Detection reagent, detection method, and quantitative method

Abstract

To provide a thiocyanate dehydrogenase, a detection reagent, a detection method, a quantification method, and a treatment method that decompose thiocyanate ions via a cyanate pathway.SOLUTION: There is provided a thiocyanate dehydrogenase that is any one of the following proteins (a) to (c): (a) a protein that contains a specific amino acid sequence and has thiocyanate dehydrogenase activity; (b) a protein that contains an amino acid sequence in which one or more amino acids are deleted, substituted, or added in a specific amino acid sequence and has thiocyanate dehydrogenase activity; and (c) a protein that contains an amino acid sequence that has 98% or more sequence identity with a specific amino acid sequence and has thiocyanate dehydrogenase activity. There is provided a detection reagent having a polynucleotide containing the thiocyanate dehydrogenase gene or a partial sequence of the base sequence of the gene product.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a detection reagent, a detection method, a quantification method, and a treatment method.

Background Art

[0002] After the physicochemical treatment of coke oven wastewater (ammonia-free water), the COD (Chemical Oxygen Demand) component, which is the effluent standard, is treated by the activated sludge method, which is a biological water treatment method, and then discharged. Thiocyanic acid [present as thiocyanate ion (SCN - )] in ammonia-free water is a COD component, so treatment is required, but the treatment tends to be unstable. For the biodegradation of thiocyanate ion, a COS pathway is known in which thiocyanate ion is decomposed via carbonyl sulfide (COS). Thiocyanate-degrading microorganisms in the COS pathway have already been isolated (Patent Document 1), and the gene sequence of the thiocyanate-degrading enzyme has also been registered in the database.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, since carbonyl sulfide is a greenhouse gas, it is desirable to decompose thiocyanate ion while suppressing the generation of carbonyl sulfide. There are two pathways for the biodegradation of thiocyanate ion. One is cyanate ion (OCN -It was known that thiocyanate ions are decomposed via the cyanate pathway (via carbonyl sulfide) and the carbonyl sulfide pathway (via carbonyl sulfide) (Figure 1). Inspired by this, the inventors hypothesized that thiocyanate ions in activated sludge could also be decomposed via the cyanate pathway. However, while methods for detecting microorganisms that decompose thiocyanate ions via the carbonyl sulfide pathway have been reported, there is no method for detecting microorganisms that decompose thiocyanate ions via the cyanate pathway.

[0005] The present invention has been made in view of the circumstances described above, and aims to provide a thiocyanate dehydrogenase that decomposes thiocyanate ions via the cyanate pathway, a detection reagent, a detection method, a quantitative method, and a processing method. [Means for solving the problem]

[0006] As a result of diligent research to solve the aforementioned problems, the inventors have identified a new thiocyanate dehydrogenase and completed the present invention. Furthermore, we discovered that water containing thiocyanate can be treated using microorganisms possessing thiocyanate dehydrogenase, thus completing the present invention. The present invention employs the following configuration as a means to solve the above problems.

[0007] [1] A thiocyanate dehydrogenase, which is one of the following proteins (a) to (c). (a) A protein comprising the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9, having thiocyanate dehydrogenase activity. (b) Proteins having thiocyanate dehydrogenase activity, which include an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9. (c) A protein having thiocyanate dehydrogenase activity, containing an amino acid sequence that has 98% or more sequence identity with the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9. [2] A detection reagent having at least one polynucleotide selected from the group consisting of (I) to (VI) below. (I) Polynucleotides comprising the gene for thiocyanate dehydrogenase described in [1] above or a partial sequence of the base sequence of the gene product thereof (II) A polynucleotide comprising the gene for the thiocyanate dehydrogenase described in [1] above, or a partial sequence of a nucleotide sequence complementary to the nucleotide sequence of said gene. (III) A polynucleotide having a base sequence in which one or more bases are deleted, substituted, or added in the subsequence of (I) above. (IV) A polynucleotide having a base sequence in which one or more bases are deleted, substituted, or added in the subsequence of (II) above. (V) A polynucleotide containing a base sequence having 98% or more sequence identity with the subsequence of (I) above. (VI) A polynucleotide containing a base sequence having 98% or more sequence identity with the subsequence of (II) above. [3] The detection reagent according to [2], comprising a primer set having a combination of primer functions capable of detecting the thiocyanate dehydrogenase gene or the gene product thereof, the set comprising at least one polynucleotide selected from the group consisting of (1) and (2) below, and at least one polynucleotide selected from the group consisting of (3) and (4) below. (1) Polynucleotides containing the base sequence represented by Sequence ID No. 11 (2) Polynucleotides containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by Sequence ID No. 11. (3) Polynucleotides containing the base sequence represented by Sequence ID No. 12 (4) Polynucleotides containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by Sequence ID No. 12. [4] A detection method for detecting the gene of thiocyanate dehydrogenase or the gene product thereof using the detection reagent described in [2] or [3] above. [5] A method for quantifying the thiocyanate dehydrogenase gene or its gene product using the detection reagent described in [2] or [3] above. [6] A processing method comprising a processing step of forming a mixed phase containing thiocyanate and the thiocyanate dehydrogenase described in [1] above, and decomposing the thiocyanate with the thiocyanate dehydrogenase. [7] A treatment step of forming a mixed phase containing water to be treated containing thiocyanate and microorganisms having thiocyanate dehydrogenase, and decomposing the thiocyanate, A processing method comprising a detection step of detecting a gene or gene product related to the cyanate pathway that decomposes the thiocyanate. [8] The treatment method according to [7], wherein the gene related to the cyanate pathway is the thiocyanate dehydrogenase gene. [9] The treatment method according to [7] or [8], wherein the microorganism is a microorganism having a thiocyanide-degrading ability that decomposes the thiocyanate via cyanate.

[10] The processing method according to any one of [7] to [9], further comprising a second detection step of detecting a gene or gene product related to the carbonyl sulfide pathway that decomposes the thiocyanate via carbonyl sulfide.

[11] The treatment method according to

[10] , wherein the gene related to the sulfide carbonyl pathway is the thiocyanate hydrolase gene.

[12] A treatment method comprising a treatment step of forming a mixed phase containing water to be treated containing thiocyanate and a microorganism having the thiocyanate dehydrogenase described in [1] above, thereby decomposing the thiocyanate.

[13] The processing method according to

[12] , comprising a detection step of detecting the gene of thiocyanate dehydrogenase or the gene product thereof using the detection reagent described in [2] or [3].

[14] The treatment method according to any one of [7] to

[13] , wherein the water to be treated containing thiocyanate is ammonia water discharged from a coke oven. [Effects of the Invention]

[0008] According to the thiocyanate dehydrogenase of the present invention, thiocyanate can be decomposed. According to the detection reagent of the present invention, the gene of thiocyanate dehydrogenase or the gene product can be detected. According to the detection method of the present invention, the gene of thiocyanate dehydrogenase or the gene product can be detected. According to the quantification method of the present invention, the gene of thiocyanate dehydrogenase or the gene product can be quantified. According to the treatment method of the present invention, thiocyanate can be decomposed. According to the treatment method of the present invention, when the water to be treated contains thiocyanate, the water to be treated containing thiocyanate can be treated by using microorganisms having thiocyanate dehydrogenase.

Brief Description of Drawings

[0009] [Figure 1] It is a diagram explaining the reaction pathway involved in the decomposition of thiocyanate. [Figure 2] It is a schematic diagram showing the configuration of the biological treatment apparatus used in the examples. [Figure 3] It is a diagram showing the results of the nitrite generation rate in the biological treatment apparatus obtained in the examples. [Figure 4] It is a diagram showing the results of the thiocyanate removal rate in the biological treatment apparatus obtained in the examples. [Figure 5] It is a calibration curve obtained by using the primer set designed in the examples. [Figure 6] It is a calibration curve obtained by using the primer set designed in the examples. [Figure 7] It is a graph showing the ratio of the thiocyanate dehydrogenase gene and the carbonyl sulfide pathway gene contained in the samples obtained from the biological treatment apparatus in the examples for each operation day.

Modes for Carrying Out the Invention

[0010] In this specification, regarding the decomposition of thiocyanate ions as shown in Figure 1, thiocyanate ions (SCN) - ) is sometimes simply called "thiocyanate," and the cyanate ion (OCN - This substance is sometimes simply called "cyanic acid."

[0011] ≪Thiocyanate Dehydrogenase≫ The following describes the thiocyanate dehydrogenase of the embodiment. The thiocyanate dehydrogenase (hereinafter also referred to as TcDH) in this embodiment is one of the following proteins (a) to (c). (a) A protein having TcDH activity, comprising the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9. (b) Proteins having TcDH activity, which include an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9. (c) A protein having TcDH activity, containing an amino acid sequence with 98% or more sequence identity with the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9.

[0012] In (a) above, the amino acid sequence represented by Sequence ID No. 1 is the amino acid sequence of TcDH1 obtained in the example described later. An example of a nucleic acid encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 1 is a nucleic acid consisting of the base sequence represented by Sequence ID No. 2.

[0013] In (a) above, the amino acid sequence represented by Sequence ID No. 3 is the amino acid sequence of TcDH2 obtained in the example described later. An example of a nucleic acid encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 3 is a nucleic acid consisting of the base sequence represented by Sequence ID No. 4.

[0014] In (a) above, the amino acid sequence represented by Sequence ID No. 5 is the amino acid sequence of TcDH3 obtained in the example described later. An example of a nucleic acid encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 5 is a nucleic acid consisting of the base sequence represented by Sequence ID No. 6.

[0015] In (a) above, the amino acid sequence represented by Sequence ID No. 7 is the amino acid sequence of TcDH4 obtained in the example described later. An example of a nucleic acid encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 7 is a nucleic acid consisting of the base sequence represented by Sequence ID No. 8.

[0016] In (a) above, the amino acid sequence represented by Sequence ID No. 9 is the amino acid sequence of TcDH5 obtained in the example described later. An example of a nucleic acid encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 9 is a nucleic acid consisting of the base sequence represented by Sequence ID No. 10.

[0017] The TcDH of the embodiment may have mutations in the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9, as long as it is a protein having TcDH activity, and may be the protein of (b) and the protein of (c) above.

[0018] In the protein of (b) above, one or more means, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0019] In the protein described in (c) above, the amino acid sequence identity may be 98% or higher, or 99% or higher. The amino acid sequence identity can be determined by a BLAST search provided on the GenBank database.

[0020] One of the proteins (a) to (c) possesses TcDH activity. As shown in Figure 1, TcDH activity is the process by which thiocyanate is converted in the OCN pathway into cyanate and elemental sulfur (S). 0 The activity may be one that degrades thiocyanate into thiocyanate. Whether a candidate TcDH protein has TcDH activity can be determined by analyzing the function of the candidate protein, for example, by the method shown in the examples below. Specifically, the gene for a candidate TcDH protein is introduced into a microorganism that does not originally possess thiocyanate degradation ability, and the candidate TcDH protein is expressed. If the microorganism then acquires thiocyanate degradation ability, the candidate protein can be determined to be TcDH.

[0021] The TcDH according to this embodiment may be chemically synthesized based on the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9, or it may be produced using a known protein expression system with the gene encoding the TcDH. Furthermore, TcDH having mutations in the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9 may be produced using genetic recombination technology to introduce known amino acid mutations based on the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9.

[0022] The TcDH according to this embodiment can be used for the decomposition of thiocyanate and can be used in various reactions such as wastewater treatment and material production.

[0023] Detection Reagents The detection reagents of the embodiment will be described below. The detection reagent of the embodiment comprises at least one polynucleotide selected from the group consisting of (I) to (VI) below. (I) Polynucleotides comprising a partial sequence of the TcDH gene or the base sequence of the gene product according to the present invention. (II) A polynucleotide comprising a partial sequence of a nucleotide sequence complementary to the nucleotide sequence of the TcDH gene or the gene product according to the present invention. (III) A polynucleotide having a base sequence in which one or more bases are deleted, substituted, or added in the subsequence of (I) above. (IV) A polynucleotide having a base sequence in which one or more bases are deleted, substituted, or added in the subsequence of (II) above. (V) A polynucleotide containing a base sequence having 98% or more sequence identity with the subsequence of (I) above. (VI) A polynucleotide containing a base sequence having 98% or more sequence identity with the subsequence of (II) above.

[0024] In this specification, "gene" may include not only the protein-coding region corresponding to the TcDH transcript on the genome, but also introns, untranslated regions, and adjacent transcriptional regulatory regions. An example of an adjacent transcriptional regulatory region is a promoter. The "gene product" of TcDH here refers to mRNA and mRNA precursors.

[0025] The TcDH gene in the detection reagent of the embodiment is the gene that encodes any of the proteins (a) to (c) above. In the detection reagent of the embodiment, the partial sequence of (I) is any consecutive part of the base sequence, not including the entire base sequence of the TcDH gene or the gene product according to the present invention, and having a length that can be used as a probe or primer as described later. In the detection reagent of the embodiment, the partial sequence of (II) is any continuous portion of the base sequence that does not include all of the base sequence complementary to the base sequence of the TcDH gene or the gene product of the present invention, and has a length that can be used as a probe or primer as described later. In the detection reagent of the embodiment, the polynucleotides (I) and (II) contain a partial sequence of the base sequence of the TcDH gene or its gene product, or a sequence complementary to the partial sequence. Therefore, their presence can be detected by hybridizing them with the TcDH gene or its gene product. It is known that hybridization between polynucleotides can occur even if the sequences are not perfectly complementary. Therefore, the polynucleotides may have mutations as long as they hybridize with the polynucleotides of (I) or (II) under stringent conditions. Examples of polynucleotides that may be included in the detection reagent of the embodiment include the polynucleotides of (III) to (VI) above.

[0026] In the above, "stringent conditions" refers to, for example, the method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). For example, hybridization can be performed by incubating in a hybridization buffer consisting of 5×SSC (composition of 20×SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 0.1% by weight N-lauroyl sarcosine, 0.02% by weight SDS, 2% by weight blocking reagent for nucleic acid hybridization, and 50% formamide at 55°C to 70°C for several hours to overnight. The washing buffer used for washing after incubation is preferably a 1×SSC solution containing 0.1% by weight SDS, and more preferably a 0.1×SSC solution containing 0.1% by weight SDS.

[0027] In the sequences described in (III) and (IV) above, one or more items may be, for example, 1 to 30 items, 1 to 20 items, 1 to 10 items, 1 to 5 items, 1 to 4 items, 1 to 3 items, or 1 to 2 items.

[0028] In the sequences of (V) and (VI) above, the sequence identity of the nucleotide sequences may be 98% or higher, or 99% or higher. The sequence identity of the nucleotide sequences can be determined by a BLAST search provided on the GenBank database.

[0029] The polynucleotides contained in the detection reagents of the embodiments can be used as primers or probes. The length of the polynucleotides contained in the detection reagents of the embodiments may be any length suitable for use as a primer or probe, and an appropriate length can be selected depending on the application. When the detection reagent is a probe, the length of the probe may be, for example, 10 to 500 bases, 20 to 200 bases, or 50 to 100 bases. When the detection reagent is a primer, the length of the primer may be, for example, 10 to 40 bases, 18 to 35 bases, or 20 to 25 bases. The polynucleotides can be produced by chemical synthesis or known genetic recombination techniques based on the base sequence of the polynucleotides.

[0030] If the detection reagent in the embodiment is a primer, the detection reagent in the embodiment may include at least one polynucleotide selected from the group consisting of (I), (III), and (V), and at least one polynucleotide selected from the group consisting of (II), (IV), and (VI).

[0031] The polynucleotide included in the detection reagent of the embodiment is preferably DNA, but may also include artificial nucleic acids such as PNA (peptide nucleic acid) or LNA (locked nucleic acid), as long as they have a similar function to DNA.

[0032] <Primer Set>

[0033] The detection reagent in this embodiment may comprise a primer set having a combination of primer functions capable of detecting the TcDH gene or its gene product, comprising at least one polynucleotide selected from the group consisting of (1) and (2) below, and at least one polynucleotide selected from the group consisting of (3) and (4) below. (1) Polynucleotides containing the base sequence represented by Sequence ID No. 11 (2) Polynucleotides containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by Sequence ID No. 11. (3) Polynucleotides containing the base sequence represented by Sequence ID No. 12 (4) Polynucleotides containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by Sequence ID No. 12.

[0034] The nucleotide sequences represented by SEQ ID NOs: 11 and 12 are subsequences that are common to the nucleotide sequences represented by SEQ ID NOs: 1, 3, 5, 7, and 9, or sequences that are complementary to those subsequences. These polynucleotide combinations (1) to (4) allow for the simultaneous amplification of five different TcDH genes, including the amino acid sequences represented by SEQ ID NOs: 1, 3, 5, 7, or 9. By detecting the amplification products, all of these TcDH genes can be detected simultaneously.

[0035] A preferred combination of polynucleotides contained in the detection reagent is, for example, a combination of polynucleotide (1) and / or (2) and polynucleotide (3) and / or (4). The polynucleotide can be produced based on its base sequence by chemical synthesis or by known genetic recombination techniques.

[0036] In the polynucleotides of (2) and (4) above, one or more may be, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Furthermore, it is preferable that there are no base deletions, substitutions, or additions in the two bases from the 3' end of these polynucleotides.

[0037] According to the detection reagent of this embodiment, the TcDH gene or its gene product can be detected with high accuracy.

[0038] ≪Detection Method≫ The detection method of the embodiment is a method for detecting the TcDH gene or its gene product using the detection reagent of the embodiment described above.

[0039] The polynucleotides contained in the detection reagent of the embodiment can be used as primers or probes. The method for detecting the TcDH gene or its gene product using the primers or probes is not particularly limited and can be applied to various methods. Examples include PCR, RT-PCR, microarrays, and sequencing analysis.

[0040] The detection method of the embodiment comprises the steps of: performing an amplification reaction of the TcDH gene from the analyte using the primer set of the embodiment described above; and detecting the amplification product amplified by the amplification reaction.

[0041] PCR is one example of an amplification reaction. The amplification product may be detected by observing the amplified product of the gene fragment, for example, by agarose gel electrophoresis. Alternatively, the gene amplification reaction may be performed in the presence of an intercalator dye such as SYBR green, and the fluorescence of the intercalator dye may be detected.

[0042] If the amplification product is detected by the detection method according to this embodiment, it can be determined that the TcDH gene is present in the analyte. Furthermore, if the TcDH gene is present in the analyte, it can be determined that a microorganism possessing the TcDH gene is present in the analyte.

[0043] Furthermore, by using a primer set having at least one polynucleotide selected from the group consisting of (1) and (2) below, and at least one polynucleotide selected from the group consisting of (3) and (4) below, five types of TcDH genes, including the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9, can be detected simultaneously. In addition, by determining the presence of the TcDH gene in the analyte, it is possible to simultaneously determine the presence of a microorganism in the analyte that has the TcDH gene containing the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9.

[0044] (1) Polynucleotides containing the base sequence represented by Sequence ID No. 11 (2) Polynucleotides containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by Sequence ID No. 11. (3) Polynucleotides containing the base sequence represented by Sequence ID No. 12 (4) Polynucleotides containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by Sequence ID No. 12.

[0045] ≪Quantitative method≫ The quantitative method of the embodiment is a method of quantifying the TcDH gene or its gene product using the detection reagent of the embodiment described above.

[0046] The quantitative method of the embodiment comprises the steps of: performing an amplification reaction of the TcDH gene from the analyte using the primer set of the embodiment described above; detecting the amplification product amplified by the amplification reaction; and quantifying the amount of the gene contained in the analyte from the amount of the detected amplification product. For example, the quantity of a gene can be quantified from the fluorescence intensity by preparing a calibration curve in advance that utilizes the correlation between the copy number of the gene corresponding to the amplification product and the fluorescence intensity.

[0047] According to the quantitative method of this embodiment, the number of copies of the TcDH gene contained in the analyte can be quantified by quantifying the amplification product. Furthermore, it is possible to estimate the number of microorganisms possessing the TcDH gene present in the analyte from the number of copies of the TcDH gene in the analyte.

[0048] Furthermore, by using a primer set comprising at least one polynucleotide selected from the group consisting of (1) and (2) below, and at least one polynucleotide selected from the group consisting of (3) and (4) below, five types of TcDH genes, including the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9, can be simultaneously quantified. In addition, it is possible to estimate the number of microorganisms present in the analyte that possess the TcDH gene containing the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9, from the copy number of the TcDH gene in the analyte.

[0049] (1) Polynucleotides containing the base sequence represented by Sequence ID No. 11 (2) Polynucleotides containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by Sequence ID No. 11. (3) Polynucleotides containing the base sequence represented by Sequence ID No. 12 (4) Polynucleotides containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by Sequence ID No. 12.

[0050] Processing Method <First Embodiment> The processing method of this embodiment includes a step of forming a mixed phase containing thiocyanate and TcDH of the above embodiment, and decomposing the thiocyanate with the TcDH. Since the TcDH of the above embodiment has thiocyanate decomposition activity, thiocyanate and TcDH react, and thiocyanate is decomposed into cyanic acid and elemental sulfur. The thiocyanate may be a thiocyanate salt or a thiocyanate ion. Examples of thiocyanates include sodium thiocyanate and potassium thiocyanate.

[0051] According to the processing method of this embodiment, thiocyanate can be decomposed by using TcDH according to the present invention.

[0052] <Second Embodiment> (Processing steps) The treatment method of this embodiment includes a treatment step of forming a mixed phase containing water to be treated that contains thiocyanate and microorganisms having TcDH, and decomposing the thiocyanate.

[0053] Examples of water to be treated that contain thiocyanate include wastewater from various factories, with ammonia discharged from coke ovens being preferred.

[0054] The water to be treated, which contains thiocyanate, is mixed with microorganisms that have TcDH to form a mixed phase containing these. For example, when water to be treated, which contains thiocyanate, is introduced into a treatment tank where activated sludge containing microorganisms with TcDH is stored, the mixed phase is formed.

[0055] The thiocyanate contained in the treated water may be a thiocyanate salt or a thiocyanate ion. Examples of thiocyanates include sodium thiocyanate and potassium thiocyanate.

[0056] Thiocyanates contained in the treated water are broken down by TcDH to form cyanic acid. The resulting cyanic acid is then broken down by microorganisms that possess cyanide-degrading enzymes.

[0057] The decomposition of thiocyanate contained in the treated water to cyanate may be carried out by microorganisms contained in the mixed phase. The microorganism having TcDH may have the ability to decompose both thiocyanate to cyanate and cyanate. That is, the microorganism having TcDH may be a microorganism that has the ability to decompose thiocyanate via cyanate.

[0058] The processing step may be carried out under anaerobic conditions, but it is preferable to carry it out under aerobic conditions.

[0059] (Detection process) The processing method of this embodiment includes a detection step for detecting a gene or gene product related to the cyanate pathway that decomposes thiocyanate into cyanate.

[0060] As shown in Figure 1, there are two pathways for the decomposition of thiocyanate: the cyanate pathway, which decomposes thiocyanate via cyanate, and the carbonyl sulfide pathway, which decomposes thiocyanate via carbonyl sulfide.

[0061] The cyanate pathway, as shown in Figure 1, is the pathway in which thiocyanate is broken down into cyanate and elemental sulfur. Genes involved in the cyanate pathway include genes for enzymes involved in the reactions in this pathway, and may also be genes for unknown enzymes, with the TcDH gene being preferred.

[0062] In the processing method of the embodiment, TcDH has thiocyanase activity. TcDH activity is the activity to decompose cyanate or cyanate ions, and as shown in Figure 1, it may be the activity to decompose thiocyanate into cyanate and elemental sulfur.

[0063] Examples of TcDH in the processing method of the embodiment include those exemplified in the above-mentioned "thiocyanate dehydrogenase". For the detection of the TcDH gene or its gene product, the detection reagent of the above embodiment may be used, and this can be carried out, for example, by the method exemplified in "<Detection Method / Quantification Method>". In this embodiment, the detection step may also detect a protein as the gene product. Various methods can be used to detect the protein, such as ELISA and Western blotting.

[0064] The processing method of this embodiment may further include a second detection step in which a gene or gene product related to the carbonyl sulfide pathway that decomposes the thiocyanate via carbonyl sulfide is detected.

[0065] The carbonyl sulfide pathway, as shown in Figure 1, is a pathway in which thiocyanate is broken down into carbon dioxide and hydrogen sulfide via carbonyl sulfide. Genes involved in the carbonyl sulfide pathway include genes for enzymes involved in the reactions in this pathway, and may be genes for unknown enzymes, carbonyl sulfide degrading enzymes, or thiocyanate hydrolase genes.

[0066] The thiocyanate hydrolase in the processing method of the embodiment has thiocyanase-degrading activity. The thiocyanate hydrolase is assigned the designation "EC 3.5.5.8". Thiocyanate hydrolase activity is the activity of hydrolyzing thiocyanate or thiocyanate ions, and as shown in Figure 1, it may be the activity of decomposing thiocyanate or thiocyanate ions into carbonyl sulfides.

[0067] The presence of thiocyanate hydrolase candidate proteins in thiocyanase can be determined by analyzing the function of the candidate proteins. For example, if a microorganism that does not originally possess thiocyanate degradation ability is given a gene for a candidate thiocyanate hydrolase protein and expressed, then the microorganism can be determined to be thiocyanate hydrolase if it acquires thiocyanate degradation ability.

[0068] The detection of the thiocyanate hydrolase gene or its gene product can be carried out by the same method as in the case of TcDH. For example, it can be carried out by the method described in the examples.

[0069] The target for detection of genes or gene products may be the mixed phase, or a sample obtained by sampling from the mixed phase. The method for extracting nucleic acids or proteins from the sample can be carried out by known methods. Sampling may be performed before or after the processing step, but it is preferable to perform it in the middle of the processing step from the viewpoint of understanding the processing status during the processing step. The detection process may be performed separately from the processing process, or simultaneously. The detection process may also be performed over time during the processing process.

[0070] According to the treatment method of this embodiment, by having a detection step to detect genes or gene products related to the cyanate pathway, it becomes possible to understand the presence and activity status of the cyanate pathway of microorganisms contained in the treated water. Furthermore, it becomes possible to understand the presence and amount of microorganisms that decompose thiocyanate in the cyanate pathway in the treated water.

[0071] Conventionally, in the treatment of wastewater containing thiocyanate, microorganisms that decompose thiocyanate via the carbonyl sulfide pathway have been isolated, and attempts have been made to understand and control the treatment status of thiocyanate by obtaining information about these microorganisms.

[0072] In this study, the inventors discovered that in the treatment of wastewater containing thiocyanate, decomposition of thiocyanate occurs via the cyanate pathway. Furthermore, they found that the cyanate pathway can be dominant over the carbonyl sulfide pathway.

[0073] According to the treatment method of this embodiment, it is possible to monitor the presence and activity status of the cyanate pathway of microorganisms contained in the treated water, as well as the presence and amount of microorganisms that decompose thiocyanate in the cyanate pathway. Therefore, treatment conditions can be controlled to improve the treatment of thiocyanate by the cyanate pathway, thereby improving the treatment efficiency of thiocyanate by the cyanate pathway.

[0074] Since carbonyl sulfide is a greenhouse gas, it is desirable to decompose thiocyanate while suppressing the generation of carbonyl sulfide. According to the treatment method of this embodiment, in addition to monitoring the cyanate pathway, by monitoring the presence and activity status of the carbonyl sulfide pathway of microorganisms contained in the treated water, and the presence and amount of microorganisms that decompose thiocyanate in the carbonyl sulfide pathway, for example, the treatment conditions can be controlled so that the treatment of thiocyanate by the cyanate pathway is improved compared to the carbonyl sulfide pathway, and thiocyanate can be treated while controlling the generation of carbonyl sulfide. [Examples]

[0075] The present invention will be described in detail below with reference to examples. However, the present invention is not limited to these examples.

[0076] [Example 1] (1) Operation of biological wastewater treatment processes, water quality analysis, calculation of decomposition rates, and collection of microbial samples. Microbial samples were obtained in accordance with the method described in Japanese Patent Publication No. 2016-112556. Artificial wastewater (treated water) was prepared by dissolving the solutes shown in Table 1 at the concentrations shown in Table 1 in a solvent obtained by mixing industrial water and natural seawater in a volume ratio of 2:3.

[0077] [Table 1]

[0078] Furthermore, as shown in Figure 2, an integrated biological treatment apparatus 20 (treatment tank) was prepared in which a biological treatment area 20a and a sedimentation area 20b were separated from each other by a partition wall 23 within a single tank, and were connected below the partition wall 23. A sponge carrier 21 [fluidized carrier (AQ-1 manufactured by Kanto Inoac)] measuring 10 mm × 10 mm × 10 mm was placed in the biological treatment area 20a of the biological treatment apparatus 20 at a volume ratio of 20% (v / v).

[0079] In each of the prepared biological treatment devices 20, the water to be treated 24 was introduced, and activated sludge was added as a microbial inoculation source. During the microbial acclimatization treatment (first stage treatment) to establish microorganisms on the sponge carrier 21, the water to be treated 24 was introduced in such a way that its hydraulic residence time was 24 hours. In addition, air aeration 22 was performed on the water to be treated 24 in each biological treatment device 20 to form an aerobic fluidized bed and acclimatize the microorganisms.

[0080] Immediately after the start of this biological treatment, the removal of thiocyanate ions was observed. However, a gradual decrease in pH was observed, and the removal of thiocyanate ions was unstable. Therefore, from the 69th day after the start of operation, the treatment was continued while adjusting the pH to around 7.5 using a 5% by mass sodium hydroxide aqueous solution. The microbial acclimatization treatment (first stage treatment) was terminated when the removal rate of thiocyanate ions stabilized at 98% or more. At the end of this microbial acclimatization treatment (first stage treatment), the amount of nitrite ions had increased.

[0081] After the completion of this microbial acclimatization treatment (first stage treatment), the thiocyanate ion concentration and nitrite ion concentration were measured in the treated water within the biological treatment area 20a of each biological treatment device 20 to monitor thiocyanate ions and nitrite ions. Furthermore, while monitoring the pH value by measuring the pH of the treated water in the biological treatment area 20a of each biological treatment device 20, the inflow rate of the treated water 24 was increased from the 90th day after the start of operation so that the hydraulic residence time in the area was 18 hours (second stage treatment). From the 111th day after the start of operation, the inflow rate of the treated water 24 was further increased so that the hydraulic residence time in the area was 12 hours (third stage treatment). Furthermore, from the 118th day after the start of operation, the inflow rate of the treated water 24 was further increased so that the hydraulic residence time in the area was 8 hours (fourth stage treatment), and operation was continued until the 175th day.

[0082] During this period, by shortening the hydraulic residence time within the region to 18 hours in the second stage of treatment, a decreasing trend in nitrite ion generation was observed while maintaining a high removal rate of thiocyanate ions. Furthermore, by shortening the hydraulic residence time within the region to 12 hours in the third stage of treatment, it was possible to almost completely suppress nitrite ion generation while maintaining a high removal rate of thiocyanate ions. Moreover, it was confirmed that even when the hydraulic residence time within the region was shortened to 8 hours in the fourth stage of treatment, it was possible to maintain a high removal rate of thiocyanate ions while suppressing nitrite ion generation.

[0083] In the biological treatment in Example 1, the daily nitrite production rate and thiocyanate removal rate were calculated according to equations (1) and (2) with respect to the number of operating days.

[0084]

number

[0085]

number

[0086] Figure 3 shows the daily nitrite production rate as a percentage of the number of operating days, and Figure 4 shows the thiocyanate removal rate. Furthermore, for microbial community analysis, sponge carriers 21 to which microorganisms were attached were collected from the biological treatment area 20a of the biological treatment device 20 on the same day as the water quality analysis or within ±1 day, and stored frozen at -20°C.

[0087] (2) DNA extraction DNA extraction was performed from a sponge carrier 21 to which microorganisms were attached within the biological treatment area 20a of the biological treatment apparatus 20. The sponge carrier was collected on day 127, when thiocyanate removal was successful. When the sponge carrier was cut in half, the periphery was brown and the center was black. Since this difference may reflect differences in the environmental conditions (dissolved oxygen content) and microbial flora inside and outside the sponge, the sponge sample was cut and divided according to the color difference. DNA was extracted and purified from both the internal and external sections of the sponge using ISOIL for Beads Beating (Nippon Gene) and the Fast Prep 24 Instrument cell disruption device (MP Biomedicals).

[0088] (3) Determination of base sequences using next-generation sequencers The extracted DNA was analyzed using a next-generation sequencer (Hiseq 2000) to determine its base sequence. This sequencing was outsourced to Takara Bio. As a result, sequence data was obtained from the samples inside and outside the sponge, as shown in Table 2.

[0089] [Table 2]

[0090] (4) Array assembly The assembly was performed using SPAdes as the assembler. The analysis was outsourced (J-Bio21 Center).

[0091] (5) Selection and functional analysis of candidate gene sequences related to thiocyan degradation TcDH derived from the Thiohalobacter thiocyaniticus strain FOKN1 is suspected to be the main enzyme that breaks down thiocyanate (genome Announcement 5:32, e00799-17, 2017). Therefore, we searched for genes with high homology to TcDH derived from the FOKN1 strain in all the contigs obtained from the assembly. As a result, we obtained five open reading frames (ORFs) with high homology to TcDH derived from the FOKN1 strain. As shown in Figure 1, TcDH is involved in thiocyanate degradation via the cyanate pathway. While methods for detecting genes in microorganisms that degrade thiocyanate via the carbonyl sulfide pathway have been reported, methods for detecting TcDH genes in microorganisms that degrade thiocyanate via the cyanate pathway have not yet been reported. The five ORFs obtained above showed high homology to the TcDH derived from the FOKN1 strain, and were therefore determined to be TcDH genes involved in thiocyanate degradation via cyanate. These five TcDH genes were designated as TcDH genes A to E. The nucleotide sequence represented by Sequence ID No. 2 is the nucleotide sequence of TcDH gene A. The nucleotide sequence represented by Sequence ID No. 4 is the nucleotide sequence of TcDH gene B. The nucleotide sequence represented by Sequence ID No. 6 is the nucleotide sequence of TcDH gene C. The nucleotide sequence represented by Sequence ID No. 8 is the nucleotide sequence of TcDH gene D. The nucleotide sequence represented by Sequence ID No. 10 is the nucleotide sequence of TcDH gene E.

[0092] Next, the base sequences of TcDH genes A to E were converted into amino acid sequences. The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of the protein encoded by TcDH gene A. The amino acid sequence represented by SEQ ID NO: 3 is the amino acid sequence of the protein encoded by TcDH gene B. The amino acid sequence represented by SEQ ID NO: 5 is the amino acid sequence of the protein encoded by TcDH gene C. The amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence of the protein encoded by TcDH gene D. The amino acid sequence represented by SEQ ID NO: 9 is the amino acid sequence of the protein encoded by TcDH gene E. These TcDH molecules, each possessing the amino acid sequences represented by sequence numbers 1, 3, 5, 7, and 9, will be hereafter referred to as "TcDH1," "TcDH2," "TcDH3," "TcDH4," and "TcDH5," respectively.

[0093] (6) Design of primers for comprehensive quantification of TcDH1-5 genes involved in thiocyanophage degradation via cyanate. Primers were designed targeting common sequences of the TcDH1-5 gene sequences. Primer3Plus was used for primer design. The designed primer sequences were searched using BLAST to confirm that only the target genes were being quantified. As a result, one set of primers, as shown in Table 3, was designed.

[0094] [Table 3]

[0095] (7) Comprehensive quantification of genes involved in thiocyanogenesis via cyanate For the comprehensive quantification of the TcDH1-5 genes involved in thiocyanophage degradation via cyanate, a calibration curve was created using the above-mentioned primer set with real-time PCR. The real-time PCR reagent used was SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio), and the real-time PCR instrument was Thermal Cycler Dice® Real Time System II MRQ (Takara Bio). The PCR reaction conditions consisted of a hot start reaction at 95°C for 30 seconds, followed by 40 cycles of reactions at 95°C for 5 seconds and 60°C for 30 seconds. As a result, as shown in Figure 5, a good calibration curve was obtained with the designed primer set, and the quantification method was established. In Figure 5, the relationship between x and y represents the equation of the calibration curve. In the equation, y is the gene copy number, x is the Ct value, and R 2 These represent the correlation coefficients, respectively.

[0096] (8) Quantification of genes involved in thiocyanogenesis via carbonyl sulfide Microorganisms that degrade thiocyanate via carbonyl sulfide have been isolated, with Thiobacillus thioparus THI 115 being the only reported example. To date, the only thiocyanate hydrolase whose activity has been evaluated is the gene from the Thiobacillus thioparus THI 115 strain (J. Bacteriol. 1998. 180.2583-2589). Thiocyanate hydrolase consists of three subunits: α, β, and γ. Of these, we designed a thiocyanate hydrolase gene (thiocyanase gene) detection primer targeting the γ subunit, which has a relatively large number of conserved residues, as shown in Table 4.

[0097] [Table 4]

[0098] To quantify the thiocyanase gene involved in thiocyan degradation via carbonyl sulfide, a calibration curve was created using the above primer set with real-time PCR. The real-time PCR reagent used was SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio), and the real-time PCR instrument was Thermal Cycler Dice® Real Time System II MRQ (Takara Bio). The PCR reaction conditions consisted of a hot start reaction at 95°C for 30 seconds, followed by 40 cycles of reactions at 95°C for 5 seconds and 60°C for 30 seconds. As a result, a good calibration curve was obtained, as shown in Figure 6. In Figure 6, the relationship between x and y represents the equation of the calibration curve. In the equation, y is the gene copy number, x is the Ct value, and R 2 These represent the correlation coefficients, respectively.

[0099] (9) Determination of the total number of microorganisms The number of eubacteria genes was quantified as the total number of microorganisms. Many methods for quantification have been reported, so any appropriate method can be used; for example, the method described in Kumar et al, 2012 Bioresource Technology, 113, 148-153 may be used.

[0100] (10) Determination of thiocyanogenic microorganisms that degrade thiocyanate via cyanate 1) DNA extraction from microorganisms attached to sponge carriers in aerobic fluidized beds After dividing the sponge carrier 21, to which microorganisms were attached, within the biological treatment area 20a of the biological treatment device 20 into four sections, DNA extraction and purification were performed using Extrap Soil DNA Plus ver.2 (J-Bio21 Center). DNA extraction was outsourced (J-Bio21 Center). Subsequently, the DNA concentration of the purified DNA solution was measured using PicoGreen dsDNA Assay Kit (Invitrogen).

[0101] The number of TcDH1-5 genes in the DNA extracted as described in 1) above was quantified. For comparison, the number of genes for thiocyanases that degrade thiocyanate via carbonyl sulfide was also quantified. In addition, since the total number of microorganisms in the collected sponge carrier and activated sludge differs, the number of indicator genes of all microorganisms obtained from the sample was separately quantified, and the quantified values ​​of the TcDH1-5 genes and the thiocyanase genes via carbonyl sulfide were normalized by dividing them by the quantified value of the total number of indicator genes of all microorganisms. In this example, the 16S rRNA gene was quantified as the indicator gene for all microorganisms. As a result, as shown in Figure 7, the number of TcDH genes in microorganisms attached to the sponge carrier in the aerobic fluidized bed increased as operation progressed. This suggests that thiocyanide-degrading microorganisms via cyanate were dominant over those via carbonyl sulfide. Furthermore, the number of TcDH genes increased sharply immediately after the start of operation, suggesting that thiocyanide-degrading microorganisms via cyanate contribute significantly to thiocyanide degradation during the startup of the water treatment system. Therefore, it was found that in the aerobic fluidized bed, thiocyanide-degrading microorganisms via cyanate were responsible for the majority of thiocyanide removal from wastewater. In other words, it was found that the number of TcDH genes contained in activated sludge is related to thiocyanide degradation performance, and it was shown that detecting TcDH genes can serve as an indicator for determining thiocyanide removal capacity.

[0102] Each configuration and combination thereof in each embodiment is merely an example, and additions, omissions, substitutions, and other modifications are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited by each embodiment, but is limited only by the scope of the claims. [Explanation of symbols]

[0103] 20...Biological treatment device, 20a...Biological treatment area, 20b...Sedimentation area, 21...Sponge carrier, 22...Air aeration, 23...Partition, 24...Water to be treated, 25...Treated water treated by the biological treatment device

Claims

1. A detection reagent comprising a set of primers comprising the following polynucleotides (1) and (3), which have primer function capable of detecting the thiocyanate dehydrogenase gene or its amplification product, consisting of the amino acid sequence represented by SEQ ID NOs: 1, 3, 5, 7, or 9. (1) Polynucleotides containing the base sequence described in SEQ ID NO: 11 (3) Polynucleotides containing the base sequence described in Sequence ID No. 12

2. A detection method for detecting the thiocyanate dehydrogenase gene or its gene amplification product using the detection reagent described in claim 1.

3. A method for quantifying the thiocyanate dehydrogenase gene or its gene amplification product using the detection reagent described in claim 1.

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