JP7817693B2 - 1,4-Dioxane-decomposing bacteria, 1,4-dioxane-decomposing agent, method for producing 1,4-dioxane-decomposing agent, and method for treating 1,4-dioxane - Google Patents

Description

NPMD NITE P-03327

The present invention relates to 1,4-dioxane-decomposing bacteria, a 1,4-dioxane-decomposing agent, a method for producing a 1,4-dioxane-decomposing agent, and a method for treating 1,4-dioxane.

1,4-Dioxane is a persistent substance used as a solvent for organic synthesis reactions in chemical plants and other facilities. Environmental standards have been set for 1,4-dioxane due to its suspected harmful properties, and there is a need to purify contaminated water and soil. A chemical decomposition method known as the advanced oxidation process is known as a method for purifying contaminated water and soil containing 1,4-dioxane. As described in Patent Documents 1 and 2, the advanced oxidation process is a method of oxidatively decomposing 1,4-dioxane in contaminated soil or water using radicals generated by ozone treatment, a combination of ozone treatment and hydrogen peroxide treatment, or a combination of ozone treatment and ultraviolet light treatment.

However, the chemicals used in advanced oxidation are relatively expensive, and ozone generators and ultraviolet light irradiation devices require large amounts of electricity to operate. Furthermore, contaminated water and soil often contain substances that inhibit the reactions of advanced oxidation or substances that competitively consume radicals as targets for decomposition, requiring enormous amounts of chemicals and electricity. This makes it cost-prohibitive. Furthermore, methods that combine ultraviolet light treatment are difficult to apply to contaminated soil that does not transmit ultraviolet light, or contaminated water that contains turbidity.

Japanese Patent Application Laid-Open No. 2005-103401 Japanese Patent Application Laid-Open No. 2005-58854

Meanwhile, microbial decomposition is known as a method for purifying contaminated water. By bringing contaminated water or the like into contact with microorganisms that have the activity of decomposing 1,4-dioxane, the microorganisms decompose 1,4-dioxane over time. When contacted with 1,4-dioxane in an environment favorable for the microorganisms, 1,4-dioxane is selectively and efficiently decomposed, which is cost-effective.

The present invention is based on the discovery of a new microorganism with excellent 1,4-dioxane decomposition capabilities, and aims to provide a new 1,4-dioxane-decomposing bacterium, a 1,4-dioxane decomposing agent, a method for producing a 1,4-dioxane decomposing agent, and a method for treating 1,4-dioxane.

In order to solve the above problems, the 1,4-dioxane-degrading bacterium of the present invention is strain 3e deposited under accession number NITE P-03327.
In order to solve the above problems, the 1,4-dioxane decomposing agent of the present invention contains the above 1,4-dioxane decomposing bacterium.

To solve the above problems, the method for producing a 1,4-dioxane decomposing agent of the present invention includes a culturing step in which 1,4-dioxane-decomposing bacteria are cultured in the presence of a cyclic ether or glycol.

In order to solve the above problems, the method for treating 1,4-dioxane of the present invention includes a contacting step of contacting the 1,4-dioxane-decomposing bacteria with 1,4-dioxane.
In the above configuration, the contact step is preferably carried out in the presence of 1,4-butanediol.

According to the present invention, 1,4-dioxene contained in contaminated water can be treated at low cost using a novel 1,4-dioxane-degrading bacterium.

FIG. 1 shows the growth curve of strain 3e in a medium containing 1,4-dioxane as the sole carbon source. FIG. 1 shows the growth curve of strain 3e in a medium containing tetrahydropyran as the sole carbon source. FIG. 1 shows the 1,4-dioxane decomposition activity of strain 3e. FIG. 1 shows the 1,4-dioxane decomposition activity of strain 3e. FIG. 1 shows the 1,4-dioxane decomposition activity of strain 3e. FIG. 1 shows the 1,4-dioxane decomposition activity of strain 219. FIG. 1 shows the change in 1,4-dioxane concentration over time in the contact step, where (a) shows the change in 1,4-dioxane concentration over time in the presence of 1,4-dioxane alone, (b) shows the change in 1,4-butanediol coexistence, and (c) shows the change in 1,4-dioxane concentration over time in the presence of tetrahydrofuran. 1 shows the effects of cyclic ethers and glycols used as 1,4-dioxane decomposition agents during the cultivation of strain 3e, and the changes in 1,4-dioxane concentration over time during the subsequent contact step. (a) shows the changes in 1,4-dioxane concentration over time when a culture solution cultured in a rich LB medium was contacted, (b) shows the changes in 1,4-dioxane concentration over time when a culture solution cultured in an LB medium supplemented with 1,4-butanediol was contacted, and (c) shows the changes in 1,4-dioxane concentration over time when a culture solution cultured in an LB medium supplemented with tetrahydrofuran was contacted.

Hereinafter, an embodiment of the present invention will be described.
<About the 1,4-dioxane-degrading bacterium strain 3e>
The 1,4-dioxane-degrading bacterium of the present invention was isolated from river water near a chemical plant. The isolated 1,4-dioxane-degrading bacterium was named strain 3e. Strain 3e was deposited at the National Institute of Technology and Evaluation (NPMD), an international depository institution (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), on November 27, 2020. Its accession number is NITE P-03327.

The isolated 3e strain was grown on a nutrient plate, and genomic DNA was extracted from the resulting bacterial cells. Genomic DNA was extracted by selecting three colonies from the 3e strain grown on a nutrient plate. The resulting genomic DNA was used as a template for PCR amplification and analysis of the 16S rRNA gene. Primers used were 27F (AGAGTTTGATCMTGGCTCAG; SEQ ID NO: 1) and 1492R (TACGGHTACCTTGTTACGACTT; SEQ ID NO: 2), which are capable of amplifying nearly the full length of the bacterial 16S rRNA gene. Partial nucleotide sequences of the 16S rRNA gene were obtained from each of the three colonies. The three partial nucleotide sequences are shown in the Sequence Listing as SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

A homology search was performed using the BLAST program on the partial nucleotide sequence of the 16S rRNA gene of strain 3e in the National Center for Biotechnology Information (NCBI) 16S ribosomal RNA sequences database. The nucleotide sequence of SEQ ID NO:5 showed 100% (1371/1371) homology with strain DSM43338, the type strain of Rhodococcus ruber. The nucleotide sequences of SEQ ID NOs:3 and 4 showed 99.93% (1371/1372) homology with strain DSM43338, the type strain of Rhodococcus ruber. It was estimated that strain 3e belongs to the genus Rhodococcus ruber (hereinafter referred to as R. ruber).

Strain 3e has the ability to decompose cyclic ethers, which have a structure in which carbon atoms in cyclic hydrocarbons are replaced with oxygen, and can grow using cyclic ethers as a carbon source. Examples of cyclic ethers include 1,4-dioxane, tetrahydrofuran, and tetrahydropyran. Strain 3e also has the ability to decompose glycols such as 1,4-butanediol and ethylene glycol, and can grow using glycols as a carbon source.

Strain 3e exhibits improved 1,4-dioxane decomposition activity in the presence of glycols such as 1,4-butanediol. On the other hand, 1,4-dioxane decomposition activity is temporarily suppressed in the presence of tetrahydrofuran.

R. ruber strains T1 and T5 (hereinafter referred to as strain T1 and strain T5) are known as 1,4-dioxane-degrading bacteria belonging to R. ruber. Strains T1 and T5 are described in the non-patent literature Journal of Water and Environmental Technology Vol. 11, No. 1, pp. 11-19, (2013). Neither strain T1 nor strain T5 can grow in a medium containing 1,4-dioxane as the sole carbon source. Both strains T1 and T5 belong to so-called cometabolic bacteria, capable of decomposing 1,4-dioxane in the presence of cyclic ethers other than 1,4-dioxane, such as tetrahydrofuran, or glycols, such as 1,4-butanediol. On the other hand, strain 3e is a bacterium capable of assimilating 1,4-dioxane as the sole carbon source. It is clearly a different bacterium from strain T1 and strain T5.

R. ruber strain 219 (hereinafter referred to as strain 219) is known as a 1,4-dioxane-degrading bacterium belonging to the genus R. ruber that can utilize 1,4-dioxane as its sole carbon source. Strain 219 is described in the non-patent document Applied Microbiology and Biotechnology Vol. 36, No. 1, pp. 120-123, (1991). Like strain 219, strain 3e is also a bacterium of the genus R. ruber that can utilize 1,4-dioxane, but has significantly higher 1,4-dioxane-degrading activity than strain 219.

<1,4-dioxane decomposing agent>
Strain 3e can be used as a 1,4-dioxane decomposer.
The form of the 1,4-dioxane decomposer is not particularly limited, but is preferably liquid or powdered from the viewpoint of ease of storage and use. Specific forms include a culture solution of the 3e strain, a concentrated solution obtained by concentrating the culture solution, bacterial cells obtained by centrifuging the culture solution, bacterial cells obtained by freezing and storing the culture solution, bacterial cells obtained by freeze-drying the culture solution, and an immobilization carrier on which bacterial cells are immobilized. Powdered bacterial cells may also be mixed with various porous substances and adsorbed to form a formulation. By contacting such a 1,4-dioxane decomposer with contaminated water such as river water, groundwater, industrial wastewater, or sewage, or contaminated soil, the 1,4-dioxane contained in the contaminated water or soil can be decomposed.

<Method of producing 1,4-dioxane decomposing agent>
The method for producing a 1,4-dioxane decomposing agent comprises a culturing step of culturing the 3e strain to obtain a required amount of cells, and, if necessary, a post-treatment step of post-treating the culture solution containing the cells.

In the culturing process, the 3e strain is preferably cultured in liquid medium or plate medium at a temperature range of 15 to 35°C for approximately half a day to one week. For example, a single colony of the streaked 3e strain is scraped off and suspended in liquid medium, followed by shaking culture at 30°C for approximately two days.

The medium used in the culture step can be appropriately selected from media capable of growing microorganisms belonging to the genus R. ruber. Specific examples include, but are not limited to, R2A medium, W medium, and LB medium.

The culture process is preferably carried out by appropriately adding a cyclic ether or glycol to these known media. Examples of cyclic ethers include tetrahydrofuran and tetrahydropyran. Examples of glycols include 1,4-butanediol and ethylene glycol. The presence of a cyclic ether or glycol in the culture medium of the 3e strain induces the enzyme 1,4-dioxane decomposition enzyme, improving the 1,4-dioxane decomposition activity during the contact step when treating 1,4-dioxane.

The concentration of the cyclic ether or glycol in the medium during the culture step is preferably adjusted to about several to several tens of mg/L.
In post-treatment steps, for example, the culture solution of the 3e strain may be concentrated, frozen, or freeze-dried. Alternatively, the bacterial cells obtained by centrifugation or the like may be immobilized on an appropriate carrier.

<Method for treating 1,4-dioxane>
The method for treating 1,4-dioxane includes a contacting step of contacting the 3e strain with 1,4-dioxane.

For example, when treating 1,4-dioxane-contaminated groundwater pumped from the ground, the contaminated groundwater is supplied to an aeration tank, and the above-mentioned forms of 1,4-dioxane decomposer are mixed into the aeration tank in appropriate proportions. This brings the 3e strain into contact with 1,4-dioxane. The contacting step is preferably carried out at a temperature range of 15 to 35°C for approximately 1 to 7 days. The contacting step may be carried out continuously or batchwise.

Contaminated groundwater contains 1,4-dioxane at levels ranging from several tens of mg/L to the environmental standard for groundwater, but the contact process can reduce the 1,4-dioxane concentration to approximately 0.05 mg/L, which is the environmental standard for groundwater. The contaminated groundwater, which has had 1,4-dioxane decomposed through the contact process, can then be returned to its original location as treated water.

In the contact step, it is preferable to mix a cyclic ether or glycol in an appropriate ratio with the 1,4-dioxane decomposition agent. Examples of cyclic ethers include tetrahydrofuran and tetrahydropyran. Examples of glycols include 1,4-butanediol and ethylene glycol. The presence of 1,4-butanediol increases the rate of 1,4-dioxane decomposition by strain 3e. Furthermore, in the presence of tetrahydrofuran, the decomposition of tetrahydrofuran tends to proceed before the decomposition of 1,4-dioxane, but the rate of 1,4-dioxane decomposition after tetrahydrofuran decomposition tends to be faster than in the coexistence of 1,4-butanediol.

According to this embodiment, the following effects can be obtained.
(1) The 1,4-dioxane-degrading bacterium is strain 3e deposited under accession number NITE P-03327. The 1,4-dioxane decomposing agent contains strain 3e, which is a 1,4-dioxane-decomposing bacterium.

As a result, strain 3e, which can grow using 1,4-dioxane as its sole carbon source, can decompose 1,4-dioxane. Compared to the accelerated oxidation method, which involves adding an oxidizing agent to contaminated soil or water, 1,4-dioxane can be decomposed at a lower cost. Furthermore, it can effectively decompose 1,4-dioxane with a higher degradation activity than the previously known strain 219.

(2) The 1,4-dioxane treatment method involves a contact step in which the 1,4-dioxane-degrading bacterium strain 3e is brought into contact with 1,4-dioxane. Therefore, 1,4-dioxane in contaminated water and soil can be decomposed to a level that can be released into the natural environment at a lower cost than with advanced oxidation methods. Furthermore, while relatively low concentrations of 1,4-dioxane are occasionally found at actual 1,4-dioxane-contaminated sites, strain 3e has high 1,4-dioxane-degrading activity even at relatively low 1,4-dioxane concentrations. Therefore, this method is highly applicable to actual contaminated sites.

(3) The method for producing a 1,4-dioxane decomposing agent includes a culturing step in which a 1,4-dioxane-decomposing bacterium is cultured in the presence of a cyclic ether or glycol. Therefore, the cyclic ether or glycol induces the 1,4-dioxane decomposing enzyme, improving the 1,4-dioxane decomposing activity in the contacting step in which the 1,4-dioxane decomposing agent is brought into contact with 1,4-dioxane.

(4) The method for treating 1,4-dioxane involves a contact step in the presence of 1,4-butanediol. This increases the rate at which strain 3e decomposes 1,4-dioxane, enabling efficient decomposition of 1,4-dioxane.

(Example 1: Isolation of 1,4-dioxane-degrading bacteria)
Long-term acclimation was performed by adding river water collected near a chemical plant to 10 ml of W medium supplemented with 1,4-butanediol as a carbon source. 1,4-butanediol was added at a concentration of 8 mg/L. Three weeks after the start of acclimation, 1 ml of the acclimation solution was used for enrichment culture.

For the enrichment culture, 1 ml of the acclimatization solution was added to 9 ml of W medium containing 20 mg/L of 1,4-butanediol, and the first culture was performed at 30°C for 3 to 4 days. From the second culture onwards, 5 ml of the culture solution was added to 5 ml of W medium containing 20 mg/L of 1,4-butanediol, and the culture was performed at 30°C for 3 to 4 days, and this process was repeated twice a week. The enrichment culture was terminated after the 10th culture, and the culture solution was frozen and stored.

The presence of 1,4-dioxane-degrading bacteria in the enrichment culture was confirmed by culturing a portion of the frozen enrichment culture in the presence of 1,4-dioxane and examining the reduction of 1,4-dioxane. A loopful of the frozen enrichment culture was taken and added to 10 ml of W medium containing 20 mg/L 1,4-butanediol. After culturing at 30°C for one week, the bacterial cells were recovered using a centrifuge. The recovered bacterial cells were suspended in 1 ml of W medium containing 10 mg/L 1,4-dioxane, sealed in a 20 ml vial, and cultured at 30°C for 20 hours. The 1,4-dioxane concentration in the gas phase was then measured. 1,4-dioxane concentration was measured using gas chromatography under the following conditions.

Column used: Agilent DB-624 column, length 30 m × inner diameter 320 μm
Temperature increase conditions: 40°C, 1 min → 5°C/min → 150°C, 5 min
FID temperature: 300℃
Flow rate: 400ml/min
Injection volume: 2.5ml
1,4-Dioxane-degrading bacteria were isolated by streak culture from the 1,4-butanediol culture described above. A loopful of frozen enrichment culture was collected and added to 10 ml of W medium containing 20 mg/L 1,4-butanediol. After incubation at 30°C for one week, the culture was streaked onto R2A plates (R2A medium plus 15 g/L agar) and incubated at 30°C for three to four days. The resulting colonies were selected by type based on color and shape, and their 1,4-dioxane degradation activity was examined. A portion of the colonies was inoculated into 10 ml of W medium containing 20 mg/L 1,4-butanediol. The bacterial cells, cultured for one week as described above, were suspended in 1 ml of W medium containing 10 mg/L 1,4-dioxane and incubated for 20 hours. The 1,4-dioxane concentration in the gas phase was then measured. Colonies that exhibited 1,4-dioxane decomposition activity were further streaked to isolate the 1,4-dioxane-decomposing bacterium strain 3e.

The isolated 3e strain was grown on an R2A plate medium, genomic DNA was extracted from the resulting cells, and the 16S rRNA gene was amplified by PCR. Genomic DNA was extracted from three colonies selected from the 3e strain grown on an R2A plate medium, as described above. The partial base sequences of the 16S rRNA genes obtained from the three colonies are shown in the sequence listing as SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

Example 2: Evaluation of substrate metabolism ability of strain 3e
The substrate metabolism ability and resistance to drugs, heavy metals, etc. of the 3e strain were evaluated using a Biolog GEN III Microplate (registered trademark). The results are shown in Table 1. The well numbers in the table correspond to the well numbers on the Biolog GEN III Microplate (registered trademark). The evaluation results are indicated by a "+" if color development of the redox reaction reagent associated with the metabolism of the substrate and growth of the 3e strain was confirmed in the well, and a "-" if no color development was confirmed.

Example 3: Confirmation that strain 3e is a 1,4-dioxane-utilizing bacterium
A single colony of the 3e strain grown on a W plate medium using 1,4-dioxane as the sole carbon source was scraped and suspended in W medium containing 10 mg/L of 1,4-dioxane as the sole carbon source to prepare a bacterial suspension. The bacterial suspension was cultured with shaking at 30°C for 1 week to obtain a preculture solution. The preculture solution was subcultured on the same W medium as above and further cultured with shaking at 30°C for 1 week to obtain a culture solution. The OD at a wavelength of 600 nm of the culture solution during the shaking culture was measured every 2 days using an ultraviolet-visible spectrophotometer (DU-800, manufactured by Beckman Coulter, Inc.) to obtain a growth curve of the 3e strain.

The growth curve of strain 3e in the presence of 1,4-dioxane is shown in Figure 1. It was confirmed that strain 3e is an assimilating bacterium that can grow using 1,4-dioxane as the sole carbon source.
Example 4: Confirmation that strain 3e can grow using other cyclic ethers as carbon sources
A colony of the 3e strain grown on a W plate medium using tetrahydropyran as the sole carbon source was inoculated into a W medium containing 20 mg/L of tetrahydropyran in the same manner as in Example 3 to obtain a preculture solution. The preculture solution was inoculated into a W medium containing 20 mg/L of tetrahydropyran and cultured with shaking at 30°C for 1 week. The OD at 600 nm of the culture solution during the shaking culture was measured in the same manner as in Example 3 to obtain a growth curve of the 3e strain.

The growth curve of strain 3e in the presence of tetrahydropyran is shown in Figure 2. It was confirmed that strain 3e can grow using tetrahydropyran as the sole carbon source. Tetrahydropyran is an organic compound consisting of a saturated six-membered ring with five carbons and one oxygen atom, and is closer in shape to 1,4-dioxane, a saturated six-membered ring with four carbons and two oxygen atoms, than to tetrahydrofuran, a saturated five-membered ring with four carbons and one oxygen atom.

However, while the OD600 after four days in Example 3 was approximately 0.015, the OD600 after four days in Example 4 was approximately 0.004. It was confirmed that the initial growth rate of strain 3e in the presence of 1,4-dioxane was faster than the growth rate in the presence of tetrahydropyran. On the other hand, in the presence of 1,4-dioxane, the growth of strain 3e remained at an OD600 of 0.016, and it was predicted that the final growth rate would exceed that of tetrahydropyran in 1,4-dioxane.

Although not shown, we also confirmed whether the strain could grow using tetrahydrofuran or ethylene glycol as a carbon source. The carbon source substrate was supplied as a gas by dripping it onto the lid of a petri dish, and the 3e strain was streaked onto a W plate and cultured at 30°C. Growth ability was assessed by comparing the size of colonies with a control where no substrate was supplied.

In the presence of tetrahydrofuran, growth was evident, although it was inferior to that in the presence of tetrahydropyran. Furthermore, in the presence of ethylene glycol, growth was suggested to exceed that in the presence of tetrahydropyran.

Example 5: Evaluation of 1,4-dioxane decomposition activity of strain 3e
A single colony was scraped from the streaked W plate containing 1,4-dioxane as the sole carbon source, suspended in R2A medium, and cultured at 30°C for 18 hours with shaking to obtain a preculture solution. The preculture solution was centrifuged (13,500 rpm, 5 min, 24°C) to remove the supernatant, and the bacterial cells were suspended in physiological saline to obtain a bacterial solution. This process was repeated twice to prevent the introduction of R2A medium. The OD at a wavelength of 680 nm of the bacterial solution after two repetitions was measured using a UV-visible spectrophotometer (Shimadzu Corporation, UV-1850). The OD at a wavelength of 680 nm was 0.034.

Subsequently, the bacterial solution was centrifuged to remove the supernatant, and the bacterial pellet was suspended in 20 ml of BSM medium to obtain a BSM medium containing the bacterial cells.
5 ml, 0.5 ml, and 0.05 ml of the BSM medium containing the bacterial cells were measured out into glass bottles, and 10 g/L of 1,4-dioxane solution and BSM medium were added to adjust the final volume to 10 ml, yielding test solutions 1 to 3. As a control, 0 ml of the BSM solution containing the bacterial cells was prepared to yield test solution 4. The final concentration of 1,4-dioxane in each test solution was 10 mg/L.

The amount of BSM medium containing the bacterial cells, the amount of 10 g/L 1,4-dioxane solution, and the amount of BSM medium in each test solution are shown in Table 2.

50 μl of each test solution was collected after 0, 5, 24, 48, and 72 hours. 100 μl of 98% ethanol was added to each collected test solution to stop the 1,4-dioxane decomposition reaction. The 1,4-dioxane concentration in each test solution was measured using GC-MS (Shimadzu Corporation, GCMS-QP2010 Ultra). The results of the change in 1,4-dioxane concentration over time are shown in Figure 3. The lower limit of quantitation for 1,4-dioxane was 0.1 mg/L.

As shown in Figure 3, it was confirmed that the 1,4-dioxane concentration decreased over time in test solutions 1 to 3, and that the 1,4-dioxane decomposition rate increased depending on the concentration of bacterial cells in test solutions 1 to 3.

Example 6: Evaluation of strain 3e as a 1,4-dioxane decomposer
The concentration of 1,4-dioxane in river water, groundwater, soil, and other 1,4-dioxane-contaminated water and soil is approximately several tens of mg/L, up to the environmental standard value for groundwater. We evaluated the 1,4-dioxane treatment ability of a 1,4-dioxane decomposer made from the culture medium of strain 3e at this level.

(Example 6-1)
A single colony of the streaked 3e strain was scraped and suspended in LB medium, followed by shaking culture at 30°C for 2 days to obtain a preculture solution. The preculture solution was centrifuged (13,500 rpm, 5 min, 4°C) to obtain a bacterial cell pellet, and the bacterial cells were then suspended in W medium containing 1,4-dioxane as the sole carbon source to obtain a culture solution. The concentration of 1,4-dioxane in the culture solution at the start of culture was adjusted to 10 mg/L. The OD of the culture solution at the start of culture was measured using a UV-visible spectrophotometer (DU-800, manufactured by Beckman Coulter, Inc.), and the OD at a wavelength of 600 nm was 1.0.

1 ml of the culture medium was dispensed into crimp vials, each sealed with a crimp cap, and then statically cultured in an incubator at 30°C. Every hour, three crimp vials were removed from the incubator, and the 1,4-dioxane concentration in the culture medium was measured using gas chromatography (Agilent Technologies, Inc., 7890A) under the conditions described above. The 1,4-dioxane concentration at each elapsed time was taken as the average of the measurements for the three crimp vials.

FIG. 4 shows the change in the concentration of 1,4-dioxane in the culture medium over time.
(Example 6-2)
A single colony of the streaked 3e strain was scraped and suspended in LB medium, followed by shaking culture at 30°C for 18 hours to obtain a preculture solution. The preculture solution was centrifuged (13,500 rpm, 5 min, 24°C) to obtain a bacterial cell pellet, and the bacterial cells were then suspended in BSM medium containing 1,4-dioxane as the sole carbon source to obtain a culture solution. The concentration of 1,4-dioxane in the culture solution at the start of culture was adjusted to 10 mg/L. The OD of the culture solution at the start of culture was measured using a UV-visible spectrophotometer (Shimadzu Corporation, UV-1850), and the OD at a wavelength of 578 nm was found to be 2.1.

The culture medium was cultured at 30°C with shaking at 100 rpm. The culture medium was sampled every hour, and the 1,4-dioxane concentration in the culture medium was measured using GC-MS (Shimadzu Corporation, GCMS-QP2010 Ultra) under the above conditions. Figure 5 shows the change in 1,4-dioxane concentration in the culture medium over time.

As shown in Figure 4, the concentration of 1,4-dioxane in the culture medium of Example 6-1 decreased almost linearly over time. 10 mg/L of 1,4-dioxane had almost completely disappeared after 7 hours and was no longer measurable. Since 10 mg/L of 1,4-dioxane had disappeared in 7 hours, the decomposition rate was calculated to be approximately 1.43 mg/L·hr. The results are shown in Table 3.

As shown in Figure 5, the 1,4-dioxane concentration in the culture medium of Example 6-2 decreased almost linearly with time for the first three hours after the start of culture, and was estimated to have almost completely disappeared and become unmeasurable between three and four hours after the start of culture. The 1,4-dioxane decomposition rate for the first three hours after the start of culture was calculated to be approximately 2.74 mg/L·hr. The results are shown in Table 3.

Note that in Examples 6-1 and 6-2, the measurement wavelengths for the culture solution at the start of cultivation were different, but it was confirmed that the OD at a wavelength of 600 nm and the OD at a wavelength of 578 nm for the culture solution of the 3e strain were approximately the same.

Figure 6, on the other hand, is a figure reproduced from Applied Microbiology and Biotechnology, Vol. 36, No. 1, pp. 120-123, (1991), the aforementioned non-patent document describing strain 219. Figure 6 shows the growth curve and 1,4-dioxane degradation activity of R. ruber strain 219. Here, R. ruber strain 219 was inoculated into a mineral medium containing 10 mM 1,4-dioxane and cultured at 30°C with shaking at 175 rpm. The OD at a wavelength of 578 nm and the 1,4-dioxane concentration in the culture medium were measured every 24 hours.

At the start of the culture, the concentration of 1,4-dioxane in the culture medium was 10 mM (881.1 mg/L), and the OD at 578 nm of the culture medium was about 4.3.
As shown in Figure 6, for the R. ruber 219 strain, the 1,4-dioxane concentration in the culture solution decreased almost linearly with time up to 72 hours after the start of cultivation, but the rate of decrease slowed after 72 hours when the 1,4-dioxane concentration in the culture solution reached approximately 0.74 mM (= 65.20 mg/L).

The concentration of 1,4-dioxane contained in 1,4-dioxane-contaminated water and soil ranges from several tens of mg/L to the groundwater environmental standard level. In other words, when considering the treatment of contaminated water or soil, it can be seen that the 1,4-dioxane degradation rate of strain 219 is extremely slow within this concentration range. From Figure 6, assuming that the 1,4-dioxane concentration in the culture medium after 96 hours is approximately 0.10 mM (= 8.81 mg/L), the decomposition rate of strain 219 between 72 and 96 hours can be calculated to be 2.35 mg/L·hr. The results are shown in Table 3.

The right column of Table 3 shows the 1,4-dioxane decomposition rates of strain 3e and strain 219 divided by the bacterial cell concentration (OD). The results for strain 3e show that the bacterial cell concentration (OD) is proportional to the 1,4-dioxane decomposition rate. Furthermore, a comparison of the decomposition rates of strain 3e and strain 219 confirmed that strain 3e has 2.4 to 2.6 times the 1,4-dioxane decomposition ability of strain 219. At the level of treating actual contaminated water and soil, strain 3e is a bacterium with significantly higher 1,4-dioxane decomposition activity than strain 219.

(Example 7: Evaluation as a 1,4-dioxane decomposing agent in the coexistence of other cyclic ethers and glycols)
(Example 7-1)
The preculture solution cultured in the same manner as in Example 6-1 was centrifuged (13,500 rpm, 5 min, 4°C) to obtain a bacterial cell pellet, and the bacterial cells were then suspended in W medium containing 1,4-dioxane and 1,4-butanediol to obtain a culture solution. The culture solution was adjusted so that the 1,4-dioxane concentration in the culture solution at the start of the culture was 10 mg/L and the 1,4-butanediol concentration was 20 mg/L. As in Example 6-1, the culture solution was dispensed into crimp vials in 1 ml aliquots, and the 1,4-dioxane concentration in three vials was measured every hour. Figure 7(b) shows the change in 1,4-dioxane concentration in the culture solution over time.

(Example 7-2)
A preculture solution cultured in the same manner as in Example 6-1 was centrifuged in the same manner to obtain a bacterial cell pellet, and the bacterial cells were then suspended in W medium containing 1,4-dioxane and tetrahydrofuran to obtain a culture solution. The culture solution was adjusted so that the 1,4-dioxane concentration in the culture solution at the start of the culture was 10 mg/L and the tetrahydrofuran concentration was 20 mg/L. As in Example 6-1, 1 ml of the culture solution was dispensed into crimp vials, and the 1,4-dioxane concentration in three vials was measured every hour. Figure 7(c) shows the change in 1,4-dioxane concentration in the culture solution over time.

The results of Example 6-1 (FIG. 4) are shown again in FIG. 7(a) as a graph showing the 1,4-dioxane treating capacity in the absence of tetrahydrofuran or 1,4-butanediol.
As shown in Figure 7(a), when 1,4-dioxane was used alone, it took 7 hours to decompose 10 mg/L of 1,4-dioxane. However, in the presence of 20 mg/L of 1,4-butanediol, as shown in Figure 7(b), 1,4-dioxane disappeared within 4 hours. It was confirmed that 1,4-butanediol enhances 1,4-dioxane decomposition activity. This is presumably due to the induction of 1,4-dioxane decomposition enzymes in the presence of 1,4-butanediol, as is known in co-metabolizing bacteria.

Furthermore, as shown in Figure 7(c), in the presence of 20 mg/L tetrahydrofuran, the 1,4-dioxane concentration initially decreased little, but then decreased rapidly after 5 hours. The 1,4-dioxane decomposition rate after 5 hours was faster than the 1,4-dioxane decomposition rate in the presence of 1,4-butanediol, as shown in Figure 7(b). In the presence of tetrahydrofuran, 1,4-dioxane is thought to be decomposed after tetrahydrofuran decomposition, and the decomposition rate was found to be faster than in the case of 1,4-dioxane alone. This is presumably due to the induction of 1,4-dioxane-decomposing enzymes in the presence of tetrahydrofuran.

(Example 8: Evaluation of 1,4-dioxane decomposing agents cultured in the presence of other cyclic ethers and glycols)
The 1,4-dioxane treating ability was evaluated when strain 3e was cultured in the absence of other cyclic ethers or glycols, and when strain 3e was pre-cultured in the co-presence of other cyclic ethers or glycols.

(Example 8-1)
A single colony of the streaked 3e strain was scraped and suspended in LB medium, followed by shaking culture at 30°C for 2 days to obtain a preculture solution. The preculture solution was centrifuged (13,500 rpm, 5 min, 4°C) to obtain a bacterial cell pellet, which was then suspended in W medium containing 1,4-dioxane as the sole carbon source to obtain a culture solution. The concentration of 1,4-dioxane in the bacterial suspension at the start of culture was adjusted to 10 mg/L.

As in Example 6-1, 1 ml of the culture medium was dispensed into crimp vials, and the 1,4-dioxane concentration in three vials was measured every hour using gas chromatography (Agilent Technologies, Inc., 7890A). Figure 8(a) shows the change in 1,4-dioxane concentration over time in the culture medium of Example 8-1.

(Example 8-2)
A single colony of the streaked 3e strain was scraped and suspended in LB medium. After shaking culture at 30°C for 1 day, 1,4-butanediol was added and the mixture was further shaken for another day to obtain a preculture solution. The concentration of 1,4-butanediol in the preculture solution was adjusted to 20 mg/L. Under the same conditions as in Example 8-1, the preculture solution was centrifuged to obtain a bacterial cell pellet, and the bacterial cells were then suspended in W medium containing 1,4-dioxane as the sole carbon source to obtain a culture solution. As in Example 6-1, the culture solution was dispensed into crimp vials in 1 ml aliquots, and the 1,4-dioxane concentration in three vials was measured every hour. Figure 8(b) shows the time course of the 1,4-dioxane concentration in the culture solution of Example 8-2.

(Example 8-3)
The same procedure as in Example 8-2 was repeated, except that tetrahydrofuran was added instead of 1,4-butanediol. The concentration of tetrahydrofuran in the preculture solution was adjusted to 20 mg/L. Figure 8(c) shows the change over time in the 1,4-dioxane concentration in the culture solution of Example 8-3.

As shown in Figure 8(a), in the culture medium in which strain 3e was cultured in the absence of 1,4-butanediol or tetrahydrofuran, it took 4 hours to decompose 10 mg/L of 1,4-dioxane. On the other hand, as shown in Figure 8(b), in the culture medium in which strain 3e was cultured using 20 mg/L of 1,4-butanediol as a carbon source, 1,4-dioxane disappeared within 1 hour. Furthermore, as shown in Figure 8(c), in the culture medium in which strain 3e was cultured using 20 mg/L of tetrahydrofuran as a carbon source, 1,4-dioxane was rapidly decomposed within 1 hour and disappeared within 2 hours.

It is presumed that culturing strain 3e in the presence of 1,4-butanediol or tetrahydrofuran induces the induction of 1,4-dioxane decomposing enzymes, which is thought to improve the processing ability of the strain as a 1,4-dioxane decomposing agent.

Claims (5)

A 1,4-dioxane-degrading bacterium, strain 3e, deposited under accession number NITE P-03327. A 1,4-dioxane decomposing agent containing the 1,4-dioxane-decomposing bacterium described in claim 1. A method for producing a 1,4-dioxane decomposing agent according to claim 2, comprising:
A method for producing a 1,4-dioxane decomposing agent, comprising a culturing step of culturing a 1,4-dioxane-decomposing bacterium in the presence of a cyclic ether or glycol. A method for treating 1,4-dioxane, comprising a contacting step of contacting the 1,4-dioxane-degrading bacteria described in claim 1 with 1,4-dioxane. The method for treating 1,4-dioxane according to claim 4, wherein the contacting step is carried out in the presence of 1,4-butanediol.