Method for culturing dehalococcoides bacteria, agent for decomposing volatile organic compounds, and process for decomposing volatile organic compounds

Abstract

To provide a method for culturing Dehalococcoides bacteria, an agent for decomposing volatile organic compounds, and a process for decomposing volatile organic compounds, while ensuring superior stability.SOLUTION: The present invention provides a method for culturing Dehalococcoides bacteria using Dehalococcoides bacteria, obligate anaerobic bacteria, and a carbon source capable of generating hydrogen through the obligate anaerobic bacteria.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for culturing Dehalococcoides bacteria, a decomposition treatment agent for volatile organic compounds, and a decomposition treatment method for volatile organic compounds. [Background technology]

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

[0003] Conventionally, bacteria of the genus Dehalococcoides have been known as VOC decomposition bacteria, as described in Patent Document 1. Dehalococcoides bacteria require hydrogen as an electron source for the reductive dehalogenation reaction, and decompose organic halogen compounds in the presence of hydrogen. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2008-131879 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, in conventional culture methods, Dehalococcoides bacteria were sometimes inactivated during the culture. It is believed that culturing Dehalococcoides bacteria stably with high reproducibility leads to excellent decomposition of volatile organic compounds. There is a demand for a culture method of Dehalococcoides bacteria with excellent stability, a decomposition treatment agent for volatile organic compounds, and a decomposition treatment method for volatile organic compounds. [Means for solving the problem]

[0006] As a result of intensive research to achieve the above-mentioned object, the inventors discovered that Dehalococcoides bacteria can be stably cultured by using obligate anaerobic bacteria and a carbon source that can produce hydrogen by the obligate anaerobic bacteria.

[0007] Various aspects for solving the above problems will be described below. The method for culturing Dehalococcoides bacteria of aspect 1 is characterized by using Dehalococcoides bacteria, obligate anaerobic bacteria, and a carbon source that can produce hydrogen by the obligate anaerobic bacteria.

[0008] A second aspect is the method for culturing Dehalococcoides bacteria according to the first aspect, wherein the obligately anaerobic bacteria is at least one selected from homoacetogens and sulfate-reducing bacteria. A third aspect is the method for culturing a Dehalococcoides bacterium according to the first or second aspect, wherein the carbon source is an organic acid.

[0009] The volatile organic compound decomposition treatment agent of aspect 4 is characterized by comprising a Dehalococcoides bacterium, an obligate anaerobic bacterium, and a carbon source capable of producing hydrogen by the obligate anaerobic bacterium, or a microorganism capable of producing the carbon source.

[0010] The method for decomposing a volatile organic compound of the fifth aspect is characterized by including a step of adding the decomposing agent for a volatile organic compound described in the fourth aspect to a pollutant containing a volatile organic compound. Effect of the Invention

[0011] According to the culture method of the present invention, bacteria of the genus Dehalococcoides can be stably cultured. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the RFLP migration pattern of the Dehalococcoides bacteria isolated in Test Example 1. [Diagram 2] FIG. 2 shows a scanning electron microscope (SEM) photograph at a magnification of 20,000 times of the Dehalococcoides bacteria isolated in Test Example 1. [Diagram 3] FIG. 3 is a graph showing the cell count of Dehalococcoides bacteria and each VOC concentration in the medium of Comparative Example 1. [Figure 4] FIG. 4 is a graph showing the cell count of Dehalococcoides bacteria and the concentration of each VOC in the medium of Example 1. [Diagram 5] FIG. 5 is a graph showing the cell count of Dehalococcoides bacteria and the concentration of each VOC in the medium of Example 2. [Figure 6] FIG. 6 is a graph showing the cell count of Dehalococcoides bacteria and the concentration of each VOC in the medium of Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] First Embodiment Hereinafter, a first embodiment of a method for culturing Dehalococcoides bacteria of the present invention (hereinafter referred to as the "cultivation method") will be described.

[0014] The culture method of the present embodiment uses Dehalococcoides bacteria, obligately anaerobic bacteria, and a carbon source that can produce hydrogen by the obligately anaerobic bacteria. (Dehalococcoides spp.) Specific examples of Dehalococcoides bacteria include Dehalococcoides mccartyi, Dehalococcoides sp., etc. Specific examples of Dehalococcoides mccartyi include Dehalococcoides mccartyi strain CBDB1, etc. These Dehalococcoides bacteria may be used alone or in combination of two or more.

[0015] Since Dehalococcoides bacteria are widely present in nature, they may be obtained from an inoculum source such as groundwater or soil by a known isolation method. The inoculum source is preferably collected from a contaminated site where purification of contamination by volatile organic compounds is progressing and Dehalococcoides bacteria are significantly proliferating during the purification process. Dehalococcoides bacteria may be detected by a PCR method using known primers for detecting Dehalococcoides bacteria.

[0016] (Obligate anaerobic bacteria) Obligate anaerobic bacteria are bacteria that do not require oxygen for growth and die when exposed to atmospheric levels of oxygen. Obligate anaerobic bacteria metabolize a carbon source added to the medium, as described below, to produce hydrogen as an electron source required for the reductive dehalogenation reaction of Dehalococcoides bacteria.

[0017] Specific examples of obligately anaerobic bacteria include homoacetogens (such as Acetobacterium bacteria), sulfate-reducing bacteria (such as Desulfovibrio bacteria, Desulfomaculum bacteria, Desulfobarbus bacteria, Desulfobacter bacteria, Desulfobacterium bacteria, Desulfococcus bacteria, Desulfosarcina bacteria, and Desulfomonas bacteria), Clostridium bacteria, Bifidobacterium bacteria, Megasphaera bacteria, Geobacter bacteria, Acetogenium bacteria, Butyrivacterium bacteria, Sporomusa bacteria, Eubacterium bacteria, Peptostreptococcus bacteria, Prevotella bacteria, and Collinsella bacteria.

[0018] These obligate anaerobic bacteria may be used alone or in combination of two or more kinds. (Carbon source) In the culture method of this embodiment, a carbon source capable of producing hydrogen through the metabolism of obligate anaerobic bacteria is used. In the steps of the culture method of this embodiment, a carbon source may be used, and a microorganism capable of producing the carbon source through metabolism may be added to the medium without directly adding the carbon source. Examples of carbon sources include sugars, organic acids, alcohols, proteins, and materials containing these. Examples of microorganisms capable of producing the carbon source include known microorganisms capable of producing these components through metabolism.

[0019] Specific examples of sugars include monosaccharides such as glucose, fructose, and galactose, disaccharides such as sucrose, lactose, and maltose, and polysaccharides such as starch, cellulose, and dextrin. Specific examples of organic acids include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, lactic acid, malic acid, citric acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, fumaric acid, maleic acid, pyruvic acid, amino acids, gluconic acid, tartaric acid, and salts thereof. Specific examples of alcohols include methanol, ethanol, propanol, and glycerin. These carbon sources may be used alone or in combination of two or more. These are appropriately selected depending on the type of obligate anaerobic bacteria. Among these, organic acids are preferred in terms of the ease of metabolism of obligate anaerobic bacteria, and organic acids having 6 or less carbon atoms are more preferably applied.

[0020] (Microbial carrier) In culturing Dehalococcoides bacteria, a microbial carrier may be used in combination from the viewpoint of promoting metabolism. Specific examples of the microbial carrier include resin polymer carriers, activated carbon, charcoal, etc. Specific examples of the resin polymer carrier include polylactic acid, styrene-divinylbenzene copolymer, DEAE (diethylaminoethylcellulose), etc. These microbial carriers may be used alone or in combination of two or more.

[0021] (Culture method) When Dehalococcoides bacteria live in contaminated soil, groundwater, or other contaminated material contaminated with volatile organic compounds such as trichloroethylene, a medium containing obligate anaerobic bacteria and the above-mentioned carbon source, etc. may be added to the contaminated soil, groundwater, or other contaminated material to culture the Dehalococcoides bacteria.

[0022] For the purpose of bioaugmentation, in which Dehalococcoides bacteria are artificially cultured to increase their numbers and then introduced into a target environment for purification to promote purification, the Dehalococcoides bacteria are cultured in a medium. The Dehalococcoides bacteria, obligate anaerobic bacteria, and the above-mentioned carbon source or a microorganism capable of producing the carbon source by metabolism may be added to the medium for culture.

[0023] It is an absolute requirement to culture the Dehalococcoides bacteria with the redox potential of the medium 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 the reducing agent include reduced glutathione, iron sulfide, sodium sulfide, L-cysteine, D-dithiothreitol, titanium (III) complexed with citrate or nitrilotriacetate, etc.

[0024] The medium used in the culture method of this embodiment is supplemented with Dehalococcoides bacteria, obligate anaerobic bacteria, and the above-mentioned carbon source or a microorganism capable of producing the carbon source by metabolism. Other components necessary for the growth and culture of Dehalococcoides bacteria or obligate anaerobic bacteria may be appropriately blended into the medium. Examples of other components include inorganic salts, trace elements, nitrogen sources, various vitamins, pH adjusters, etc. Specific examples of nitrogen sources include casitone, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, etc. Specific examples of inorganic salts include phosphates, magnesium salts, calcium salts, sodium salts, potassium salts, etc. Specific examples of trace elements include iron, cobalt, copper, zinc, boron, nickel, molybdenum, manganese, selenium, tungsten, etc.

[0025] The medium may contain chlorinated ethylenes, such as tetrachloroethylene, trichloroethylene (TCE), dichloroethylene (DCE), and chloroethylene (VC).

[0026] As the gas phase, nitrogen gas or a mixed gas of nitrogen and carbon dioxide is preferably used. 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. However, in this embodiment, hydrogen is supplied by the metabolism of the obligate anaerobic bacteria, so the addition of hydrogen is not necessarily required. The volume ratio of nitrogen to carbon dioxide is appropriately set, but is preferably 60:40 to 90:10, more preferably 70:30 to 85:15.

[0027] The culture temperature is appropriately set depending on the type of obligate anaerobic bacteria, but 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 appropriately set depending on the type of obligate anaerobic bacteria, but is preferably near neutral, weakly acidic to weakly alkaline, more preferably pH 6 to 8, and even more preferably pH 6.5 to 7.5.

[0028] (action) In the culture method of this embodiment, Dehalococcoides bacteria, obligate anaerobic bacteria, and the above-mentioned carbon source were used. Dehalococcoides bacteria require hydrogen as an electron source for reductive dehalogenation reaction, and decompose organic halogen compounds in the presence of hydrogen. Dehalococcoides bacteria are inactivated by even a small amount of oxygen contamination, so it has not been easy to culture Dehalococcoides bacteria. There are legal restrictions on the installation of hydrogen gas cylinders, and hydrogen gas generated by a hydrogen generating device is often used for culture, but the purity of hydrogen is not 100% and there are cases where a small amount of oxygen is contaminated. Therefore, when culturing by the conventional method of introducing hydrogen, Dehalococcoides bacteria may be inactivated during the culture.

[0029] By co-culturing Dehalococcoides bacteria with obligate anaerobic bacteria that can grow under the same conditions as Dehalococcoides bacteria, it is possible to stably culture Dehalococcoides bacteria without using hydrogen gas. This is because hydrogen is generated from a carbon source by the metabolism of the obligate anaerobic bacteria, and the Dehalococcoides bacteria can directly incorporate the generated hydrogen into their metabolism. In addition, the obligate anaerobic bacteria effectively functions as a barrier against oxygen during the culture process, allowing Dehalococcoides bacteria to be cultured stably with high reproducibility.

[0030] The effects of the first embodiment will be described. (1-1) In the culture method of the first embodiment, Dehalococcoides bacteria, obligate anaerobic bacteria, and the above-mentioned carbon source are used. Dehalococcoides bacteria can be cultured stably with high reproducibility. This is expected to lead to excellent decomposition of volatile organic compounds.

[0031] (1-2) There is no need to actively add hydrogen to the culture medium of Dehalococcoides bacteria. Therefore, there is no need to handle hydrogen, which improves the ease of handling the material and culturing, and also allows for inexpensive cultivation.

[0032] (1-3) When at least one type of microbial carrier selected from a resin polymer carrier, activated carbon, and charcoal is further added to the medium, a highly anaerobic area is formed during co-culture, and the activity of the Dehalococcoides bacteria can be further enhanced.

[0033] <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.

[0034] The decomposition method of the present embodiment uses a decomposition treatment agent for volatile organic compounds, which contains Dehalococcoides bacteria, obligate anaerobic bacteria, and the above-mentioned carbon source or a microorganism capable of producing the carbon source. The decomposition treatment method includes a step of adding the decomposition treatment agent for volatile organic compounds to contaminants containing volatile organic compounds.

[0035] The Dehalococcoides bacteria, the obligate anaerobic bacteria, and the carbon source or the microorganism capable of producing the carbon source are the same as the respective components described in the first embodiment. Specific examples of volatile organic compounds include tetrachloroethylene, trichloroethylene, dichloroethylene, and chloroethylene.

[0036] The pollutants are not particularly limited, but examples include river water, groundwater, industrial wastewater, industrial effluent, domestic wastewater, industrial waste, contaminated soil, sludge, etc. contaminated with volatile organic compounds (VOCs) such as trichloroethylene.

[0037] 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.

[0038] The effects of the second embodiment will be described. (2-1) In the decomposition treatment method of the second embodiment, a decomposition treatment agent for volatile organic compounds, which contains Dehalococcoides bacteria, obligate anaerobic bacteria, and the above-mentioned carbon source or a microorganism capable of producing the carbon source, is administered to contaminated material containing volatile organic compounds. Therefore, an excellent decomposition effect of volatile organic compounds can be exerted. In addition, a highly stable culture is expected, and the efficiency of the treatment of contaminants can be improved. In addition, since there is no need to actively use hydrogen, the workability at the contaminant treatment site can be improved.

[0039] (2-2) In the decomposition method of the present embodiment, the addition of hydrogen as an electron source for the reductive dehalogenation reaction is not essential, and an organic acid such as lactic acid can be used as a carbon source, so that contaminants can be decomposed at low cost.

[0040] 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. In the above embodiment, it is not necessary to use hydrogen in the cultivation of the Dehalococcoides bacteria. However, this does not prevent the addition of hydrogen in the cultivation of the Dehalococcoides bacteria. EXAMPLES

[0041] 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.

[0042] <Test Example 1: Isolation and Characterization of Dehalococcoides Bacteria> (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.

[0043] [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 5 The 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.

[0044] [Table 2]

[0045] [Table 3]

[0046] [Table 4]

[0047] [Table 5]

[0048] [Table 6] (3) Confirmation of isolation achievement The culture medium after repeated limiting dilution culture was observed under a fluorescent microscope. When only a single bacterium was observed, microbial DNA was extracted and RFLP analysis was performed using the restriction enzymes HaeIII and HhaI. The results are shown in Figure 1. 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. The results of SEM observation are shown in Figure 2. As shown in Figure 2, only disc-shaped cells with a diameter of about 0.5 μm, which are thought to be Dehalococcoides bacteria, were observed, confirming the achievement of isolation. In Figure 2, the disc-shaped cells represent the isolated strain. The black circles represent holes in the base in the background, and the fine particles represent the residue of the bacteria bursting.

[0049] (4) Identification method (a) Morphological properties The isolated Dehalococcoides bacteria are non-motile and non-sporulating.

[0050] (b) Physiological characteristics It is strictly anaerobic and obtains energy through dehalogenation respiration. It uses hydrogen as an electron donor and chloroethylenes as electron acceptors. It has the ability to dechlorinate trichloroethylene (TCE), cis-1,2-dichloroethylene (DCE), and chloroethylene (VC), and was able to dechlorinate 500 μM trichloroethylene (TCE) to ethylene in 21 days. It also assimilated acetate as a carbon source. The optimal growth temperature was 28°C. The pH of the medium was neutral.

[0051] (c) Genetic characteristics Genomic DNA was extracted from the isolated Dehalococcoides bacteria, and the base sequence of the DNA encoding the 16S rRNA of the isolated strain was determined by single molecule real-time sequencing.

[0052] Identification was performed using the NCBI program BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ), and the base sequence of the 16S rRNA gene region was 100% identical to that of known Dehalococcoides species, such as Dehalococcoides mccartyi strain CBDB1.

[0053] In addition, it possessed 27 dechlorination enzyme genes. Based on the above, the isolated Dehalococcoides bacterium is considered to be closely related to Dehalococcoides mccartyi CBDB1 strain.

[0054] <Test Example 2: Evaluation of decomposition treatment of volatile organic compounds using Dehalococcoides bacteria> (Materials) The Dehalococcoides bacterium isolated in Test Example 1 was used as the Dehalococcoides bacterium.

[0055] The obligate anaerobic bacteria used were a homoacetogen (Acetobacterium carbinolicum, obtained from NITE) and a sulfate-reducing bacterium (Humidesulfovibrio idahonensis, owned by Nagoya Institute of Technology).

[0056] (Test Method) Dehalococcoides bacteria were cultured using the composition combinations shown in Table 7. The composition of the culture medium used in each example is shown in Table 8.

[0057] First, 20 mL of DHB-CO3 medium was placed in a 50 mL vial, and after gas replacement with a mixed gas of N2:CO2 = 4:1 in each case, the vial was closed with a fluoro-ETFE liner rubber stopper and an aluminum cap, and sterilized in an autoclave. Each component in Table 8 was added aseptically in a clean bench, and the vial was cultured in an incubator at 28 ° C. The gas phase of the positive control was replaced again with a mixed gas of H2:CO2 = 4:1 before addition. The culture solution was sampled at the start of the experiment and 2, 4, and 6 weeks later, and the pH was measured. The VOC concentration of TCE, as well as cis-1,2-DCE and VC, which are decomposition products of TCE, were measured by PID gas chromatography to determine the concentration in the culture solution. In addition, the number of 16S rRNA genes of Dehalococcoides bacteria in the culture solution was measured by real-time PCR to determine the number of bacteria. In the experiment, multiple specimens were prepared under the same conditions, and the same specimen was crushed after measurement in weeks 0 and 2, and the remaining specimen was measured in weeks 4 and 6. The results of Comparative Example 1 are shown in Figure 3, and the results of Examples 1 to 3 are shown in Figures 4 to 6, respectively.

[0058] [Table 7]

[0059] [Table 8] As shown in Comparative Example 1, when only Dehalococcoides bacteria were used without adding hydrogen gas or obligate anaerobic bacteria, the number of Dehalococcoides bacteria decreased over time. When co-cultured with obligate anaerobic bacteria of each Example, the number of Dehalococcoides bacteria decreased to about 10 6 copies / mL to about 10 7 copies / mL. In addition, during the culture period in each example, VC, a decomposition product of TCE, tended to increase. It was confirmed that the decomposition of TCE also progressed with the proliferation of Dehalococcoides bacteria.

[0060] The pH of the medium was maintained in the range of pH 7 to 7.5 throughout the implementation period in each of the Examples and Comparative Examples.

Claims

1. A method for culturing Dehalococcoides bacteria using a Dehalococcoides genus bacterium, an obligate anaerobic bacterium, and a carbon source that can produce hydrogen by the obligate anaerobic bacterium.

2. The method for culturing Dehalococcoides bacteria according to claim 1, wherein the obligate anaerobic bacteria is at least one species selected from homoacetogenic bacteria and sulfate-reducing bacteria.

3. The method for culturing a Dehalococcoides bacterium according to claim 1 , wherein the carbon source is an organic acid.

4. A decomposition treatment agent for volatile organic compounds, comprising a bacterium of the genus Dehalococcoides, an obligate anaerobic bacterium, and a carbon source capable of producing hydrogen by the obligate anaerobic bacterium, or a microorganism capable of producing the carbon source.

5. A method for decomposing and treating volatile organic compounds, comprising the step of adding the volatile organic compound decomposing agent according to claim 4 to pollutants containing volatile organic compounds.

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