Method for culturing bacteria of the genus Dehalococcoides and method for purifying chlorinated ethylenes

Abstract

To provide a method for stably mass-culturing Dehalococcoides genus bacteria using a keg-like metal pressure-resistant culture vessel.SOLUTION: This method for culturing Dehalococcoides genus bacteria uses a metal pressure-resistant culture vessel to culture Dehalococcoides genus bacteria in a culture solution while maintaining ion concentrations in the range of (a) 1 mM or less of iron-containing ions, (b) 23 μM or less of chromium-containing ions, and (c) 1.5 mM or less of nickel-containing ions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for culturing bacteria of the genus Dehalococcoides, and more specifically, to a method for efficiently culturing large quantities of Dehalococcoides bacteria and purifying chlorinated ethylenes using these large-scale cultivated Dehalococcoides bacteria. [Background technology]

[0002] Bacteria of the genus Dehalococcoides are capable of reductively dechlorinating chlorinated ethylenes such as tetrachloroethylene (PCE) and trichloroethylene (TCE), which are contaminants of groundwater and soil, to harmless ethylene. Therefore, bioremediation using these Dehalococcoides bacteria to purify soil and groundwater contaminated with chlorinated ethylenes (CEs) is anticipated. For example, Patent Document 1 by the present inventor discloses the Dehalococcoides mccartyi NIT01 strain as a Dehalococcoides bacterium useful for bioremediation, and it is stated that this NIT01 strain can decompose trichloroethylene and 1,1,2-trichloroethane into ethylene.

[0003] To implement bioremediation using bacteria of the genus Dehalococcoides, it is necessary to prepare large quantities of Dehalococcoides cultures and transport them to the contaminated site. Since Dehalococcoides bacteria are obligate anaerobic bacteria, anaerobic culture can be stably performed on a small scale of several tens of milliliters, but stable anaerobic culture on a large scale of several liters or more is extremely difficult. Furthermore, transporting these cultures to the contaminated site while maintaining anaerobic conditions is also a problem.

[0004] Therefore, Non-Patent Document 1 proposes using commercially available metal beer kegs, which are pressure-resistant containers, for the mass cultivation of obligate anaerobic bacteria. By using these metal beer kegs as culture vessels, it is possible to autoclave sterilize the culture vessel with the liquid culture medium inside, gas replacement after autoclaving is easy, and the culture can be transported along with the culture vessel after cultivation. As an example, Patent Document 2 discloses the mass cultivation of bacteria of the genus Dehalococcoides using a 5-gallon beer keg (Cornelius type). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2021-31 [Patent Document 2] Japanese Patent Publication No. 2021-16368 [Non-patent literature]

[0006] [Non-Patent Document 1] "Large-scale cultivation using commercially available pressure-resistant containers such as beer kegs," [online], National Institute of Technology and Evaluation website, [Accessed September 1, 2022], Internet<URL:https: / / www.nite.go.jp / nbrc / industry / support / cultivation.html> [Overview of the project] [Problems that the invention aims to solve]

[0007] However, the inventors of this application, while repeatedly performing large-scale cultures of bacteria of the genus Dehalococcoides using commercially available stainless steel beer kegs as pressure-resistant culture vessels for application in bioremediation, discovered that the dechlorination of chlorinated ethylenes stopped, and the activity and growth of Dehalococcoides bacteria also ceased.

[0008] Accordingly, the present invention has been made in view of the above points, and an object of the present invention is to provide a method that enables stable mass culture of Dehalococcoides bacteria when culturing said bacteria using a keg-shaped metal pressure-resistant culture vessel. [Means for Solving the Problem]

[0009] The inventor of the present application noted that commercially available autoclaveable beer kegs are formed of stainless steel, and the keg that the inventor of the present application had tested as a mass culture vessel was also formed of SUS304 steel. The inventor hypothesized that components eluted or peeled from this type of stainless steel container might have affected the culture of Dehalococcoides bacteria, and proceeded with research. The present invention has been completed based on the findings obtained as a result.

[0010] In order to solve the above problems, the method for culturing Dehalococcoides bacteria of the present invention is a method for culturing Dehalococcoides bacteria using a metal pressure-resistant culture vessel, wherein Dehalococcoides bacteria are cultured while maintaining the following concentration ranges in a culture solution: (a) 1 mM or less of iron-containing ions, (b) 23 μM or less of chromium-containing ions, and (c) 1.5 mM or less of nickel-containing ions. Accordingly, even when a metal pressure-resistant culture vessel is used, the dechlorination of chlorinated ethylenes by Dehalococcoides bacteria is less likely to be inhibited. As a result, dechlorination of chlorinated ethylenes in the culture solution proceeds while the number of bacteria increases, enabling stable mass culture.

[0011] In the above culture method, it is also preferable to culture the bacteria while maintaining the following concentration ranges in the culture solution: (a) 300 μM or less of iron-containing ions, (b) 10 μM or less of chromium-containing ions, and (c) 1 mM or less of nickel-containing ions. Accordingly, even when a metal pressure-resistant culture vessel is used, the dechlorination of chlorinated ethylenes by Dehalococcoides bacteria is even less likely to be inhibited. As a result, dechlorination of chlorinated ethylenes in the culture solution proceeds while the number of bacteria increases, enabling stable mass culture.

[0012] In addition, in the culture method of the present invention, it is also preferable to use a pressure-resistant culture vessel made of titanium as the metal pressure-resistant culture vessel. This allows a suitable material to be selected as the metal material of the pressure-resistant culture vessel which can maintain the aforementioned concentration ranges without affecting the concentration ranges of various metal ions in the culture solution.

[0013] In addition, in the culture method of the present invention, it is also preferable that the Dehalococcoides bacterium is Dehalococcoides mccartyi NIT01 strain identified under accession number NITE P-02893. This allows a suitable Dehalococcoides bacterium to be selected in the present invention. Since this NIT01 strain can dechlorinate not only chlorinated ethylenes but also chlorinated ethanes, it is a strain useful for bioremediation of contaminated sites polluted by chlorinated aliphatic compounds.

[0014] In addition, in the culture method of the present invention, it is also preferable that the iron-containing ion is Fe 2+ and the chromium-containing ion is Cr 3+ and the nickel-containing ion is Ni 2+ This allows particularly suitable metal ions to be selected as the metal ions whose predetermined concentration range should be maintained in the culture solution.

[0015] Furthermore, the method for remediating chlorinated ethylene contamination according to the present invention is carried out by transporting the Dehalococcoides bacterium cultured by the aforementioned culture method together with the metal pressure-resistant culture vessel to a contaminated site polluted by chlorinated ethylenes, and supplying the Dehalococcoides bacterium taken out from the pressure-resistant culture vessel to this contaminated site. This allows stable mass culture of the Dehalococcoides bacterium in the pressure-resistant culture vessel, and the whole pressure-resistant culture vessel can be transported to the contaminated site, so that the Dehalococcoides bacterium in the pressure-resistant culture vessel can be easily and quickly supplied to the contaminated site, and the chlorinated ethylenes in the contaminated site can be dechlorinated and remediated. Effects of the Invention

[0016] According to the present invention, it is possible to provide a method for culturing bacteria of the genus Dehalococcoides, which has the following excellent effects, and a method for purifying chlorinated ethylenes using the same. (1) By keeping the concentration range of each metal ion in the culture medium below a predetermined concentration, the dechlorination of chlorinated ethylenes by bacteria of the genus Dehalococcoides is less likely to be inhibited. As a result, the dechlorination of chlorinated ethylenes in the culture medium progresses, the number of bacteria increases, and stable large-scale cultivation becomes possible. (2) By selecting a titanium container as the pressure-resistant culture vessel, the concentration range of various metal ions in the culture medium is not affected, so the dechlorination of chlorinated ethylenes in the culture medium is not inhibited, and stable large-scale culture can be repeated. (3) By stably culturing large quantities of bacteria of the genus Dehalococcoides in a metal pressure-resistant culture vessel, the culture vessel can be transported to the contaminated site. This allows for easy and rapid supply of bacteria of the genus Dehalococcoides in the culture vessel to the contaminated site, thereby dechlorinating and purifying the chlorinated ethylenes at the contaminated site. [Brief explanation of the drawing]

[0017] [Figure 1] This graph shows the concentration of chlorinated ethylenes (CEs) and cell density in the culture medium with (A) iron metal pieces added and (B) SUS316 metal pieces added in Example 1. [Figure 2] This graph shows the concentration of chlorinated ethylenes (CEs) and cell density in the culture medium to which SUS304 metal pieces were added in Example 1. [Figure 3] This graph shows the concentration of chlorinated ethylenes (CEs) and cell density in the culture medium to which titanium metal pieces were added in Example 1. [Figure 4] These are photographs showing the appearance of the metal pieces in each culture medium after the culture test in Example 1. [Figure 5] These are photographs showing the surface condition of each metal piece before and after the culture test in Example 2. [Figure 6]This graph shows the concentrations of chlorinated ethylenes in the control group (A) and in the culture solutions to which iron ions were added at various concentrations (B) to (E) in Example 4. [Figure 7] This graph shows the concentrations of chlorinated ethylenes in culture solutions to which chromium ions were added at various concentrations in Example 4. [Figure 8] This graph shows the concentrations of chlorinated ethylenes in culture solutions to which nickel ions were added at various concentrations in Example 4. [Figure 9] This is a photograph showing the titanium pressure-resistant culture vessel used in Example 5. [Figure 10] This graph shows the concentration of chlorinated ethylenes in the culture medium and the cell density when large-scale cultures were repeatedly performed in a titanium pressure-resistant culture vessel in Example 5. [Modes for carrying out the invention]

[0018] The method for culturing bacteria of the genus Dehalococcoides according to the present invention will be described in detail below. The method for culturing bacteria of the genus Dehalococcoides according to this embodiment is a method for culturing bacteria of the genus Dehalococcoides using a metal pressure-resistant culture vessel, wherein the bacteria of the genus Dehalococcoides are cultured such that the iron-containing ions, chromium-containing ions, and nickel-containing ions in the culture medium each maintain a predetermined concentration range.

[0019] The Dehalococcoides bacteria used in this embodiment can be any Dehalococcoides sp. capable of dehalogenating organic halogens such as chlorinated ethylenes or chlorinated ethanes. More specifically, Dehalococcoides bacteria that obtain energy and proliferate through dehalogenation respiration of this type of organic halogen are preferred. In this embodiment, there are no particular limitations, but Dehalococcoides mccartyi can be suitably used. Of these, Dehalococcoides mccartyi strain NIT01 (accession number: NITE P-02893), Dehalococcoides mccartyi strain CBDB1, or mutants thereof are preferred, with Dehalococcoides mccartyi strain NIT01 being particularly preferred. Furthermore, as the Dehalococcoides bacteria, it is possible to culture a single strain or a single species, or to co-culture multiple strains or multiple species. Furthermore, it is possible to co-culture bacteria other than those of the genus Dehalococcoides with bacteria, as long as the effects of the present invention are not diminished.

[0020] The Dehalococcoides mccartyi NIT01 strain (hereinafter also referred to as "NIT01 strain") described above is a bacterium of the genus Dehalococcoides that possesses the ability to decompose 4 mM trichloroethylene (TCE) into ethylene, and the ability to decompose 1,1,2-trichloroethane into ethylene. This NIT01 strain does not possess the ability to reduce nitrate, nitrite, sulfuric acid, sulfite, thiosulfate, and oxygen, and is a strain that can only grow through the reductive dechlorination of organic halogens such as chlorinated ethylenes. This NIT01 strain has been deposited in Japan with the patent microorganism depositary as follows. (1) Accession number: NITE P-02893 (2) Entrustment date: February 25, 2019 (3) Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Depository Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan)

[0021] In the present invention, chlorinated ethylenes include, but are not limited to, tetrachloroethylene (PCE), trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC), and chlorinated ethanes include, but are not limited to, 1,2-dichloroethane, 1,1,2-trichloroethane, and 1,1,1-trichloroethane.

[0022] In this embodiment, Dehalococcoides bacteria are cultured using a metal pressure-resistant culture vessel, and it is important to carry out the culture such that the concentration ranges of iron-containing ions, chromium-containing ions, and nickel-containing ions in the culture solution are maintained at 1 mM or less, 23 μM or less, and 1.5 mM or less, respectively. If these metal ions are contained in the culture solution in excess of these concentration ranges, the dechlorination of chlorinated ethylenes by Dehalococcoides bacteria is inhibited, and the growth of the bacteria is impaired. Specifically, the concentration of iron-containing ions in the culture solution is preferably maintained at 1 mM or less, more preferably 300 μM or less. Further, the concentration of chromium-containing ions in the culture solution is preferably maintained at 23 μM or less, more preferably 10 μM or less. The concentration of nickel-containing ions in the culture solution is preferably maintained at 1.5 mM or less, and more preferably maintained at 1 mM or less. The iron-containing ions are ion species containing iron, and are Fe 2+ or Fe 3+ , with Fe 2+ being more preferred. Further, the chromium-containing ions are ion species containing chromium, and are Cr 3+ , CrO4 2- or Cr2O7 2- , with Cr 3+ being more preferred. Further, the nickel-containing ions are ion species containing nickel, and are Ni 2+ or Ni 3+ , with Ni 2+ being more preferred.

[0023] In this invention, bacteria of the genus Dehalococcoides are cultured using a metal pressure-resistant culture vessel. Using a metal pressure-resistant culture vessel has the advantages of allowing autoclaving sterilization with the liquid culture medium inside the vessel, facilitating gas replacement after autoclaving, and allowing the culture to be transported along with the culture vessel after cultivation. Although not particularly limited, a so-called keg-like metal pressure-resistant vessel is preferred, as introduced in Non-Patent Document 1, and it is preferable that the structure be modified to include a sampling hole in the lid. Here, commercially available kegs are made of iron or stainless steel, and as shown in the examples described later, it was found that when autoclaving and cultivation are repeated multiple times using a culture vessel made of iron or stainless steel, metal ion components contained in the iron or stainless steel leach into the culture medium, inhibiting the growth of bacteria of the genus Dehalococcoides. Therefore, it is preferable to use a metal pressure-resistant culture vessel that can maintain a concentration range of 1 mM or less for iron-containing ions, 23 μM or less for chromium-containing ions, and 1.5 mM or less for nickel-containing ions in the culture medium, and it is more preferable to use a vessel that can maintain a concentration range of 300 μM or less for iron-containing ions, 10 μM or less for chromium-containing ions, and 1 mM or less for nickel-containing ions in the culture medium.

[0024] More specifically, as a metal pressure-resistant culture vessel, it is preferable to use a pressure-resistant culture vessel made of titanium, as shown in Example 5 described later. Pure titanium is preferred as the titanium, and for example, pure titanium of JIS standards types 1, 2, 3, and 4 can be suitably used. This provides excellent corrosion resistance and ensures that the concentration range of iron-containing ions in the culture medium is kept below 1 mM, chromium-containing ions below 23 μM, and nickel-containing ions below 1.5 mM, thus enabling stable large-scale cultivation of Dehalococcoides bacteria. Furthermore, since the culture vessel is made of titanium, the vessel itself is lightweight, which has the advantage of making it easier to transport the culture along with the culture vessel to a contaminated site. As mentioned above, titanium alloys can be used as well as pure titanium, as long as they can maintain the concentration range of iron-containing ions below 1 mM, chromium-containing ions below 23 μM, and nickel-containing ions below 1.5 mM in the culture medium.

[0025] As the culture medium for Dehalococcoides bacteria, in addition to the DHB-CO3-Br medium used in Example 1 described later, any medium with any composition used as a culture medium for Dehalococcoides bacteria can be used. Furthermore, the composition of the culture medium can be appropriately modified depending on the type of Dehalococcoides bacteria to be inoculated and the culture conditions. The culture medium preferably contains a carbon source, a reducing agent, an electron acceptor source, an electron donor source, inorganic salts, trace elements, and vitamins. Of these, it is preferable to include chlorinated ethylenes as the electron acceptor source, depending on the decomposition characteristics of chlorinated ethylenes of the Dehalococcoides bacteria to be inoculated. For example, the NIT01 strain mentioned above has the characteristic of decomposing trichloroethylene (TCE) into ethylene, so trichloroethylene can be added as an electron acceptor. The pH of the culture medium is adjusted to a pH suitable for the growth of the inoculated Dehalococcoides bacteria, but a pH of approximately neutral, typically between 6.5 and 7.5, is usually preferred.

[0026] Since bacteria of the genus Dehalococcoides are obligate anaerobic bacteria, cultivation is carried out anaerobically. Therefore, it is preferable to cultivate them in a mixed gas atmosphere of hydrogen and carbon dioxide. The hydrogen gas in the mixed gas of hydrogen and carbon dioxide is used as an electron donor source for the Dehalococcoides bacteria. If hydrogen gas is not used as an electron donor source and the bacteria are mixed with other microorganisms that generate hydrogen from organic acids, cultivation can be carried out in a mixed gas atmosphere of nitrogen and carbon dioxide. The culture temperature should be set to a temperature suitable for the growth of the inoculated Dehalococcoides bacteria, but it is usually preferable to use a temperature of 25°C to 35°C, and more preferably a room temperature of around 25 to 30°C.

[0027] When inoculating Dehalococcoides bacteria into a culture medium, it is preferable to perform a pre-culture and then add the pre-cultured culture solution to the new culture medium for inoculation. The culture period can be set as appropriate and can be cultured until the desired number of Dehalococcoides bacteria (bacterial density) is reached. For example, a period of 1 week to 2 months is preferable, and 2 weeks to 1.5 months is more preferable.

[0028] Examples of contaminated sites to which Dehalococcoides bacteria cultured by the culture method of the present invention are supplied include soil, groundwater, waste treatment plants, wastewater pits, water treatment plants, and rivers. Furthermore, there are no particular limitations on the method of supplying Dehalococcoides bacteria of the present invention to a contaminated site, but depending on the form of the contaminant, the Dehalococcoides bacteria may be directly added to or mixed with the contaminant, or a purifying agent containing Dehalococcoides bacteria may be sprayed on the contaminant, or the Dehalococcoides bacteria may be immobilized or supported on a support. The amount of Dehalococcoides bacteria supplied can be arbitrarily set depending on the concentration of chlorinated ethylenes at the contaminated site, for example, 10 5 It is preferable to add approximately cells / mL. Furthermore, it is possible to determine the appropriate supply amount of Dehalococcoides bacteria for the contaminated site through preliminary experiments.

[0029] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited in any way by these examples. [Examples]

[0030] [Example 1] 1. Examination of the growth status of Dehalococcoides bacteria in culture medium with added metal fragments. In this example, when the inventor used a stainless steel beer barrel as a culture vessel, inhibition of the growth of bacteria of the genus Dehalococcoides was observed. Therefore, metal pieces of iron plate, stainless steel plate (2 types), and titanium plate were each placed in the culture medium to investigate whether they affected the growth state of bacteria of the genus Dehalococcoides. The growth state of bacteria of the genus Dehalococcoides was confirmed by the progress level of dechlorination of chlorinated ethylenes (CEs) added to the culture medium and the bacterial density.

[0031] The culture of bacteria of the genus Dehalococcoides was performed using the test media shown in Table 1. Specifically, DHB-CO3-Br medium with the compositions shown in Tables 2 to 4 was prepared, and 20 mL was placed in each 50 mL vial. Then, four new metal pieces of the same type, as shown in Table 5, were added to each vial to form the test group, while the control group did not contain any metal pieces. This liquid medium was replaced with N2:CO2=4:1 (v / v) gas to create anaerobic conditions. After that, the DHB-CO3-Br medium in each test group and the control group, including the metal pieces, was autoclaved and cooled. Of the components of the DHB-CO3-Br medium, the vitamin solution and the Ti(III)-nitrilotriacetic acid aqueous solution were added to the DHB-CO3-Br medium after cooling by sterilization by filtration. To the DHB-CO3-Br medium in each cooled vial, acetic acid was added as a carbon source to a final concentration of 5 mM, and then trichloroethylene (TCE) was added as an electron acceptor to a final concentration of 1 mM. To supply H2 as an electron donor, the headspace gas in each vial was replaced with H2:CO2=4:1 (v / v) to prepare test media with the compositions shown in Table 1 below.

[0032] [Table 1]

[0033] [Table 2]

[0034] [Table 3]

[0035] [Table 4]

[0036] [Table 5]

[0037] In each vial of the control and test groups, a pre-cultured Dehalococcoides bacterium was inoculated at a concentration of 5 v / v% of the test medium in the vial, and static culture was performed at room temperature. The Dehalococcoides bacterium used was Dehalococcoides mccartyi strain NIT01 (accession number: NITE P-02893).

[0038] After the start of cultivation, headspace gas from the vial was collected at regular intervals using a gas-tight syringe, and the concentrations of chlorinated ethylenes (CEs), including trichloroethylene (TCE), added to the test medium were measured by gas chromatography (model number: GC-2014, Shimadzu Corporation). Culture medium samples were also taken, and the cell density of Dehalococcoides bacteria in the culture medium was measured. Cell density was measured by direct microscopy using a fluorescence microscope with nucleic acid staining using a fluorescent dye (SYBR® Green I, Thermo Fisher Scientific). The condition of the culture medium and metal pieces in each test group was also visually inspected.

[0039] For the test groups where dechlorination of chlorinated ethylenes was completed (test groups 3 and 4), four metal pieces were removed from the culture medium, and these metal pieces were placed back into a vial containing 20 mL of newly prepared DHB-CO3-Br medium. The test medium shown in Table 1 was then prepared using the same materials and methods as described above, and a second culture test of Dehalococcoides bacteria was performed. The concentration of chlorinated ethylenes (CEs) and the cell density of Dehalococcoides bacteria in the culture medium were measured at regular intervals using the same method as in the first culture test. In addition, the condition of the culture medium and metal pieces in each test group was visually inspected.

[0040] Figures 1 to 3 show the results of the culture tests. Figure 1(A) shows the concentration (μM) of chlorinated ethylenes (CEs) and cell density (cells / mL) in the culture medium of test group 1 (iron), and Figure 1(B) shows the concentration (μM) and cell density (cells / mL) in the culture medium of test group 2 (SUS316). Figure 2(A) shows the data from the first culture test of test group 3 (SUS304), and Figure 2(B) shows the data from the second culture test of the same test group 3. Furthermore, Figure 3(A) shows the data from the first culture test of test group 4 (titanium), and Figure 3(B) shows the data from the second culture test of the same test group 4. In each graph, "NC" indicates the concentration of chlorinated ethylenes and cell density in the culture medium of the control group.

[0041] As shown in Figures 1(A) and (B), in the culture medium to which iron or SUS316 metal pieces were added, the dechlorination of chlorinated ethylenes stopped midway. Also, as shown in Figure 2(A), in the first culture test to which SUS304 metal pieces were added, it took longer to complete the dechlorination of chlorinated ethylenes compared to the control group (NC), but bacterial growth was observed to be similar to that of the control group. When a second culture test was performed using metal pieces removed from this culture medium, as shown in Figure 2(B), the dechlorination of chlorinated ethylenes stopped midway. On the other hand, as shown in Figures 3(A) and (B), in the culture medium to which pure titanium metal pieces were added, no delay or stoppage of dechlorination of chlorinated ethylenes was observed, and dechlorination of chlorinated ethylenes and bacterial growth were observed to be similar to that of the control group (NC) culture medium.

[0042] Furthermore, Figure 4 shows photographs illustrating the state of the metal fragments in the culture medium after the completion of the culture tests in test sections 1 to 4. The photographs for iron (test section 1) and SUS316 (test section 2) are from after the first culture test, while the photographs for SUS304 (test section 3) and titanium (test section 4) are from after the second culture test. As shown in the photographs in Figure 4, a black precipitate was observed in the culture medium to which iron, SUS316, and SUS304 metal fragments were added, but no precipitate was observed in the culture medium to which titanium metal fragments were added. This black precipitate was presumed to be metal sulfides formed by the reaction of metal ions such as iron eluted from the metal fragments with sulfide ions.

[0043] [Example 2] 2. Observation of the surface condition of metal pieces added to the culture medium of bacteria of the genus Dehalococcoides. The surface shape of the metal pieces from each test group added to the culture medium in Example 1 described above was photographed before and after culturing using a scanning electron microscope (JSM-7800F, manufactured by JEOL Ltd.). The metal pieces from Test Group 1 (iron) and Test Group 2 (SUS316) in Example 1 were taken after the first culturing test, while the metal pieces from Test Group 3 (SUS304) and Test Group 4 (titanium) in Example 1 were taken after the second culturing test. The acceleration voltage of the electron gun of the scanning electron microscope was set to 5.0 kV, and the microscope magnification was set to 1000x. The metal pieces before culturing were washed with acetone to remove any oil or other substances adhering to the surface before observation. The metal pieces after culturing were washed with detergent before observation.

[0044] Figure 5 shows the results of scanning electron microscopy observation of each metal piece before and after culturing. The iron metal piece after culturing showed an increase in surface irregularities compared to before culturing, indicating a significant change in surface shape due to culturing. SUS316 had crack-like irregularities even before culturing, and no significant change was observed after culturing. SUS304 only showed linear irregularities before culturing, but after culturing, a crack-like shape change was observed on the surface. No change in surface shape was observed for the titanium metal piece before and after culturing. Of these, the significant changes in surface shape before and after culturing of the iron and SUS304 metal pieces suggest that components constituting the metal pieces leached into the culture medium during culturing. Considering the components constituting the iron plate and the stainless steel shown in Table 6, at least iron ions leached out, and it is also possible that chromium and nickel ions leached out.

[0045] [Table 6]

[0046] [Example 3] 3. Measurement of total iron ion concentration in culture medium with added iron-based metal fragments. In Example 2 described above, changes were observed on the surfaces of the iron and stainless steel metal pieces, suggesting that at least iron ions may have leached from the metal pieces into the culture medium. To confirm this, the total iron ion concentration in the culture medium after the culture tests of the control group and test groups 1-3 in Example 1 was measured.

[0047] As shown in Table 7, the culture solutions for Test Section 1 (Iron) and Test Section 2 (SUS316) in Example 1 are from the first culture test, and the culture solution for Test Section 3 (SUS304) in Example 1 is from the second culture test. In addition, the total iron ion concentration of the DHB-CO3-Br medium shown in Table 2 was also measured as the liquid medium itself in which Dehalococcoides bacteria were not cultured. The total iron ion concentration was measured as follows: To 0.2 to 2.5 mL of sample solution obtained by filtering each culture solution, hydroxylammonium chloride was added to a final concentration of 144 mM, and then 1,10-phenanthroline hydrochloride was added to a final concentration of 0.82 mM. Then, 1 mL of acetate buffer was added and the volume was made up to 5 mL with Milli-Q water. After stirring this mixture, the absorbance was measured at a wavelength of 510 nm using a micro spectrophotometer. The results are shown in Table 7 below.

[0048] [Table 7]

[0049] The culture medium for culturing bacteria of the genus Dehalococcoides generally contains approximately 7-10 μM of iron ions as a trace element (see SL-10 trace element solution shown in Table 4). As shown in Table 7, the total iron ion concentration in the DHB-CO3-Br medium alone was 9.2 μM, while the total iron ion concentration in the culture medium of the control group without added metal fragments was 8.5 μM. This suggests that bacteria of the genus Dehalococcoides consume almost no iron during the dechlorination of chlorinated ethylenes.

[0050] On the other hand, as shown in Table 7, the total iron ion concentration in the culture medium of Test Group 1, to which iron metal pieces were added, was approximately 1 mM; the total iron ion concentration in the culture medium of Test Group 2, to which SUS316 metal pieces were added, was 67 μM; and the total iron ion concentration in the culture medium of Test Group 3, to which SUS304 metal pieces were added, was 276 μM. The results for Test Group 1 (iron) suggest that at least at a total iron ion concentration of 1 mM in the culture medium, the dechlorination of chlorinated ethylenes by Dehalococcoides bacteria is inhibited, affecting their growth. On the other hand, the total iron ion concentrations in the culture medium of Test Group 2 (SUS316) and Test Group 3 (SUS304) were significantly lower (approximately 7% to 27%) than those in the culture medium of Test Group 1 (iron), indicating that even at such low total iron ion concentrations, the dechlorination of chlorinated ethylenes is inhibited. From this, both possibilities were considered: (i) the dechlorination of chlorinated ethylenes may be inhibited by other metal components constituting the stainless steel, or (ii) the dechlorination of chlorinated ethylenes may be inhibited by a total iron ion concentration of 67 μM. In particular, regarding (i), considering the components constituting the stainless steel shown in Table 6, the high content of chromium and nickel suggests that the dechlorination of chlorinated ethylenes may be inhibited by the elution of these metal ions. Based on the measurement results of the total iron ion concentration, assuming that iron, chromium, and nickel eluted from the metal piece in Test Section 2 (SUS316) in the same proportions as the component composition of SUS316 in Table 6, it is possible that 67 μM of iron ions, 19 μM of chromium ions, and 45 μM of nickel ions eluted into the culture solution of Test Section 2. Furthermore, assuming that iron, chromium, and nickel leached from the metal piece in test section 3 (SUS304) in the same proportions as the component composition of SUS304 in Table 6, it is possible that 276 μM of iron ions, 72 μM of chromium ions, and 45 μM of nickel ions leached into the culture medium of test section 3.

[0051] [Example 4] 4. Examination of the growth status of Dehalococcoides bacteria in culture media supplemented with metal ions. To confirm the effects of iron, chromium, and nickel on the growth of Dehalococcoides bacteria, culture tests of Dehalococcoides bacteria were conducted by adding these metal ions to the culture medium. Except that the metal ions shown in Table 8 were added to the DHB-CO3-Br medium at the concentrations shown in the table below to prepare the test media for each test group 4-1 to 4-10, the culture tests of Dehalococcoides bacteria were conducted in the same manner as in Example 1, except that the metal ions shown in Table 8 were added to the DHB-CO3-Br medium at the concentrations shown in the table. As in Example 1, the Dehalococcoides mccartyi NIT01 strain was used as the Dehalococcoides bacteria. Static incubation was performed at room temperature, and the headspace gas from the vial was collected at regular intervals using a gas-tight syringe. The concentrations of chlorinated ethylenes (VC: vinyl chloride, cis-DCE: cis-1,2-dichloroethylene, TCE: trichloroethylene) and ethylene (ETH) were measured by gas chromatography (model number: GC-2014, Shimadzu Corporation).

[0052] [Table 8]

[0053] Figures 6-8 show the results of the culture test. Figure 6(A) is the control group, and Figure 6(B) is the test group 4-1(Fe 2+ (68 μM), Figure 6(C) shows test section 4-2 (Fe 2+ , 273M), Figure 6(D) shows test section 4-3 (Fe 2+ , 1 mM) and Figure 6(E) are from test section 4-4 (Fe 2+ Figure 7(A) shows the concentration (μM) of chlorinated ethylenes in the culture medium (5 mM). Also, Figure 7(A) shows the concentration (Cr) in test section 4-5. 3+ , 23 μM), Test section 4-6 (Cr 3+ , 72 μM) and test section 4-7 (Cr 3+ Figure 8(A) shows the concentration (μM) of chlorinated ethylenes in the culture medium (500 μM). Also, Figure 8(A) shows the concentration (μM) of test section 4-8 (Ni 2+ , 1 mM), Test section 4-9 (Ni 2+ , 2 mM) and test section 4-10 (Ni 2+The graphs show the concentrations (μM) of chlorinated ethylenes in a culture medium of 5 mM. In each graph, the square markers represent trichloroethylene (TCE), the diamond markers represent cis-1,2-dichloroethylene (cis-DCE), the triangular markers represent vinyl chloride (VC), and the circular markers represent ethylene (ETH).

[0054] According to Figures 6-8, Fe 2+ , Cr 3+ Ni 2+ When added to the culture medium, it became clear that the inhibition of dechlorination of chlorinated ethylenes increased as the concentration of the added substance increased. Among these, iron ions (Fe 2+ Regarding the above, no inhibition of dechlorination of chlorinated ethylenes was observed at addition concentrations of 68 μM and 273 μM (Figures 6(B) and (C)), but an inhibitory trend was observed at an addition concentration of 1 mM (Figure 6(D)), and dechlorination of chlorinated ethylenes was completely inhibited at an addition concentration of 5 mM (Figure 6(E)).

[0055] Also, chromium ions (Cr 3+ Regarding ), a weak dechlorination of chlorinated ethylenes was observed at an addition concentration of 23 μM (Figure 7(A)), but at addition concentrations of 72 μM and 500 μM, only a slight increase in cis-DCE was observed, confirming inhibition of dechlorination (Figures 7(B) and (C)). In addition, nickel ions (Ni 2+ Regarding the additive concentration of 1 mM, dechlorination was completed (Figure 8(A)), but at additive concentrations of 2 mM and 5 mM, dechlorination did not proceed at all (Figures 8(B) and (C)).

[0056] The results of this example suggest that the main components and their concentrations that inhibit the growth of Dehalococcoides bacteria, i.e., inhibit the dechlorination of chlorinated ethylenes, are iron ions at a concentration of more than 1 mM when iron metal pieces are added to the culture medium, and chromium ions at a concentration of more than 23 μM when stainless steel metal pieces are added to the culture medium.

[0057] [Example 5] 5. Mass cultivation of Dehalococcoides bacteria using titanium pressure-resistant culture vessels. Instead of using stainless steel beer kegs as pressure-resistant culture vessels, a keg-like pressure-resistant culture vessel was fabricated from pure titanium (Titanium Grade 2 according to JIS standards) (Figure 9). The capacity was set to 18 L. As shown in Non-Patent Literature 1, a sampling port was provided on the top lid. 10 L of the DHB-CO3-Br medium shown in Table 2 was placed in this titanium pressure-resistant culture vessel, and anaerobic conditions were established by replacing the gas with a N2:CO2=4:1 (v / v) mixed gas. The entire pressure-resistant culture vessel was then autoclaved. After autoclaving, the pressure-resistant culture vessel was cooled by immersing it in ice water, and the headspace gas was replaced with H2:CO2=4:1 (v / v). The vitamin solution and Ti(III)-nitrilotriacetic acid aqueous solution, which are components of the DHB-CO3-Br medium, were added to the cooled medium by filtration sterilization. Acetic acid was added to DHB-CO3-Br medium to a final concentration of 5 mM as a carbon source, and trichloroethylene (TCE) was added to a final concentration of 1 mM as an electron acceptor. A 10 L test medium with the same composition as in Example 1, as shown in Table 1, was prepared. Dehalococcoides mccartyi NIT01 strain, which had been cultured in advance, was inoculated into this 10 L test medium at a concentration of 16 v / v%, and static culture was performed at room temperature. After the start of culture, the concentration of chlorinated ethylenes (CEs) and the cell density of Dehalococcoides bacteria in the culture medium were measured at regular intervals using the same method as in Example 1.

[0058] The first culture test was considered complete when the dechlorination of chlorinated ethylenes in the titanium pressure-resistant culture vessel was finished. The same large-scale culture test was then repeated six times using the same pressure-resistant culture vessel. In the sixth culture test, the test was not considered complete when the dechlorination of chlorinated ethylenes in the pressure-resistant culture vessel was finished. Instead, trichloroethylene (TCE) was added to a final concentration of 1 mM, and the culture was continued. Subsequently, the concentration of chlorinated ethylenes in the pressure-resistant culture vessel and the cell density of Dehalococcoides bacteria in the culture medium were measured.

[0059] Figure 10 shows the results of the culture test. Figure 10(A) shows the concentration (μM) and cell density (cells / mL) of chlorinated ethylenes in the culture medium for the first test, Figure 10(B) for the second test, Figure 10(C) for the fourth test, Figure 10(D) for the fifth test, and Figure 10(E) for the sixth test. In each graph, the square markers represent the concentration of trichloroethylene (TCE), the diamond markers represent the concentration of cis-1,2-dichloroethylene (cis-DCE), the triangular markers represent the concentration of vinyl chloride (VC), and the circular markers represent the concentration of ethylene (ETH), while the cross-shaped markers represent the cell density.

[0060] As shown in Figure 10, it was found that stable large-scale culture of Dehalococcoides bacteria can be achieved by culturing them in titanium pressure-resistant culture vessels. Dechlorination of 1 mM trichloroethylene by Dehalococcoides mccartyi NIT01 strain was completed in only about 12 to 28 days, and healthy growth was observed. Furthermore, the cell density reached 2.8 to 7.7 × 10⁶ upon completion of dechlorination. 7 It increased to [a certain level]. Furthermore, as shown in Figure 10(E), in the sixth culture test, when 1 mM trichloroethylene was added to the culture medium in which the dechlorination of 1 mM trichloroethylene had been completed, the dechlorination of the added trichloroethylene was completed in just 8 days, and the cell density increased to 3.5 × 10⁶. 7 From 4.0 x 10 7 It increased to [a certain value].

[0061] Thus, it was found that using titanium pressure-resistant culture vessels for culturing Dehalococcoides bacteria does not inhibit the dechlorination of chlorinated ethylenes, unlike conventional stainless steel or iron pressure-resistant culture vessels, and allows for stable large-scale cultivation even when the same vessel is used repeatedly. [Industrial applicability]

[0062] The method for culturing bacteria of the genus Dehalococcoides according to the present invention is useful for bioremediation of contaminated sites contaminated with chlorinated ethylenes and the like, and can be used for the purification of groundwater and soil. [Accession Number]

[0063] Accession number: NITE P-02893, Dehalococcoides mccartyi strain NIT01, Date of admission: February 25, 2019, Depositing institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan)

Claims

1. A method for culturing bacteria of the genus Dehalococcoides using a metal pressure-resistant culture vessel, The aforementioned metal pressure-resistant culture vessel is a titanium pressure-resistant culture vessel. Dehalococcoides bacteria in the culture medium (a) Iron-containing ions are 300 μM or less, (b) Chromium-containing ions are 10 μM or less, (c) A method for culturing bacteria of the genus Dehalococcoides, characterized by culturing in such a way that nickel-containing ions are maintained in a concentration range of 1 mM or less.

2. The method for culturing a bacterium of the genus Dehalococcoides according to claim 1, characterized in that the Dehalococcoides bacterium is strain Dehalococcoides mccartyi NIT01, identified by accession number: NITE BP-02893.

3. The aforementioned iron-containing ions are Fe ２＋ And, The chromium-containing ion is Cr ３＋ And, The nickel-containing ions are Ni ２＋ A method for culturing bacteria of the genus Dehalococcoides according to claim 1 or 2, characterized in that...

4. A method for purifying chlorinated ethylenes at a contaminated site by transporting Dehalococcoides bacteria cultured by the culture method described in claim 1 or 2, along with the titanium pressure-resistant culture container, to a contaminated site contaminated with chlorinated ethylenes, and supplying the Dehalococcoides bacteria removed from the titanium pressure-resistant culture container to the contaminated site.

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