JP2015226896A - Wastewater treatment system and wastewater treatment method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a system and method for wastewater treatment capable of supporting evaluation in effectivity of the wastewater treatment by detecting the existence of microbes contributing to decomposition and removal of ammonia nitrogen.SOLUTION: A wastewater treatment system includes an active sludge apparatus and a detection device. In the active sludge apparatus, an aerobic tank which introduces the wastewater including at least ammonia nitrogen and an active sludge and generates a nitrification material by nitrifying the ammonia nitrogen, and an anaerobic tank which decomposes the nitrification material to generate a nitrogen gas are arranged so as to enable circulation of the wastewater between both tanks. The detection device enables detection of a microbe in which the base sequence of 16S rRNA gene indicates the homologousness of 97% or more with the base sequence described in a sequence No. 1 from the wastewater at least in the active sludge apparatus.

Description

  The present invention relates to a wastewater treatment system and a wastewater treatment method for decomposing ammonia nitrogen in wastewater with activated sludge.

The activated sludge method is widely used as a wastewater treatment method containing ammonia nitrogen. In the activated sludge method, ammonia contained in the wastewater is nitrified (oxidized) under aerobic conditions by the action of microorganisms present in the activated sludge, and oxidized nitrogen compounds are reduced and removed under anaerobic conditions. Nitrogen decomposes and removes ammonia nitrogen.
Furthermore, based on such an activated sludge method, a method for adjusting the pH of a biological reaction tank in which the microorganisms are active (see Patent Document 1), a method for adjusting the dissolved oxygen concentration in the biological reaction tank (see Patent Document 2), Various ideas such as adjustment of the amount of sludge treatment in the biological reaction tank (see Patent Document 3) have been proposed, and efforts for effective wastewater treatment are still underway.

However, even with such a device, many of the microorganisms that perform the nitrification and the microorganisms that perform the denitrification have not been clarified, and it is unclear what kind of microorganisms should be proliferated preferentially. Depending on the waste water and the activated sludge used in the treatment, there is a problem that the ecological environment of the target microorganism is not prepared, and as a result, the waste water treatment is not as expected.
The wastewater is not limited to containing human waste, and even in organic wastewater, ammonia nitrogen is generated by the decomposition of amino acids (protein degradation products) in organic matter, which is harmful to the destination ecosystem. In addition, since it may cause odors, it should be treated reliably before release. For this treatment, by identifying microorganisms that contribute to the decomposition and removal of ammonia nitrogen, the ecology involved in the treatment of wastewater can be identified. It is expected to clarify the environment.

JP-A-53-070551 JP-A-61-090796 Japanese Patent Laid-Open No. 05-253597

  As a result of intensive studies, the present inventors have succeeded in identifying microorganisms that contribute to the decomposition and removal of ammonia nitrogen. If such microorganisms can be identified, the effectiveness of wastewater treatment can be evaluated before the discharge of the wastewater by detecting whether the corresponding microorganisms are present in the wastewater and the activated sludge, A reliable wastewater treatment can be expected.

  The present invention solves the above-mentioned problems in the prior art, and detects the presence of microorganisms that contribute to the decomposition and removal of ammonia nitrogen, thereby enabling the wastewater treatment system and wastewater treatment method to support the evaluation of the effectiveness of wastewater treatment. The purpose is to provide.

Means for solving the problems are as follows. That is,
<1> Waste water containing at least ammonia nitrogen and activated sludge are introduced, and each of an anaerobic tank that nitrifies the ammonia nitrogen to generate nitrate and an anaerobic tank that decomposes the nitrate to generate nitrogen gas are provided. An activated sludge apparatus in which the waste water can be circulated between tanks, and at least the base sequence of 16S rRNA gene from the waste water in the activated sludge apparatus has a homology of 97% or more with the base sequence described in SEQ ID NO: 1 A wastewater treatment system comprising: a detection device capable of detecting a microorganism to be displayed.
<2> The wastewater treatment system according to <1>, wherein the detection device is capable of detecting a microorganism in which the base sequence of the 16S rRNA gene has 97% or more homology with the base sequence described in SEQ ID NO: 2.
<3> Any one of <1> to <2> above, wherein the detection device is capable of detecting a microorganism in which the base sequence of the 16S rRNA gene has 97% or more homology with the base sequence described in SEQ ID NO: 3 The wastewater treatment system described in 1.
<4> Any one of <1> to <3>, wherein the detection apparatus is capable of detecting a microorganism in which the base sequence of the 16S rRNA gene further shows 97% or more homology with the base sequence described in SEQ ID NO: 4 The wastewater treatment system described in 1.
<5> Any one of <1> to <4>, wherein the detection apparatus is capable of detecting a microorganism in which the base sequence of the 16S rRNA gene further shows 97% or more homology with the base sequence set forth in SEQ ID NO: 5 The wastewater treatment system described in 1.
<6> The detection value of the microbial abundance at a time point before or after the start of the operation of the activated sludge device, and the microorganism at the next time point when the activated sludge device is operated for a certain period from the temporary point. The detected value of the abundance is compared with any one of <1> to <5>, in which it is possible to detect that the abundance of the microorganism is increased at the next time point from the temporary point. Wastewater treatment system.
<7> Waste water containing at least ammonia nitrogen and activated sludge are introduced, and each of an anaerobic tank that nitrifies the ammonia nitrogen to generate nitrate and an anaerobic tank that decomposes the nitrate to generate nitrogen gas A wastewater treatment method for treating the wastewater by using an activated sludge apparatus that can circulate the wastewater between tanks, wherein at least the base sequence of the 16S rRNA gene from the wastewater in the activated sludge apparatus is SEQ ID NO: 1. A wastewater treatment method comprising a detection step of detecting a microorganism having a homology of 97% or more with the base sequence described in 1.
<8> The wastewater treatment method according to <7>, wherein the detection step is a step of detecting a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence described in SEQ ID NO: 2.
<9> The method according to any one of <7> to <8>, wherein the detection step is a step of further detecting a microorganism in which the base sequence of the 16S rRNA gene has 97% or more homology with the base sequence described in SEQ ID NO: 3. The wastewater treatment method described in 1.
<10> The method according to any one of <7> to <9>, wherein the detection step is a step of detecting a microorganism in which the base sequence of the 16S rRNA gene further shows 97% or more homology with the base sequence described in SEQ ID NO: 4 The wastewater treatment method described in 1.
<11> The method according to any one of <7> to <10>, wherein the detection step is a step of detecting a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence set forth in SEQ ID NO: 5 The wastewater treatment method described in 1.
<12> The detection step includes the detection value of the microbial abundance at one time point before starting the operation of the activated sludge apparatus or after the start of the operation, and the microorganism at the next time point when the activated sludge apparatus is operated for a certain period from the temporary point. The method according to any one of <7> to <11>, further including a step of detecting that the microbial abundance is increased at the next time point from the temporary point by comparing with a detection value of the abundance. Wastewater treatment method.

  According to the present invention, the above-mentioned problems in the prior art can be solved, and wastewater treatment capable of supporting the evaluation of the effectiveness of wastewater treatment by detecting the presence of microorganisms that contribute to the decomposition and removal of ammonia nitrogen. A system and a wastewater treatment method can be provided.

It is a figure which shows an example of the activated sludge apparatus applicable to this invention. It is explanatory drawing which shows schematic structure of the activated sludge apparatus which concerns on an Example. It is a figure which shows transition of the measurement result of ammonia concentration in a verification driving | operation period. It is a figure which shows the daily change of the relative abundance of OTU1-10 whose increase rate is the top 1 rank-the 10th rank. It is a figure which shows the daily change of the relative presence rate of OTU11-20 whose increase rate is the 11th top 20th.

(Waste water treatment system)
The wastewater treatment system of the present invention includes an activated sludge device and a detection device, and may include any device as necessary.

<Activated sludge device>
The activated sludge apparatus is equipped with an aerobic tank in which waste water containing at least ammonia nitrogen and activated sludge are introduced, nitrifying the ammonia nitrogen to generate nitrate, and anaerobic reducing the nitrate to generate nitrogen gas It is set as the structure by which a tank is distribute | arranged so that the said wastewater can be circulated between each tank.

  The waste water is not particularly limited as long as it contains the ammonia nitrogen, and examples thereof include sewage containing human waste and various waste water containing organic matter.

  There is no restriction | limiting in particular as said activated sludge, The arbitrary activated sludge applied to a well-known activated sludge method is contained. That is, in the wastewater treatment system according to the present invention, the activated sludge introduced into the tank contains microorganisms that contribute to the decomposition and removal of the ammonia nitrogen, and further, the environment in the tank contains the microorganisms. Whether it is suitable for ecology is confirmed through detection of the microorganisms by a detection device described later, and it is evaluated whether the activated sludge device is effective for decomposing and removing ammonia nitrogen. As a result of the detection of the microorganisms by the detection device, if the microorganisms that contribute to the decomposition and removal of the ammonia nitrogen are not included, the activated sludge and microorganism coagulant containing such microorganisms are introduced. Thus, the effectiveness of the activated sludge apparatus can be ensured. Moreover, when the environment in the tank is not suitable for the ecology of the microorganism, the environment in the tank can be adjusted by adjusting the aeration conditions in the tank.

  The aerobic tank is not particularly limited, and can be configured similarly to an aerobic tank applied to a known activated sludge method. For example, the aerobic tank includes a reaction tank in which an aeration blower is disposed on the bottom side. can do.

  There is no restriction | limiting in particular as said anaerobic tank, It can be set as the structure similar to the anaerobic tank applied to the well-known activated sludge method. Here, in the present specification, the anaerobic tank indicates a tank having a region in which the dissolved oxygen concentration is lower than that of the aerobic tank in the wastewater introduced into the tank. Examples of the anaerobic tank include a reaction tank having a configuration in which an aeration blower is not disposed, and a porous cylindrical structure in which the waste water and the activated sludge can flow into the inside while the inflow of bubbles is difficult. And a reaction vessel into which is introduced.

The activated sludge apparatus is not particularly limited as long as it is a reaction tank having such an aerobic tank and an anaerobic tank, and further, the waste water purified water and the activated sludge / microorganisms are solid-liquid separated in the tank. What is provided with a separation membrane is preferable.
In the reaction tank applied to the activated sludge method, the activated sludge is precipitated, so that a large facility area is required, and it is difficult to maintain the microorganisms at a high concentration in the tank.
On the other hand, according to the membrane separation activated sludge method (MBR method) in which the separation membrane is disposed, the apparatus can be saved by drawing the purified water through the separation membrane disposed in a tank. Space can be made, and the microorganisms are filtered by the separation membrane, so that the microorganisms can be maintained at a high concentration in the tank.
When conforming to such a membrane separation activated sludge method, the activated sludge apparatus can be constituted by a two-tank reaction tank in which the separation membrane is arranged in either the aerobic tank or the anaerobic tank, In addition to the aerobic tank and the anaerobic tank, these tanks, the waste water, and the activated sludge are connected in a circulatory manner, and a membrane separation tank in which the separation membrane is arranged may be provided.
The separation membrane is not particularly limited, and a known microfiltration (MF) membrane or ultrafiltration (UF) membrane can be used.

  The schematic configuration of the activated sludge apparatus will be described with reference to FIG. In addition, this activated sludge apparatus is a standard apparatus comprised according to the said MBR method. Moreover, FIG. 1 is explanatory drawing which shows an example of the said activated sludge apparatus applicable to this invention.

The activated sludge apparatus 10 is composed of a two-tank reaction tank of an anaerobic tank 11 and an aerobic tank 13. A stirrer 12 is arranged in the anaerobic tank 11 so that the waste water and the activated sludge introduced into the tank can be stirred. The aerobic tank 13 is connected to the bottom side with an aeration blower 14 so that the inside of the tank can be made an aerobic environment, and the membrane separation unit 15 allows the purified water of the wastewater and the activated sludge. The solid can be separated from the microorganism, and the purified water can be discharged out of the system.
The anaerobic tank 11 and the aerobic tank 13 are connected to be able to pass water, and the waste water introduced into the anaerobic tank 11 is passed through the aerobic tank 13 while consuming dissolved oxygen in the aerobic tank 13. The nitrification (oxidation) of the ammonia nitrogen by the microorganism is performed, and the nitrification (oxide) returned from the aerobic tank 13 to the anaerobic tank 11 is reduced by the microorganism under anaerobic conditions to form nitrogen gas. Is done.

  Of the activated sludge that flows into the aerobic tank 13 from the anaerobic tank 11 together with the waste water, the surplus can be discharged out of the system by the pump 16. The activated sludge flowing from the anaerobic tank 11 into the aerobic tank 13 can be returned from the aerobic tank 13 to the anaerobic tank 11 by the pump 17.

  The membrane separation unit 15 includes the separation membrane, and performs the solid-liquid separation. A membrane cleaning liquid tank 18 is connected to the membrane separation unit 15, and when the separation membrane is clogged, the solid-liquid separation is stopped, and the separation membrane is removed by the membrane cleaning liquid drawn by the pump 19. It can be washed.

The flow rate of the waste water introduced into the anaerobic tank 11 is preferably adjusted in advance according to the treatment performance of the activated sludge apparatus 10. In this example, the flow rate adjusting apparatus 50 is connected to the activated sludge apparatus 10. .
The flow rate adjusting device 50 includes a fine screen 51 and a flow rate adjusting tank 52, and stores the waste water in the flow rate adjusting tank 52 in a state where solid matter in the waste water is removed by the fine screen 51. In the flow rate adjusting tank 52, stirring by the stirrer 53 is possible, and the waste water can be fed to the anaerobic tank 11 of the activated sludge apparatus 10 in a state where the flow rate is adjusted by the pump 54.

<Detection device>
Next, the detection device will be described.
The detection device can detect a microorganism in which the base sequence of the 16S rRNA gene has at least 97% homology with the base sequence shown in SEQ ID NO: 1 from the wastewater in the activated sludge device. Furthermore, it is preferable that the detection apparatus is capable of detecting a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence shown in SEQ ID NOs: 2 to 20. The microorganisms related to the base sequences described in SEQ ID NOs: 1 to 20 relate to microorganisms that increase corresponding to a decrease in the ammonia concentration in the tank in the examples described later, and contribute to the decomposition and removal of the ammonia nitrogen. Among them, as discussed in Examples below, since the microorganisms are strongly involved in the decomposition and removal of the ammonia nitrogen in order of increasing sequence numbers, the base sequence of the 16S rRNA gene is changed to SEQ ID NOs: 2 to 10. More preferably, a microorganism having 97% or more homology with the base sequence described above can be detected, and the base sequence of the 16S rRNA gene is 97% or higher with the base sequence described in SEQ ID NOs: 2 to 5 It is particularly preferable that a microorganism exhibiting the above can be detected.
In addition, in addition to the microorganisms related to the base sequences described in SEQ ID NOs: 1 to 20, the detection target includes a microorganism in which the base sequence of the 16S rRNA gene exhibits 97% or more homology to the base sequence of the microorganism. Including.
The microorganisms having such homology are considered to be the same “species” as described in Reference Document 1 below, and contribute to the decomposition and removal of the ammonia nitrogen.
Reference 1: P. Vandamme et al., Microbiological Reviews, 1996, p. 407-438

The detection device is not particularly limited as long as it is a device that can detect the microorganism, but it is preferable to use a high-performance sequencer called a “next-generation sequencer”.
In the next-generation sequencer, by registering the base sequences described in SEQ ID NOs: 1 to 20, the microorganism identification and homology search can be performed in large quantities at a time, and further, in a short time. it can.
Such a next-generation sequencer is not particularly limited, but for example, MiSeq manufactured by Illumina can be used, and the detection can be performed according to the attached instructions.

The detection device is not particularly limited, but the activated sludge device is operated for a certain period from the detected value of the microbial abundance at a point in time before or after the start of the operation of the activated sludge device. It is preferable that it is possible to detect that the microbial abundance is increased at the next time point from the temporary point by comparing the detected value of the microbial abundance at the next time point. If such detection can be performed, it can be confirmed that an environment suitable for the ecology of microorganisms contributing to the decomposition and removal of the ammonia nitrogen is formed in the tank, and the activated sludge is more effective. The effectiveness of the wastewater treatment of the equipment can be evaluated.
Such an increase in the microorganisms over time can be detected using the next-generation sequencer. However, it is preferable to use a PC and analysis software for obtaining more accurate and detailed systematic information. In addition, as the analysis software, for example, various software described later in the column of Examples can be used.
The microbial abundance may be detected as the absolute abundance (MLSS; mg / L) of the microorganisms contained in the activated sludge, or the relative abundance (%) of all microorganisms in the activated sludge. ) May be detected. In these cases, the detection device can be constructed according to each abundance to be detected. When the relative abundance is detected, the detection device is constructed using the next-generation sequencer, the PC, and the analysis software. In addition, when detecting the absolute abundance, in addition to these, a known MLSS measuring instrument for measuring MLSS in wastewater can be used.

(Waste water treatment method)
The wastewater treatment method of the present invention is a wastewater treatment method for treating wastewater using an activated sludge apparatus, and includes a detection step for detecting microorganisms.

<Activated sludge device>
The activated sludge apparatus includes an aerobic tank in which waste water containing at least ammonia nitrogen and activated sludge are introduced, and the ammonia nitrogen is nitrified to produce nitrate, and anaerobic that reduces the nitrate and generates nitrogen gas. A tank is arranged so that the waste water can be circulated between the tanks.
The specific contents are the same as those of the activated sludge apparatus in the wastewater treatment system of the present invention, and thus the description thereof is omitted.

<Detection process>
The detection step is a step of detecting a microorganism in which the base sequence of 16S rRNA gene shows 97% or more homology with the base sequence described in SEQ ID NO: 1 from at least the wastewater in the activated sludge apparatus, and The 16S rRNA gene is preferably a step of detecting a microorganism in which the nucleotide sequence of the 16S rRNA gene shows 97% or more homology with the nucleotide sequence of SEQ ID NOs: 2 to 20.

In addition, the detection step is not particularly limited, but the detection value of the microbial abundance at a point in time before or after the start of the operation of the activated sludge device and the activated sludge device for a certain period from the temporary point. It is preferable to include a step of comparing the detected value of the microbial abundance at the next time point when the operation is performed and detecting that the microbial abundance is increasing at the next time point from the temporary point.
When such a detection step is executed, it can be confirmed that an environment suitable for the ecology of microorganisms contributing to the decomposition and removal of the ammonia nitrogen is formed in the tank, and the activated sludge is more effective. The effectiveness of the wastewater treatment of the equipment can be evaluated.

  There is no restriction | limiting in particular as the implementation method of the said detection process, It can implement using the said detection apparatus in the said wastewater treatment system of this invention.

Waste water treatment was performed using the activated sludge apparatus according to the example produced as an experimental MBR apparatus. FIG. 2 shows a schematic configuration of the activated sludge apparatus according to the embodiment.
As shown in FIG. 2, the activated sludge apparatus 100 is a three-tank MBR apparatus having an aerobic tank 101, an anaerobic tank 102, and a membrane separation tank 103. Each tank has a capacity of 91L, 80L, and 57L, respectively. And is formed of polycarbonate.

Here, as the activated sludge, standard activated sludge distributed from Kinu Aqua Station of the West Basin Sewerage Office in Ibaraki Prefecture was used.
Moreover, organic wastewater was used as waste water. In this artificial sewage, as sewage components, 5.30 g / L CH ₃ COONa, 0.751 g / L NH ₄ Cl, 0.217 g / L KH ₂ PO ₄ , 1.41 g / L Peptone, 1 Contains .57 mg / L FeCl ₃ .6H ₂ O, 3.13 mg / L CaCl ₂ , 3.13 mg / L MgSO ₄ , 3.13 mg / L KCl, and 3.13 mg / L NaCl.

The artificial sewage and the standard activated sludge are introduced into the aerobic tank 101 from outside the system. An aeration blower (not shown) is connected to the aerobic tank 101, and the inside of the tank is adjusted to an aerobic environment.
The artificial sewage introduced into the aerobic tank 101 is introduced into the anaerobic tank 102 together with a part of the standard activated sludge in the aerobic tank 101 via the connecting portion 104. The anaerobic tank 102 is provided with a cylindrical hollow container (not shown) containing a microorganism carrier. On the surface of this hollow container, a hole with a diameter of several millimeters is opened, and the standard activated sludge and the artificial sewage can flow into the inside. An anaerobic environment is formed. Further, as the microorganism carrier, a pumice-like porous material was used as a scaffold for colonization of microorganisms.

  The artificial sewage introduced into the anaerobic tank 102 is introduced into the membrane separation tank 103 together with a part of the standard activated sludge in the anaerobic tank 102 via the connecting portion 105. In the membrane separation tank 103, a membrane separation unit 106 for separating the standard activated sludge and the artificial sewage is disposed. The membrane separation unit 106 has four separation membranes that separate the standard activated sludge from the artificial sewage. Here, as the separation membrane, a flat and bag-shaped flat membrane made of PAN (polyacrylonitrile) of 150 mm × 300 mm (Awa Paper Co., Ltd., pore size 0.07 μm) is used. The filtration is performed by drawing water into the bag, and the filtrate can be discharged out of the system as purified water. Further, the filtered standard activated sludge is returned to the aerobic tank by a pump (not shown) connected to the membrane separation tank 103 together with a part of the artificial sewage.

  For the activated sludge apparatus 100 configured as described above, the inflow speed into the artificial sewage system, the outflow speed of the purified water out of the system, and the standard from the membrane separation tank 103 to the aerobic tank 101 The return rate of the artificial sewage containing activated sludge was set to 115 L / day, and the hydraulic sewage hydraulic residence time (HRT) in the activated sludge apparatus 100 was set to 2 days. During the operation period, the standard activated sludge was constantly aerated by the aeration blower.

Further, in this embodiment, the activated sludge apparatus 100 is continuously operated under the above conditions, and when the ammonia concentration in the tank rises, a microbial flocculant (manufactured by CNT, BRcnt-5E) is added into the anaerobic tank 102. Then, the wastewater treatment was performed for the purpose of decomposing and removing ammonia nitrogen by continuously performing the verification operation for 7 days.
The microbial flocculant relates to a flocculated microbial species present in a natural environment in order to select a microorganism that decomposes and removes the ammonia nitrogen.

Regarding this example, MLSS (Mixed Liquid Suspended Solids), temperature and pH of the artificial sewage, the water quality and ammonia concentration of the artificial sewage and the purified water are measured as physicochemical parameters, and the activated sludge apparatus 100 The changes in the physicochemical parameters according to the operating conditions were observed.
Here, the measurement of the MLSS was performed on the artificial sewage near 30 cm from the sludge interface using an MLSS measuring device (Iijima Electronics Co., Ltd., IM-50P).
Moreover, each measurement of the said temperature and the said pH was performed with respect to the said artificial sewage of 30 cm vicinity from the said sludge interface using the pH measuring device (the product made by HORIBA, D-55).
The water quality of the artificial sewage is measured using a potassium permanganate acid method (COD-) on the supernatant portion solid-liquid separated from the standard activated sludge by a centrifuge (manufactured by TOMY, MX-307). Mn) was used to measure COD (Chemical Oxygen Demand) in the supernatant portion as an index.
The quality of the purified water was measured by measuring the COD in the purified water by the potassium permanganate acidic method and using this as an index.
The ammonia concentration in the artificial sewage was measured on the supernatant using a capillary electrophoresis apparatus (Agilent technologies, Agilent CE).
Finally, the ammonia concentration in the purified water was measured using the capillary electrophoresis apparatus (Agilent Technologies, Agilent CE).
These measurements were made at 5 time points during the verification operation period of 7 days.

The standard activated sludge at the 5 time points was collected, and microbial flora analysis was performed on the standard activated sludge using a next-generation sequencer (MilSeq, manufactured by Illumina).
Here, the microbial flora analysis in the next-generation sequencer was performed according to the following procedure.
First, with respect to the sludge sample (centrifugal sediment) of the standard activated sludge obtained by centrifugation, mixed beads of zirconia and silica having an average particle diameter of 0.1 mm (Zirconia / Silica Beads) were added. The sample is subjected to a beater (Shake Master, manufactured by Biomedical Science), and the cells of the microorganisms contained in the sludge sample are crushed.
Next, chromosomal DNA is extracted and purified from the obtained cell lysate by a phenol / chloroform extraction method.
Next, based on the universal primer targeting 16S rRNA of the chromosomal DNA, PCR (Polymerase Chain Reaction) using the primer described in Reference 2 below to which a barcode sequence for MiSeq manufactured by Illumina was added. The 16S rRNA gene was amplified by the method.
(Reference 2: J Gregory Caporaso et al., The ISME Journal (2012) 6, 1621-1624)

Unreacted primers and primer dimers were removed from the obtained PCR amplification product using AMPure magnetic beads (Beckmann coulter, A63881) and a purification magnet stand (Beckmann coulter, A32782). The purified product was fractionated by agarose gel electrophoresis, and only the PCR amplification product having the desired fragment length was purified using a spin column (Promega, Wizard SV Gel and PCR Clean-up System).
Next, the DNA concentration in the DNA sample was quantified using a fluorescent reagent kit for DNA staining Quant-iT PicoGreen dsDNA Assay Kit (manufactured by Thermo scientific, P11496) and a fluorescence spectrometer for trace amounts (manufactured by Thermo scientific, NanoDrop 3300). The amount was subjected to MiSeq Reagent Kits v2 (manufactured by Illumina, MS-102-2003), and DNA sequence analysis was performed using the next-generation sequencer.
As a result, sequence data of about 50,000 to 100,000 reads was obtained from the DNA sample.
The obtained sequence data is linked with gene sequence information using software ea-utils-1.1.2-301 (free software available on the Internet), and further software version 1. After removing the chimera sequence using 31.2, systematic analysis of the gene sequence was performed with software QIIME version 1.6.0, and the increase or decrease in relative abundance of each microbial species and the physicochemical parameters (ammonia The relationship with the decrease in concentration was considered. Note that the following reference 3 can be referred to for details of the software “Moture version 1.31.2”, and the following reference 4 can be referred to for details of the software QIIME version 1.6.0.
Reference 3: Schloss P. D et al., Appl. Environ. Microbiol (2009) 75, 7537-7541.
Reference 4: Caporaso J. G et al., Nat. Methods (2010) 7, 335-336.

<Discussion results>
Hereinafter, discussion results will be described.
During the operation period of the activated sludge apparatus 100, the temperature of the artificial sewage in the anaerobic tank 102 changes between 26.0 ° C and 33.3 ° C, and the pH changes between 8.39 and 8.75. , MLSS transitioned between 6,750 mg / L and 10,400 mg / L. Moreover, COD-Mn of the said purified water changed below 55.0 ppm, and it confirmed that the process condition by the activated sludge apparatus 100 was favorable, without changing with the process condition of a general MBR apparatus.
During the steady operation period of the activated sludge apparatus 100, the ammonia concentration in the anaerobic tank 102 gradually increased to 25.95 mM, and at this point, the above-described microbial flocculant was added. The transition of the measurement result of ammonia concentration during the verification operation period for 7 days is shown in FIG.
As shown in FIG. 3, the ammonia concentration gradually decreased after the addition of the microbial flocculant, and decreased to 13.64 mM at the seventh day.

In order to observe the transition of the microbial community structure during these 7 days, as described above, the standard activated sludge was subjected to analysis by the next-generation sequencer, and the microbial community was classified.
As a result, it was found that the relative abundance of some microbial species increased rapidly during this period.
Next, the increase rate was calculated by comparing the relative abundance between the first day and the seventh day of the seven days.
FIG. 4 and FIG. 5 show the transition of the relative abundance of OTUs (Operational Taxonomic Units) 1 to 20 of the microbial species whose increase rate was in the top 20 as a result of analysis. 4 and 5 are numbered in descending order of the increase rate, and these numbers are the same as the numbers in SEQ ID NOS: 1-20. That is, these numbers are common numbers. For example, the microbial species of OTU “1” is the microbial species having the base sequence of 16S rRNA gene described in SEQ ID NO: “1”, and the OTU “2” The microbial species indicates a microbial species having the base sequence of 16S rRNA gene described in SEQ ID NO: “2”. Moreover, FIG. 4 is a figure which shows the daily change of the relative abundance rate of OTU1-10 whose said increase rate is a top 1 rank-10th, and FIG. It is a figure which shows the daily change of the relative abundance rate of a certain OTU11-20, and in each figure, the area | region enclosed with the dotted line in the left side column is expanded and shown in the right side column.
The related species information, system classification information, etc. of these OTU1 to 20 are shown in Table 1 and Table 2 below.

  The increase rate of the relative abundance of each microorganism of OTU1 to 20 shown in FIG. 4 and FIG. 5 is 1,486.7 times for the OTU1 microorganism having the highest increase rate, and 9 for the OTU20 microorganism having the lowest increase rate. .6 times. The specific increase rate of each OTU is as follows. OTU1: 1,486.7 times, OTU2: 91.0 times, OTU3: 89.8 times, OTU4: 58.9 times, OTU5: 38.7 times, OTU6: 37.0 times, OTU7: 23.0 times OTU8: 21.9 times, OTU9: 20.2 times, OTU10: 20.2 times, OTU11: 19.3 times, OTU12: 18.5 times, OTU13: 14.7 times, OTU14: 14.1 times, OTU15: 13.5 times, OTU16: 13.1 times, OTU17: 11.8 times, OTU18: 10.1 times, OTU19: 10.1 times, OTU20: 9.6 times.

As described above, each of the OTU1 to 20 microorganisms has a low relative abundance before the start of the verification operation, but suddenly corresponds to a decrease in ammonia concentration (see FIG. 3) after the start of the verification operation. (See FIGS. 4 and 5).
In this respect, it is not certain how all the microorganisms of OTU1 to 20 act on the artificial sewage containing ammonia, but each microorganism of OTU8, 9, 12, and 14 has a denitrification ability. (Anaerobic breathing mode that reduces oxidized nitrogen compounds such as nitric acid to nitrogen gas) It shows extremely high homology and is considered to have the above-mentioned denitrification ability, and these microorganisms tend to increase Indirectly indicates that microorganisms other than these that produce the oxidized nitrogen compound from ammonia exist.
Therefore, if it can confirm that each microorganism of OTU8, 9, 12, 14 exists in the tank in the activated sludge apparatus 100, the denitrification performance of the activated sludge apparatus 100 can be positively evaluated, and the increase The presence of microorganisms that assimilate ammonia or oxidize the oxidized nitrogen compound can be positively evaluated.

On the other hand, each microorganism other than OTU8, 9, 12, and 14 in OTU1 to 20 is a microorganism that oxidizes ammonia to the oxidized nitrogen compound, a microorganism that has the denitrification ability, or these microorganisms and These microorganisms are symbiotic and assist these functions, and any microorganism is considered to contribute to the decomposition and removal of ammonia.
That is, if the microorganism oxidizes ammonia to the oxidized nitrogen compound, the ammonia treatment performance of the activated sludge apparatus 100 can be positively evaluated, and the increase is evaluated as improving the ammonia treatment performance. be able to. In addition, as described above, the microorganisms having the denitrification ability can positively evaluate the denitrification performance of the activated sludge apparatus 100, and the increase oxidizes ammonia to the oxidized nitrogen compound. In addition to positively evaluating the presence of microorganisms, it can be evaluated that the denitrification performance is improved. In addition, if the microorganisms assist the action of these microorganisms, the ammonia treatment performance and the denitrification performance can be positively evaluated, and the increase is an improvement in the ammonia treatment performance and the denitrification performance. Can be evaluated.
Therefore, any microorganism can be positively evaluated for its ability to decompose and remove ammonia nitrogen in wastewater.

In this example, as shown in FIGS. 4, 5, 1, and 2, microorganisms contained in the Bacteroidetes gate were preferentially confirmed as microorganisms with a high increase rate (OTU 1 to 6, 11). 13, 15, 16).
Among the microorganisms of OTU1 to 20, at least each microorganism of OTU1 to 6, 11, 13, 15, and 16 has not been reported to have ammonia oxidizing ability, and as a result of assimilating ammonia, ammonia is oxidized as described above. It can be inferred that it is a microorganism that is oxidized to a nitrogen-type nitrogen compound.
Therefore, in the experimental system according to the present embodiment, although not necessarily known, the oxidized nitrogen compound is generated from the ammonia in the artificial sewage by microorganisms containing OTU1-6, 11, 13, 15, 16 and It is considered that nitrogen gas was generated from the oxidized nitrogen compound by microorganisms including OTU8, 9, 12, and 14, and these microorganisms are preferentially generated in the tank of the activated sludge apparatus 100. It can be considered that ammonia nitrogen in the artificial sewage is reduced and nitrogen gas which is a decomposition product thereof is discharged out of the system.

  As described above, since each microorganism of OTU1-20 (each microorganism described in SEQ ID NOs: 1-20) is a microorganism involved in the decomposition and removal of ammonia nitrogen in wastewater, the base sequences of these microorganisms are detected. If setting (registration etc.) is performed so as to be detected by the apparatus, and detection work is performed, it is possible to evaluate the decomposition and removal performance of ammonia nitrogen of the activated sludge apparatus 100 at that time. Further, by performing the detection operation for a certain period, it is possible to evaluate the decomposition and removal performance of ammonia nitrogen of the activated sludge apparatus 100 over time.

DESCRIPTION OF SYMBOLS 10,100 Activated sludge apparatus 11,102 Anaerobic tank 12,53 Stirrer 13,101 Aerobic tank 14 Aeration blower 15,106 Membrane separation unit 16,17,19,54 Pump 18 Membrane cleaning liquid tank 50 Flow rate adjustment apparatus 51 Fine Screen 52 Flow adjustment tank 103 Membrane separation tank 104, 105 Connection

Claims (12)

Waste water containing at least ammonia nitrogen and activated sludge are introduced, and an anaerobic tank that nitrifies the ammonia nitrogen to generate nitrified substances and an anaerobic tank that decomposes the nitrites to generate nitrogen gas between the tanks. An activated sludge apparatus arranged to circulate the waste water, and
A detection device capable of detecting a microorganism in which the base sequence of 16S rRNA gene has at least 97% homology with the base sequence described in SEQ ID NO: 1 from at least the wastewater in the activated sludge device;
A wastewater treatment system characterized by comprising:   The wastewater treatment system according to claim 1, wherein the detection device can further detect a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence shown in SEQ ID NO: 2.   The wastewater treatment system according to any one of claims 1 to 2, wherein the detection device can further detect a microorganism in which the base sequence of the 16S rRNA gene has a homology of 97% or more with the base sequence of SEQ ID NO: 3. .   The wastewater treatment system according to any one of claims 1 to 3, wherein the detection device can further detect a microorganism in which the base sequence of the 16S rRNA gene has 97% or more homology with the base sequence of SEQ ID NO: 4. .   The wastewater treatment system according to any one of claims 1 to 4, wherein the detection device can further detect a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence shown in SEQ ID NO: 5. .   The detected value of the microbial abundance at a time point before or after the start of the operation of the activated sludge apparatus, and the microbial abundance at the next time point when the activated sludge apparatus is operated for a certain period from the temporary point. The wastewater treatment system according to any one of claims 1 to 5, wherein it is possible to detect that the microbial abundance is increased at the next time point from the temporary point by comparing with a detection value. Waste water containing at least ammonia nitrogen and activated sludge are introduced, and an anaerobic tank that nitrifies the ammonia nitrogen to generate nitrified substances and an anaerobic tank that decomposes the nitrites to generate nitrogen gas between the tanks. A wastewater treatment method for treating the wastewater using an activated sludge device that is arranged to circulate the wastewater,
A wastewater treatment method comprising a detection step of detecting a microorganism having a base sequence of 16S rRNA gene having 97% or more homology with the base sequence described in SEQ ID NO: 1 from at least the wastewater in the activated sludge apparatus .   The wastewater treatment method according to claim 7, wherein the detection step is a step of further detecting a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence shown in SEQ ID NO: 2.   The wastewater treatment method according to any one of claims 7 to 8, wherein the detection step is a step of detecting a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence shown in SEQ ID NO: 3. .   The wastewater treatment method according to any one of claims 7 to 9, wherein the detection step is a step of further detecting a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence set forth in SEQ ID NO: 4. .   The wastewater treatment method according to any one of claims 7 to 10, wherein the detection step is a step of further detecting a microorganism in which the base sequence of the 16S rRNA gene shows 97% or more homology with the base sequence shown in SEQ ID NO: 5. .   The detection step is the detection value of the microbial abundance at one time point before the start of operation of the activated sludge apparatus or after the start of the operation, and the microbial abundance at the next time point when the activated sludge apparatus is operated for a certain period from the temporary point. The wastewater treatment method according to any one of claims 7 to 11, further comprising a step of detecting that the microbial abundance is increased at the next time point from the temporary point by comparing with a detection value.