Method for reducing selenium in selenium-contaminated samples

Abstract

A method for reducing selenium in a selenium-contaminated sample using selenium-reducing bacteria is provided. [Solution] A method for reducing selenium in a selenium-contaminated sample, in which the selenium-contaminated sample containing selenium-reducing bacteria is left to stand or stirred under anaerobic conditions in the presence of lactic acid, wherein the selenium-reducing bacteria is at least one species selected from the group consisting of bacteria A having a 16S rRNA gene that is 97% or more identical to a specific base sequence, and bacteria B having a 16S rRNA gene that is 97% or more identical to a specific base sequence different from the above base sequence.

Description

[Technical Field]

[0001] The present invention relates to a method for reducing selenium in a selenium-contaminated sample using selenium-reducing bacteria. [Background technology]

[0002] Selenium is a naturally occurring element found in sulfide or sulfur deposits. While essential for the human body, selenium poses a health risk if ingested in large quantities. Therefore, it is designated as a Type 2 specified hazardous substance under the Environmental Basic Act and the Environmental Quality Standards for Water Pollution under the Soil Contamination Countermeasures Act. Therefore, in-situ purification and insolubilization treatment of contaminated water may be implemented as a countermeasure for contaminated groundwater exceeding the standard level or contaminated soil from which selenium has leached. Cases where naturally occurring selenium can be a problem include the treatment of excavation debris generated during tunnel construction, wastewater generated from tunnels due to spring water, etc., measures to prevent contamination of surrounding soil, groundwater, and rivers caused by scattering or leakage from excavation debris during removal, transportation, and storage, and the treatment of wastewater at industrial waste treatment plants and sludge treatment plants.

[0003] However, existing treatment materials are only slightly effective against selenium, and complex treatment equipment is required to purify contaminated water. Therefore, there is a need for a treatment technology that is highly effective against selenium-contaminated samples. The form of selenium in the natural environment is tetravalent selenite (SeO3 2- ) or hexavalent selenium (SeO4 2- ), and are often found mixed together in the natural environment. The difficulty in reducing hexavalent selenium is thought to be what makes selenium treatment difficult. When treating hexavalent selenium, it is common to first reduce it to tetravalent selenium before treatment (see, for example, Patent Document 1), but because this requires a two-stage treatment, a reduction method that is effective for all valences is desired. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-205697 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for reducing selenium in a selenium-contaminated sample using selenium-reducing bacteria. [Means for solving the problem]

[0006] The present inventors have conducted extensive research into selenium reduction treatments and have discovered that by leaving a selenium-contaminated sample containing specific selenium-reducing bacteria to stand or stir under anaerobic conditions in the presence of lactic acid, the selenium-reducing bacteria, which are present in extremely low relative abundance (%), become active and can reduce tetravalent and hexavalent selenium. They have also discovered that selenium reduction treatment of selenium-contaminated samples is possible by bioaugmentation using a culture containing the selenium-reducing bacteria. Based on these findings, the present invention has been completed.

[0007] That is, the present invention relates to the following 1) to 10). 1) A method for reducing selenium in a selenium-contaminated sample, in which the selenium-contaminated sample containing selenium-reducing bacteria is left to stand or stirred under anaerobic conditions in the presence of lactic acid, wherein the selenium-reducing bacteria is at least one species selected from the group consisting of bacteria A having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 1 and bacteria B having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 2. 2) The method according to 1), wherein the selenium-contaminated sample further comprises a bacterium C having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO:3. 3) A method for reducing selenium in a selenium-contaminated sample, comprising the steps of applying a culture of selenium-reducing bacteria to the selenium-contaminated sample and allowing it to stand or stir in the presence of lactic acid under anaerobic conditions, wherein the selenium-reducing bacteria include at least one species selected from the group consisting of bacteria A having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 1 and bacteria B having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 2. 4) The method according to 3), wherein the selenium-reducing bacteria are bacteria collected from a selenium-contaminated sample. 5) The method according to 1) or 3), wherein the bacterium A is Paradesulfitobacterium aromaticivorans or a microorganism phylogenetically identical thereto, and the bacterium B is Intrasporangium calvum or a microorganism phylogenetically identical thereto. 6) The method according to 1) or 3), wherein the standing or stirring under anaerobic conditions is standing or stirring under anaerobic conditions at 10 to 40°C for 12 hours or more. 7) The selenium-contaminated sample contains bacteria D having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 4, bacteria E having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 5, bacteria F having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 6, bacteria G having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 7, bacteria H having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 8, bacteria I having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 9, bacteria J having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 10, bacteria K having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 11, bacteria K having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 12, bacteria K having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 13, bacteria K having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 14, bacteria K having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 15, bacteria K having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 16, bacteria K having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 17 The method according to 3), wherein the bacteria include one or more species selected from the group consisting of bacterium L having a 16S rRNA gene, bacterium M having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 13, bacterium N having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 14, bacterium O having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 15, bacterium P having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 16, bacterium Q having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 17, bacterium R having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 18, bacterium S having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 19, and bacterium T having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 20. 8) The method according to 1) or 3), wherein the selenium-contaminated sample is one or more selected from excavated waste, soil, sludge, water, incineration ash, and industrial wastewater containing selenium. 9) The method according to 1) or 3), which comprises the step of adding one or more selected from phosphoric acid, organic acids other than lactic acid, alcohols and sugars to the selenium-contaminated sample. 10) A method for evaluating the selenium-reducing ability of a selenium-contaminated sample, comprising a step of confirming the presence in the sample of at least one species selected from the group consisting of bacteria A having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 1 and bacteria B having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 2. [Effects of the Invention]

[0008] According to the present invention, by utilizing the selenium reducing ability of selenium-reducing bacteria, it is possible to reduce selenium in selenium-contaminated samples. [Brief explanation of the drawings]

[0009] [Figure 1] Bioaugmentation test flow. [Figure 2] Microbial abundance (16S rRNA gene copy number per gram of excavated waste) at the start and end of the bioaugmentation test. DETAILED DESCRIPTION OF THE INVENTION

[0010] In the present invention, the range "X to Y" means "at least X and at most Y." Unless otherwise specified, the operations and measurements of physical properties are performed under the conditions of room temperature cv (20 to 25°C) and relative humidity 40 to 50% RH.

[0011] In the present invention, unless otherwise specified, the term "identity to a base sequence" refers to the percentage of identical nucleotides shared between two base sequences when the two sequences are optimally aligned. That is, identity (%) can be calculated as follows: (number of identical positions / total number of positions) × 100, and can be calculated using commercially available algorithms. Such algorithms are incorporated into the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215 (1990) 403-410. More specifically, searches and analyses of base sequence identity can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, ClustalW, etc.). When using a program, parameters can be appropriately set by those skilled in the art, or the default parameters of each program can be used. Specific techniques for these analysis methods are also well known to those skilled in the art.

[0012] In the present invention, "OTU (operational taxonomic unit)" refers to a unit obtained when classifying the base sequences of essential bacterial genes (16S ribosomal RNA genes) on a computer using their similarity as an index.

[0013] In the present invention, the term "selenium-reducing bacteria" refers to bacteria that can reduce tetravalent and hexavalent selenium as electron acceptor substrates.

[0014] As described in the Examples below, we investigated the relationship between the state of microbial communities in excavation waste extracted from a tunnel construction site and their activity in reducing tetravalent and hexavalent selenium. We found that the relative abundance of these microorganisms increased when lactic acid was added under anaerobic conditions, and identified useful microorganisms involved in selenium reduction (selenium-reducing bacteria A and B). Therefore, by utilizing the selenium-reducing ability of the selenium-reducing bacteria, it is possible to reduce selenium in selenium-contaminated samples, and the presence of the selenium-reducing bacteria in a sample can be used as an indicator to evaluate the selenium-reducing ability of the sample.

[0015] In one aspect of the present invention, there is provided a method for reducing selenium in a selenium-contaminated sample, the method comprising the step of allowing or stirring a selenium-contaminated sample containing at least one selenium-reducing bacterium selected from the group consisting of bacterium A having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 1 and bacterium B having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 2 in the presence of lactic acid under anaerobic conditions (treatment method 1). According to this method, by utilizing the selenium reducing ability of selenium-reducing bacteria in a selenium-contaminated sample, selenium reduction treatment becomes possible without using any special treatment material.

[0016] The bacterium having a 16S rRNA gene consisting of the nucleotide sequence of SEQ ID NO: 1 is a selenium-reducing bacterium identified as OTU9931, and its closest relative has been determined to be Paradesulfitobacterium aromaticivorans (also referred to as "P. aromaticivorans") (16S rRNA gene sequence identity is 99.6%). Therefore, in the present invention, Bacterium A (also referred to as "Selenium-reducing bacterium A") having a 16S rRNA gene with 97% or more identity to the nucleotide sequence of SEQ ID NO: 1 includes P. aromaticivorans and microorganisms phylogenetically identical to P. aromaticivorans. Furthermore, a bacterium having a 16S rRNA gene consisting of the base sequence of SEQ ID NO: 2 is a selenium-reducing bacterium identified as OTU13678, and its closest relative has been determined to be Intrasporangium calvum (also referred to as "I. calvum") (16S rRNA gene sequence identity is 100%). Therefore, in the present invention, bacterium B (also referred to as "selenium-reducing bacterium B") having a 16S rRNA gene with 97% or more identity to the base sequence of SEQ ID NO: 2 includes I. calvum or microorganisms phylogenetically identical thereto. P. aromaticivorans is a selenium-reducing bacterium belonging to the phylum Desulfitobacteriia, and I. carbam is a selenium-reducing bacterium belonging to the phylum Actinobacteriota. P. aromaticivorans is registered in the GenBank of the National Center for Biotechnology Information (NCBI) under the accession number NR_116427, and I. carbam under the accession number NR_115639. Both are available from the German Collection of Microorganisms and Cell Cultures (DSMZ).

[0017] The selenium-contaminated sample used in treatment method 1 may contain either selenium-reducing bacteria A or B, but preferably contains selenium-reducing bacteria A, and more preferably contains both selenium-reducing bacteria A and B. In addition to selenium-reducing bacteria A and B, the selenium-contaminated sample may also contain bacteria C, etc., which have a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 3. Here, the bacterium having a 16S rRNA gene consisting of the base sequence of SEQ ID NO: 3 is a selenium-reducing bacterium identified as OTU12242, and its closest relative has been determined to be Rubrivivax gelatinosus (also referred to as "R. gelatinosus") (16S rRNA gene sequence identity is 99.2%). Therefore, bacterium C (also referred to as "selenium-reducing bacterium C") having a 16S rRNA gene with 97% or more identity to the base sequence of SEQ ID NO: 3 includes R. gelatinosus or microorganisms phylogenetically identical to R. gelatinosus.

[0018] In the present invention, the "selenium-contaminated sample" is not particularly limited as long as it contains selenium, and may be any of groundwater, soil such as ground or bedrock (specifically, excavated waste from constructing underground structures such as tunnels), soil and sludge generated during the construction of underground structures, wastewater, incineration ash, and other waste materials.

[0019] In another aspect of the present invention, there is provided a method for reducing selenium in a selenium-contaminated sample (Treatment Method 2), which comprises applying a culture of selenium-reducing bacteria, the culture comprising at least one bacterium selected from the group consisting of Bacterium A having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 1 and Bacterium B having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 2, to the selenium-contaminated sample and allowing the sample to stand or stir under anaerobic conditions in the presence of lactic acid. Such selenium reduction treatment in which a culture of selenium-reducing bacteria is applied to a selenium-contaminated sample is also referred to as bioaugmentation. Here, the term "culture of selenium-reducing bacteria" refers to a culture solution or a dried product thereof obtained by cultivating selenium-reducing bacteria containing at least one species selected from the group consisting of Bacterium A and Bacterium B. The culture also includes a microbial culture collected from a treated product obtained by treating a selenium-contaminated sample containing at least one species of selenium-reducing bacteria selected from the group consisting of Bacterium A and B shown in Treatment Method 1 under anaerobic conditions in the presence of lactic acid. The microbial culture may be subcultured in an anaerobic medium containing selenium and lactic acid as components.

[0020] In bioaugmentation, the selenium-contaminated sample to which the culture is applied may be a selenium-contaminated sample containing at least one selenium-reducing bacterium selected from the group consisting of the above-mentioned bacteria A and bacteria B, but it may also be a different selenium-contaminated sample (for example, a selenium-contaminated sample not containing selenium-reducing bacteria, or a selenium-contaminated sample containing selenium-reducing bacteria different from bacteria A and bacteria B). Such bioaugmentation allows selenium-reducing bacteria (including at least one of bacteria A and B) in the culture and selenium-reducing bacteria (including, for example, selenium-reducing bacteria different from bacteria A and B) in the selenium-contaminated sample to grow actively.

[0021] Examples of selenium-contaminated samples containing selenium-reducing bacteria different from bacteria A and bacteria B include those containing one or more species selected from the group consisting of bacteria C to T below. Bacterium C having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 3 (also referred to simply as "selenium-reducing bacterium C" in this specification), Bacterium D having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 4 (also referred to simply as "selenium-reducing bacterium D" in this specification), Bacterium E having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 5 (also referred to simply as "selenium-reducing bacterium E" in this specification), Bacterium F having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 6 (also referred to simply as "selenium-reducing bacterium F" in this specification), Bacterium G having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 7 (also referred to simply as "selenium-reducing bacterium G" in this specification), Bacterium H having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 8 (also referred to simply as "selenium-reducing bacterium H" in this specification), Bacterium I having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 9 (also referred to simply as "selenium-reducing bacterium I" in this specification), Bacterium J having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 10 (also referred to simply as "selenium-reducing bacterium J" in this specification), Bacterium K having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 11 (also referred to simply as "selenium-reducing bacterium K" in this specification), Bacterium L having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 12 (also referred to simply as "selenium-reducing bacterium L" in this specification), Bacterium M having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 13 (also referred to simply as "selenium-reducing bacterium M" in this specification), Bacterium N having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 14 (also referred to simply as "selenium-reducing bacterium N" in this specification), Bacterium O having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 15 (also referred to simply as "selenium-reducing bacterium O" in this specification), Bacterium P having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 16 (also referred to simply as "selenium-reducing bacterium P" in this specification), Bacterium Q having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 17 (also referred to simply as "selenium-reducing bacterium Q" in this specification), Bacterium R having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 18 (also referred to simply as "selenium-reducing bacterium R" in this specification), Bacterium S having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 19 (also referred to simply as "selenium-reducing bacterium S" in this specification), Bacterium T (also referred to herein simply as "selenium-reducing bacterium T") having a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 20.

[0022] Table 1 shows selenium-reducing bacteria having 16S rRNA genes consisting of the base sequences of SEQ ID NOs: 3 to 20. These selenium-reducing bacteria were identified in the Examples described below.

[0023] [Table 1]

[0024] In the present invention, the selenium-contaminated sample to be subjected to selenium reduction treatment may contain approximately 4,000 to 5,000 microbial species. However, the relative abundance of the selenium-reducing bacteria of the present invention (e.g., bacteria A to T) in this microbial community is not particularly limited and may be extremely low compared to the total microbial species.

[0025] The selenium-reducing bacteria are present in extremely small amounts in samples, making their isolation technically difficult. Therefore, they have not been deposited with a depository institution. However, the applicant guarantees that selenium-contaminated samples that are the subject of the selenium reduction treatment of the present invention will be distributed to third parties if they fall under any of the items of Article 27-3 of the Enforcement Regulations of the Patent Act of Japan, provided that the relevant laws and regulations are observed.

[0026] In the present invention, selenium-reducing bacteria in a sample containing multiple microorganisms (for example, selenium-contaminated soil) can be identified by the following steps A to C. Step A: recovering nucleic acids from an uncultured sample containing selenium; and analyzing the base sequence of a target gene present in the nucleic acid using a next-generation sequencer to determine the relative abundance (1) of each microorganism. Step B: Initiating the cultivation of the sample in the presence of tetravalent and hexavalent selenium and lactic acid to obtain a culture; recovering nucleic acids from the culture; and analyzing the base sequence of the target gene present in the nucleic acid using a next-generation sequencer to determine the relative abundance (2) of each microorganism. Step C: A step of determining that a microorganism having a higher relative abundance (2) than the relative abundance (1) is a selenium-reducing bacterium.

[0027] Selenium-reducing bacteria that have reduced tetravalent and hexavalent selenium are more abundant relative to the amount before reduction. Specifically, an uncultured sample and a sample cultured with tetravalent and hexavalent selenium and lactate are prepared, and the DNA obtained from each sample is analyzed and compared. In samples in which selenium reduction and a decrease in lactate concentration are observed, microorganisms whose relative abundance in the sample cultured with tetravalent and hexavalent selenium and lactate is greater than that in the uncultured sample are considered to be microorganisms that have grown by reducing tetravalent and hexavalent selenium in the presence of lactate. This allows the identification of microorganisms that reduce tetravalent and hexavalent selenium in the presence of lactate (selenium-reducing bacteria).

[0028] In steps A and B, the following three steps are carried out to determine the relative abundance of microorganisms in a selenium-containing sample. (i) supplementing a selenium-containing sample with tetravalent and hexavalent selenium and lactic acid, and culturing microorganisms contained in the sample; (ii) recovering nucleic acids (e.g., DNA) from samples before and after incubation with tetravalent and hexavalent selenium and lactate; (iii) Analyzing the base sequence of the target gene (e.g., 16S rRNA gene) present in the recovered nucleic acid using a next-generation sequencer.

[0029] The above items (i) to (iii) will be specifically explained below. (i) Adding tetravalent and hexavalent selenium and lactic acid to a selenium-containing sample and culturing the microorganisms contained in the sample. It is preferable to add water to the selenium-containing sample used for the culture in order to improve the reactivity between the microorganism and tetravalent and hexavalent selenium. The microbial culture is carried out in an airtight container after aeration with CO2 / N2 or N2 to create a reducing atmosphere, i.e., anaerobic conditions, which induces biological selenium reduction.

[0030] The selenium-containing sample is mixed with tetravalent and hexavalent selenium and lactic acid, and the microorganism is cultured in the presence of tetravalent and hexavalent selenium and lactic acid. Microbial culture can be performed by adding lactic acid to a selenium-containing sample along with tetravalent and hexavalent selenium, from the viewpoint of maintaining the state of the microbial community. However, other components such as phosphoric acid may also be added as needed. In particular, the addition of phosphoric acid (e.g., potassium hydrogen phosphate), organic acids other than lactic acid (e.g., acetic acid), alcohols (e.g., ethanol), and sugars (e.g., glucose) is preferred for the growth of selenium-reducing bacteria.

[0031] The concentrations of tetravalent and hexavalent selenium can be adjusted appropriately, taking into account concentration fluctuations in actual environments (0.000015 to 0.04 mM). In one embodiment of the present invention, the culture of the plurality of microorganisms is initiated in the presence of 0.000127 to 0.02 mM of tetravalent and hexavalent selenium. When the tetravalent and hexavalent selenium concentrations are 0.000127 mM or higher before culture, the selenium-reducing bacteria can reduce tetravalent and hexavalent selenium even during the preferred culture period described below. When the tetravalent and hexavalent selenium concentrations are 0.02 mM or lower, changes in the state of the microbial community can be suppressed. Therefore, in the method of identifying selenium-reducing bacteria of the present invention, it is preferable to initiate the culture of the microorganisms in the presence of 0.000127 to 0.02 mM of tetravalent and hexavalent selenium. Furthermore, 1.5 M of lactic acid is required stoichiometrically to reduce 1 M of hexavalent selenium to zero, but it is preferable to add lactic acid to a contaminated sample in an amount 1 to 500 times the stoichiometrically required concentration.

[0032] The culture temperature of the microorganisms may be set taking into account annual temperature changes in the actual environment. From the viewpoint of being able to detect selenium-reducing bacteria active in the actual environment, the culture temperature of the microorganisms is preferably in the range of 10 to 40°C, more preferably 20 to 30°C. A culture temperature of 10°C or higher allows for confirmation of the reduction of tetravalent and hexavalent selenium. A culture temperature of 40°C or lower allows for suppression of changes in the microbial community.

[0033] During the cultivation of the microorganisms, the culture solution may be left standing or stirred. Conventionally known culture equipment can be used for culturing the microorganisms. The culture time for the microorganism is, for example, 12 hours or more, preferably 24 hours or more.

[0034] (ii) recovering nucleic acids from the culture; The method for recovering nucleic acids from the culture obtained in (i) is not particularly limited, and any conventionally known method can be used. The nucleic acids to be recovered may be either DNA or RNA. Methods for recovering DNA include disrupting microbial cells using, for example, physical disruption with beads (glass beads, mixed zirconia and silica beads, etc.), ultrasonic disruption, disruption using a French press, or disrupting a sample frozen in liquid nitrogen with a mortar and pestle, followed by removing proteins using conventional methods (such as phenol-chloroform extraction), digesting RNA with RNase, and recovering DNA.

[0035] (iii) Analyzing the base sequence of the target gene present in the collected nucleic acid using a next-generation sequencer. The target gene used to identify selenium-reducing bacteria is preferably the V4 region of the 16S rRNA gene, as this is suitable for large-scale phylogenetic analysis. The term "next-generation sequencer" is used in contrast to "first-generation sequencer," a fluorescent capillary sequencer that uses the Sanger method. It refers to a device that uses sequential DNA synthesis with DNA polymerase or DNA ligase to comprehensively analyze fragments with read lengths of tens to thousands of base pairs for tens to hundreds of millions of DNA fragments in a massively parallel manner. Next-generation sequencers use a different sequencing principle than first-generation sequencers, which use the Sanger method, which uses dideoxynucleotides to stop DNA polymerase elongation. Examples of such principles include synthetic sequencing, pyrosequencing, and ligase reaction sequencing. To date, many companies and research institutions have provided a variety of next-generation sequencers, including HiSeq2500 (Illumina Inc.), MiSeq (Illumina Inc.), 5500xl SOLiD (registered trademark) (Thermo Fisher Scientific), Ion Proton (registered trademark) (Thermo Fisher Scientific), Ion PGM (registered trademark) (Thermo Fisher Scientific), and GS FLX+ (Roche Diagnostics). Analysis using a next-generation sequencer can be performed according to the accompanying instructions, allowing identification of tens of thousands to hundreds of thousands of microbial species (OTUs) and analysis of the relative abundance (%) of each microbial species.

[0036] The determination of whether the microorganism is a selenium-reducing bacterium in step C can be made by comparing the relative abundance of the microorganism before culture (1) with the relative abundance of the microorganism after culture with the addition of tetravalent and hexavalent selenium and lactic acid (2). For example, among the OTUs, microorganisms whose abundance after cultivation has increased to more than twice that at the start of cultivation in a culture in which selenium reduction has been observed are identified. In the cultures in which reduction of tetravalent and hexavalent selenium was observed, microorganisms whose levels increased by more than two-fold compared to the uncultured state reduced tetravalent and hexavalent selenium in the presence of lactic acid and grew, and therefore such microorganisms can be determined to be selenium-reducing bacteria.

[0037] The selenium reduction treatment of a selenium-contaminated sample containing the selenium-reducing bacteria of the present invention is carried out in the presence of lactic acid. The amount of lactic acid used is not limited as long as it allows the selenium-reducing bacteria of the present invention to sufficiently grow in the contaminated sample and exert their reducing power, but it is preferable to carry out the treatment so that the lactic acid content in the contaminated sample (in a dry state) is 0.014% by mass or more. For example, the content of lactic acid in a contaminated sample is preferably 0.014 to 0.70 mass %, more preferably 0.14 to 0.70 mass %.

[0038] There are no particular limitations on the method for adding lactic acid to a contaminated sample, and methods that can be used include spraying lactic acid directly onto the contaminated sample using a sprayer, or dissolving lactic acid in a solvent such as water and spraying the solution onto the contaminated sample using a spray nozzle, irrigation machine, etc. When lactic acid is dissolved in a solvent such as water, it is preferable to prepare a solution having a concentration of, for example, 0.9 to 90.08 g / L, preferably 0.9 to 45.0 g / L, and more preferably 9.0 to 45.0 g / L.

[0039] The treatment conditions may be any conditions that allow the selenium-reducing bacteria in the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention to grow and exert their reducing power, and may be left standing or stirred under anaerobic conditions. Examples include aeration with CO2 / N2 or N2, followed by leaving standing or stirring in an airtight container, preferably at 10 to 40°C, more preferably at 20 to 30°C, for 12 hours or more, preferably 18 hours or more, more preferably 24 hours or more, and preferably 4500 hours or less, more preferably 1500 hours or less. Furthermore, the selenium-contaminated sample may be converted into a reduced state by adding iron salts, sulfites, oxalic acid, or other reducing agents to facilitate the exertion of the reducing power of the selenium-reducing bacteria.

[0040] For example, selenium in a selenium-contaminated sample containing the selenium-reducing bacteria of the present invention can be reduced by adding lactic acid to the sample and then leaving it to stand or stirring for 12 hours or more at 10 to 40° C. Furthermore, by adding another selenium-contaminated sample to a tank containing the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention and lactic acid, the selenium in the other selenium-contaminated sample can also be reduced.

[0041] In addition, other components such as phosphoric acid may be added to the reduction treatment as needed. In particular, it is preferable to include a step of adding one or more of phosphoric acid (e.g., potassium hydrogen phosphate), organic acids other than lactic acid (e.g., acetic acid), alcohols (e.g., ethanol), and sugars (e.g., glucose) in order to promote the growth of selenium-reducing bacteria.

[0042] In yet another aspect of the present invention, there is provided a method for evaluating the selenium-reducing ability of a selenium-contaminated sample, comprising the step of confirming the presence in the sample of at least one species selected from the group consisting of Bacterium A having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO:1 and Bacterium B having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO:2.

[0043] The means for identifying the selenium-reducing bacteria is not particularly limited, but a preferred method is to analyze the bacterial species present in a sample based on the base sequence of the 16S rRNA gene contained in the bacterial flora genomic DNA. That is, as described above, nucleic acid is recovered from a sample, and the base sequence of the target gene present in the nucleic acid is analyzed using a next-generation sequencer to confirm the relative abundance (%).

[0044] This allows the selenium-reducing ability of a selenium-contaminated sample to be evaluated. Here, the selenium-reducing ability of a sample refers to the selenium-reducing ability of the sample due to the selenium-reducing bacteria. [Example]

[0045] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0046] Example 1: Search for selenium-reducing bacteria (1) Selection of excavated waste with selenium reducing ability Five types of tunnel excavation muck (N, G, H, Z, N2) generated during construction work in various locations were prepared (Table 2), and test solutions were prepared in accordance with the method described in Environment Agency Notification No. 18, "Establishing Measurement Methods for Soil Leaching Surveys," to determine the leaching characteristics of selenium, arsenic, lead, and fluorine, and measurements were made in accordance with "JIS K 0102:2013 (Factory Wastewater Testing Methods)."

[0047] [Table 2]

[0048] The following test was carried out to evaluate the selenium reduction ability of the selenium-reducing bacteria contained in each excavated waste. 1) Preculture Five grams of drilling debris was placed in a 50 ml glass vial, and sterile water or Widdel medium was added. N2CO2 (N2 / CO2 = 80 / 20) was bubbled through for 5 minutes to aerate the mixture. The vial was then immediately sealed and allowed to stand in a dark place at 25°C for at least one week.

[0049] 2) Main culture The pre-cultured excavation waste was dispensed into vials (2-3 ml) using a spatula, and the culture medium was Widdel medium. The gas phase of the vial was aerated to N2CO2 (N2 / CO2 = 80 / 20), and the vial was immediately sealed. A lactic acid solution (500 mM sodium lactate solution) was added through the vial's rubber stopper using a syringe, bringing the lactic acid concentration in the culture to 10 mM. Similarly, a hexavalent selenium solution (500 mg / L) was added, bringing the selenium concentration in the culture to 5 mg / L. The culture was then statically cultured for 3 weeks in a dark place at 25°C. After 3 weeks, the selenium concentration of the culture was measured according to the method described in the Ministry of the Environment Notification No. 18, "Establishing the measurement method for soil elution amount survey." The results are shown in Table 3.

[0050] [Table 3]

[0051] Se reduction was the fastest in excavation waste N, with selenium levels decreasing to low levels. Therefore, excavation waste N was subjected to enrichment culture.

[0052] 3) Enrichment culture 20 ml of Widdel freshwater medium was placed in a 50 ml vial, and the gas phase was replaced with N2CO2 (N2 / CO2 = 80 / 20), then the vial was sealed. A bacterial colony (first-generation culture) derived from excavation waste N was inoculated into the medium at 5 vol%, and a lactic acid solution and a hexavalent selenium solution were added. Static culture was then carried out in a dark place at 25°C for 7 to 14 weeks. This culture was designated the second-generation culture. After 7 to 14 weeks, the selenium concentration of the culture was measured, and it was confirmed to have decreased to approximately 0.03 mg / L. The second culture was grown under the same conditions for 10 -1 ~10 -4The bacteria were inoculated at a dilution ratio of 1000 and cultured statically in a dark place at 25°C for 7 to 14 weeks. After 7 to 14 weeks, the selenium concentration of the culture was measured and confirmed to have decreased sufficiently. This culture was designated as the third generation. 3rd generation dilution ratio 10 -4 The culture was grown under the same conditions for 10 -1 ~10 -6 The bacteria were inoculated at a dilution ratio of 10 and cultured statically in a dark place at 25°C for 7 to 14 weeks. After 7 to 14 weeks, the selenium concentration of the culture was measured and confirmed to have decreased sufficiently. This culture was used as the fourth generation and was diluted 10 times. -2 Microbial DNA analysis was performed on the cultures.

[0053] 4) Microbial phylogenetic analysis 4th generation dilution ratio 10 -2 The culture samples were centrifuged to obtain a centrifugal precipitate. Zirconia / Silica beads (average particle size 0.1 mm) were added to the centrifugal precipitate, and the microbial cells in the solid phase were disrupted using a Shake Master Auto (Biomedical Science Co., Ltd.). Coexisting proteins were then removed through a purification step using phenol and chloroform. RNA was then digested using RNase A (Beckman) and DNA was purified. Using this purified DNA as a template, Q5 High-Fidelity DNA Polymerase was used to target the V4 region of the 16S rRNA gene (approximately 300 base pairs). Polymerase chain reaction (PCR) was performed using DNA polymerase (NEB). The resulting PCR products were purified using the AMPure XP kit (Beckman Coulter) and the Wizard® SV gel and PCR clean-up kit (Promega). The concentrations of the PCR products were measured using the Quant-iT PicoGreen dsDNA Reagent Kit (Thermo Fisher Scientific) and a Nanodrop 3300 fluorometer (Thermo Fisher Scientific).

[0054] The optimal amount of purified product was subjected to large-scale base sequence analysis using 300 cycles of the MiSeq Reagent Kit and a next-generation sequencer, MiSeq (Illumina, Inc.). Tens of thousands of gene fragment reads were decoded from each sample, and nonspecific sequences were removed using the software mothur (ver. 1.31.2). Microbial phylogenetic analysis was performed using the software QIIME (ver. 1.6.0), and microbial species (OTU: Operational Taxonomic Unit) were identified and their relative abundance (%) was analyzed.

[0055] The top 10 microbial species by relative abundance are shown in Table 4. The gene sequence identity of the OTUs to their related species was determined using the BLAST program in the NCBI (National Center for Biotechnology Information) base sequence database.

[0056] [Table 4]

[0057] As shown in Table 4, the microbial species in the culture were OTU9931 (belonging to selenium-reducing bacteria A), OTU13678 (belonging to selenium-reducing bacteria B), and OTU12242 (belonging to selenium-reducing bacteria C), which accounted for 96.6% of the total, with selenium-reducing bacteria A and B accounting for 92.77% of the total. The closely related species of OTU9931 is Paradesulfitobacterium aromaticivorans (16S rRNA gene sequence identity: 99.6%). The closely related species of OTU13678 is Intrasporangium calvum (16S rRNA gene sequence identity: 100%). The closest relative of OTU12242 is Rubrivivax gelatinosus (16S rRNA gene sequence identity: 99.2%).

[0058] Example 2 Treatment of selenium-contaminated samples by bioaugmentation (1) Seed culture The fourth-generation dilution ratio of the microorganisms derived from the excavated debris N obtained in Example 1 was 10 -2 In order to ensure the bacterial count of the culture, cultivation was carried out. 20 ml of Widdel freshwater medium was placed in a 50 ml vial, and the gas phase was replaced with N2CO2 (N2 / CO2 = 80 / 20), and the vial was sealed. -2 The culture was inoculated into the medium at a concentration of 5%, and a lactic acid solution and a hexavalent selenium solution were added. A total of six cultures were prepared. They were then statically cultured at 25°C in a dark place for 8 weeks. After 8 weeks, the selenium concentration of the culture was measured and confirmed to have decreased to approximately 0.03 mg / L.

[0059] (2) Bioaugmentation Test Five grams of excavation debris from four species (G, Z, N, and H) that were not found to have selenium-reducing ability in Example 1 were placed in a vial, diluted to 10 mL with sterile water, and anaerobicized with N2CO2 aeration for 11 days. Subsequently, 1 mL or 7.5 mL of the culture prepared in (1) and a lactic acid solution (5 mM lactic acid, 0.5 ppm Se or 10 mM lactic acid, 5 ppm Se) were added and cultured for 20 days (Figure 1). For convenience, 1 mL of the precipitated microorganisms was collected and added to the 7.5 mL culture. (3) Measurement of selenium concentration The selenium concentrations in each excavated waste before and after the bioaugmentation test were measured in the same manner as in Example 1, and the results are shown in Table 5. Selenium reduction was observed at almost all levels.

[0060] [Table 5]

[0061] (4) Measurement of microbial abundance 1) DNA extracted from the added culture medium and the excavated waste before and after the bioaugmentation test was subjected to quantitative PCR (qPCR) using the GoTaq qPCR kit (Promega) and a real-time PCR detection system (CFX Opus: Bio-Rad). The primer set "515f / 806r" was used to quantify 16S rRNA derived from 16S rRNA derived from all bacteria. To create a calibration curve, the 16S rRNA gene was amplified from E. coli DNA using the primer set "B27f / B907r," and the resulting fragment was analyzed at different concentrations (10 2 -10 7 A DNA solution containing 1000 copies / μl of 1000 ribonucleotides was prepared. Melting curve analysis from 60°C to 95°C confirmed that no nonspecific PCR products were produced.

[0062] 2) The number of 16S rRNA gene copies per gram of excavated debris is shown in Figure 2. In the figure, ND indicates a value below the detection limit [1 × 10 4 copies / 1g shear From Figure 2, the microbial 16S rRNA gene concentration is 1.60 × 10 8 In other words, the concentrations of microorganisms added in the bioaugmentation test were 1.60 × 10 copies / ml at 1 ml and 7.5 ml, respectively. 8 copies / 5g shear, 1.2 x 10 9 It was copies / 5g. The detection limit is 1.03 x 10 4 The concentration of N in the excavated muck was below the detection limit at the start of the bioaugmentation test. The concentration of N in the other excavated muck was 1.03 × 10 7 to 1.03 × 10 8 At the end of the test (day 20), the 16S rRNA gene concentration was 10 1 From 10 3An increase in the 16S rRNA gene concentration of copies / g of excavated debris (1 to 2 orders of magnitude) was observed, indicating that the microorganisms in the added culture medium or excavated debris were actively growing.

[0063] (5) Microbial phylogenetic analysis As in Example 1, microbial phylogenetic analysis was performed on the excavation waste before and after the bioaugmentation test, and microbial species (OTU: Operational taxonomic unit) were identified and their relative abundance (%) was analyzed. Tables 6-1 to 6-4 show the changes in relative abundance before and after cultivation of three dominant species (selenium-reducing bacteria A, B, and C) of microorganisms (inoculation source) derived from excavated debris N used in the augmentation test. As shown in Tables 6-1 to 6-4, when the culture medium was added, an increase in the abundance of selenium-reducing bacteria A, B, and C in the culture medium was observed before and after the test.

[0064] [Table 6-1]

[0065] [Table 6-2]

[0066] [Table 6-3]

[0067] [Table 6-4]

[0068] Tables 7-1 and 7-2 show the results of microbial analysis (relative abundance (%)) 20 days after the start of the test. As shown in Table 4, bacteria A, B, and C contained in the culture medium added to all the wastes proliferated. The increase in bacteria A and B was particularly significant, and they are thought to have mainly contributed to selenium reduction.

[0069] Table 7-1

[0070] Table 7-2

Claims

1. A method for reducing selenium in a selenium-contaminated sample, comprising allowing the selenium-contaminated sample containing selenium-reducing bacteria to stand or stir under anaerobic conditions in the presence of lactic acid, wherein the selenium-reducing bacteria are at least one species selected from the group consisting of bacteria A having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 1 and bacteria B having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO:

2.

2. The method of claim 1, wherein the selenium-contaminated sample further contains Bacterium C having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO:

3.

3. A method for reducing selenium in a selenium-contaminated sample, comprising the steps of applying a culture of selenium-reducing bacteria to the selenium-contaminated sample and allowing the culture to stand or stir under anaerobic conditions in the presence of lactic acid, wherein the selenium-reducing bacteria comprise at least one species selected from the group consisting of bacteria A having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 1 and bacteria B having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO:

2.

4. The method according to claim 3, wherein the selenium-reducing bacteria are bacteria collected from a selenium-contaminated sample.

5. 4. The method according to claim 1, wherein the bacterium A is Paradesulfitobacterium aromaticivorans or a microorganism phylogenetically identical thereto, and the bacterium B is Intrasporangium calvum or a microorganism phylogenetically identical thereto.

6. The method according to claim 1 or 3, wherein the standing or stirring under anaerobic conditions is performed under anaerobic conditions at 10 to 40°C for 12 hours or more.

7. The selenium-contaminated sample was found to contain bacteria D having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 4, bacteria E having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 5, bacteria F having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 6, bacteria G having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 7, bacteria H having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 8, bacteria I having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 9, bacteria J having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 10, bacteria K having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 11, bacteria K having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 12, bacteria K having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 13, bacteria K having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 14, bacteria K having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 15, bacteria K having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 16, bacteria K having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 17, bacteria K having a 16S rRNA gene that The method according to claim 3, comprising one or more bacteria selected from the group consisting of: bacterium L having a 16S rRNA gene; bacterium M having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 13; bacterium N having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 14; bacterium O having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 15; bacterium P having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 16; bacterium Q having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 17; bacterium R having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 18; bacterium S having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 19; and bacterium T having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO:

20.

8. 4. The method according to claim 1, wherein the selenium-contaminated sample is one or more selected from the group consisting of excavated waste, soil, sludge, water, incineration ash, and industrial wastewater containing selenium.

9. The method according to claim 1 or 3, further comprising the step of adding one or more selected from phosphoric acid, organic acids other than lactic acid, alcohols, and sugars to the selenium-contaminated sample.

10. A method for evaluating the selenium-reducing ability of a selenium-contaminated sample, comprising a step of confirming the presence in the sample of at least one species selected from the group consisting of bacteria A having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 1 and bacteria B having a 16S rRNA gene that is 97% or more identical to the base sequence of SEQ ID NO: 2.

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