JP2009060795A - Method for analyzing bacterium contained in activated sludge - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing bacteria contained in activated sludge, by which the kinds and population of the bacteria in the activated sludge can be known quickly. <P>SOLUTION: This method for analyzing the bacteria contained in the activated sludge is characterized by extracting RNAs of the bacteria contained in the activated sludge, subjecting the extracted RNAs to a reverse transcription to obtain the reverse transcription DNAs, and then hybridizing the obtained reverse transcription DNAs with the DNA fragments of the following DNA chip. The DNA chip is such that a DNA chip is obtained, by immobilizing DNA fragments containing the DNA base sequences of Nocardia filamentous bacteria, Rhodococcus filamentous bacteria, Beggiatoa filamentous bacteria, Thiothrix filamentous bacteria, Desulfobacter sulfur metabolism bacteria, Desulfotomaculum sulfur metabolism bacteria, Desulfovibrio sulfur metabolism bacteria, Desulfomicrobium sulfur metabolism bacteria, Desulforhopalus sulfur metabolism bacteria, Nitrosomonas nitrifying bacteria, Nitrosospira nitrifying bacterium, and Nitrospira nitrifying bacterium on a substrate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a method for analyzing bacteria contained in activated sludge.

The so-called activated sludge method, which uses activated sludge to biologically decompose pollutants contained in wastewater, such as organic carbon, by various bacteria that inhabit it, is a wastewater treatment method that purifies wastewater. Is widely used as a biological wastewater treatment method for treating wastewater biologically [Patent Document 1: JP-A-5-237488, Patent Document 2: JP-A 2000-27998.
No. 2 publication etc.].

In general, bacteria contained in activated sludge each selectively decompose specific pollutants. For example, sulfur-metabolizing bacteria selectively decompose inorganic sulfur compounds and the like, but do not decompose organic carbon, ammonia nitrogen, and the like. Moreover, when bacteria in activated sludge break down pollutants,
The number of inhabitants increases according to the amount of decomposition, but on the other hand, if there is no pollutant to be decomposed, it will gradually die and decrease.

In addition, the types and concentrations of pollutants contained in the drainage from the same drainage source are usually within a certain range over a long period of time. For example, domestic wastewater discharged from households usually has organic carbon as the main pollutant, and its concentration is generally within a certain range.

For this reason, the type of bacteria in the activated sludge and the number of inhabitants are usually more appropriate for the treatment of the wastewater than the treatment of the wastewater from the same wastewater source for a long time.

However, the bacteria in the activated sludge may all be killed due to various causes such as high temperatures in summer, sudden contamination with poisonous substances, and sudden increase in pollutants. In such a case, the activated sludge used to treat wastewater from other wastewater sources may be thrown in and wastewater treatment resumed.
Such activated sludge is not necessarily suitable for pollutants contained in the wastewater to be treated. For this reason, there was a problem that the pollutants were not efficiently decomposed and the wastewater treatment was delayed.

In order to solve such a problem, it is only necessary to select and input activated sludge containing an appropriate type of bacteria with an appropriate number of inhabitants for the wastewater to be treated, and restart the wastewater treatment. For this reason, there is a need for a method that can quickly and accurately know the types of bacteria in activated sludge and the number of inhabitants.

As a method for analyzing bacteria in activated sludge, a method of observing the shape and number of bacteria with a microscope has been conventionally known [Patent Document 3: Japanese Patent Laid-Open No. Hei 5-14691].

However, the conventional analysis method using a microscope cannot analyze bacteria quickly and accurately.

JP-A-5-237488 JP 2000-279982 A Japanese Patent Laid-Open No. 5-146791 SCAS NEWS 2005-II (Sumitomo Analysis Center Co., Ltd. (August 22, 2005), pages 3-6)

Therefore, the present inventor has intensively studied to quickly know the type and number of bacteria in the activated sludge, resulting in the present invention.

That is, the present invention extracts bacterial RNA [ribonucleic acid] contained in activated sludge, performs reverse transcription of the extracted RNA to obtain reverse transcription DNA [reverse transcription deoxyribonucleic acid], and obtains the reverse transcription DNA obtained The present invention provides a method for analyzing bacteria, which is characterized by crossing with a DNA fragment of the following DNA chip.

DNA chip: on the substrate,
Nocardia spp., Rhodococcus spp.,
Beggiatoa filamentous bacteria sulfur metabolizing bacteria, Thiothrix filamentous bacteria sulfur metabolizing bacteria,
Desulfobacter genus sulfur-metabolizing bacteria, Desulfotomaculum genus sulfur-metabolizing bacteria, Desulfovibrio genus sulfur-metabolizing bacteria, Desulfomicrobium genus sulfur-metabolizing bacteria, Desulforhopalus genus sulfur-metabolizing bacteria,
Nitrosomonas nitrifying bacteria, Nitrosospira nitrifying bacteria, and a DNA chip on which DNA fragments containing DNA base sequences of Nitrospira genus nitrifying bacteria are fixed

The analysis method of the present invention is suitable for analysis of nitrifying bacteria among bacteria contained in activated sludge.

According to the analysis method of the present invention, the type of bacteria in the activated sludge can be known and the analysis for estimating the number of inhabitants can be performed quickly and accurately.

[Substrate constituting the DNA chip]
The substrate constituting the DNA chip used in the analysis method of the present invention usually includes an inorganic glass plate. As the size of the substrate, one having a thickness of 0.5 mm to 2 mm, a short side of 10 mm to 20 mm, and a long side of 50 mm to 100 mm is usually used because it is easy to handle. As such a substrate, a slide glass widely used for optical microscope observation is preferably used because it is easily available.

[Bacteria]
The DNA fragment fixed on such a substrate contains the DNA base sequences of the filamentous bacteria, sulfur-metabolizing bacteria and nitrifying bacteria described above.

[Filamentous bacteria]
Filamentous bacteria are bacteria that can hinder the degradation of pollutants by activated sludge. Among the filamentous bacteria, those of the genus Nocardia and Rhodococcus mentioned above are considered to be prone to hindrance to decomposition by activated sludge as a cause of the phenomenon called activated sludge that rises when excessively propagated. Can be included.

The DNA chip may be fixed with a DNA fragment containing a DNA base sequence of a filamentous bacterium other than the filamentous bacterium described above, and among them, the genus Sphaerotilus can be contained in a relatively large amount of activated sludge. Therefore, it is preferable that a DNA fragment containing the DNA base sequence is fixed.

[Sulfur metabolizing bacteria]
A sulfur-metabolizing bacterium is a bacterium that can oxidize sulfur or an inorganic sulfur compound, and is abundant in activated sludge when waste water contains sulfur or an inorganic sulfur compound. Among the sulfur-metabolizing bacteria, the genus Desulfobacter, Desulfotomaculum, Desulfovibrio, and Desulfomicrobium described above
The genus and those of the genus Desulforhopalus can vary greatly depending on the activated sludge.

The DNA chip may be fixed with a DNA fragment containing a DNA base sequence of a sulfur-metabolizing bacterium other than the above-described sulfur-metabolizing bacterium, among which, Desulfococcus genus sulfur-metabolizing bacteria, Desulfomonas genus sulfur-metabolizing bacteria, Desulfuromonas genus sulfur-metabolizing bacteria, Desulfurella genus sulfur-metabolizing bacteria, Desulfomonile genus sulfur-metabolizing bacteria, Desulfosarcina genus sulfur-metabolizing bacteria, and Lawsonia genus sulfur-metabolizing bacteria can vary in amount by activated sludge. It is preferably fixed.

[Filamentous bacteria sulfur-metabolizing bacteria]
Filamentous bacteria sulfur-metabolizing bacteria are bacteria having both the properties of filamentous bacteria and the properties of sulfur-metabolizing bacteria, and examples thereof include Beggiatoa filamentous bacteria, sulfur-metabolizing bacteria, and Thiothrix genus filamentous bacteria.

[Nitrifying bacteria]
Nitrifying bacteria are bacteria that oxidize ammonia nitrogen or nitrite nitrogen, and are abundant in activated sludge when wastewater contains ammonia nitrogen, and ammonia that oxidizes ammonia nitrogen to nitrite Oxidizing bacteria or nitrite oxidizing bacteria that oxidize nitrite to nitric acid. Nitrosomonas genus nitrifying bacteria (ammonia-oxidizing bacteria), Nitrosospira genus nitrifying bacteria (
Ammonia-oxidizing bacteria) and Nitrospira genus nitrifying bacteria (nitrite-oxidizing bacteria) vary greatly depending on the activated sludge.

The DNA chip may have a DNA fragment containing a DNA base sequence of a nitrifying bacterium other than those described above, among which Nitrosolobus genus nitrifying bacteria (ammonia oxidizing bacteria), Nitrobacter genus nitrifying bacteria (nitrite oxidizing bacteria), Since the amount of activated sludge changes relatively greatly, it is preferable that the DNA fragment containing the DNA base sequence is fixed.

[Other bacteria]
The DNA chip may have a DNA fragment containing a DNA base sequence of bacteria other than the filamentous bacteria, sulfur-metabolizing bacteria, and nitrifying bacteria described above. Such a bacterium may be an aerobic bacterium or an anaerobic bacterium, and examples thereof include Acinetobacter genus polyphosphate accumulating bacteria, Arthrobacter genus polyphosphate accumulating bacteria, Microlunatas genus polyphosphate accumulating bacteria and the like. Other examples include filamentous bacteria, sulfur-metabolizing bacteria, filamentous bacteria, sulfur-metabolizing bacteria, and nitrification cells.

[Preparation of DNA chip]
Such a DNA chip is, for example,
(1) a DNA fragment preparation step for preparing a DNA fragment containing the DNA base sequence of each of the above bacteria;
(2) It can be prepared through a DNA fragment fixing step in which the DNA fragment obtained in the DNA fragment preparing step is fixed on a substrate.

[DNA fragment preparation step]
The DNA fragment can be prepared by an ordinary method, for example, a method for lysing a target bacterium, extracting DNA, and amplifying the obtained DNA, for example, a polymerase chain reaction (PCR reaction). Both ends of the DNA fragment may be fixed with a DNA primer.

[DNA fragment fixing step]
The DNA fragment obtained in the DNA fragment preparation step can be obtained by a conventional method, for example, D obtained above.
The NA fragment may be diluted with a solvent, attached to the substrate in a spot shape, and then dried.

The obtained DNA chip is usually stored at 0 ° C. or lower, preferably −15 ° C. or lower.

[Bacteria analysis]
The method of the present invention analyzes the type and amount of bacteria contained in activated sludge using such a DNA chip.

In particular,
(1) Bacterial extraction process for extracting bacteria from activated sludge,
(2) Lysis / RNA extraction process for extracting RNA after lysis of bacteria obtained in the bacterial extraction process,
(3) Reverse transcription step of reverse transcription of RNA extracted in the lysis / RNA extraction step to obtain reverse transcription DNA
(4) a labeling step in which the reverse transcription DNA obtained in the reverse transcription step is labeled with a fluorescent dye, and
(5) Bacteria contained in the activated sludge can be analyzed through a hybridization step in which the reverse-transcribed DNA labeled in the labeling step is crossed with a DNA fragment on the DNA chip.

[Bacteria extraction process]
In order to extract bacteria from the activated sludge, for example, the activated sludge may be diluted with physiological saline and then centrifuged. By centrifugation, the bacteria settle to become a precipitate, so that the bacteria can be obtained as a precipitate after centrifugation.

[Bacteria / RNA extraction process]
In order to lyse the obtained bacteria, for example, the bacteria may be shaken with a grinding medium in a solvent. As a grinding medium. For example, glass beads are used. By shaking, the bacteria are destroyed, for example, by colliding with the grinding medium, and the RNA in the bacteria is dispersed in the solvent. Thereafter, the grinding medium is separated, the solid content is removed by centrifugation, sodium chloride and ethanol are added to precipitate RNA, and RNA can be obtained as a precipitate by centrifugation.

In addition, since the DNA in bacteria is contained in the sediment obtained above, normally, the DNA in the sediment is degraded by contacting the sediment with a DNA degrading enzyme [Dnase].

[Reverse transcription process]
Reverse transcription of RNA obtained in the lysis / RNA extraction step can be performed by, for example, an ordinary method for purifying reverse transcription DNA using the obtained RNA as a template.

[Labeling step]
In order to label the reverse transcription DNA, for example, a primer labeled with a fluorescent dye such as indocarbocyanine (Cy) dye may be bound by the PCR method.

[Mating process]
In order to cross-link the reverse transcribed DNA after labeling with the DNA fragment on the DNA chip, the reverse transcribed DNA may be applied on the DNA chip. The application is performed, for example, by dispersing reverse-transcribed DNA in a solvent, dripping and then drying. By application, reverse transcribed DNA binds to the corresponding DNA fragment.

Bacteria can be analyzed by removing excess reverse-transcribed DNA that was not bound by washing with water after the hybridization step, and observing fluorescence under ultraviolet irradiation. From the presence or absence of fluorescence, activated sludge Can be analyzed for the presence or absence of bacteria, and the amount of the bacteria can be analyzed from the fluorescence intensity.

According to the analysis method of the present invention, since the type and amount of bacteria contained in the activated sludge, preferably nitrifying bacteria, can be analyzed quickly and accurately, for example, when the bacteria in the activated sludge are killed, In resuming wastewater treatment using other activated sludge, preferably wastewater containing ammonia nitrogen or nitrite nitrogen, the optimum activated sludge can be quickly selected.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by this Example.

[Example 1]
[Preparation of DNA chip]
A DNA chip was prepared using the following filamentous bacteria, filamentous bacteria, sulfur-metabolizing bacteria, sulfur-metabolizing bacteria, and nitrifying bacteria.

In addition, although the genus is clear in the used bacterium, but the species is not clear, each such bacterium was marked with (*). In addition, the bacteria described as two types indicate that DNAs of two types of bacteria that have been found to be different species were used, respectively.

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Filamentous bacteria ────────────────────────────────────────
Nocardia genus: Nocardia 1 (*), Nocardia 2 (*), Nocardia 3 (*), Nocardia 4 (*)
Rhodococcus genus: Rhodococcus 1 (*), Rhodococcus 2 (*)
Thiothrix genus: eikelbloomii 1, eikelbloomii 2, fructosivorans,
Thiothrix sp.1, EJ1M-B (*), unzii, Eikelbloom Type 1701
Genus Sphaerotilus: Sphaerotilus (*)
Other genera: Microthrix, Haliscomenobacter, Nostocoida 1,
Nostocoida 2 (*), Nostocoida 3 (*), Achromatium 1 (*),
Achromatium 2 (*)
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Filamentous bacteria Sulfur metabolizing bacteria ─────────────────────────────────────────
Beggiatoa genus: Beggiatoa1, Beggiatoa2, Beggiatoa3, Beggiatoa4, Beggiatoa5,
Beggiatoa6
Thiothrix genus: Thiothrix1, Thiothrix2, Thiothrix3, Thiothrix4, Thiothrix5,
Thiothrix6
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Sulfur metabolizing bacteria────────────────────────────────────────
Desulfobacter genus: Desulfobacter1 (*), Desulfobacter2 (*), Desulfobacter3 (*),
Desulfobacter4 (*)
Genus Desulfotomaculum: D. acetoxidance, D. aeronauticum, D. alkaliphilum,
D. auripigmentum, D. guttoideum, D. halophilum,
D. kuznetsovii, D. nigrificans 1, D. nigrificans 2,
D. reducens, D. ruminis, D. thermobenzoicum
Desulfovibrio genus: Desulfovibrio 1 (2 types) (*), Desulfovibrio 2 (2 types) (*)
, Desulfovibrio 3 (2 types) (*), Desulfovibrio 4 (*),
Desulfovibrio 5 (*), Desulfovibrio 6 (*), Desulfovibrio 7 (*)
Desulfomicrobium genus: Desulfomicrobium, D. apsheronum, D. norvegicum
Desulforhopalus genus: Desulforhopalus 1 (*), Desulforhopalus 2 (*),
Desulforhopalus 3 (*)
Desulfococcus genus: Desulfococcus 1 (biacutus) (*), Desulforhopalus 2 (*)
Genus Desulfomonas: Desulfomonas
Desulfuromonas genus: Desulfuromonas acetoxidance, D. chloroethanica,
Desulfuromonas 1 (*), Desulfuromonas 2 (*),
Desulfuromonas 3 (*), Desulfuromonas 4 (*),
Desulfuromonas 5 (*), Desulfuromonas 6 (*)
Desulfurella genus: Desulfurella
Desulfomonile genus: Desulfomonile limimaris, Desulfomonile sp. (*),
Desulfomonile sp.1 (*), Desulfomonile sp.2 (*),
Desulfomonile sp.3 (*)
Desulfosarcina genus: Desulfosarcina
Lawsonia genus: Lawsonia intracellularis
Other genera: Bilophila wadsworthia, Desulfobotulus,
Desulfobulbus 1 (*), Desulfobulbus 2 (*),
Desulfobulbus 3 (*), Desulfobulbus 4 (*),
Desulfocapsa 1 (*), Desulfocapsa 2 (*), Desulfocapsa 3 (*),
Desulfokalobium, Desulfonatronum,
Desulfonosporus thiosulfogenes, Desulfotalea 1 (*),
Desulfotalea 2 (*), Desulfuromusa 1 (*),
Desulfuromusa 2 (*), Desulfuromusa 3 (*),
Thermosulfovibrio, Thermoterrabacterium ferrireducens
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Nitrifying bacteria (ammonia oxidizing bacteria)
────────────────────────────────────────
Nitrosomonas genus: Nitrosomonas, Nitrosomonas sp. (2 types) (*),
N. aestuarii, N. communis, N. cryotolerans, N. europaea,
N. halophila, N. nitrosa, N. oligotropha, N. ureae
Nitrosospira genus: Nitrosospira, N. briensis, N. multiformis
Nitorosolobus genus: Nitrosolobus
Other genera: Nitrosococcus, Nitrosococcus 1 (*), Nitrosococcus 2 (*),
Nitrosococcus 3 (*), Anammoxoglobus 1 (*),
Anammoxoglobus 2 (*), Brocadia, Kuenenia, Scalindua 1 (*),
Scalindua 2 (*), Scalindua 3 (*)
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Nitrifying bacteria (nitrite oxidizing bacteria)
────────────────────────────────────────
Nitrospira genus: Nitrospira, N. marina, N. moscoviensis
Nitrobacter genus: Nitrobacter, Nitrobacter 1 (*), Nitrobacter 2 (*),
Nitrobacter 3 (*), Nitrobacter 4 (*)
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[Preparation of DNA fragment]
The DNA fragments of each bacterium are 16S rRNA (
It was obtained by selecting a unique gene region from ribosomal RNA) base sequence information and synthesizing its clone.

[Immobilization of DNA fragments]
Each of the DNA fragments obtained above is attached to a predetermined position on a slide glass in a spot shape,
The DNA chip was obtained by fixing by drying. The obtained DNA chip was stored at −20 ° C. until used for the following hybridization.

[Bacteria analysis]
Using the DNA chip obtained above, the number and type of bacteria contained in each activated sludge were analyzed by the following procedure using 4 activated sludge samples collected from the biological wastewater treatment facility of the chemical factory. . For activated sludge, the temperature of the wastewater flowing into the biological wastewater treatment facility is expected to change (1st to 3rd collected samples), and the temperature of the biological wastewater treatment facility is several degrees Celsius. Each sample was collected when expected to have risen (fourth sample collected).

[Bacteria extraction]
After adding 36 mL of physiological saline [Otsuka Pharmaceutical Co., Ltd.] to 4 mL of activated sludge, it was shaken. afterwards,
After separating 2 mL and centrifuging at 15,000 rpm, the supernatant was separated to obtain a precipitate, and a buffered aqueous solution [“Lysis Buffer” manufactured by AMR Co., Ltd. (Gifu City)] 150
μL was added to dilute. All instruments used were sterilized.

[Bacterial lysis and RNA extraction]
After adding zirconia beads (manufactured by AMR Co., Ltd. (Gifu City)) to the precipitate after the above dilution and holding at 90 ° C. for 10 minutes, the cell crusher (“Multibeads Shocker” manufactured by Yasui Kikai Co., Ltd.) is used at 2500 rpm. Shake for 60 seconds. Then, 200 μL of sodium dodecyl sulfate (SDS) aqueous solution (concentration 0.2% by mass) was added, vortexed for 1 minute, centrifuged at 15000 rpm for 5 minutes, and the supernatant was collected. 0.05 volume times sodium chloride aqueous solution [5 mol / L] and 2 volume times ethanol [100%] were added to the supernatant and vortexed for 30 seconds. Thereafter, the precipitate was collected by centrifugation at 15000 rpm for 5 minutes, and dried by holding at 55 ° C. for 5 minutes to obtain a precipitate containing RNA. This precipitate was suspended in 200 μL of RNase free sterilized distilled water [sterilized distilled water not containing RNase] to prepare a suspension.

Next, 5 μL of a buffer aqueous solution [10 × DNAase buffer (manufactured by Invitrogen)] and 2 μL of an aqueous solution of DNA-degrading enzyme [DNAase I (manufactured by Invitrogen)] are added to 43 μL of this suspension and kept at 37 ° C. for 2 hours. DNA was degraded. Thereafter, the temperature was raised to 55 ° C., the temperature was maintained for 5 minutes to deactivate the DNA-degrading enzyme, and then cooled to 4 ° C.

[Reverse transcription]
RNase free sterilized distilled water (13.5 μL) was added to 5 μL of the mixture after degrading DNA as described above, and a commercially available PCR reaction solution [“TaKaRa-One Step SYBR RT-PCR Kit” (Takara Shuzo)] and “ 16SrRNA8UA-519B primer set "(Sigma Aldrich Japan Co., Ltd.) in a 50 μL reaction system, held at 42 ° C. for 60 minutes, held at 98 ° C. for 5 minutes, and then cooled to 4 ° C. to perform reverse transcription. A mixture containing the copied DNA was obtained.

[Labeling]
The reverse transcription DNA was amplified by the PCR method of 100 μL using the commercially available “16SrRNA8UA-519B primer set” (manufactured by Sigma Aldrich Japan).

Thereafter, reverse transcription DNA was labeled with a fluorescent dye by PCR in a 100 μL reaction system using “Cy Dye 8UA-519B primer set” (manufactured by Sigma-Aldrich Japan) (fluorescent dye).

Thereafter, 0.05 vol. Times aqueous sodium chloride solution [5 mol / L] and 2 vol. Times ethanol [100%] were added to the mixture after the reverse transcription DNA was bound to the fluorescent dye, and vortexed for 30 seconds. Stirring was performed. Thereafter, the precipitate was collected by centrifugation at 15000 rpm for 5 minutes, suspended in 20 μL of PCR grade pure water, and then stored at −20 ° C.

[Crossing]
The previously prepared DNA chip is a mixed aqueous solution of sodium chloride (0.15 mol / L) -sodium citrate (0.015 mol / L) [hereinafter referred to as SSC aqueous solution. ] 2 capacitors and S
The mixture was washed with 1 part by volume of a DS aqueous solution (concentration: 0.2% by mass) at room temperature for 15 minutes, further washed at 95 ° C. for 5 minutes, and then washed with ultrapure water three times. Thereafter, it was centrifugally dried at 800 rpm.

On the other hand, pure water was added to the suspension obtained in the above [labeling] to dilute the DNA concentration to 100 ng / μL, and 28 μL was separated, and 10 μL of SSC aqueous solution and SDS aqueous solution (concentration 0.
(2% by mass) was added dropwise to the DNA chip, and then covered with a cover glass so that bubbles did not enter, and held at 50 to 65 ° C. for 12 to 16 hours. Thereafter, the DNA chip was immersed in an aqueous solution of sodium chloride (0.3 mol / L), sodium citrate (0.03 mol / L) and SDS (concentration 0.2% by mass) at 37 ° C. for 1 minute. The cover glass was removed, and then shaken in an aqueous solution of sodium chloride (0.3 mol / L), sodium citrate (0.03 mol / L) and SDS (concentration 0.2% by mass) for 5 minutes. After washing with an aqueous solution, it was centrifugally dried at 800 rpm.

[Measurement of fluorescence intensity]
After drying the DNA chip, the laser scanner [GSI LUMONICS]
Relative intensity was obtained by irradiating ultraviolet rays with “Scan Array 4000” manufactured by the company, measuring the intensity of the fluorescent signal.

For the measurement of fluorescence intensity, image analysis software ("DNASYS ARRAY" manufactured by Hitachi Software Co., Ltd.)
Was used. The fluorescence intensity is calculated by taking the total intensity of all the detected fluorescence signals as 100%.
A fluorescence intensity of less than 1% was not detected.

The RNA measurement results of various bacterial groups of the activated sludge collected four times are shown in FIGS. The measurement results for the nitrifying bacteria group, the filamentous bacteria group, and the sulfur-metabolizing bacteria group, respectively, the columnar graph indicates the number of detected bacterial species, and the line graph indicates the total value of the relative intensities of the fluorescent signals. Filamentous and sulfur-metabolizing bacteria were counted in both. As shown in these figures, there was a possibility that the quality of the influent wastewater changed or the water temperature increased for the biological treatment equipment, but the activity of these bacteria in the activated sludge According to the measurement result of RNA that can be regarded as a degree, it has been found that the function of the activated sludge of the biological treatment apparatus is generally kept constant both in the number of bacterial species and the detection level.

[Example 2]
The biological treatment test tank shown in FIG. 4 [aeration part volume 1 L, sedimentation part volume 0.3 L] (1) in the activated sludge of chemical factory [concentration 5000 mg-MLSS / L, MLSS means suspended suspended solids] (2) was placed and stored in a temperature-controlled room (incubator) (not shown) where the temperature could be adjusted arbitrarily. This biological treatment test tank (1) is of a continuous water flow type, and is supplied with air (A) by an air stone (diameter of about 2 cm) (3) and an aeration section having a stirring function by this air [ Only a volume of 1 L, an effective depth of about 40 cm] (11), and a lower part (from the bottom to about 2 cm height) (12) are composed of an aeration part and a settling part (13) opened. The settling part (13) is partitioned from the aeration part (11) by a partition plate (14). In this structure, waste water (B) flows continuously from the upper part of the aeration unit, and treated water (C) is discharged from the discharge port (15) opened on the upper side surface of the settling part (13). The biological treatment test tank is made of a transparent vinyl chloride resin.

The aerobic treatment was performed by continuously flowing waste water from the factory (mainly containing organic substances and ammoniacal substances as pollutants) into the test tank at a flow rate of 78 mL / hour. As an index of processing results, TOC (total organic carbon) concentration is measured for organic pollutants, and ammonia nitrogen concentration is measured for ammonia pollutants using TOC meter ("TOC-5000" manufactured by Shimadzu Corporation). Each was measured over time by an ultraviolet absorptiometry (JIS Japanese Industrial Standard K0102 45.2). First, the operation of the test tank was started at a treatment water temperature of 36 ° C., and the treatment water temperature was raised to 42 ° C. when it was considered that the treatment of contaminants was generally stable. The activated sludge was collected from the test tank immediately before the temperature rise, 1 day after the rise, and at 3 time points after 20 days after the temperature rise that the activated sludge is considered to be generally adapted (acclimated) to the condition of 42 ° C. RNA was extracted by the procedure described in 1, reverse transcription was performed, and nitrifying bacteria, filamentous bacteria, and sulfur-metabolizing bacteria were analyzed using a DNA chip.

FIG. 5 shows the TOC of the test tank, the ammonia nitrogen (NH 4 -N) load transition and the treatment water temperature change. On the 17th day, the temperature rose from about 36 ° C to about 42 ° C.

FIG. 6 shows the transition of the TOC removal rate and NH4-N removal rate of the test tank. The water temperature was increased from about 36 ° C to 42 ° C on the assumption that the treatment of pollutants was almost stable on the 17th day, and activated sludge was collected immediately before the water temperature rise (17th day) and 1 day later (18th day). They were named X-1 and X-2, respectively. As described in JP-A-2006-263642, when the water temperature of the biological treatment apparatus reaches a temperature region exceeding 40 ° C., the quality of the treated water of the organic waste water is temporarily deteriorated. It is known to recover gradually with this. In FIG. 6, the TOC removal rate, which was about 60% one day after the water temperature rose to 42 ° C., recovered to about 80% on the 38th day after about 20 days from the water temperature rise. Was collected and designated X-3. The NH4-N removal rate was about 50% before the water temperature rose but decreased to around 20% after the water temperature rose, and unlike the TOC removal rate, no recovery was seen over time. .

The measurement result by the DNA chip of RNA extracted from each collected activated sludge is shown in FIGS. The measurement results for the nitrifying bacteria group, the filamentous bacteria group, and the sulfur-metabolizing bacteria group, respectively, the columnar graph indicates the number of detected bacterial species, and the line graph indicates the total value of the relative intensities of the fluorescent signals. Filamentous and sulfur-metabolizing bacteria were counted in both.

In the nitrifying bacteria (FIG. 7), both the number of detected bacteria and the relative intensity of the fluorescence signal were reduced one day after the water temperature rise (X-2) compared to before the water temperature rise (sample name X-1). No recovery was observed even after (X-3), and a result almost reflecting the transition of the NH4-N removal rate shown in FIG. 6 was obtained. Filamentous bacteria (FIG. 8) and sulfur-metabolizing bacteria (FIG. 9) do not necessarily include all bacterial groups that perform organic decontamination, but immediately after the water temperature rises (X-2) Although there is a slight increase in the relative intensity of the fluorescence signal, there is no extreme change due to fluctuations in the water temperature, and the trend of supporting the relatively early adaptation (acclimation) of the organic pollution treatment function of activated sludge to a high water temperature environment of 42 ° C Is shown.

The change of nitrifying bacteria group RNA for every collection sample in the biological treatment apparatus of a chemical factory is shown. The horizontal axis represents the activated sludge sample collected 1 to 4 times, the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total value of the relative intensities of fluorescent signals. The change of filamentous bacteria group RNA for every collection sample in the biological treatment apparatus of a chemical factory is shown. The horizontal axis represents the activated sludge sample collected 1 to 4 times, the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total value of the relative intensities of fluorescent signals. The change of the sulfur metabolism bacteria group RNA for every collection sample in the biological treatment apparatus of a chemical factory is shown. The horizontal axis represents the activated sludge sample collected 1 to 4 times, the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total value of the relative intensities of fluorescent signals. It is a figure which shows typically the continuous flow type biological treatment test tank used in Example 2. FIG. The TOC of the biological treatment test tank, the ammonia nitrogen (NH4-N) load transition and the treatment water temperature change are shown. The horizontal axis is the number of days elapsed from the start of the test, the left vertical axis is the load of TOC and NH4-N (grams per day), and the right vertical axis is the treated water temperature (° C.). The transition of the TOC removal rate and NH4-N removal rate of the biological treatment test tank is shown. The horizontal axis is the number of days elapsed from the start of the test, and the vertical axis is the removal rate (%). The change of nitrifying bacteria group RNA for every collection sample in a biological treatment test tank is shown. The horizontal axis represents sample names (X-1, X-2, and X-3), the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total relative intensity of fluorescent signals. The change of filamentous bacteria group RNA for every collection sample in a biological treatment test tank is shown. The horizontal axis represents sample names (X-1, X-2, and X-3), the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total relative intensity of fluorescent signals. The change of the sulfur metabolism bacteria group RNA for every collection sample in a biological treatment test tank is shown. The horizontal axis represents sample names (X-1, X-2, and X-3), the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total relative intensity of fluorescent signals.

Explanation of symbols

1: Biological treatment tank
11: Aeration part 12: Lower part 13: Settling part 14: Partition plate 15: Drainage port 2: Activated sludge 3: Air stone A: Air B: Drainage C: Treated water

Claims (2)

Extracting bacterial RNA contained in activated sludge, performing reverse transcription of the extracted RNA to obtain reverse transcription DNA, and crossing the obtained reverse transcription DNA with DNA fragments of the following DNA chip The method for analyzing bacteria.
DNA chip: on the substrate,
Nocardia spp., Rhodococcus spp.,
Beggiatoa filamentous bacteria sulfur metabolizing bacteria, Thiothrix filamentous bacteria sulfur metabolizing bacteria,
Desulfobacter genus sulfur-metabolizing bacteria, Desulfotomaculum genus sulfur-metabolizing bacteria, Desulfovibrio genus sulfur-metabolizing bacteria, Desulfomicrobium genus sulfur-metabolizing bacteria, Desulforhopalus genus sulfur-metabolizing bacteria,
Nitrosomonas nitrifying bacteria, Nitrosospira nitrifying bacteria, and a DNA chip on which DNA fragments containing DNA base sequences of Nitrospira genus nitrifying bacteria are fixed The analysis method according to claim 1, wherein the bacteria contained in the activated sludge are nitrifying bacteria.

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