KR102120425B1 - Phthalate-degrading novosphingobium sp. abrd hk-2 and method for water purification using the same - Google Patents

Abstract

The present invention relates to a phthalate-degrading strain of Novosphingobium sp. ABRD HK-2, and a water purification method for freshwater using the same. More specifically, the strain of the present invention exhibits excellent degrading capability with respect to dibutyl phthalate (DBP), dimethyl phthalate (DMP), and diethyl phthalate (DEP), and particularly, exhibits excellent degrading capability with respect to DBP in high concentrations (1,000-4,000 ppm). In addition, the strain is capable of degrading DBP without forming by-products in a minimal salt medium having a composition similar to freshwater, and thus may be utilized for a water purification method for freshwater environments.

Description

ABRD HK-2 strain in the genus Novosfingobium decomposing phthalate and a method for purifying water using the same PHTHALATE-DEGRADING NOVOSPHINGOBIUM SP. ABRD HK-2 AND METHOD FOR WATER PURIFICATION USING THE SAME}

The present invention is a genus decomposing phthalate (phthalate) Novosphingobium ABRD HK-2 ( Novosphingobium sp. ABRD HK-2) strains and water purification method using the same.

Phthalate is a chemical additive used to soften plastics, and has been widely used in cosmetics containers, toys, and household flooring materials, but is currently prohibited as a toxic substance.

Phthalates are chemical additives used to soften plastics, and are used in a wide range of applications, from cosmetic containers to toys and household flooring.

Phthalate types are dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalte (DBP), bis(2-ethylhexyl) phthalate (bis(2-ethylhexyl) phthalate, DEHP ), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP) and polyethylene terephthalate (polyethyleneterephthalate, PET).

In particular, dibutyl phthalate is a substance that limits its use in the manufacture of toys in Korea because it causes reproductive toxicity and affects the sexual development of the human body.

In addition, in 2015, the European Union's Environmental Sciences Committee found that four phthalates of dibutylphthalate, diisobutyl phthalate (DIBP), benzylbutyl phthalate and bis(2-ethylhexyl) phthalate have carcinogenicity and mutation. It has been reported to cause toxicity and regenerative toxicity.

To date, in order to remove phthalates exposed to the environment, a method of decomposing by treating ozone, ultrasonic waves or light, and a method of removing using an adsorbent have been used.

However, these methods have a disadvantage of generating or removing toxic substances such as methyl propyl phthalate or phthalate ester as a by-product of decomposition.

Therefore, there is a need for research on a method capable of efficiently removing phthalates without generating decomposition by-products.

On the other hand, phthalates exposed to the environment can be biodegraded using microorganisms such as the genus Rhodococcus , genus Gordonia , and genus Variovorax (Non-Patent Document 1). .

Accordingly, the present inventors while researching for microorganisms capable of biodegrading phthalates, Novrd Phingobium genus ABRD HK-2 ( Novosphingobium sp. ABRD HK-2) The strain was found to efficiently decompose phthalates without generating decomposition by-products, thereby completing the present invention.

Looking at the prior art related to the genus Novospinggobium, Korean Registered Patent Publication No. 10-1636849 “Soil purification method and soil purification using lubricating microorganisms” includes Novosphingobium capsule OLu-2 ( Novosphingobium capsulatum OLu-2) ) Using a strain to describe the method of purifying soil contaminated with lubricant.

However, when comparing the prior art with the present invention, it is similar in that it uses the genus Novospinggobium, but the present invention is used in order to purify water quality contaminated with phthalate using the Novospinggobium ABRD HK-2 strain. The effect is different.

Republic of Korea Registered Patent Publication No. 10-1636849 (2016.06.30.)

Marianna A. Patrauchan, Christine Florizone, Manisha Dosanjh, William W. Mohn, Julian Davies, Lindsay D. Eltis (2005) Catabolism of Benzoate and Phthalate in Rhodococcus sp. Strain RHA1: Redundancies and Convergence. Journal of Bacteriology. 187 (12) 4050-4063.

The present invention provides an ABRD HK-2 strain ABRD HK-2 of the genus Novosfingobium that decomposes phthalates and a method for purifying water using the same.

More specifically, it has excellent resolution for dibutyl phthalate, dimethyl phthalate, and diethyl phthalate, especially for high concentrations (1000 to 4000 ppm) of dibutyl phthalate, has excellent resolution, and is a by-product in a minimal salt medium similar in composition to fresh water. Since it decomposes dibutyl phthalate without formation, it can be used as a water purification method in a fresh water environment.

As a means for solving the above problems, the present invention seeks to solve the technical problems by providing the strain ABRD HK-2 (Accession No. KACC 81079BP) in the genus Novospinggobium that decomposes phthalates.

The phthalate is characterized by dibutyl phthalate, dimethyl phthalate and diethyl phthalate.

The concentration of the dibutyl phthalate is characterized in that 100 to 4000 ppm.

In addition, the present invention is to solve the technical problem by providing a water purification method using the strain ABRD HK-2 and the genus Novospinggobium decomposing phthalate.

The ABRD HK-2 strain is characterized by decomposing phthalates present in a fresh water environment.

The ABRD HK-2 strain of the genus Novospinggobium that decomposes phthalates according to the present invention has excellent decomposition for dibutyl phthalate, dimethyl phthalate and diethyl phthalate, especially for high concentrations (1000 to 4000 ppm) of dibutyl phthalate It is excellent and has the effect of decomposing dibutyl phthalate without generating by-products in a minimal salt medium having similar components to fresh water.

Figure 1 is a phylogenetic tree for the ABRD HK-2 strain of Novosfingobium, showing the results of 16S rRNA gene sequence analysis.
2 is a photograph of a single colony of the strain ABRD HK-2 of the genus Novosfingobium.
Figure 3 is a picture observed by the transmission electron microscope ABRD HK-2 strain in the genus Novospinggobium.
Figure 4 is a graph showing the water purification effect through the verification of the dibutylphthalate resolution of the strain ABRD HK-2 in the genus Novosfingobium.

The terms or words used in the present specification and claims should not be construed as being limited to ordinary or lexical meanings, and the inventor can appropriately define the concept of terms in order to best describe his or her invention. Based on the principles, it should be interpreted as meanings and concepts consistent with the technical spirit of the present invention.

Therefore, the embodiments described in this specification are only the most preferred embodiments of the present invention, and do not represent all of the technical spirit of the present invention, there are various equivalents and modifications that can replace them at the time of application. It should be understood that it can.

Example 1. Isolation and identification of ABRD HK-2 strains of the genus Novosfingobium

1. Isolation of the ABRD HK-2 strain from Novospingobium

The strain ABRD HK-2 of the genus Novospingobium provided by the present invention was directly isolated and identified by the inventors to achieve the object of the invention to safely remove phthalates, substances that cause toxicity.

After adding 500 ppm of dibutylphthalate to 100 ml of river water and 10 g of sediment collected from the Nakdong River, and incubating for 3 weeks at 28°C.

2 ml of the culture solution was inoculated into a mixed solution of 100 ml of river water and 500 ppm dibutyl phthalate, and subcultured, and 200 ul of the subculture was plated on R2A agar medium (BD) and incubated at 28°C.

A single colony was separated from the cultured R2A agar medium, and incubated in the R2A liquid medium for 3 days, followed by centrifugation (10,000 rpm, 10 minutes) to remove the R2A liquid medium component.

The cells from which the medium components were removed were inoculated in a minimal salt medium (MSM) and suspended to 10 x 10 ⁹ cells/ml.

Minimal salt medium was used as a freshwater mimetic medium to confirm the phthalate resolution of the isolated ABRD HK-2 strain.

The composition of the minimum salt medium is shown in the table below.

Minimum salt medium composition Solution A Solution B Solution C K ₂ HPO ₄ 10.5 g/L NH ₄ Cl 3g/L Fe ₂ (SO ₄ ) ₃ 0.03 g/L KH ₂ PO ₄ 4.5g/L CaCl ₂ 0.09 g/L MgSO ₄ 8.1 g/L

The minimum salt medium was prepared by preparing solution A, solution B, and solution C according to the composition of Table 1, and sterilizing the solution A, solution B, and solution C in a volume ratio of 1:1:1.

The minimum salt medium may be prepared together with solutions A and B as necessary, and the amount of the composition contained in the two solutions may be adjusted.

Dibutyl phthalate concentration was added to the minimum salt medium of 10 ml to be 500 ppm, mixed, and then dispensed into a serum bottle. 100 ul of the isolated strain suspension was inoculated to make 1 x 10 ⁸ cells/ml, and 28°C. Incubated for 5 days to confirm the dibutylphthalate resolution.

2. Identification of ABRD HK-2 strains in the genus Novosfingobium

In order to confirm the growth range of the strain in which dibutylphthalate resolution was confirmed, the growth temperature, pH and NaCl% of the strain were analyzed using R2A medium.

For the morphological analysis of the strains in which dibutylphthalate resolution was confirmed, the colonies generated on the R2A agar medium were observed, and the shape of the strains was observed with a transmission electron microscope (TEM).

For the phylogenetic analysis of the strain in which dibutylphthalate resolution was confirmed, the sequencing of the 16S rRNA gene was analyzed.

Figure 1 is a phylogenetic tree for the ABRD HK-2 strain of Novosfingobium, showing the results of 16S rRNA gene sequencing.

2 is a photograph of a single colony of the strain ABRD HK-2 of the genus Novosfingobium.

Figure 3 is a photograph of the observation of the strain ABRD HK-2 in the genus Novospinggobium with a transmission electron microscope.

As a result, the growth temperature, pH, and NaCl% of the isolated strains are shown in Table 2 below.

Growth range Condition Growth scope pH 5-8 Temperature 15℃~40℃ NaCl ∼1.0%

As a result of sequencing the 16S rRNA sequence, the strain having confirmed phthalate resolution was a new microorganism belonging to the genus Novosfingobium.

In addition, the single colony of the ABRD HK-2 strain was yellowish, glossy, and 1-2 mm in size.

As a result of confirming the morphology of the ABRD HK-2 strain through a transmission electron microscope, it was a long oval shape with an average size of 6 um.

Accordingly, the present inventor patented the strain of ABRD HK-2 of the genus Novosfingobium to the Korean Agricultural Culture Collection (KACC) of the National Academy of Agricultural Science, and was given the accession number of KACC 81079BP on November 19, 2018.

Example 2 Verification of resolution according to the phthalate type of ABRD HK-2 strain of Novosfingobium

In order to determine whether the ABRD HK-2 strain possesses resolution for phthalates other than dibutylphthalate, phthalate resolution was verified using dimethylphthalate and diethylphthalate.

After inoculating and incubating the ABRD HK-2 strain in a minimal salt medium to which 200 ppm of dimethylphthalate, diethylphthalate, and dibutylphthalate were added, and measuring the amount of phthalate remaining in the culture medium using a GC chromatograph system. Did.

To recover the residual dibutylphthalate from the culture solution, 5 ml of dimethyl chloride was added to extract the culture solution.

A portion of the extracted dimethyl chloride layer was transferred to a glass bottle dedicated to a 1.5 ml GC chromatograph system, and the amount of dimethylphthalate remaining in the culture was measured using an HP-5 column.

The minimum salt medium to which 200 ppm of three phthalates were added without inoculating the ABRD HK-2 strains was added to the control groups 1 to 3, and the minimum of 200 ppm of three phthalates were respectively added after inoculating the strains of ABRD HK-2. The salt medium was used as experimental groups 1 to 3.

As a result, the ABRD HK-2 strain decomposed dimethylphthalate and diethylphthalate as well as dibutylphthalate.

Example 3. Verbosphingobium genus ABRD HK-2 strain verification of the resolution according to the concentration of dibutylphthalate

In order to verify the resolution according to the concentration of dibutylphthalate of the ABRD HK-2 strain, it was verified using 100 to 4000 ppm of dibutylphthalate.

The amount of phthalate remaining in the culture medium after inoculation and culture by inoculating the ABRD HK-2 strain in a minimal salt medium to which 100, 200, 400, 1000, 2000 and 4000 ppm of dibutylphthalate were added, respectively. Was measured.

In order to recover the dibutylphthalate remaining from the culture solution, 5 ml of dimethyl chloride was added to extract the culture solution.

A portion of the extracted dimethyl chloride layer was transferred to a glass bottle dedicated to a 1.5 ml GC chromatograph system, and the amount of phthalate remaining was measured using an HP-5 column.

The minimum salt medium inoculated with the ABRD HK-1 strain of the genus Rhodococcus sp. with phthalate resolution was the control group, and the minimum salt medium inoculated with the ABRD HK-2 strain was used as the experimental group.

The amount of phthalate remaining in the control culture medium and the amount of phthalate remaining in the experimental group culture were compared and expressed as a decomposition rate (%).

As a result, the decomposition rate according to the phthalate concentration of the ABRD HK-2 strain was shown in Table 3 below.

Decomposition rate by phthalate concentration Dibutylphthalate concentration (ppm) Decomposition after 7 days 100 100% 200 100% 400 100% 1000 95% 2000 90% 4000 90%

The ABRD HK-2 strain was generally excellent in decomposing dibutylphthalate of 100 to 4000 ppm.

In addition, it was found that the ABRD HK-2 strain has excellent ability to decompose high concentrations (1000 to 4000 ppm) of dibutylphthalate.

Example 4. Analysis of water purification effect through verification of dibutylphthalate resolution

The degree of decomposition of dibutylphthalate of 1000 ppm over time was observed to verify that the ABRD HK-2 strain can effectively purify contaminated water quality by effectively removing phthalates spilled into the freshwater environment.

In a 250 ml glass test tube, 10 ml of a minimum salt medium similar in composition to fresh water with 1000 ppm of dibutylphthalate was dispensed.

A strain of 1.0 x 10 ⁵ cells/ml was inoculated into 10 ml of minimal salt medium, and cultured under aerobic conditions at a rate of 220 rpm at 28°C.

The amount of dibutyl phthalate remaining in the culture was analyzed using a GC chromatograph system in the same manner as in Example 3 to measure the debutyl phthalate decomposition rate of each strain over time.

In addition, a gas chromatography-mass spectrometry (GC-MS) was used to detect and analyze by-products generated during decomposition of dibutyl phthalate for each time period.

In order to extract the produced by-products, dimethyl chloride was added to the culture solution to extract the produced by-products, and after adding a derivative that binds the by-products to the extract, it was analyzed with a gas chromatograph mass spectrometer.

The minimum salt medium inoculated with ABRD HK-1 strain of Rhodococcus genus having phthalate resolution is Control 1, the minimum salt medium inoculated with ABRD HK-4 strain of Acidovorax sp. having phthalate resolution is Control 2, ABRD The minimal salt medium inoculated with the HK-2 strain was used as the experimental group.

Figure 4 is a graph showing the water purification effect through the verification of the dibutylphthalate resolution of the strain ABRD HK-2 in the genus Novosfingobium.

As a result, the strain ABRD HK-2 in the genus Novospingobium degraded dibutylphthalate at a rate as fast as the strain ABRD HK-1 in the genus Rhodococcus, and despite the inoculation of a small amount (1.0 x 10 ⁵ cells/ml) of the strain, 160 After time (about 7 days), it was confirmed that most of the dibutylphthalate of 1000 ppm decomposed.

In addition, in the case of the strain ABRD HK-1 of the genus Rhodococcus that was used as a control as a result of detecting and analyzing by-products generated during the diphthalate decomposition process, by-products such as methyl propyl phthalate and phthalate ester were used. While accumulating, the ABDR HK-2 strain of the experimental group Novosfingobium did not produce by-products.

Therefore, when synthesizing the results of the Examples, the strains of ABRD HK-2 in the Novosfingobium genus isolated and identified by the present inventors have excellent resolution for dibutylphthalate, dimethylphthalate and diethylphthalate, and particularly high concentration (1000~ It is confirmed that it can be used as a water purification method in a fresh water environment because it has excellent resolution for dibutylphthalate of 4000 ppm), and dibutylphthalate is decomposed without generating by-products in a minimal salt medium having similar components to fresh water.

Microbiological Bank of the National Academy of Agricultural Science KACC81079BP 20181119

Claims (5)

Novosphingobium sp. ABRD HK-2 strain that degrades phthalate (Accession No. KACC 81079BP).
The method according to claim 1,
The phthalate is dibutyl phthalate (dibuthyl phthalate, DBP), dimethyl phthalate (dimethyl phthalate, DMP) and diethyl phthalate (diethyl phthalate, DEP), characterized in that Novospinggobium genus ABRD HK-2 strain (Accession number KACC 81079BP).
The method according to claim 2,
The concentration of the dibutyl phthalate is characterized in that 100 to 4000 ppm, Novospinggobium genus ABRD HK-2 strain (Accession number KACC 81079BP).
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