Methanogenic archaea with tetramethylammonium hydroxide resolution

Abstract

To provide a methanogen having an ability to decompose tetramethylammonium hydroxide.SOLUTION: The present invention discloses a methanogen having an ability to decompose tetramethylammonium hydroxide.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a methane-producing archaea having tetramethylammonium hydroxide-degrading ability.

Background Art

[0002] In the manufacturing process of electronic components such as semiconductors, wastewater containing highly toxic organic chemicals such as tetramethylammonium hydroxide (TMAH) is discharged. Conventionally, industrial wastewater containing TMAH has been treated by aerobic treatment using the activated sludge method. However, aerobic treatment requires a large amount of electric power for oxygen supply (aeration), and a large amount of excess sludge is generated, resulting in a high environmental load.

[0003] On the other hand, anaerobic treatment using methane fermentation not only can solve the above problems in aerobic treatment but also has the advantage of recovering methane gas with utilization value as bioenergy. Non-Patent Document 1 reports an example of anaerobic treatment of wastewater containing TMAH.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, since only a very small amount of bacteria capable of decomposing TMAH exists in the inoculum sludge for methane fermentation treatment, it is necessary to acclimatize the sludge to TMAH for a long time in advance, which is not efficient. In order to efficiently and stably treat TMAH in wastewater by methane fermentation, it was essential to isolate and obtain methanogenic archaea capable of decomposing TMAH.

[0006] The present invention has been made in view of the above problems, and an object thereof is to provide methanogenic archaea having tetramethylammonium hydroxide-degrading ability.

Means for Solving the Problems

[0007] The methanogenic archaea according to one aspect of the present invention have tetramethylammonium hydroxide-degrading ability. The methanogenic archaea may be methanogenic archaea belonging to the genus Methanomethylovorans and may be capable of growing at a temperature of 5 to 37°C. The methanogenic archaea may have the following physiological properties (1) to (15): (1) Gram staining: negative, (2) Growth temperature: 18 to 37°C, (3) Growth pH: 6.5 to 7.5, (4) Growth NaCl concentration: less than 0.1 M, (5) TMAH utilization: positive, (6) Trimethylamine utilization: positive, (7) Dimethylamine utilization: positive, (8) Monomethylamine utilization: positive, (9) Methanol utilization: positive, (10) Dimethyl sulfide utilization: positive, (11) Methanethiol utilization: positive, (12) Isopropyl alcohol utilization: slightly positive, (13) Formate utilization: negative, (14) Acetate utilization: negative, and (15) 1 w / v% SDS solubility: negative. The methanogenic archaea may have an accession number of NITE AP-03370.

[0008] A method for treating wastewater according to one aspect of the present invention includes a step of adding the above-mentioned methanogenic archaea to wastewater containing tetramethylammonium hydroxide and decomposing the tetramethylammonium hydroxide in the wastewater by methanogenesis.

[0009] A method for purifying an environment contaminated with tetramethylammonium hydroxide according to one aspect of the present invention includes a step of adding the above-mentioned methanogenic archaea to an environment contaminated with tetramethylammonium hydroxide and decomposing the tetramethylammonium hydroxide in the environment by methanogenesis.

[0010] A wastewater treatment apparatus according to one aspect of the present invention includes a methanogenic reactor, a raw water supply unit that supplies wastewater to the methanogenic reactor, and a methanogenic archaea supply unit that supplies the above-mentioned methanogenic archaea to the methanogenic reactor.

Advantages of the Invention

[0011] According to the present invention, methanogenic archaea having the ability to decompose tetramethylammonium hydroxide are provided.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Embodiments for Carrying Out the Invention

[0013] <Methanogenic archaea> The methanogenic archaea according to one aspect of the present invention have the ability to decompose TMAH. The methanogenic archaea may be methanogenic archaea belonging to the genus Methanomethylovorans.

[0014] The growth temperature of the methanogenic archaea may be, for example, 5°C or higher, 10°C or higher, 15°C or higher, 16°C or higher, 17°C or higher, 18°C or higher, 19°C or higher, or 20°C or higher, and 37°C or lower, and may be 18 - 37°C or 20°C - 37°C. Wastewater generated during the production of electronic components such as semiconductors is discharged at room temperature (generally around 20°C). However, in conventional wastewater treatment technologies that utilize anaerobic microorganisms including methanogenic archaea, a treatment temperature of 30°C or higher is required to maintain the growth of the microorganisms. According to methanogenic archaea that can grow at a low temperature around 20°C, wastewater at room temperature can be treated at room temperature without heating, enabling reduction of the energy related to the operation of the wastewater treatment device.

[0015] The growth pH of the methanogenic archaea may be, for example, 6.0 - 9.0, 6.0 - 8.0, or 6.5 - 7.5. Known methanogenic archaea can generally grow in the range of 6.0 - 8.0.

[0016] The growth NaCl concentration of the methanogenic archaea is not particularly limited and may be, for example, less than 0.1M or 0M.

[0017] Methanogenic archaea may further have the ability to degrade (assimilate) at least one, preferably all substances selected from the group consisting of trimethylamine, dimethylamine, monomethylamine, methanol, dimethyl sulfide, methanethiol, and isopropyl alcohol. Also, methanogenic archaea may not have the ability to degrade formate and acetate.

[0018] Methanogenic archaea are preferably not lysed (lysed) with 1 w / v% or more or 1 w / v% of SDS.

[0019] Methanogenic archaea may be Gram-negative bacteria.

[0020] The GC content of the genome of methanogenic archaea is not particularly limited, but may be, for example, 44.7 mol% or more.

[0021] Methanogenic archaea are preferably the NY-STAYD strain deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (postal code 292-0818, 122, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE AP-03370 (date of deposit: January 28, 2021). The NY-STAYD strain has a 16S rRNA gene sequence containing the nucleotide sequence shown in SEQ ID NO: 1. Also, the NY-STAYD strain has the following physiological properties (1) to (15): (1) Gram staining: negative, (2) Growth temperature: 18 - 37 °C or 20 °C - 37 °C (optimum 25 - 30 °C), (3) Growth pH: 6.5 - 7.5 (optimum 6.5 - 7.0), (4) Growth NaCl concentration: less than 0.1 M or 0 M, (5) TMAH assimilation: positive, (6) Trimethylamine assimilation: positive, (7) Dimethylamine assimilation: positive, (8) Monomethylamine assimilation: positive, (9) Methanol assimilation: positive, (10) Dimethyl sulfide assimilation: positive, (11) Methanethiol utilization: Positive, (12) Isopropyl alcohol utilization: Slightly positive, (13) Formate utilization: Negative, (14) Acetate utilization: Negative, and (15) Solubility in 1 w / v% SDS: Negative.

[0022] The methanogenic archaea according to this aspect can be obtained, for example, by accumulating methanogenic archaea in granular sludge formed using sludge (for example, granular sludge) used in industrial wastewater treatment, paddy field soil, etc. as a seeding source, and then separating and culturing the accumulated methanogenic archaea. The accumulation, separation, and culturing of methanogenic archaea can be carried out, for example, as follows. First, at the above-mentioned temperature (for example, about 18°C to 25°C) at which the methanogenic archaea according to this aspect can grow, wastewater containing TMAH is supplied to a methane fermentation wastewater treatment device containing sludge as a seeding source to acclimatize the sludge to TMAH. During the period until the acclimatization to TMAH is sufficiently carried out, in order to maintain a low oxidation-reduction potential environment (for example, -250 mV or less), which is the growth environment of anaerobic microorganisms (methanogenic archaea group, etc.), other easily decomposable organic substances (lower fatty acids, sugars, proteins, alcohols, etc.) and / or reducing agents (cysteine hydrochloride, sodium sulfide, etc.) may be supplied simultaneously. After confirming the decomposition of TMAH by the sludge, limit dilution and subculture of the bacterial group in the sludge are carried out using a medium added with TMAH. The methanogenic archaea according to this aspect can be separated by repeating the limit dilution and subculture until it is confirmed that there are no bacteria other than the methanogenic archaea according to this aspect in the bacterial community.

[0023] The medium used for cultivation may be, for example, Widdel medium. The concentration of TMAH added to the medium may be, for example, 200 to 1000 mg COD / L. Other substrates (organic substances) and reducing agents useful for the growth of methanogenic archaea, and / or substances that inhibit the growth of other bacterial species may be added to the medium. Examples of other substrates include yeast extract. The concentration of yeast extract may be, for example, 40 mg COD / L or less. Examples of reducing agents include cysteine and sodium sulfide. The substance that inhibits the growth of other bacterial species may be, for example, an antibiotic.

[0024] The temperature, pH, and NaCl concentration of the medium and the culture solution may be the above-mentioned temperature, pH, and NaCl concentration at which the methanogenic archaea according to this aspect can grow. Also, the cultivation is preferably carried out under anaerobic conditions, for example, in an atmosphere of a mixed gas of 80% N 2 and 20% CO 2 . Other culture conditions can follow the conditions for culturing known methanogenic archaea. For example, the redox potential of the medium and the culture solution may be -250 mV or less.

[0025] The presence of the methanogenic archaea according to this aspect in the culture solution can be confirmed by observing the decomposition of TMAH by methanogenesis, and for example, by observing the autofluorescence of the F420 coenzyme specific to methanogenic archaea, by microbial community structure analysis using a generation sequencer, and / or by fluorescence in situ hybridization (FISH) method. In particular, according to the FISH method using a probe specific to the methanogenic archaea according to this aspect, discrimination from other methanogenic archaea is also possible.

[0026] Since the methanogenic archaea according to this aspect can decompose TMAH by methanogenesis, it can be used not only for the treatment of electronic industrial wastewater containing TMAH but also for bioremediation such as the purification of soil and groundwater contaminated with TMAH.

[0027] <Wastewater treatment method> A method for treating wastewater according to one aspect of the present invention includes a step of adding the methanogenic archaea according to the above aspect of the present invention to the wastewater containing TMAH and decomposing TMAH in the wastewater by methanogenesis.

[0028] The wastewater containing TMAH is not particularly limited as long as it contains TMAH, and may be, for example, electronic industry wastewater. The wastewater containing TMAH may contain other organic substances such as monoethanolamine and isopropyl alcohol.

[0029] The temperature, pH, and NaCl concentration of the wastewater containing TMAH are preferably the above-mentioned temperature, pH, and NaCl concentration at which the methanogenic archaea according to the above aspect of the present invention can grow. When the temperature, pH, and NaCl concentration of the wastewater containing TMAH are not the above-mentioned temperature, pH, and NaCl concentration, a step of adjusting the temperature, pH, and NaCl concentration of the wastewater may be performed so as to be the above-mentioned temperature, pH, and NaCl concentration. Further, the redox potential of the wastewater containing TMAH is preferably -250 mV or less. When the redox potential of the wastewater containing TMAH is not -250 mV or less, a step of adjusting the redox potential of the wastewater containing TMAH to -250 mV or less may be performed. The redox potential of the wastewater containing TMAH can be adjusted to -250 mV or less, for example, by adding an organic substance (lower fatty acid, sugar, protein, alcohols, yeast extract, etc.) and / or a reducing agent (cysteine hydrochloride, sodium sulfide, etc.) to the wastewater.

[0030] The methanogenic archaea added to the wastewater is preferably in the form of a culture solution. Therefore, the method for treating wastewater according to this aspect may further include a step of culturing the methanogenic archaea to obtain a culture solution of the methanogenic archaea. The composition of the medium used for culturing, the temperature, pH, and NaCl concentration of the medium and the culture solution, and other culture conditions are as described above.

[0031] The method for treating the wastewater according to this aspect may further include a step of adding an organic substance and / or a reducing agent useful for the growth of the methanogenic archaea according to the above aspect to the wastewater. The substrates and reducing agents useful for the growth of the methanogenic archaea are as described above.

[0032] The method for treating the wastewater according to this aspect can be applied to a conventional wastewater treatment apparatus that utilizes methane fermentation. Specifically, in a conventional wastewater treatment apparatus that utilizes methane fermentation, by adding the methanogenic archaea according to the above aspect of the present invention to the wastewater (raw water) supplied to the methane fermentation tank or the wastewater in the methane fermentation tank, TMAH in the wastewater can be decomposed. The methane fermentation tank is not particularly limited and may be a methane fermentation tank using a known method such as the upflow anaerobic sludge bed (UASB) method, the expanded granular sludge bed (EGSB) method, the fluidized bed method, the fixed bed method, the fluidized carrier method, the anaerobic membrane bioreactor (AnMBR) method, or the CSTR method. It is preferable that granular sludge is retained in the methane fermentation tank. The granular sludge is not particularly limited and may be, for example, granular sludge used for industrial wastewater treatment or granular sludge formed using paddy soil or the like as a seeding source.

[0033] The timing for adding the methanogenic archaea to the wastewater is preferably at the startup of the wastewater treatment apparatus. The startup of the wastewater treatment apparatus means any point in time from the start of operation of the wastewater treatment apparatus (i.e., the start of the inflow of the wastewater containing TMAH into the methane fermentation tank) to the point in time when the wastewater treatment apparatus can stably and sufficiently decompose TMAH (for example, the point in time when it can stably decompose about 70% to 80% or more of the inflowing TMAH). The methanogenic archaea are preferably added at least at the start of operation of the wastewater treatment apparatus. When treating wastewater containing TMAH using a conventional wastewater treatment apparatus that utilizes methane fermentation, it takes time to acclimatize the granular sludge to TMAH, so the period from the start of operation of the apparatus until it can stably and sufficiently decompose TMAH (i.e., the startup period) is long. By adding the methanogenic archaea according to the above aspect of the present invention to the wastewater at the startup of the wastewater treatment apparatus, the methanogenic archaea can be fixed and accumulated in the granular sludge, and the startup period of the wastewater treatment apparatus can be shortened.

[0034] <Environmental purification method> A method for purifying an environment contaminated with TMAH according to one aspect of the present invention includes a step of adding methanogenic archaea according to the above aspect of the present invention to the environment contaminated with TMAH and decomposing TMAH in the environment by methane fermentation.

[0035] The environment contaminated with TMAH is not particularly limited as long as it is an environment in which methanogenic archaea can grow (for example, an environment satisfying the above-described temperature, pH, and NaCl concentration in which methanogenic archaea can grow), and any environment such as soil, groundwater, rivers, lakes, and the ocean can be mentioned. Further, in order for the environment contaminated with TMAH to be an environment suitable for the growth of methanogenic archaea (anaerobic and low redox potential), a reducing agent and / or an organic substance may be added to the environment contaminated with TMAH, and an inert gas such as nitrogen gas may be supplied.

[0036] The method for adding methanogenic archaea to the environment is not particularly limited, and a conventionally known method such as injection at natural pressure, pressure injection, stirring injection, spraying and stirring, etc. can be appropriately selected according to the environment.

[0037] <Wastewater treatment device> The wastewater treatment apparatus according to one aspect of the present invention includes a methane fermentation tank, a raw water supply unit, and a methane-producing archaea supply unit. The raw water supply unit supplies wastewater (raw water) containing TMAH to the methane fermentation tank. The methane-producing archaea supply unit supplies the methane-producing archaea according to the above aspect to the methane fermentation tank. The methane-producing archaea supply unit may supply the methane-producing archaea to the methane fermentation tank by allowing the methane-producing archaea to flow into the wastewater supplied to the methane fermentation tank, or may directly supply the methane-producing archaea to the methane fermentation tank independently of the supply of the wastewater. In the methane fermentation tank, the methane-producing archaea held in the methane fermentation tank (more specifically, for example, sludge or a carrier in the methane fermentation tank) perform methane fermentation using TMAH in the wastewater as a substrate, whereby TMAH in the wastewater is decomposed. Details of the wastewater containing TMAH and the methane-producing archaea are as described above. The methane fermentation tank may be a known methane fermentation tank such as a UASB reactor or an EGSB reactor described above.

[0038] Hereinafter, with reference to FIG. 1, a wastewater treatment apparatus according to an embodiment will be described, but the wastewater treatment apparatus according to this aspect is not limited to the following embodiment.

[0039] The wastewater treatment apparatus 100 shown in FIG. 1 includes a raw water supply unit, an organic matter and reducing agent supply unit, a methane-producing archaea supply unit, and a methane fermentation tank. The raw water supply unit includes a raw water tank 11 and a raw water supply line 12. The organic matter and reducing agent supply unit includes an organic matter and reducing agent storage tank 21 and an organic matter and reducing agent supply line 22. The methane-producing archaea supply unit includes a methane-producing archaea storage tank 31 and a methane-producing archaea supply line 32. The methane fermentation tank 40 is provided with a treated water discharge line 41, a gas-liquid-solid separation unit 42, and a biogas discharge line 43.

[0040] The original water tank 11 stores the wastewater containing TMAH, and the wastewater is supplied to the bottom of the UASB-type methane fermentation tank 40 through the raw water supply line 12. The wastewater supplied to the bottom of the methane fermentation tank 40 flows upward by the upflow. Granular sludge is retained in the lower part of the methane fermentation tank 40, and methane-producing archaea are accumulated in the granular sludge (described later). TMAH in the wastewater is decomposed by the methane-producing archaea accumulated in the granular sludge, and methane gas is generated. The gas-solid-liquid separation part 42 provided in the upper part of the methane fermentation tank 40 separates methane gas, floating granular sludge, and treated water. The methane gas is discharged out of the wastewater treatment device 100 through the biogas discharge line 43, and the treated water is discharged out of the wastewater treatment device 100 through the treated water discharge line 41. The floating granular sludge settles again to the lower part of the methane fermentation tank 40.

[0041] A methane-producing archaea storage tank 31 is connected to the raw water supply line 12 via a methane-producing archaea supply line 32, and the methane-producing archaea related to the above side are stored in the methane-producing archaea storage tank 31. The methane-producing archaea in the methane-producing archaea storage tank 31 are supplied to the methane fermentation tank 40 through the methane-producing archaea supply line 32 and the raw water supply line 12, and settle and accumulate in the granular sludge in the methane fermentation tank 40. The start and stop of the supply of methane-producing archaea can be carried out at any time. For example, the supply of methane-producing archaea may be carried out constantly during the operation of the wastewater treatment device 100, or may be carried out at the start-up of the wastewater treatment device 100. More specifically, the supply of methane-producing archaea may be started at the start of the operation of the wastewater treatment device 100, and the supply of methane-producing archaea may be stopped when stable decomposition of TMAH or sufficient settlement and accumulation of methane-producing archaea in the granular sludge are recognized. Also, when the decomposition of TMAH becomes unstable, the supply of methane-producing archaea may be restarted as appropriate.

[0042] An organic matter and reducing agent storage tank 21 is further connected to the raw water supply line 12 via an organic matter and reducing agent supply line 22. The organic matter and reducing agent storage tank 21 stores the above-mentioned organic matter and reducing agent useful for the growth of methanogenic archaea, as well as the organic matter and reducing agent for maintaining a low redox potential. These organic matter and reducing agent are supplied to the methane fermentation tank 40 through the organic matter and reducing agent supply line 22 and the raw water supply line 12.

[0043] By providing a methanogenic archaea supply unit, the wastewater treatment apparatus 100 can supply methanogenic archaea to the methane fermentation tank 40 at the startup of the wastewater treatment apparatus 100, so that the startup can be completed in a short period of time.

[0044] Note that the wastewater treatment apparatus 100 is only one embodiment, and modified forms of the wastewater treatment apparatus 100 are also included in the scope of the wastewater treatment apparatus according to this aspect. For example, the raw water supply unit may supply wastewater from outside the wastewater treatment apparatus 100 instead of from the raw water tank 11. The organic matter and reducing agent supply unit may be divided into an independent organic matter supply unit and a reducing agent supply unit. The organic matter and reducing agent supply line 22 and the methanogenic archaea supply line 32 may be directly connected to the methane fermentation tank 40. The methane fermentation tank 40 may be another known methane fermentation tank other than the UASB reactor.

Example

[0045] <Example 1. Isolation and culture> Granular sludge collected from industrial wastewater treatment facilities was introduced into a UASB-type methane fermentation tank, and wastewater containing TMAH and other easily decomposable organic substances that serve as substrates for methane-fermenting bacterial groups, such as lower fatty acids and isopropyl alcohol, was supplied at 18 - 19 °C to acclimatize the granular sludge to TMAH. After confirming the decomposition of TMAH by the granular sludge, the granular sludge was added to Widdel medium (pH 7.0) supplemented with substrates (TMAH and yeast extract) and a reducing agent, and limiting dilution was performed. Table 1 shows the composition of the Widdel medium used in Examples 1 - 4, and the substrates and reducing agents appropriately added to the Widdel medium. Tables 2 and 3 show the compositions of the trace element solution and vitamin solution contained in the Widdel medium, respectively.

[0046]

Table 1

Table 2

Table 3

[0047] The bacterial solution was added to a 6-well plate of the above Widdel medium supplemented with substrates (TMAH and yeast extract) and a reducing agent, and cultured at 20 °C to form colonies (first passage). The colonies were inoculated into the same Widdel medium, and limiting dilution and subculture were repeated 4 times. The second subculture was performed at 20 °C, and the third to fifth subcultures were performed at 37 °C. In the fourth subculture, 40 μg / mL of vancomycin was added to the medium for the purpose of inhibiting the growth of eubacteria. All cultures were carried out under N 2 / CO 2This was carried out under a gas atmosphere. When the microbial community structure was analyzed using the next-generation sequencer MiSeq (registered trademark) system (Illumina), it was confirmed that the proportion of Methanomethylovorans archaea, which accounted for approximately 62% of all microorganisms in the third subculture, reached 100% in the fifth subculture, indicating successful isolation and culture of Methanomethylovorans archaea. The isolated strain was named NY-STAYD strain.

[0048] <Example 2. Analysis of Taxonomic Properties> DNA was extracted from the culture broth of the NY-STAYD strain, and molecular phylogenetic analysis based on the 16S rRNA gene sequence (SEQ ID NO: 1) was performed using the ARB Software package (Ludwig W, et al., ARB: a software environment for sequence data, Nucleic Acids Research, 2004, 32(4), 1363-1371.). The molecular phylogenetic tree is shown in Figure 2. In Figure 2, Mmv_isolate is the NY-STAYD strain. The NY-STAYD strain showed homology of 99.3%, 98.8%, and 97.8% with the known species of Methanomethylovorans, M. uponensis, M. hollandica, and M. thermophila, respectively.

[0049] M. uponensis, M. hollandica, and M. thermophila were obtained from the Biotechnology Center (NBRC), National Institute of Technology and Evaluation. M. uponensis and M. hollandica were acclimated in Widdel medium containing trimethylamine and yeast extract as substrates, and M. thermophila was acclimated in Widdel medium containing methanol and yeast extract as substrates. The NY-STAYD strain was cultured in Widdel medium containing TMAH and yeast extract as substrates. These Methanomethylovorans archaea were inoculated into Widdel medium containing TMAH and yeast extract and statically cultured at 37°C or 50°C for 40 days. The growth of each archaeon was determined by the optical density (OD) of the culture broth at 660 nm 660)It was examined by measurement. The results are shown in Fig. 3. As shown in Fig. 3, significant growth was observed only in the NY-STAYD strain 3 weeks after the start of cultivation. Since the proportion of yeast extract in all substrates in the medium was only 3.8%, it was shown that the NY-STAYD strain mainly grew using TMAH as a substrate. On the other hand, since no growth tendency was observed even after culturing the known species of the genus Methanomethylovorans for 40 days, it was shown that these known species do not have the ability to degrade TMAH.

[0050] Since the NY-STAYD strain has a clearly different phenotypic trait from the known species of the genus Methanomethylovorans in that it has the ability to degrade TMAH, it was found that the NY-STAYD strain is a new species. Therefore, the NY-STAYD strain was deposited with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Accession No.: NITE AP-03370, Date of receipt: January 28, 2021).

[0051] <Example 3. Analysis of Physiological Properties> The staining property, growth temperature, growth pH, growth NaCl concentration, substrate assimilation ability, sodium dodecyl sulfate (SDS) solubility, and GC content of the NY-STAYD strain were analyzed as follows.

[0052] The staining property was examined using a Gram staining kit (manufactured by Nippon Becton Dickinson Co., Ltd.). The Rhodococcus equi JCM311 strain was used as a positive control, and the Escherichia coli K12 strain was used as a negative control. The stained strains were observed by bright-field microscopy at 400-fold or 1000-fold magnification. The results are shown in Table 4.

[0053] The growth temperature was measured in Widdel medium (without resazurin) (pH 7.0) supplemented with a reducing agent, TMAH, and yeast extract, under N 2 / CO 2It was examined by culturing the NY-STAYD strain under an atmosphere. The culturing was carried out at temperatures of 15°C, 20°C, 25°C, 30°C, 37°C, 40°C, 45°C, and 55°C. Growth was confirmed by measuring the OD 660 of the culture solution using a spectrophotometer V-650 (manufactured by JASCO Corporation). OD 660 was measured once every 4 days, then once every 3 days, such as on the 0th day, 4th day, 7th day, 11th day..., and measured until approximately the 50th day. The results are shown in Table 4 and Figure 4. In Figure 4, (A) shows the growth rate, and (B) shows the specific growth rate (1 / day).

[0054] The growth pH was examined by culturing the NY-STAYD strain in Widdel medium (without resazurin) supplemented with a reducing agent, TMAH, and yeast extract at 37°C under an N 2 / CO 2 atmosphere. The pH of the medium was adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 using NaCl. Growth was confirmed by measuring the OD 660 of the culture solution using a spectrophotometer V-650. OD 660 was measured on the 0th day, 7th day, 14th day, 24th day, and 32nd day. The results are shown in Table 4 and Figure 5. In Figure 5, (A) shows the growth rate, and (B) shows the specific growth rate (1 / day).

[0055] The growth NaCl concentration was examined by culturing the NY-STAYD strain in Widdel medium (pH 6.5 - 7.0) supplemented with a reducing agent, TMAH, yeast extract, and NaCl at 25°C under an N 2 / CO 2 atmosphere. The concentration of NaCl was adjusted to 0 M, 0.1 M, 0.2 M, and 0.3 M. Growth was confirmed by visually observing or observing the autofluorescence of the F420 coenzyme specific to methanogenic archaea every other week. The results are shown in Table 4.

[0056] The substrate utilizability was examined in Widdel medium (pH 6.5 - 7.0) supplemented with a reducing agent, yeast extract, and other substrates at 25°C under an N 2 / CO2 It was examined by culturing NY-STAYD strain under an atmosphere. As other substrates, 5 mM of TMAH, 5 mM of trimethylamine, 5 mM of dimethylamine, 5 mM of monomethylamine, 5 mM of methanol, 2 mM of dimethyl sulfide, 200 μM of methanethiol, 5 mM of isopropyl alcohol, 5 mM of formate, and 5 mM of acetate were used. Growth was visually confirmed. The results are shown in Table 4.

[0057] The SDS solubility was examined by dropping 1% SDS and 0.1% SDS onto a slide glass on which a bacterial solution of NY-STAYD strain was dropped and air-dried, allowing it to stand for 5 to 10 minutes, and then performing phase-contrast observation using an epi-fluorescence microscope. The results are shown in Table 4.

[0058] The analysis of the GC content was entrusted to Technosuga Lab Co., Ltd. The results are shown in Table 4.

[0059]

Table 4

[0060] Table 4 is a table comparing the physiological properties of NY-STAYD strain and known species of the genus Methanomethylovorans.

[0061] As shown in Table 4 and Figure 4, the NY-STAYD strain grew in the temperature range of 20 to 37°C. The higher the culture temperature, the higher the specific growth rate, but the lysis was also faster. On the other hand, the lower the culture temperature, the less likely it was to lyse, and the cell yield was higher. From the specific growth rate and cell yield, it was determined that 25 to 30°C, especially 25°C, was the optimal growth temperature for the NY-STAYD strain. The NY-STAYD strain did not grow at 15°C, but as shown in Example 1, since the NY-STAYD strain was a strain isolated and cultured from a UASB reactor at 18 to 19°C, it is considered that it can also grow at 18 to 19°C.

[0062] As shown in Table 4 and Figure 5, the NY-STAYD strain grew at a pH of 6.5 to 7.5. This is generally consistent with the growth pH (6.0 to 8.0) of methanogenic archaea. The optimal growth pH is considered to be 6.5 to 7.0. However, the NaOH used to adjust the pH to 8.0 to 9.0 caused a neutralization reaction with the HCl used to adjust the pH to 7.0 during the preparation of the Widdel medium, forming NaCl in the medium, which may have inhibited the growth of the NY-STAYD strain during cultivation at pH 8.0 to 9.0. Therefore, the NY-STAYD strain may be able to grow even at pH values above 8.0.

[0063] Since the GC content of the NY-STAYD strain is high compared to known species, it is speculated that the DNA encoding genes related to physiological properties showing differences from known species (i.e., genes related to growth temperature and substrate utilization) contains a large amount of guanine and cytosine.

[0064] <Example 4. TMAH Decomposition Test at Low Temperature> A wastewater treatment device equipped with a UASB-type methane fermentation tank as shown in Figure 1 was prepared, and 32 g VSS of granular sludge with a treatment history of acetic acid, propionic acid, and isopropyl alcohol was seeded. Wastewater containing 600 to 700 mg COD / L of TMAH was supplied from the raw water tank to the methane fermentation tank. The temperature in the methane fermentation tank was maintained at 18 to 19 °C. In addition, in order to maintain the degree of reduction (-250 mV or less) in the wastewater treatment device, isopropyl alcohol was allowed to flow into the raw water supply line through the organic matter and reducing agent supply lines so that the isopropyl alcohol concentration in the methane fermentation tank would be 600 to 700 mg COD / L.

[0065] On the first and second days from the start of operation of the wastewater treatment device, a culture solution of the NY-STAYD strain in an amount of 0.63 w / w% of the granular sludge in the methane fermentation tank (a total of 1.26 w / w%, 0.403 g VSS) was allowed to flow into the raw water supply line through the methanogenic archaea supply line. After the third day, the supply of the NY-STAYD strain culture solution was stopped. During the operation period of the wastewater treatment device, the TMAH concentration in the treated water was measured over time.

[0066] For comparison, a wastewater treatment device similar to the above wastewater treatment device was operated under the same conditions as above except that NY-STAYD strain was not supplied, and the concentration of TMAH in the treated water was measured over time.

[0067] The results are shown in Fig. 6. In the wastewater treatment device without the addition of NY-STAYD strain, the decomposition of TMAH did not occur even after 50 days, and TMAH remained in the treated water. On the other hand, in the wastewater treatment device with the addition of NY-STAYD strain, due to the addition of NY-STAYD strain, the concentration of TMAH in the treated water significantly decreased on the 15th day, and the TMAH concentration decreased to a concentration below the detection limit (5 mg COD / L) on the 20th day. At the same time, an increase in methane gas production accompanying the decomposition of TMAH was confirmed. Also, thereafter, the wastewater treatment device with the addition of NY-STAYD strain could continue to operate stably for more than 200 days, and the establishment and accumulation of NY-STAYD strain in the granular sludge were also confirmed by electron microscope observation and analysis of the 16S rRNA gene.

[0068] As shown by the above results, when starting up a wastewater treatment device capable of decomposing TMAH using normal granular sludge collected from wastewater treatment facilities, etc., a long period (more than 50 days) is required to acclimatize the granular sludge to TMAH until it can actually decompose TMAH. On the other hand, it was shown that by separately adding NY-STAYD strain to normal granular sludge, the required acclimatization period was significantly shortened, and the wastewater treatment device could be started up in a short period.

Explanation of symbols

[0069] 11 ··· Raw water tank, 12 ··· Raw water supply line, 21 ··· Organic matter and reducing agent storage tank, 22 ··· Organic matter and reducing agent supply line, 31 ··· Methanogenic archaea storage tank, 32 ··· Methanogenic archaea supply line, 40 ··· Methane fermentation tank, 41 ··· Treated water discharge line, 42 ··· Gas-liquid-solid separation section, 43 ··· Biogas discharge line, 100 ··· Wastewater treatment device.

Claims

**Claim 1** A methanogenic archaeon having a tetramethylammonium hydroxide resolution and a receipt number of NITE AP-03370. **Claim 2** A method for treating wastewater, comprising the step of adding the methanogenic archaeon according to Claim 1 to wastewater containing tetramethylammonium hydroxide and decomposing the tetramethylammonium hydroxide in the wastewater by methane fermentation. **Claim 3** A method for purifying an environment contaminated with tetramethylammonium hydroxide, comprising the step of adding the methanogenic archaeon according to Claim 1 to the environment contaminated with tetramethylammonium hydroxide and decomposing the tetramethylammonium hydroxide in the environment by methane fermentation. **Claim 4** A methane fermentation tank, A raw water supply unit for supplying wastewater to the methane fermentation tank, A wastewater treatment apparatus comprising a methanogenic archaeon supply unit for supplying the methanogenic archaeon according to Claim 1 to the methane fermentation tank.

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