Method for culturing Dehalococcoides bacteria and medium for Dehalococcoides bacteria

Abstract

To provide a novel method of culturing Dehalococcoides bacteria, particularly, a culturing method with reduced hydrogen gas concentrations, and a medium for use in the culturing method.SOLUTION: The present invention provides a method of culturing Dehalococcoides bacteria using a medium comprising pyruvate or salt thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for culturing bacteria of the genus Dehalococcoides and a medium for culturing bacteria of the genus Dehalococcoides. [Background technology]

[0002] Purification technologies using microorganisms are widely used in wastewater treatment, such as activated sludge and anaerobic treatment. 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.

[0003] 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. [ka]

[0004] Dehalococcoides, a type of anaerobic dechlorinator, is the only bacterium reported to be capable of dechlorinating chlorinated ethylenes beyond cis-DCE. 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. Even if Dehalococcoides bacteria are present, their bacterial mass in the environment is very small, and their growth rate is slow, resulting in a prolonged remediation period.

[0005] To solve these problems, a method known as bioaugmentation is anticipated, 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 such strain in Japan, and proposed a method for purifying chlorinated ethylenes using this UCH007 strain. Dehalococcoides bacteria have previously been believed to utilize only hydrogen as an electron donor (Non-Patent Document 1). Therefore, Dehalococcoides bacteria have been cultivated exclusively in the presence of hydrogen. However, hydrogen-based cultivation poses a problem, as the cultivation scale increases, increasing the amount of hydrogen used, which increases the risk of explosion during handling. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-108061 [Non-patent literature]

[0007] [Non-Patent Document 1] Loffler, FE, Yan, J., Ritalahti, KM, Adrian, L., Edwards, EA, Konstantinidis, KT, Muller, JA, Fullerton, H., Zinder, SH & Spormann, AM " 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. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention addresses the problem of providing a novel method for culturing Dehalococcoides bacteria, particularly a culture method using a low hydrogen gas concentration, and a culture medium to be used in this culture method. [Means for solving the problem]

[0009] The means for solving the problems of the present invention are as follows. 1. A method for culturing Dehalococcoides bacteria, characterized by 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 Dehalococcoides bacterium is a strain of the Dehalococcoides genus strain having accession number NITE P-14 73. The method for culturing according to 1. or 2., wherein the strain is UCH007 deposited as reference number 1 or a mutant thereof. 4. A medium for Dehalococcoides bacteria, comprising pyruvic acid or a salt thereof. 5. The culture medium according to 4, characterized in that the dissolved hydrogen concentration is 0.16 ppm or less. [Effects of the Invention]

[0010] 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. Pyruvic acid acts not only as a carbon source but also as an electron donor. While hydrogen was previously the only electron donor known for culturing Dehalococcoides bacteria, the culture method of the present invention uses pyruvic acid as an electron donor, eliminating the need for hydrogen gas and reducing the risk of using hydrogen gas during culture medium preparation. [Brief explanation of the drawings]

[0011] [Figure 1] 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. [Figure 2] Graph showing the change over time in chlorinated ethylenes when TCE was decomposed in a 15 L medium in Experiment 2. DETAILED DESCRIPTION OF THE INVENTION

[0012] 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, which is used for said culturing.

[0013] Dehalococcoides spp. 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. While there are no particular limitations on the Dehalococcoides bacteria cultured in the present invention, when the cultured Dehalococcoides bacteria are used for purifying chlorinated ethylenes by bioaugmentation or the like, a strain capable of completely dechlorinating chlorinated ethylenes to ethylene is preferred. Examples include Dehalococcoides sp. UCH007 and its mutants. In this specification, a mutant refers to a strain with 98% or greater homology in the nucleotide sequence of its 16S rRNA gene. The homology of the 16S rRNA gene is preferably 99% or greater, more preferably 99.5% or greater, and even more preferably 100%.

[0014] The UCH007 strain was deposited on November 22, 2012, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (postal code 292-0818)) under accession number NITE P-1471. The mycological properties of the UCH007 strain are as follows: (a) Morphological properties Non-motile, non-sporulating, discoidal (difficult to observe with a light microscope). (b) An example of a 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 H2 / CO2 mixture (80:20).

[0015] (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 is vitamin B12-requiring. It is sensitive to high concentrations of sulfide (used as a reducing agent). Growth temperature: 30°C. Optimal pH: 7.2±0.3.

[0016] In the present invention, Dehalococcoides bacteria may be cultured alone, or isolated pure strains may 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, thereby promoting their growth. Examples of Sulfurospirillum bacteria include Sulfurospirillum sp. UCH001 strain (hereinafter also referred to as UCH001 strain) and / or Sulfurospirillum sp. UCH003 strain (hereinafter also referred to as UCH003 strain), or mutants thereof.

[0017] The UCH001 and UCH003 strains were deposited on November 22, 2012, at the National Institute of Technology and Evaluation, Patent Microorganisms 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 characteristics, culture characteristics, and physiological characteristics of the UCH001 and UCH003 strains are as follows, and are common to both strains.

[0018] (a) Morphological properties Curved or spiral rods. It has flagella, is motile, and does not form spores.

[0019] (b) An example of a culture medium Absolute anaerobic. Uses fumarate, lactate, and acetate. Bicarbonate buffer. pH 7.2. Gas replacement in the culture medium and culture vessel is performed with a H2 / CO2 mixture (80:20).

[0020] (c) Physiological characteristics Anaerobic. Obtains energy through respiration using nitrate, fumarate, etc. as electron acceptors. Utilizes hydrogen, formate, pyruvate, lactic acid, etc. as electron donors. Utilizes acetate, etc. as a carbon source. Growth temperature: 30℃ Growth pH: pH7.2

[0021] ·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 shaking 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.

[0022] Dehalococcoides bacteria are strictly anaerobic. As mentioned above, Dehalococcoides bacteria have traditionally been cultured in an H2 / CO2 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 hydrogen gas. The culture method of the present invention does not require the use of hydrogen gas, but a H2 / CO2 mixed gas can be used, as in conventional culture methods. However, due to the risk of explosion, hydrogen gas is preferably used in small amounts when used in a culture atmosphere. The hydrogen gas concentration in the culture atmosphere is preferably 10% by volume or less, more preferably 5% by volume or less, and most preferably no hydrogen gas is used at all. When hydrogen gas is not used, a N2 / CO2 mixed gas can be used when bicarbonate is used as a pH buffer, and N2 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.

[0023] 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 are preferably subjected to gas exchange using sterile nitrogen gas or filled with a reducing agent to completely remove air bubbles. Furthermore, gas exchange and filter sterilization are performed on vitamin solutions and other materials that cannot be heated. Sterilization filters are also preferably subjected to gas exchange using sterile nitrogen gas or filled with a reducing agent to remove air, followed by passing the gas-exchanged vitamin solution 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 excellent durability in the reduced state.

[0024] Culture medium 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, 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.

[0025] Pyruvic acid acts as a carbon source and an electron donor. The culture method of the present invention can contain, as carbon sources other than pyruvic acid, alcohols such as methanol and ethanol, and organic acids such as acetic acid and lactic acid. However, the ratio of pyruvic acid to the total carbon sources 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.

[0026] 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.

[0027] 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 to be cultured. The chlorinated ethylenes are preferably any one of PCE, TCE, cis-DCE, and VCM, or a combination of two or more of these.

[0028] 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 of 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. [Example]

[0029] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0030] "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. [Table 1]

[0031] 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 air with N2 gas, sealing it 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.

[0032] [Table 2]

[0033] [Table 3]

[0034] [Table 4]

[0035] [Table 5]

[0036] 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. 6103518, except that the H2 / CO2 (80 / 20) mixed gas was replaced with N2 / CO2 (80 / 20) mixed gas and other substrates were 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 culture solution of the UCH007 strain, which had been separately cultured, was inoculated into the medium at a volume of 1 / 100 of the medium using a syringe that had been treated with nitrogen gas substitution. Static culture was performed at 30°C for 52 days.

[0037] The concentrations of chlorinated ethylenes after cultivation were quantified using GCMS (Shimadzu GCMS-QP2010 Ultra). The concentrations of chlorinated ethylenes are shown in Figure 1.

[0038] Only when sodium pyruvate was used as a substrate was cis-DCE not detected, indicating that dechlorination of chlorinated ethylenes had progressed and the number of Dehalococcoides bacteria had increased. This is because the Dehalococcoides bacteria used sodium pyruvate as an electron donor.

[0039] "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.

[0040] Although large amounts of anaerobic gas are required for medium preparation, we used N2 gas, which is readily available and inexpensive, rather than gas mixtures such as N2 / CO2, which are time-consuming and costly to produce. Furthermore, to use N2 gas, we did not use bicarbonate as a pH buffer in the medium, but instead used 3-Morpholinopropanesulfonic acid (MOPS) (when using bicarbonate as a pH reducer in the medium, the pH tends to shift toward the alkaline side if carbon dioxide is not present in the gas phase of the culture vessel, making the medium pH unstable). Furthermore, to maintain a stable anaerobic state, we used an airtight beer barrel as the culture vessel, added 5 mM reduced glutathione to the medium as a reducing agent, and performed gas substitution and autoclave sterilization. Additionally, an appropriate amount of cyanocobalamin (vitamin B12) was added as one of the vitamins, preferably at 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).

[0041] As a culture vessel for mass cultivation, a 5-gallon beer barrel (Cornelius type) was used, which had been modified to have a sampling port on the top (lid) of the tube that enabled collection of bacterial liquid and gas. The sampling port was created by drilling a hole approximately 12 mm in diameter in the lid of the beer keg, creating a screw port in the partition using a nut-tightened φ12 electrode port (product number: C00000-C005) manufactured by Able Co., Ltd. and a butyl rubber O-ring, and then attaching a butyl rubber stopper for freeze-drying, product number 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 Co., Ltd. Bacterial liquid and gas samples were collected by puncturing the butyl rubber stopper with the needle of a sterilized syringe.

[0042] 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 composition shown in Table 6 is 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 syringes previously treated with sterile nitrogen gas purging, the vitamin solution shown in Table 4 (vitamin B12 was adjusted to a final concentration of 25 μg / L after addition to the medium) and TCE or cis-DCE stock solution were added aseptically and anaerobically. After preparing the medium, a co-culture of strains UCH007 and UCH001, which had been cultivated separately, was inoculated into the medium at 1 / 100th the volume using a syringe that had been purged with nitrogen gas, and static cultivation was initiated at room temperature. During cultivation, 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.

[0043] [Table 6]

[0044] In Experiment 2, it was confirmed that Dehalococcoides bacteria could be stably cultivated regardless of whether the chlorinated ethylenes added to the medium were TCE or cis-DCE, and regardless of whether the 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 medium.

Claims

1. A method for culturing Dehalococcoides bacteria, comprising using a medium containing pyruvic acid or a salt thereof as an electron donor for Dehalococcoides bacteria.

2. 2. The culture method according to claim 1, wherein the concentration of hydrogen gas in the culture atmosphere is 10% by volume or less.

3. 3. The culture method according to claim 1, wherein the Dehalococcoides bacterium is the UCH007 strain deposited under accession number NITE P-1471 or a mutant thereof.

4. A culture medium for a pure strain of Dehalococcoides bacteria, comprising pyruvic acid or a salt thereof as an electron donor for the bacteria.

5. 5. The culture medium according to claim 4, wherein the dissolved hydrogen concentration is 0.16 ppm or less.

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