JP6126935B2 - Microorganism and wastewater treatment method - Google Patents

Description

  The present invention relates to a microorganism and a method for treating wastewater, and more particularly to a microorganism having a high nonionic surfactant resolving power and a method for treating wastewater using the microorganism.

  Wastewater treatment by biological treatment such as the activated sludge method is widely performed. However, there are many problems in treating the surfactant by the activated sludge method. That is, the surfactant is hardly decomposable and is difficult to process by the activated sludge method.

Various studies have been made on biological treatment of nonionic surfactants. Although the removal rate of primary decomposition (partial decomposition) is 90-96% in one day in the removal investigation of nonionic surfactant with activated sludge (Non-patent Document 1), 74% removal in four weeks in complete decomposition However, it is known that complete removal in a short period is difficult.
As countermeasures, non-ionic surfactant-degrading bacteria can be added as microbial preparations. Although there have been reports of separation of microorganisms that partially degrade the substance, a single carbon source has been reported. There are few reports of microorganisms that completely decompose as (Non-patent Document 3).
Furthermore, there are no cases where the acquired degrading bacteria can be applied to biological treatment methods such as activated sludge (examples where complete decomposition at the total organic carbon level has been achieved).

  In addition, several microorganisms having the ability to degrade nonionic surfactants have been found (for example, Patent Documents 1 and 2). However, it cannot be said that it is sufficiently applied to the treatment of wastewater containing nonionic surfactants.

JP 2000-354891 A JP 2000-270852 A

Prats, D., Ruiz, F., Vazquez, B. and Rodriguez-Pastor, M. (1997) Removal of anionic and nonionic surfactans in a water treatment plant with anaerobic digestion.A comparative study.Water Research., 31,1925 -1930. National Institute of Technology and Evaluation, 2007, Initial Risk Assessment for Chemical Substances, Ver.1.0 No. 89 Maki, H., Masuda, N., Fujiwara, Y., Ike, M. and Fujita, M. (1994) Degradation of Alkylphenol Ethoxylates by Pseudomonas sp. Strain TR01. Appl. Environ. Microbiol. 60, 2265-2271

  The problems of the prior art in the biological treatment of nonionic surfactants are summarized as follows. First, when a non-ionic surfactant that is hardly degradable is used as a treatment target, a conventional biological treatment technique represented by an activated sludge method cannot obtain a sufficient removal rate required when designing a treatment facility. In the case of a treatment environment in which easily decomposable general organic substances coexist, activated sludge consumes it and may not decompose a degradable substance such as a nonionic surfactant (or treatment efficiency may be reduced). . Furthermore, the physical adsorption removal method using a porous material such as activated carbon has a problem of cost because it requires periodic material replacement.

  In view of the above-mentioned present situation, an object of the present invention is to provide a novel microorganism excellent in nonionic surfactant resolution and to provide a biological treatment technology of waste water using the microorganism.

  The present inventors screened microorganisms using the rhizosphere of Spirodela polyrrhiza as a separation source in order to isolate novel microorganisms having higher nonionic surfactant resolution. As a result, we succeeded in isolating a novel microorganism that is particularly excellent in resolution for polyoxyethylene alkyl ether, which is a kind of nonionic surfactant. And the biological treatment method of the polyoxyethylene alkyl ether containing waste water using the said microorganisms was developed, and this invention was completed. The present invention for solving the above-described problems is as follows.

  The invention described in claim 1 is a microorganism that is the S45Y strain (NITE P-01636).

  The microorganism of the present invention has a high resolution for polyoxyethylene alkyl ethers. According to the microorganism of the present invention, polyoxyethylene alkyl ether-containing wastewater can be biologically treated with high efficiency.

  The invention according to claim 2 is characterized in that the microorganism according to claim 1 is brought into contact with waste water containing polyoxyethylene alkyl ether to decompose the polyoxyethylene alkyl ether in the waste water. This is a wastewater treatment method.

  The present invention relates to a method for treating wastewater, and includes a step of bringing the above-described microorganism of the present invention into contact with wastewater containing polyoxyethylene alkyl ether. According to the wastewater treatment method of the present invention, polyoxyethylene alkyl ether can be biologically treated with high efficiency. For example, polyoxyethylene alkyl ether can be efficiently decomposed even in an environment where easily decomposable general organic substances coexist. It is also possible to completely decompose the polyoxyethylene alkyl ether.

  The invention according to claim 3 is the wastewater treatment method according to claim 2, wherein the microorganism is incorporated into activated sludge.

  With this configuration, application to the activated sludge method becomes possible.

  According to the present invention, wastewater containing polyoxyethylene alkyl ether can be efficiently biologically treated.

It is a graph showing the time-dependent change of TOC in an activated sludge method.

  The microorganism of the present invention is a microorganism that is the S45Y strain (NITE P-01636). The microorganism is deposited at the Patent Microorganism Depositary Center for Product Evaluation Technology, an independent administrative agency. Details of the deposit are shown below.

Display: S45Y strain Accession number: NITE P-01636
Date of receipt: June 19, 2013

  The mycological properties of the S45Y strain (NITE P-01636) are as follows. “+” Indicates positive and “−” indicates negative.

[Form etc.]
-Cell morphology: Neisseria gonorrhoeae (2.0-2.5 μm × 0.5-1.5 μm)
-Gram stainability:-
・ Spore presence:
・ Mobility:-
Colony morphology (medium: LB agar, culture time: 48 hours, culture temperature: 28 ° C.)
Diameter: 0.1-1.5mm
Color: Yellow Shape: Circular Protrusion: Hemispherical (convex)
Surface shape, etc .: Smooth Transparency: Opaque

[Aerobic, anaerobic]
・ Aerobic

[Usability, etc.]
Catalase reaction: +
・ Oxidase reaction: +
・ Urease:-
.Beta.-galactosidase:-
・ Indole test: +
・ Nitrate reduction:-
・ Mannitol:-
・ Sorbitol: +
・ Cellobiose: +
・ Trehalose: +
・ Xylose: +
・ Fructose: +
・ Sucrose: +
・ Raffinose: +
Acid production from glucose: +
Acid production from galactose: +

・ Homology of 16rDNA base sequence
Sphingomonas paucimobilis: 100%

  As a method for culturing the microorganism of the present invention, a general method for culturing aerobic microorganisms can be employed as it is. For example, using a liquid medium containing an appropriate carbon source or the like, it can be cultured with aeration and agitation. As culture | cultivation temperature, the range of 5-40 degreeC, for example, Preferably 15-40 degreeC, More preferably, 20-35 degreeC can be selected. When polyoxyethylene alkyl ether (hereinafter sometimes abbreviated as “POE alkyl ether”) is added to the medium as a carbon source, the concentration is, for example, 750 mg / L or less, preferably 500 mg / L or less, more preferably Can select a range of 100 mg / L or less.

  The microorganism of the present invention has a high resolution for POE alkyl ethers. As an evaluation method of POE alkyl ether resolution, for example, when the microorganism of the present invention is cultured in a medium containing POE alkyl ether, it can be evaluated based on the rate of decrease of POE alkyl ether in the culture solution. The rate of decrease of POE alkyl ether is calculated by, for example, detecting a change in the amount of POE alkyl ether in the medium with a change in total organic carbon (TOC) using a medium containing POE alkyl ether as the sole or main carbon source. can do. In addition, the POE alkyl ether can be quantified by the potassium thiocyanate method (see Examples described later).

  The wastewater treatment method of the present invention comprises contacting S45Y strain (NITE P-01636) with wastewater containing POE alkyl ether to decompose POE alkyl ether in the wastewater.

  In the wastewater treatment method of the present invention, examples of the POE alkyl ether to be treated include POE alkyl ethers having 12 to 15 carbon atoms in the alkyl group. In addition, each POE alkyl ether used in the below-mentioned Example is mentioned.

  The wastewater treatment method of the present invention can be applied to all methods of aerobic treatment, for example, activated sludge method and biofilm method. When applied to the activated sludge method, the POE alkyl ether-containing wastewater can be treated with high efficiency by adding the microorganism of the present invention to the activated sludge and allowing it to coexist with other microorganisms. The microorganism of the present invention can exhibit a high decomposing ability for POE alkyl ethers even in a processing environment in which other microorganisms coexist.

  When applying to the biofilm method, a known method such as a rotating disk method or a trickling filter method can be applied as it is. The microorganisms of the present invention can be immobilized on a carrier and fluidized bed type treatment can be performed. In addition, the wastewater treatment method of the present invention can be applied to both batch and continuous treatment methods.

  In the wastewater treatment method of the present invention, the amount of microorganisms to be brought into contact with the POE alkyl ether-containing wastewater (for example, addition amount or addition cycle) is appropriately selected according to the properties of the wastewater to be treated, the configuration of the wastewater treatment facility, and the like. That's fine. Further, the contact time between the POE alkyl ether-containing wastewater and the microorganism can be appropriately selected according to the type and amount of the POE alkyl ether contained in the wastewater, the properties of the wastewater to be treated, and the like. The temperature at the time of wastewater treatment may be within the range in which the microorganism of the present invention can grow, for example, the range of 5 to 40 ° C., preferably 15 to 40 ° C., more preferably 20 to 35 ° C. exemplified as the culture temperature. You can choose. Similarly, as the concentration of the POE alkyl ether, for example, a range of 750 mg / L or less, preferably 500 mg / L or less, more preferably 100 mg / L or less can be selected.

  According to the microorganism of the present invention, POE alkyl ether can be decomposed and removed in a short time of about 12 hours. Moreover, it has a high decomposition activity with respect to POE alkyl ether even in a processing environment where easily decomposable general organic substances coexist. Furthermore, it can exhibit high decomposing activity for POE alkyl ethers even in a processing environment in which various general microorganisms coexist. As a result, it is possible to utilize a biological treatment system stock facility such as an activated sludge method that is most widely used as a wastewater treatment method. Furthermore, it is possible to provide a water purification method capable of removing POE alkyl ether released into public water areas without being completely treated by conventional methods at a low cost.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited only to these Examples.

  First, the nonionic surfactant used in this example is shown below.

[POE alkyl ether]
・ Polyoxyethylene (7) lauryl ether (POE (7) LE) (manufactured by Wako Pure Chemical Industries, Ltd.)
・ Polyoxyethylene (9) lauryl ether (POE (9) LE) (manufactured by Wako Pure Chemical Industries, Ltd.)
・ Polyoxyethylene (23) lauryl ether (POE (23) LE) (manufactured by Wako Pure Chemical Industries, Ltd.)
・ Polyoxyethylene (7) oleyl ether (POE (7) OE) (manufactured by Wako Pure Chemical Industries, Ltd.)
・ Polyoxyethylene alkyl (12-14) ether (BT-7)
BT-7 has a similar structure to POE (7) LE.

  Table 1 shows the chemical structural characteristics of each POE alkyl ether.

[Other nonionic surfactants]
・ Polyethylene polypropylene glycol monobutyl ether ・ Polyoxyalkylene ether

(1) Screening of novel microorganisms Duckweed having POE (7) LE degrading activity in the rhizosphere was used as a source of separation of novel microorganisms. The primary screening was performed by direct separation by plate culture method or separation by enrichment culture using POE (7) LE degradation activity as an index. Next, the strain selected in the primary screening was screened again by the same method to obtain a secondary screening. About the strain selected by the secondary screening, the tertiary screening was performed using the POE (7) LE degrading activity in the presence of an easily degradable organic substance as an index. By the third screening, one strain which was particularly excellent in POE (7) LE degrading activity was selected and named S45Y strain.

  The S45Y strain was subjected to colony PCR using the primers shown in SEQ ID NOs: 1 and 2 to amplify the 16S rRNA gene, and the nucleotide sequence was analyzed. The obtained sequence data was compared with a base sequence registered in the NCBI database using a homology search program (BLAST search). As a result, the homology with Sphingomonas paucimobilis was 100%.

  S45Y strain was deposited at the Patent Microorganism Depositary Center for Product Evaluation Technology (NITE P-01636).

(2) Evaluation of POE (7) LE degradation activity of S45Y strain (during secondary screening)
To a 48-well microplate, 500 L of A & HPB medium (Table 3) was added, and POE (7) LE was added to a final concentration of 50 mg / L. After inoculating each hole with about a sesame grain strain, it was cultured with shaking at 25 ° C. under dark conditions. On the 0th day (before the start of culture), 2nd day, and 5th day, 100 μL of the culture solution was sampled. About the sampled culture solution, the density | concentration of POE (7) LE which remains in a culture medium was measured by the potassium thiocyanate method (a procedure is mentioned later). The residual rate (%) of POE (7) LE on the fifth day of culture was calculated by the following formula.

  Residual rate (%) = POE (7) LE concentration (day 5) / POE (7) LE concentration (day 0) × 100

  As a result, the residual rate of POE (7) LE in the S45Y strain was 10% or less. Thus, the S45Y strain had high degradation performance against POE (7) LE.

  The compositions of Modified Arnon & Hoagland medium (A & H medium) and A & HPB medium are shown in Table 2 and Table 3, respectively.

Quantification by the potassium thiocyanate method was performed according to the following procedure.
50 μL of test water was diluted 4 times with distilled water. Add 200 μL of diluted test water (sample for preparing calibration curve to POE (7) LE concentration 0, 4, 8, 12, 16, 20 mg / mL) into a 1.5 mL sampling tube and add 0.8 g / mL potassium thiocyanate 50 μL of the solution, 12.5 μL of 0.4 g / mL iron (III) nitrate solution, and 150 μL of 1,2-dichloroethane were added. The mixture was shaken for 5 minutes with a shaker and allowed to stand for 5 minutes. The organic layer was separated and the absorbance (A510 nm) was measured. The polyoxyethylene alkyl ether concentration of the test water was calculated from the approximate formula derived from the calibration curve.

(3) POE in the presence of readily decomposable organic substances (7) LE degradation activity (during 3rd screening)
The bacterial strain selected in the secondary screening is cultured in an R2A medium (Table 4) supplemented with POE (7) LE to a final concentration of 50 mg / L, and POE (7) LE degradation in the presence of readily degradable organic matter. The activity was examined.
Specifically, 5 mL of R2A medium was placed in a 25 mL screw-cap test tube and added so that POE (7) LE had a final concentration of 50 mg / L. The POE (7) LE-degrading bacterium was inoculated so that the initial cell concentration was OD ₆₆₀ = 0.02. The culture was reciprocally shaken at 160 rpm, 25 ° C. under dark conditions for 5 days. 200 μL of the culture solution was sampled at 0 hour culture (before the start of culture), 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, and 120 hours. With respect to the sampled culture solution, the concentration of POE (7) LE remaining in the medium was measured by the potassium thiocyanate method. The residual rate (%) of POE (7) LE at 120 hours of culture (5th day) was calculated by the following formula.

  Residual rate (%) = POE (7) LE concentration (day 5) / POE (7) LE concentration (day 0) × 100

  As a result, the residual rate of POE (7) LE in the S45Y strain was 20% or less. Thus, the S45Y strain efficiently decomposed POE (7) LE even in the presence of readily decomposable organic substances.

The composition of R2A medium is shown in Table 4.

(4) Substrate specificity The degradation performance of nonionic surfactants other than POE (7) LE was examined in the same manner as (2) above. As a result, the residual rate is 20% or less for POE (9) LE, the residual rate is 10% or less for POE (23) LE, POE (7) OE, and BT-7, and the same high decomposition as POE (7) LE. Performance was recognized. On the other hand, polyethylene polypropylene glycol monobutyl ether and polyoxyalkylene ether were hardly decomposed. Thus, it was found that the S45Y strain has a high degradation performance for POE alkyl ethers.

(5) Addition experiment to activated sludge (evaluation as microbial preparation)
A 300 mL Erlenmeyer flask was charged with 200 mL of R2A medium, 1 platinum ear of S45Y strain was inoculated, and cultured under rotary conditions at 160 rpm, 25 ° C. in the dark until the late logarithmic growth phase (preculture). The cultured cells were washed twice with A & H medium and suspended in the same medium. In a 25 mL screw mouth test tube, activated sludge (final concentration 1500 mg / L), bacterial cells (final concentration 500 mg / L), carbon source (POE (7) LE (final concentration 50 mg / L)), and artificial sewage (Table 5). ) (115 μL) was added to make a total volume of 5 mL, which was cultured by reciprocal shaking for 1 day at 160 rpm in the dark, at 0 hour (before the start of culture), 3 hours, 6 hours, 12 hours, and At 24 hours, 200 μL of the culture solution was sampled.

  The composition of artificial sewage is shown in Table 5.

  About the sampled culture solution, POE (7) LE was measured by the potassium thiocyanate method, and decomposition | disassembly evaluation of POE (7) LE was performed. The residual rate (%) of POE (7) LE at 12 hours and 24 hours of culture was calculated by the following formula. As a control, the same experiment was performed using only activated sludge without adding the S45Y strain.

  Residual rate (%) = POE (7) LE concentration (12th or 24th hour) / POE (7) LE concentration (0th hour) × 100

  As a result, the residual rate of POE (7) LE was 30% or less at both the 12th and 24th hours, indicating high POE (7) LE decomposition performance. On the other hand, POE (7) LE was hardly decomposed by the control using only activated sludge.

  Moreover, TOC was measured with the TOC meter about the sampled culture solution, and decomposition | disassembly evaluation of all the organic substances was performed. As a control, without using the S45Y strain, artificial sludge and POE (7) LE were added to activated sludge (control 1), and activated sludge added with artificial sewage (control 2). The experiment was conducted. The results are shown in FIG.

  As shown in FIG. 1, when the S45Y strain was used, almost all organic substances were decomposed at 12 hours from the start of the culture. On the other hand, in the case of only activated sludge, the organic matter of artificial sewage was decomposed (Control 2), but POE (7) LE was hardly decomposed (Control 1).

  As described above, it was shown that by incorporating S45Y strain into activated sludge, about 50 ppm of POE (7) LE can be decomposed and removed in a short time of 12 hours. Moreover, it was shown that S45Y strain has a high treatment capacity for POE (7) LE even in an environment where various general microorganisms coexist. Furthermore, it was shown that the S45Y strain has a high degrading activity against POE (7) LE even in a processing environment in which easily degradable general organic substances coexist.

Claims (3)

  A microorganism which is S45Y strain (NITE P-01636).   A method for treating waste water, comprising contacting the microorganism according to claim 1 with waste water containing polyoxyethylene alkyl ether to decompose the polyoxyethylene alkyl ether in the waste water.   The method for treating wastewater according to claim 2, wherein the microorganism is used by being incorporated in activated sludge.

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