of the electronic medium substance 5, the type of the hydrogen bacterium 2 to be used, the cell density, etc., but is generally 0.5 to 10 mM, preferably It may be 0.5 to 5 mM, more preferably 0.5 to 3 mM, and even more preferably about 2 mM. When the addition amount of the electron medium substance 5 is too small, electrons are not sufficiently extracted from the hydrogen bacteria 2 and it becomes difficult to obtain a metabolic control effect. 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 hydrogen bacteria 2 may be inhibited.

  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.

  The hydrogen bacterium 2 reduces the oxidized electron medium material 5 to convert it into the reduced electron medium material 5 (in other words, supplies electrons to the oxidized electron medium material 5 to reduce the reduced electron medium material 5. Has a function to convert to Therefore, by applying an oxidation potential to the electrode 9, electrons are extracted from the reduced electron medium material 5 and converted into the oxidized electron medium material 5. By these series of flows, electrons are continuously extracted electrochemically from the hydrogen bacterium 2, and the metabolism of the hydrogen bacterium 2 is controlled.

  Here, the value of the oxidation potential applied to the electrode 9 can be obtained based on the cyclic voltammogram of the electronic medium material 5. For example, when neutral red is used as the electronic medium material 5, a potential of about +0.4 V may be applied to the electrode 9. When anthraquinone-2,6-disulfonic acid disodium is used as the electronic medium material 5, a potential of about +0.6 V may be applied to the electrode 9.

  By controlling the metabolism of hydrogen bacteria 2 by the metabolic control method of the present invention, the proportion of useful substances contained in the metabolites of hydrogen bacteria 2 can be increased. Specifically, when the hydrogen bacterium 2 is a hydrogenobacter themophilus, the production amounts of amino acids and fatty acids can be increased.

  Here, when the hydrogen bacterium 2 is Hydrogenobacter themophilus, when it is desired to increase the amount of lysine and arginine as amino acids, the metabolic control of the present invention is controlled against Hydrogenobacter themophilus in the stationary phase or late logarithmic phase. It is preferred to apply the method. In addition, regarding the period of culturing while extracting electrons, if it is too long, the number of bacteria may decrease or the effect of increasing the amount of desired metabolites may be reduced. For example, it is shorter than 850 minutes, preferably It is 500 minutes or less, more preferably 300 minutes or less, and even more preferably about 150 minutes.

  In addition, when the hydrogen bacterium 2 is made into Hydrogenobacter themophilus, propionic acid, butyric acid, hexanoic acid, heptanoic acid, octylic acid, pelargonic acid, decanoic acid, undecanoic acid, which are straight chain saturated fatty acids having 3 to 12 carbon atoms, in particular, When it is desired to increase the amount of lauric acid (in particular, undecanoic acid and lauric acid having 11 and 12 carbon atoms), the growth phase (before the logarithmic phase, early or middle of the logarithmic phase, preferably before the logarithmic phase) It is preferable to apply the metabolic control method of the present invention to Hydrogenobacter themophilus. The effect of increasing the linear saturated fatty acid is easily obtained by culturing until the logarithmic growth phase is obtained and the stationary growth phase is reached while extracting electrons.

  By the metabolic control method of the present invention, the production amount of useful substances of hydrogen bacteria 2 can be increased. When hydrogen bacterium 2 is made into Hydrogenobacter themophilus, the amount of amino acids and fatty acids can be increased. Therefore, by using the metabolic control method of the present invention, it becomes possible to efficiently produce useful substances using hydrogen bacteria.

  In addition, according to the metabolic control method of the present invention, the amount of useful substances accumulated in hydrogen bacteria 2 can be increased more than usual (that is, when normal culture is performed without extracting electrons from hydrogen bacteria). That is, the hydrogen bacterium 2 in which the accumulated amount of useful substances is increased than usual is obtained. Therefore, this hydrogen bacterium 2 can be used as a microbial protein resource (SCP).

  It is also possible to provide a microorganism-containing composition containing hydrogen bacteria 2 in which the amount of useful substances accumulated is greater than usual. Examples of the microorganism-containing composition include livestock feed. By using as a livestock feed a microorganism-containing composition containing Hydrogenobacter themophilus in which the accumulation amounts of amino acids and fatty acids are increased more than usual, livestock feed with high nutritional value can be provided. In addition, it can be provided as a supplement or food for the purpose of ingesting amino acids or the like.

  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 electronic medium material 5 is oxidized electrochemically using the electrode 9, but the oxidation of the electronic medium material 5 is not limited to an electrochemical method. For example, since most of the electrochemically oxidizable compounds can be oxidized by oxygen, the electronic medium material 5 is oxidized by supplying oxygen efficiently by bubbling or the like in the culture medium 4. You can also. That is, by continuously supplying oxygen into the culture medium 4, it is possible to carry out the culture while extracting electrons from the hydrogen bacteria 2 through the electron medium substance 5, and to carry out the metabolic control method of the present invention.

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

(Used cells)
Hydrogenobacter thermophilus (TK-6) obtained from the Department of Applied Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, was used as a hydrogen bacterium. In the following description, this hydrogen bacterium is sometimes simply referred to as TK-6.
TK-6 can also be obtained from the RIKEN BioResource Center Microbial Materials Development Office.

  The composition of the medium used for the culture of TK-6 is shown below. In the culture test described later, a medium having the following composition was used.

[Medium composition (in 1 L of deionized water)]
(NH ₄ ) ₂ SO ₄ 3 g
KH ₂ PO ₄ 1g
K ₂ HPO ₄ 2g
NaCl 0.25g
FeSO ₄ · _7H 2 O 0.014g
MgSO ₄ · 7H ₂ O 0.5g
CaCl ₂ 0.03 g
Trace element solution 500μL

[Trace element solution (in 1 L of deionized water)]
MoO ₃ 4mg
ZnSO ₄ · _7H 2 O 28mg
CuSO ₄ · _5H 2 O 2mg
H ₃ BO ₃ 4 mg
MnSO ₄ · 5H ₂ O 4mg
CoCl ₂ · 6H ₂ O 4mg

TK-6 was subjected to a culture test described below after pre-culture. That is, using the above-mentioned medium, under anaerobic conditions (the gas composition of the gas phase part in the glass vial as a culture container is controlled to H ₂ : N ₂ : CO ₂ = 75: 10: 15 (1.5 kPa)) Then, after culturing with shaking at 70 ° C., it was subjected to a culture test described later.

Example 1
The effect of the electronic medium material on the growth of TK-6 was examined.

TK-6 was added to 10 mL of the above medium contained in a 100 mL glass vial (Duran) so that the cell density was 1 × 10 ⁷ cells / mL. Further, NaNO ₃ was added to the medium so as to be 60 mM, and the following electronic medium substance (oxidized substance) was added so as to be 2 mM. Then, the gas phase gas composition of the vial is H ₂ : N ₂ : CO ₂ = 75: 10: 15 (1.5 kPa), the vial is closed, cultured at 70 ° C. with shaking, and a batch test is performed. Carried out. In addition, a culture test in which no electronic medium substance was added was performed as a control test.

[Electronic material]
Anthraquinone-2,6-disulfonic acid disodium anthraquinone-1,5-disulfonic acid disodium salt Fe (III) -EDTA
Remazol Brilliant Blue

  The cell density after 60 hours of culture is shown in FIG. From the results shown in FIG. 2, it was confirmed that the addition of the electronic medium material does not inhibit the growth of TK-6.

  In addition, when anthraquinone-2,6-disulfonate dipotassium and anthraquinone-1,5-disulfonate disodium are used as the electron medium substances, the medium color is colorless due to the reduction of these electron medium substances by TK-6. It was also confirmed that the color changed from (transparent) to a reduced color (red).

(Example 2)
It was examined whether TK-6 utilizes an electron medium substance as a final electron acceptor (respiratory substrate).

The examination conditions were as follows.
(A) Oxygen breathing condition (b) Nitric acid breathing condition (c) Electron medium material condition 1: Anthraquinone-2,6-disulfonic acid disodium (d) Electron medium material condition 2: Anthraquinone-1,5-disulfonic acid disodium (E) Electronic medium material condition 3: Fe (III) -EDTA

(A) Oxygen breathing condition TK-6 was added to 10 mL of medium contained in a 100 mL glass vial (Duran) so that OD ₅₄₀ = 0.1, and the gas phase gas composition of the vial was changed to H. ₂ : CO ₂ : O ₂ = 75: 15: 10 (1.5 kPa) was cultured with shaking at 70 ° C., and a batch test was performed. That is, under this condition, the final electron acceptor is oxygen.

(B) Nitric acid respiration conditions TK-6 was added to 10 mL of medium contained in a 100 mL glass vial (Duran) so that OD ₅₄₀ = 0.1. Further, NaNO ₃ was added to the medium so as to be 60 mM. And the gas phase gas composition of the vial is H ₂ : CO ₂ : N ₂ = 75: 15: 10 (1.5 kPa), the lid of the vial is closed, cultured at 70 ° C. with shaking, and a batch test is performed. Carried out. In addition, a culture test in which no electronic medium substance was added was performed as a control test. That is, in this condition, the final electron acceptor is nitric acid.

(C) to (e) Electronic medium material conditions TK-6 was added to 10 mL of medium contained in a 100 mL glass vial (Duran) so that OD ₅₄₀ = 0.1. Furthermore, an electronic medium substance was added to the medium so as to be 2 mM. And the gas phase gas composition of the vial is H ₂ : CO ₂ : N ₂ = 75: 15: 10 (1.5 kPa), the lid of the vial is closed, cultured at 70 ° C. with shaking, and a batch test is performed. Carried out.

  The results of the culture test are shown in FIG. Under the oxygen respiration condition (a) and the nitric acid respiration condition (b), good growth of TK-6 was confirmed. However, TK-6 did not grow under the electronic medium material conditions (c) to (e). From this, it has been clarified that TK-6 cannot grow by respiration using the above-mentioned electron medium substance as a final electron acceptor.

  Here, in Example 1, when anthraquinone-2,6-disulfonic acid disodium and anthraquinone-1,5-disulfonic acid disodium are used as the electronic medium substances, reduction of these electronic medium substances by TK-6. It was confirmed that the medium color changed from colorless (transparent) to reduced color (red). Further, in this example, it was confirmed that these electronic medium substances did not function as the final electron acceptor of TK-6. Comprehensively judging from these results, it was suggested that by using an electronic medium substance, electrons in metabolic processes in TK-6 cells could be extracted outside the cells.

(Example 3)
In order to efficiently transfer electrons from the inside of the TK-6 cells to the outside of the cells, it is considered that the use of the cell-adsorbing type electron medium material is preferable to the use of the medium-dissolved type electronic medium substance. It was. Therefore, cell adsorption type electronic medium materials were examined.

TK-6 cell suspension was added to the following nine kinds of electronic medium substance solutions, and the adsorption of TK-6 of the electronic medium substance was evaluated after 6 hours.
-Potassium ferricyanide-PMS (phenazine methosulfate)
・ Methyl viologen ・ Neutral red ・ Fe (III) -EDTA
・ Indigo carmine ・ Methylene blue ・ DCPIP (2,6-dichlorophenol-indophenol)
・ Remazol Brilliant Blue

  As a result, the neutral red concentration of the solution tended to decrease. From this, it was estimated that neutral red was adsorbed on TK-6 and settled together with TK-6.

Example 4
The effect of electrochemically extracting electrons from TK-6 adsorbed with neutral red via neutral red was examined. In other words, the influence by electrochemically changing the electron flow (redox balance) in the TK-6 cells was examined.

  In this example, in order to extract electrochemical electrons via neutral red from TK-6 adsorbed with neutral red (specifically, hydrogen → intra-TK-6 cell → neutral red → Electrons were collected by the flow of the electrodes), and the electroculture apparatus 1 shown in FIG. This electric culture apparatus 1 used a 100 mL screw cap bottle (Duran) (hereinafter referred to as container 20) 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 as a counter electrode tank was formed into a bag shape (hereinafter also referred to as a bag 21) by molding the ion exchange membrane 6. Specifically, a cation exchange membrane (manufactured by DuPont, Nafion K) was processed by thermocompression bonding with a heat sealer, and the upper part was filled with a silicon adhesive and sealed. The area on one side of the anion exchange membrane 6 corresponds to 10.5 cm ² . The bag 21 contained the electrolyte solution 4a and the counter electrode 10 and was immersed in the electrolyte solution 4a. The electrolyte solution 4a was the above-described medium described in the column of the cells used.

  100 mL of the culture solution 4 (the above-described medium described in the column of cells used) was stored 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 pierced into the container 20 from the lid 30 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. The size of the working electrode 9 was 6 cm × 2.3 cm. The size of the counter electrode 10 was 5 cm × 1 cm.

  TK-6 adsorbed with neutral red is obtained by converting the cells (late logarithm phase) collected by carrying out the pre-culture (under nitrate respiration conditions) described in the column of used cells into a solution containing 2 mM neutral red. After suspending and adsorbing neutral red on the cell surface, the cells were washed with a medium to wash away excess neutral red and then subjected to a culture test.

TK-6 to which neutral red was adsorbed by the above procedure was added to the culture solution 4 contained in the container 20 of the electric culture apparatus 1 (OD ₅₄₀ = 2.3), and the change in current value over time was measured. In addition, the addition amount of TK-6 in a present Example is the high microbial cell density conditions (stationary microbe conditions) in which a microbial cell does not propagate.

The test conditions were the following three conditions. Further, the gas composition in the gas phase portion of the container _{20 H 2: N 2: CO} 2 = 75: 10: Controls the 15 (1.5 kPa), the culture temperature was 70 ° C..
(1) TK-6 with neutral red adsorbed added, NaNO ₃ added (60 mM)
(2) Add TK-6 with neutral red adsorbed, no NaNO ₃ added (3) No bacterial cells added, neutral red added (2 mM), NaNO ₃ added

  The applied potential to the working electrode 9 was + 0.4V.

  In this example, and in the following examples, in order to accurately evaluate the effect of electrochemical oxidation, the electron medium substance is oxidized on the electrode other than on the electrode, or oxygen reacts on the electrode. The test was conducted under anaerobic conditions (nitrate respiration conditions).

  The change with time of the current value is shown in FIG. In both conditions (1) and (2), the current value derived from the bacterial cells was detected in the vicinity of 200 minutes. Moreover, since the current value was such that the condition (1) <the condition (2), in the condition (2), the electrons that should flow to the nitric acid through the respiratory chain originally flowed to the electrode through the neutral red. Estimated. In the condition (1), the nitric acid concentration was reduced to 37.1 mM after the end of the experiment (after 600 minutes).

Next, after the completion of the experiment under the condition (1), NAD ⁺ (oxidized substance) and NADH (reduced substance), which are important substances used for electron transfer reaction in intracellular metabolism, were measured. Specifically, 1 mL of the culture solution after completion of the experiment was collected, and then the cells were collected by centrifugation (15000 rpm, 4 ° C., 10 minutes), and the collected cells were collected using NAD ⁺ extraction buffer (0.5 M perchloric acid). ) Or NADH extraction buffer (50 mM sodium hydroxide, 1 mM EDTA), and sonication was repeated 5 times for 30 seconds to obtain a metabolite extract. About this extract, NAD ⁺ and NADH were measured using CycLex NAD ⁺ / NADH Colorimetric assay kit. In addition, as a control test, the same test was performed when the condition (1) was performed without energization, and NAD ⁺ and NADH were measured.

The results are shown in FIG. FIG. 7 shows a conversion result of NAD ⁺ / NADH with energization when NAD ⁺ / NADH is set to 1 for the result without energization. From the results shown in FIG. 6 and FIG. 7, it became clear that the ratio of NAD ⁺ that is an oxidant is increased by energization. Therefore, it has been clarified that the extraction of electrons from the cell to the electrode causes an increase in important NAD ⁺ (oxidant) / NADH (reductant) related to electron exchange in metabolism. This also suggested the possibility of electrochemically controlling metabolism of TK-6.

(Example 5)
The metabolome analysis was performed about the sample after performing an electric culture test (stationary bacteria test).

Culturing was performed under the same conditions as in Example 4 (1) and with the condition (1) in Example 4 de-energized, and the cultures were collected 150 minutes and 850 minutes after the start of culture. The bacterial cells contained in the collected culture solution were collected on a filter (Millipore Isopore Membrane Filter HTTP 0.4 μm pore 47 mm diameter, Millipore) using a suction filtration device. The bacterial cells collected on the filter were washed twice with 10 mL of MilliQ water, and the filter was placed in a sealed petri dish containing 2 mL of methanol (for LC / MS, Wako) containing the internal standard (H3304-1002, HMT). The filter was immersed in methanol with the bacterial cell adhesion side down. A filter immersed in methanol was subjected to ultrasonic treatment for 30 seconds to obtain a metabolite extract. The number of collected bacteria was as follows.
(A) 150 minutes energization: 6.7 × 10 ⁹ cells
(B) 150 minutes de-energization: 6.7 × 10 ⁹ cells
(C) 850 minutes energization: 3.4 × 10 ⁹ cells
(D) No energization for 850 minutes: 5.8 × 10 ⁹ cells

  The collected cells were stirred by adding 1600 μL of chloroform and 640 μL of MilliQ water, and centrifuged (2300 × g, 4 ° C., 5 minutes). After centrifugation, 325 μL × 4 aqueous layers were transferred to an ultrafiltration tube (MILLIPORE, Ultra Free MC PLHCC HMT centrifugal filter unit 5 kDa). This was centrifuged (9100 × g, 4 ° C., 120 minutes) and subjected to ultrafiltration treatment. The filtrate was dried and dissolved again in 50 μL of Milli-Q water and used for measurement of metabolites (bacterial cell sample).

  In addition, 40 μL of the culture solution was collected 850 minutes after the start of the culture, and 10 μL of an aqueous solution prepared so that the concentration of the internal standard substance was 1000 μM was added to the 40 μL sample and stirred, and the ultrafiltration tube (MILLIPORE) was stirred. , 325 μL × 4 pieces were transferred to an ultra-free MC PLHCC HMT centrifugal filter unit (5 kDa). This was centrifuged (9100 × g, 4 ° C., 120 minutes) and subjected to ultrafiltration treatment. The filtrate was dried and dissolved again in 50 μL of Milli-Q water, and used for metabolite measurement (supernatant sample).

  For the analysis of metabolites, CE-TOF MS system (Agilent) was used, and cationic substances and anionic substances were measured in the cation mode and the anion mode, respectively. The measurement conditions for the cation mode and the anion mode are shown below.

[Cation mode measurement conditions]
Run buffer: Cation Buffer Solution (p / n: H3301-1001)
Rinse buffer: Cation Buffer Solution (p / n: H3301-1001)
Sample injection: Pressure injection 50 mbar, 10 sec
CE voltage: Positive, 27 kV
MS ionization: ESI Positive
MS capillary voltage: 4,000 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)

[Measurement conditions for anion mode]
Run buffer: Anion Buffer Solution (p / n: H3302-1021)
Rinse buffer: Anion Buffer Solution (p / n: H3302-1022)
Sample injection: Pressure injection 50 mbar, 25 sec
CE voltage: Positive, 30 kV
MS ionization: ESI Negative
MS capillary voltage: 3,500 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)

Peaks detected by CE-TOF MS were automatically integrated with MasterHands ver. The sample was automatically extracted using 2.9.0.9 (developed by Keio University), and mass-to-charge ratio (m / z), migration time (MT) and peak area value were obtained as peak information. The obtained peak area value was converted into a relative area value using the following [Equation 1] (for cell samples, correction was made based on the number of collected cells. That is, the number of samples in the following equation [1]. Was calculated as the number of cells collected.) 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. However, since there were substance-specific adducts and fragments, it was not possible to examine all of them. With respect to the examined peaks, the peaks between the samples were collated and aligned based on the values of m / z and MT.
Relative area value = Area value of target peak / (Area value of internal standard substance × Sample amount) ... [Formula 1]

The detected peak was collated and searched with all substances registered in the HMT metabolite database based on the values of m / z and MT. The tolerance for the search was ± 0.5 min for MT and ± 10 ppm for m / z.
Mass error (ppm) = (actual value−theoretical value × 106) / actual value

  108 substances including amino acids, organic acids, sugar phosphates and nucleic acids were analyzed as major metabolites. For the calibration curve, the peak area corrected by the internal standard substance was used, and the concentration was calculated as one inspection quantity of 100 μM (internal standard substance 200 μM) for each substance.

  As a result of the metabolome analysis, 130 peaks (cation 42, anion 88) were detected in the cell sample, and 62 substances (cation 28, anion 34) among them could be quantified. For the supernatant sample, 43 peaks (cation 25, anion 18) were detected, and 33 substances (cation 24, anion 9) among them could be quantified.

  Table 1 shows the metabolites of the bacterial cell sample (150 minutes) that increased with energization than with non-energization.

  In the pyrimidine group, CTP (cytidine triphosphate), CDP (cytidine diphosphate), and CMP (cytidine monophosphate) increased.

  In the purine group, Guanine, GMP (guanosine monophosphate), GDP (guanosine diphosphate), and GTP (guanosine triphosphate) increased.

  In the amino acid group, Trp (tryptophan), His (histidine), Glu (glutamic acid), Arg (arginine), Lys (lysine), Asp (aspartic acid) increased.

  In addition, propionic acid (propionic acid), 2-aminodipic acid (2-aminoadipic acid), and CoA (coenzyme A) increased.

  Next, Table 2 shows the metabolites of the bacterial cell sample (850 minutes) that were increased during energization than during non-energization.

  In the pyrimidine group, CMP (cytidine monophosphate), CDP (cytidine diphosphate), CTP (cytidine triphosphate), dCMP (deoxycytidine monophosphate), dCDP (deoxycytidine diphosphate), dCTP (deoxycytidine) Triphosphate), dTMP (thymidine monophosphate), dTDP (thymidine diphosphate), dTTP (thymidine triphosphate), and UDP (uridine diphosphate) increased.

  In the purine group, GMP (guanosine monophosphate), GDP (guanosine diphosphate), GTP (guanosine triphosphate), IMP (inosine monophosphate), adenine (adenine), ADP (adenosine diphosphate), dADP (Deoxyadenosine diphosphate) and dATP (deoxyadenosine triphosphate) increased.

  In the amino acid group, Lys (lysine), Arg (arginine), Ala (alanine), Trp (tryptophan), Tyr (tyrosine), Phe (phenylalanine), Ser (serine), Thr (threonine), Gly (glycine), His (Histidine), Pro (proline), Uric acid (uric acid), Oxoproline (oxoproline) increased.

  In the TCA cycle group, citric acid (citric acid), cis-aconic acid (cis-aconitic acid), and succinic acid (succinic acid) increased.

  In the gluconeogenic group, AcCoA (acetyl coenzyme A), Lactic acid (lactic acid), Glyceric acid (glyceric acid), and F6P (fructose-6-phosphate) increased.

  In addition, Choline (choline), Ethanolamine (ethanolamine), and Urea (urea) increased.

  From the results shown in Table 1 and Table 2, with respect to the bacterial cell samples, CTP (cytidine triphosphate), CDP (cytidine diphosphate), CMP (cytidine monophosphate), guanine (guanine), in particular, by energization. , GMP (guanosine monophosphate), GDP (guanosine diphosphate), GTP (guanosine triphosphate), Trp (tryptophan), His (histidine), Arg (arginine), and Lys (lysine) increase. Of these, it has been clarified that the increased amounts of Arg (arginine) and Lys (lysine) are remarkably increased.

  Table 3 shows comparison data of concentration between electrification and non-energization (Control) for Arg (arginine) and Lys (lysine), in which the amount of increase by energization was significantly large.

  Judging from the results shown in Table 3, a larger amount of Arg (arginine) and Lys (lysine) is obtained when the energization is performed for 150 minutes than when the energization is performed for 850 minutes and the cells are collected. It was thought that it could be recovered.

  Next, regarding the supernatant sample, the metabolites that increased when energized than when not energized are shown in Table 4.

  In the pyrimidine group, UDP (uridine diphosphate), CMP (cytidine monophosphate), and UMP (uridine monophosphate) increased.

  In the purine group, GMP (guanosine monophosphate) and AMP (adenosine monophosphate) increased.

  In the amino acid group, Gly (glycine), Val (valine), Leu (leucine), lle (isoleucine), Asn (asparagine), Gln (glutamine), His (histidine), Tyr (tyrosine), Pro (proline), Phe (Phenylalanine), Ser (serine), Arg (arginine), Lys (lysine), 5-Oxoproline (5-oxoproline) increased.

  In the TCA cycle group, succinic acid (succinic acid) and malic acid (malic acid) increased.

  In the fatty acid group, butyric acid (butyric acid), isovaleric acid (isovaleric acid), adipic acid (adipic acid), trans-ferulic acid (trans-ferulic acid), and sebacic acid (sebacic acid) increased.

(Example 6)
In Example 4, a culture test was carried out under high cell density conditions (stationary bacteria conditions). In this example, a culture test similar to that in Example 4 was carried out under growth conditions to examine the controllability of metabolism.

Specifically, anthraquinone-2,6-disulfonic acid disodium is dissolved in the culture solution 4 (2 mM) as an electron medium material without adsorbing neutral red on TK-6, and the applied potential of the working electrode 9 is +0. The culture test was performed under the same conditions as the condition (1) of Example 4 except that the initial cell density was 1.0 × 10 ⁷ cells / mL. In addition, the same culture test was conducted with no current applied.

  The cell density after completion of the culture test is shown in FIG. 8, and the nitric acid concentration of the culture solution is shown in FIG. It was clarified that the energization increased the cell density after completion of the culture, and the nitric acid concentration correspondingly decreased.

  Next, FIG. 10 shows the results of examining the temporal change of the current value depending on the presence or absence of bacterial cells under energization conditions. When cells were present, the current value increased with the growth of the cells. Therefore, it became clear that the electric current through anthraquinone-2,6-disulfonic acid disodium was detected with the proliferation of a microbial cell.

(Example 7)
Metabolome analysis was performed on the sample after the electroculture test (growth conditions).

A culture test was performed under the same conditions as in Example 6, and the cells were collected after 3000 minutes from the start of the culture in which an increase in current value was observed. The number of collected bacteria was as follows.
(A) Energization: 5.0 × 10 ⁹ cells
(B) Non-energization: 2.8 × 10 ⁹ cells

  The bacterial cell collection method and metabolomic analysis conditions were the same as in Example 5.

  As a result of the metabolomic analysis, 96 peaks were detected, and 30 metabolites were increased by energization. Metabolites of amino acid groups and amino acid biosynthesis groups increased by energization are shown in Table 5, and metabolites such as linear saturated fatty acid groups increased by energization are shown in Table 6. FIG. 11 shows chemical structural formulas of various fatty acids increased by energization.

  In the amino acid group, Gln (glutamine), Val (valine), Glu (glutamic acid), Pro (proline), lle (isoleucine), Asp (aspartic acid), Ala (alanine), Tyr (tyrosine), Leu (leucine) Increased.

  In the amino acid biosynthesis group, betaine (betaine), 2,6-diaminopimeric acid (2,6-diaminopimelic acid), 2-aminodipic acid (2-aminoadipic acid), 2-hydroxyisobutyric acid (2-hydroxyisobutyric acid), Glycerol 3-phosphate (glycerol 3-phosphate) increased.

  In the linear saturated fatty acid group, undecanoic acid (undecanoic acid), Lauric acid (lauric acid), Decanoic acid (decanoic acid), butyric acid (butyric acid), octanic acid (octylic acid), propionic acid (propionic acid), pelic acid (Pelargonic acid), Heptanoic acid (heptanoic acid), Hexanoic acid (hexanoic acid) increased.

  In addition, Isovaleric acid (isovaleric acid) and Tiglycic acid (tiglic acid) increased.

  From the above results, it was clarified that the amount of amino acid was increased by energization in the growth condition as well as in the stationary bacteria condition. In addition, the growth conditions were characterized by an increase in the amount of linear saturated fatty acids having 3 to 12 carbon atoms. In particular, it has been clarified that the effect of increasing the straight chain saturated fatty acids (undecanoic acid and lauric acid) having a long chain length of 11 and 12 carbon atoms is large.

(Summary)
The metabolic mechanism of hydrogen bacteria estimated from the results of this example is shown in FIG. By extracting electrons electrochemically, a reduced form of the electron medium material (NR _red (reduced form of neutral red) or 2,6-AQDS _red (reduced form of anthraquinone-2,6-disulphonic acid disodium salt)) And oxidized to an oxidant (NR _ox (neutral red oxidant) or 2,6-AQDS _ox (anthraquinone-2,6-dipotassium oxidant)). In hydrogen bacteria cells, NAD ⁺ is reduced to NADH by electrons derived from hydrogen, but the reaction of NADH being oxidized to NAD ⁺ by the extraction of electrons through the electron medium material by the electrode becomes dominant, and it is against NADH. The amount of NAD ⁺ increases. That is, it is presumed that the redox balance in hydrogen bacteria cells shifts to oxidation, and the productivity of amino acids and fatty acids is improved. Hydrogen bacteria are very fast growing organisms, and the fixed carbon dioxide is used to efficiently build their own cells. For this reason, it is considered that there is almost no significant accumulation of useful substances in the cells under normal culture conditions. On the other hand, according to the present invention, amino acids and fatty acids can be significantly accumulated by controlling metabolism. It can be said that this is an extremely superior technique compared to a normal culture method in that useful substances can be significantly accumulated in cells.

2 Hydrogen bacteria 4 Medium 5 Electron medium material 9 Electrode

Claims (4)

A substance that functions as a final electron acceptor of hydrogen bacterium, carbon dioxide, and hydrogen bacterium, while extracting electrons through an electron medium substance that can take both oxidant and reductant forms for hydrogen bacteria in the medium; There line of the culture giving,
The extraction of the electrons is performed by one or more of the following (1) to (4):
(1) Adsorbing the electron medium substance on the cell surface of the hydrogen bacterium and bringing the electrode into contact with the medium to apply an oxidation potential to the electrode
(2) Adding the electron medium substance to the medium and bringing an electrode into contact with the medium and applying an oxidation potential to the electrode
(3) The electron medium substance is adsorbed on the cell surface of the hydrogen bacterium and oxygen is continuously supplied to the medium.
(4) Adding the electron medium substance to the medium and continuously supplying oxygen to the medium
A method for controlling metabolism of hydrogen bacteria, characterized in that the hydrogen bacteria are Hydrogenogenacter themophilus . A method for producing one or more useful substances selected from the group consisting of amino acids and fatty acids, comprising a step of performing the metabolic control method according to claim 1 . A method for producing Hydrogenobacter themophilus in which amino acids and fatty acids are accumulated more than usual , comprising a step of performing the metabolic control method according to claim 1 . A method for producing a microorganism-containing composition comprising Hydrogenobacter themophilus according to claim 3 .