KR20220075014A - Strains of Paracoccus sp. with 3,3'-Iminodipropionitrile Degradation Activity, and Uses thereof - Google Patents

Abstract

The present invention relates to a paracoccus sp. strain having iminodipropionitrile decomposition activity, a composition for biodegradation of iminodipropionitrile comprising the same, and a method for removing the toxicity of iminodipropionitrile from wastewater using the same.
Paracoccus communis NK1007 strain of the present invention is capable of continuous self-proliferation even in a wastewater environment containing 100,000 mg/L of ultra-high concentration iminodipropionitrile, and is capable of biodegrading iminodipropionitrile. Because of its excellent activity, it can be usefully used to reduce or remove the toxicity of iminodipropionitrile-contaminated wastewater.

Description

Paracoccus sp. strain having iminodipropionitrile degrading activity and use thereof {Strains of Paracoccus sp. with 3,3'-Iminodipropionitrile Degradation Activity, and Uses thereof}

The present invention relates to a paracoccus sp. strain having iminodipropionitrile decomposition activity, a composition for biodegradation of iminodipropionitrile comprising the same, and a method for removing the toxicity of iminodipropionitrile from wastewater using the same.

With the development of scientific and technological civilization, the standard of living and convenience of mankind has improved and it has been possible to maintain the current standard of living, which has been improved compared to the past. In particular, water pollution is the most important problem that poses an important threat to human survival along with air and soil pollution.

Therefore, not only environmental groups and governments, but also general companies are trying to develop process technology and produce products in a way that does not damage the environment as much as possible, and most companies in the chemical industry do not cause water pollution. There is a lot of interest in how to purify wastewater without wastewater, and related technologies are being developed.

Regarding the wastewater treatment method using microorganisms, it is divided into bioremediation, in situ biorestoration, bioaugmentation, and biostimulation. This is a technology that increases the treatment efficiency of environmental pollutants.

The application of the bio-enhancement method in wastewater treatment is made in a way to maintain or increase the biodegradability of environmental pollutants present in wastewater by manufacturing a microbial preparation using selective isolates distributed in nature and using this. The bio-enhancement method is applied in the field for the purposes of i) increase in BOD removal efficiency, ii) reduction of wastewater sludge volume, iii) bio-treatment of hydrocarbon waste, and iv) bio-treatment of organic waste. There is a case where it was applied to biodegradation of phenolic compounds or cyanide compounds, or a case in which brown lignin compounds present in effluent were removed using white humus in a paper mill.

On the other hand, iminodipropionitrile (C ₆ H ₉ N ₃ , 3,3'-Iminobispropanenitrile, CAS No. 111-94-4) is a viscous, transparent liquid compound having a molecular weight of 123.1558. Copper compounds are raw materials used to manufacture various chemical products, such as pesticides or solvents for dyes, or substances generated during the manufacture of chemical products. Copper compound acts as a strong oxidizing agent, and is toxic if inhaled orally, is an irritant that causes skin irritation and serious eye damage, and is a toxic substance causing irritation and damage to the respiratory system. Therefore, if iminodipropionitrile is used as a raw material, it is separated and removed from wastewater and discharged into sewage without discharging it, thereby causing serious pollution to the ecosystem. However, the application of a physicochemical method to remove iminodipropionitrile by applying a physicochemical method in an industrial complex where chemical factories are dense requires a complicated facility process and expensive cost for operating it. There is a problem.

Therefore, it is necessary to discover microorganisms necessary for the removal of iminodipropionitrile compounds that destroy the ecosystem and apply them to a biological purification method. There is no research on microorganisms having biodegradable activity.

Accordingly, the present inventors have made intensive research efforts to overcome the problems of the prior art, and as a result, the Paracoccus communis NK1007 strain identified in the wastewater of the Yeosu Industrial Complex is an ultra-high concentration iminodipropionitrile of 100,000 mg/L. It was confirmed that it can be usefully utilized to reduce or remove the toxicity of wastewater contaminated with iminodipropionitrile while growing in medium conditions, and thus the present invention has been completed.

Chinese Patent Publication No. 110255717, Degrading bacteria of methoxy acrylate type bactericide and application of same Korean Patent Laid-Open No. 10-2002-0092860, Oh, a device for manufacturing microorganism contact media for wastewater purification and its products Korean Patent Registration No. 10-0511769, Sphingobium strain, a microorganism that decomposes chlorinated biphenyl compounds, and environmental purification method using the same

Accordingly, the main object of the present invention is to provide an iminodipropionitrile (IDPN, 3,3'-Iminodipropionitrile) biodegradable Paracoccus communis ( Paracoccus communis ) NK1007 strain.

Another object of the present invention is a composition for biodegradation of iminodipropionitrile and a composition for biodegradation of iminodipropionitrile comprising Paracoccus communis NK1007 strain (accession number: KCTC 18866P) or a culture thereof for biodegradation of iminodipropionitrile and wastewater using the same To provide a method for alleviating or eliminating IDPN (3,3'-Iminodipropionitrile) toxicity.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides iminodipropionitrile (IDPN, 3,3'-Iminodipropionitrile) for biodegradation of Paracoccus communis NK1007 strain (accession number: KCTC 18866P).

While the present inventors were conducting various studies to develop a method for purifying wastewater near industrial complexes contaminated with iminodipropionitrile, the present inventors were As a result, attention was paid to microorganisms capable of metabolizing iminodipropionitrile to remove iminodipropionitrile compounds present in wastewater. The microorganism can remove iminodipropionitrile while growing and proliferating in wastewater contaminated with iminodipropionitrile, so that it is possible to economically purify wastewater contaminated with iminodipropionitrile. did

To this end, the present inventors collected a sample from wastewater contaminated with iminodipropionitrile and secured a total of 84 independent colonies through the process of thickening and culturing. By culturing in the compound-added medium, the viability of the strain and the efficiency of decomposing the iminodipropionitrile compound were measured, and strains with excellent degradation efficiency were isolated and identified. In addition, in order to confirm that the strain exhibiting the activity of biodegrading the iminodipropionitrile compound is a known strain, the sequence of 16S RNA obtained from the strain was analyzed, and the growth characteristics of the strain were analyzed. When cultured in BCM 660 medium or TSB (Tryptic Soy Broth) medium, it was confirmed that the growth rate and the activity of biodegrading iminodipropionitrile compounds can be significantly increased.

Accordingly, the present inventors named the strain Paracoccus communis NK1007 strain, and deposited it at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on November 12, 2020 and was given an accession number KCTC 18866P.

The present invention Paracoccus communis ( Paracoccus communis ) NK1007 strain (Accession No.: KCTC 18866P) has a 16S rRNA sequence of SEQ ID NO: 1.

As demonstrated in one embodiment of the present invention, according to one aspect of the present invention, Paracoccus communis NK1007 strain (Accession No.: KCTC 18866P) of the present invention is iminodipropionitrile as a carbon source In addition, it can grow in a minimally nutrient medium excluding other carbon sources.

In addition, the Paracoccus communis NK1007 strain (Accession No.: KCTC 18866P) of the present invention is characterized in that it is possible to grow in an ultra-high concentration iminodipropionitrile medium condition of 100,000 mg/L.

In an embodiment of the present invention, it was observed that the Paracoccus communis cells were able to grow on M9 medium containing a high concentration of IDPN compound within 3 days (see FIG. 2), especially at an ultra-high concentration (100,000 mg/L) ), it has been observed that the turbidity of the culture body increases without growth inhibition within 7 days even under the medium condition.

According to another aspect of the present invention, the present invention provides an iminodipropionitrile biodegradation composition comprising Paracoccus communis NK1007 strain (Accession No.: KCTC 18866P) or a culture solution thereof for biodegradation of iminodipropionitrile provides

Paracoccus communis of the present invention NK1007 strain (accession number: KCTC 18866P) is characterized by having excellent iminodipropionitrile biodegradation activity, in a preferred embodiment of the present invention, each 80,000 mg / L and This Paracoccus communis NK1007 strain cultured under 15,000mg/L IDPN compound concentration condition was diluted at a ratio of 1/50 and inoculated in M9 medium (100mg/L IDPN compound added) and cultured for 9 days. As a result, it was confirmed that 88.35% and 87.83% of IDPN compounds were decomposed, respectively (see FIG. 4 ). In addition, it was confirmed that the final number of restricted individuals of the Paracoccus communis NK1007 strain increases depending on the concentration of the input IDPN compound. And, Paracoccus communis of the present invention ( Paracoccus communis ) NK1007 strain shows optimal biodegradation activity at an IDPN compound concentration of 1,000 mg/L, and a maximum of 80,000 mg/L IDPN compound at a concentration of 80,000 mg/L during a 9-day culture period. It was measured to be able to remove about 88%. Therefore, considering that the concentration of IDPN compound in wastewater generated in large amounts in chemical production and treatment facilities such as latex production is actually about 1,200 mg/L, Paracoccus communis NK1007 strain of the present invention is an IDPN compound It is considered to be an optimal microbial resource that can be completely treated biologically.

The iminodipropionitrile biodegradation composition of the present invention is Paracoccus communis NK1007 strain (Accession No.: KCTC 18866P) may be cultured in BCM 660 medium or TSB (Tryptic Soy Broth) medium. The present inventors prepare a mass culture medium to secure the biomass of the Paracoccus communis NK1007 strain so that the strain of the present invention can be applied to the actual sewage treatment facility for the application of the bioaugmentation method. studied. For this purpose, BCM660 medium and TSB (Tryptic Soy Broth) medium were selected in consideration of the number and economic feasibility of securing the Paracoccus communis NK1007 strain of the present invention. The strain growth rate was evaluated according to the total oxygen consumption rate (AOU) measurement value for each medium concentration. As a result, a short lag-time was observed in both of the BCM 660 medium or the TSB (Tryptic Soy Broth) medium, and a relatively high oxygen consumption rate was observed. In addition, when cultured using BCM 660 medium, cell proliferation of 2X10 ⁹ level was observed, and cell proliferation of 6X10 ⁸ level was observed in TSB (Tryptic Soy Broth) medium. Paracoccus communis ) It was confirmed that it can be used to secure the biomass of the NK1007 strain.

The composition for biodegradation of iminodipropionitrile of the present invention may be used to purify contaminated wastewater. In order to confirm this, the present inventors implemented a real wastewater environment and conducted a process operation study based on the bioaugmentation method, and Paracoccus communis of the present invention When the NK1007 strain culture was inoculated, total organic It has been confirmed that the lag-time for carbon (TOC) reduction can be reduced by about 1 day, and the final organic carbon (TOC) concentration can also be maintained significantly lower than that of the untreated negative control group. In addition, in the decomposition activity experiment on the IDPN compound in the activated sludge, it was confirmed that there was a difference in the amount of decrease in the IDPN compound of about 100 ppm in the daily culture process depending on whether or not this strain was added. Through these experimental results, Paracoccus communis ( Paracoccus communis ) When the bio-enhancement process using the NK1007 strain is applied to the sewage treatment process or facility of the actual plant, it is analyzed that the decomposition efficiency of the IDPN compound can be substantially improved. do.

According to another aspect of the present invention, the present invention treats the iminodipropionitrile-contaminated wastewater with the iminodipropionitrile biodegradation composition containing the Paracoccus communis NK1007 strain, A method for alleviating or eliminating toxicity of nodipropionitrile (3,3'-Iminodipropionitrile) is provided. As described above, the composition for biodegradation of iminodipropionitrile of the present invention can survive in wastewater containing iminodipropionitrile at an ultra-high concentration, and since it exhibits excellent activity in biodegradation of iminodipropionitrile compounds, Propinitrile (3,3'-Iminodipropionitrile) It can be used to reduce or eliminate toxicity and to purify contaminated wastewater. The Paracoccus communis ( Paracoccus communis ) NK1007 strain, its culture medium characteristics, and iminodipropionitrile biodegradation characteristics have been described in detail above, so that the specification is omitted here to avoid overcomplicating it.

The features and advantages of the present invention are summarized as follows:

(A) The present invention provides a composition for biodegradation of iminodipropionitrile comprising the paracoccus communis NK1007 strain having iminodipropionitrile decomposition activity.

(b) Paracoccus communis NK1007 strain of the present invention is capable of continuous self-proliferation even in a wastewater environment containing 100,000 mg/L ultra-high concentration iminodipropionitrile, iminodipropionitrile It has excellent biodegradation activity, so it can be usefully used to reduce or remove the toxicity of iminodipropionitrile-contaminated wastewater.

1 is a photographic sample of a colony of candidate strains generated by inoculating tertiary enriched cultures separated from wastewater on a solid medium.
2 is a diagram showing a phylogenetic tree for the Paracoccus communis NK1007 strain of the present invention.
3 is a Paracoccus communis strain of the present invention in a medium to which IDPN compound is added at a concentration of 100mg/L on 100ml M9 liquid medium, 1 day, 2 days, and 3 days after inoculation This is a photograph taken after observing the growth pattern.
4 is a graph showing the results of evaluating the biodegradation activity of the IDPN compound of the Paracoccus communis NK1007 strain of the present invention.
5 is a graph showing the measurement of the growth curve of the Paracoccus communis NK1007 strain of the present invention according to the change in IDPN concentration.
6 is a graph comparing and analyzing the biodegradation activity of the Paracoccus communis NK1007 strain of the present invention according to the change in IDPN concentration.
7 is a graph showing the simulation of the maximum decomposition rate of the IDPN compound per cell based on the OD value and the IDPN compound concentration observed in the culture process according to the change in the IDPN concentration.
8 is a graph showing the inoculation of the Paracoccus communis NK1007 strain of the present invention in M9 medium (M9 + IDPN 2000ppm) to which the IDPN compound is added and measuring the growth rate.
9 is a graph showing the Paracoccus communis NK1007 strain of the present invention inoculated twice in a semi-continuous manner in M9 medium (M9 + IDPN 2000ppm) to which the IDPN compound is added, and the growth rate is measured.
10 is a Paracoccus communis NK1007 strain of the present invention in BCM 660 medium (Glucose + Yeast Extract combination medium) and TSB (Tryptic Soy Broth) medium and the total oxygen consumption (Accumulated Oxygen Uptake) is measured. This is the graph shown.
11 is a graph showing the measurement of the total oxygen consumption (Accumulated Oxygen Uptake) of the Paracoccus communis NK1007 strain of the present invention according to the change in BCM 660 medium concentration.
12 is a photograph of observing appearance characteristics after inoculating and culturing a culture of Paracoccus communis NK1007 strain in a wastewater sample.
13 is a graph showing the change in total organic carbon (TOC) value after inoculating and culturing the Paracoccus communis NK1007 strain in a wastewater sample.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Isolation and identification of candidate strains

1-1. sampling

Iminodipropionitrile (IDPN, 3,3'-Iminodipropionitrile, hereinafter referred to as IDPN), which is a compound to be degraded in the present invention, is a neurotoxic substance. Therefore, the present inventors preferentially isolated candidate strains by collecting wastewater from Yeosu Industrial Complex in order to find bacteria that can survive and decompose IDPN compounds even in a condition containing a large amount of IDPN compounds.

Samples were collected by 1L each including raw water, and immediately after collection, they were stored in an ICE box and transported to the laboratory in order to prevent deterioration of the samples as much as possible. After arriving at the laboratory, 50 ml of each was aliquoted and stored refrigerated at 4°C. The sample for DNA extraction was pelleted down using a centrifuge and stored in a -20°C freezer.

1-2. Microbial enrichment culture

In order to isolate and identify biodegradable microorganisms of the target nervous system toxic substance IDPN, candidate microorganisms collected from samples collected from wastewater in the Yeosu Industrial Complex were cultured through a thickening culture method.

In the present invention, a liquid medium for thickening culture was prepared based on M9 medium, a minimal nutrient medium excluding other carbon sources, so that the neurotoxic substance IDPN becomes a pure substrate of the microorganism to be isolated and identified. The composition of M9 minimally nutrient medium is shown in [Table 1].

[Table 1]

In the process of preparing the medium for enriched culture, it was observed that the IDPN compound is a hydrophilic material and has high solubility, so that it dissolves well in the liquid medium. To 5 ml of the prepared M9 liquid medium, IDPN compounds, which are toxic substances, were added to 1,000 mg/L and 100 mg/L, respectively. The collected samples were diluted and inoculated at 1/50 (40ul), cultured in an incubator at 30° C. for 7 days, and then a part of the concentrated culture medium (1/50 concentration) was inoculated into a new medium and newly concentrated culture was performed. After repeating this process three times, the microorganisms concentrated and cultured by inoculation on a solid medium were separated and selected by colony.

The presence or absence of growth of the thickened culture was judged based on the turbidity observed during the culture process, and the test conditions with increased turbidity among the thickened cultures of each sample were selected and the process of culturing in a new medium was performed.

1-3. Microbial Isolation and Identification

The solid medium was prepared by including 20% agar in M9 liquid medium, and after autoclaving, in the process of cooling to a temperature of 60 ° C, IDPN compound, a neurotoxic substance, was added at a concentration of 1000 mg/L. On the prepared solid medium, the tertiary enriched culture was diluted to a concentration of 1×10 ⁶ CFU/ml and inoculated on a solid medium, and colony formation was confirmed through a 7-day culture process in an incubator at 30° C. did

Thereafter, each colony was isolated using a wood stick, inoculated into a new solid medium, and a total of 84 independent colonies were selected through the same culture process (see FIG. 1). .

The selected microorganism was identified for the 16S rRNA gene.

To identify the strain, the DNA of the isolated microorganism was extracted using a commercial kit, etc. to analyze the phylogenetic tree. did For the amplified PCR product, primer dimers and the like were removed through a purification kit, and the gene sequence was obtained.

The obtained sequence was identified by using the global alignment analysis method using the EzTaxon program to identify the closest strain isolated so far, and a phylogenetic tree was created by collecting the adjacent sequences (FIG. 2).

When creating a phylogenetic tree, based on the neighbor-joining provided in the MEGA6 program, minimum-evolution and maximum-likelihood algorithms were used if necessary.

In addition, in order to select a strain capable of applying the bioaugmentation technique to the wastewater contaminated with the IDPN compound, which is the final object of the present invention, a strain with rapid growth on the liquid medium was selected, and the independent biodegradability of the IDPN compound using the representative strain Measurement experiments were performed.

In the process of culturing an independent individual, it was observed that the culture named Paracoccus communis in [Table 2] is capable of growth on M9 medium containing a high concentration of IDPN compound within 3 days, especially at ultra-high concentration (100,000mg/L), the phenomenon of increasing the turbidity of the culture was observed without growth inhibition within 7 days even in the medium condition.

[Table 2]

Through these experimental results, Paracoccus communis NK1007 strain was selected as a strain that biodegrades the neurotoxic IDPN compound, and the biodegradation characteristics of the IDPN compound for the same strain were performed below.

Example 2. Analysis of biodegradation properties of IDPN compounds of Paracoccus comunis strain

IDPN concentration in M9+ medium containing IDPN compound, a neurotoxic substance, was measured using Humas' CODcr kit. After purifying 2ml of a sample (M9 liquid medium containing IDPN) using a 0.22um syringe filter, it was added to a vial provided in the COD Kit and heated at 150° C. for 2 hours. After cooling the vial at room temperature, the concentration of the IDPN compound in the sample was measured using a spectrophotometer provided by the manufacturer.

As a result of the pretest, the M9 medium did not contain any carbon components, and biodegradation characteristics were analyzed using the CODcr concentration measured in the “M9 + IDPN” medium as a reference value.

Growth characteristics for calculating the IDPN compound degradation characteristics were performed on the Paracoccus communis strain NK1007 of the present invention isolated and identified in Examples 1-3.

To this end, the IDPN compound was added at a concentration of 100mg/L on 100ml M9 broth. After that, the pre-cultured Paracoccus communis ( Paracoccus communis ) culture of the strain NK1007 of the present invention was diluted at a ratio of 1/50 and inoculated, while stirring and culturing in a 30 ℃ stirred incubator, 3ml of sample was prepared at intervals of 24 hours collected.

As a result of the experiment, the Paracoccus communis strain of the present invention showed a plateau for about 3 days after inoculation, and turbidity was observed to increase from the 4th day of inoculation (see FIG. 3 ). Therefore, biodegradation activity was maximized through strain growth for a sufficient period up to 9 days of inoculation, and then the supernatant was purified using a 0.22um syringe filter, and then used for COD measurement experiments. IDPN compound measured as COD value on “M9+IDPN” medium inoculated with IDPN compound at a concentration of 100 mg/L was measured at a concentration of about 85,000 mg/L.

Paracoccus communis ( Paracoccus communis ) Since strain NK1007 can grow even at high IDPN compound concentration, Paracoccus communis cultured at about 80,000 mg/L IDPN compound concentration condition Paracoccus communis ) Based on the culture medium of the NK1007 strain By measuring the COD value, the biodegradation activity of the IDPN compound of the same strain was evaluated. In addition, the biodegradation activity of the IDPN compound of the same strain was evaluated by applying the same measurement technique based on the culture of Paracoccus communis strain NK1007 cultured at about 15,000 mg/L IDPN compound concentration condition.

As a result of the experiment, referring to FIG. 4 , even under ultra-high concentration of IDPN, the Paracoccus communis NK1007 strain was observed to exhibit about 88% of the IDPN compound degradability during the 9-day culture period. Specifically, the COD measurement value of the M9I-100 medium prepared by adding the IDPN compound at a concentration of 100 mg/L on 100 ml of M9 medium was measured to be 85,000 ppm, and on 100 ml of M9 medium with a concentration of 10 mg/L. The COD measurement value of the M9I-10 medium prepared by adding the IDPN compound was measured to be 8,500 ppm.

Therefore, based on the COD measurement value of the M9I-100 medium and the COD measurement value of the M9I-10 medium, the strain culture of the present invention that was cultured at 80,000 mg/L IDPN compound concentration condition was diluted by a factor of 1/50 and the It was confirmed that approximately 88.35% of the IDPN compound was decomposed through the COD measurement value of the experimental group (left of Fig. 4) inoculated in M9I-100 medium and cultured for 9 days, and similarly, it was cultured at 15,000 mg/L IDPN compound concentration condition. It was confirmed that approximately 87.83% of the IDPN compound was decomposed through the COD measurement of the experimental group (the right side of FIG. 4) inoculated with the strain culture of the present invention in the M9I-100 medium and cultured for 9 days.

Based on these experimental results, it was confirmed that the Paracoccus communis strain NK1007 of the present invention has excellent activity in decomposing IDPN compounds.

Example 3. Analysis of biodegradation characteristics according to kinetic parameters of microbial growth

Paracoccus communis of the present invention ( Paracoccus communis ) Growth characteristics observation study for calculating the microorganism growth kinetic parameters (kinetic parameters) related to the IDPN compound degradation characteristics and biodegradation properties targeting strain NK1007 was performed as follows.

First, IDPN compounds were added at concentrations of 0, 100, 500, 1,000, 3,000, and 6,000 mg/L, respectively, on 100 ml of M9 liquid medium. Then, the pre-cultured Paracoccus communis ( Paracoccus communis ) The culture of the NK1007 strain was inoculated at a ratio of 1/50, and while stirring and culturing in a 30° C. stirred incubator, a sample of 3 ml was collected every 24 hours. The OD value was measured by aliquoting 1ml from the collected sample, and 2ml of the sample was filtered using a 0.22um syringe filter, and then total organic carbon (TOC, total organic carbon) was measured using SHIMADZU's TOC measuring equipment. ) values were measured.

Growth curves observed in the culture process for a total of 120 hours are summarized in [FIG. 5]. Referring to [Fig. 5], in the negative control group (IDPN 0mg/L) to which the IDPN compound was not added, the concentration of the initial strain was maintained until the end of the culture, and the growth of the Paracoccus communis NK1007 strain was not observed. didn't On the other hand, Paracoccus communis NK1007 strain was observed to show a growth retardation period of about 3 days during the culture process under the conditions in which the IDPN compound was added, but showed a rapid population increase within about 48 hours from the time of passing the retardation period. observed.

In addition, Paracoccus communis ( Paracoccus communis ) The final number of restricted individuals of the NK1007 strain increases depending on the concentration of the input IDPN compound, which could be confirmed as an increase in the final OD value.

Example 4. Quantitative analysis of biodegradation activity of IDPN compounds through total organic carbon (TOC) analysis

Using a TOC measuring device (Shimazu, Japan), the IDPN concentration in the sample collected at 24 hour intervals in the concentration-specific culture process was quantitatively analyzed, and the biodegradation activity of the Paracoccus communis NK1007 strain on the IDPN compound. was measured.

In consideration of the measurable concentration limit of the device, the analysis was performed by diluting the concentration of the IDPN compound in each sample to a final 20 mg/L. The sample was diluted using tertiary distilled water provided in a dedicated vial of the TOC measuring device, and the final volume was adjusted to 30ml. The operation of the TOC measuring device was performed using the established NTOC measuring method (for measuring non-volatile substances), and the measured concentration value of the sample after the measurement was completed was converted to the final concentration value by applying the final dilution factor.

6, as a result of comparative analysis of the biodegradation activity of the Paracoccus communis NK1007 strain administered for each IDPN concentration, the IDPN concentration conditions of 100, 500, 1,000, 3,000, and 6,000 mg/L, respectively Under, Paracoccus communis ( Paracoccus communis ) NK1007 strain showed a final removal rate for IDPN compounds of 80.9%, 86.1%, 89.8%, 49.6%, and 37.1%, respectively.

In addition, Paracoccus communis ( Paracoccus communis ) The maximum specific proliferation rate (umax (OD)) and limiting substrate half-saturation concentration (Ks) values of cells, which are parameters of the evaluation of the biodegradation activity of the NK1007 strain, were 0.09 and 2039.12, respectively. was measured with

This means that Paracoccus communis NK1007 strain can grow at a rate of up to 0.09OD/hr, and the half-saturation concentration for maximum growth is about 2,039 mg/L.

In addition, as a result of evaluating the degradation rate of the maximum IDPN compound per cell based on the OD value and IDPN compound concentration observed during the culture process, the maximum degradation rate of the IDPN compound per cell of Paracoccus communis NK1007 strain was It was observed that the maximum IDPN degradation rate per cell was observed at a concentration of 1,000 mg/L of the IDPN compound, and a culture condition of 96 to 120 hours (see FIG. 7 ).

Through these experimental results, Paracoccus communis NK1007 strain shows optimal biodegradation activity at an IDPN compound concentration of 1,000 mg/L, and a maximum concentration of 80,000 mg/L IDPN compound during a 9-day culture period It was measured to be able to remove about 88%. In addition, considering that the concentration of IDPN compound in wastewater generated in large amounts in chemical production and treatment facilities such as latex production is actually about 1,200 mg/L, Paracoccus communis NK1007 strain of the present invention is an IDPN compound It is considered to be an optimal microbial resource that can be completely treated biologically.

Example 5. Development of a medium for mass growth

For the application of the bioaugmentation method, to secure the biomass of the Paracoccus communis NK1007 strain so that it can be applied to actual sewage treatment facilities, it is cultured using a microbial mass culture system (BCS system) A study was conducted.

In the “M9+IDPN” medium condition, which was prepared by adding IDPN to the conventional M9 basic medium, the growth rate is remarkably slow and it is difficult to secure sufficient biomass to be applied to sewage treatment facilities. Munis ( Paracoccus communis ) A medium that can be used for mass culture of NK1007 strain was developed.

In addition, in order to evaluate the adequacy of the selected medium, the measured value of total oxygen consumption over time observed in the culture process was compared with the previously tested measured value in M9 medium (M9 + IDPN 2000ppm) to which the IDPN compound was added.

As a result of the experiment, the lag-time of the Paracoccus communis NK1007 strain of the present invention in M9 medium to which the IDPN compound was added was measured to be approximately 120 hours (see FIG. 8 ). If the lag-time of the strain used in the bio-enhancement process is long, the indigenous use of the useful strain does not occur due to the short operation time (HRT) of the sewage treatment facility, and there is a risk of loss. Therefore, the Paracoccus communis NK1007 strain of the present invention decomposes the IDPN compound into a single carbon source and can be propagated, but due to the relatively long lag-time, it is confirmed that there is a need for new medium development. could

In addition, in order to shorten the relatively long lag-time for direct application in sewage treatment plants, strains grown in M9 medium (M9 + IDPN 2000 ppm) added with IDPN compounds were newly added to M9 medium with IDPN compounds. (M9 + IDPN 2000ppm) A semi-continuous culture study was conducted to induce secondary proliferation. As a result, the lag-time was reduced to approximately 20 hours, but still A relatively long lag-time was observed for application to the treatment facility site (see FIG. 9).

In order to overcome this, a method was attempted to produce an excess of biomass as a mixed medium rather than a minimal nutrient medium such as M9 medium, and directly inject it into a sewage treatment facility reactor. To this end, the Paracoccus communis NK1007 strain of the present invention is inoculated and cultured, respectively, in two types of medium: BCM 660 medium (Glucose + Yeast Extract combination medium) and TSB (Tryptic Soy Broth) medium, and the respiratory rate measuring device (EZ-Oxyro Respirometer) and BOD Trak II were used to measure the total oxygen consumption over time (Accumulated Oxygen Uptake).

As a result of the experiment, referring to FIG. 10 , a short lag-time was observed in both media, and a relatively high oxygen consumption rate was observed. In addition, when cultured using the BCM 660 medium, cell proliferation at the 2X10 ⁹ level was observed, indicating that the proliferation efficiency was high compared to the TSB (Tryptic Soy Broth) medium in which the cell proliferation level of 6X10 ⁸ was observed.

Finally, in the culturing process, the BCM 660 medium was finally selected in consideration of the number and economic feasibility of the Paracoccus communis NK1007 strain of the present invention, and each medium concentration to optimize the bacterial culture rate according to the medium concentration. The strain growth rate was evaluated according to the total oxygen consumption (AOU, Accumulated Oxygen Uptake Unit) measurement value.

Referring to FIG. 11 , it was observed that the proliferation efficiency of the Paracoccus communis NK1007 strain according to the concentration of the BCM 660 medium was increased, but the proliferation efficiency did not increase in proportion to the input concentration of the medium under conditions of a certain concentration or more. didn't Considering the medium input concentration and strain growth rate, it is analyzed that the optimal culture conditions for applying this strain to the sewage treatment facility site using the bioaugmentation process are culturing using 0.4% BCM 660 medium. became

As a result of performing mass culture of this strain using a commercialized incubator (BCS System) based on the medium conditions selected as described above, the concentration of the grown bacteria was applied to the reactor in the sewage treatment facility using the bioaugmentation method. It is considered to meet the theoretical quantitative criteria for

Example 6. IDPN decomposition activity in sewage treatment facilities

In order to apply the decomposition activity of the IDPN compound of the Paracoccus communis NK1007 strain of the present invention to an actual sewage treatment facility, a process operation study based on the bioaugmentation method was performed by realizing an actual wastewater environment. , and through this, the degradation ability of this strain for IDPN compounds present in wastewater was evaluated.

In order to exclude the influence of other microorganisms distributed in the sample collected from the activated sludge reactor of the sewage treatment facility, the wastewater sample was sterilized using a 0.22um syringe filter.

In order to secure the biomass of the Paracoccus communis NK1007 strain, O/N culture was performed on a mixed medium, and the culture medium was centrifuged (4500rpm, 20min), and the purified water sample was was washed 3 times, and the previously sterilized wastewater sample was inoculated at a final inoculation concentration of 1×10 ⁸ cells.

Paracoccus communis ( Paracoccus communis ) The test group inoculated with the NK1007 strain and the untreated negative control group were cultured for about 10 days at 25°C and 200rpm conditions, and samples were collected at intervals of 1 day, and the samples were 0.22um. Purification by syringe filter.

In order to observe the change in the concentration of the IDPN compound in the reaction solution from the purified samples obtained as above, it was quantified using the total organic carbon (TOC) analysis value.

As a result of the experiment, in the process of bioaugmentation, the medium inoculated with the culture medium of Paracoccus communis NK1007 strain maintained a certain turbidity (OD=~0.2), whereas no turbidity change was observed in the negative control group. not (see FIG. 12).

In addition, referring to the total organic carbon (TOC) analysis result of FIG. 13, when the sterilized wastewater is inoculated with the Paracoccus communis NK1007 strain culture of the present invention, the total organic carbon (TOC) reduction It was observed that the lag-time can be reduced by about 1 day, and the final organic carbon (TOC) concentration can also be maintained significantly lower than that of the untreated negative control.

Through the above results, the Paracoccus communis NK1007 strain of the present invention can be applied to actual sewage treatment facilities, and a bioaugmentation process for decomposing IDPN compounds, which are neurotoxic substances distributed in sewage It was confirmed that it can be usefully used for

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

Iminodipropionitrile (IDPN, 3,3'- Iminodipropionitrile) for biodegradation Paracoccus communis ( Paracoccus communis ) NK1007 strain (Accession No.: KCTC 18866P). According to claim 1,
The strain is Paracoccus communis characterized in that it has the 16S rRNA sequence of SEQ ID NO: 1 NK1007 strain (Accession No.: KCTC 18866P). According to claim 1,
The strain is Paracoccus communis, characterized in that it can grow in a minimal nutrient medium in which other carbon sources are excluded in addition to iminodipropionitrile as a carbon source. Paracoccus communis NK1007 strain (accession number: KCTC 18866P). The method of claim 1,
The strain is Paracoccus communis characterized in that it is possible to grow in ultra-high concentration iminodipropionitrile medium conditions of 100,000mg/L ( Paracoccus communis ) NK1007 strain (Accession No.: KCTC 18866P). Paracoccus communis NK1007 strain (Accession No.: KCTC 18866P) for biodegradation of iminodipropionitrile of claim 1 or a composition for iminodipropionitrile biodegradation comprising a culture solution thereof. 6. The method of claim 5,
The Paracoccus communis ( Paracoccus communis ) NK1007 strain (accession number: KCTC 18866P) is iminodipropionitrile biodegradation composition, characterized in that it is cultured in BCM 660 medium or TSB (Tryptic Soy Broth) medium. 6. The method of claim 5,
The composition is iminodipropionitrile biodegradable composition, characterized in that used for purification of wastewater contaminated with iminodipropionitrile. Method for reducing or removing iminodipropionitrile (3,3'-Iminodipropionitrile) toxicity of wastewater by treating wastewater contaminated with iminodipropionitrile with one or more compositions selected from the group consisting of claims 5 to 7 .

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