JPH0928372A - Culturing of chemical autotrophic bacterium by potential control - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a culturing system capable of directly or indirectly feeding an electrical energy to a chemical autotrophic bacterium and promoting the proliferation. SOLUTION: This method for culturing is to carry out the culturing of a chemical autotrophic bacterium (an iron oxidizing bacterium, etc.) by applyihg a potential within the range suitable for proliferation of bacterium in a culture medium containing an electron donor. The method for culturing of the iron oxidizing bacterium (e.g. Thiobacterium ferrooxidans) comprises applying 0.5 to -0.4V vs. Ag/AgCl or -0.4 to -1.0V vs. Ag/AgCl potential into the culture medium containing Fe<2+> . Thereby, the culturing of the bacterium (a sulfur oxidizing bacterium, a denitrifying bacterium, etc.) is carried out by applying the potential within the range suitable for the proliferation of the bacterium into the culture medium containing the electron donor to directly utilize the electrical energy applied to the culture medium as a living body energy for the bacterium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for culturing chemoautotrophic bacteria by controlling potential. More specifically, the present invention relates to a culturing method for promoting the growth of chemoautotrophic bacteria such as iron-oxidizing bacteria by directly or indirectly supplying electric energy to the bacteria.

[0002]

2. Description of the Related Art Substance production by microorganisms such as bacteria is used as a fermentation method in various fields such as the pharmaceutical industry, food industry, and fermentation industry. However, mainly used for this purpose are heterotrophic microorganisms that produce substances using plant-derived organic matter as an energy source, and the energy utilization efficiency of these microorganisms is 1% when sunlight is converted to the first energy source. Below, very low. On the other hand, autotrophic microorganisms can directly extract energy from light or reducing compounds, and can grow using trace elements and carbon dioxide.

[0003] Photoautotrophic microorganisms (photosynthetic microorganisms) that can directly use sunlight have a relatively high energy use efficiency of about 5%. However, since photosynthetic microorganisms grow slowly and it is difficult to supply light energy efficiently, they are rarely used for substance production. In the case of artificial culture,
At night, light energy must be generated and supplied from electrical energy, so that the energy use efficiency of the entire material production using photosynthetic microorganisms is reduced.

When a solar cell is used, the energy efficiency when converting sunlight into electric energy is 15 to 20%. Therefore, if electrical energy can be supplied directly or indirectly to microorganisms, and the microorganisms can utilize it,
A microbial culture system or substance production system with high energy utilization efficiency can be constructed. Here, a group of bacteria called chemoautotrophic bacteria are inorganic compounds such as hydrogen sulfide and other reducing sulfur compounds, ammonia, nitrite, carbon monoxide,
It can grow by the oxidation of hydrogen molecules, ferrous ions and the like. That is, chemoautotrophic bacteria grow by reducing electrons by obtaining electrons from the above substances called electron donors, converting them into high-energy substances such as ATP and NADH, and using them as bioenergy. Therefore, it is conceivable to electrochemically generate the above-mentioned reduced-state inorganic substance and indirectly supply electric energy to the bacteria via the reduced-state inorganic substance. In addition, if electric energy can be directly supplied to the cells without passing through such a reduced inorganic substance, it is considered that the energy use efficiency is further improved. If a bacterial culture system or substance production system utilizing these electric energy is established, it can be a new bacterial culture technique or substance production technique that replaces the conventional fermentation method.

As a microorganism that can be cultivated by using electric energy, cultivation of chemoautotrophic bacteria such as hydrogen bacterium and iron-oxidizing bacterium has been studied so far. Since hydrogen bacteria have a high protein content in their cells, the United States Aeronautics and Space Administration (NASA) started research on their culture as a candidate for a long-term food source in space. Can be cultivated by producing hydrogen gas electrochemically [Schlegel, HG et al., Nature, 205: 308-309]
(1965)]. However, there are many problems that need to be solved, such as generation of superoxide and hydrogen peroxide during culturing, which hinders bacterial growth. In addition, since it is necessary to generate hydrogen gas, which has a high risk of explosion, the current situation is that mass culture by supplying electric energy has not been studied.

On the other hand, iron-oxidizing bacteria are useful microorganisms used for desulfurizing coal and recovering rare metals such as gold and uranium. This iron-oxidizing bacterium grows using Fe ²⁺ as an energy source, and extracts electrons (e ⁻ ) when oxidizing Fe ²⁺ to Fe ³⁺ as energy. However, since Fe ³⁺ generated after oxidation forms a precipitate in the medium, the concentration of Fe ^{2+ in} the medium cannot be increased, which hinders large-scale culture. Therefore, conventionally, culturing by electrochemically regenerating Fe ³⁺ into Fe ²⁺ has been studied. Conventional culturing of iron-oxidizing bacteria by such an electrochemical method is performed by a constant current electrolysis method (for example, Yunker, SB et al., Biotechnol. Bioeng., 28: 1
867-1875 (1985)] or constant voltage electrolysis [eg Deni
Sov, GB, Microbiologiya, 50: 919-923 (1981)], it was completely unknown how the electrochemical reduction reaction was involved in bacterial growth.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made in consideration of such a situation, and provides a culturing method in which chemoautotrophic bacteria are directly or indirectly supplied with electric energy to make them usable. An object of the present invention is to provide a culturing method for promoting the growth of the bacterium using electric energy, and a method for using the bacterium, the growth of which is promoted, for the production of a useful substance or a substance conversion element.

[0008]

In order to investigate how the electrochemical reduction reaction is involved in the growth of chemoautotrophic bacteria, the inventors of the present invention have studied the electron donation for each bacterium. When the relationship between the electrode reaction and the growth of bacteria was examined by controlling the electrode potential in the presence of the body, the conditions under which bacteria could be cultivated with high energy utilization efficiency were found, and after further diligent study The present invention has been completed.

That is, the present invention is characterized in that a chemoautotrophic bacterium is cultured by applying a potential within a range suitable for the growth of the bacterium in a medium containing an electron donor of the bacterium. The present invention relates to a method for culturing a vegetative bacterium.

In the present invention, the chemoautotrophic bacterium is one which oxidizes an electron donor by a chemical dark reaction to obtain energy, for example, an iron-oxidizing bacterium [representative bacterium Thiobacillus
(Thiobacillus)], sulfur-oxidizing bacteria (representative bacterium Thiobacillus), denitrifying bacteria (representative bacterium Paracoccus),
Hydrogen bacteria (representative bacterium Alcaligenes), ammonia-oxidizing bacteria (representative bacterium Nitrosomona
s)], nitrite-oxidizing bacteria (representative bacterium Nitroba
cter)] and the like.

In the present invention, the electron donor is a substance that can be used by chemoautotrophic bacteria as an energy source and oxidizes it to obtain energy, and must be electrochemically renewable. No, it depends on each chemoautotrophic bacterium. For example, ferrous iron (Fe ²⁺ ) and / or reducing sulfur molecules are used for iron-oxidizing bacteria and sulfur-oxidizing bacteria, and hydrogen molecules (H ₂ ) are used for denitrifying bacteria and hydrogen-producing bacteria, and ammonia oxidizing is used. It is an ammonium ion (NH ₄ ⁺ ) for bacteria and a nitrite ion (NO ₂ ⁻ ) for nitrite-oxidizing bacteria. It should be noted that electron donors for chemoautotrophic bacteria other than the above can be easily found in literatures and the like in the relevant field.

The medium used in the present invention is not particularly limited with respect to other components as long as it contains each of the above-mentioned electric donors for chemoautotrophic bacteria, and the growth of chemoautotrophic bacteria to be cultured is not limited. It goes without saying that it can be contained as long as it is necessary and does not have an adverse effect such as inhibiting the oxidation / reduction of the above-mentioned electric donor. The above medium is usually a liquid, but if the potential control in the medium is possible and the free movement of the electron donor in the medium is not hindered, even if it is in a mixed state of solid and liquid, it is in a solid state. May be

In addition, the potential of the chemoautotrophic bacterium in the present invention during culture needs to be controlled within a range suitable for the growth of the bacterium. Normally, the potential is controlled by arranging a working electrode, a counter electrode, and a reference electrode in a medium and applying a specific potential to the working electrode. At this time, in order to prevent the re-oxidation of the electron donor, it is preferable that the working electrode and the counter electrode are separated by an ion exchange membrane. In addition, it is preferable that the materials of the above-mentioned electrodes, particularly the material of the working electrode, etc. are selected according to the bacteria to be cultured so that desired electron transfer can be performed. The applied potential can be easily determined in an appropriate range depending on each chemoautotrophic bacterium and the electrode used, but is usually 1.0 to −1.0 V vs. It is in the range of Ag / AgCl.

In addition, the culture according to the present invention can be carried out under appropriate pH and temperature except that the applied potential is controlled as described above.
Preferably under the optimal conditions of the bacteria, under aeration or shut off the air according to the bacteria, injecting an inert gas appropriately,
It can be carried out in the above-mentioned medium similarly to the culture of general chemoautotrophic bacteria. If the above conditions are satisfied,
The culture may be performed in a batch system or a continuous system.

The present invention also comprises culturing iron-oxidizing bacteria in a medium containing ferrous iron (Fe ²⁺ ) in an amount of 0.5 to −.
0.4V vs. Ag / AgCl or -0.4 to -1.0 V vs. The present invention relates to a method for culturing iron-oxidizing bacteria, which is performed by applying a potential of Ag / AgCl.

Here, the iron-oxidizing bacterium is a thiobacillus, for example, Thiobacillus ferrooxidans (Thi) as described above.
obacillus ferrooxidans), as well as Siderocapsa (Sider
ocapsa), Siderococcus and the like.

Further, according to the present invention, the chemoautotrophic bacterium is cultured in a medium containing an electron donor of the bacterium by applying an electric potential in a range suitable for the growth of the bacterium, and an electric current added to the medium is applied. It relates to a method for culturing chemoautotrophic bacteria, characterized in that energy is directly used as bioenergy of the bacteria.

[0018]

EXAMPLES The present invention will be described below with reference to examples.
The present invention is not limited to these examples. Example 1 Electron Transfer to Bacteria via Electron Carriers The materials used in this example and the method of the test performed are first described and then the results of the test are described. A. Materials and Methods (1) Culture of iron-oxidizing bacteria The iron-oxidizing bacteria are Thiobacillus ferrooxidans (Thiob).
acillus ferrooxidans) ATCC 23270 was used.
For culture, m9K + CaMg medium [(NH ₄ ) ₂ S
O ₄ ; 3.0 g / l, KH ₂ PO ₄ ; 0.05 g / l,
_{MgSO 4 · 7H 2 O; 0.5g} / l, Ca (NO 3)
_{2 · 4H 2 O; 0.01g /} l ] to FeSO ₄ · 7H ₂
The one to which 22.2 g / l of O was added was used. PH of medium
Was adjusted to 1.5 with sulfuric acid. This medium was sterilized by an autoclave and then used for culture. Culture temperature is 27 ±
It was carried out under aeration at 5 ° C. The cells after 3 to 4 days of culture were subjected to the following tests. Also, water is a pure water generator (Millipore,
The one manufactured under the trade name Milli-Q) was used in the following tests.

[0019] (2) As Fe ²⁺ concentration in the affected media Fe ²⁺ concentration in the medium for iron oxidizing bacteria growth is 0～700MM Fe
It was added SO ₄ · 7H ₂ O, were cultured iron oxidizing bacteria. The inoculum amount was set to 10%. The other culture conditions were the same as in the above (1). After culturing for 4 days, the bacterial concentration was measured using a bacteria counter. The pH of the medium was measured using a glass electrode (trade name: pH meter 220, manufactured by Corning Incorporated).

(3) Cultivation of iron-oxidizing bacteria by medium exchange The iron-oxidizing bacteria were cultured in m9K + CaMg medium containing 80 mM Fe ²⁺ . After 3 to 4 days of culture,
The medium was exchanged by centrifugation at 10,000 rpm and subculturing in a new medium containing 80 mM Fe ²⁺ . The second and subsequent medium exchanges were performed about every day in the same procedure.

(4) Electrochemical Measurement (Cyclic Voltammetry) Cyclic voltammetry is an electrochemical measuring instrument (B
The product was manufactured by AS, trade name BAS100B / W). The working electrode was a platinum electrode (diameter: 1.6 mm, manufactured by BAS, trade name 11-2013), and the counter electrode was a platinum wire (BAS).
(Trade name: 51-2233), reference electrode is a silver / silver chloride electrode (Ag / AgCl, manufactured by BAS, trade name: 11-202)
0) was used. The electrolyte is m9K + CaMg medium (pH
1.5) was used. The Fe ²⁺ concentration was 80 mM. In the cyclic voltammetry of the electrolytic solution in which the bacterial cells were suspended, the above-mentioned electrolytic solution containing the iron-oxidizing bacteria added thereto at a concentration of 10 ¹¹ cells / ml was used.

(5) Cultivation of iron-oxidizing bacteria by electric potential control (electroculturing) Electroculturing uses the electroculturing apparatus 1 shown in FIG. 1, and the test bacteria are placed in a culture tank 2 filled with a medium (culture solution) 3. It was suspended at a predetermined concentration. Here, a platinum mesh electrode (50 × 100 mm, 80 mesh) was used as the working electrode 4 in the culture device 1,
A carbon fiber is provided as the counter electrode 5, and a silver / silver chloride electrode (Ag / AgCl, manufactured by Toa Denpa Co., Ltd., trade name HS-205C) is provided as the reference electrode 6, and both electrodes are connected to the potential control device 7. The working electrode 4 and the counter electrode 5 are separated by an anion exchange membrane (manufactured by Asahi Kasei, trade name A-202) 8 to prevent re-oxidation of Fe ²⁺ at the counter electrode 5. Further, air is sent from the air supply pipe 9 in the direction of the arrow to be aerated in the medium 3, and the stirrer 10 stirs the medium 3. The electrocultivation was performed by culturing the iron-oxidizing bacteria for 2 days in a medium containing 80 mM of Fe ²⁺ , and then applying a constant potential to the working electrode 4 immersed in the medium.

(6) Rotating ring disk electrode (RRD
Reduction of iron-oxidizing bacterial medium with E) The measurement by RRDE was carried out using a rotating electrode device (Hokuto Denko KK,
(Trade name HR-103A), dual potentiostat (manufactured by Hokuto Denko, trade name HR-101B) and function generator (manufactured by Hokuto Denko, trade name HB-104). RRDE of platinum was used for the working electrodes (ring electrode and disk electrode). The counter electrode is a platinum wire, and the reference electrode is a silver-silver chloride electrode (Ag / AgCl, manufactured by Toa Denpa,
The brand name HS-205C) was used. The potential sweep of the disk electrode is 1.0 V vs. Starting from Ag / AgCl, the speed was 10 mV / sec to the negative side. The potential of the ring electrode is 0.8 V vs. Fe ²⁺ for detecting Fe ^2+. Ag /
A constant potential of AgCl was applied. For the test, the supernatant after culturing the iron-oxidizing bacteria, that is, the supernatant obtained by centrifuging the stationary-phase culture solution obtained by culturing the above-mentioned iron-oxidizing bacteria for 3 days in a medium containing about 80 mM Fe ²⁺ at 10000 rpm, was used. I was there.

B. Results (1) were examined in the Fe ²⁺ concentration in the medium on growth of impact test試鉄oxidizing bacteria Fe ²⁺ concentration in the medium on the growth of iron-oxidizing bacteria. As a result, as shown in FIG. 2, [black circle (●) in FIG. 2], up to 360 mM, the bacterial cell concentration increased with the increase of the Fe ²⁺ concentration. From this, 360m
It can be seen that up to M, the supply of Fe ²⁺ , that is, the energy supply is the rate-determining factor of the growth. Furthermore, when the Fe ²⁺ concentration was raised to 720 mM, the bacterial cell concentration decreased. From this, it is understood that if the Fe ²⁺ concentration is too high, some kind of growth inhibitory action occurs. Such an inhibitory action is described in, for example, Barron, JLA et al., Appl. Environ. Micro.
Biol., 56: 2801-2806 (1990), etc. are also reported.

The increase in pH of the medium due to the growth of iron-oxidizing bacteria is considered as a factor for inhibiting growth. So the pH in the medium
Was measured [white circle (◯) in FIG. 2]. F up to 180 mM
The pH increased with increasing e ²⁺ concentration. At this time, the color of the medium changed from a light blue color derived from Fe ²⁺ to an orange color derived from Fe ^{3+ as} the cells ^grew . This is expressed by the following formula (1) in which Fe ²⁺ which is an energy source is consumed and Fe ³⁺ is generated.
The reaction was catalyzed by bacteria, resulting in an increase in pH. 4Fe ²⁺ + O ₂ + 4H ⁺ → 4Fe ³⁺ + 2H ₂ O + (ATP) (1) However, at 180 mM or more, the pH did not change although the Fe ³⁺ concentration in the medium increased. Also,
At 180 mM or higher, precipitation occurred in the medium. Therefore, if the pH was increased, the reaction of the following formula (2): Fe ³⁺ + 3OH ⁻ → Fe (OH) ₃ (2) proceeded to increase the pH. Will stop. 18
Since pH was about 2.2 at 0 to 720 mM, it is considered that the growth inhibitory effect at 720 mM is not due to an increase in pH, but is due to substrate inhibition by excess Fe ²⁺ or product inhibition by Fe ^3+. To be From the above results, the large-scale production of iron-oxidizing bacteria is high in Fe ²⁺ concentration.
Cultivation with feeding proved difficult.

(2) Cultivation of iron-oxidizing bacteria by changing the medium It was examined whether or not the iron-oxidizing bacteria could be efficiently cultured when the iron-oxidizing bacteria were cultured at a low concentration of Fe ²⁺ . That is,
When subcultured in a medium containing 80 mM Fe ²⁺ ,
The bacterial cell concentration was measured, and the presence or absence of the growth inhibitory effect was tested. As a result, as shown in FIG. 3, the bacterial cell concentration increased every time the medium was changed at the time indicated by the arrow in the figure. When the results shown in FIG. 3 were converted into the relationship between the total iron supply amount and the bacterial cell concentration [black circle (●) in FIG. 4], even if the total fed Fe ²⁺ concentration exceeded 700 mM, Body concentration is 8.0
It increased to × 10 ⁸ cells / ml, and no growth inhibition was observed. For comparison, the same graph is shown when Fe ²⁺ is supplied all at once [white circle (◯) in FIG. 4]. in short,
For iron-oxidizing bacteria, it is better to supply low concentration Fe ²⁺ little by little.
It became clear that it can be efficiently used as an energy source.

(3) Analysis of Electron Transfer Reaction to Cell by Cyclic Voltammetry The result of the cyclic voltammetry to examine whether or not electrons are supplied from the electrode to the iron-oxidizing bacteria by using Fe ²⁺ as an electron carrier. As shown in FIG. From these results, when the cells were not included (FIG. 5A), a reversible redox response of iron ions was exhibited, while when iron-oxidizing bacteria were added (FIG. 5A).
B), it was found that an irreversible response of disappearance of oxidation current and increase of reduction current was exhibited. This phenomenon is conceptually shown in FIG.
As shown in (B), since Fe ²⁺ produced by the electrochemical reduction reaction is biochemically oxidized by iron-oxidizing bacteria, the concentration of Fe ^{2+ on} the electrode surface is low. Since the oxidation current (Fe ²⁺ → Fe ³⁺ + e ⁻ ) decreases and the Fe ³⁺ concentration on the electrode surface increases due to the same mechanism, the reduction current (Fe ³⁺ + e ⁻ → Fe ²⁺ ) decreases. It is an increase. That is, as shown in the upper diagram of FIG. 6B, it was shown that electrons from the electrodes can be supplied to the fungus body by using Fe ²⁺ as an electron carrier. In addition, this electron transfer reaction is performed at 0.5 V v, where Fe ³⁺ is reduced to Fe ^2+.
s. It became clear that it occurs below Ag / AgCl.

(4) Analysis of reduction reaction of iron-oxidizing bacterial medium by RRDE RRDE measurement was carried out using the supernatant of the culture solution in the stationary phase. As shown in FIG. 7, 0.5 V vs. A reduction current was observed below Ag / AgCl. The reduction current is -0.2 V vs. Ag / AgCl
Gradually increases to −0.2 to −0.6 V vs. Ag
/ AgCl became almost constant. The amount of Fe ²⁺ produced on the disk electrode at this time increased as the reduction current increased. From this, it was found that the reduction current at the disk electrode was mainly used for the production of Fe ²⁺ . On the other hand, -0.6V vs. Below Ag / AgCl, the amount of Fe ²⁺ produced on the disk electrode decreased even though the reduction current further increased. Since no generation of hydrogen gas was observed on the electrode surface, it is considered that the increase in reduction current observed here is mainly due to the formation of metallic iron.

(5) Proliferation of Iron-oxidizing Bacteria by Applying Electric Potential Using Fe ²⁺ as an electron carrier, it was confirmed whether or not iron-oxidizing bacteria were proliferated by supplying electrons from the electrodes to the bacterial cells. That is, 0.3 V vs. generated by Fe ^2+. Ag
A potential of / AgCl was applied to examine whether or not the iron-oxidizing bacteria proliferate (the operating conditions were the same as those described in the section of the electroculturing). As shown in FIG. 8, when no electric potential was applied, the bacterial cell concentration increased to about 1.5 × 10 ⁸ cells / ml with consumption of the added Fe ²⁺ , and thereafter, the increase in growth stagnated. [White circle (O) in FIG. 8]. It is considered that this is because Fe ²⁺ in the medium, which is an energy source, was exhausted. In this state, 0.3V is applied to the working electrode immersed in the medium.
vs. It was shown that the cell concentration was further increased by applying the potential of Ag / AgCl (arrow in FIG. 8) [black circle (●) in FIG. 8]. This is 0.3 V vs. Ag
This is because Fe ^{2+, which} is an energy source of bacterial cells, was regenerated in the medium by applying the potential of / AgCl. That is, as shown in FIG. 6B, it was proved that iron-oxidizing bacteria can be cultured and the proliferation can be promoted by using iron ions as an electron carrier and supplying electrons from the electrodes to the bacterial cells.

(6) Effect of Applied Potential on Cell Concentration The effect of applied potential on the growth of iron-oxidizing bacteria was examined. 0.5V vs. When the potential of Ag / AgCl was applied, the cell concentration was about 2.8 × 10 ⁸ cells / ml, which was at the same level as when no potential was applied. When a lower potential was applied, a two-step growth promoting effect was observed depending on the difference in the applied potential, as shown in FIG. First, the acceleration effect of the first stage is 0.5 to −0.4 V vs. It was observed at the potential of Ag / AgCl. In this potential range, Fe ²⁺ is electrochemically regenerated and utilized by the bacterial cells, so as the applied potential decreases,
The cell concentration is increased. On the other hand, the promoting effect in the second stage is -0.4 V vs. It was observed at a potential below Ag / AgCl. From the analysis of the electrode reaction product using RRDE, although the production amount of Fe ²⁺ acting as an electron carrier is not changed (see FIG. 7), the promoting effect is recognized (FIG. 9), It is considered that this promoting effect is not due to only the electrochemical regeneration of Fe ²⁺ . In the above-mentioned reference of Yunker et al., -0.4V v
s. It has been suggested that there is a growth-promoting effect that does not depend on electron transfer, as seen with Ag / AgCl or less, and it also has a growth-promoting effect on cultured plant cells as an electrical effect on the living body, and a transfer of a membrane protein of animal cells. The potential for accelerating effects has been previously presented. However, it has not been mentioned at all about the mechanism of promoting effect in iron-oxidizing bacteria, and other possibilities have not been considered.

Therefore, in order to investigate the electron transfer from other substances, the present inventor used a medium containing no iron ions.
0.6V vs. When the potential of Ag / AgCl was applied and the cells were electrocultured, no bacterial growth was observed, so it was concluded that there was no energy donation from substances other than iron ions. Iron-oxidizing bacteria use iron-oxidizing enzymes to obtain energy from Fe ²⁺ ,
When a certain oxidase is present in the vicinity of the electrode, the activity of the enzyme can be controlled by the electrode potential and iron ions are required for this promoting effect, so iron-oxidizing bacteria receive electrons from Fe ^2+. It is considered that there is some electrical promotion effect by the potential application with respect to the efficiency (Fig. 10).

Example 2: Direct electron transfer to bacteria A. Materials and Methods Thiobacillus ferrooxidans (Thiob
acillus ferrooxidans) ATCC 23270, Paracoccus denitrificans
JCM6892 and Thiobacillus thiooxidans
(Thiobacillus thiooxidans) ATCC8085,
Each was cultured according to the following procedure: (a) Thiobacillus ferrooxidans ATCC23
270: Cultured for 4 days in m9K + CaMg medium containing 80 mM or 10 g / l of Fe ²⁺ or elemental sulfur, respectively. (B) Paracoccus denitrificans JCM689
2: Culture was carried out for 12 hours in a medium for hydrogen bacteria in a gas phase containing 80% hydrogen gas. (C) Thiobacillus thiooxidans ATCC808
5: Cultured in 9K medium containing 10 g / l of elemental sulfur for 7 days.

Cyclic voltammetry for each test strain after culturing was carried out by an electrochemical measuring instrument (BAS).
(Manufactured by the company, trade name BAS100B / W). A carbon electrode (GC, BPG-Edge, BPG
-Basal, GC-BPG), platinum wire (BA
S company, product name 51-2233), reference electrode is silver-silver chloride electrode (Ag / AgCl, BAS product, product name 11-202)
0) was used. The electrolyte is m9K + CaMg medium (pH
1.5) was used. In the cyclic voltammetry of the electrolytic solution in which the bacterial cells were suspended, the above-mentioned electrolytic strain to which the above-mentioned test strain was added at a concentration of 10 ¹¹ cells / ml was used.

B. Results The measurement results of cyclic voltammetry for each of the test strains are shown in FIGS. 11 to 13. In each of these figures, a is the one to which bacteria were added, and the others are those to which bacteria were not added. A peak, which was not seen without the addition of bacteria, appeared again with the addition of bacteria. If there is no oxygen gas,
No such peak was seen in the presence of bacteria (data not shown). From these results, it was revealed that direct electron transfer from the carbon electrode to the respiratory chain (electron transfer system) of the bacterium was carried out in each of the test strains. This phenomenon is conceptually shown in FIG. 14, and by the direct electron transfer, an electron (e ⁻ ) is directly incorporated into the bacterium from the electrode through the unknown substance X in the outer membrane of the bacterium, and the electron Is processed in the electron transfer system of bacteria to generate bioenergy. In addition, when the reaction of the above-mentioned test strains was analyzed using various respiratory inhibitors, electrons directly transferred were part of the respiratory chain [cyt
c → cyt aa3 (cyt c oxidase, cyt
c oxidase) → O ₂ ].
This suggests that the generation of a proton concentration gradient associated with the direct electron transfer reaction and the ATP production thereby are performed. Thus, the direct electron transfer reaction between the electrode and the electron transfer system of bacteria indicates that there is a novel protein that transfers electrons into the periplasmic space in the extracellular membrane of bacteria. Is. That is,
Bacteria can generate bioenergy from electrical energy by direct electron transfer reaction. Therefore, there is a possibility that an energy supply system, which is a problem in the field of nanomechatronics, can be constructed by applying the system that causes direct electron transfer to bacteria by controlling potential according to the present invention.

[0035]

INDUSTRIAL APPLICABILITY As described in detail above, the method for culturing chemoautotrophic bacteria of the present invention can efficiently supply electric energy to bacterial cells and utilize the electric energy for the growth of the bacterial cells. Therefore, it became possible to grow the chemoautotrophic bacterium which utilizes electric energy with high efficiency. As described above, the method of the present invention is a culture system of chemoautotrophic bacteria, which has never been reported before, which enables highly efficient conversion of electric energy into bioenergy, and efficiently converts electric energy. It is possible to mass-culture the bacterium by utilizing it. Further, the present invention provides that an iron-oxidizing bacterium, which is a type of chemoautotrophic bacterium, is cultured by utilizing electric energy with high efficiency even in the presence of a low concentration of ferrous iron (Fe ²⁺ ). Made it possible to get.
Furthermore, it was found for the first time that two-step growth promotion was observed by controlling the electrode potential. For this reason,
By controlling the potential, iron-oxidizing bacteria can be cultivated with better efficiency. Furthermore, the present invention makes it possible to directly carry out an electron transfer reaction in chemoautotrophic bacteria without interposing an electron carrier, that is, to directly incorporate an electron (e ⁻ ) from an electrode into a bacterium. Is. As a result, the utilization efficiency of electric energy is further enhanced, and it becomes possible to culture chemoautotrophic bacteria with higher efficiency, and to simplify the culture system of the bacteria. Further, this direct electron transfer reaction means that bacteria can directly generate bioenergy from electric energy, which greatly contributes to the construction of an energy supply system in the field of nanomechatronics.

As described above, the present invention enables large-scale culture of the bacteria with high energy efficiency by supplying electric energy to chemoautotrophic bacteria such as iron-oxidizing bacteria and hydrogen bacteria. . Therefore, according to the culturing method of the present invention, a large amount of hydrogen bacteria capable of utilizing the bacterium itself as food can be supplied by utilizing electric energy, and iron oxidation useful for coal desulfurization and rare metal recovery. Bacteria can be provided in large quantities with high energy efficiency. Furthermore, after introducing a useful substance producing gene by applying a conventional gene manipulation technique to chemoautotrophic bacteria,
By carrying out the highly efficient culture of the present invention, it becomes possible to produce useful substances such as enzymes and pharmaceuticals from carbon dioxide as a raw material with good energy efficiency and at low cost.

[Brief description of drawings]

FIG. 1 is a drawing schematically showing an electroculture apparatus used in Examples of the present invention.

FIG. 2 is a graph showing the effect of Fe ²⁺ concentration on the growth and pH of iron-oxidizing bacteria. (●); concentration of iron-oxidizing bacteria, (◯); pH.

FIG. 3 is a graph showing the effect of medium exchange on the growth of iron-oxidizing bacteria. The arrow in the graph indicates the time when the medium was replaced.

FIG. 4 is a graph showing the effect of total fed Fe ²⁺ concentration on the growth of iron-oxidizing bacteria. (○); When Fe ²⁺ was supplied at once, (●): When subcultured in a medium containing 80 mM Fe ²⁺ .

FIG. 5 is a graph showing the effect of adding iron-oxidizing bacteria on the cyclic voltammetry of iron ions.

FIG. 6 is a drawing conceptually showing a reaction mechanism in the cyclic voltammetry shown in FIG. (A) is the case without addition of bacteria, and (B) is the case with addition of bacteria.

FIG. 7 is a graph showing a result of electrochemically examining a redox substance in a medium by a rotating ring disc electrode.

FIG. 8 is a graph showing changes in growth of iron-oxidizing bacteria due to application of a reduction potential. In the graph, the arrow indicates the time when the potential was applied. (●); When reducing potential is applied,
(◯): When no reduction potential was applied.

FIG. 9 is a graph showing the influence of the magnitude of the applied potential on the growth of iron-oxidizing bacteria.

FIG. 10 is a drawing conceptually showing a mechanism of a growth promoting effect of iron-oxidizing bacteria by applying a potential. (A) and (B) are applied voltages of 0.5 to -0.4 V vs. Ag
/ AgCl or -0.4 to -1.0 V vs. Ag /
The mechanism of the growth promotion effect in AgCl is shown.

FIG. 11 is a graph showing the effect of addition of iron-oxidizing bacteria (Thiobacillus ferrooxidans) on cyclic voltammetry of iron ions and sulfur. A shows the case where bacteria were added.

FIG. 12 is a graph showing the effect of addition of denitrifying bacteria (Paracoccus denitrificans) on cyclic voltammetry of hydrogen. A shows the case where bacteria were added.

FIG. 13 is a graph showing the effect of addition of a sulfur-oxidizing bacterium (Thiobacillus thiooxidans) on cyclic voltammetry of sulfur. A shows the case where bacteria were added.

FIG. 14 is a diagram conceptually showing direct electron transfer to chemoautotrophic bacteria upon application.

Claims (3)

[Claims] 1. A method of culturing chemoautotrophic bacteria, which comprises culturing chemoautotrophic bacteria by applying an electric potential within a range suitable for the growth of the bacteria in a medium containing an electron donor of the bacteria. Method. 2. A ferrous ion (Fe
²⁺ ) in a medium containing 0.5 to −0.4 V v
s. Ag / AgCl or -0.4 to -1.0V
vs. A method for culturing iron-oxidizing bacteria, which is performed by applying a potential of Ag / AgCl. 3. A chemoautotrophic bacterium is cultured by applying an electric potential within a range suitable for the growth of the bacterium in a medium containing an electron donor of the bacterium, and the electric energy applied to the medium is applied to the bacterium. A method for culturing chemoautotrophic bacteria, which is characterized in that it is directly utilized as the bioenergetics of.