Dehalococcoides bacteria and process for decomposing volatile organic compound

Abstract

To provide novel Dehalococcoides bacteria that exhibit a superior capability for decomposing chloropropene compounds, and processes for decomposing volatile organic compounds.SOLUTION: The present invention provides the Dehalococcoides mccartyi NIT-OBY strain deposited under an accession number NITE AP-03891, or a mutant or a related strain of the Dehalococcoides mccartyi NIT-OBY strain capable of dechlorinating chloropropenes.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a novel Dehalococcoides bacterium and a method for decomposing volatile organic compounds using the bacterium. [Background technology]

[0002] Generally, a method is known for purifying contaminated soil, groundwater, and other materials contaminated with volatile organic compounds (VOCs) such as trichloroethylene by using VOC-degrading bacteria to decompose and render them harmless. One purification method known is biostimulation, which provides nutrients to VOC-degrading bacteria living in the ground to activate them and decompose and render VOCs harmless. Another method known is bioaugmentation, which involves isolating VOC-degrading bacteria from soil or groundwater where they exist, culturing them in large quantities outside, and injecting them into the ground.

[0003] Conventionally, known VOC decomposition bacteria include the Dehalococcoides bacteria described in Patent Document 1. The Dehalococcoides bacteria described in Patent Document 1 decomposes trichloroethylene into ethylene and also decomposes 1,1,2-trichloroethane into ethylene. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-31 Summary of the Invention [Problem to be solved by the invention]

[0005] However, no Dehalococcoides bacteria capable of decomposing chloropropenes, a type of volatile organic compounds, have been known so far. The present invention provides a novel Dehalococcoides bacterium that exhibits excellent decomposition activity for chloropropenes, and a method for decomposing volatile organic compounds. [Means for solving the problem]

[0006] Means for Solving the Problems The present inventors have conducted extensive research to achieve the above object and have discovered a novel bacterium belonging to the genus Dehalococcoides that exhibits an excellent ability to decompose chloropropenes. Various aspects for solving the above problems will be described below.

[0007] The Dehalococcoides bacterium of embodiment 1 is the Dehalococcoides mccartyi NIT-OBY strain deposited under accession number NITE AP-03891, or a mutant or closely related strain of the Dehalococcoides mccartyi NIT-OBY strain having the ability to dechlorinate chloropropenes.

[0008] In a second aspect, the Dehalococcoides bacterium according to the first aspect is used for decomposing volatile organic compounds. A third aspect is the Dehalococcoides bacterium according to the second aspect, wherein the volatile organic compound is at least one selected from the group consisting of chloroethylenes, chloroethanes, and chloropropenes.

[0009] The method for decomposing volatile organic compounds according to the fourth embodiment uses the Dehalococcoides bacterium according to the first embodiment. A fifth aspect is the method for decomposing a volatile organic compound according to the fourth aspect, wherein the volatile organic compound is at least one selected from the group consisting of chloroethylenes, chloroethanes, and chloropropenes. Effect of the Invention

[0010] According to the present invention, an excellent decomposition effect of chloropropenes can be exhibited. [Brief description of the drawings]

[0011] [Figure 1]Figure 1 shows a phylogenetic tree of the Dehalococcoides mcCartii NIT-OBY strain and other Dehalococcoides bacteria, constructed based on the nucleotide sequence of the 16S rRNA gene region. [Diagram 2] Figure 2 shows a scanning electron microscope (SEM) photograph of Dehalococcoides mccartii NIT-OBY strain at a magnification of 20,000 times. [Diagram 3] FIG. 3 shows the RFLP migration pattern of the Dehalococcoides bacteria isolated in Test Example 1. [Figure 4] FIG. 4 shows a graph illustrating the dechlorination behavior of trichloroethylene (TCE) by Dehalococcoides mccarti NIT-OBY strain. [Diagram 5] Figure 5 shows a graph illustrating the dechlorination behavior of cis-1,3-dichloropropene (cis-1,3-D) by Dehalococcoides mccarti NIT-OBY strain. [Figure 6] Figure 6 shows a graph illustrating the dechlorination behavior of 1,1,2-trichloroethane (1,1,2-TCA) by Dehalococcoides mccarti NIT-OBY strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] First Embodiment Hereinafter, a first embodiment of the Dehalococcoides bacterium of the present invention will be described. (Dehalococcoides spp.) The Dehalococcoides bacterium of the present embodiment is a novel microorganism belonging to the genus Dehalococcoides that can decompose chloropropenes into harmless propylene. More specifically, it is a microorganism that has the ability to dechlorinate 1,3-dichloropropene, which is a chloropropene, into propylene.

[0013] The Dehalococcoides bacteria of the present invention were isolated from a mixture of soil and groundwater collected from a site of soil and groundwater contamination by volatile organic compounds. The isolated Dehalococcoides bacteria was named Dehalococcoides mccartyi NIT-OBY strain (hereinafter referred to as NIT-OBY strain). The NIT-OBY strain was received by the National Institute of Technology and Evaluation (NPMD), an international depository institution (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on May 23, 2023. Its receipt number is NITE AP-03891.

[0014] Genomic DNA was extracted from the isolated NIT-OBY strain cells. Using the obtained genomic DNA, the base sequence of the DNA encoding the 16S rRNA of the NIT-OBY strain and the entire genome sequence were determined by single molecule real-time sequencing. SEQ ID NO: 1 shows the base sequence of the 16S rRNA gene region of the NIT-OBY strain. SEQ ID NO: 2 shows the entire genome sequence of the NIT-OBY strain.

[0015] Identification was performed using the NCBI program BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ), and the nucleotide sequence of the 16S rRNA gene region was 100% identical to known Dehalococcoides genus bacteria such as Dehalococcoides mccartyi strain CBDB1 (Saiyari et al. (2018) Sustainable Environment Research, 28(4), 149-157), as shown in the phylogenetic tree in Figure 1. The NIT-OBY strain belongs to the Pinellas subgroup (Hendrickson et al. (2002) Applied and Environmental Microbiology, 68(2), 485-495) (Loffler et al. (2013) International Journal of Systematic and Evolutionary Microbiology, 63(2), 625-635).

[0016] When comparing the whole genome, the highest homology was 98.82% with Dehalococcoides mccartyi strain 195. Therefore, it was estimated that the NIT-OBY strain belongs to Dehalococcoides mccartyi. In addition, it possessed 27 dechlorinase genes. Other characteristics are shown below.

[0017] (a) Morphological properties The NIT-OBY strain is non-motile and non-sporulating. As shown in Figure 2, the NIT-OBY strain has a disk-shaped morphology and a diameter of about 0.5 μm. In Figure 2, the disk-shaped cells represent the NIT-OBY strain. The black circles represent holes in the base in the background, and the fine particles represent the residue of ruptured bacteria.

[0018] (b) Characteristics on culture medium Cultivation of the NIT-OBY strain is strictly anaerobic. Hydrogen is used as the electron donor. For example, TCE (trichloroethylene) and cis-1,2-DCE (cis-1,2-dichloroethylene) are used as electron acceptors, and acetate is used as the carbon source. Other ingredients include inorganic salts, trace elements, vitamins, etc. The pH of the medium is neutral. Gas replacement is performed on the medium and the gas phase in the incubator with a mixed gas of H2:CO2 = 4:1.

[0019] (c) Physiological characteristics The NIT-OBY strain is an obligate anaerobe. It obtains energy through dehalogenation respiration. It uses hydrogen as an electron donor. It uses VOCs (volatile organic compounds) as electron acceptors. Specifically, it has the ability to dechlorinate chloroethylenes such as TCE, cis-1,2-DCE, and VC (chloroethylene), chloropropenes such as cis-1,3-D (cis-1,3-dichloropropene) and trans-1,3-D (trans-1,3-dichloropropene), and chloroethanes such as 1,1,2-TCA (1,1,2-trichloroethane). For example, it is capable of dechlorinating 500 μM of TCE to ethylene in 21 days. For example, it is capable of dechlorinating 550 μM of cis-1,3-D to propylene in 19 days. It utilizes acetate as a carbon source. The optimal growth temperature is 28°C. In particular, a bacterium of the genus Dehalococcoides having the ability to dechlorinate dichloropropene to propene has not been isolated so far and was discovered for the first time by the present inventors.

[0020] (closely related strains or mutants) The Dehalococcoides bacteria of the present invention include not only the NIT-OBY strain, but also mutants or closely related strains of the Dehalococcoides mccarti NIT-OBY strain that have the ability to dechlorinate chloropropenes. Microorganisms having a genome that has 99% or more homology, preferably 99.5% or more homology, and more preferably 99.9% or more homology to the nucleotide sequence set forth in SEQ ID NO: 2, which is the entire genome sequence of the NIT-OBY strain described below, are included in strains closely related to the NIT-OBY strain.

[0021] Mutants can be obtained by mutagenesis using ethyl methanesulfonate, a mutagen that has been widely used in the past, treatment with other chemicals such as nitrosoguanidine and methyl methanesulfonate, or exposure to ultraviolet light. Mutants can also be obtained without treatment with a mutagen, that is, by natural mutation.

[0022] (Culture method) It is an absolute requirement to culture the NIT-OBY strain in a medium with a redox potential as low as possible. For example, the redox potential can be confirmed by using the fact that resazurin, an redox indicator added to the medium, becomes colorless as an indicator. It is also necessary to prevent air from being mixed in as much as possible from the preparation of the medium to the completion of the culture. For example, it is preferable to carry out the treatment under a nitrogen gas atmosphere or to use a reducing agent to completely remove air bubbles. Specific examples of reducing agents include reduced glutathione, iron sulfide, sodium sulfide, L-cysteine, D-dithiothreitol, titanium (III) complexed with citrate or nitrilotriacetate, etc.

[0023] Other than the above conditions, the bacteria of the genus Dehalococcoides can be cultured according to a conventional method for culturing the bacteria of the genus Dehalococcoides. The medium used for the culture is not particularly limited as long as it contains an electron acceptor, a carbon source, a nitrogen source, inorganic salts, trace elements, various vitamins, a pH adjuster, etc., which are necessary for the growth of the microorganism, and either a solid medium or a liquid medium can be used.

[0024] A specific example of the carbon source is acetic acid. A specific example of the nitrogen source is peptone, casitone, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, various amino acids, etc. A specific example of the inorganic salt is phosphate, magnesium salt, calcium salt, sodium salt, potassium salt, etc. A specific example of the trace element is iron, cobalt, copper, zinc, boron, nickel, molybdenum, manganese, selenium, tungsten, etc. The above-mentioned chloroethylenes, chloroethanes, chloropropenes, etc. may be added to the medium as an electron acceptor.

[0025] The culture can be performed under various culture conditions such as static culture, shaking culture, etc. Since Dehalococcoides bacteria can only utilize hydrogen as an electron donor, a mixed gas of hydrogen and carbon dioxide is usually used for the culture.

[0026] The culture temperature is preferably 15 to 40° C., more preferably 20 to 35° C., and even more preferably 25 to 30° C. The pH of the medium is preferably weakly acidic to weakly alkaline, near neutral, more preferably pH 6 to 8, and even more preferably pH 6.5 to 7.5.

[0027] The effects of the first embodiment will be described. (1-1) The NIT-OBY strain of the first embodiment is a novel Dehalococcoides bacterium that exhibits an excellent decomposition effect of chloropropenes. Therefore, it can detoxify not only volatile organic compounds such as chloroethylenes and chloroethanes, but also pollutants contaminated with chloropropenes.

[0028] <Second embodiment> A second embodiment of the method for decomposing volatile organic compounds (hereinafter referred to as the "decomposition method") of the present invention will be described below.

[0029] The decomposition method of this embodiment uses the NIT-OBY strain described in the first embodiment. The NIT-OBY strain of the present invention can be used to purify contaminated materials contaminated with volatile organic compounds (VOCs) such as chloroethylenes, chloroethanes, and chloropropenes. The contaminated materials are not particularly limited, and examples thereof include river water, groundwater, industrial wastewater, industrial wastewater, domestic wastewater, industrial waste, contaminated soil, sludge, etc. contaminated with volatile organic compounds (VOCs) such as chloroethylenes, chloroethanes, and chloropropenes.

[0030] Specific examples of chloroethylenes to be purified include tetrachloroethylene, trichloroethylene, dichloroethylene, vinyl chloride monomer, etc. Specific examples of chloroethanes include trichloroethane, dichloroethane, chloroethane, etc. Specific examples of chloropropenes include hexachloropropene, pentachloropropene, tetrachloropropene, trichloropropene, dichloropropene, chloropropene, etc. Contaminants may contain one type of these volatile organic compounds, or two or more types of compounds.

[0031] The decomposition treatment method involves preparing the NIT-OBY strain as a decomposition treatment agent for volatile organic compounds (hereinafter simply referred to as "decomposition treatment agent") and administering the decomposition treatment agent to the contaminated material. The decomposition treatment agent may be the NIT-OBY strain itself, or a culture obtained by the culture method described in the first embodiment section may be used. The culture may be a culture of the NIT-OBY strain of the present invention as is, or the culture may be subjected to concentration, extraction, or purification treatment such as filtration or centrifugation.

[0032] The decomposition processing agent of the present invention may also be blended with additives such as a carrier (filler), a surfactant, and an auxiliary. The dosage form of the decomposition processing agent is not particularly limited, and examples thereof include liquids, powders, granules, and tablets. The method of administering the decomposition processing agent is not particularly limited, and methods such as addition, spraying, mixing, and injection can be used depending on the form of the contaminated material. The dosage of the decomposition processing agent to the contaminated material is appropriately set depending on conditions such as the content of volatile organic compounds, outside temperature, components contained in the contaminated material, and pH.

[0033] The effects of the second embodiment will be described. (2-1) In the decomposition method of the second embodiment, the NIT-OBY strain is used, which is capable of exerting an excellent decomposition effect on volatile organic compounds including chloropropenes.

[0034] (2-2) In particular, when the NIT-OBY strain is artificially cultured and increased, and then introduced into the target environment for purification (bioaugmentation) purposes, it can efficiently purify pollutants containing volatile organic compounds, including chloropropenes.

[0035] The above embodiment may be modified as follows: The above embodiment and the following modifications may be combined with each other to the extent that no technical contradiction occurs. The NIT-OBY strain of the above embodiment can detoxify chloropropenes. However, this does not prevent the application to contaminants that do not contain chloropropenes but contain chloroethylenes or chloroethanes. EXAMPLES

[0036] The present invention will be described in more detail below with reference to examples. Note that the present invention is not limited to the following examples, and various modifications can be made without departing from the spirit of the present invention.

[0037] <Test Example 1: Isolation of a novel Dehalococcoides bacterium> (1) Enrichment culture 20 L of a mixture of soil and groundwater collected from a site of soil and groundwater contamination by volatile organic compounds (VOCs) was placed in a 20 L plastic container. The enrichment culture solution was prepared by adding 100 mL of saturated TCE aqueous solution, 55.8 g of sodium gluconate, 3.6 g of urea, 0.6 g of sodium dihydrogen phosphate, 20 g of sodium bicarbonate, and 200 mL of a trace element enrichment solution with the composition shown in Table 1 below. The enrichment culture solution was cultured stationary in a room maintained at 22-23°C. Half the culture solution (10 L) was transferred to 10 L of tap water every 1-4 months for about 10 years.

[0038] [Table 1] (2) Limiting dilution culture A 50 mL vial containing 20 mL of DHB-CO3 medium shown in Table 3 was filled with H2:CO2 = 4:1 gas and sealed with a fluorine resin-coated butyl rubber stopper and an aluminum seal, and the components shown in Table 2 below were added to prepare a medium for limiting dilution culture. 100 μL of enrichment culture solution was added to the medium for limiting dilution culture, and 10 1 10x diluted sample 1 100 μL of diluted sample was added to 10 2 The samples were diluted 10-fold and then 5The samples were diluted 1:1 and cultured at 28°C. After the culture, all TCE was dechlorinated to ethylene, and the culture medium was repeatedly subcultured in the same manner. The compositions of the DHB-CO3 medium, the SL-10 trace element solution, the selenium-tungsten solution, and the 1000-fold concentrated vitamin mixture are shown in Tables 3 to 6, respectively.

[0039] [Table 2]

[0040] [Table 3]

[0041] [Table 4]

[0042] [Table 5]

[0043] [Table 6] (3) Confirmation of isolation achievement The culture medium after repeated limiting dilution culture was observed under a fluorescent microscope, and when only a single-shaped bacterium was observed, the microbial DNA was extracted and RFLP analysis was performed using the restriction enzymes HaeIII and HhaI. The results are shown in Figure 3. The RFLP migration patterns of both HaeIII and HhaI were specific to Dehalococcoides bacteria (Yoshida et al., (2018), 24th Research Meeting on Groundwater and Soil Contamination and Its Prevention Measures, pp. 588-589), and no contamination bands were observed, confirming the achievement of isolation. In addition, the results of observation by SEM are shown in Figure 2. As shown in Figure 2, only disc-shaped cells thought to be Dehalococcoides bacteria were observed, confirming the achievement of isolation.

[0044] (4) NIT-OBY strain Genomic DNA was extracted from the isolated Dehalococcoides bacteria. Using the obtained genomic DNA, the base sequence of the DNA encoding the 16S rRNA of the NIT-OBY strain and the entire genome sequence were determined by single molecule real-time sequencing. SEQ ID NO: 1 shows the base sequence of the 16S rRNA gene region of the NIT-OBY strain. SEQ ID NO: 2 shows the entire genome sequence of the NIT-OBY strain.

[0045] Identification was performed using the NCBI program BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ). As shown in the phylogenetic tree in Figure 1, the base sequence of the 16S rRNA gene region was 100% identical to known Dehalococcoides bacteria, such as Dehalococcoides mccartyi strain CBDB1. When compared with the whole genome, the highest homology was Dehalococcoides mccartyi strain 195, which had a homology of 98.82%. Therefore, it was presumed that the isolated Dehalococcoides bacteria belong to Dehalococcoides mccartyi. The isolated Dehalococcoides bacteria was named Dehalococcoides mccartyi strain NIT-OBY.

[0046] <Test Example 2: Evaluation of decomposition treatment of volatile organic compounds using novel Dehalococcoides bacteria> (1) TCE dechlorination experiment A 50 mL vial containing 20 mL of DHB-CO3 medium was purged with H2:CO2 = 4:1 gas, sealed with a fluorine resin-coated butyl rubber stopper and aluminum seal, and each component shown in Table 7 below was added and cultured statically at 28°C. The VOC concentration in the gas phase of the vial was measured at regular intervals using a gas chromatograph (Shimadzu GC-2014).

[0047] [Table 7] FIG. 4 shows a graph illustrating the TCE dechlorination behavior by the NIT-OBY strain.

[0048] As shown in Figure 4, it was confirmed that 500 μM TCE was completely dechlorinated to ethylene via cis-1,2-DCE and VC in 21 days. (2) Dechlorination experiment of 1,3-D and 1,1,2-TCA The pure culture of the NIT-OBY strain was dispensed from the vial into a centrifuge tube, and the lid was closed while aerating and replacing the culture and gas phase with nitrogen gas. This was centrifuged at 4°C and 10,000 rpm for 10 minutes using a large centrifuge (Kubota Shoji Co., Ltd., high-speed large-capacity refrigerated centrifuge 7000). After centrifugation, the supernatant was discarded while aerating with nitrogen gas, and the cells were resuspended in the reaction solution shown in Table 8. While continuing the aeration with nitrogen gas, the cell density in the resuspended solution was measured by direct microscopy, and the reaction solution was added so that the cell density was about 10 times that of the original pure culture solution. This was dispensed into vials in 10 mL portions, sealed with fluorine resin-coated butyl rubber stoppers and aluminum seals, and 0.5 μL of 1,3-D (a mixture of cis and trans isomers) or 1,1,2-TCA was added. The VOC concentration in the gas phase of the vials was measured by gas chromatography at regular intervals.

[0049] [Table 8] Graphs showing the dechlorination behavior of cis-1,3-D and 1,1,2-TCA by the NIT-OBY strain are shown in Figures 5 and 6, respectively.

[0050] As shown in Figure 5, cis-1,3-D was completely dechlorinated to propylene in 19 days. It has also been confirmed that trans-1,3-D can also be dechlorinated to propylene (data not included).

[0051] As shown in FIG. 6, it was confirmed that 1,1,2-TCA could also be dechlorinated to ethylene.

Claims

1. A Dehalococcoides bacterium which is a Dehalococcoides mccartyi NIT-OBY strain deposited under accession number NITE AP-03891, or a mutant or closely related strain of the Dehalococcoides mccartyi NIT-OBY strain having the ability to dechlorinate chloropropenes.

2. The Dehalococcoides bacterium according to claim 1, which is used for decomposing volatile organic compounds.

3. The Dehalococcoides bacterium according to claim 2 , wherein the volatile organic compound is at least one selected from the group consisting of chloroethylenes, chloroethanes, and chloropropenes.

4. A method for decomposing volatile organic compounds using the Dehalococcoides bacterium according to claim 1.

5. 5. The method for decomposing a volatile organic compound according to claim 4, wherein the volatile organic compound is at least one selected from the group consisting of chloroethylenes, chloroethanes, and chloropropenes.

Images