JP7452794B2 - Selenium reduction treatment method using selenium-contaminated samples containing selenium-reducing bacteria - Google Patents

Description

The present invention relates to a selenium reduction treatment method using a selenium-contaminated sample containing a novel selenium-reducing bacterium.

Selenium is a naturally occurring element found in sulfides or sulfur deposits. While this element is one of the essential elements for the human body, it can cause health damage if ingested in large quantities, so it is classified as a Class 2 Specified Hazardous Substance under the Environmental Standards for Water Pollution under the Basic Environment Law and the Soil Contamination Countermeasures Law. specified. Therefore, as a countermeasure for contaminated groundwater that exceeds the standard value or contaminated soil from which selenium is leached, in-situ purification treatment or insolubilization treatment of contaminated water may be taken. One of the cases where naturally occurring selenium becomes a problem is the excavation slag generated during tunnel construction, tunnel wastewater treatment generated by spring water, etc., and the surrounding area due to scattering or leakage from excavation slag during removal, transportation, and storage. These include countermeasures against pollution of soil, groundwater, and rivers, and treatment of wastewater when treated at industrial waste treatment plants, sludge treatment plants, etc. However, existing treatment materials have low effects on selenium, and complicated treatment equipment is required to purify contaminated water, so there is a need for highly effective treatment techniques for selenium-contaminated samples.

The form of selenium in the natural environment is tetravalent selenite (SeO ₃ ^2- ) or hexavalent selenic acid (SeO ₄ ^2- ), and in many cases they are mixed in the natural environment. It is believed that the difficulty in reducing hexavalent selenium makes it difficult to process selenium. When treating hexavalent selenium, it is common to first reduce it to tetravalent selenium and then treat it (for example, Patent Document 1), but since it requires two stages of treatment, it is not effective for either valence. A reduction treatment method is desired.

JP2017-205697A

An object of the present invention is to provide a method for reducing selenium using a selenium-contaminated sample containing a novel selenium-reducing bacterium.

The present inventors conducted intensive studies on selenium reduction treatment, and found that by leaving selenium-contaminated samples containing novel selenium-reducing bacteria standing or stirring under anaerobic conditions in the presence of lactic acid, the We obtained the knowledge that selenium with a valence of 4 and 6 can be reduced by the activity of the selenium-reducing bacteria, which is the relative abundance (%), and based on this knowledge, we completed the present invention.

That is, the present invention relates to the following 1) to 7).
1) A selenium reduction treatment method for a selenium-contaminated sample, in which the selenium-contaminated sample containing selenium-reducing bacteria is left standing or stirred under anaerobic conditions in the presence of lactic acid, wherein the selenium-contaminated sample contains selenium-reducing bacteria, SEQ ID NO: 1 selenium-reducing bacteria A having a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 2; selenium-reducing bacteria B having a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 2; Selenium-reducing bacterium C having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 3, selenium having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 4 Selenium-reducing bacterium D, a 16S rRNA gene having an identity of 97% or more to the nucleotide sequence of SEQ ID NO: 5; Selenium-reducing bacterium E having a 16S rRNA gene having an identity of 97% or more to the nucleotide sequence of SEQ ID NO: 6; Selenium-reducing bacterium F having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 7.Selenium-reducing bacteria G having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 8. At least one species selected from the group consisting of selenium-reducing bacteria H having a 16S rRNA gene that is There is a method.
2) The method according to 1), which includes a step of standing or stirring at 10 to 40°C under anaerobic conditions for 12 hours or more.
3) The selenium-reducing bacteria has a 16S rRNA gene that has a 16S rRNA gene that has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 1, and has a 16S rRNA gene that has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 2. A selenium-reducing bacterium B having a 16S rRNA gene, a selenium-reducing bacterium C having a 16S rRNA gene with an identity of 97% or more to the nucleotide sequence of SEQ ID NO: 3, and a selenium-reducing bacterium C having a 16S rRNA gene having an identity of 97% or more to the nucleotide sequence of SEQ ID NO: 5. The method according to 1) or 2), comprising at least one species selected from the group consisting of selenium-reducing bacteria E having 97% or more of the 16S rRNA gene.
4) The selenium-reducing bacteria has a 16S rRNA gene that has a 16S rRNA gene that has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 2, and has a 16S rRNA gene that has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 7. 1) containing at least one species selected from the group consisting of a selenium-reducing bacterium G having a certain 16S rRNA gene and a selenium-reducing bacterium I having a 16S rRNA gene having 97% or more identity with SEQ ID NO: 9; The method described in any of 3).
5) The method according to any one of 1) to 4), wherein the selenium-contaminated sample to be treated is one or more selected from selenium-containing excavation slag, earth and sand, sludge, water, incineration ash, and factory wastewater.
6) The method according to any one of 1) to 5), which includes 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 to be treated.
7) A method for evaluating the selenium reducing ability of a selenium-contaminated sample, comprising: a selenium-reducing bacterium A having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 1 in the sample; : Selenium-reducing bacteria B having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 2 and selenium-reducing bacteria having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 3. C, a selenium-reducing bacterium having a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 4; D, a 16S rRNA gene that has 97% or more identity with the base sequence SEQ ID NO: 5; Selenium-reducing bacteria E having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 6.Selenium-reducing bacteria F having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 7. Selenium-reducing bacteria G having a 16S rRNA gene, selenium-reducing bacteria H having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 8, and selenium-reducing bacteria H having 97% or more identity with the nucleotide sequence of SEQ ID NO: 9. A method comprising the step of confirming the presence of at least one species selected from the group consisting of selenium-reducing bacteria I having 97% or more of the 16S rRNA gene.

According to the present invention, by utilizing the selenium-reducing power based on selenium-reducing bacteria in a selenium-contaminated sample, selenium reduction treatment can be performed without using a treatment material.

Figure 3 shows the decrease in selenium concentration in a selenium-contaminated sample to which lactic acid has been added. Relative abundance of four microorganisms in a selenium-contaminated sample with added lactic acid (contaminated sample 1). Relative abundance of seven microorganisms in a lactic acid-added selenium-contaminated sample (contaminated sample 2).

In this specification, the range "X to Y" means "more than or equal to X and less than or equal to Y." Further, unless otherwise specified, operations and physical properties are measured under conditions of room temperature CV (20 to 25° C.)/relative humidity 40 to 50% RH.

As described in the Examples below, we investigated the relationship between the state of the microbial community in excavated excavations from two different tunnel construction sites and the reduction activity of tetravalent and hexavalent selenium. Nine types of microorganisms (selenium-reducing bacteria), including three new bacterial species involved in reduction, were identified. These selenium-reducing bacteria had an extremely low relative abundance (for example, 1% or less) with respect to the entire microbial community, but the presence of lactic acid increased their relative abundance, and the selenium-reducing activity of the drilling slag increased. was found to increase.
Therefore, the selenium-contaminated sample containing nine types of selenium-reducing bacteria is considered to have selenium-reducing ability, and it is possible to reduce selenium by utilizing the selenium-reducing power of the selenium-reducing bacteria in the sample. Become. Furthermore, the selenium reducing ability of the sample can be evaluated using the presence of selenium-reducing bacteria in the sample as an indicator.

[Selenium reducing bacteria]
In the present invention, selenium-reducing bacteria refers to bacteria that can reduce tetravalent and hexavalent selenium as electron acceptor substrates.
The selenium-reducing bacterium according to the present invention includes Bacterium A having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 1 (herein also simply referred to as "Selenium-reducing bacterium A"). , Bacterium B having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 2 (herein also simply referred to as "Selenium-reducing bacterium B"), and the nucleotide sequence of SEQ ID NO: 3. Bacterium C (herein also simply referred to as "Selenium-reducing bacterium C") having a 16S rRNA gene with an identity of 97% or more to the base sequence of SEQ ID NO: 4, which has an identity of 97% or more to the nucleotide sequence of SEQ ID NO: 4. Bacterium D having a 16S rRNA gene (herein also simply referred to as "Selenium-reducing bacterium D"), Bacterium E having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 5 (in this specification). Bacteria F, which has a 16S rRNA gene having a 97% or more identity with the base sequence of SEQ ID NO: 6 (also simply referred to as "Selenium-reducing bacteria E" in the specification); F), Bacterium G having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 7 (herein also simply referred to as "Selenium-reducing bacterium G"), SEQ ID NO: Bacterium H having a 16S rRNA gene that has 97% or more identity with the nucleotide sequence of SEQ ID NO: 8 (herein also simply referred to as "Selenium-reducing bacterium H"), and identity with the nucleotide sequence of SEQ ID NO: 9. Examples include bacteria selected from the group consisting of bacteria I (herein also simply referred to as "selenium-reducing bacteria I") having a 16S rRNA gene of 97% or more.

In the present invention, identity with a nucleotide sequence refers to the identity shared between two nucleotide sequences when the two sequences are aligned in an optimal manner, unless otherwise specified. It means the percentage of the number of nucleotides. That is, it can be calculated as identity (%) = (number of matched positions/total number of positions) x 100, and can be calculated using a commercially available algorithm. Such algorithms have also been incorporated into the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215 (1990) 403-410. More specifically, searches and analyzes regarding the identity of base sequences can be performed using algorithms or programs (eg, BLASTN, ClustalW, etc.) well known to those skilled in the art. Parameters when using a program can be appropriately set by those skilled in the art, and default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art.

Table 1 shows selenium-reducing bacteria having a 16S rRNA gene containing the base sequence of SEQ ID NOs: 1 to 9. These selenium-reducing bacteria were identified by the method described in "Identification of selenium-reducing bacteria" below. In Table 1 below, "homology (%)" was determined for the nucleotide sequences of SEQ ID NOs: 1 to 9 using the BLAST program of the nucleotide sequence database of NCBI (The National Center for Biotechnology Information).

The selenium-reducing bacteria (OTU3234, OTU12206, and OTU7833) having a 16S rRNA gene containing the base sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 are Bacillus thioparans, which are known as arsenate-resistant bacteria, respectively. (Bacillus thioparans), Pelosinus fermentans, which is known as a lactic acid-fermenting bacterium, and Brevibacillus ginsengisoli, which is known as an acetic acid-utilizing bacterium.

Selenium-reducing bacteria (OTU26314, OTU16025) having a 16S rRNA gene containing the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 are known sulfate-reducing bacteria Desulfosporosaenus burensis and Desulfosporosaenas burensis, respectively. It is considered to be a microorganism that is phylogenetically the same as or closely related to Desulfosporosinus nitroreducens.
Furthermore, the selenium-reducing bacterium (OTU24704) that has a 16S rRNA gene containing SEQ ID NO: 8 is phylogenetically related to Desulfitobacterium hafniense, which is known as a known heavy metal-reducing bacterium. It is thought that it is a microorganism.

Selenium-reducing bacteria (OTU33786, OTU31065, OTU9628) that have a 16S rRNA gene containing the base sequence of SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 9 are The homology is 94.1%, 88.3%, and 93.7%, respectively. According to analysis using the base sequence of the 16S rRNA gene, if it shows 97% or more homology with known species, it can be defined as the same species. In other words, these selenium-reducing bacteria are considered to be microorganisms different from known microbial species.

The selenium-contaminated sample containing selenium-reducing bacteria of the present invention contains the above-mentioned selenium-reducing bacteria in its microbial community.
The origin of the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention is not limited as long as it contains the selenium-reducing bacteria of the present invention, but the selenium-contaminated sample contains selenium and is maintained in an anaerobic state in an environment suitable for the growth of the selenium-reducing bacteria. These include groundwater obtained, soil such as ground and bedrock (specifically, excavation slag when constructing underground structures such as tunnels), and earth, sand, sludge, and wastewater generated during construction work of underground structures. The selenium-contaminated sample containing selenium-reducing bacteria of the present invention may contain filamentous fungi and protozoa in addition to bacteria, as long as it contains a microbial community containing the selenium-reducing bacteria.

From the viewpoint of selenium-reducing ability, the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention is preferably a selenium-reducing bacterium having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 1. A, a selenium-reducing bacterium having a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 2; B, a 16S rRNA gene that has 97% or more identity with the base sequence SEQ ID NO: 3; Examples include those containing at least one species selected from the group consisting of selenium-reducing bacteria C having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 5.

Furthermore, in a more preferred embodiment, the selenium-contaminated sample contains selenium-reducing bacteria A, more preferably selenium-reducing bacteria A, selenium-reducing bacteria B, and selenium-reducing bacteria C, from the viewpoint of selenium-reducing ability. or those containing selenium-reducing bacteria A, selenium-reducing bacteria C, and selenium-reducing bacteria E. More preferred are those containing selenium-reducing bacteria A, selenium-reducing bacteria B, selenium-reducing bacteria C, and selenium-reducing bacteria E.

In another embodiment, the selenium-contaminated sample is a selenium-reducing bacterium B having a 16S rRNA gene that has a nucleotide sequence of 97% or more identical to the nucleotide sequence of SEQ ID NO: 2; At least one species selected from the group consisting of selenium-reducing bacteria G having a 16S rRNA gene with a 16S rRNA gene having a identity of 97% or more and selenium-reducing bacteria I having a 16S rRNA gene having a 97% or more identity with SEQ ID NO: 9. Examples include those containing.

The selenium-contaminated sample containing the selenium-reducing bacteria of the present invention contains about 4,000 to about 5,000 microbial species, and among these microbial species, the relative abundance of the selenium-reducing bacteria of the present invention is It is not particularly limited and may be extremely low relative to all microbial species. The relative abundance (%) of selenium-reducing bacteria is, for example, 0.001% or less to 0.025% with respect to the entire microbial community. The value of relative abundance is a value determined by the method described in Examples.

In addition, since the selenium-reducing bacteria described above exist in extremely small amounts in the sample, it is difficult to isolate them for technical reasons. Therefore, it has not been deposited with a depositary institution. However, in cases where the selenium-contaminated sample containing the selenium-reducing bacteria according to the present invention falls under Article 27-3 of the Japanese Patent Law Enforcement Regulations, the applicant may request that a third party provide the We guarantee that the property will be distributed to

[Identification of selenium-reducing bacteria]
Identification of selenium-reducing bacteria in a sample containing multiple microorganisms (for example, selenium-contaminated soil) can be performed by steps A to C shown below.
Step A: Recovering nucleic acid from an uncultured sample containing selenium; and analyzing the base sequence of the target gene present in the nucleic acid using a next-generation sequencer to determine the relative abundance (1) of each microorganism. ,
Step B: Starting anaerobic culture of the sample in the presence of tetravalent and hexavalent selenium and lactic acid to obtain a culture; Recovering the nucleic acid from the culture; and base of the target gene present in the nucleic acid. Analyzing the sequence with a next-generation sequencer and determining the relative abundance (2) of each microorganism,
Step C: A step of determining that microorganisms in which the relative abundance (2) is greater than the relative abundance (1) are selenium-reducing bacteria.

The relative abundance of selenium-reducing bacteria that reduced tetravalent and hexavalent selenium becomes greater than before the reduction occurs. That is, a sample at the time of unculture and a sample cultured with tetravalent and hexavalent selenium and lactic acid are prepared, and the DNA obtained from each sample is analyzed and compared. In the sample in which selenium reduction was observed, microorganisms whose relative abundance in the sample after culture with tetravalent and hexavalent selenium and lactic acid were larger than the relative abundance in the DNA of the sample at the time of unculture, were It is understood that it is a microorganism that grows by reducing hexavalent selenium. This makes it possible to identify microorganisms (selenium-reducing bacteria) that reduce tetravalent and hexavalent selenium in the presence of lactic acid.

In Step A and Step B, the following three steps are performed to determine the relative abundance of microorganisms in the selenium-containing sample.
(i) additionally adding tetravalent and hexavalent selenium and lactic acid to a selenium-containing sample and culturing microorganisms contained in the sample;
(ii) recovering nucleic acids (e.g., DNA) from each sample before culture and after culture with the addition of tetravalent and hexavalent selenium and lactic acid;
(iii) Analyzing the base sequence of the target gene (eg, 16S rRNA gene) present in the recovered nucleic acid using a next-generation sequencer.

Below, the above (i) to (iii) will be specifically explained.
(i) Adding 4- and 6-valent selenium and lactic acid to a selenium-containing sample and culturing the microorganisms contained in the sample. From this point of view, it is preferable to add water.
The cultivation of the microorganisms is carried out in airtight containers after aeration with CO ₂ /N ₂ or N ₂ with a view to creating a reducing atmosphere, ie anaerobic conditions. This induces biological selenium reduction.

Cultivation of microorganisms is performed in the presence of tetravalent and hexavalent selenium and lactic acid by mixing the selenium-containing sample with tetravalent and hexavalent selenium and lactic acid.
From the perspective of maintaining the state of the microbial community, microorganisms can be cultured by adding lactic acid to selenium-containing samples along with tetravalent and hexavalent selenium. Other components such as acids may be added as appropriate. In particular, it is preferable for the growth of selenium-reducing bacteria to add phosphoric acid (potassium hydrogen phosphate, etc.), organic acids other than lactic acid (acetic acid, etc.), alcohols (ethanol, etc.), and sugars (glucose, etc.).

Tetravalent and hexavalent selenium can be adjusted as appropriate based on concentration fluctuations in the actual environment (0.000015 to 0.04mM). One embodiment of the present invention includes starting the culture of the plurality of microorganisms in the presence of 0.000127 to 0.02 mM of tetravalent and hexavalent selenium. By setting the concentration of 4- and 6-valent selenium to 0.000127 mM or more before culturing, the selenium-reducing bacteria can reduce 4- and 6-valent selenium even during the preferred culture time described below. When the concentration of tetravalent and hexavalent selenium is 0.02 mM or less, changes in the state of the microbial community can be suppressed. Therefore, in the method for identifying selenium-reducing bacteria of the present invention, it is preferable to start culturing the microorganism in the presence of 0.000127 to 0.02 mM of tetravalent and hexavalent selenium.
In addition, the lactic acid required to reduce 1M of hexavalent selenium to zero valence is 1.5M in terms of stoichiometry, but the addition of lactic acid to the contaminated sample is less than the required stoichiometric concentration. It is preferable to increase the amount by 1 to 500 times.

The culture temperature of microorganisms may be set based on annual temperature changes in the actual environment. The culture temperature of the microorganism is preferably in the range of 10 to 40°C, more preferably 20 to 30°C, from the viewpoint of detecting selenium-reducing bacteria active in a real environment. By setting the culture temperature to 10° C. or higher, reduction of tetravalent and hexavalent selenium can be confirmed. By setting the culture temperature to 40° C. or lower, changes in the microbial community can be suppressed.

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

(ii) Recovering nucleic acids from the culture The method for recovering nucleic acids from the culture obtained in (i) is not particularly limited, and conventionally known methods can be used. The nucleic acid to be recovered may be either DNA or RNA.
Methods for recovering DNA include, for example, physical crushing using beads (glass beads, mixed beads of zirconia and silica, etc.), crushing using ultrasonication, crushing using a French press, and crushing a sample frozen with liquid nitrogen in a mortar. Microbial cells are destroyed using a method such as crushing with a pestle and a pestle, and then proteins are removed using conventionally known methods (phenol/chloroform extraction, etc.), RNA is digested using RNase, and DNA is extracted. One example is to collect the

(iii) Analyzing the base sequence of the target gene present in the recovered nucleic acid using a next-generation sequencer From the viewpoint of being suitable for large-scale phylogenetic analysis, the target gene used to identify selenium-reducing bacteria is preferably 16S rRNA gene V4 region.
Next-generation sequencer is a term used in contrast to "first-generation sequencer," which is a fluorescent capillary sequencer that uses the Sanger method. It refers to a device that determines base sequences in massively parallel manner by comprehensively analyzing fragments with read lengths of several tens to several thousand bp for tens of millions to hundreds of millions of DNA fragments. Next-generation sequencers use a different sequencing principle from first-generation sequencers, which use the Sanger method, which uses dideoxynucleotides to stop DNA polymerase elongation. Such principles include synthetic sequencing methods, pyrosequencing methods, ligase reaction sequencing methods, and the like. To date, many companies and research institutes have provided a variety of next-generation sequencers, such as HiSeq2500 (Illumina Corporation), MiSeq (Illumina Corporation), 5500xl SOLiD (registered trademark) (Thermo Fisher Science Ion Proton (registered trademark) (Thermo Fisher Scientific), Ion PGM (registered trademark) (thermo Fisher Scientific), GS FLX+ (Roche Diagnostics), and the like.
The analysis method using a next-generation sequencer can be carried out according to the attached instructions. This makes it possible to identify tens of thousands to hundreds of thousands of microbial species (OTU) and to analyze the relative abundance (%) of each microbial species.

The judgment that it is a selenium-reducing bacterium in Step C is made by comparing the relative abundance of microorganisms at the time of unculture (1) and the relative abundance of microorganisms after culturing with the addition of tetravalent and hexavalent selenium and lactic acid (2). This can be done by doing this.
For example, among OTUs, in a culture in which selenium reduction has been observed, microorganisms whose selenium reduction has significantly increased by 70 times or more after culture than at the time of culture initiation are identified.
In the cultures in which 4- and 6-valent selenium reduction was observed, the microorganisms whose reduction was significantly 70 times or more compared to the uncultured state grew by reducing 4- and hexavalent selenium in the presence of lactic acid. Such microorganisms can be determined to be selenium-reducing bacteria.

[Selenium reduction treatment of selenium-contaminated samples]
According to the present invention, there is provided a method for reducing selenium in a selenium-contaminated sample, which includes a step of allowing or stirring the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention under anaerobic conditions in the presence of lactic acid. be done.
Here, the selenium-contaminated sample to be processed may be the selenium-contaminated sample itself containing the selenium-reducing bacteria described above, or it may be another selenium-contaminated sample (for example, another selenium-contaminated sample that does not contain selenium-reducing bacteria). , another selenium-contaminated sample containing selenium-reducing bacteria different from the selenium-reducing bacteria of the present invention). In addition, the isolated, extracted and cultured selenium-reducing bacteria may be used in a selenium-contaminated sample (for example, another selenium-contaminated sample that does not contain selenium-reducing bacteria, or another selenium-contaminated sample that contains selenium-reducing bacteria different from the selenium-reducing bacteria of the present invention). It may also be added to a contaminated sample. Here, the selenium-contaminated sample is not particularly limited as long as it contains selenium, and may be any waste such as excavation slag, earth and sand, sludge, water, incineration ash, factory wastewater, etc.

The selenium reduction treatment of such a selenium-contaminated sample is performed in the presence of lactic acid. The amount of lactic acid used is not limited as long as the selenium-reducing bacteria of the present invention in the contaminated sample can sufficiently proliferate and exert its reducing power, but if the lactic acid content in the contaminated sample (dry state) is 0. It is preferable that the amount is 000285% by mass or more.
For example, the content of lactic acid in the contaminated sample is preferably 0.000285 to 0.23% by mass, more preferably 0.000285 to 0.17% by mass, and even more preferably 0.00285 to 0.114% by mass. There are many things you can do.

There are no particular restrictions on the method of adding lactic acid to the contaminated sample, and there are methods such as spraying lactic acid directly onto the contaminated sample using a sprayer, or adding a solution of lactic acid dissolved in a solvent such as water using a water spray nozzle, irrigation machine, etc. A method of spraying contaminated samples with water can be adopted.
When lactic acid is dissolved in a solvent such as water, the concentration is, for example, 0.005 to 1.14 g/L, preferably 0.005 to 0.856 g/L, more preferably 0.005 to 0.57 g/L. Preferably, it is a solution.

The treatment conditions may be any conditions as long as the selenium-reducing bacteria in the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention can proliferate and exhibit reducing power, and may be left standing or stirring under anaerobic conditions, but preferably , in an anaerobic tank, 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 is 4500 hours or less, more preferably 1500 hours or less.
In addition, iron salts, sulfites, oxalic acid, or other reducing agents may be added to the selenium-contaminated sample to change it to a reduced state so that the reducing power of selenium-reducing bacteria can be easily exerted.

For example, a selenium-contaminated sample containing the selenium-reducing bacteria of the present invention is placed in an anaerobic tank, lactic acid is added thereto, and then the selenium in the sample is removed by standing or stirring at 10 to 40°C for 12 hours or more. Reduction processing is possible. Furthermore, by adding another selenium-contaminated sample to the anaerobic tank containing the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention and lactic acid, the selenium-contaminated sample can also be subjected to selenium reduction treatment. It is possible.

In addition, other components such as phosphoric acid may be appropriately added to the reduction treatment, if necessary. In particular, the process of adding one or more of phosphoric acid (potassium hydrogen phosphate, etc.), organic acids other than lactic acid (acetic acid, etc.), alcohols (ethanol, etc.), and sugars (glucose, etc.) It is preferable from the viewpoint of promoting proliferation.

[Evaluation of selenium reduction ability of selenium-contaminated samples]
According to one embodiment of the present invention, there is provided a method for evaluating the selenium reducing ability of a selenium-contaminated sample, which comprises selenium-reducing bacteria A, selenium-reducing bacteria B, selenium-reducing bacteria C, selenium-reducing bacteria D, A method is provided that includes a step of confirming the presence of at least one species selected from the group consisting of selenium-reducing bacteria E, selenium-reducing bacteria F, selenium-reducing bacteria G, selenium-reducing bacteria H, and selenium-reducing bacteria I.

The means for confirming the selenium-reducing bacteria is not particularly limited, but preferably includes a method of analyzing the bacterial species present in the sample based on the base sequence of the 16S rRNA gene contained in the bacterial flora genomic DNA described above. It will be done.
That is, as described above, the relative abundance (%) can be confirmed by collecting a nucleic acid from a sample and analyzing the base sequence of a target gene present in the nucleic acid using a next-generation sequencer.

Thereby, the selenium reduction ability of the selenium-contaminated sample can be evaluated. Here, the selenium-reducing ability of a sample means the selenium-reducing ability based on the selenium-reducing bacteria that the sample has.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

<Evaluation of selenium contaminated samples>
The amount of selenium eluted from a selenium-contaminated sample is determined by preparing a test solution in accordance with the method described in the Environment Agency Notification No. 18, "Measurement Methods for Surveys of Soil Elution Amounts," and using the "JIS K 0102:2013. (Industrial wastewater test method)”.

Example 1 Identification of selenium-reducing bacteria (1) Cultivation of microorganisms in sample contaminated with hexavalent selenium Two types of tunnel excavation debris from different production areas generated during construction work were used as samples.
Specifically, 5 g of the contaminated sample was diluted to 10 ml with sterile water or sterile water to which lactic acid (10 mM) had been added, and placed in a 50 ml glass vial. The gas phase portion was N ₂ CO ₂ (N ₂ /CO ₂ =80/20). A hexavalent selenium solution (sodium selenite; manufactured by Wako Pure Chemical Industries, Ltd.) was added to the contaminated sample so that the final concentration of hexavalent selenium was 0.0127 mM. Thereafter, static culture was performed for 2 weeks at 25°C in the dark.

(2) Chemical analysis The liquid phase was collected at the start of the culture and from the hexavalent selenium culture and subjected to chemical analysis using the method described above to measure the selenium concentration.
The results are shown in Figure 1. In contaminated sample 1 and contaminated sample 2, in the test plot where only selenium was added, the selenium concentration tended to decrease after two weeks of culture, but in the test plot where lactic acid and selenium were added, the selenium concentration decreased significantly after two weeks of culture. decreased to
This confirmed that selenium reduction occurred due to microorganisms present in the excavation slag, and that the reduction reaction was accelerated by the addition of lactic acid.

(3) Microbiological analysis The samples were centrifuged at the time of unculture and after culture with hexavalent selenium to obtain centrifuged precipitates. Mixed beads of zirconia and silica (Zirconia/Silica Beads; average particle diameter 0.1 mm) were added to the centrifugal precipitate, and microbial cells in the solid phase were isolated using Shake Master Auto (manufactured by Biomedical Science Co., Ltd.). After crushing, coexisting proteins were removed by a purification step using phenol and chloroform. Thereafter, RNA was digested by treatment with RNase A (manufactured by Beckman), and DNA was purified.
Using this purified DNA as a template and targeting the 16S rRNA gene V4 region (approximately 300 base pairs), Q5 High-Fidelity DNA Polymerase Q5
Gene amplification reaction (Polymerase chain reaction [PCR]) using DNA polymerase (NEB) was performed. The obtained PCR product was used with AMPure XP kit (manufactured by Beckman Coulter) and Wizard (registered trademark) SV gel.
and PCR clean-up kit (manufactured by Promega), and its concentration was determined using a Quant-iT PicoGreen dsDNA reagent kit (manufactured by Thermo Fisher Scientific) and a fluorescence quantification device Nanodrop 3300 (manufactured by Thermo Fisher Scientific). Co., Ltd.).

The optimal amount of the purified product was subjected to large-scale base sequence analysis using MiSeq Reagent kit 300 cycles and next-generation sequencer MiSeq (manufactured by Illumina Corporation). Tens of thousands of reads of gene fragments were decoded from each sample, non-specific sequences were removed using the software mothur (ver 1.31.2), and the software QIIME (ver 1.6.0) was used to Microbial phylogenetic analysis was performed, and microbial species (OTU: Operational taxonomic unit) were identified and relative abundances (%) were analyzed.

Among the obtained OTUs, in a culture cultured with the addition of lactic acid and selenium in which selenium reduction was observed, the relative abundance significantly increased by 70 times or more after culture compared to before culture, and the relative abundance We identified nine types of microorganisms belonging to the phylum Firmicutes in which the amount of microorganisms increased to 1% or more (Figures 2 and 3). Gene sequence homology of the OTU with related species was determined using the BLAST program of the NCBI nucleotide sequence database.

OTU3234 (belongs to selenium-reducing bacteria D), OTU12206 (belongs to selenium-reducing bacteria E), and OTU7833 (belongs to selenium-reducing bacteria F) are Bacillus thioparans, which is known as an arsenate-resistant bacterium, and lactic acid fermenter. It was phylogenetically identical to Pelosinus fermentans, which is known as a bacterium, and Brevibacillus ginsengisoli, which is known as an acetic acid-utilizing bacterium (16S rRNA gene sequence homology). (all showed 100%).

OTU26314 (belongs to selenium-reducing bacteria A) and OTU16025 (belongs to selenium-reducing bacteria C) are both Desulfosporosaenas burensis and Desulfosporosaenas nitro, which are known as sulfate-reducing bacteria. It was phylogenetically identical or closely related to Desulfosporosinus nitroreducens (16S rRNA gene sequence homology was 99.6% and 98.4%, respectively).

OTU24704 (belonging to selenium-reducing bacteria H) was phylogenetically related to Desulfitobacterium hafniense, which is known as a reducing bacterium of heavy metals, etc. (16S rRNA gene sequence homology was 97.2%).

OTU33786 (belongs to selenium-reducing bacteria B) and OTU9628 (belongs to selenium-reducing bacteria I) are Symbiobacterium terraclitae, Symbiobacterium turbinis, which are the most closely related microbial species in the database. turbinis) showed low sequence homology (16S rRNA gene sequence homology was 94.1% and 93.7%, respectively), suggesting that it is a phylogenetically novel microorganism. .

OTU31065 (belonging to selenium-reducing bacteria G) shows extremely low sequence homology (88.3%) to the most closely related microbial species in the database, Ruminiclostridium cellobioparum, which has not been previously reported. It was suggested that this microorganism is phylogenetically different from any other microorganisms that have been studied, and is an extremely novel microorganism.

In the microbial communities of contaminated sample 1 and contaminated sample 2, the relative abundance of selenium-reducing bacteria at the start of culture was 1% or less (contaminated sample 1: from 0.001% or less to 0.025%; contaminated sample 2: It was revealed that it is a rare microbial species with a concentration of 0.001% to 0.022%).

In contaminated sample 1, OTU26314 had the highest relative abundance after 2 weeks of culture (67.6% of the total). Next, OTU33786 had the highest relative abundance (16.8% of the total).

In contaminated sample 2, OTU26314 had the highest relative abundance after 2 weeks of culture (63.7% of the total). OTU12206 had the next highest relative abundance (12.1% of the total).

From the above, it is strongly suggested that selenium-reducing bacteria in contaminated samples exposed to an anaerobic atmosphere dramatically increase in each sample and in the presence of lactic acid, and are involved in the reduction of tetravalent and hexavalent selenium. Ru.

Claims (8)

A selenium-reducing method for a selenium-contaminated sample, in which the selenium-contaminated sample containing selenium-reducing bacteria is left standing or stirred under anaerobic conditions in the presence of lactic acid, wherein the selenium-contaminated sample contains the base of SEQ ID NO: 1. A method, wherein the selenium-reducing bacterium A has a 16S rRNA gene having a sequence identity of 97% or more. The selenium-reducing bacterium further has a 16S rRNA gene that has a 16S rRNA gene that has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 2, and a selenium-reducing bacterium B that has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 3. Selenium-reducing bacteria C having a 16S rRNA gene, which has an identity of 97% or more with the base sequence of SEQ ID NO: 4. Selenium-reducing bacteria D having a 16S rRNA gene, which has an identity of 97% with the base sequence of SEQ ID NO: 5. % or more of the 16S rRNA gene, selenium-reducing bacteria F having a 16S rRNA gene that has 97% or more identity with the nucleotide sequence of SEQ ID NO: 6, and the nucleotide sequence of SEQ ID NO: 7. Selenium-reducing bacteria G having a 16S rRNA gene with an identity of 97% or more, selenium-reducing bacteria H having a 16S rRNA gene having a 97% or more identity with the base sequence of SEQ ID NO: 8, and SEQ ID NO: 9. 2. The method according to claim 1, comprising at least one species selected from the group consisting of selenium-reducing bacteria I having a 16S rRNA gene that has a nucleotide sequence of 97% or more in identity. The method according to claim 1 or 2 , comprising a step of standing or stirring at 10 to 40°C under anaerobic conditions for 12 hours or more. The selenium-reducing bacterium further has selenium-reducing bacterium B having a 16S rRNA gene that has a 16S rRNA gene that has 97% or more identity with the nucleotide sequence of SEQ ID NO: 2, and 97% identity with the nucleotide sequence of SEQ ID NO: 3. At least one species selected from the group consisting of selenium-reducing bacteria C having a 16S rRNA gene as described above, and selenium-reducing bacteria E having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 5. 2. The method of claim 1, comprising: The method according to any one of claims 1 to 4, wherein the selenium-contaminated sample to be treated is one or more selected from excavation debris, earth and sand, sludge, water, incineration ash, and industrial wastewater containing selenium. The method according to any one of claims 1 to 5, 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 to be treated. A method for evaluating the selenium-reducing ability of a selenium-contaminated sample, the method comprising confirming the presence of selenium-reducing bacteria A having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 1 in the sample. A method, including a process. Furthermore, a selenium-reducing bacterium B having a 16S rRNA gene having an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 2, and a 16S rRNA gene having an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 3. Selenium-reducing bacteria C having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 4.Selenium-reducing bacteria D having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 5. Selenium-reducing bacteria E having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 6.Selenium-reducing bacteria F having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 7. % or more, selenium-reducing bacteria G having a 16S rRNA gene having an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 8, and selenium-reducing bacteria H having a 16S rRNA gene having an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 9. 8. The method according to claim 7, comprising the step of confirming the presence of at least one species selected from the group consisting of selenium-reducing bacteria I having a 16S rRNA gene with an identity of 97% or more.