Method for producing microorganisms, microorganisms, microbial preparations, and processing methods

Abstract

One of the objectives of the present invention is to provide a method for producing thiocyanogenic bacteria having improved thiocyanogenic degradation ability. [Solution] One aspect of the present invention is a method for producing sulfur-based COD component degrading bacteria, comprising the step of culturing thiocyanate-degrading bacteria in a culture medium containing thiocyanate or a salt thereof and a copper compound.

Description

Technical Field

[0001] One aspect of the present invention relates to a method for producing a microorganism, a microorganism, a microbial preparation, and a method for treating treated water.

Background Art

[0002] As a biological treatment method applied to wastewater containing a sulfur-based COD component, that is, a reducing sulfur component, a treatment method using sulfur-oxidizing bacteria is known, and the optimal aeration conditions and optimal pH for decomposition vary depending on the bacterial species (Patent Documents 1 to 8, Non-Patent Documents 1 to 4).

[0003] In the treatment method under aerobic sulfur-oxidizing conditions, the sulfur-based COD component serves as an electron donor, and molecular oxygen serves as an electron acceptor, resulting in a sulfur-oxidizing reaction. For example, aerobic sulfur-oxidizing bacteria belonging to the genus Pseudomonas (Patent Document 9) or the genus Paracoccus (Non-Patent Documents 1, 5) perform this reaction. On the other hand, in the treatment method under anoxic sulfur-oxidizing conditions, the sulfur-based COD component serves as an electron acceptor, and nitrite ions or nitrate ions serve as electron acceptors, resulting in a sulfur denitrification reaction. The nitrite ions or nitrate ions that have become electron acceptors are vaporized as nitrogen gas, enabling the simultaneous removal of the sulfur-based COD component and the nitrogen component. For example, denitrifying sulfur-oxidizing bacteria belonging to the genus Thiobacillus (Patent Documents 5, 8, 10) or the genus Thioalkalivirio (Non-Patent Documents 2, 3) perform this reaction. However, there is a problem that the sulfur-oxidizing reaction and the culture rate by this anoxic sulfur-oxidizing bacterium are slower than those of aerobic sulfur-oxidizing bacteria.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

[0005] [Non-licensed Document 1] Katayama, et al., Paracoccus thiocyanatus sp. nov., a new species ofthiocyanate-utilizing facultative chemolithotroph, and transfer of Thiobacillusversutus to the genus Paracoccus as Paracoccus versutus comb. nov. withemendation of the genus, Microbiobgy, 141, 1469-1477, 1995 [Non-licensed Document 2] Sorokin, DY. et al., Denitrification at extremely high pH values ​​by thealkaliphilic, obligately chemolithoautotrophic, sulfur-oxidizing bacteriumThioalkalivibrio denitrificans ALJD, Archives of Microbiology, 175, 94-101, 2001 [Non-licensed Document 3] Sorokin, DY. et al., Thioalkalivibrio nitratireducens sp. Nov., a nitrate-reducing member of an autotrophic denitrifying consortium from a soda lake., International Journal of Systematic and Evolutionary Microbiology, 53, 1779-1783, 2003 [Non-Patent Document 4] Jiang Xiaolin et al., Development of a novel ion chromatography stationary phase for the quantitative determination of trace inorganic anions in seawater, Analytical Chemistry, 57, 991-999, 2008. [Non-Patent Document 5] Understanding the simultaneous biodegradation of thiocyanate and salicylic acid by Paracoccus thiocyanatus and Pseudomonas putida. Int. J. Environ. Sci. Technol. (2016)13:649-662. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Among the genus Paracoccus, microorganisms such as Paracoccus thiocyanatus are known to decompose sulfur-based COD components. Paracoccus thiocyanatus is a thiocyanide-degrading bacterium that can decompose at least thiocyanate.

[0007] Here, wastewater from plants and other sources is treated in treatment facilities such as aeration tanks, where sulfur-based COD components such as thiocyanate are decomposed. In the aeration tank, the decomposition reaction takes place under aerobic conditions in the presence of activated sludge, and sulfur-based COD components are mainly decomposed by the action of microorganisms in the activated sludge. However, the ability to decompose thiocyanate may decrease due to various factors such as reduced oxygen supply due to foaming or a sudden increase in raw water load. In such cases, the ability to decompose thiocyanate can be improved by adding thiocyanate-decomposing bacteria, thereby restoring decomposition efficiency. This restores the ability to decompose sulfur-based COD components such as thiocyanate, ensuring the stability of the entire wastewater treatment process.

[0008] In this case, by adding microorganisms with high thiocyanide degradation capabilities, rapid degradation of thiocyanide can be expected.

[0009] Therefore, one object of the present invention is to provide a method for producing thiocyanogenic bacteria having improved thiocyanogenesis. Another object of the present invention is to provide thiocyanogenic bacteria having improved thiocyanogenesis. Another object of the present invention is to provide a microbial preparation containing thiocyanogenic bacteria having improved thiocyanogenesis. Another object of the present invention is to provide a method for treating water to be treated using thiocyanogenic bacteria having improved thiocyanogenesis. [Means for solving the problem]

[0010] Through diligent research, the inventors discovered that improved thiocyanide-degrading bacteria can be cultured by culturing thiocyanide-degrading bacteria in the presence of thiocyanic acid or its salt and copper compounds, leading to the present invention.

[0011] One aspect of the present invention is described as follows: [1] A method for producing sulfur-based COD-degrading bacteria, comprising the step of culturing thiocyanate-degrading bacteria in a culture medium containing thiocyanate or a salt thereof and a copper compound. [2] The method according to [1], wherein the copper compound is a copper organic acid, a copper inorganic acid, or a copper complex. [3] The method according to any one of [1] to [2], wherein the copper organic acid contains at least one selected from copper citrate, copper gluconate, copper thiocyanate, copper oleate, copper acetate, copper formate, copper tartrate, copper naphthenate, copper neodecanoate, copper teanuzoneate, copper isobutyrate, copper oxalate, copper thiophene-2-carboxylate, copper tetrafluoroborate, copper pyrophosphate, copper propionate, copper benzoate, copper adipate, copper isophthalate, copper stearate, and hydrates thereof. [4] The method according to any one of [1] to [3], wherein the copper inorganic acid contains at least one selected from copper molybdate, copper phosphate, copper sulfate, copper sulfite, copper nitrate, copper carbonate, copper chloride, copper bromide, copper iodide, copper aluminum oxide, copper iodate, copper perchlorate, and hydrates thereof. [5] Copper complexes include bis(2,4-pentanedionate)copper, bis(8-quinolinolate)copper, copper trifluoromethanesulfonate, bis(hexafluoroacetylacetonate)copper, copper ethylacetoacetate, copper benzoylacetonate, copper disodium ethylenediaminetetraacetate, copper bromide-dimethyl sulfide, copper methacrylate, copper hexafluoro-2,4-pentadionate 1,5-cyclooctadiene, tetrakis(acetonitrile)copper hexafluorophosphate, copper trifluoroacetate, copper hexafluoro-2,4-pentanedione, copper bis(2,2,6,6-tetramethyl-3,5-heptanedione), copper hydrogenated triphenylphosphine, copper trifluoroacetylacetonate, bis(cyclohexa The method according to any one of [1] to [4], comprising at least one selected from copper (butyrate), copper chlorophyllin, tetrasodium copper phthalocyanine tetrasulfonate, copper bis(2-ethylhexanoate), copper bis(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato), copper tetrafluoroborate (acetonitrile), copper oxyxin, copper dimethyldithiocarbamate, copper bis-3,5-diisopropylsalicylate, copper bis(acetylacetonate), copper terephthalate, copper acetylacetonate, copper bis(acetylacetone), copper boron hydride bis(triphenylphosphine), copper 3-methylsalicylate, and copper trifluoromethanethiolate. [6] The method according to any one of [1] to [5], wherein the copper compound comprises at least one selected from copper sulfate, copper nitrate, copper carbonate, copper chloride, and hydrates thereof. [7] The method according to any one of [1] to [6], wherein the content of the copper compound in the culture medium is 0.01 mg / L or more and 500 mg / L or less. [8] The method according to any one of [1] to [7], wherein the copper compound comprises an organic copper acid or an inorganic copper acid, and the content of the copper compound in the culture medium is 1 mg / L or more and 500 mg / L or less. [9] A thiocyanide-degrading bacterium is a method of any one of [1] to [8] that degrades at least thiocyanide.

[10] The method according to any one of [1] to [9], wherein the thiocyanate-degrading bacterium is a bacterium belonging to the genus Paracoccus, Afipia, Pseudomonas, or Thiobacillus.

[11] The method according to any one of [1] to

[10] , wherein the thiocyanate-degrading bacterium is Paracoccus thiocyanatus.

[12] The method according to any one of [1] to

[11] , wherein the thiocyanate-degrading bacterium is Paracoccus thiocyanatus strain THI011 (P. Thiocyanatus THI011, NBRC14569).

[13] The method according to any one of [1] to

[12] , wherein the thiocyanate-degrading bacterium contains any one of the following proteins (1) to (3): (1) A protein containing the amino acid sequence represented by SEQ ID NO: 1 (2) Thiocyanate hydrolase containing an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 (3) Thiocyanate hydrolase containing an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 AVHDATGNGTYNNNKKNKGNVNNNNGKYSGTVSANDNSVNNTNAWYNYGRAGVDNNNNHHNAAMNSADNYKGNDNYNTMNNNGANYVNNNSNNWRDRGDNKMYKMRYDGSGTNNSN SVVNDNSATTGMANGVHVSNGVGNNANKYVANADGNKDMVNNTTNDDDNKNVKANRADYDNNARNNTNNHNNNDASTGRNDYNGRKGMKTSHNAMNGNNNMNNADNTAVNVDAWTWH NKNNNGANNNRRHGCCVNVNTNTWNNNANNSTAKGANDHNNNVRNNGTSWTYSNNSVNTNNHNAGNNTSGNNNNACNNVNNNNNAVYRSNDNDNMKWKKNANVNGNGDKYNNNHMGNVNDSRWVNNTNWARKNNNGNNCKVDNKTWKVVANWDTGNDNHTCDCTTDGKYNTTVYSGNNSGNSGNVVNDADTDNNVARNNSNAGHHDHVVVNNSWNGNKASRSTSV(Sequence ID 1).

[14] A thiocyanophilic bacterium obtained by the manufacturing method described in any one of [1] to

[13] .

[15] A thiocyanogenic bacterium as described in

[14] , having a thiocyanogenic activity of 1.0 mg / L-SCN / OD / hr or higher. A microbial preparation containing thiocyanogenic bacteria as described in any one of

[16]

[14] to

[15] . A method for producing the microbial preparation described in

[17] and

[16] , comprising the step of freeze-drying the thiocyanophilic bacteria described in any one of

[14] to

[15] . A method for treating water to be treated, comprising a decomposition step of contacting the water to be treated containing sulfur-based COD components with a thiocyanide-degrading bacterium described in any one of

[14] to

[15] or a microbial preparation described in

[16] to decompose the sulfur-based COD components.

[19] The method according to

[18] , wherein the decomposition step is carried out in the presence of activated sludge.

[20] The method according to

[19] , wherein the decomposition step is carried out under aerobic conditions. [Effects of the Invention]

[0012] One embodiment of the present invention provides a method for producing thiocyanogenic bacteria having improved thiocyanogenesis. Another embodiment of the present invention provides thiocyanogenic bacteria having improved thiocyanogenesis. Another embodiment of the present invention provides a microbial preparation containing thiocyanogenic bacteria having improved thiocyanogenesis. Another embodiment of the present invention provides a method for treating water to be treated using thiocyanogenic bacteria having improved thiocyanogenesis. [Brief explanation of the drawing]

[0013] [Figure 1] This graph shows the time course of the OD660 of the bacterial cells obtained in the main culture in Example 1 (using copper sulfate). [Figure 2] This graph shows the time course of the OD660 of the bacterial cells obtained in the main culture in Example 3 (using copper nitrate). [Figure 3] This graph shows the time course of the OD660 of the bacterial cells obtained in the main culture in Example 4 (using copper gluconate). [Figure 4] This graph shows the results of a decomposition test when the bacterial cells from Example 5 were added to factory wastewater. [Figure 5] This graph shows the results of thiocyanate decomposition by P. thiocyanatus in factory wastewater under the presence of sludge with reduced oscianate decomposition capacity in Example 6. [Figure 6] This graph shows the results of the degradation test when the bacterial cells were formulated by freeze-drying in Example 7. [Modes for carrying out the invention]

[0014] This embodiment is a method for producing sulfur-based COD component-degrading bacteria, comprising the step of culturing thiocyanate-degrading bacteria in a culture medium containing thiocyanate or a salt thereof and a copper compound.

[0015] This embodiment provides a method for producing thiocyan-degrading bacteria having improved thiocyanogenesis. The reason why thiocyan-degrading bacteria with improved thiocyanogenesis can be obtained by culturing in the presence of thiocyanic acid or its salt and a copper compound is presumed to be as follows: Thiocyan-degrading bacteria can grow in a culture medium containing assimilated organic matter. By including a copper ion compound in the culture medium, copper ions bind to the thiocyanase, making the thiocyanase activatable. By adding thiocyanic acid or its salt to a bacterial cell solution in this state, thiocyan-degrading bacteria with improved thiocyanogenesis can be cultured.

[0016] Furthermore, thiocyanate ions (SCN) produced by microorganisms - The decomposition pathways of ) include one that goes via carbonyl sulfide (COS) and one that goes via cyanate ( - There is a pathway that goes through OCN. In the carbonyl sulfide pathway, thiocyanate ion (SCN) - ) is broken down by thiocyanate hydrolase, and it is known that genera such as Thiobacillus (e.g., Thiobacillus thioparus), Afipia, Pseudomonas, and Paracoccus (e.g., Paracoccus thiocyanatus) possess this enzyme. In addition, in the cyanate pathway, it is broken down by thiocyanate dehydrogenase (TcDH), and it is known that genera such as Thioalkalivibrio, Thiohalobacter, and Guyparkeria possess thiocyanate dehydrogenase, which is activated when copper ions bind to 2 or 3 sites per molecule of thiocyanate dehydrogenase.

[0017] The configuration of this embodiment will be described below.

[0018] (Thiocyanide-degrading bacteria and manufacturing method) Thiocyanide-degrading bacteria refer to microorganisms that have the ability to break down thiocyanate. Thiocyanate is a harmful compound that is often produced in the chemical industry, mining, and wastewater treatment processes. Thiocyanide-degrading bacteria play an important role in environmental protection and wastewater treatment because they break down thiocyanate into harmless substances.

[0019] In this specification, "thiocyanate" mainly refers to thiocyanate ions (SCN - ) refers to, and in some cases includes thiocyanic acid or thiocyanate.

[0020] There are no particular restrictions on the thiocyanide-degrading bacteria, but examples include the genera Paracoccus, Afipia, Pseudomonas, Thiobacillus, or Thioalkalivibrio. Of these, Paracoccus, Afipia, Pseudomonas, or Thiobacillus are preferred. Among these, Paracoccus or Afipia are preferred. The thiocyanide-degrading bacteria may be included individually or in combination of two or more species.

[0021] As thiocyanophilic bacteria, the genus Paracoccus is preferred, and Paracoccus thiocyanatus is preferred. Among these, Paracoccus thiocyanatus THI011 (NBRC 14569) and Paracoccus thiocyanatus SST are preferred, with Paracoccus thiocyanatus THI011 (NBRC 14569) being preferred.

[0022] The thiocyanogenic bacteria preferably contain one of the following proteins (1) to (3): (1) A protein containing the amino acid sequence represented by Sequence ID No. 1, (2) A thiocyanate hydrolase containing an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by Sequence ID No. 1, (3) Thiocyanate hydrolases containing an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence represented by Sequence ID No. 1: AVHDATGNGTYNNNKKNKGNVNNNNGKYSGTVSANDNSVNNTNAWYNYGRAGVDNNNNHHNAAMNSADNYKGNDNYNTMNNNGANYVNNNSNNWRDRGDNKMYKMRYDGSGTNNSN SVVNDNSATTGMANGVHVSNGVGNNANKYVANADGNKDMVNNTTNDDDNKNVKANRADYDNNARNNTNNHNNNDASTGRNDYNGRKGMKTSHNAMNGNNNMNNADNTAVNVDAWTWH NKNNNGANNNRRHGCCVNVNTNTWNNNANNSTAKGANDHNNNVRNNGTSWTYSNNSVNTNNHNAGNNTSGNNNNACNNVNNNNNAVYRSNDNDNMKWKKNANVNGNGDKYNNNHMGNVNDSRWVNNTNWARKNNNGNNCKVDNKTWKVVANWDTGNDNHTCDCTTDGKYNTTVYSGNNSGNSGNVVNDADTDNNVARNNSNAGHHDHVVVNNSWNGNKASRSTSV(Sequence ID 1).

[0023] Here, "sequence identity" refers to the percentage obtained by aligning two amino acid sequences to be compared so that as many residues as possible match, and then dividing the number of matching residues by the total number of residues. During the alignment process, gaps are inserted into one or both of the two sequences being compared as needed. Such sequence alignment can be performed using well-known programs such as BLAST, FASTA, or CLUSTALW. When gaps are inserted, the total number of residues is calculated by counting each gap as one residue. If the total number of residues counted in this way differs between the two sequences being compared, the identity (%) is calculated by dividing the number of matching residues by the total number of residues in the longer sequence.

[0024] In this specification, "(amino acid) substitution" preferably refers to substitutions within a group of conserved amino acids that have similar properties such as charge, side chain, polarity, and aromaticity among the 20 amino acids that make up natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). It is known that amino acid substitutions within these groups are unlikely to cause changes in the properties of polypeptides.

[0025] (2) For amino acid sequences specified in (2), sequence identity is 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more. Preferably, the percentages are 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0026] With respect to the amino acid sequences defined in (3), "multiple" means 2 to 90, for example, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0027] The protein consisting of the amino acid sequence of SEQ ID NO: 1 is a thiocyanogenic protein derived from the Paracoccus thiocyanatus THI011 strain, and contains thiocyanate (SCN). - ) is hydrolyzed to produce carbonyl sulfide (COS), ammonia (NH3), and hydroxide ions (HO3). - It has the function of converting to ).

[0028] The activity of microorganisms possessing this enzyme in thiocyanogenesis can be evaluated by the following method. A bacterial strain containing activated thiocyanhydrolase is added to an aqueous solution of thiocyanate and reacted at 30°C for 1 to 5 hours. After the reaction, the thiocyanate ion concentration of the supernatant obtained by centrifugation is quantified. Quantification is performed by a colorimetric method using iron(III) thiocyanate complex. Specifically, 50% of the color reaction solution is added to the reaction solution, and the absorbance at 460 nm is immediately measured at room temperature. The gravimetric concentration is quantified using a calibration curve prepared in advance with an aqueous potassium thiocyanate solution (0 to 200 ppm). The colorimetric reaction solution can be prepared by adding 0.69 g of concentrated nitric acid (1.38%) to 10 mL of 0.1 g / mL iron(III) nitrate nonahydrate, and then adding 9.5 mL of MQ water. After preparation, this reaction solution can be stored in a dark room at room temperature.

[0029] For example, Paracoccus thiocyanatus SST is a microorganism that possesses a protein whose amino acid sequence has more than 80% sequence identity with the thiocyanase protein (thiocyanate hydrolase) derived from the Paracoccus thiocyanatus THI011 strain.

[0030] Furthermore, as mentioned above, in addition to the genus Paracoccus, the genus Afipia can also be used as a thiocyanide-degrading bacterium. Bacteria of the genus Afipia can degrade thiocyanate (SCN) under specific environmental conditions. -Because it has the ability to decompose ) it plays an important role in environmental pollution remediation and wastewater treatment. An example of the genus Afipia is Afipia felis NBRC 106669.

[0031] The culture medium used for culturing thiocyanogenic bacteria is not particularly limited, as long as it contains thiocyanic acid or its salt and copper compounds and is a culture medium capable of culturing thiocyanogenic bacteria.

[0032] The nutrient medium used for culturing thiocyanogenic bacteria can be any medium containing a carbon source, nitrogen source, and inorganic salts necessary for bacterial growth, and the addition of vitamins may be preferable. Furthermore, the addition of amino acids, nucleic acid bases, etc., may also be preferable. As a nutrient medium, for example, a complex medium component derived from natural products containing a nitrogen source and a carbon source can be used. Examples of such complex medium components include peptone (e.g., phyton peptone), yeast extract, meat extract, and corn stippler. Other materials that may be used include dried yeast and soybean meal.

[0033] As a carbon source, in addition to the above-mentioned complex culture medium components, sugars (e.g., glucose, lactose, sorbitol, mannitol, etc.) and organic acids (lactic acid, formic acid, acetic acid, etc.) can be added, and it is preferable to use mannitol as the carbon source. Sugars may be used individually or in combination of two or more. The sugar content in the culture medium is preferably 5 g / L or more, preferably 6 g / L or more, preferably 7 g / L or more, preferably 8 g / L or more, preferably 9 g / L or more, and preferably 10 g / L or more. In the culture of thiocyanophilic bacteria (especially Paracoccus species), by using mannitol as a carbon source, the thiocyanophilic bacteria can be cultured to a high density, and in particular, even when thiocyanic acid or its salt and copper compounds are included, as in this embodiment, the thiocyanophilic bacteria can be cultured to a high density. In one embodiment of this model, thiocyanate-degrading bacteria can be cultured to a high density by adding thiocyanate or its salt and a copper compound to a culture medium containing a high concentration of nutrients, and the thiocyanate-degrading ability can be improved during cultivation. In one embodiment, the culture medium contains 3 g / L to 50 g / L of yeast extract, 1 g / L to 30 g / L of peptone, 3 g / L to 50 g / L of mannitol, and optionally further contains 1 g / L to 30 g / L of meat extract. In another embodiment, the culture medium contains 3 g / L to 30 g / L of yeast extract, 1 g / L to 20 g / L of peptone, and 3 g / L to 30 g / L of mannitol, and optionally further contains 1 g / L to 15 g / L of meat extract.

[0034] In addition to the above-mentioned complex culture medium components, other nitrogen sources such as potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, or urea can be added. These may be used individually or in combination of two or more. The addition ratio varies depending on the type of nitrogen source and should be adjusted as appropriate, for example, between 0.1 g / L and 10 g / L.

[0035] When adding vitamins, the amount of vitamins varies depending on the type of vitamin and should be adjusted as appropriate. Typically, it is 0.1 to 1000 mg per liter of culture medium, preferably 1 to 100 mg. Vitamins may be used individually or in combination of two or more.

[0036] Examples of inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, calcium carbonate, and ammonium heptamolybdate. Inorganic salts may be used individually or in combination of two or more.

[0037] Thiocyanate or its salt is also added to the culture medium. Examples of thiocyanates include sodium thiocyanate and potassium thiocyanate. By adding thiocyanate or its salt to the culture medium, thiocyan-degrading bacteria can be selectively grown. These may be used individually or in combination of two or more. The content of thiocyanate or its salt in the culture medium is not particularly limited as long as an effect of improving thiocyan degradation is obtained. The content of thiocyanate or its salt in the culture medium is, for example, 100 mg / L or more, preferably 200 mg / L or more, and preferably 300 mg / L or more. Alternatively, the content of thiocyanate or its salt in the culture medium is, for example, 3000 mg / L or less, preferably 2000 mg / L or less, preferably 1800 mg / L or less, preferably 1600 mg / L or less, preferably 1400 mg / L or less, preferably 1200 mg / L or less, and preferably 1000 mg / L or less. It is preferable to add thiocyanic acid or its salt all at once to the culture medium at the start of cultivation, but it may also be added as needed during cultivation.

[0038] Copper compounds are also added to the culture medium. By adding copper compounds to the culture medium, copper ions bind to the active site of thiocyanate hydrolase, a thiocyan-degrading enzyme, thereby activating thiocyanate hydrolase and improving the thiocyan-degrading ability of thiocyan-degrading bacteria during cultivation.

[0039] Examples of copper compounds include organic copper acids, inorganic copper acids, or copper complexes. These may be used individually or in combination of two or more.

[0040] Organic copper acids may be at least one selected from, for example, copper citrate, copper gluconate, copper thiocyanate, copper oleate, copper acetate, copper formate, copper methoxyl, copper tartrate, methacryloxyethyl acetacetate, copper ethoxide, capryethylenediamine, copper naphthenate, copper neodecanoate, copper theanuzonate, copper isobutyrate, copper isopropoxide, copper oxalate, copper thiophene-2-carboxylate, copper tetrafluoroborate, copper pyrophosphate, copper propionate, copper benzoate, copper adipate, copper isophthalate, copper stearate, and their hydrates. Inorganic copper acids may be at least one selected from, for example, copper molybdate, copper phosphate, copper sulfate, copper sulfite, copper nitrate, copper carbonate, copper chloride, copper bromide, copper iodide, aluminum copper oxide, copper iodate, copper perchlorate, and their hydrates. Examples of copper complexes include bis(2,4-pentanedionate)copper, bis(8-quinolinolate)copper, copper trifluoromethanesulfonate, bis(hexafluoroacetylacetonate)copper, copper ethylacetoacetate, copper benzoylacetonate, copper disodium ethylenediaminetetraacetate, copper bromide-dimethyl sulfide, copper methacrylate, copper hexafluoro-2,4-pentadionate 1,5-cyclooctadiene, tetrakis(acetonitrile)copper hexafluorophosphate, copper trifluoroacetate, copper hexafluoro-2,4-pentanedione, copper bis(2,2,6,6-tetramethyl-3,5-heptanedione), copper hydrogenated triphenylphosphine, and copper trifluoroacetylacetonate. It may be at least one selected from bis(cyclohexanebutyrate)copper, chlorophyllin copper, copper phthalocyanine tetrasulfonate tetrasodium, bis(2-ethylhexanoate)copper, bis(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato)copper, tetrakis(acetonitrile)copper tetrafluoroborate, oxine copper, dimethyldithiocarbamate copper, bis-3,5-diisopropylsalicylate copper, bis(acetylacetonate)copper, terephthalate copper, acetylacetonate copper, bis(acetylacetone)copper, boron hydride bis(triphenylphosphosphine)copper, 3-methylsalicylate copper, and trifluoromethanethiolate copper.

[0041] As the copper compound, organic copper oxides or inorganic copper oxides are preferred. Among these, the inorganic copper oxide is preferably at least one selected from copper sulfate, copper nitrate, copper carbonate, copper chloride, and their hydrates.

[0042] The content of the copper compound in the culture medium is not particularly limited as long as an improvement in thiocyanide decomposition is obtained. For example, the content of the copper compound in the culture medium is 0.01 mg / L or more and 500 mg / L or less. Preferably, the content of the copper compound in the culture medium is 1 mg / L or more, preferably 2 mg / L or more, preferably 3 mg / L or more, preferably 4 mg / L or more, preferably 5 mg / L or more, preferably 6 mg / L or more, preferably 7 mg / L or more, preferably 8 mg / L or more, preferably 9 mg / L or more, preferably 10 mg / L or more, preferably 11 mg / L or more, preferably 12 mg / L or more, preferably 13 mg / L or more, preferably 14 mg / L or more, and preferably 15 mg / L or more. When the content of the copper compound is 1 mg / L or more, an improvement in thiocyanide decomposition can be effectively obtained. Furthermore, the content of the copper compound in the culture medium is preferably 200 mg / L or less, preferably 150 mg / L or less, preferably 100 mg / L or less, and preferably 80 mg / L or less. When the content of the copper compound is 200 mg / L or less, thiocyan-degrading bacteria can be cultured without or substantially without being affected by the growth inhibitory effect of the copper compound on thiocyan-degrading bacteria.

[0043] The molar concentration of copper (Cu) in the culture medium is not particularly limited, but is preferably 6 μM or more, preferably 8 μM or more, preferably 10 μM or more, preferably 12 μM or more, preferably 14 μM or more, preferably 16 μM or more, preferably 18 μM or more, preferably 20 μM or more, preferably 30 μM or more, preferably 40 μM or more, preferably 50 μM or more, and preferably 60 μM or more.

[0044] When preparing the culture medium, all components should be kept sterile. Sterilization methods include, for example, heat sterilization using an autoclave (121°C, 20 minutes) or filtration sterilization (0.22 μm filter). Since there is a concern about reactions between culture medium components during heat sterilization, copper compounds, thiocyanic acid or its salts, and sugars may be sterilized individually before being mixed.

[0045] The pH of the culture medium is not particularly limited, but is for example 5 to 10, preferably 6 to 9.5. The culture temperature is not particularly limited, but is for example 15 to 40°C, preferably 20 to 35°C, preferably 25 to 32°C. The culture time is for example 8 to 60 hours, preferably 10 to 48 hours, preferably 12 to 36 hours, preferably 14 to 30 hours, preferably 16 to 24 hours. Longer culture times result in a higher OD660, making it easier to culture bacteria at high density, but the thiocyanogenesis activity of the resulting thiocyan-degrading bacteria may decrease. Therefore, a culture time of 24 hours or less is preferable, but those skilled in the art can select it as appropriate.

[0046] The culture may be carried out under aerobic or anaerobic conditions, but it is preferable to carry it out under aerobic conditions. Examples of aerobic conditions include shaking culture or aeration stirring culture. It is preferable to control the dissolved oxygen concentration within a certain range. The dissolved oxygen concentration can be controlled, for example, by changing the stirring speed, aeration rate, internal pressure, etc. The dissolved oxygen concentration is, for example, 0.1 to 20 ppm, preferably 0.3 to 10 ppm, preferably 0.5 to 7 ppm, and preferably 1 to 5 ppm.

[0047] The cultivation of thiocyanide-degrading bacteria is not particularly limited, but can be carried out using liquid culture, solid culture, or a combination thereof. From the viewpoint of obtaining a large quantity of bacterial cells, cultivation is preferably carried out using liquid culture.

[0048] The culture is carried out in an appropriate culture vessel. The culture vessel can be selected as appropriate depending on the culture volume, and examples include test tubes, flasks, and fermenters.

[0049] There are no particular restrictions on the culture method; for example, batch, half-batch, fed-batch, or continuous methods may be used.

[0050] In this embodiment, the number of thiocyan-degrading bacteria or the concentration of thiocyanocytes after cultivation can be measured or determined by OD660. In this embodiment, the OD660 of the culture solution after cultivation is preferably 7.0 or higher, preferably 7.5 or higher, and preferably 8.0 or higher. In one embodiment, thiocyan-degrading bacteria can be cultured under conditions where the thiocyanocyte-degrading bacteria are cultured at a concentration of 1 g / L or higher, and the thiocyanocyte-degrading ability of the thiocyanocyte-degrading bacteria is 4.0 g-SCN / kg-DCW / hr or higher. Such conditions have not been reported conventionally.

[0051] The thiocyanogenesis activity (mg / L-SCN / OD / hr) of thiocyanogenic bacteria can be measured, for example, by the method described in "Evaluation of Thiocyanogenesis Degradation" in [Examples]. The obtained degradation activity can then be divided by the converted value of turbidity and dry bacterial weight (0.401 in the case of Paracoccus Thiocyanatu THI011) to calculate the thiocyanogenesis degradation capacity (g-SCN / kg-DCW / hr).

[0052] In this embodiment, the thiocyanogenesis activity of the obtained thiocyan-degrading bacteria is preferably 1.0 mg / L-SCN / OD / hr or higher, preferably 2.0 mg / L-SCN / OD / hr or higher, preferably 3.0 mg / L-SCN / OD / hr or higher, preferably 4.0 mg / L-SCN / OD / hr or higher, preferably 5.0 mg / L-SCN / OD / hr or higher, preferably 6.0 mg / L-SCN / OD / hr or higher, preferably 7.0 mg / L-SCN / OD / hr or higher, preferably 8.0 mg / L-SCN / OD / hr or higher, preferably 9.0 mg / L-SCN / OD / hr or higher, and preferably 10.0 mg / L-SCN / OD / hr or higher. The thiocyanogenesis activity of the thiocyan-degrading bacteria can be measured, for example, by the method described in "Evaluation of Thiocyanogenesis Degradation Ability" in [Examples].

[0053] Cultured thiocyanogenic bacteria can be obtained in forms such as cell concentrate, wet cells, or dried cells. Cell concentrate can be obtained by centrifuging or membrane filtration concentration of the culture solution. Wet cells can be obtained by centrifuging or filtering the culture solution. Dried cells can be obtained by drying the wet cells or cell concentrate using a general drying method.

[0054] The obtained bacterial cells can be recovered by centrifugation, washed with sterile phosphate buffer (PBS), and recovered again by centrifugation. In this state, they can be frozen and stored for more than 3 months at -80°C.

[0055] (Microbial preparations) The microbial preparation according to this embodiment contains thiocyanide-degrading bacteria obtained by the manufacturing method according to this embodiment. The thiocyanide-degrading bacteria obtained by the manufacturing method according to this embodiment have improved thiocyanide-degrading activity.

[0056] In addition to the thiocyanide-degrading bacteria mentioned above, the microbial preparation may include other bacterial cells, additives, and / or carriers as needed. Specific examples of microbial preparations include those containing the thiocyanide-degrading bacteria, other bacterial cells, additives, and carriers.

[0057] Other bacterial organisms are not particularly limited as long as they do not render the thiocyanide-degrading bacteria inoperable, but examples include bacteria that contain thiocyanide and have the ability to decompose target components in wastewater. One type of bacteria may be used alone, or two or more types may be used in combination.

[0058] The additives are not particularly limited, but examples include protective agents (e.g., cryoprotectants), surfactants, dispersants, and auxiliary agents. The type and concentration of the additives can be appropriately determined under conditions that do not kill the thiocyanide-degrading bacteria or cause loss of thiocyanide-degrading ability. Additives may be used individually or in combination of two or more types.

[0059] Examples of protective agents include skim milk, trehalose, dimethyl sulfoxide (DMSO), polyethylene glycol, propylene glycol, glycerin, polyvinylpyrrolidone, sorbitol, or dextran. These may be used individually or in combination of two or more.

[0060] The carrier is not particularly limited, but examples include inorganic fine particle carriers. The inorganic fine particle carrier may be a metal and its inorganic salt or oxide, or it may contain carbon or be chemically classified as an inorganic substance. One type of carrier may be used alone, or two or more types may be used in combination.

[0061] In this embodiment, the means for formulating the thiocyan-degrading bacteria are not particularly limited as long as the thiocyan-degrading ability is not lost, and known formulation methods can be used. The microbial formulation may be in liquid or solid form (including capsule form, agar form, powder form, etc.), or it may be a frozen or lyophilized form. If the microbial formulation is liquid, it may be a bacterial suspension suspended in a culture medium, buffer solution, physiological saline, etc. The liquid may be acidic or neutral. If the microbial formulation is solid or lyophilized, for example, cultured bacteria may be concentrated and then dried or lyophilized appropriately to obtain a solid or lyophilized form. Excipients may be added at that time.

[0062] In one embodiment, the microbial preparation may include powdered bacterial cells obtained by pulverizing thiocyanide-degrading bacteria. One type of thiocyanide-degrading bacteria may be pulverized alone, or two or more types may be combined and pulverized. Alternatively, powdered bacterial cells obtained by pulverizing different strains separately may be mixed together. When pulverizing the bacterial cells, known methods such as spray drying, fluidized bed drying, and freeze-drying can be used as appropriate. When pulverizing, the bacterial cells may be mixed with a base material (also called a protective agent or dispersant) such as sugars, skim milk, or chelating agents before pulverization.

[0063] A method for producing a microbial preparation may include, for example, a step of culturing the bacterial cells and then recovering the bacterial cells from the culture. The recovery of the bacterial cells may also be carried out by methods known in the art, for example, by centrifuging the culture and then recovering the resulting precipitate. In a method for producing a microbial preparation, it is preferable to freeze-dry the recovered bacterial cells (thiocyanide-degrading bacteria). It is preferable to use an additive as a cryoprotective agent, and the method may further include a step of mixing the recovered bacterial cells with the additive and then freeze-drying the resulting mixture. Freeze-drying may also be carried out by methods known in the art. The freeze-dried bacterial cells obtained by freeze-drying may be in a viable state. Furthermore, the freeze-dried bacterial cells may contain a cryoprotective agent, which can suppress the decrease in the viable state of the bacterial cells.

[0064] (Processing method) The method for treating water according to this embodiment includes a decomposition step in which thiocyan-degrading bacteria obtained by the manufacturing method according to this embodiment, or a microbial preparation according to this embodiment, are brought into contact with water to be treated containing sulfur-based COD components to decompose the sulfur-based COD components.

[0065] The conditions for the decomposition process can be appropriately set by those skilled in the art. For example, the temperature may be 15-40°C or 20-35°C. Also, the pH may be 6.0-10.0 or 6.5-9.5.

[0066] The decomposition process is preferably carried out in the presence of activated sludge. Furthermore, the decomposition process is preferably carried out under aerobic conditions, for example, by using an aeration tank.

[0067] The treatment method according to this embodiment can perform the decomposition step by, for example, bringing the water to be treated, activated sludge containing microorganisms capable of decomposing COD components, and the thiocyanide-degrading bacteria or microbial preparation obtained by the manufacturing method according to this embodiment into contact in an aeration tank. For example, the thiocyanide-degrading bacteria or microbial preparation obtained by the manufacturing method according to this embodiment can be added to an aeration tank containing the water to be treated and activated sludge containing microorganisms capable of decomposing COD components. The timing of addition is not particularly limited and can be appropriately determined by those skilled in the art, for example, by monitoring the thiocyanide concentration of the water to be treated and adding the product when the thiocyanide concentration exceeds a specified value.

[0068] The amount of thiocyanide-degrading bacteria and microbial preparation according to this embodiment added to the activated sludge can be appropriately set considering the conditions of the water to be treated, such as thiocyanide concentration, the surrounding environment, the volume of the reaction system, etc.

[0069] There are no particular restrictions on the types of water to be treated, but examples include domestic wastewater and industrial wastewater. Furthermore, groundwater or environmental water contaminated with domestic wastewater or industrial wastewater may also be included as treated water. Environmental water refers to water from public water bodies (rivers, lakes, harbors, coastal waters, public ditches, irrigation canals, and other water bodies or canals used for public purposes (excluding sewers)). While there are no particular restrictions on industrial wastewater, examples include food processing wastewater, beverage manufacturing wastewater, fermentation and brewing wastewater, pharmaceutical industry wastewater, chemical industry wastewater, textile industry wastewater, scouring and dyeing wastewater, oil and fat industry wastewater, sewage, or community wastewater.

[0070] In the aeration tank, oxygen is supplied to the tank, and the thiocyanide-degrading bacteria and aerobic microorganisms in the activated sludge decompose organic matter in the treated water. This reduces the COD of the treated water.

[0071] After treatment in the aeration tank, the mixture of activated sludge and treated water, with reduced COD, is supplied to the sedimentation tank, where sedimentation and separation treatment is performed. Specifically, in the sedimentation tank, the supernatant liquid is flowed down as treated water for subsequent treatments, and the settled sludge is discharged from the bottom as withdrawn sludge.

[0072] Through the above process, treated water with reduced COD can be produced from the water to be treated. [Examples]

[0073] The present invention will be described in more detail below with reference to examples. The present invention is not limited to the following examples, and modifications using conventional techniques in the art of the present invention are possible.

[0074] [Bacterial cell stock solution] For cultivation, the microorganism (P. Thiocyanus strain THI011) was inoculated into PtSCN medium (5 g / L meat extract, 5 g / L peptone, 5 g / L yeast extract, 2.5 g / L sodium chloride, 0.5 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium thiocyanate), cultured at 30°C for 24 hours, and then glycerol was added to a final concentration of 10% to prepare a bacterial stock solution. The bacterial stock solution was stored at -80°C. During cultivation, the bacterial stock solution was added to the culture medium.

[0075] [Evaluation of thiocyanate resolution] The thiocyanide degradation ability of bacterial cells was evaluated by the following method. After culturing, the bacterial cells were collected by centrifugation and washed with phosphate buffer. Next, a certain amount of washed bacterial cells (the amount of cells that resulted in an OD660 of 7.5 when suspended in 2 mL of phosphate buffer) was subjected to artificial wastewater containing 300-800 mg / L of potassium thiocyanate (e.g., 400 mg / L) (0.5 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate heptahydrate, 0.01 g / L iron(III) chloride hexahydrate, 0.01 g / L calcium chloride dihydrate, 0.01 g / L yeast extract). After contacting the mixture (25 g / L, zinc sulfide heptahydrate 0.08 mg / L, copper sulfate pentahydrate 0.04 mg / L, boric acid 0.04 mg / L, disodium molybdate dihydrate 0.04 mg / L, cobalt(II) chloride hexahydrate 0.04 mg / L, pH 8.0) in a 14 mL tube, 150 μL was taken and centrifuged (20,000 g, 4 °C, 3 min), and the resulting supernatant was used as the pre-reaction sample. The remainder after taking the pre-reaction sample was shaken at 30 °C and 180 rpm for 3 hours. After 3 hours, the reaction mixture was centrifuged (20,000 g, 4 °C, 3 min), and the supernatant was collected to remove bacterial cells from the artificial wastewater. Subsequently, 50 μL of thiocyanate color reaction solution was added to 100 μL of the collected pre- and post-reaction artificial wastewater, and the absorbance at 460 nm was immediately measured. In addition, potassium thiocyanate aqueous solutions ranging from 0 to 200 mg / L (0, 50, 100, 150, and 200 mg / L) were measured simultaneously, and a calibration curve was created and used to quantify the thiocyanate ions in the reaction solution.

[0076] The color reaction solution was prepared by dissolving 1 g of iron(III) nitrate nonahydrate in 10 mL of MQ water, adding 0.69 g of 1.38% concentrated nitric acid, and finally adding 9.5 mL of MQ water. The prepared color reaction solution was stored in a dark room at room temperature and used within six months of preparation.

[0077] The difference in thiocyanate concentration in the reaction solution before and after the reaction (mg / L) was divided by the cell mass (OD660 = 7.5) and reaction time (3 hours) to calculate the degradation activity of the thiocyan-degrading bacteria (mg / L-SCN / OD / hr). The calculated degradation activity was then divided by the conversion value (0.401) of the OD value and dry cell weight (DCW) of the Paracoccus Thiocyanatus THI011 strain to calculate the degradation capacity of the thiocyan-degrading bacteria (g-SCN / kg-DCW / hr).

[0078] (Example 1) In Example 1, P. Thiocyanatus strain THI011 was cultured and evaluated using a medium containing a copper compound (copper sulfate pentahydrate).

[0079] The bacterial stock solution was added to 20 mL of pre-culture medium (2.5 g / L sodium chloride, 0.9 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate heptahydrate, 10 g / L yeast extract, 5 g / L phyton peptone, 10 g / L D-mannitol) in a 200 mL flask, and the cells were inoculated. Pre-culture was then performed at 30°C and 200 rpm for 30 hours.

[0080] Next, pre-culture solution was added to 20 mL of the main culture medium in a 200 mL flask (2.5 g / L sodium chloride, 0.9 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate heptahydrate, 10 g / L yeast extract, 5 g / L phyton peptone, 10 g / L D-mannitol, 1 g / L potassium thiocyanate, and copper sulfate pentahydrate (0 mg / L, 3 mg / L, 4 mg / L, 8 mg / L, or 16 mg / L)) so that the absorbance at 660 nm (OD660) was 0.05, and the culture was incubated at 30°C and 200 rpm for 18 to 24 hours. Figure 1 shows the change in OD660 of the bacterial cells obtained in this culture over time.

[0081] After the main culture, each cell was collected, washed with phosphate buffer, and then pelletized by centrifugation. The resulting cell pellets were frozen at -80°C. The cell pellets were formed so that the OD660 was 7.5 when suspended in 2 mL of phosphate buffer. After freezing, each cell pellet was resuspended in 2 mL of artificial wastewater (400 mg / L potassium thiocyanate) to bring the cells into contact with the artificial wastewater, and reacted at 30°C and 180 rpm for 3 hours. Subsequently, the concentration of thiocyanate in the artificial wastewater was quantified, and the thiocyanate degradation rate was calculated as the thiocyanate degradation activity. The thiocyanate degradation activity and culture cell concentration (OD660) are shown in Table 1.

[0082] [Table 1]

[0083] From Example 1, it was confirmed that when bacteria were cultured in a culture medium containing thiocyanate and a copper compound (copper sulfate pentahydrate), bacterial cells with improved thiocyanogenesis were obtained. Furthermore, it was confirmed that the thiocyanogenesis of the obtained bacterial cells improved with increasing content of the copper compound (copper sulfate pentahydrate) in the culture medium. Specifically, when the content of the copper compound (copper sulfate pentahydrate) in the culture medium was 8 mg / L or more, P. Thiocyanatus with a thiocyanogenesis of 5 mg / L or more per hour per OD could be cultured to an OD660 of 7.5 or more. In addition, a tendency for thiocyanogenesis to decrease with longer culture times was observed, but when the content of the copper compound in the culture medium was 16 mg / L, an effect of suppressing the decrease in thiocyanogenesis due to long-term culture was confirmed. Therefore, from the viewpoint of obtaining an effect of suppressing the decrease in thiocyanogenesis due to long-term culture, it was found that a high content of the copper compound is desirable, for example, 10 mg / L or more is desirable.

[0084] Furthermore, in this embodiment, it was confirmed that when the Paracoccus thiocyanatus THI011 strain (NBRC 14569), isolated from thiocyanate-concentrated activated sludge in Japan, was added to a natural culture medium containing mannitol, bacterial cells with an OD660 of 10 or higher could be obtained within 24 hours of incubation.

[0085] In another study, adding a certain amount of copper ion compounds such as copper sulfate along with potassium thiocyanate improved the thiocyanate degradation ability during culture, and we were able to obtain bacterial cells with a degradation ability of 25 g-SCN / kg-DCW / hr at a concentration of 2.8 g / L.

[0086] (Example 2) In Example 2, the effect of a copper compound (copper sulfate pentahydrate) on the growth of P. thiocyanatus THI011 strain was investigated.

[0087] 2 mL of a culture medium prepared to contain copper sulfate pentahydrate between 0 and 100 mg / L (sodium chloride 2.5 g / L, dipotassium hydrogen phosphate 0.9 g / L, magnesium sulfate heptahydrate 0.05 g / L, yeast extract 10 g / L, phyton peptone 5 g / L, D-mannitol 5 g / L, potassium thiocyanate 400 mg / L) was placed in a 14 mL test tube. Next, a bacterial stock solution was added to the culture medium and inoculated, and the cells were incubated at 30°C and 180 rpm for 16 hours. Table 2 shows the cell concentration (OD660) of P. thiocyanatus after incubation and the relative inhibition ratio of growth for copper compounds (copper sulfate pentahydrate). The relative inhibition ratio (%) is the ratio (%) of "OD660 at the target copper compound concentration" to "OD660 without copper compound (0 mg / L)".

[0088] [Table 2]

[0089] From Example 2, it was confirmed that bacterial growth was inhibited as the content of copper compounds (copper sulfate pentahydrate) in the culture medium increased, but bacterial growth was still observed even when the copper compound content was 100 mg / L.

[0090] (Example 3) The bacterial cells were cultured and evaluated in the same manner as in Example 1, except that copper nitrate trihydrate was used instead of copper sulfate pentahydrate, and the content of copper nitrate trihydrate in the culture medium was adjusted to 0-64 μM (0 μM, 8 μM, 16 μM, 32 μM, 64 μM). Figure 2 shows the change in OD660 of the bacterial cells obtained in this culture over time. Table 3 shows the thiocyanogenesis activity and the concentration of cultured bacterial cells (OD660). Note that 8 μM of copper nitrate trihydrate corresponds to approximately 1.9 mg / L.

[0091] [Table 3]

[0092] From Example 3, it was confirmed that even when copper nitrate trihydrate was used as the copper compound, bacterial cells with improved thiocyanide decomposition ability could be obtained. Furthermore, it was confirmed that even when copper nitrate trihydrate was used as the copper compound, the thiocyanide decomposition ability of the obtained bacterial cells improved as the content of the copper compound in the culture medium increased.

[0093] (Example 4) Except for using copper gluconate instead of copper sulfate pentahydrate and adjusting the copper gluconate content in the culture medium to 0-64 μM, the bacterial cells were cultured and evaluated in the same manner as in Example 1. The bacterial cell concentration after this culture is shown in Figure 3. The thiocyanogenesis activity and cultured bacterial cell concentration (OD660) are shown in Table 4.

[0094] [Table 4]

[0095] From Example 4, it was confirmed that even when copper gluconate was used as the copper compound, bacterial cells with improved thiocyanide decomposition ability could be obtained. Furthermore, it was confirmed that even when copper gluconate was used as the copper compound, the thiocyanide decomposition ability of the obtained bacterial cells improved as the content of the copper compound in the culture medium increased.

[0096] (Example 5) In Example 5, a decomposition test (OD=10+ flask, single run) was performed when bacterial cells were added to factory wastewater.

[0097] Bacterial cells were collected from the culture medium obtained in the same manner as in Example 1 (copper sulfate pentahydrate 16 mg / L) and washed with phosphate buffer. After washing, a bacterial cell suspension in 10 mL of phosphate buffer (OD660=10) was obtained. 10 mL of factory wastewater containing thiocyanate was added to the bacterial cell suspension, transferred to a 100 mL flask, and the decomposition reaction was carried out at 30°C and 180 rpm. As shown in Figure 4, complete decomposition of thiocyanate in the wastewater was confirmed on the 5th day.

[0098] (Example 6) In Example 6, a decomposition test (OD=10 + sludge + flask, single run) was conducted when bacterial cells were added to factory wastewater together with activated sludge. Activated sludge from a factory wastewater treatment facility was collected in a bench facility, and factory wastewater was continuously fed into it with the aeration blocked. The condition was maintained for more than two weeks under which the thiocyanate concentration in the influent wastewater reached 100% and was discharged into the wastewater after sludge passage, thereby preparing activated sludge with reduced thiocyanate decomposition ability.

[0099] Using bacterial cells recovered from a culture solution obtained in the same manner as in Example 1 (copper sulfate pentahydrate 16 mg / L), the ability to degrade thiocyanate in factory wastewater in the presence of deactivated sludge was tested. Specifically, the decomposition reaction was carried out in the same manner as in Example 5, except that 10 mL of factory wastewater containing deactivated sludge equivalent to MLSS = 17,600 ppm was added instead of the 10 mL of factory wastewater containing thiocyanate used in Example 5. Figure 5 shows the results of thiocyanate degradation by P. thiocyanatus in factory wastewater in the presence of sludge with reduced thiocyanate degradation ability.

[0100] As shown in Figure 5, thiocyanide decomposition by thiocyanide-degrading bacteria (P. Thiocyanatus) was observed 4 days after the start of the reaction. On the other hand, thiocyanide decomposition was not observed with only deactivated sludge, confirming that the above-mentioned thiocyanide-degrading bacteria can decompose thiocyanide in factory wastewater even in the presence of deactivated sludge.

[0101] (Example 7) In Example 7, a degradation test was conducted on bacterial cells formulated by freeze-drying. Bacterial cells recovered from the culture medium obtained by the method described in Example 1, in which the copper sulfate pentahydrate concentration was changed to 20 mg / L, were concentrated fivefold by centrifugation, and then frozen. After freezing, the cells were dried in a freeze-dryer for more than 7 hours. The resulting powdered thiocyanide-degrading bacteria were stored under refrigerated or frozen conditions for one week, and a degradation test was conducted one week later under the same conditions as in Example 1. It was confirmed that the degradation activity was maintained at more than 80% even after formulation (Figure 6).

[0102] The upper and / or lower limits of the numerical ranges described herein can be arbitrarily combined to define a preferred range. For example, the upper and lower limits of the numerical ranges can be arbitrarily combined to define a preferred range, the upper limits of the numerical ranges can be arbitrarily combined to define a preferred range, and the lower limits of the numerical ranges can be arbitrarily combined to define a preferred range.

[0103] The claims following this disclosure are expressly incorporated herein into this disclosure, and each claim stands independently as a separate embodiment. This disclosure includes all instances in which an independent claim is replaced by its dependent claim. Furthermore, any additional embodiments derived from the independent claims and subsequent dependent claims are also expressly incorporated herein into this specification.

[0104] Although this embodiment has been described in detail above, the specific configuration is not limited to this embodiment, and any design changes that do not depart from the gist of this disclosure are also included in this disclosure. [Sequence Listing Free Text]

[0105] SEQ ID NO: 1: Thiocyanide-degrading protein of Paracoccus thiocyanatus THI011

Claims

1. A method for producing sulfur-based COD component-degrading bacteria, comprising the step of culturing thiocyanate-degrading bacteria in a culture medium containing thiocyanate or a salt thereof and a copper compound.

2. The method according to claim 1, wherein the copper compound is an organic copper acid, an inorganic copper acid, or a copper complex.

3. The method according to claim 1, wherein the organic copper acid comprises at least one selected from copper citrate, copper gluconate, copper thiocyanate, copper oleate, copper acetate, copper formate, copper tartrate, copper naphthenate, copper neodecanoate, copper theanuzonate, copper isobutyrate, copper oxalate, copper thiophene-2-carboxylate, copper tetrafluoroborate, copper pyrophosphate, copper propionate, copper benzoate, copper adipate, copper isophthalate, copper stearate, and hydrates thereof.

4. The method according to claim 1, wherein the inorganic copper oxide comprises at least one selected from copper molybdate, copper phosphate, copper sulfate, cuprous sulfite, copper nitrate, copper carbonate, copper chloride, copper bromide, copper iodide, aluminum copper oxide, copper iodate, copper perchlorate, and hydrates thereof.

5. The copper complexes include bis(2,4-pentanedionate)copper, bis(8-quinolinolate)copper, copper trifluoromethanesulfonate, bis(hexafluoroacetylacetonate)copper, copper ethylacetoacetate, copper benzoylacetonate, copper disodium ethylenediaminetetraacetate, copper bromide-dimethyl sulfide, copper methacrylate, copper hexafluoro-2,4-pentadionate 1,5-cyclooctadiene, tetrakis(acetonitrile)copper hexafluorophosphate, copper trifluoroacetate, copper hexafluoro-2,4-pentanedione, copper bis(2,2,6,6-tetramethyl-3,5-heptanedionate), copper hydrogenated triphenylphosphine, copper trifluoroacetylacetonate, and bis(s The method according to claim 1, comprising at least one selected from copper chlorohexanobutyrate, copper chlorophyllin, tetrasodium copper phthalocyanine tetrasulfonate, copper bis(2-ethylhexanoate), copper bis(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato), copper tetrakis(acetonitrile) tetrafluoroborate, copper oxine, copper dimethyldithiocarbamate, copper bis-3,5-diisopropylsalicylate, copper bis(acetylacetonate), copper terephthalate, copper acetylacetonate, copper bis(acetylacetone), copper boron hydride bis(triphenylphosphine), copper 3-methylsalicylate, and copper trifluoromethanethiolate.

6. The method according to claim 1, wherein the copper compound comprises at least one selected from copper sulfate, copper nitrate, copper carbonate, copper chloride, and hydrates thereof.

7. The method according to claim 1, wherein the content of the copper compound in the culture medium is 0.01 mg / L or more and 500 mg / L or less.

8. The method according to claim 1, wherein the copper compound comprises an organic copper acid or an inorganic copper acid, and the content of the copper compound in the culture medium is 1 mg / L or more and 500 mg / L or less.

9. The method according to claim 1, wherein the thiocyanide-degrading bacteria degrade at least thiocyanide.

10. The method according to claim 1, wherein the thiocyanide-degrading bacteria belong to the genera Paracoccus, Aphipia, Pseudomonas, or Thiobacillus.

11. The method according to claim 1, wherein the thiocyanide-degrading bacterium is Paracoccus thiocyanatus.

12. The method according to claim 1, wherein the thiocyanide-degrading bacterium is Paracoccus thiocyanatus THI011 strain (P. Thiocyanatus THI011, NBRC14569).

13. The method according to claim 1, wherein the thiocyanogenic bacteria contain any of the following proteins (1) to (3): (1) A protein containing the amino acid sequence represented by Sequence ID No. 1, (2) A thiocyanate hydrolase containing an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by Sequence ID No. 1, (3) Thiocyanate hydrolases containing an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence represented by Sequence ID No. 1: AVHDATGNGTYNNNKKNKGNVNNNNGKYSGTVSANDNSVNNTNAWYNYGRAGVDNNNNHHNAAMNSADNYKGNDNYNTMNNNGANYVNNNSNNWRDRGDNKMYKMRYDGSGTNNSN SVVNDNSATTGMANGVHVSNGVGNNANKYVANADGNKDMVNNTTNDDDNKNVKANRADYDNNARNNTNNHNNNDASTGRNDYNGRKGMKTSHNAMNGNNNMNNADNTAVNVDAWTWH NKNNNGANNNRRHGCCVNVNTNTWNNNANNSTAKGANDHNNNVRNNGTSWTYSNNSVNTNNHNAGNNTSGNNNNACNNVNNNNNAVYRSNDNDNMKWKKNANVNGNGDKYNNNHMGNVNDSRWVNNTNWARKNNNGNNCKVDNKTWKVVANWDTGNDNHTCDCTTDGKYNTTVYSGNNSGNSGNVVNDADTDNNVARNNSNAGHHDHVVVNNSWNGNKASRSTSV (Sequence ID 1).

14. Thiocyanide-degrading bacteria obtained by the manufacturing method described in claim 1.

15. The thiocyanogenic bacterium according to claim 14, wherein the thiocyanogenic activity is 1.0 mg / L-SCN / OD / hr or more.

16. A microbial preparation containing the thiocyanide-degrading bacteria described in claim 14.

17. A method for producing the microbial preparation described in claim 16, comprising the step of freeze-drying the thiocyanide-degrading bacteria described in claim 14.

18. A method for treating water to be treated, comprising a decomposition step of contacting the water to be treated containing sulfur-based COD components with a thiocyanide-degrading bacterium according to claim 14 or 15 or a microbial preparation according to claim 16, thereby decomposing the sulfur-based COD components.

19. The method according to claim 18, wherein the decomposition step is carried out in the presence of activated sludge.

20. The method according to claim 19, wherein the decomposition step is carried out under aerobic conditions.

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