JP2018057356A - Activated sludge containing 1,4-dioxane degrading microorganisms and methods of waste water treatment using the same, and methods for identifying 1,4-dioxane degrading microorganisms - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel activated sludge excellent in 1,4-dioxane degrading ability.SOLUTION: The invention provides an activated sludge comprising at least one microorganism selected from 1,4-dioxane degrading microorganisms A, B and C, where the 16S rRNA gene of microorganism A has 97% or more identity to the base sequence of SEQ ID NO: 1, the 16S rRNA gene of microorganism B has 97% or more identity to the base sequence of SEQ ID NO: 2, and the 16S rRNA gene of microorganism C has 97% or more identity to the base sequence of SEQ ID NO: 3.SELECTED DRAWING: None

Description

  The present invention relates to an activated sludge containing 1,4-dioxane-degrading bacteria, a wastewater treatment method using the same, and a method for identifying 1,4-dioxane-degrading bacteria.

  Regarding 1,4-dioxane, uniform drainage standards (0.5 mg / L or less) have been set in Japan since May 25, 2012.

  1,4-Dioxane is a biologically degradable substance. It is said that 1,4-dioxane cannot be sufficiently removed by an activated sludge method, an activated carbon adsorption method, or the like used in a conventional wastewater treatment facility. Thus, accelerated oxidation treatments such as ozone treatment, a combination of ozone treatment and hydrogen peroxide treatment, and a combination of ozone treatment and ultraviolet treatment are known as methods for removing 1,4-dioxane (Patent Document 1). And Patent Document 2).

JP-A-2005-103401 JP 2005-58854 A

  However, in the accelerated oxidation treatment, if organic substances other than 1,4-dioxane are present, these organic substances are also decomposed, so that the required ozone injection amount, the amount of chemicals, and the like are increased, which may increase the processing cost.

  Therefore, an object of the present invention is to provide a novel activated sludge excellent in 1,4-dioxane resolution, a wastewater treatment method using the same, and a method for identifying 1,4-dioxane degrading bacteria.

  The present inventors have intensively studied to solve the above problems. In the process, surprisingly, in the activated sludge, the knowledge that 1,4-dioxane can be decomposed by a predetermined microorganism having an extremely low relative abundance (%) with respect to the microbial community contained therein, Based on this finding, the present invention has been completed.

  That is, according to the first embodiment of the present invention, 1,4-dioxane-degrading bacterium A having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 1 is 97% or more, the base of SEQ ID NO: 2 1,4-dioxane-degrading bacterium B having a 16S rRNA gene having 97% or more identity with the sequence and 1,4 having a 16S rRNA gene having 97% or more identity to the base sequence of SEQ ID NO: 3 -An activated sludge comprising at least one selected from the group consisting of dioxane-degrading bacteria C is provided.

  In one embodiment of the present invention, there is provided a method for treating 1,4-dioxane-containing wastewater, which comprises bringing activated sludge according to the first aspect of the present invention into contact with 1,4-dioxane-containing wastewater.

According to the second aspect of the present invention, there is provided a method for identifying 1,4-dioxane degrading bacteria in a sample containing a plurality of microorganisms, wherein the plurality of the plurality of microorganisms are present in the presence of ¹³ C-labeled 1,4-dioxane. Initiating microbial culture and obtaining a culture; recovering nucleic acid from the culture; ultracentrifuging the nucleic acid to obtain a high-density fraction; and a target present in the high-density fraction Analyzing the base sequence of the gene with a next-generation sequencer to determine the relative abundance (1) of each microorganism, and starting the culture of the plurality of microorganisms in the presence of ¹³ C-unlabeled 1,4-dioxane Obtaining a culture; recovering nucleic acid from the culture; ultracentrifuging the nucleic acid to obtain a high-density fraction; and base sequence of a target gene present in the high-density fraction By generation sequencer Analyzing and determining the relative abundance (2) of each microorganism, including step B, and determining that the relative abundance (1) is greater than the relative abundance (2) as 1,4-dioxane-degrading bacteria A method is provided.

  According to the present invention, it is possible to provide a novel activated sludge excellent in 1,4-dioxane resolution, a wastewater treatment method using the same, and a method for identifying 1,4-dioxane degrading bacteria.

FIG. 1 shows a method for measuring 1,4-dioxane concentration. FIG. 2 shows chemical analysis data for full year operation of the full scale activated sludge system. (A) 1,4-dioxane concentration of inflow / outflow water, 1,4-dioxane removal rate, (B) temperature of aeration tank, (C) DO of aeration tank, (D) pH of aeration tank, (E) TOC of inflow / outflow water, (F) COD-Cr of aeration tank. FIG. 3 shows the annual transition of the microbial community in the aeration tank of the full-scale activated sludge system (system classification at the gate / class level). FIG. 4 shows measurement data in the culture process of the method for identifying 1,4-dioxane degrading bacteria according to the present invention. ( ¹³ C-labeled section: solid line, non-labeled section: dotted line): (A) CO ₂ concentration, (B) ¹³ CO ₂ concentration (●), ¹³ C atom% of CO ₂ (◯), (C) 1, 4 -Dioxane concentration, (D) COD-Cr. FIG. 5 shows the identification results of 1,4-dioxane degrading bacteria identified by the method for identifying 1,4-dioxane degrading bacteria according to the present invention. (A) Microbial community structure (phylogenetic classification at the gate / class level) in RNA density fractions (1H, 2H, 3H, L) in ¹³ C-labeled and unlabeled sections, (B), (C) and (D ) Species whose relative abundance is significantly increased in the high density fraction (1H (B), 2H (C) and 3H (D)) of the ¹³ C-labeled group compared to the equivalent density fraction (2H) of the non-labeled group (OTU: operational taxonomic unit), the relative abundance of OTU in the ¹³ C-labeled group and the unlabeled group is indicated by a circle (●) and a triangle (▲), respectively. Fig. 6-1 shows annual fluctuations in the full-scale activated sludge system of 1,4-dioxane degrading bacteria identified by the method for identifying 1,4-dioxane degrading bacteria according to the present invention (solid line: aeration tank derived, dotted line). : Derived from the return line). (A) OTU2230, (B) OTU8474, (C) OTU12266, (D) OTU5104, (E) OTU13856, (F) OTU2197. FIG. 6-2 shows annual fluctuations in the full-scale activated sludge system of 1,4-dioxane-degrading bacteria identified by the method for identifying 1,4-dioxane-degrading bacteria according to the present invention (solid line: derived from aeration tank, dotted line). : Derived from the return line). (H) OTU100 (I) OTU8385 (J) OTU8532.

  Embodiments according to one embodiment of the present invention will be described below. The present invention is not limited only to the following embodiments.

  In this specification, “X to Y” indicating a range means “X or more and Y or less”. Unless otherwise specified, measurement of operation and physical properties is performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50% RH.

<Activated sludge>
The first aspect of the present invention is a 1,4-dioxane-degrading bacterium A having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 1 is 97% or more, identical to the base sequence of SEQ ID NO: 2. 1,4-dioxane-degrading bacterium B having a 16S rRNA gene having a sexiness of 97% or more and 1,4-dioxane-degrading bacterium having a 16S rRNA gene having an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 3 It is activated sludge containing at least one selected from the group consisting of C.

  The activated sludge generally refers to a mud-like substance obtained by continuously agitating and agitating sewage, drainage, etc., and proliferating various aerobic cells having high ability to assimilate and oxidize those contained organic substances. . The activated sludge contains a microbial community and may contain filamentous fungi and protozoa in addition to bacteria. In this specification, the microbial community contained in activated sludge is also called "sludge microbial community."

The present inventors examined the correlation between the state of microbial communities in activated sludge and 1,4-dioxane degradation. In addition, according to the method for identifying 1,4-dioxane degrading bacteria described below, in the high-density fractions (1H and 2H) from the sludge microbial community, seven kinds of ¹³ C cultures that are significantly higher than the unlabeled cultures 1,4-Dioxane degrading bacteria were identified. These 1,4-dioxane degrading bacteria had a very low relative abundance (for example, 1.5% or less) with respect to the entire sludge microbial community. Surprisingly, it has been found that the 1,4-dioxane treatment performance of the activated sludge varies in conjunction with the variation of the relative abundance of 1,4-dioxane degrading bacteria.

  The activated sludge of the present invention contains a predetermined 1,4-dioxane degrading bacterium in the microbial group concentration. By using such activated sludge, 1,4-dioxane can be stably decomposed.

  The activated sludge of the present invention systematically characterized about 30,000 to about 110,000 microbial species. In these microbial species, the relative abundance of 1,4-dioxane degrading bacteria of the microbial species may be extremely low relative to the entire sludge microbial community. The relative abundance of 1,4-dioxane degrading bacteria is, for example, 0.001% to 1.500% with respect to the entire sludge microbial community.

  In the present invention, as described later, a sludge microbial community structure was analyzed by performing high-speed and large-scale base sequence decoding using a next-generation sequencer. This makes it possible to identify 1,4-dioxane-degrading bacteria with extremely low relative abundance relative to the entire sludge microbial community. Since the following 1,4-dioxane degrading bacteria are extremely small in the activated sludge, it is difficult to isolate them for technical reasons. Therefore, no deposit has been made with the depository. However, the applicant, for activated sludge containing 1,4-dioxane-degrading bacterium according to the present invention, falls under each item of Article 27-3 of the Japanese Patent Law Enforcement Regulations, subject to compliance with each law. Guarantee to sell to third parties.

[1,4-dioxane degrading bacteria]
In the present specification, 1,4-dioxane-degrading bacterium means a bacterium that can be decomposed and assimilated using 1,4-dioxane as a single carbon source, or a bacterium that can decompose and assimilate 1,4-dioxane.

  Hereinafter, bacteria that can decompose and assimilate using 1,4-dioxane as a single carbon source, and bacteria that can decompose and assimilate 1,4-dioxane will be described.

(Bacteria that can be decomposed and assimilated using 1,4-dioxane as a single carbon source)
In the 1,4-dioxane-degrading bacterium according to the present invention, a bacterium that can be decomposed and assimilated using 1,4-dioxane as a single carbon source (hereinafter, also simply referred to as “dioxane-assimilating bacterium”) is described later. In particular, in the high-density fraction of 1H and 2H, the relative abundance of the ¹³ C culture is higher than that of the unlabeled culture. Are microorganisms that are significantly elevated. Examples of the dioxane-assimilating bacterium include a bacterium having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In the activated sludge according to the present invention, dioxane assimilating bacteria are not limited to these, and the identity with the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 is 97% or more. A bacterium having a 16S rRNA gene and a bacterium selected from conventionally known dioxane-assimilating bacteria may be further included.

  In the present specification, the identity with a base sequence means a coincidence shared between two sequences when the two sequences are aligned in an optimal manner in any base sequence, unless otherwise specified. Means the percentage of the number of nucleotides formed. That is, identity (%) = (number of matched positions / total number of positions) × 100, and can be calculated using a commercially available algorithm. Such an algorithm is also described in Altschul et al. , J. et al. Mol. Biol. 215 (1990) 403-410, which is incorporated into the NBLAST and XBLAST programs. More specifically, the search / analysis regarding the identity of the base sequence can be performed by an algorithm or program (for example, BLASTN, ClustalW, etc.) well known to those skilled in the art. Those skilled in the art can appropriately set parameters when using a program, and may use default parameters of each program. Specific techniques for these analysis methods are also well known to those skilled in the art.

  A dioxane assimilating bacterium having a 16S rRNA gene comprising the base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 is shown. It is shown in 1. In Table 1 below, “homology (%)” was determined for the nucleotide sequences of SEQ ID NOs: 1 to 7 using the BLAST program of the nucleotide sequence database of NCBI (The National Center for Biotechnology Information).

  1,4-Dioxane-degrading bacterium (dioxane-utilizing bacterium) (OTU2230, OTU8474, OTU5104 or OTU13856) having a 16S rRNA gene comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5 ) Have a sequence homology of 88.6%, 94.1%, 94.9% or 96.9%, respectively, for the closest microbial species on the database. In the analysis using the base sequence of 16S rRNA gene, when it shows 97% or more homology with a known species, it can be defined as the same species. That is, these 1,4-dioxane degrading bacteria (dioxane assimilating bacteria) are considered to be microorganisms having different species from known microbial species.

  A 1,4-dioxane-degrading bacterium (dioxane-utilizing bacterium) (OTU12266) having a 16S rRNA gene containing the base sequence of SEQ ID NO: 3 is a nitrosomonas ureae known as a degrading bacterium of aromatic compounds such as benzene and toluene. The sequence homology with respect to (Nitrosomonas ureae) is 97.6%, and is considered to be a related species of Nitrosomonas ureae.

  1,4-dioxane degrading bacteria (dioxane assimilating bacteria) (OTU2197 or OTU6825) having a 16S rRNA gene containing the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7 are respectively Pseudocardia dioxanovolans (Pseudocardia). Dioxanivorans) or Micrococcus luteus. Pseudocardia dioxanovorans is a known 1,4-dioxane degrading bacterium.

(Bacteria capable of degrading and assimilating 1,4-dioxane)
The activated sludge of the present invention can contain bacteria capable of decomposing and assimilating 1,4-dioxane as 1,4-dioxane-degrading bacteria in addition to the dioxane-assimilating bacteria.

In the 1,4-dioxane-degrading bacterium according to the present invention, a bacterium capable of degrading and assimilating 1,4-dioxane (in the present specification, simply referred to as “dioxane anabolic bacterium”) refers to “1,4-dioxane” described later. Specifically, microorganisms whose relative abundance is significantly increased in ¹³ C cultures compared to unlabeled cultures in the high density fraction of 3H. is there. Examples of the dioxane assimilating bacterium include a bacterium having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

  Table 2 shows dioxane assimilating bacteria having a 16S rRNA gene comprising the nucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. In Table 2 below, “homology (%)” is determined using the BLAST program of the nucleotide sequence database of NCBI (The National Center for Biotechnology Information) for the nucleotide sequences of SEQ ID NOs: 8 to 10 in the same manner as described above. did.

  1,4-Dioxane-degrading bacterium (dioxane anabolic bacterium) (OTU8385 or OTU8532) having a 16S rRNA gene containing the base sequence of SEQ ID NO: 9 or SEQ ID NO: 10 is the most closely related microbial species on the database. , The sequence homology is 95.7% or 88.9%, respectively. As described above, in the analysis using the base sequence of the 16S rRNA gene, it can be defined as the same species when showing 97% or more homology with a known species. That is, these 1,4-dioxane-degrading bacteria (dioxane assimilating bacteria) are considered to be microorganisms having different species from known microbial species.

  A 1,4-dioxane-degrading bacterium (dioxane assimilating bacterium) (OTU100) having a 16S rRNA gene containing the base sequence of SEQ ID NO: 8 is considered to be Rhodopseudomonas palustris.

  In one embodiment of the present invention, the activated sludge is a 1,4-dioxane-degrading bacterium A having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 1 is 97% or more (herein, simply “ 1,4-dioxane-degrading bacterium A ”), 1,4-dioxane-degrading bacterium B having a 16S rRNA gene whose identity to the nucleotide sequence of SEQ ID NO: 2 is 97% or more (in this specification, simply (Also referred to as “1,4-dioxane-degrading bacterium B”) and 1,4-dioxane-degrading bacterium C having a 16S rRNA gene whose identity to the nucleotide sequence of SEQ ID NO: 3 is 97% or more (herein, And at least one selected from the group consisting of “1,4-dioxane-degrading bacterium C”). The activated sludge preferably contains at least one of 1,4-dioxane degrading bacteria A and 1,4-dioxane degrading bacteria B, more preferably 1,4-dioxane degrading bacteria A, from the viewpoint of 1,4-dioxane degrading ability. 1,4-dioxane degrading bacteria B and 1,4-dioxane degrading bacteria C are all included.

  In a preferred embodiment of the present invention, activated sludge has an identity with the base sequence of SEQ ID NO: 4 of 97 from the viewpoint of 1,4-dioxane degrading bacteria in addition to the 1,4-dioxane degrading bacteria of the above embodiment. % 1,4-dioxane-degrading bacterium D having a 16S rRNA gene of not less than 1% (also referred to simply as “1,4-dioxane-degrading bacterium D” in the present specification), and the identity with the base sequence of SEQ ID NO: 5 1,4-dioxane-degrading bacterium E having a 16S rRNA gene of 97% or more (herein simply referred to as “1,4-dioxane-degrading bacterium E”), and the same as the base sequence of SEQ ID NO: 6 1,4-dioxane-degrading bacterium F (also referred to simply as “1,4-dioxane-degrading bacterium F” in the present specification) having a 16S rRNA gene having a sex of 97% or more is further included.

  In a further preferred embodiment of the present invention, in addition to the 1,4-dioxane-degrading bacterium of the above embodiment, from the viewpoint of more efficiently degrading 1,4-dioxane, the activated sludge has the base sequence of SEQ ID NO: 7. 1,4-dioxane-degrading bacterium having a 16S rRNA gene whose identity is 97% or more (hereinafter, also simply referred to as “1,4-dioxane-degrading bacterium G”), base sequence of SEQ ID NO: 8 1,4-dioxane-degrading bacterium having a 16S rRNA gene whose identity is 97% or more (hereinafter, also simply referred to as “1,4-dioxane-degrading bacterium H”), base sequence of SEQ ID NO: 9 1,4-dioxane-degrading bacterium having a 16S rRNA gene having an identity of 97% or more (hereinafter also simply referred to as “1,4-dioxane-degrading bacterium I”) and the salt of SEQ ID NO: 10 It is selected from the group consisting of 1,4-dioxane degrading bacteria having a 16S rRNA gene whose identity to the sequence is 97% or more (also simply referred to as “1,4-dioxane degrading bacteria J” in the present specification) Contains at least one. More preferably, the activated sludge according to the present embodiment is a 1,4-dioxane-degrading bacterium H having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 8 and the base of SEQ ID NO: 9 It includes at least one of 1,4-dioxane-degrading bacteria I having a 16S rRNA gene whose identity to the sequence is 97% or more.

  In the 1,4-dioxane-degrading bacterium contained in the activated sludge of the present invention, the relative abundance (%) of the dioxane-assimilating bacterium (1,4-dioxane-degrading bacterium AG) is as long as the effects of the present invention can be expressed. Although it does not restrict | limit especially, it is 0.001-1.500% with respect to the whole sludge microbial community. In addition, the relative abundance (%) of dioxane assimilating bacteria (1,4-dioxane degrading bacteria H to J) is not particularly limited as long as the effects of the present invention can be expressed. 001-5.700%. The value of relative abundance is a value obtained by the method described in the examples.

[Treatment of 1,4-dioxane-containing wastewater]
According to one Embodiment of this invention, the processing method of the 1, 4- dioxane containing waste water which has making the activated sludge which concerns on the said 1st form contact a 1, 4- dioxane containing waste water is provided.

  As a method for treating 1,4-dioxane-containing wastewater, any conventionally known method can be used as long as the activated sludge is brought into contact with 1,4-dioxane-containing wastewater. For example, 1,4-dioxane can be decomposed by adding 1,4-dioxane-containing wastewater to an aeration tank containing the activated sludge. In addition, 1,4-dioxane can be decomposed by adding the activated sludge to a container containing 1,4-dioxane-containing wastewater.

  The 1,4-dioxane-containing wastewater is not particularly limited as long as 1,4-dioxane is contained. The 1,4-dioxane-containing waste water may contain an organic compound, a nitrogen compound, a sulfur compound, and the like.

  When 1,4-dioxane-containing waste water is added to an aeration tank containing activated sludge, MLSS (active sludge suspended solids) is preferably 4,000 mg / dioxide from the viewpoint of 1,4-dioxane resolution. L to 12,000 mg / L.

  In an aeration tank containing activated sludge, operation and management conditions may be adjusted as appropriate, and conventional knowledge can be used. For example, the pH may be 6 to 8, the temperature may be 20 to 40 ° C., and the dissolved oxygen amount (DO: Dissolved Oxygen) may be 2 to 10 mg / L.

  The concentration of 1,4-dioxane contained in the 1,4-dioxane-containing wastewater is not particularly limited. By using the activated sludge of the present invention a plurality of times, 1,4-dioxane can be sufficiently removed.

<Method for Identifying 1,4-Dioxane Degrading Bacteria>
A second aspect of the present invention is a method for identifying 1,4-dioxane-degrading bacteria in a sample containing a plurality of microorganisms, wherein the plurality of microorganisms in the presence of ¹³ C-labeled 1,4-dioxane. Starting the culture and obtaining the culture; collecting the nucleic acid from the culture; ultracentrifugating the nucleic acid to obtain a high-density fraction; and the target gene present in the high-density fraction The base sequence is analyzed by a next-generation sequencer, and the cultivation of the plurality of microorganisms is started in the presence of the step A in which the relative abundance (1) of each microorganism is obtained and the presence of ¹³ C-unlabeled 1,4-dioxane. Recovering nucleic acid from the culture; ultracentrifugating the nucleic acid to obtain a high-density fraction; and next-generation sequencer to determine the base sequence of the target gene present in the high-density fraction Analyzed by And determining the relative abundance (2) of each microorganism, and determining that the microorganism having a relative abundance (1) greater than the relative abundance (2) is a 1,4-dioxane-degrading bacterium It is.

  Examples of the sample containing a plurality of microorganisms include those containing about tens of thousands of microorganisms, such as the activated sludge described above. Hereinafter, a method for identifying 1,4-dioxane-degrading bacteria contained in an activated sludge sample will be described using activated sludge collected from an actual environment as an example of a sample containing a plurality of microorganisms. However, the invention according to this embodiment is not limited.

[Step of determining the relative abundance of microorganisms]
In the method of identifying the 1,4-dioxane-degrading bacteria of the present invention, using a ¹³ C-labeled 1,4-dioxane in the step A, using ¹³ C unlabeled 1,4-dioxane in the step B. As described above, in the present specification, 1,4-dioxane-degrading bacterium refers to a bacterium that can decompose and assimilate using 1,4-dioxane as a single carbon source, or a bacterium that can decompose and assimilate 1,4-dioxane. means. ¹³ C-labeled 1,4-dioxane incorporating 1,4-dioxane degrading bacteria is heavier than the captured 1,4-dioxane degrading bacteria to ¹³ C unlabeled 1,4-dioxane. For example, ¹³ C-labeled 1,4-dioxane by culture ⁽¹³ C-labeled culture) and ¹³ C unlabeled 1,4-dioxane by culture (unlabeled cultures) were prepared, respectively culture The nucleic acid (RNA) in the high-density fraction obtained from can be analyzed and compared. Microorganisms with a higher relative abundance in the ¹³ C labeled culture than the relative abundance in the high density fraction of the unlabeled culture took up ¹³ C and became heavier (more easily transferred to the higher density fraction). ) It is understood that it is a microorganism. Thereby, a microorganism (1,4-dioxane degrading bacterium) that remarkably takes in ¹³ C can be identified.

  In one embodiment according to the identification method of this embodiment, the relative abundance of microorganisms is determined by the following four steps. In Step A and Step B, the following steps (ii) to (iv) are common.

(I) ¹³ C-labeled 1,4-dioxane (step A) or ¹³ C unlabeled 1,4-dioxane (step B) was added to the activated sludge sample, culturing a microorganism contained in a sample ;
(Ii) recovering nucleic acid (RNA) from each culture;
(Iii) separating the recovered nucleic acids by ultracentrifugation to obtain a high-density fraction; and (iv) using a next-generation sequencer to determine the base sequence of the target nucleic acid (RNA) (eg, 16S rRNA) present in the high-density fraction. To analyze.

In the T-RFLP (Terminal restriction fragment length polymorphism) method conventionally used in microbial community structure analysis, in order to obtain ¹³ C-labeled RNA sufficient for detection, a high concentration of substrate and a long incubation time are often set. It was. This creates a gap between the real environment condition and the culture condition, which makes it difficult to identify microorganisms having metabolic activity in the real environment.

  The present inventors have found that by using a next-generation sequencer, labeled RNA can be detected with higher sensitivity even under conditions closer to the actual environment without the need for addition of a high concentration of substrate or a long incubation time. The invention according to this embodiment has been completed.

  Hereinafter, the above (i) to (iv) will be specifically described.

(I) ¹³ with the addition of C-labeled 1,4-dioxane or ¹³ C unlabeled 1,4-dioxane in activated sludge samples, the activated sludge sample used in the culture by culturing a microorganism contained in a sample, From the viewpoint of suppressing changes in the state of the sludge microbial community, the cultivation of microorganisms is preferably started within 10 hours, more preferably within 6 hours, and even more preferably within 4 hours from the collection of the activated sludge sample.

The microorganism is cultured in the presence of 1,4-dioxane by mixing the activated sludge sample with ¹³ C-labeled 1,4-dioxane or ¹³ C-unlabeled 1,4-dioxane. In the present specification, ¹³ C-labeled 1,4-dioxane was added to the cultures cultivated ^"13 C culture", the ¹³ C unlabeled 1,4-dioxane cultures were incubated with the addition of This is referred to as “unlabeled culture”.

Cultivation of the microorganism is from the viewpoint of capable of maintaining a state of sludge microbial community, active against sludge sample, ¹³ C-labeled 1,4-dioxane or ¹³ C unlabeled 1,4-dioxane only was added It is preferable to culture. However, other components such as ethylene glycol may be added as necessary.

¹³ C-labeled or ¹³ C unlabeled 1,4-dioxane concentration, based on the density variation in a real environment (5~70mg / L), it can be appropriately adjusted. In one embodiment of the invention according to this embodiment, the culture of the plurality of microorganisms is started in the presence of 100 to 300 mg / L of the 1,4-dioxane. When the concentration of 1,4-dioxane is 100 mg / L or more at the start of culture, 1,4-dioxane-degrading bacteria decompose and assimilate 1,4-dioxane even in the following preferable culture time. Or can be disassembled and assimilated. When the 1,4-dioxane concentration is 300 mg / L or less, the state change of the sludge microbial community can be suppressed. Therefore, in the method for identifying 1,4-dioxane degrading bacteria of the present invention, the cultivation of the plurality of microorganisms may be started in the presence of 100 to 300 mg / L of the ¹³ C-labeled 1,4-dioxane. preferable.

  The culture temperature of microorganisms may be set based on the annual temperature change in the actual environment. The culture temperature of the microorganism is preferably in the range of 20 to 40 ° C. from the viewpoint that 1,4-dioxane degrading bacteria active in the real environment can be detected. When the culture temperature is 20 ° C. or higher, the degradation of 1,4-dioxane by 1,4-dioxane degrading bacteria can be confirmed. Moreover, culture time can be made into the following preferable range. When the culture temperature is 40 ° C. or lower, the state change of the sludge microbial community can be suppressed.

The culture | cultivation time of microorganisms is 12 hours or less, for example, Preferably it is 6 to 10 hours. When the cultivation time is 6 hours or more, a sufficient amount of dioxane decomposition for ¹³ C labeling of microorganisms can be secured. When the incubation time is less than 10 hours, ¹³ can recover nucleic acid before C-labeled 1,4-dioxane ¹³ C from microorganisms captured by decomposing is released into the liquid phase, thus between the ¹³ C microorganisms Noise due to the food chain (detection of non-1,4-dioxane degrading bacteria) can be suppressed.

  While culturing the microorganism, the culture solution may be stirred or shaken.

For culturing microorganisms, conventionally known culture devices can be used, but considering the following points, it is preferable to use a vial with a sufficiently secured gas phase volume. There is an advantage that contact between air and activated sludge can be promoted by sufficiently securing the gas phase volume. In addition, by culturing in a closed system called a vial, there is an advantage that changes in the gas phase (such as the production of ¹³ CO ₂ ) can be closely tracked. In particular, when culturing microorganisms, it is necessary to measure the oxygen concentration in the gas phase portion to confirm that a sufficient amount of oxygen is secured. In the identification method of the present embodiment, in the culture of microorganisms, in order to identify aerobic 1,4-dioxane-degrading bacteria, the above-mentioned advantages can be obtained by using a vial bottle usually used for anaerobic bacteria culture. Can be obtained.

That is, in one embodiment of the second aspect of the present invention, the method for identifying 1,4-dioxane-degrading bacteria comprises aerobically culturing aerobic microorganisms in an airtight container in step A and step B, Preferably, the method includes measuring a gas phase CO ₂ concentration in the container. By measuring the CO ₂ concentration in the gas phase, 1,4-dioxane decomposition (mineralization) can be specified.

Moreover, it is preferable that the method for identifying 1,4-dioxane degrading bacteria further includes measuring the ¹³ CO ₂ concentration in the gas phase in Step A and Step B. By measuring the ¹³ CO ₂ concentration in the gas phase, decomposition (mineralization) of ¹³ C-labeled 1,4-dioxane can be specified.

(Ii) Recovering nucleic acid from culture The method for recovering nucleic acid from ¹³ C-labeled culture or unlabeled culture obtained in (i) is not particularly limited, and a conventionally known method can be used. . The nucleic acid to be recovered may be either DNA or RNA, but is preferably RNA from the viewpoint of detecting the metabolic activity of a microorganism with high sensitivity even in a short time.

  Hereinafter, a method for recovering RNA will be described.

  Examples of methods for recovering RNA include physical crushing methods using beads (glass beads, mixed beads of zirconia and silica, etc.), crushing methods using ultrasonic treatment, crushing methods using a French press, and samples frozen in liquid nitrogen in a mortar. Destroy microbial cells using a method such as crushing with a pestle. Thereafter, proteins are removed using a conventionally known method (phenol / chloroform extraction or the like), DNA is digested using DNase, and RNA is recovered.

(Iii) Separating the recovered RNA by ultracentrifugation to obtain a high-density fraction In the RNA recovered in (ii), ultracentrifugation (for example, 128,000 × g, 20 ° C., 42 to 65 hours) Thus, a nucleic acid density gradient (density fraction) can be obtained.

  The RN density fraction is determined by the buoyant density using a refractometer. Specifically, RNA exhibiting a buoyant density of 1.803 to 1.808 g / ml, 1.796 to 1.800 g / ml, 1.788 to 1.793 g / ml, and 1.769 to 1.771 g / ml Fractions are defined as 1H, 2H, 3H, and L fractions, respectively. High density fractions indicate 1H, 2H and 3H.

(Iv) Analyzing the base sequence of the target nucleic acid (RNA) present in the high-density fraction with a next-generation sequencer In the identification method of this embodiment, the target nucleic acid (RNA used for identifying 1,4-dioxane-degrading bacteria ) Is preferably 16S rRNA from the viewpoint of suitability for large-scale phylogenetic analysis.

  The next generation sequencer is a term used in contrast to the “first generation sequencer”, which is a fluorescent capillary sequencer using the Sanger method, and a sequential DNA synthesis method using DNA polymerase or DNA ligase, It means an apparatus for determining base sequences in a massively parallel manner by comprehensively analyzing fragments with a read length of several tens to several thousand bps from tens of millions to hundreds of millions of DNA fragments. The next generation sequencer uses a sequencing principle different from the first generation sequencer using the Sanger method that uses dideoxynucleotides to stop the elongation of DNA polymerase. Such principles include synthetic sequencing methods, pyrosequencing methods, ligase reaction sequencing methods, and the like. To date, a variety of next-generation sequencers have been provided by many companies and research institutes, such as HiSeq 2500 (Illumina Corporation), MiSeq (Illumina Corporation), 5500xl SOLiD (registered trademark) (Thermo Fisher Scientific) Tiffic Co., Ltd.), Ion Proton (registered trademark) (Thermo Fisher Scientific Co., Ltd.), Ion PGM (registered trademark) (Thermo Fisher Scientific Co., Ltd.), GS FLX + (Roche Diagnostics) Is mentioned.

  The analysis method using the next-generation sequencer can be carried out according to the attached instructions. Thereby, tens of thousands to hundreds of thousands of microbial species (OTU) can be identified per sample, and the relative abundance (%) of each microbial species can be analyzed.

[Judgment method of 1,4-dioxane degrading bacteria]
To determine 1,4-dioxane-degrading bacteria from a sample containing multiple microorganisms, the relative abundance (1) of microorganisms in the high-density RNA fraction in ¹³ C culture and the high-density RNA in unlabeled culture This can be done by comparing the relative abundance (2) of microorganisms in minutes.

Specifically, among OTUs, microorganisms that are significantly elevated in the high density fractions (1H, 2H, and 3H) in the ¹³ C culture compared to the unlabeled culture are identified as Student's t-test (Student's t-test). t-test).

  Student's t-test is one of parametric tests. The t-test is a method for testing whether there is a difference in the average value between two data, data X and data Y. Student's t-test is a method used particularly when there is no correspondence between two data, and equivariance can be assumed in the distribution of the two data.

Microorganisms in which the relative abundance (1) is significantly higher than the relative abundance (2) in the high-density fraction takes up, degrades and assimilates ¹³ C-labeled 1,4-dioxane, or By decomposing and assimilating, since it has ¹³ C-labeled and heavy RNA, such a microorganism can be determined to be a 1,4-dioxane-degrading bacterium (see FIG. 5).

  Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the present invention is not limited to these.

<Evaluation of activated sludge>
[Chemical analysis]
Industrial catalyst wastewater containing 1,4-dioxane is treated for about one year by the full-scale activated sludge system at Nippon Shokubai Kawasaki Works Ukishima Plant (10-12 Ukishimacho Kawasaki-ku, Kawasaki-shi, Kanagawa). (May 2015-April 2016: Of which, the system was suspended from August 20, 2015 to September 23, 2015). Sludge residence time (SRT) and hydraulic retention time (HRT) were set to 1 day. Moreover, it adjusted suitably so that MLSS might be about 7,000-10,000 mg / L. The dissolved oxygen (DO) was measured with a DO meter DO5509 (manufactured by FUSO Corporation), the temperature was measured with a thermometer (manufactured by Yokogawa Electric Corporation), and the pH was measured with a pH meter pH200S (manufactured by Yokogawa Electric Corporation). Total organic carbon (TOC) of inflow / outflow water in the aeration tank was determined by TOC-4110 (manufactured by Shimadzu Corporation). Chemical oxygen demand (COD: Chemical oxygen demand) -Cr was measured using a TNT821 kit (manufactured by Hack) and a COD analyzer (DRB200; manufactured by Hack).

  The 1,4-dioxane concentration of inflow / outflow water in the aeration tank was determined by the method and conditions of FIG.

  The results of chemical analysis are shown in FIG.

  As shown in FIG. 2, the concentration of 1,4-dioxane in the influent in the aeration tank varied from 5 to 70 mg / L throughout the year. As a result of the measurement, 1,4-dioxane treatment was good from the start of the test to July 13, 2015 (removal rate: about 97%), but then the period during which the temperature in the aeration tank rose (July 2015) From 21st to August 10th, a decrease in DO (<1 mg / L) and a decrease in 1,4-dioxane removal rate (<68%) were observed. Although the deterioration of 1,4-dioxane treatment was observed when the system was restarted after the suspension period (September 24 to October 26, 2015) (removal rate: 0% to 74%), then recovered. Good 1,4-dioxane treatment was achieved from November 2, 2015 to March 22, 2016 (removal rate: 87% -99%). Since then, a decreasing trend of 1,4-dioxane treatment performance was observed (removal rate: <72%). In addition, during the period when stable 1,4-dioxane treatment performance was observed, other physicochemical parameters also remained unchanged.

[Microbiological analysis]
Microbiological analysis was performed using 41 activated sludge samples collected over time from the activated sludge system.

  The return sludge sample in the aeration tank was centrifuged (20,400 × g, 4 ° C., 3 to 5 minutes) to obtain a centrifugal precipitate. To the centrifugal precipitate, mixed beads of zirconia and silica (Zirconia / Silica Beads; average particle size 0.1 mm) are added, and microbial cells in the solid phase using Shake Master Auto (manufactured by Biomedical Science Co., Ltd.) After crushing, a coexisting protein was removed by a purification step using phenol and chloroform. Then, RNA was digested by performing Type II-A RNase (manufactured by Sigma-Aldrich), and DNA was purified.

  Using purified DNA as a template and targeting 16S rRNA gene V4 region (about 300 base pairs), DNA amplification reaction (polymerase chain reaction: PCR) by Q5 Hot Start High-Fidelity DNA Polymerase (manufactured by New England Biolabs) was performed. It was.

  The obtained PCR product was purified using AMPure XP kit (manufactured by Beckman Coulter) and Wizard (registered trademark) SV gel and PCR clean-up kit (manufactured by Promega), and the concentration was Quant-iT PicoGreen dsDNA reagent Measurement was performed with a kit (manufactured by Thermo Fisher Scientific Co., Ltd.) and a fluorescence quantification apparatus 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 300 cycles of the MiSeq Reagent kit and the next-generation sequencer MiSeq (manufactured by Illumina). Decode tens of thousands of gene fragments from each sample, remove non-specific sequences using software motur (ver 1.31.2), and use software QIIME (ver 1.7.0), Microbial system analysis was performed, and identification of microbial species (OTU) and analysis of relative abundance (%) were performed.

  In the analysis by the next-generation sequencer, 41 libraries were created, and a total of about 2800000 gene sequences were analyzed (68616 sequences / library). As a result of phylogenetic analysis of the next-generation sequence data at the higher microbial classification level (gate or class), it can be seen that the sludge microbial community has changed significantly throughout the year (Fig. 3).

<Identification of 1,4-dioxane degrading bacteria>
[Culture of sludge microbial communities with 1,4-dioxane]
Microorganisms contained in the activated sludge sample were cultured using the activated sludge sample collected in the morning of December 1, 2015 at the Ukishima Plant of Nippon Shokubai Kawasaki Factory. The time from collection of the activated sludge sample to the start of culture was within 4 hours.

Specifically, 20 ml of the collected activated sludge sample was accommodated in a 100 ml glass vial. The gas phase portion was air. ¹³ C-labeled 1,4-dioxane solution (Sigma-Aldrich) or unlabeled 1,4-dioxane solution (Wako Pure Chemical Industries, Ltd.) is used as the final concentration of 1,4-dioxane for the activated sludge sample. It added so that it might become 200 mg / L. Thereafter, the cells were cultured for 8 hours in a dark place at 25 ° C. with a shaking speed of 150 rpm. Furthermore, after autoclave sterilization (121 ° C., 3 times of treatment for 1 hour), a test section to which an unlabeled 1,4-dioxane solution was added was prepared. Hereinafter, the test section to which the ¹³ C-labeled 1,4-dioxane solution was added was “ ¹³ C-labeled section”, the test section to which the unlabeled 1,4-dioxane solution was added was “non-labeled section”, and autoclave sterilization was performed. The test section is referred to as “autoclave section”.

[Chemical analysis]
^{In the 13} C-labeled group and unlabeled group, the gas phase and liquid phase at 0, 2, 4, 6, and 8 hours of culture were collected and subjected to chemical analysis. The total concentrations of CO ₂ and O _{2 in} the gas phase were measured with GC-14B (manufactured by Shimadzu Corporation) equipped with a Shin Carbon ST column (manufactured by Shinwa Kako Co., Ltd.), and the ¹³ C fraction of CO ₂ was determined by GC IsoLink. GC-C-IRMS (manufactured by Thermo Fisher Scientific Co., Ltd.) consisting of ConFlo IV and Delta v plus IRMS. The 1,4-dioxane concentration in the liquid phase was determined by GC-2010plus (manufactured by Shimadzu Corporation) equipped with a DB-1 column (manufactured by Agilent Technologies). In the autoclave system, the 1,4-dioxane concentration was measured only after 8 hours of culture. COD-Cr was determined using a TNT821 kit (manufactured by Hack) and a COD analyzer (DRB200; manufactured by Hack).

^{In the 13} C-labeled group and the non-labeled group, the total CO ₂ concentration in the gas phase increased from the start of the test, and at the end, the total CO ₂ concentration reached about 1.7 mmol / L (FIG. 4 (A)). In the ¹³ C-labeled section, the concentration of ¹³ CO ₂ increased to about 150 μmol / L by 8 hours of culture (FIG. 4B).

^{In the 13} C-labeled group and the unlabeled group, the 1,4-dioxane concentration in the liquid phase showed a decrease of about 50 mg / L (FIG. 4C). Along with this, a decrease in COD was also observed (FIG. 4D).

  On the other hand, the autoclave system in which the sludge microbial community was sterilized did not show a decrease in 1,4-dioxane concentration (data not shown).

From the above, it is considered that the added 1,4-dioxane was microbially decomposed to CO ₂ .

[Microbiological analysis]
^{For the 13} C-labeled group and the non-labeled group, the sludge sample after 8 hours of culture was centrifuged (20,400 × g, 4 ° C., 3 to 5 minutes) to obtain a centrifugal precipitate. To the centrifugal precipitate, mixed beads of zirconia and silica (Zirconia / Silica Beads; average particle size 0.1 mm) are added, and microorganisms in the solid phase are used using Shake Master Auto (manufactured by Biomedical Science Co., Ltd.). After disrupting the cells, the coexisting proteins were removed through a purification step using phenol and chloroform. Then, DNA was digested by processing RQ1 DNase (manufactured by Promega), and RNA was purified. The total concentration of RNA was determined using a RiboGreen (registered trademark) RNA quantification kit (manufactured by Thermo Fisher Scientific Co., Ltd.) and a microplate reader (SH-900Lab, manufactured by Corona Electric Co., Ltd.).

  500 ng of RNA was dissolved in cesium trifluoroacetic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.), formamide (manufactured by Wako Pure Chemical Industries, Ltd.), gradient buffer (Tris 0.1 M; KCl 0.1 M; and EDTA 1 mM). The sample was subjected to ultracentrifugation at 000 × g and 20 ° C. for 60 hours.

  Thereafter, the RNA density gradient was collected using a peristaltic pump, and the floating density of each fraction was determined using a refractometer AR200 (manufactured by Leichhardt). RNA fractions having a buoyant density of 1.803 to 1.808 g / ml, 1.796 to 1.800 g / ml, 1.788 to 1.793 g / ml, and 1.769 to 1.771 g / ml, respectively, Defined as 1H, 2H, 3H, and L fractions.

  Using these purified RNAs as templates and targeting the 16S rRNA gene V4 region (about 300 base pairs), reverse transcription and gene amplification reaction (Reverse transcription-polymerase chain reaction) using Access Quick 1-step RT-PCR system (Promega) : RT-PCR).

  The obtained RT-PCR product was purified using AMPure XP kit (manufactured by Beckman Coulter) and Wizard (registered trademark) SV gel and PCR clean-up kit (manufactured by Promega), and the concentration was Quant-iT PicoGreen. It was measured with a dsDNA reagent / kit (manufactured by Thermo Fisher Scientific Co., Ltd.) and a fluorescence quantification apparatus 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 300 cycles of the MiSeq Reagent kit and the next-generation sequencer MiSeq (manufactured by Illumina). Decode tens of thousands of gene fragments from each sample, remove non-specific sequences using software motur (ver 1.31.2), and use software QIIME (ver 1.7.0), Microbial phylogenetic analysis was performed, and identification of microbial species (OTU) and analysis of relative abundance (%) were performed.

Among the obtained OTUs, at least one fraction of 1H, 2H and 3H was identified by Student's t-test (Student's t-test), which was significantly elevated in the ¹³ C-labeled group than in the unlabeled group. . The homologous species of OTU and gene sequence homology were determined by the BLAST program of NCBI nucleotide sequence database.

21 libraries were created from RNA density fractions (1H, 2H, 3H and L) and a total of about 1340000 gene sequences were analyzed (63999 sequences / library). In the unlabeled 1H fraction (floating density 1.806 g / ml), the amount of recovered RNA was low, and RT-PCR amplification was not observed (FIG. 5 (A)). As a result of phylogenetic analysis of the next-generation sequence data at a higher microorganism classification level (gate or class), the microbial community structure change with the increase in density was remarkable in the ¹³ C-labeled group compared to the non-labeled group.

Next, when analyzed at the microbial species (OTU) level, the relative abundance was significantly increased in the high density fractions (1H2H and 3H) of the ¹³ C-labeled section compared to the equivalent density fraction (2H) of the non-labeled section. Ten types of microorganisms could be identified (FIGS. 5B, 5C and 5D).

  OTU2197 (belonging to 1,4-dioxane degrading bacterium F) and OTU13856 (belonging to 1,4-dioxane degrading bacterium E) are pseudo 1,4-dioxane degrading bacterium belonging to the Actinobacterium gate. It was phylogenetically identical or closely related to Nocardia dioxanovorans (NR074465) (representing 16S rRNA gene sequence homology 100% and 96.9%, respectively).

  OTU12266 (belonging to 1,4-dioxane-degrading bacterium C) was closely related to nitrosomonas ureae (AF272414), which is known as a bacterium that decomposes aromatic compounds such as benzene and toluene (gene sequence homology). : 97.6%).

  OTU5104 (belonging to 1,4-dioxane-degrading bacterium D) and OTU6825 (belonging to 1,4-dioxane-degrading bacterium G) are methyl tert-butyl ether degrading bacterium Methylloversatillis universalis; KC5757607 ) And Micrococcus luteus (LN 998081), respectively, had 94.9% and 100% gene sequence homology.

  OTU8474 (belonging to 1,4-dioxane-degrading bacterium B) belongs to the family Burkholderiaceae where many aromatic compound-degrading bacteria are classified, and was closely related to Chitinimonas korensis; AB682437 (Sequence homology: 94.1%).

  OTU2230 (belonging to 1,4-dioxane-degrading bacterium A) has a very low sequence homology (88.6) to the most closely related microbial species on the database, Aciditrix ferrooxidans (KC208497). %), Suggesting that it is a novel microorganism that is phylogenetically different from any of the microorganisms reported so far.

  OTU100 (belonging to 1,4-dioxane-degrading bacterium H) was phylogenetically identical to the microbial species Rhodopseudomonas palustris (KT180194) having benzoic acid resolution (16S rRNA gene sequence homology: 100%).

  OTU8385 (belonging to 1,4-dioxane-degrading bacterium I) was closely related to Blastochoris viridis that can grow even in the presence of 1,4-dioxane (16S rRNA gene sequence homology: 95 .7%).

  OTU8532 (belonging to 1,4-dioxane-degrading bacterium J) has a very low sequence homology (88.9%) to the microbial species Vampirovibrio chlorellavorus that shows the degradation potential of benzoic acid. It was suggested that the microorganism is systematically extremely novel.

  From the above, 10 kinds of 1,4-dioxane degrading bacteria (7 kinds of dioxane assimilating bacteria and 3 kinds of dioxane assimilating bacteria) among tens of thousands of kinds of microorganisms present in the activated sludge system are identified and known 1 It can be seen that not only Pseudonocardia dioxanivorans, which is a 1,4-dioxane-degrading bacterium, but also phylogenetically new microorganisms are included in the activated sludge.

  Furthermore, about 10 types of 1, 4- dioxane decomposing bacteria from the sludge microbial community, those relative abundance was extracted. The annual fluctuations in relative abundance in the aeration tank and return line are shown in FIGS. 6-1 and 6-2. The relative abundance (%) of OTU6825 was very low and below the detection limit. The detection limit means that the relative abundance is at least less than 0.003% in the analysis by the next-generation sequencer.

  The relative abundance is obtained by dividing the number of sequences of a certain microorganism species (one OTU) by the total number of sequences obtained in the library (the entire sludge microbial community) when one sample (library) is targeted. The value obtained by multiplying the number by 100. The detection limit of less than 0.003% means that when one sample (library) having the smallest total sequence number (33,436) in the example is used as a target, the number of the microorganism species for which one sequence number is detected. The relative abundance was determined from a number less than (1/33496) × 100≈0.003% (less than 0.003%). This is because the total number of sequences varies from library to library, and the detection limit varies depending on the number of sequence analyzes (about 30,000 to about 110,000) in adjusting the sample supplied to the next-generation sequencer. In other words, the relative abundance of microbial species may be detected up to 0.001%, but in this example, 0.003% was set as the threshold and less than 0.003% was set as the detection limit.

  As shown in Fig. 6-1, in the sludge microbial community, 1,4-dioxane degrading bacteria (dioxane-utilizing bacteria) are all rare microbial species with a relative abundance of 1.5% or less throughout the year. It was revealed.

  OTU2230 (Fig. 6-1 (A)) is present in the activated sludge system as the most major 1,4-dioxane degrading bacterium, and its relative is particularly during the period when the 1,4-dioxane concentration increases by restarting the activated sludge system. The abundance has increased significantly. This strongly suggested that the decomposing bacteria are involved in the degradation of accumulated high concentration 1,4-dioxane.

  In addition, at the end of the test in which a trend toward deterioration in 1,4-dioxane treatment performance was observed, in addition to OTU2230, the relative abundance of OTU12266 (FIG. 6-1 (C)) and 8474 (FIG. 6-1 (B)) It is considered that the activation of 1,4-dioxane degrading bacteria C and 1,4-dioxane degrading bacteria B is likely to contribute to the stability of the activated sludge system.

  Furthermore, OTU2197 (FIG. 6-1 (F)), OTU5104 (FIG. 6-1 (D)) and OTU13856 (FIG. 6-1 (E)) are extremely rare, with relative abundances of 0.05% or less. Although they were microbial species, they showed different transition patterns.

  As shown in FIG. 6-2, in the sludge microbial community, the relative abundance of 1,4-dioxane-degrading bacteria (dioxane assimilating bacteria) is greater than that of dioxane-assimilating bacteria, but all have a relative abundance of 5 throughout the year. It became clear that it was less than 7%.

OTU100 (FIG. 6-2 (H)), OTU8385 ((FIG. 6-2 (I)), and OTU8532 (FIG. 6-2 (J)), which are dioxane assimilating bacteria, have ¹³ C accumulation compared to dioxane-assimilating bacteria. The reason is that these dioxane assimilating bacteria do not decompose only 1,4-dioxane in an actual activated sludge system, but monoethylene glycol (MEG) coexists in the system. These dioxane assimilating bacteria are also considered to decompose the decomposition intermediate of 1,4-dioxane, in particular, OTU100 and OTU8385 are dioxane The relative abundance was higher than that of the bacterium, that is, OTU100 and OTU8385 are not only 1,4-dioxane, It can be inferred that the superiority in the activated sludge system is ensured by decomposing other organic substances richer in energy than 1,4-dioxane.

  From the above, 1,4-dioxane degrading bacteria in activated sludge changed dramatically in conjunction with changes in the physicochemical parameters of the activated sludge system, and complementarily involved in maintaining 1,4-dioxane treatment performance. It is strongly suggested that

Claims (12)

  1,4-dioxane-degrading bacterium A having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 1 is 97% or more, 16S rRNA whose identity to the base sequence of SEQ ID NO: 2 is 97% or more At least selected from the group consisting of 1,4-dioxane-degrading bacteria B having a gene and 1,4-dioxane-degrading bacteria C having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 3 is 97% or more Activated sludge containing one species.   1,4-dioxane-degrading bacterium A having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 1 is 97% or more and 16S rRNA whose identity to the base sequence of SEQ ID NO: 2 is 97% or more The activated sludge according to claim 1, comprising at least one of 1,4-dioxane degrading bacteria B having a gene.   1,4-dioxane-degrading bacterium A having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 1 is 97% or more, 16S rRNA whose identity to the base sequence of SEQ ID NO: 2 is 97% or more The 1,4-dioxane-degrading bacterium B having a gene and the 1,4-dioxane-degrading bacterium C having a 16S rRNA gene having an identity of 97% or more with SEQ ID NO: 3 Activated sludge.   1,4-dioxane-degrading bacterium D having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 4 is 97% or more, 16S rRNA whose identity to the base sequence of SEQ ID NO: 5 is 97% or more And further comprising a 1,4-dioxane-degrading bacterium E having a gene and a 1,4-dioxane-degrading bacterium F having a 16S rRNA gene whose identity to the nucleotide sequence of SEQ ID NO: 6 is 97% or more. Activated sludge of any one of -3.   1,4-dioxane-degrading bacterium G having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 7 is 97% or more, 16S rRNA whose identity to the base sequence of SEQ ID NO: 8 is 97% or more 1,4-dioxane-degrading bacterium H having a gene, 1,4-dioxane-degrading bacterium I having a 16S rRNA gene whose identity with the base sequence of SEQ ID NO: 9 is 97% or more, and the base sequence of SEQ ID NO: 10 The activity according to any one of claims 1 to 4, comprising at least one selected from the group consisting of 1,4-dioxane-degrading bacteria J having a 16S rRNA gene whose identity is 97% or more Sludge.   1,4-Dioxane-degrading bacterium H having a 16S rRNA gene whose identity to the base sequence of SEQ ID NO: 8 is 97% or more and 16S rRNA whose identity to the base sequence of SEQ ID NO: 9 is 97% or more The activated sludge according to claim 5, comprising at least one of 1,4-dioxane degrading bacteria I having a gene.   The processing method of a 1, 4- dioxane containing wastewater including making the activated sludge of any one of Claims 1-6 contact a 1, 4- dioxane containing waste water. A method for identifying 1,4-dioxane degrading bacteria in a sample containing a plurality of microorganisms,
Initiating cultivation of the plurality of microorganisms in the presence of ¹³ C-labeled 1,4-dioxane to obtain a culture; recovering nucleic acid from the culture; ultracentrifuging the nucleic acid, and high density Obtaining a fraction; and analyzing the base sequence of the target gene present in the high-density fraction with a next-generation sequencer to determine the relative abundance (1) of each microorganism, and
Initiating culture of the plurality of microorganisms in the presence of ¹³ C-unlabeled 1,4-dioxane to obtain a culture; recovering nucleic acid from the culture; ultracentrifuging the nucleic acid, and high density Obtaining a fraction; and analyzing the base sequence of the target gene present in the high-density fraction with a next-generation sequencer, and determining the relative abundance (2) of each microorganism,
A method in which a microorganism having a relative abundance (1) greater than the relative abundance (2) is determined to be a 1,4-dioxane-degrading bacterium. The method according to claim 8, wherein the culture of the plurality of microorganisms is started in the presence of 100 to 300 mg / L of the ¹³ C-labeled 1,4-dioxane.   The method according to claim 8 or 9, wherein the culture in the step A and the step B is performed at 20 to 40 ° C for 6 to 10 hours. 11. The process according to claim 8, wherein the process A and the process B include aerobically culturing the plurality of microorganisms in an airtight container and measuring a gas phase CO ₂ concentration in the container. The method described in 1. The method of claim 11, further comprising measuring the ¹³ CO ₂ concentration in the gas phase.