JP7349051B2 - Method for improving sustainability of reduced state of reducing agent, reducing agent with improved sustainability, medium containing this reducing agent, and method for culturing anaerobic microorganisms using this medium - Google Patents

Description

NPMD NITE P-1469 NPMD NITE P-1470 NPMD NITE P-1471

The present invention provides a method for improving the sustainability of the reduced state of a reducing agent, specifically, a method for improving the sustainability of the reduced state in which reduced glutathione is heat-treated in an anaerobic state, and a reducing agent with improved sustainability of the reduced state. , a medium containing this reducing agent, and a method for culturing anaerobic microorganisms using this medium.

Purification technology using microorganisms is widely used in activated sludge treatment, anaerobic treatment, etc. in wastewater treatment. Furthermore, in recent years, technology for purifying soil and groundwater contaminated with hazardous chemicals using microorganisms (bioremediation) has been attracting attention as a purification method with low environmental impact and low purification costs.

Chlorinated ethylenes such as tetrachlorethylene (PCE) and trichlorethylene (TCE) are used in a wide range of fields, including the metal industry, semiconductor industry, and dry cleaning shops, as inexpensive solvents with strong cleaning power against oil. On the other hand, there have been many reports of soil and groundwater contamination by chlorinated ethylenes, which has become a social problem.
Bioremediation of chlorinated ethylenes involves the reductive dechlorination reaction of "tetrachlorethylene (PCE) → trichlorethylene (TCE) → cis-1,2-dichloroethylene (cis-DCE) → vinyl chloride monomer (VCM) → ethylene." Anaerobic dechlorinating bacteria are used to render chlorinated ethylene harmless.

Bacteria of the genus Dehalococcoides, which is a type of anaerobic dechlorinating bacteria, are the only bacteria reported to be able to proceed with dechlorination after cis-DCE. That is, in bioremediation of chlorinated ethylenes, if Dehalococcoides bacteria are not present in the soil to be treated, dechlorination can only be achieved up to cis-DCE. Furthermore, even if Dehalococcoides bacteria exist, the amount of Dehalococcoides bacteria in the environment is very small and their growth rate is slow, resulting in a long purification period. There is.

In order to solve these problems, there is hope for a method (bioaugmentation) in which Dehalococcoides bacteria are artificially cultivated and increased, and then introduced into the environment to be purified to promote purification. . However, bacteria of the genus Dehalococcoides are obligately anaerobic and have extremely severe operating conditions, such as using only hydrogen as an electron donor, making it difficult to isolate and culture them. In Patent Document 1, the present inventors isolated Dehalococcoides sp. UCH007 strain (NITE P-1471, hereinafter also referred to as UCH007 strain) for the first time in Japan, and developed a method for producing chlorinated ethylene using this UCH007 strain. A purification method is proposed.

In order to stably cultivate anaerobic bacteria, the culture medium is placed in a highly airtight container, and the gas phase inside the container is purged and replaced with a gas that does not contain oxygen, such as nitrogen gas. ing. In the case of obligate anaerobic bacteria that require higher anaerobic conditions, such as Dehalococcoides bacteria, a reducing agent is added to the culture medium to lower the oxidation-reduction potential (ORP). There is a need to. There are various types of reducing agents, and there are differences in the ORP reached, the rate of arrival, and the durability of the effect.The type and amount of reducing agent added depends not only on the characteristics of the reducing agent, but also on the The decision must be made taking into account the susceptibility of

As reducing agents for culturing Dehalococcoides bacteria, iron sulfide (0.5mM (set concentration)), sodium sulfide (0.2-0.5mM) + L-cysteine (0.2mM) have been used so far. Combinations of +DL-dithiothreitol (0.5-1mM), titanium(III) complexed with citrate or nitrilotriacetate (0.2-1.5mM), etc. are known (non-patent literature). 1, paragraph 0050 of Patent Document 1).
These reducing agents allow stable cultivation of Dehalococcoides bacteria only when cultured in a highly airtight culture container, but the problem is that if even a small amount of air gets in, the growth of the bacteria will be inhibited. there were. When the culture scale is increased, it is difficult to maintain high airtightness and there is a high possibility that a small amount of air may be mixed in, so it has been difficult to culture Dehalococcoides bacteria in large quantities.

Japanese Patent Application Publication No. 2014-108061

Loffler, F.E., Yan, J., Ritalahti, K.M., Adrian, L., Edwards, E.A., Konstantinidis, K.T., Muller, J.A., Fullerton, H., Zinder, S.H. & Spormann, A.M. " Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi." Int. J. Syst. Evol. Microbiol. (2013) 63: p625-635.

It is an object of the present invention to provide a method for improving the sustainability of a reducing agent, a reducing agent with improved sustainability of a reduced state, a medium containing this reducing agent, a method for producing the medium, and a method for culturing anaerobic microorganisms using this medium. Take it as a challenge.

Means for solving the problems of the present invention are as follows.
1. A method for improving the sustainability of a reduced state, which comprises heat-treating reduced glutathione under anaerobic conditions.
2. A reducing agent consisting of reduced glutathione heated under anaerobic conditions.
3.2. A medium comprising the reducing agent described in .
4. 1. A method for producing a culture medium, which comprises replacing a culture medium containing reduced glutathione with anaerobic gas in a sealed container and subjecting it to heat treatment.
5. 4. The concentration of the reduced glutathione is 1 mM or more and 10 mM or less. A method for producing the medium described in .
6.3. A method for culturing anaerobic microorganisms, characterized by using the medium described in .
7. 6. The anaerobic microorganism is a bacterium belonging to the genus Dehalococcoides. The culture method described in.
8. 6. The concentration of the reduced glutathione is 1 mM or more and 10 mM or less. or 7. The culture method described in.
9. 6. The medium has a capacity of 10 L or more. ~8. The culture method according to any one of.
10. 7. The Dehalococcoides bacterium is the UCH007 strain deposited under accession number NITE P- 1471 or a mutant strain thereof.7. ~9. The culture method according to any one of.

The present invention is based on the novel finding that the sustainability of the reduced state is significantly improved by heating distilled water or culture medium in which reduced glutathione is dissolved in an anaerobic state.
The sustainability of the reduced state can be improved by a simple method of heat-treating reduced glutathione in an anaerobic state. The reducing agent of the present invention, which has improved sustainability of the reducing state, can maintain the reducing state even if some air (oxygen) is mixed in, and the culture medium of the present invention containing this reducing agent can maintain the reducing state under anaerobic conditions. is easy. Therefore, the culture method of the present invention using this medium can more stably culture anaerobic microorganisms. The culture method of the present invention is suitable for culturing in a large capacity, and is suitable for culturing in a large capacity of 10 L or more, since it is possible to maintain a reduced state even if some air is mixed in.

A diagram showing the color of resazurin in Experiment 1. A diagram showing the color of resazurin in Experiment 2. Graph showing changes in chlorinated ethylene concentration over time in Experiment 4. Graph showing changes in chlorinated ethylene concentration over time in Experiment 5. 12 is a graph showing changes over time in the concentration of chlorinated ethylenes when TCE is decomposed in a medium volume of 15 L in Example 14.

The present invention is based on the novel finding that when distilled water or a culture medium in which reduced glutathione is dissolved is heat-treated in an anaerobic state, the sustainability of the reduced state is significantly improved.
The present invention will be explained in order below.

"Reducing agent"
The reducing agent of the present invention contains reduced glutathione that has been heat-treated under anaerobic conditions.
Glutathione is a tripeptide consisting of three amino acids: glutamic acid, cysteine, and glycine, and is also called "reduced glutathione." In reduced glutathione, the thiol group derived from the cysteine structure is responsible for the reducing ability, and when oxygen is mixed in, the thiol group is oxidized to disulfide, resulting in oxidized glutathione in which two glutathione molecules are bonded by a disulfide bond.

The temperature when heating reduced glutathione in an anaerobic state is preferably 80°C or higher, more preferably 100°C or higher, and even more preferably 110°C or higher. The upper limit of the heating temperature is about 140°C or lower. Further, the temperature at which the heat treatment is performed in an anaerobic state is preferably 10 minutes or more, more preferably 20 minutes or more. An example of the heat treatment conditions is 115° C. to 125° C. for 15 to 25 minutes.

The reason why reduced glutathione heat-treated under anaerobic conditions has significantly improved sustainability of the reduced state compared to reduced glutathione that has not been heat-treated under anaerobic conditions is currently unknown. The present inventors discovered that heat treatment under anaerobic conditions decomposes reduced glutathione (tripeptide) to produce monopeptides and dipeptides with sulfide bonds, which form a mixture, and when oxygen is mixed in, these sulfide bonds break down. It is speculated that this is due to the formation of disulfide bonds between monopeptides, dipeptides, and tripeptides having . Note that even when cysteine is heat-treated under anaerobic conditions, no change is observed in the sustainability of the reduced state.

"Culture medium"
The culture medium of the present invention contains reduced glutathione that has been heat-treated under anaerobic conditions. The culture medium of the present invention may contain reduced glutathione heat-treated under anaerobic conditions, and may also contain an electron acceptor source, a carbon source necessary for the growth of microorganisms, a nitrogen source, inorganic salts, trace elements, yeast extract, etc. It can be either a solid medium or a liquid medium.

Examples of carbon sources include alcohols such as methanol and ethanol, and organic acids such as pyruvic acid, acetic acid, and lactic acid. Since pyruvic acid also acts as an electron donor, it is preferable to use pyruvic acid.
Examples of nitrogen sources include peptone, cacitone, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, and various amino acids.
Inorganic salts include phosphates, magnesium salts, and calcium salts.
Trace elements include iron, cobalt, copper, zinc, boron, nickel, and molybdenum.
The carbon sources, nitrogen sources, inorganic salts, and trace elements exemplified above may be used alone or in combination of two or more.
Furthermore, appropriate amounts of vitamins such as cyanocobalamin, yeast extract, malt extract, etc. may be added as nutritional sources for promoting the growth of the microorganisms of the present invention.

The method for producing the culture medium of the present invention is not particularly limited, and can be produced according to conventional methods. For example, it can be manufactured by methods 1 and 2 below.
Method 1: A method in which all components including reduced glutathione are mixed and then heated under anaerobic conditions.
Method 2: A method in which an aqueous solution containing components other than reduced glutathione is gas replaced and sterilized, and then a reduced glutathione aqueous solution that has been separately gas replaced and heat treated is added anaerobically.
Conditions for heat treatment (sterilization treatment) of the medium may be appropriately selected depending on the type of medium, and may be, for example, 115° C. to 125° C. for 15 to 25 minutes.

The medium produced by either method 1 or method 2 has improved sustainability of the reduced state, and can maintain the reduced state even if some oxygen is mixed in. However, whereas the medium produced by method 1 is in a reduced state after heat treatment, the culture medium produced by method 2 may be contaminated with oxygen during addition. Therefore, it is preferable to use Method 1, which allows medium preparation in a short time and easily.

"Culture method"
In the culture method of the present invention, anaerobic microorganisms are cultured in a medium containing reduced glutathione that has been heat-treated under anaerobic conditions. The anaerobic microorganisms to be cultured in the culture method of the present invention are not particularly limited, and include anaerobic bacteria (Dehalococcoides sp. bacteria, etc.), methane bacteria, fermentation bacteria, etc. Further, the culture medium and culture conditions used in the culture method of the present invention can be adjusted as appropriate depending on the type of anaerobic microorganism to be cultured.
The culture of Dehalococcoides bacteria will be described below as an example.

Bacteria of the genus Dehalococcoides have the ability to dechlorinate chlorinated ethylenes and chlorinated ethanes. Even with Dehalococcoides bacteria, the chlorinated ethylenes that can be decomposed differ depending on the type of bacteria. When cultured Dehalococcoides bacteria are used to purify chlorinated ethylene by bioaugmentation etc., it is preferable that the strain is a strain that can completely dechlorinate chlorinated ethylene to ethylene. Examples include Dehalococcoides sp. UCH007 strain or a mutant strain thereof. In addition, in this specification, a mutant strain means a strain in which the base sequence of the 16S rRNA gene has 98% or more homology. The homology of the 16S rRNA gene is preferably 99% or more, more preferably 99.5% or more, and even more preferably 100%.

Strain UCH007 has been deposited with the accession number NITE P-1471 at the National Institute of Technology and Evaluation, Patent Microorganism Depositary (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (zip code 292-0818)). It was deposited on November 22, 2012.
The mycological properties of UCH007 strain are as follows.
(a) Morphological characteristics Non-motile, non-spore forming, disc-shaped form (difficult to observe with an optical microscope).
(b) Example of culture medium Absolute anaerobic. Hydrogen is used as an electron donor, TCE or cis-DCE is used as an electron acceptor, and acetate is used as a carbon source. bicarbonate buffer. pH 7.2. The inside of the culture medium/culture container is replaced with a mixed gas of H ₂ /CO ₂ (80:20).

(c) Physiological characteristics: Absolute anaerobic. Obtain energy through dehalogenated respiration. Hydrogen is used as the electron donor and chlorinated ethylenes are used as the electron acceptor.
It has the ability to dechlorinate trichlorethylene (TCE), cis-1,2-dichloroethylene (cis-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride monomer (VCM). Assimilates acetate as a carbon source. Vitamin B12 requirement. Sensitive to high concentrations of sulfides (used as reducing agents).
Growth temperature: 30°C.
Optimum pH: 7.2±0.3.

In the present invention, the Dehalococcoides bacterium may be cultured alone, or the isolated pure strain may be co-cultured as a bioconsortium (composite microbial system) containing other bacteria. For example, it is preferable to co-culture with bacteria of the genus Sulfurospirillum. Bacteria of the genus Sulfurospirillum have the ability to promote the dechlorination reaction of bacteria of the genus Dehalococcoides. It can help obtain energy through oxidation and promote the growth of Dehalococcoides bacteria. Sulfurospirillum bacteria include Sulfurospirillum sp. UCH001 strain (hereinafter also referred to as UCH001 strain) and Sulfurospirillum sp. UCH003 strain (hereinafter also referred to as UCH003 strain). ), or mutant strains thereof.

Strains UCH001 and UCH003 have been deposited with the National Institute of Technology and Evaluation (NPMD) (2-5 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession numbers NITE P-1419 and NITE P-1470, respectively. 8 (zip code 292-0818)) on November 22, 2012.
The morphological properties, medium characteristics, and physiological characteristics of the UCH001 strain and the UCH003 strain are as follows, and are common to both strains.

(a) Morphological properties Curved or spiral bacilli.
Has flagella. It has motility. Non-spore forming.

(b) Example of culture medium Absolute anaerobic. Use fumarate, lactate, acetate. bicarbonate buffer. pH 7.2. The inside of the culture medium/culture container is replaced with a mixed gas of H ₂ /CO ₂ (80:20).

(c) Physiological characteristics Anaerobic. Obtains energy through respiration using nitrates, fumarates, etc. as electron acceptors. Hydrogen, formic acid, pyruvic acid, lactic acid, etc. are used as electron donors. Assimilates acetate, etc. as a carbon source.
Growth temperature: 30℃
Growth pH: pH7.2

When culturing Dehalococcoides bacteria, one or more types of chlorinated ethylenes can be mixed and added to the medium depending on the characteristics of the Dehalococcoides bacteria to be cultured. The chlorinated ethylenes are preferably any one of PCE, TCE, cis-DCE, and VCM, or a combination containing a plurality of them.

Since Deharococcoides bacteria are absolutely anaerobic, it is important to reduce air contamination as much as possible during culture medium preparation, reagent addition, bacterial solution inoculation, sampling, etc. For example, sterilized syringes used for adding easily volatile reagents such as chlorinated ethylene or for inoculating bacterial solutions should be replaced with sterile nitrogen gas or filled with a reducing agent to completely eliminate air bubbles. It is preferable to take steps to expel them. In addition, vitamin solutions that cannot be heated are subjected to gas replacement and filter sterilization treatment, but sterilization filters are also subjected to gas replacement treatment using sterile nitrogen gas, or the inside is filled with a reducing agent and air is expelled. After that, it is preferable to further perform a treatment to expel the reducing agent by passing a gas-substituted vitamin solution. Also in these treatments, it is preferable to use reduced glutathione that has been heat sterilized as a reducing agent.

Cultivation can be performed by various culture methods such as static culture and shaking culture. Culture conditions such as culture method, temperature, pH, period, etc. can be appropriately selected depending on the Dehalococcoides bacteria to be cultured. For example, the culture temperature is usually 25°C or higher and 35°C or lower, the pH is in the range of 6.5 to 7.5, and the culture period is about 2 months. In addition, as the gas for culturing, a mixed gas of H ₂ /CO ₂ is usually used, but when using pyruvic acid as a carbon source, a mixed gas of N ₂ /CO ₂ (pH buffer (when using bicarbonate as a pH buffer) or _N2 gas (when using a pH buffer other than bicarbonate). The volume ratio of H ₂ and CO ₂ is not particularly limited, but is preferably 70:30 to 90:10. Further, the volume ratio of N ₂ and CO ₂ is not particularly limited, but is preferably 70:30 to 90:10.

Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the present invention is not limited to these.

"Experiment 1: Confirmation of the ability of a reduced glutathione aqueous solution heat-treated under anaerobic conditions to maintain anaerobic conditions"
Aqueous solutions 1 to 3 were prepared in the following manner, and the anaerobic state of each aqueous solution was evaluated by observing the change in the color of resazurin. Note that resazurin is transparent in a reduced state and takes on a pink color in the presence of oxygen.

"Example 1" Aqueous solution 1 (5mM reduced glutathione aqueous solution subjected to anaerobic heat treatment)
Dissolve reduced glutathione to 5mM (1.53g/L) in distilled water containing 10mg/L of resazurin, put 10ml of the reduced glutathione aqueous solution into a 20ml vial, and bubble with nitrogen gas for 7 minutes. After purging with fresh gas, the container was sealed with a butyl rubber stopper and an aluminum seal, and heat-treated (autoclave at 121° C. for 20 minutes).

"Example 2" Aqueous solution 2 (anaerobic/heat-treated reduced glutathione concentrate was added to distilled water to make a 5mM reduced glutathione aqueous solution)
Pour 10 ml of distilled water in which 10 mg/L of resazurin was dissolved into a 20 ml vial, bubble with nitrogen gas for 7 minutes to replace the gas, seal with a butyl rubber stopper and an aluminum seal, and heat-treat (autoclave at 121°C for 20 minutes). )did. 100 μl of a separately prepared 500 mM reduced glutathione aqueous solution that had been heat-treated under anaerobic conditions was sealed in this vial using a syringe that had been purged with nitrogen gas. To prepare this 500mM reduced glutathione aqueous solution, put 10ml of distilled water into a 20ml vial, mix with 1.53g of reduced glutathione corresponding to a concentration of 500mM, bubble with nitrogen gas for 7 minutes to replace the gas, and then insert a butyl rubber stopper. It was prepared by sealing it with an aluminum seal and heat-treating it (autoclave at 121°C for 20 minutes).

"Comparative Example 1" Aqueous Solution 3 (Anaerobically treated reduced glutathione concentrate was added to distilled water to make a 5mM reduced glutathione aqueous solution)
Pour 10 ml of distilled water in which 10 mg/L of resazurin was dissolved into a 20 ml vial, bubble with nitrogen gas for 7 minutes to replace the gas, seal with a butyl rubber stopper and an aluminum seal, and heat-treat (autoclave at 121°C for 20 minutes). )did. Into this vial, 300 μl of a separately prepared 167 mM reduced glutathione aqueous solution that had been subjected to only anaerobic treatment was sealed using an anaerobically treated syringe. To prepare this 167mM reduced glutathione aqueous solution, put 10ml of distilled water into a 20ml vial, mix with 0.51g of reduced glutathione corresponding to a concentration of 167mM, bubble with nitrogen gas for 7 minutes to replace the gas, and then insert a butyl rubber stopper. It was prepared by sealing it with an aluminum seal.

Anaerobic conditions were evaluated by observing changes in the color of resazurin for up to 5 hours after production. Furthermore, after 5 hours had elapsed, 10 ml of air was sealed using a syringe, and the anaerobic state of each aqueous solution was evaluated by observing the change in the color of resazurin after 1 hour. The results are shown in Table 1 and Figure 1.
Shown below.

Aqueous solution 1 became colorless after heat treatment (after autoclave treatment) and remained colorless even after 5 hours. In addition, it remained colorless even after air was encapsulated, and the reduced state had excellent sustainability.
Aqueous solution 2 was deep pink immediately after adding reduced glutathione, but the color became lighter over time and became transparent after 5 hours. In addition, it remained colorless even after air was encapsulated, and the reduced state had excellent sustainability.
Aqueous solution 3 was deep pink immediately after adding reduced glutathione. The color gradually became lighter until 2 hours later, but after that, the color remained pink and did not become colorless. It remained pink even after air was encapsulated.

"Experiment 2: Comparison of the ability to maintain anaerobic conditions when a reduced glutathione aqueous solution and a cysteine aqueous solution are heat-treated under anaerobic conditions"
Aqueous solutions 4 and 5 were prepared in the following manner, and the anaerobic state of each aqueous solution was evaluated by observing the change in the color of resazurin.

"Example 3" Aqueous solution 4 (5mM reduced glutathione aqueous solution subjected to anaerobic heat treatment)
Dissolve reduced glutathione to 5mM (1.53g/L) in distilled water containing 10mg/L of resazurin, put 10ml of the reduced glutathione aqueous solution into a 20ml vial, and bubble with nitrogen gas for 7 minutes. After purging with fresh gas, the container was sealed with a butyl rubber stopper and an aluminum seal, and heat-treated (autoclave at 121° C. for 20 minutes).

"Comparative Example 2" Aqueous solution 5 (5mM cysteine hydrochloride aqueous solution subjected to anaerobic heat treatment)
Dissolve L-cysteine hydrochloride monohydrate to 5mM (0.878g/L) in distilled water in which 10mg/L of resazurin has been dissolved, and put 10ml of the cysteine hydrochloride aqueous solution into a 20ml vial. After bubbling with nitrogen gas for 7 minutes to replace the gas, the tube was sealed with a butyl rubber stopper and an aluminum seal, and heat-treated (autoclave at 121° C. for 20 minutes).

After the vial had cooled to room temperature after autoclaving, 10 ml of air was sealed using a syringe, and the anaerobic state of each aqueous solution was evaluated by observing the color change of resazurin after 17 hours. . The results are shown in Table 2 and Figure 2.

Aqueous solution 4 in which reduced glutathione was heat-treated under anaerobic conditions became colorless after the heat treatment (after autoclave treatment). In addition, it remained colorless even after air was encapsulated, and the reduced state had excellent sustainability.
On the other hand, aqueous solution 5 in which cysteine was heat-treated under anaerobic conditions was pale pink after the heat treatment (after autoclave treatment). Immediately after air was encapsulated, the same light pink color remained, but as time passed, the color of resazurin gradually became darker, and became dark pink after 17 hours.

"Experiment 3: Confirmation of the relationship between heating temperature and anaerobic retention capacity of reduced glutathione aqueous solution"
Pour 10 ml of distilled water into a 20 ml vial, mix with 0.51 g of reduced glutathione corresponding to a concentration of 167 mM, bubble with nitrogen gas for 7 minutes to replace the gas, then seal with a butyl rubber stopper and aluminum seal for anaerobic treatment. A reduced glutathione aqueous solution that had not been subjected to heat treatment was prepared.
Pour 10 ml of distilled water in which 10 mg/L of resazurin was dissolved into a 20 ml vial, bubble with nitrogen gas for 7 minutes to replace the gas, seal with a butyl rubber stopper and an aluminum seal, and heat-treat (autoclave at 121°C for 20 minutes). )did. 300 μl of the 167 mM reduced glutathione aqueous solution prepared above was sealed in this vial using an anaerobically treated syringe.

“Comparative Example 3”
The vial containing the reduced glutathione aqueous solution that had been subjected to the above anaerobic treatment was allowed to stand for 5 hours after preparation, and after confirming that the color of resazurin remained deep pink and did not change, it was left at room temperature for 1 hour and 50 minutes. I left it still. After the tube was allowed to stand still, 10 ml of air was sealed using a syringe.

“Comparative Example 4”
The same procedure as Comparative Example 3 was carried out except that the sample was allowed to stand at 40° C. for 20 minutes and then at room temperature for 1.5 hours.
"Example 4"
The same procedure as Comparative Example 3 was carried out except that the sample was allowed to stand at 80° C. for 20 minutes and then at room temperature for 1.5 hours.
“Example 5”
The same procedure as Comparative Example 3 was carried out except that the sample was allowed to stand at 120° C. for 20 minutes and then at room temperature for 1.5 hours.

Anaerobic conditions were evaluated by observing color changes before and after culturing. Furthermore, the anaerobic state of each aqueous solution was evaluated by observing the change in color of resazurin one hour after air was encapsulated. The results are shown in Table 3.

It was confirmed that the reducing ability of reduced glutathione was improved by heat treatment at 80° C. or higher under anaerobic conditions. It was confirmed that by heating from 80°C to 120°C, a reduced state in which resazurin could remain colorless even after air was encapsulated was confirmed.

"Experiment 4: Culture test for different reducing agents"
Table 4 shows the bicarbonate buffer medium used (He, J., Holmes, VF, Lee, PK, Alvarez-Cohen, L. "Influence of Vitamin B12 and Cocultures on the Growth of Dehalococcoides Isolates in Defined Medium." Appl The composition of Mineral salts medium (partially modified from Environ. Microbiol. (2007) 73: p2847-2853) is shown. Further, the compositions of the trace element solution and selenite/tungstate solution in Table 4 are shown in Tables 5 and 6 below, respectively. The bicarbonate buffer medium was prepared in the same manner as in Example 2 of Patent No. 6103518.

Add 50 ml of the medium shown in Table 4 (containing 0.1% resazurin sodium) to a glass vial with a total volume of 70 ml, and add the reducing agent shown in Table 7 to a concentration of 0.5 g/L or 1.0 g/L. The culture medium was sterilized by purging with nitrogen gas for 10 minutes (121°C, 20 minutes). After cooling, add 10 ml of the vitamin solution shown in Table 8 and TCE solution (500 mg/L, 1 ml) aseptically and anaerobically using a syringe whose gas inside the syringe has been replaced with sterile nitrogen gas. Created. All of the prepared media containing resazurin were transparent, indicating a reduced state.

Each medium was inoculated with 1 ml of a co-culture solution of separately cultured UCH007 strain and UCH001 strain (UCH007 strain: 1.32×10 ⁶ copies/ml), and static culture was started in a thermostatic chamber at 28° C. The concentration of chlorinated ethylenes in the culture solution was periodically determined by GCMS (GCMS-QP2010 Ultra, manufactured by Shimadzu Corporation). Figure 3 shows the change in chlorinated ethylene concentration over time.

From Comparative Examples 5 and 6, the amount of cysteine heat-treated under anaerobic conditions was 5.7mM (1.0g/L), and from Comparative Examples 7 and 8, the amount of sodium sulfide heat-treated under anaerobic conditions was 4.2mM (1.0g/L). It was confirmed that dechlorination of chlorinated ethylenes was inhibited at an addition amount of 0.5 g/L).
In contrast, the reduced glutathione heat-treated under anaerobic conditions used in Examples 1 to 4 did not inhibit the dechlorination of chlorinated ethylenes even at a concentration of 3.3 mM (1.0 g/L). Furthermore, it was confirmed that even when reduced glutathione heat-treated under anaerobic conditions and sodium sulfide were used in combination, dechlorination was not inhibited at sodium sulfide of 0.25 mM (0.06 g/L) or less.

"Experiment 5: Confirmation of the effect of the concentration of reduced glutathione heated under anaerobic conditions on the culture of Dehalococcoides bacteria"
We confirmed how the progress of the TCE dechlorination reaction of bacteria of the genus Dehalococcoides is affected by differences in the concentration of reduced glutathione contained in the culture medium and heated under anaerobic conditions. A comparison was also made with conditions using a conventional reducing agent, ``a mixed solution of sodium sulfide, L-cysteine, and DL-dithiothreitol (mixed reducing agent of three types).''

Table 9 shows the bicarbonate buffer medium used (He, J., Holmes, V.F., Lee, P.K., Alvarez-Cohen, L. "Influence of Vitamin B12 and Cocultures on the Growth of Dehalococcoides Isolates in Defined Medium." Appl The composition of the partially modified Mineral salts medium of Environ. Microbiol. (2007) 73: p2847-2853 is shown below. The composition of the three mixed reducing agents in Table 9 is shown in Table 10 below.

Table 11 shows the final concentrations of reduced glutathione and three mixed reducing agents added to the medium. Reduced glutathione and the three types of mixed reducing agent are added to the medium at different times; reduced glutathione is mixed with other medium components from the beginning, whereas with the three types of mixed reducing agent, a medium containing no reducing agent is prepared. Finally, it was added aseptically and anaerobically using a sterile syringe.

Preparation of bicarbonate buffered medium was performed as follows.
Components other than the trichlorethylene stock solution, vitamin solution, and three-part mixed reducing agent were mixed (reduced glutathione was mixed from the beginning), and the pH was adjusted to about 7.2. Dispense 10 ml of the culture medium into 20 ml vials, bubble with H ₂ /CO ₂ (80/20) mixed gas for 7 minutes to replace the gas, seal with a PTFE liner rubber stopper and an aluminum seal, and autoclave (121 ℃, 20 minutes). Using a syringe that had previously replaced the gas in the syringe with sterilized nitrogen gas, add the following separately prepared trichlorethylene stock solution, vitamin solution, and three-part reducing agent to this culture container in an aseptic and anaerobic manner. A carbonate buffered medium was prepared (note that under the conditions of using reduced glutathione, the three mixed reducing agents were not added).

The trichlorethylene stock solution was prepared by placing 20 ml of distilled water and a stirrer bar in a 50 ml light-shielding vial, purging it with _N2 gas, closing it with a PTFE liner rubber stopper and an aluminum seal, autoclaving it, and using a gas-tight syringe for 4 hours. It was prepared by adding .5 μl of trichlorethylene and stirring overnight.
For the three types of reducing agents, prepare a solution with the composition shown in Table 10 below, dispense 10 ml each into 20 ml vials, bubble with _N2 gas for 7 minutes to replace the gas, and then seal the solution with a butyl rubber stopper and an aluminum seal. Autoclave treatment (121°C, 15 minutes) was performed.

Dispense 10 ml of the above bicarbonate buffer medium into a 20 ml vial (for gas chromatography, PTFE-treated butyl rubber stopper), and use a sterilized syringe to collect the co-culture solution of UCH007 strain and UCH001 strain used in Experiment 4 above. 100 μl of the cells were inoculated, and static culture was started in a thermostatic chamber at 30°C. On the 14th day of culture, the concentration of chlorinated ethylenes in the culture solution was measured using a gas chromatography system (Model 6890N, Agilent Technologies) equipped with a capillary column (Agilent J&W GC column DB-624, 0.32 mm x 30 m) and FID. Quantification was carried out using the headspace method. The results are shown in Figure 4.

It was shown that dechlorination of reduced glutathione proceeded within the range of 1 to 10 mM, and the growth of Dehalococcoides bacteria was promoted. It was confirmed that when 5mM of reduced glutathione was added, the decomposition of TCE and cis-DCE progressed, and the number of bacteria of the genus Deharococcoides increased significantly. Furthermore, Dehalococcoides bacteria could be cultured even with reduced glutathione at 10 mM, indicating that it is possible to maintain a higher reduced state by using a high concentration of reduced glutathione during culture.
Note that the UCH007 strain belongs to the Victoria group, which is a phylogenetic group within the genus Dehalococcoides. We have confirmed that strains belonging to the Cornell group, which is different from the Victoria group, can be stably cultured using the method based on the present invention, and from this, this culture method can be applied to all bacteria of the genus Dehalococcoides. It is considered possible.

"Experiment 6 Experiment showing that obligately anaerobic Dehalococcoides bacteria can be cultured in a large-capacity container by using the present invention"
When the culture scale is increased, it is difficult to maintain high airtightness and there is a high possibility that air will get mixed in, so it has been difficult to culture Dehalococcoides bacteria, which require a high degree of anaerobism.
It was confirmed whether Dehalococcoides bacteria can be stably cultivated in large quantities by the method based on the present invention, in which reduced glutathione is heat-treated under anaerobic conditions. A comparison was also made with conditions using a conventional reducing agent, ``a mixed solution of sodium sulfide, L-cysteine, and DL-dithiothreitol (mixed reducing agent of three types).''

As a culture vessel for mass culture, a 5-gallon beer barrel (Cornelius type) was used, which had been modified to include a sampling port on the tube head (lid) to allow collection of bacterial liquid and gas.
For the sampling port, a hole of approximately 12 φ mm was made in the lid of the beer barrel, and a screw port was installed on the bulkhead using a nut tightening type φ 12 electrode port made by ABLE Co., Ltd. (product number: C00000-C005) and a butyl rubber O-ring. It was manufactured by attaching a butyl rubber stopper B (large) for freeze drying manufactured by Nichiden Rika Glass Co., Ltd., and tightening and fixing it with a φ12 cap nut (product number: C00000-C008) manufactured by ABLE Co., Ltd. Bacterial fluid and gas were collected by puncturing the butyl rubber stopper with a sterilized syringe.

"Example 14" Mass culture using a medium containing reduced glutathione 10L and 15L of the medium containing reduced glutathione shown in Table 12 was placed in a culture container consisting of a 5-gallon beer barrel with a modified lid as described above. Or, one containing 18L was prepared. In addition, the composition shown in Table 12 is a composition per 1 L of distilled water. The procedure for preparing a medium containing reduced glutathione was performed as follows. (1) The culture medium was placed in a beer barrel and replaced with nitrogen gas for 1 hour. (2) Autoclave sterilization (121°C, 20 minutes). (3) Immediately after autoclaving, the container was replaced with nitrogen gas for 1 hour, and then the container was further immersed in ice water, and the container was replaced with gas until the temperature of the entire container became below room temperature (about 30 minutes). (4) Using a syringe that had been previously gassed with sterilized nitrogen gas, the vitamin solutions shown in Table 8 and the stock solution of TCE or cis-DCE were added aseptically and anaerobically.
After preparing the medium, a co-culture solution of UCH007 strain and UCH001 strain, which had been cultured separately, was inoculated using a syringe that had been replaced with nitrogen gas in an amount of 1/100 of the medium, and static culture was started at room temperature. . During the culture, the concentration of chlorinated ethylenes in the culture solution was periodically measured using a gas chromatography system (Model 6890N, Agilent Technologies) equipped with a capillary column (Agilent J&W GC column DB-624, 0.32 mm x 30 m) and FID. Quantification was carried out using the headspace method.

“Comparative Example 10” A conventional reducing agent “sodium sulfide, L-cysteine, DL-dithiothreitol mixture (mixture of three types of reducing agents)” was added to the medium for mass culture 5-gallon beer barrel with modified lid 10 L of the medium shown in Table 12 excluding reduced glutathione was added, and the medium was prepared in the same manner as steps (1) to (4) for preparing a medium containing reduced glutathione, except for step (4). In this step, a three-part mixed reducing agent containing sodium sulfide, L-cysteine, and DL-dithiothreitol was added aseptically and anaerobically to a final concentration of 0.2 mM, 0.2 mM, and 0.5 mM. The concentration of this three-type mixed reducing agent is an appropriate concentration for culturing Dehalococcoides bacteria (Non-patent Document 1), and in a culture scale such as putting 10 ml of medium into a 20 ml vial, it is difficult to It has been confirmed that the color of the resazurin that was added becomes transparent, and Dehalococcoides bacteria can be cultured without any problems.

In Comparative Example 10, even when a reducing agent was added, the color of resazurin contained in the medium remained pink, and the redox potential could not be lowered to a state where Dehalococcoides bacteria could be cultured.
On the other hand, in Example 14, in which the medium containing reduced glutathione was gas replaced and autoclaved, the color of resazurin was always colorless after the medium was prepared, regardless of whether the type of VOC added to the medium was TCE or cis-DCE. Furthermore, it was confirmed that Dehalococcoides bacteria could be stably cultured regardless of the medium volume of 10 L, 15 L, or 18 L. As an example, FIG. 5 shows the change over time in the concentration of chlorinated ethylenes in the case of a medium volume of 15 L using TCE. It was confirmed that by using a reducing agent consisting of reduced glutathione heat-treated under anaerobic conditions, bacteria of the genus Dehalococcoides can be stably cultured even in large-scale culture using 5-gallon beer barrels.

Claims (10)

A method for improving sustainability of a reduced state, which comprises heat-treating reduced glutathione under anaerobic conditions at 80°C or more and 125°C or less for 15 minutes or more and 25 minutes or less . A reducing agent consisting of reduced glutathione that has been heat-treated under anaerobic conditions at 80°C or more and 125°C or less for 15 minutes or more and 25 minutes or less . A culture medium comprising the reducing agent according to claim 2. A method for producing a culture medium, which comprises replacing a culture medium containing reduced glutathione with anaerobic gas in a sealed container, and heat-treating the culture medium at 80°C or more and 125°C or less for 15 minutes or more and 25 minutes or less . 5. The method for producing a culture medium according to claim 4, wherein the concentration of the reduced glutathione is 1 mM or more and 10 mM or less. A method for culturing anaerobic microorganisms, comprising using the medium according to claim 3. 7. The culture method according to claim 6, wherein the anaerobic microorganism is a bacterium belonging to the genus Dehalococcoides. 8. The culture method according to claim 6, wherein the concentration of the reduced glutathione is 1 mM or more and 10 mM or less. The culture method according to any one of claims 6 to 8, characterized in that the volume of the medium is 10 L or more. The culture method according to any one of claims 7 to 9, wherein the Dehalococcoides bacterium is strain UCH007 deposited under accession number NITE P- 1471 or a mutant strain thereof.