JP7723238B2 - Method for culturing Dehalococcoides bacteria and medium for Dehalococcoides bacteria - Google Patents

Description

NPMD NITEP-1471

The present invention relates to a method for culturing bacteria of the genus Dehalococcoides and a culture medium for bacteria of the genus Dehalococcoides.

Purification technologies using microorganisms are widely used in wastewater treatment, such as the activated sludge method and anaerobic treatment method. In recent years, bioremediation, a technology that uses microorganisms to purify soil and groundwater contaminated with hazardous chemicals, has been attracting attention as a purification method with low environmental impact and purification costs.

Chlorinated ethylenes such as tetrachloroethylene (PCE) and trichloroethylene (TCE) are inexpensive solvents with strong cleaning properties for oil and are used in a wide range of fields, including the metal industry, semiconductor industry, and dry cleaners. However, there have been many reports of soil and groundwater contamination by chlorinated ethylenes, which has become a social problem.
For bioremediation of chlorinated ethylenes, anaerobic dechlorinating bacteria are used, which detoxify chlorinated ethylenes through the reductive dechlorination reaction: tetrachloroethylene (PCE) → trichloroethylene (TCE) → cis-1,2-dichloroethylene (cis-DCE) → vinyl chloride monomer (VCM) → ethylene.

Dehalococcoides bacteria, a type of anaerobic dechlorinating bacteria, are the only bacteria reported to be capable of dechlorinating chlorinated ethylenes to cis-DCE and beyond. In other words, in bioremediation of chlorinated ethylenes, if Dehalococcoides bacteria are not present in the soil being treated, dechlorination will only be possible up to cis-DCE. Furthermore, even if Dehalococcoides bacteria are present, their bacterial mass in the environment is very small, and their growth rate is slow, resulting in the problem of prolonged remediation periods.

To solve these problems, a method known as bioaugmentation is expected to be developed, in which Dehalococcoides bacteria are artificially cultivated and multiplied, and then introduced into the target environment to promote purification. In Patent Document 1, the present inventors isolated Dehalococcoides sp. UCH007 (NITE P-1471, hereinafter also referred to as UCH007 strain), the first strain in Japan, and proposed a method for purifying chlorinated ethylenes using this UCH007 strain. Dehalococcoides bacteria have previously been thought to utilize only hydrogen as an electron donor (Non-Patent Document 1). For this reason, Dehalococcoides bacteria have traditionally been cultivated only in the presence of hydrogen. However, hydrogen-based cultivation poses the problem of increased risk of explosion and other problems during handling, as the larger the cultivation scale, the more hydrogen is used.

JP 2014-108061 A

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.

The objective of this study is to provide a novel method for culturing Dehalococcoides bacteria, particularly a culture method using a low hydrogen gas concentration, and a culture medium for use in this culture method.

The means for solving the problems of the present invention are as follows.
1. A method for culturing Dehalococcoides bacteria, which comprises using a medium containing pyruvic acid or a salt thereof.
2. The culture method according to 1., wherein the hydrogen gas concentration in the culture atmosphere is 10% by volume or less.
3. The culture method according to 1. or 2., wherein the Dehalococcoides bacterium is the UCH007 strain deposited under accession number NITE P- 1471 or a mutant thereof.
4. A medium for Dehalococcoides bacteria, comprising pyruvic acid or a salt thereof.
5. The culture medium according to 4., wherein the dissolved hydrogen concentration is 0.16 ppm or less.

The method for culturing Dehalococcoides bacteria of the present invention is characterized by the use of pyruvic acid or a salt thereof, and is a novel method not previously known. Pyruvate acts not only as a carbon source but also as an electron donor. Previously, hydrogen was the only known electron donor used in culturing Dehalococcoides bacteria. However, in the culture method of the present invention, pyruvic acid acts as the electron donor, eliminating the need for hydrogen gas and reducing the risk of using hydrogen gas when preparing the culture medium.

1 is a graph showing the concentration of chlorinated ethylenes after culturing a pure strain of Dehalococcoides bacteria using various substrates in the absence of hydrogen in Experiment 1. 1 is a graph showing the change over time in chlorinated ethylenes when TCE was decomposed in a medium volume of 15 L in Experiment 2.

The present invention relates to a method for culturing bacteria of the genus Dehalococcoides, characterized by using a medium containing pyruvic acid or a salt thereof, and to a medium for Dehalococcoides bacteria containing pyruvic acid or a salt thereof used for this culturing.

Dehalococcoides bacteria have the ability to dechlorinate chlorinated ethylenes and chlorinated ethanes. Even among Dehalococcoides bacteria, the chlorinated ethylenes they can decompose vary depending on the species. There are no particular limitations on the Dehalococcoides bacteria cultured in the present invention. However, when the cultured Dehalococcoides bacteria are used to purify chlorinated ethylenes by bioaugmentation or the like, it is preferable that the strain be capable of completely dechlorinating chlorinated ethylenes down to ethylene. An example is Dehalococcoides sp. UCH007 or a mutant thereof. In this specification, a mutant refers to a strain whose 16S rRNA gene base sequence 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%.

The UCH007 strain was deposited on November 22, 2012, at the National Institute of Technology and Evaluation, National Patent Microorganism Depositary (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818)) under the accession number NITE P-1471.
The mycological properties of the UCH007 strain are as follows:
(a) Morphological characteristics: Non-motile, non-sporulating, discoidal (difficult to observe with an optical microscope).
(b) Example of culture medium: Absolute anaerobic. Hydrogen is used as the electron donor, TCE or cis-DCE as the electron acceptor, and acetate as the carbon source. Bicarbonate buffer. pH 7.2. Gas replacement in the culture medium and culture vessel is performed with a _{H 2} /CO ₂ mixed gas (80:20).

(c) Physiological characteristics: Strictly anaerobic. Obtains energy through dehalogenative respiration. Uses hydrogen as an electron donor and chlorinated ethylenes as electron acceptors.
It has the ability to dechlorinate trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride monomer (VCM). It utilizes acetate as a carbon source. It requires vitamin B12. It is sensitive to high concentrations of sulfide (used as a reducing agent).
Growth temperature: 30°C.
Optimum pH: 7.2±0.3.

In the present invention, Dehalococcoides bacteria may be cultured alone, or an isolated pure strain can be co-cultured with other bacteria as a bioconsortium (complex microbial system). For example, co-culture with Sulfurospirillum bacteria is preferred. Sulfurospirillum bacteria have the ability to promote the dechlorination reaction of Dehalococcoides bacteria. Co-culturing them with Dehalococcoides bacteria helps the Dehalococcoides bacteria acquire energy through dechlorination, promoting the growth of the Dehalococcoides bacteria. Examples of bacteria of the genus Sulfurospirillum include either or both of the Sulfurospirillum sp. UCH001 strain (hereinafter also referred to as the UCH001 strain) and the Sulfurospirillum sp. UCH003 strain (hereinafter also referred to as the UCH003 strain), or mutant strains of these.

The UCH001 and UCH003 strains were deposited on November 22, 2012, at the National Institute of Technology and Evaluation, National Patent Microorganism Depositary (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (postal code 292-0818)) under accession numbers NITE P-1419 and NITE P-1470, respectively.
The morphological properties, culture characteristics, and physiological characteristics of the UCH001 and UCH003 strains are as follows, and are common to both strains.

(a) Morphological characteristics: curved or spiral rod.
It has flagella, is motile, and does not form spores.

(b) Example of culture medium: Absolute anaerobic. Fumarate, lactate, and acetate are used. Bicarbonate buffer. pH 7.2. Gas replacement inside the culture medium and culture vessel is performed with a _{H 2} /CO ₂ mixed gas (80:20).

(c) Physiological characteristics: Anaerobic. Energy is obtained through respiration, with nitrate, fumarate, etc. as electron acceptors. Hydrogen, formate, pyruvic acid, lactate, etc. are used as electron donors. Acetate, etc. is utilized as a carbon source.
Growth temperature: 30°C
Growth pH: pH7.2

Culture Method The culture method of the present invention is characterized by using a medium containing pyruvic acid or a salt thereof.
Culturing can be carried out by various culture methods such as static culture and shake culture. Culture conditions such as the culture method, temperature, pH, and period can be appropriately selected depending on the Dehalococcoides bacterium 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.

Dehalococcoides bacteria are strictly anaerobic. As mentioned above, Dehalococcoides bacteria have traditionally been cultured in an H 2 /CO ₂ mixed gas atmosphere because only hydrogen is believed to be available as an electron donor. However, pyruvic acid used in the culture method of the present invention also functions as an electron donor, allowing culture without the use of hydrogen gas. The culture method of the present invention does not require the use of hydrogen gas, but a H ₂ /CO ₂ mixed gas can also be used, as in conventional culture methods _. However, because hydrogen poses an explosion risk, if hydrogen gas is used, it is preferable to use a small amount. The hydrogen gas concentration in the culture atmosphere is preferably 10% by volume or less, more preferably 5% by volume or less, and it is most preferable not to use hydrogen gas at all. If hydrogen gas is not used, a N ₂ /CO ₂ mixed gas can be used when bicarbonate is used as a pH buffer, or N ₂ gas can be used when a pH buffer other than bicarbonate is used. The volume ratio of _N2 to _CO2 in the mixed gas is not particularly limited, but is preferably 70:30 to 90:10.

During cultivation, it is important to minimize air (oxygen) contamination during medium preparation, reagent addition, bacterial inoculation, sampling, and other processes. For example, sterilized syringes used for adding volatile reagents such as chlorinated ethylenes or for inoculating bacterial solutions should preferably be gas-purged with sterile nitrogen gas or filled with a reducing agent to completely remove air bubbles. Furthermore, vitamin solutions and other products that cannot be heated are subjected to gas-purging and filter sterilization. Sterilizing filters should also be gas-purged with sterile nitrogen gas or filled with a reducing agent to remove air, and then the gas-purged vitamin solution should be passed through the filter to remove the reducing agent. Examples of reducing agents that can be used include reduced glutathione, iron sulfide, a combination of sodium sulfide, L-cysteine, and DL-dithiothreitol, and titanium(III) complexed with citrate or nitrilotriacetate. Among these, heat-treated reduced glutathione is preferred due to its superior durability in the reduced state.

The culture medium used in the culture method of the present invention is characterized by containing pyruvic acid or a salt thereof. This medium may contain pyruvic acid or a salt thereof, and may also contain an electron donor source, a carbon source, a nitrogen source, inorganic salts, trace elements, yeast extract, etc., and may be either a solid medium or a liquid medium.

Pyruvate acts as a carbon source and electron donor. The cultivation method of the present invention can include alcohols such as methanol and ethanol, and organic acids such as acetic acid and lactic acid as carbon sources other than pyruvate. However, the ratio of pyruvate to the total carbon source is preferably 60% by weight or more, more preferably 80% by weight or more, even more preferably 90% by weight or more, and most preferably 100% by weight.

Examples of nitrogen sources include peptone, casitone, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, and various amino acids.
Examples of inorganic salts include phosphate salts, magnesium salts, and calcium salts.
Examples of 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 appropriate combination of two or more.
Furthermore, as a nutrient source for promoting the growth of the microorganism of the present invention, an appropriate amount of vitamins such as cyanocobalamin, yeast extract, malt extract, etc. may be added.

When culturing Dehalococcoides bacteria, one or a mixture of two or more chlorinated ethylenes can be added to the medium depending on the characteristics of the Dehalococcoides bacteria being cultured. The chlorinated ethylenes are preferably PCE, TCE, cis-DCE, or VCM, or a combination of two or more of these.

As mentioned above, pyruvic acid also acts as an electron donor. The solubility of hydrogen gas at room temperature and pressure is 1.6 ppm. When cultured under conditions with a low hydrogen gas concentration, the dissolved hydrogen concentration in the medium is low; for example, when the hydrogen gas concentration is 10% by volume or less and 5% by volume or less, the dissolved hydrogen concentration is 0.16 ppm or less and 0.08 ppm or less, respectively.

The present invention will be explained in more detail below using examples. However, the present invention is not limited to these examples.

"Experiment 1: Examination of organic matter that can be assimilated by Dehalococcoides bacteria as a carbon source and used as an electron donor"
We investigated organic substances that Dehalococcoides bacteria can use alone as a carbon source and electron donor in the absence of hydrogen.
The composition of the bicarbonate-buffered medium used (a partial modification of the medium described in He, J., Ritalahti, KM, Yang, KL, Koenigsberg, SS & Loffler, FE, "Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium," Nature (2003) July 3, 424: pp. 62-65) is shown in Table 1. This bicarbonate-buffered medium was prepared in the same manner as in Example 2 of Japanese Patent No. 6,103,518.

The cis-1,2-dichloroethylene stock solution was prepared by placing 20 ml of distilled water and a stir bar in a light-shielding vial, replacing the atmosphere with _N2 gas, sealing the vial with a PTFE-liner rubber stopper and an aluminum seal, autoclaving, and then adding 3 μl of cis-1,2-dichloroethylene (cis-DCE) using a gas-tight syringe and stirring overnight.

Substrates alternative to hydrogen (electron donor) and sodium acetate (carbon source ₎ were investigated under the same conditions as in Example 2 of Japanese Patent No. 6,103,518, except that the H ₂ /CO ₂ (80/20) mixed gas was replaced with an N ₂ /CO 2 (80/20) mixed gas and another substrate was used instead of sodium acetate. The substrates used were sodium lactate, sodium pyruvate, disodium succinate, disodium malate, sodium formate, sodium butyrate, sodium propionate, glucose, disodium glutarate, ethanol, methanol, trisodium citrate, monosodium 2-ketoglutarate, disodium fumarate, sodium aspartate, and crotonic acid (all at a final concentration of 5 mM). After medium preparation, a separately cultured culture solution of the UCH007 strain was inoculated into the medium at 1/100th the volume using a syringe treated with nitrogen gas substitution, and static culture was performed at 30°C for 52 days.

The concentration of chlorinated ethylenes after cultivation was quantified using GCMS (Shimadzu GCMS-QP2010 Ultra). The concentrations of chlorinated ethylenes are shown in Figure 1.

Only when sodium pyruvate was used as a substrate was cis-DCE not detected, confirming that dechlorination of chlorinated ethylenes had progressed and that the number of Dehalococcoides bacteria had increased. This is because the Dehalococcoides bacteria used sodium pyruvate as an electron donor.

"Experiment 2: Experiment demonstrating that Dehalococcoides bacteria can be cultured in large quantities using the present invention"
The present invention does not require hydrogen, which was previously required for culturing Dehalococcoides bacteria, and enables safe cultivation without the risk of explosion. Here, we confirmed whether the amount of culture solution required to introduce purification bacteria into contaminated groundwater, etc., could be actually cultivated stably in a large container without using hydrogen.

Although large amounts of anaerobic gas are required for medium preparation, _N2 gas was used, which is more readily available and inexpensive than gas mixtures such as _N2 / _CO2, which are time-consuming and costly to produce. Furthermore, in order to use _N2 gas, bicarbonate was not used as the medium's pH buffer, but 3-morpholinopropanesulfonic acid (MOPS) was used instead. (When bicarbonate is used as a pH reducing agent in the medium, the pH tends to shift toward the alkaline side if carbon dioxide is not contained in the gas phase of the culture vessel, making the medium pH unstable.) Furthermore, to maintain a stable anaerobic state, an airtight beer barrel was used as the culture vessel, and 5 mM reduced glutathione was added to the medium as a reducing agent. Gas replacement and autoclave sterilization were then performed. Additionally, an appropriate amount of cyanocobalamin (vitamin B12) was added as one of the vitamins, in accordance with literature information that recommends a final concentration of 25 μg/L (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. Environ. Microbiol. (2007) 73: pp. 2847-2853).

As a culture vessel for mass culture, a 5-gallon beer barrel (Cornelius type) was used, which had been modified to have a sampling port at the top (lid) of the tube to enable sampling of bacterial liquid and gas.
The sampling port was fabricated by drilling a hole of approximately 12 mm diameter in the lid of a beer keg, creating a screw opening in the partition using a nut-tightening φ12 electrode port (product number: C00000-C005) manufactured by Able Corporation and a butyl rubber O-ring, and then attaching a butyl rubber stopper for freeze-drying, B (large) manufactured by Nichiden Rika Glass Co., Ltd., and fastening it in place with a φ12 cap nut (product number: C00000-C008) manufactured by Able Corporation. Bacterial liquid and gas samples were collected by puncturing the butyl rubber stopper with a sterilized syringe needle.

Culture vessels consisting of 5-gallon beer barrels with modified lids were filled with 10 L, 15 L, or 18 L of the reduced glutathione-containing medium shown in Table 6. The compositions shown in Table 6 are based on the composition per liter of distilled water. The procedure for preparing the reduced glutathione-containing medium was as follows: (1) The medium was placed in a beer barrel and nitrogen gas purging was performed for 1 hour. (2) The medium was sterilized by autoclaving (121°C, 20 minutes). (3) After nitrogen gas purging for 1 hour immediately after autoclaving, the container was immersed in ice water and gas purging was continued until the temperature of the entire container was below room temperature (approximately 30 minutes). (4) Using a syringe previously subjected to gas purging using sterilized nitrogen gas, the vitamin solution shown in Table 4 (vitamin B12 was changed so that the final concentration after adding the medium was 25 μg/L) and the TCE or cis-DCE stock solution were added aseptically and anaerobically.
After the medium was prepared, a co-culture of strains UCH007 and UCH001, which had been cultured separately, was inoculated into the medium in an amount of 1/100 of the medium using a syringe that had been subjected to nitrogen gas replacement, and static culture was initiated at room temperature. During the culture, the concentration of chlorinated ethylenes in the culture medium was periodically quantified by the headspace method using a gas chromatograph (model 6890N, Agilent Technologies) equipped with a capillary column (Agilent J&W GC column DB-624, 0.32 mm x 30 m) and FID.

In Experiment 2, it was confirmed that Dehalococcoides bacteria could be stably cultivated regardless of whether the chlorinated ethylenes added to the culture medium were TCE or cis-DCE, and regardless of whether the culture medium volume was 10 L, 15 L, or 18 L. As an example, Figure 2 shows the change in chlorinated ethylene concentration over time when TCE was used in a 15 L culture medium.

Claims (5)

A method for culturing bacteria of the genus Dehalococcoides, characterized by using a medium containing pyruvic acid or a salt thereof as an electron donor for bacteria of the genus Dehalococcoides. The culture method described in claim 1, characterized in that the hydrogen gas concentration in the culture atmosphere is 10% by volume or less. The culture method according to claim 1 or 2, characterized in that the Dehalococcoides bacterium is the UCH007 strain deposited under accession number NITE P-1471 or a mutant thereof. A culture medium for a pure strain of Dehalococcoides bacteria, comprising pyruvic acid or a salt thereof as an electron donor for the bacteria. The culture medium according to claim 4, characterized in that the dissolved hydrogen concentration is 0.16 ppm or less.