WO2019151799A1 - Method for treating polychlorinated biphenyls using microorganisms - Google Patents

Abstract

The present invention relates to a method for treating polychlorinated biphenyls (PCBs) in an insulating oil containing PCBs, in which biodegradation can be carried out by administering a certain microbial cell and culturing same under certain conditions such that the PCBs of the insulating oil can be effectively treated.

Description

Treatment Method of Polychlorinated Biphenyls Using Microorganisms

The present invention relates to a method for treating polychlorinated biphenyls (PCBs) using certain microorganisms.

PolyChlorinated Biphenyls (PCBs) refer to compounds in which 2 to 10 of the 10 hydrogen atoms of biphenyl are linked with two benzene rings and substituted with chlorine atoms. This compound is insoluble in water, good in solubility in organic solvents, stable in acids and alkalis, low in volatility, high in viscosity and very high in thermal resistance, so PCBs are used in insulating oils in transformers and accumulators or in heat exchangers. It has been used as a medium and has been widely used in industries such as paint, ink and pesticides.

However, since the PCBs are known to have serious adverse health and environmental impacts in the mid-1970s, much research has been made on the development of methods to effectively process PCBs that have already been produced and must be disposed of.

Polychlorinated Biphenyls (PCBs) are contained in insulating oils such as transformers and condensers used in power plants.In accordance with the Act on Persistent Organic Pollutants Control (2008.1.27), the use of PCBs contaminated equipment insulating oil is prohibited and until 2015. Although it is mandatory to handle it, it is not completely finished due to technical and environmental problems.

The United States, Japan and Europe have already installed and operated incineration and chemical treatment facilities for processing PCBs in their own countries. Although small chemical treatment facilities have been installed in Korea, there are problems such as PCBs processing efficiency, which is being supplemented for operation.

In order to install and operate domestic PCBs processing facilities under the auspices of the Ministry of Environment, we have conducted research and demonstration demonstration projects for incineration and chemical treatment, but the incineration method has not been evaluated for PCBs decomposition rate and the environmental impact from dioxin emissions. In addition, a high-temperature incineration demonstration project is planned, and the chemical treatment method cannot deal with PCBs contaminants such as solid waste and soil.

Domestic treatment trends are as follows. At present, Korea should be treated by high temperature incineration or chemical treatment in accordance with the Waste Management Act and the Residual Organic Pollutants Control Act. In case of less than 6 seconds and less than 6% of surplus oxygen in the incinerator), toxic gases such as dioxins are more likely to be generated, which is why they are not operated due to opposition from environmental groups and citizens. Specifically, there are domestic technologies as follows.

end. Refractory waste treatment technology using supercritical hydroxide process

KEPCO has conducted R & D projects on budget of about 2 billion won from March 2005. The decomposition process was carried out by diluting waste insulating emulsion (3%), preheating pressurized oxygen-added mixed supercritical oxidation (1020 4000psi) Is made of. According to the dilution of the insulating oil (about 3%), the waste to be treated increases by 33 times, and the decomposition rate is slow, the processing cost is high due to the addition of a large amount of oxygen, and the safety problems and the emitted gas and There is a problem that additional secondary equipment for condensate treatment is required.

I. PCBs processing technology using E-Beam

It is a technology developed by Jeongeup Radiation Research Institute of Nuclear Research Institute. It is a technology that removes and processes a large amount of chlorine constituting PCBs at room temperature and high pressure using strong energy of electron beam. However, such a treatment technique is estimated to be less than the international level (99.9999%) and not suitable for large scale treatment.

All. Chemical Catalyst Method, NaCl-Atomic Energy Research Institute

In addition, there are chemical catalyst methods, but facility costs, treatment capacity, and problems of the results are scattered.

Overseas treatment trends are as follows. Overseas, R & D activities such as decomposition treatment technology, alternative treatment technology, and recycling technology are actively progressed by PCBs processing technology, and most countries such as USA, Canada, and Europe prefer high temperature incineration treatment methods. Incineration is the most reliable method, but when the combustion conditions are poor, there is a high possibility of generating toxic gases such as dioxins, and various incineration technologies and prevention facilities have been studied to solve this problem. As a result, incineration technology is being developed or developed using 1) Grate incinerate method, 2) Fluidized bed method, 3) Pure oxygen supply liquid incineration method, 4) Rotary-kiln incinerator method, etc. (Pyroliser) decomposition method, 2) UV (infrared furnace) method, 3) molten salt-glasser treatment method, 4) plasma technology method, 5) microbial decomposition method, 1) catalyst Dechlorination, 2) oxidative UV light treatment, and 3) MC (methylene chloride) treatment are researched and developed.

In the US, EPA recognizes alternative technologies such as high-temperature combustion technology, chemical dechlorination technology, landfill technology and pyrolysis as PCBs processing technology.Incineration is also preferred in Europe. Decontamination and Sodium Reduction technology are applied. In the case of Japan, a total of 17 technologies are specified as treatment guidelines, such as dechlorination, hydrothermal and oxidative decomposition, reduction and thermochemical decomposition, photolysis, and plasma decomposition.

However, as mentioned above, domestic and foreign PCBs processing techniques remain unproven for PCBs decomposition rate, dioxin emissions, and inability to deal with PCBs contaminants such as solid waste and soil.

Accordingly, the inventors of the present invention have come to know that the processing efficiency and reproducibility of PCBs contaminated insulating oil using the PCBs purification treatment technology using a specific microorganism can be developed into a treatment facility.

[Preceding technical literature]

[Patent Documents]

(Patent Document 1) Republic of Korea Patent No. 10-0848137

(Patent Document 2) Republic of Korea Patent No. 10-0612225

(Patent Document 3) Korean Patent Registration No. 10-0864632

(Patent Document 4) Republic of Korea Patent Registration 10-1021690

(Patent Document 5) Republic of Korea Patent Registration 10-1085553

(Patent Document 6) Republic of Korea Patent Publication No. 10-2006-0036261

(Patent Document 7) Republic of Korea Patent Registration 10-0782543

(Patent Document 8) Republic of Korea Patent No. 10-0798410

One aspect of the present invention is to provide a new PCBs processing method that solves the above problems.

An aspect of the present invention is to provide a method for administering a specific bacterial community in the PCBs processing method of insulating oil containing Polychlorinated Biphenyls (PCBs).

In addition, an aspect of the present invention is to provide a PCBs processing method comprising a biodegradation step of specific conditions in the PCBs processing method of insulating oil containing polychlorinated Biphenyls (PCBs), after bacterial community administration.

In order to achieve the above object,

In one aspect of the present invention is a method for processing PCBs of insulating oil containing PCBs (Polychlorinated Biphenyls),

Administering a PCBs treated bacterial community,

The PCBs treated bacterial community is Bacillus sp. Cy106 strain, Pseudomonas sp. Cy100 strain, Brebundimonas vesicularis Cy101 selected from NBC2000 bacterial community with accession number KCTC 10623 BP. Strain, Brebundimonas vesicularis Cy102 strain, Brebunundimonasvesicularis Cy103 strain, Bacillus stearothermophilus Cy104 strain, Bacillus sp. Cy107 strain, Capital One or more of Pseudomonas aeruginosa Tnh strain, W24 strain which is an oil degradation gram negative bacterium and Nz2001 strain which is a sulfur strain; At least one of Bacillus cereus EBC106 strain and Pseudomonas sp. EBC107 strain selected from EBC1000 bacterial community with an accession number of KCTC 0652 BP; To provide.

In one aspect of the invention, the PCBs treated bacterial community is Bacillus sp. Cy106 strain, Pseudomonas sp. Cy100 strain, Brebundimonas vesicularis Cy101 strain, Brebundi Monas Veciculis Cy102 strain, Brebundimonas vesicularis Cy103 strain, Bacillus stearothermophilus Cy104 strain, Bacillus sp. Cy107 strain, Pseudomonas aruginosa (Pseudomonas aeruginosa) PCBs treatment of insulating oils, including Tnh strains, W24 strains of oil degradation gram negative bacteria, Nz2001 strains of sulfur strains, Bacillus cereus EBC106 strains and Pseudomonas sp. EBC107 strains Provide a method.

In one aspect of the invention, the PCBs treated bacterial community provides a method for treating the PCBs of insulating oil, which is administered after incubating at 10 ⁹ CFU / ml or more of the cell number.

In one aspect of the invention, the culture is incubated for 4 to 5 days with bacto-yeast extract in a Luria-Bertani nutrient medium, to provide a PCBs treatment method of insulating oil do.

In one aspect of the invention, the method of processing the PCBs of the insulating oil further provides a method of processing the PCBs of the insulating oil, further comprising the step of biodegradation after administration of the PCBs treatment bacterial community.

In one aspect of the invention, the step of biodegrading is a temperature condition of 24 ℃ to 26 ℃; pH conditions of pH 5.3 to 8.5; And DO conditions with a dissolved oxygen concentration of 73% to 95%; The present invention provides a method for treating PCBs of insulating oil, which is a step of decomposing while satisfying any one or more of the conditions.

In one aspect of the invention, the step of biodegradation is a temperature condition of 24 ℃ to 25 ℃; pH conditions of pH 6-8; And DO conditions in which the concentration of dissolved oxygen is 80% to 90%; The present invention provides a method for treating PCBs of insulating oil, which is a step of decomposing while satisfying any one or more of the conditions.

In one aspect of the invention, the step of biodegrading further comprises a condition for stirring using a stirrer of 150 rpm to 250 rpm during biodegradation, to provide a method for processing PCBs of insulating oil.

In one aspect of the invention, the step of biodegrading has a biodegradation time of 50 days or more, to provide a method for processing PCBs of insulating oil.

In one aspect of the invention, the step of biodegrading has a biodegradation time of 70 days to 200 days, to provide a method for processing PCBs of insulating oil.

In one aspect of the invention, the insulating oil is an insulating oil contained in a transformer, to provide a method for processing PCBs of insulating oil.

In one aspect of the invention, the method is to provide a method for processing PCBs of insulating oil, reducing the concentration of PCBs in the insulating oil to 50ppm or less.

In one aspect of the present invention, the method is to reduce the concentration of PCBs in the insulating oil to 50ppm or less and at the same time to provide a method for treating the PCBs of the insulating oil to generate dioxin below the concentration of 3 ng-TEQ / g.

The method according to one aspect of the present invention can significantly reduce the concentration of PCBs in the insulating oil while at the same time keeping the emission of dioxins much lower than the legal standard.

The method according to an aspect of the present invention can significantly reduce the concentration of PCBs in insulating oil without contamination problems such as solid waste and soil.

The method according to an aspect of the present invention can significantly reduce the concentration of PCBs in the insulating oil at an economical facility cost.

The method according to one aspect of the present invention can reduce the concentration of PCBs in a large amount of insulating oil at once.

The method according to an aspect of the present invention can reduce the concentration of PCBs in the insulating oil to the same level as the international level.

The method according to one aspect of the present invention can reduce the concentration of PCBs in the insulating oil in a relatively faster time than the conventional technology.

The method according to an aspect of the present invention provides a method for processing PCBs in insulating oil that can be used directly in the field such as a transformer without additional installation of secondary equipment.

1A to 1D are graphs of changes in pH, temperature, DO, and stirring speed of a reaction tank.

Figure 2a is a result of the observation of the main reaction tank GX-1 containing the high concentration PCBs, (a) is a photo before the mixing of the strain, the color of the yellow and transparent insulating oil appeared, (b) is a photograph of when the strain was stirred immediately after mixing the strain a little Turbidity (microbial proliferation) and microbial community were observed, (c) 69 days after the strain turbidity was increased, microbial community was observed inside the reactor, (d) 161 days after the strain turbidity Became thicker, and a large number of microbial communities were formed inside the reactor.

Figure 2b is a low concentration of PCBs containing insulating oil main reaction tank GX-2 observation results, (a) is a photograph before the strain mixing, the color of the yellow and transparent insulating oil appeared, (b) is a photograph when agitated immediately after mixing the strain a little strain Turbidity and microbial community were observed, (c) the 66-day old photos showed turbidity of the strain, the microbial community was observed inside the reactor, (d) 161 days the strain turbidity was increased, The color changed from light yellow to white, and microbial community was formed inside the reactor.

Figure 2c is a result of observation of the pre-reactor GR-1 containing the high concentration of PCBs containing oil, (a) is a picture of the strain before mixing the yellow and transparent insulating oil, (b) is a picture of a slight strain when the image is stirred immediately after mixing the strain Turbidity and microbial community were observed, (c) was a 51-day picture of the turbidity of the strain was increased, the microbial community was observed inside the reactor, (d) 84 days of the picture was taken around September 15 It turned brown.

Figure 2d is a result of observation of the preliminary reactor GR-2 containing a high concentration PCBs insulating oil control, (a) is a photograph immediately after the initial charge, (b) is a photograph after 51 days, (c) is a photograph after 84 days.

Figure 3 is a graph showing the change in PCBs concentration (main reactor-GX) with the reaction time applied GC-ECD analysis.

4A is a non-PCBs insulating oil GC-ECD method peak pattern (lab frontier).

Figure 4b is a non-PCBs insulating oil + microbial strain GC-ECD method peak pattern (Lab Frontier).

In the present specification, when a range is described for a variable, it will be understood that the variable includes all values within the described range including the listed endpoints of the range. For example, the range "5 to 10" includes any subrange such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, as well as values of 5, 6, 7, 8, 9, and 10. And any value between integers that are within the scope of the described range, such as 5.5, 6.5, 7.5, 5.5-8.5, 6.5-9, and the like. Also for example, the range of “10% to 30%” ranges from 10% to 15%, 12% to 10%, 11%, 12%, 13%, etc. as well as all integers including up to 30%. It will be understood to include any subranges such as 18%, 20% to 30%, etc., and to include any value between valid integers within the range of the stated range, such as 10.5%, 15.5%, 25.5% and the like.

Hereinafter, the present invention will be described in detail.

In one aspect of the present invention is a method for processing PCBs of insulating oil containing PCBs (Polychlorinated Biphenyls),

The step of administering a PCBs treated bacterial community, the PCBs treated bacterial community is Bacillus sp. Cy106 strain, Pseudomonas sp. Cy100 strain selected from NBC2000 bacterial community with accession number KCTC 10623 BP , Bredundimonas vesicularis Cy101 strain, Bredundimonas vesicularis Cy102 strain, Bredundimonasvesicularis Cy103 strain, Bacillus stearothermophilus Bacillus stearothermophilus strain Or any one or more of Bacillus sp. Cy107 strain, Pseudomonas aeruginosa Tnh strain, W24 strain which is an oil degradation gram negative bacterium and Nz2001 strain which is a sulfur strain; At least one of Bacillus cereus EBC106 strain and Pseudomonas sp. EBC107 strain selected from EBC1000 bacterial community with an accession number of KCTC 0652 BP; To provide.

In one aspect of the invention, the PCBs treated bacterial community is Bacillus sp. Cy106 strain, Pseudomonas sp. Cy100 strain, Brebundimonas vesicularis Cy101 strain, Brebundi Monas Veciculis Cy102 strain, Brebundimonas vesicularis Cy103 strain, Bacillus stearothermophilus Cy104 strain, Bacillus sp. Cy107 strain, Pseudomonas aruginosa (Pseudomonas aeruginosa) PCBs treatment of insulating oils, including Tnh strains, W24 strains of oil degradation gram negative bacteria, Nz2001 strains of sulfur strains, Bacillus cereus EBC106 strains and Pseudomonas sp. EBC107 strains Provide a method.

In one aspect of the invention, the PCBs treated bacterial community, Pseudomonas sp. Cy100 strain (hereinafter abbreviated as "Cy100"), Pseudomonas sp. EBC107 strain (hereinafter, "EBC107 Abbreviated as " Pseudomonas aeruginosa " Tnh strain (hereinafter abbreviated as "Tnh"), Brevundimonas vesicularis Cy101 strain (hereinafter abbreviated as "Cy101") , Brevundimonas vesicularis Cy102 strain (hereinafter abbreviated as "Cy102"), Brevundimonas vesicularis Cy103 strain (hereinafter abbreviated as "Cy103"), as Bacillus stea Bacillus stearothermophilus Cy104 strain (hereinafter abbreviated as "Cy104"), Bacillus sp. Cy107 strain (hereinafter abbreviated as "Cy107"), Bacillus cereus EBC106 strain (hereinafter referred to as "Cy104") , Abbreviated as "EBC106", the Nz2001 strain which is a sulfur strain Characterized in that consisting of abbreviated as "Nz2001" abbreviated as) and oil decomposition gram-negative bacteria strain W24 (hereinafter, "W24").

In one aspect of the invention, the PCBs treatment bacterial community, essentially include Cy106, Cy100, Cy101, Cy102, Cy103, Cy104, Cy107, Tnh, EBC106, W-24, EBC107 and NZ2001 It characterized in that it further comprises one or more strains selected from the group consisting of.

In one aspect of the present invention, the PCBs treated bacterial community provides a method for treating the PCBs of insulating oil, which is administered after culturing at 10 ⁹ CFU / ml or more. If the cell number is satisfied, the PCBs can be effectively decomposed.

In one aspect of the invention, the culture is incubated for 4 to 5 days with bacto-yeast extract in a Luria-Bertani nutrient medium, to provide a PCBs treatment method of insulating oil do. When the culture conditions are satisfied, PCBs can be effectively decomposed.

In one aspect of the invention, the method of treating the PCBs of the insulating oil further provides a method of processing the PCBs of the insulating oil after the biodegradation, after administration of the bacterial community.

In one aspect of the invention, the step of biodegrading is a temperature condition of 24 ℃ to 26 ℃; pH conditions of pH 5.3 to 8.5; And DO conditions with a dissolved oxygen concentration of 73% to 95%; The present invention provides a method for treating PCBs of insulating oil, which is a step of decomposing while satisfying any one or more of the conditions. When the biodegradation conditions are satisfied, PCBs can be effectively decomposed.

In one aspect of the invention, the step of biodegradation is a temperature condition of 24 ℃ to 25 ℃; pH conditions of pH 6-8; And DO conditions in which the concentration of dissolved oxygen is 80% to 90%; The present invention provides a method for treating PCBs of insulating oil, which is a step of decomposing while satisfying any one or more of the conditions.

In one aspect of the invention, the step of biodegrading further comprises a condition for stirring using a stirrer of 150 rpm to 250 rpm during biodegradation, to provide a method for processing PCBs of insulating oil. Preferably, a stirrer with a speed of 190 rpm to 210 rpm during biodegradation may be used. When the stirring conditions are satisfied, PCBs can be effectively decomposed.

In one aspect of the invention, the step of biodegrading has a biodegradation time of 50 days or more, to provide a method for processing PCBs of insulating oil. When the biodegradation time is satisfied, PCBs can be effectively decomposed.

In one aspect of the invention, the step of biodegrading has a biodegradation time of 70 days to 200 days, to provide a method for processing PCBs of insulating oil. Preferably the biodegradation time may be 150 days to 170 days. When the biodegradation time is satisfied, PCBs can be effectively decomposed.

In one aspect of the invention, the insulating oil is an insulating oil contained in a transformer, to provide a method for processing PCBs of insulating oil.

In one aspect of the invention, the method is to provide a method for processing PCBs of insulating oil, reducing the concentration of PCBs in the insulating oil to 50ppm or less.

In one aspect of the present invention, the method is to reduce the concentration of PCBs in the insulating oil to 50ppm or less and at the same time to provide a method for treating the PCBs of the insulating oil to generate dioxin below the concentration of 3 ng-TEQ / g. Preferably the concentration of dioxins may be 0.5 to 0.7 ng-TEQ / g concentration.

Hereinafter, the isolation, identification and activity of the NBC2000 bacterial community and the EBC1000 bacterial community included in the PCBs-treated bacterial community will be described in detail.

Isolation, Identification and Activity of NBC2000 Bacterial Communities

One. Isolation of a New Bacterial Community That Can Decompose Environmental Hormones

(1) Isolates bacteria that degrade PCBs, PCP, dioxin, PCE, toluene, taric acid, etc. from soil collected in Korea, Southern Europe, Oceania, etc.

One gram of the collected soil was shaken for 2 to 3 days in Luria-Bertani liquid medium (10 g of Bacterium-Tryptone, 5 g of Bakto yeast extract, 10 g of NaCl + 1 liter of demineralized water), and then 1 ml was taken to take 1 ml of Luria-Bertani agar medium ( Each colony was isolated from 10 g of Bakto-Tryptone, 5 g of Bakto yeast extract, 10 g of NaCl, 1.5% of agar, and 950 ml of demineralized water. Select each colony that appeared at this time and select the minimum liquid medium containing PCBs, PCP, PCE, toluene and taric acid (K ₂ HPO ₄ 0.065g, KH ₂ PO ₄ 0.017g, MgSO ₄ 0.1g, NaNO ₃ 0.5g + Demineralized water 1 Liters) were sequentially inoculated and shaken at 25 to 30 ^o C for at least 3 days. In addition to confirming the reduction of each harmful substance, 1 ml of shaking culture solution was taken and sequentially inoculated into Luria-Bertani agar medium and incubated at 25 to 30 ^o C for 3 to 5 days.

Each colony separated from pure water was reinoculated into the above-described minimal liquid medium, followed by shaking culture, followed by different cultures of colonies of different shapes formed in the Luria-Bertani solid medium, and the same colonies appearing in the passage of the colonies. About 50 species were separated.

(2) The bacteria isolated from the above inoculated sequentially in a minimal medium containing PCBs, PCP, PCE, toluene, tartaric acid at a high concentration step by step and repeat in the same manner as in (1) to survive at a higher concentration And 26 species were separated according to morphological differences.

The 26 bacteria obtained here were composed of a community named NBC2000, and each of the bacteria constituting the bacterial community was Cy100, Cy101, Cy102, Cy103, Cy104, Cy105, Cy106, Cy107, Ntar1, Ntar2, Ntar3, Gc300, Gc500, Gc501, Bs100, Aeng17, Aeng18, Sp300, Tnh, Djhc, Pcpts, EBC106, EBC107, Bs101, W24 and Nz2001.

The bacterial community NBC2000 according to the present invention was deposited on April 16, 2004 at the Genetic Resource Center of the Korea Research Institute of Bioscience and Biotechnology under accession number KCTC 10623 BP.

 2. Establishment of Optimum Conditions for Growth of Bacterial Community NBC2000

Luria-Bertani nutritional medium [10 g of bacto-tryptone, 5 g of bacto-yeast extract, 10 g of sodium chloride (NaCl) / 1 liter of demineralized water] 8, temperature 25 ~ 30 ^o C, shaking (shaking) rotation at 80 ~ 120rpm 48 ~ 96 hours incubation for optimum growth, even in subcultures grow well under the same conditions.

3. Biochemical Identification of Bacterial Community NBC2000

23 bacteria were identified by API Kits API20E, API20NE, API50CH and API50CHB purchased from bioMerieux (bioMerieux sa 69280 Marcy I'Etoile / France). To Table 5).

Test Item ＼ Strain	Cy100	Aeng18	Djhc	Ntar3	Ntar2	Ebc107
Ortho-nitro-phenyl-β-D-galactopyranoside	-	+	-	-	+	-
Arginine	+	+	+	+	+	+
Lysine	-	+	-	-	+	-
Ornithine	-	+	-	-	+	-
Sodium Citrit	+	+	+	+	+	+
Sodium Thiosulfate	_	-	-	-	-	-
Urea	_	-	+	-	-	-
Tryptophan	+	+	+	+	+	+
Indole	_	-	-	-	-	-
Sodium Pyruvate	_	+	-	+	+	-
Corn gelatin	_	+	-	-	-	-
Glucose	_	-	-	-	+	-
Mannitol	_	+	-	-	+	-
Inositol	_	+	-	-	+	-
Sorbitol	_	+	-	-	+	-
Rhamnos	_	-	-	-	-	-
Sucrose	_	+	-	-	+	-
Melibiose	_	-	-	-	-	-
Amygdalin	_	+	-	-	+	-
Arabinos	_	-	-	-	-	-
Oxidase	+	-	+	+	-	+
Reduction of Nitrate to Nitrite	-	+	-	+	+	-
Reduction of Nitrate to Nitrogen Gas	+	-	+	-	-	+
motility	+	+	+	+	+	+
Maconki Badge	+	+	+	+	+	+
Gram strain	-	-	-	-	-	-
Identification result	Pseudomonas sp.	Serratia sp.	Pseudomonas sp.	Pseudomonas sp.	Serratia sp.	Pseudomonas sp.

Test Item ＼ Strain	Tnh	Aeng17	Pcpts	Ntar1	Sp300
Potassium Nytreth (NO3-NO2)	+	+	-	+	+
Tryptophan (Indole)	-	+	-	-	-
Glucose	-	+	-	+	-
Arginine	-	+	-	+	-
Urea	+	+	+	+	+
Escurin	+	+	+	+	+
gelatin	+	+	+	+	+
p-nitrophenyl-β-D-galactopyranoside	-	+	-	+	-
Glucose (Fairy Tale)	+	+	+	-	+
Arabinos	-	-	-	-	-
Mannos	-	+	-	+	-
Mannitol	+	+	+	-	+
N-acetylglucosamine	+	+	+	+	+
Maltose	-	+	-	+	-
Gluconate	+	+	+	-	+
Caprit	+	+	+	-	+
Adipart	-	-	+	-	-
Malate	+	+	+	+	+
Site rate	+	+	+	+	+
Phenyl Acetate	-	+	-	-	-
Tetramethyl-p-phenylenediamine (oxidase)	+	+	+	-	+
motility	+	+	+	+	+
Maconki Badge	+	+	+	+	+
Gram strain	-	-	-	-	-
Identification result	Pseudomonas aeruginosa	Aeromonas hydrophila	Pseudomonas aeruginosa	Stenotrophomonas maltophilia	Pseudomonas aeruginosa

Test Item ＼ Strain	Gc501	Gc500	Gc300	Cy101	Cy102	Cy103
Potassium Nytreth (NO3-NO2)	+	+	+	-	-	-
Tryptophan (Indole)	-	-	-	-	-	-
Glucose (acidification)	+	-	+	-	-	-
Arginine	-	-	+	-	-	-
Urea	-	-	+	-	-	-
Escurin	+	+	+	+	+	+
gelatin	-	-	-	-	-	-
p-nitrophenyl-β-D-galactopyranoside	+	+	+	-	-	-
Glucose (Fairy Tale)	+	+	+	-	-	-
Arabinos	+	+	+	-	-	-
Mannos	+	+	+	-	-	-
Mannitol	+	+	+	-	-	-
N-acetyl glucosamine	+	+	+	-	-	-
Maltose	+	+	+	-	-	-
Gluconate	+	+	+	-	-	-
Caprit	-	-	-	-	-	-
Adipart	-	-	-	-	-	-
Malate	+	-	+	-	-	-
Site rate	+	+	+	-	-	-
Phenyl acetate	+	-	+	-	-	-
Tetramethyl-p-phenylenediamine	-	-	-	+	+	+
motility	+	+	+	+	+	+
Maconki Badge	+	+	+	+	-	+
Gram strain	-	-	-	-	-	-
Identification result	Chryseomonas luteola	Chryseomonas sp.	Chryseomonas luteola	Brevundimonas vesicularis	Brevundimonas vesicularis	Brevundimonas vesicularis

Test Item ＼ Strain	Bs100	Cy104	Cy105	Cy106	Cy107	Ebc106
Control	-	-	-	-	-	-
Glycerol	+	-	+	-	-	-
Erythritol	-	-	-	-	-	-
D-Arabinose	-	-	-	-	-	-
L-Arabinose	-	-	+	-	-	-
Ribose	-	-	-	-	-	+
D-Xylose	-	-	+	-	-	-
L-xylose	-	-	-	-	-	-
Adonitol	-	-	-	-	-	-
Beta Methyl-D-Xylose	-	-	+	-	-	-
Galactose	+	-	-	-	-	-
D-glucose	+	-	+	-	-	+
D-fractose	+	+	+	-	-	+
D-Mannose	-	+	+	-	-	+
L-sorbos	-	-	-	-	-	-
Rhamnos	-	-	-	-	-	-
Dulcitol	-	-	-	-	-	-
Inositol	-	-	-	-	-	-
Mannitol	-	+	+	-	-	-
Sorbitol	-	-	-	-	-	-
Alpha-methyl-D-mannoside	-	-	-	-	-	-
Alpha-Methyl-D-Glucoside	-	-	-	-	-	-
N-acetyl-glucosamine	+	-	+	-	-	-
Amygdarin	-	-	-	-	-	-
Arbutin	-	-	-	-	-	+
Escurin	+	+	+	+	+	+
Salinity	-	-	-	-	-	+
Cellobiose	-	+	-	-	-	-
Maltose	+	+	+	-	-	+
Lactose	+	-	-	-	-	-
Melibiose	-	-	+	-	-	-
Sucrose	+	+	+	-	-	-
Trihalose	+	+	+	-	-	+
Inulin	-	-	-	-	-	-

Melittose	-	-	+	-	-	-
D-Raffinose	-	+	+	-	-	-
Starch	-	+	+	-	-	+
Glycogen	-	+	+	-	-	+
Xylitol	-	-	-	-	-	-
Genthiobios	-	-	-	-	-	-
D-Tranose	+	-	+	-	-	-
D-recross	-	-	-	-	-	-
D-tagatose	-	-	-	-	-	-
D-fucose	-	-	-	-	-	-
L-fucose	-	-	-	-	-	-
D-Arabitol	-	-	-	-	-	-
L-Arabitol	-	-	-	-	-	-
Gluconate	-	-	-	-	-	-
2-keto-gluconate	-	-	-	-	-	-
5-keto-gluconate	-	-	-	-	-	-
motility	+	+	+	+	+	+
Maconki Badge	-	-	-	-	-	-
Gram strain	+	+	+	+	+	+
Identification result	Bacillus stearothermophilus	Bacillus stearothermophilus	Bacillus sp.	Bacillus sp.	Bacillus sp.	Bacillus cereus

4. Isolation of each bacteria constituting bacterial community NBC2000

1) Strain isolation and method

Each strain consists of NBC2000 purely separated from soils such as Korea, New Zealand, Sweden, and Cyprus, all of which have mobility characteristics and can be applied to a wide variety of soils. The method of morphological characterization of individual strains in bacterial community NBC2000 is as follows.

(A) Cultivation in Luria-Bertani agar medium (10 g of Bacterium-Tryptone, 5 g of Bacterial yeast extract, 10 g of NaCl, 1.5% agar, 950 ml of demineralized water)

Cy100: After 48 hours of incubation, the surface appears as an uneven irregular yellow colony and grows to a size of 4 mm.

Cy101: Appears as translucent ivory convex colonies after 48 hours of incubation and grows to a size of 2.5 mm.

Cy102: Appears as yellow round colonies after 48 hours of incubation and grows to 2 mm in size.

Cy103: After 48 hours of incubation, it appears as an ivory round and flat colony, growing to a size of 4 mm.

Cy104: Appears as translucent ivory round colonies after 48 hours of incubation and grows to 4 mm in size.

Cy105: Appears as ivory round colonies after 48 hours of incubation and grows to a size of 2.5 mm.

Cy106: After 48 hours of incubation, it appears as a round colony with a matt white color and grows to a size of 1.5 mm.

Cy107: After 48 hours of incubation, it appears as a matt white round colony and grows to a size of 1 mm.

Ntar1: After 24 hours of incubation, it appears as a translucent yellowish, round, glossy colony, growing to a size of 1 mm.

Ntar2: After 24 hours of incubation, it appears as a round, glossy colony of beige and ivory, growing to a size of 2 mm.

Ntar3: After 48 hours of incubation, it appears as an ivory-colored round and glossy colony, growing to a size of 1.5 mm.

Gc300: After 24 hours of cultivation, it appears as an ivory round, glossy convex colony and grows to a size of 3 mm.

Gc500: After 24 hours of incubation, it appears as an ivory round, glossy convex colony, growing to a size of 1.5 mm.

Gc501: After 24 hours of cultivation, it appears as an ivory round, glossy convex colony and grows to a size of 2.5 mm.

Bs100: Appears as bright ivory round colonies after incubation for 24 hours and grows to 2 mm in size.

Aeng17: After 24 hours of incubation, it appears as round or irregular colonies of red or ivory color and grows to 3mm in size.

Aeng18: After 24 hours of incubation, it appears as round or irregular colonies of red or ivory color and grows to 3mm in size.

Sp300: After 24 hours of incubation, it appears as round or irregular colonies of beige and brown color, growing to a size of 3 mm.

Tnh: After 40 hours of incubation, the translucent ivory surface appears as an uneven metallic colony and grows to 4 mm in size. Over time, the medium changes color to indigo blue.

Djhc: After 24 hours of cultivation, the ivory is a round, smooth surface with viscous, glossy colonies that grow to a size of 3 mm.

Pcpts: After 24 hours of incubation, they appear as beige and brown round or irregular colonies and grow to a size of 3 mm.

EBC106: Appears as a flat matte colony that grows to an uneven surface after culturing for 24 hours and grows to a size of 7 mm.

EBC107: After 40 hours of incubation, the semi-transparent ivory-colored surface appears as uneven irregular colonies and grows to a size of 4 mm.

Bs101: After culturing for 24 hours, it is yellowish ivory, rounded and surrounded by a rim, growing to a size of 5 mm. It is a gram-positive bacterium that has motility

W24: Pale yellow, round, glossy and convex colonies that grow to a size of 1.5 mm after 48 hours of incubation. Gram-negative bacteria are motility.

Nz2001: Appears as white matte colonies after 48 h of incubation and grows to 2.5 mm. During prolonged incubation, hyphae appear at the edges of the colony, and there is motility.

(B) Maconkey solid medium [MacConkey agar: 17g peptone, Proteose peptone 3g, 10g lactose, 1.5g Bil Salts No.3, 5g sodium chloride, 13.5g agar, 0.03g neutral red, 0.001 g of crystal violet, 1 liter of demineralized water, pH7.3 ~ 7.5]

Cy100 appears in light brown small colonies,

Ntar1 is transparent light brown with uneven surface,

Ntar2 is a small beige colony,

Ntar3 is a light brown transparent brown colony,

Gc300 is a dark pink colony with beige edges,

Gc500 is most of the beige and pink colonies,

Gc501 is a dark pink colony with beige edges.

Aeng17 and Aeng18 are dark red colonies,

Sp300 is a beige colony

Tnh is a dark khaki colony,

Djhc is pink in the center of the colony and beige at the edges,

Pcpts appear in light khaki

EBC107 appears as a very pale pink colony,

Nz2001 appears as a pink beige colony,

Cy101, Cy102, Cy103, Cy104, Cy105, Cy106, Cy107, Bs100, EBC106, Bs101, W24 strains do not show colonies even after 48 hours of incubation.

(C) Desoxycholate agar [Desoxycholate agar: Protez peptone 10g, lactose 10g, sodium desoxycholate 0.5g, sodium chloride 5g, sodium citrate 2g, agar 15g, neutral red 0.03g, deionized water 1 liter, pH7.3 ... 7.5] colony color after 48 h incubation

Cy101, Cy102, Cy103, Cy104, Cy105, Cy106, Cy107, Bs100, EBC106, Bs101, 24 strains do not show colonies even after 48 hours of culture.

Cy100 is a pale pink colony that looks like a flower

Ntar1, Ntar2, and Ntar3 are transparent orange colonies,

Gc300 and Gc501 are dark pink colonies with beige edges,

Gc500 is a light reddish beige colony,

Aeng17 and Aeng18 are dark red colonies,

Sp300 is a transparent light brown colony,

Tnh is a light brown colony metallic,

Djhc is a mix of pink and beige

Pcpts are transparent brown colonies,

EBC107 appeared as a yellow colony.

Nz2001 appeared as a red beige colony.

Bs101, sulfur strain Nz2001, and oil degradation strain W24, which decompose tar, were identified as unidentified strains because it was difficult to identify by the present method.

5. Scope of Environmental Hormone Degradation in Individual Strains of NBC2000

1) Range of environmental hormone degradation of each isolate

PCBs, dioxins, PCE, toluene and taric acid are measured by the bacterial community constituent bacteria under optimal conditions in the laboratory, and sulfur and PCP are average values measured at individual strain levels.

Pseudomonas sp. Cy100: 700 ppm PCBs;

Serratia sp. Aeng18: PCP 500 ppm, taric acid, dioxin 100 ng / kg;

Serratia sp. Ntar2: taric acid;

Pseudomonas sp. Djhc: 1000 ppm PCP, 300 ng / kg dioxin;

Pseudomonas sp. Ntar 3: taric acid;

Pseudomonas sp. EBC107: 700 ppm PCBs, 100 ppm PCP, dioxin 100 ng / kg, PCE 50,000 mg / kg;

Pseudomonas sp. Tnh: 700 ppm PCBs, 500 ppm PCP, dioxin 300 ng / kg, PCE 50,000 mg / kg, taric acid;

Aeromonas sp. Aeng17: taric acid, PCP 100 ppm, dioxin 50 ng / kg;

Pseudomonas sp. Pcpts: PCP 1,000 ppm, dioxin 500 ng / kg;

Stenotrophomonas sp. Ntar1: Taric acid;

Pseudomonas sp. Sp300: taric acid, PCP 700 ppm, dioxin 100 ng / kg;

Chryseomonas sp. Gc501: PCP 500 ppm, dioxin 100 ng / kg;

Chryseomonas sp. Gc500: PCP 500 ppm, dioxin 100 ng / kg;

Chryseomonas sp. Gc300: PCP 300 ppm, dioxin 100 ng / kg;

Brevundimonas sp. Cy101: PCBs 150 ppm;

Brebundimonas sp. Cy102: PCBs 150 ppm;

Brevundimonas sp. Cy103: 700 ppm PCBs;

Bacillus sp. Bs100: Taric acid;

Bacillus sp. Cy104: PCBs 800 ppm;

Bacillus sp. Cy105: 700 ppm PCBs;

Bacillus sp. Cy106: 1,000 ppm PCBs;

Bacillus sp. Cy107: 150 ppm PCBs;

Bacillus sp. EBC106: 700 ppm PCBs, 1,000 ppm PCP, dioxin 300 ng / kg, taric acid, PCE 50,000 mg / kg, toluene 50,000 mg / kg;

Gram positive bacterium Bs101: taric acid;

Gram negative bacteria W24: 100 ppm TPH, 100 ppm toluene;

Sulfur Strain Nz2001: Sulphur, Taric Acid, Dioxin 50 ng / kg

Isolation, Identification and Activity of EBC1000 Bacterial Communities

Hereinafter will be described in detail the isolation, identification and activity of the EBC1000 bacterial community according to the present invention.

1. Isolation of a Novel Bacterial Community That Can Decompose Hardly Degradable Toxic Wastes

(1) separating 40 kinds of microorganisms by shaking culture of samples collected from the soil and wastewater, such as Ulsan Industrial Complex, in a liquid medium mixed with difficult-to-dissolve wastewater

1g of soil collected from Ulsan Industrial Complex, 10ml of wastewater, waste acid, waste alkaline wastewater liquid medium (K ₂ HPO ₄ 0.65g, KH ₂ PO ₄ 0.17g, MgSO ₄ 0.1g, NaNO ₃ 0.5g and pharmaceutical industry A medium formed by diluting 10% of waste acid alkaline wastes containing hardly decomposable chemicals from waste and petrochemical wastes in 1l of deionized water, pH 0 ~ 14) and chlorine compound liquid medium (K ₂ HPO ₄ 0.65g , KH ₂ PO ₄ 0.17g, MgSO ₄ 0.1g, NaNO ₃ 0.5g and 50 ppm of pentachlorophenol (PCP) dissolved in 1l of deionized water, pH 7.2) were inoculated sequentially and 20 ~ 30 Shake culture was carried out for 5 days or more.

Take 1 ml of the shake culture solution, waste acid, waste alkali solid medium (waste acid, waste alkali, medium formed by adding only 1.5% of agar (agar) to medium, pH neutralization) and chlorine compound solid medium (BTB 20 in chlorine compound liquid medium) ㎎, medium formed by adding 1.5% agar) was sequentially inoculated and incubated at 20-30 ° C for 3-10 days.

Each colony was purified by re-inoculation on the same medium as above, and then shaken and cultured, and 40 kinds of useful bacteria having separate colonies of different shapes and the same colonies when subcultured were isolated.

(2) Separating 9 strong bacteria by culturing the bacteria isolated in step (1) in a medium gradually increasing the concentration of the hardly degradable waste liquid

Bacteria isolated in step (1) were used for pharmaceutical wastes and petrochemical wastes. Inoculated sequentially in each of the minimum medium was added and repeated in the same manner as in step (1), nine species were isolated according to colony type mainly viable strain at a higher concentration.

The nine bacteria obtained here were composed of one community and named EBC1000, and each bacteria constituting the bacterial community was named EBC100, 101, 103, 104, 105, 106, 107, 108 and 109, respectively.

The bacterial community EBC1000 according to the present invention was deposited internationally with the accession number KCTC 0652 BP at the Genetic Resource Center in Korea Biotechnology Research Institute on August 12, 1999.

2. Establish Optimal Conditions for Growth of Bacterial Community EBC1000

PH 5-8 at Luria-Bertani nutrient medium (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 10 g of NaCl / l of demineralized water), temperature 25-35 ℃, shaking (shaking) 50 ~ 100rpm per minute incubation for 24 to 48 hours shows the optimum growth, even in subcultures grow well under the same conditions.

3. Identification of Bacterial Community EBC1000

EBC 100, 101, 103, 104, 105, 106, 107, 108, and 109 strains of the bacterial community EBC1000 were mixed with Gram-negative bacteria and Gram-positive bacteria. After 24 hours of incubation in LB solid medium, EBC100 is a circular colony with a diameter of 1 mm, EBC101 is a large round about 2 mm, EBC103 is a thick round about 2 mm, EBC104 and EBC105 are a small round about 0.5 mm, and EBC106 is 3 mm EBC107 is about 1.2mm brown colony, EBC108 is about 1mm yellow colony, EBC109 is about 2mm double circle. Nine strains are aerobic and breathable strains, and EBC100, 101, 103, 106, 107, and 108 have strong viability even under strong acid (pH3 ~ 4) and strong alkali (pH9 ~ 11) conditions, and EBC104, 105, and 109 grow. This was slow. Motility is shown in EBC100, 104, 105, and 109.

Looking at the genus of the strains that make up the bacterial community EBC1000, EBC100, 101 and 103 belong to the genus Klebsiella, EBC105 belong to the genus Providencia, EBC104 and EBC109 belong to the genus Escherichia, EBC106 belong to the genus Bacillus, EBC107 is Gram-negative bacteria and EBC108 is Gram-positive bacteria.

To summarize the characteristics of each strain as described above are shown in Table 6 and Table 7.

sample		EBC 100	EBC 101	EBC 103
Gram strain	-	-	-
Catalase	+
Oxidase	-
Urease	+
Citrase utilizat.	+
Glucose utilizat.	+
V-P test	+
Lysine decarboxylase	+
Ornithine decarboxylase	-

sample		EBC 100	EBC 101	EBC 103
Inositol	+		+
Arabinos	+	+	+
Mannitol	+	+	+
Rhamnos	+	+	+
Glucose	+	+	+
Sorbitol	+	+	+
α-cyclodextrin	-	-	-
dextrin	+	+	-
Glycogen			-
Adonitol	+	+
D-Arabitol		+	+
Cellobiose		+	+
D-fructose	+	+	+
L-fucose	+	+	+
D-galactose	+	+	+
α-lactose			-
Maltose		+	+
D-Raffinose		+	+
D-trehalose	+	+	+
Methyl-pyruvate	-	+	+
Citric acid	+	+	+
Formic acid			+
Malonic acid	-	-
Succinic acid			+
D-alanine			+
L-alanine	+		+
L-glutamic acid		+	+
L-serine	+	+	+
D, L-lactic acid	+	+	+
motility	+	+	+
D-Mannose	+	+	+

sample	EBC 104	EBC 105	EBC 106
Gram strain	-	-	-
ONPG	+	-	+
Arginine	-	-	-
Lysine	+	-	-
Ornithine	-	-	-
Sodium citrate	-	-	-
Sodium thiosulfate	+	-	+
Urea	-	+	-
Tryptophan	-	+	-
Indole	+	+	+
Pyruvate creatine sodium	-	-	-
Kohn Charcoal Gelatin	-	-	-
Glucose, potassium nitrate	+	+	+
Manantiol	+	+	+
Inositol	-	-	-
Rhamnos	+	-	+
Sucrose	+	+	+
Melibiose	-	-	-
Amigdalin	-	-	-
Arabinos	-	-	-
Oxidase	+	-	+
Reduction of Nitrate to Nitrite	-	-	-
Reduction of Nitrate to N 2 Gas	+	+	+
Mobility	-	-	-
Oxidation of glucose	+	+	+
Fermentation of glucose	+	+	+
Antibiotic resistance	+	-	+
	Km R	Km S	Km S
	Ap S	Ap R	Ap R
	Tc S	Tc R	Tc R

Meanwhile, fatty acid methyl esters (FAMEs) of each strain were analyzed by gas chromatography.

Hewlett Packard series II Gas Chromatograph model 5890A; Microbial ID. Inc., Delaware, USA was used for the analysis of FAMEs.The separation column was 25m × 0.22mm × 0.33㎛ methylphenylsilicone. This fused capillary column (HP 19091B-102) was used.

In the gas chromatography, the carrier gas is hydrogen, the column head pressure is 10 psi, the split ratio is 100: 1, and the split vent is 50 ml / min, Septum Purge is 5 ml / min, FID hydrogen is 30 ml / min, FID nitrogen is 30 ml / min, FID air is 400 ml / min, initial temperature is 170 ° C., The program rate is 5 ° C./min, the final temperature is 270 ° C., the FID temperature is 300 ° C., the injection port is 250 ° C. and the injection volume is 2 μl.

The FAMEs graph used Microbial Identification System Software (Microbial ID, Inc., Delaware, USA) and peak identification and stagnation compared to a standard calibration mixture (Microbial ID, Inc., Delaware, USA). The time, peak area, and peak percentage were obtained.

As a result of analysis of FAMEs for each strain of EBC100, 101 and 103, the cellular fatty acids composition of EBC100 was C12: 0, C14: 0, C16: 0, C16: 1, C17: 0 cyclo, C14: 0 3OH, EBC101 is C12: 0, C14: 0, C15: 0, C16: 0, C17: 0 cyclo, C14: 0 3OH. EBC103 was found to be C14: 0, C15: 0, C16: 0, C17: 0 cyclo, C14: 0 3OH.

4. Isolation of each bacteria that make up the bacterial community EBC1000

The method of separating individual strains from bacterial community EBC1000 is as follows.

EBC106 (genus Bacillus), EBC107 (Gram-negative bacteria) and EBC108 (Gram-positive bacteria) were identified as Luria-Bertan agar (10 g of Bacterium-Tryptone, 5 g of Bacterium-Yeast Extract, 10 g of NaCl, 1.5% of agar, 950 ml of demineralized water) When cultured in nutrient solid medium of EBC106, the typical Bacillus-shaped thick wrinkles, EBC107 can be distinguished by the morphological features of brown colony, EBC108 is yellow colony.

Other strains include Desoxycholate agar; 10 g of bactopeptone, 10 g of bactolactose, 1 g of sodium deoxyoxylate, 5 g of sodium chloride, 2 g of dipotassium, 1 g of iron citrate, 1 g of sodium citrate, 15 g of bacto agar, neutral The colony morphology of each strain can be confirmed by diluting the bacterial community EBC1000 in 0.03 g of red red / deionized water 1-Difco manual, 1984]. After incubation in desoxycholate agar for 24 hours, EBC100, 101, and 103 show reddish white viscosities and slight differences in size. EBC104 is red and white and viscous, EBC105 is brown, EBC106, 107 is light brown, EBC108 is colorless, and EBC109 is red (Dictionary of Microbiology and Molecular Biology, 2nd, Paul Singleton Diana Sainsbury 1987).

5. Characteristics of Bacterial Community EBC1000

(1) Degradation rate of sodium alkylarylnaphthalene sulfonate (Tamol-SN) by bacterial community EBC1000

Tamol-SN at 500, 1000, 2000, 4000 ppm in a minimum medium (pH 7.2) made by dissolving 0.065 g of K ₂ HPO ₄ , 0.017 g of KH ₂ PO ₄ , 0.1 g of MgSO ₄ , and 0.5 g of NaNO _{3 in} 1 L of demineralized water. After gradually increasing the dose and inoculating the bacterial community EBC1000, the absorbance and the concentration of Tamol were measured over time.

Tamol-SN (ppm)	Degradation rate (mg / l / h)
500	1.3
1000	3.8
2000	5.2
4000	4.0

As shown in Table 9, the degradation rate of tamol per hour was 1.3 (mg / l / h) at 500 ppm and 4.0 (mg / l / h) at 4000 ppm. (2) Bacterial Community EBC1000 Degradation Rate of Digested Phenol Compounds (PCP) by

After dissolving BTB 20mg in 1L of demineralized water under the minimum liquid medium conditions as shown in (1), the contaminated digestive phenolic compound (PCP) was administered as shown in Table 4, and the inoculation of bacterial community EBC1000 was followed by absorbance and contaminated digestive phenolic compound over time. The concentration of (PCP) was measured. The results are shown in Table 10.

Digestive Phenolic Compounds (PCP) (ppm)	Degradation rate (mg / l / h)
200	0.9
500	6.5
1000	5.0
2000	4.5

Degradation Characteristics of Environmental Hormone by Combination of Bacterial Community NBC2000 and EBC1000

Although the resolution of individual strains is limited, treatment of the bacterial community of the whole NBC2000 or EBC1000 or each combination of its constituent strains in a sample or contaminated environment results in a more extensive and efficient degradation effect. This is a synergistic effect of emergence due to mutual cooperation as a bacterial community rather than the function of individual strains constituting NBC2000 and EBC1000. None of the strains can degrade complex PCBs with a single strain, but depending on the combination of each strain of NBC2000 and each strain of EBC1000 (KCTC0652 BP), the strains can be effectively complemented by complementary strains and exchange of genes in the community. Will be displayed. In order to prove this, the inventor performed the following experiment.

Hereinafter, the present invention will be described in detail based on the following experimental examples, but the present invention is not limited thereto.

Determination of PCBs Degradation Effect in Insulating Oil of 12 Mixed Bacterial Communities Selected from NBC2000 Bacteria and EBC1000 Bacteria

Verse 1. Design of PCBs Decomposition Reactor

1. Requirements and selection of reactor

The reactor design is a decomposition reaction tank made of strong glass material that can see the internal reaction process from the outside, and is designed in such a way as to control the inlet and the outlet through equipping facilities and equipment for temperature, pH, oxygen, and agitation.

In order to select Liflus GX and Liflus GR models of fermenters from Biotron, a domestic company, two main reactors (GX) and two preliminary reactors (GR) were installed, and experimental reactors (flasks) needed for studies such as preliminary experiments and reproducibility. And incubators, etc.) were installed and operated. In the case of the main reactor (Liflus GX), the stirring speed, temperature, pH and DO measurement, air injection amount setting, etc. can be periodically performed, and data on conditions and changes can be periodically stored and acquired through a computer. The preliminary reactor (Liflus GR) allowed only the setting of temperature and stirring speed (11).

2. Composition of reactor and correlation with experiment

end. Outline of reactor

A reaction tank is a device that incubates microorganisms in insulating oil containing PCBs and feeds PCBs and other nutrients to reduce the concentration of PCBs according to the growth of microorganisms. It was designed in the same form as a fermentation tank to grow microorganisms.

These reactors basically consist of PCBs decomposition strain, PCBs containing insulating oil, and agitator for effective mixing of injected air, aeration device for air injection, and cooling and heat transfer device for temperature control. It is composed of dissolved oxygen (DO) control (measurement) device and pH control (measurement) device to control.

The reason why such various kinds of control devices are installed in the reactor is to provide the strains with an effective degradation environment of the PCBs of interest, so as to obtain the maximum degradation efficiency within a given time.

I. Main body of reactor

The reactor was designed in consideration of problems such as heat resistance and other chemical corrosiveness in the biodegradation experiments with microbial strains and insulating oil containing PCBs.

The main body of the reactor consists mainly of stainless steel STS 316 and STS 304. It is resistant to corrosion, does not rust and minimizes reactivity with other materials, making it easy for durability and stability. Among them, especially in the case of STS 316, it is used for the inside of the medium or the water-contacting part, and STS 304 is mainly used for other external places.

STS 304 and 316 are different depending on the content of chromium and nickel, and 316 has a relatively weak strength but is known to have excellent chemical resistance and corrosion resistance.

The glass material is made of borosilicate, borosilicate glass instead of silicic acid. It contains at least 5% of boric acid, and the expansion coefficient decreases by adding boron, which increases chemical resistance, especially acid resistance and weather resistance. It is characterized by abundant impact properties. Used as glass for chemical resistant heat-resistant container.

All. pH measuring device

Looking at the environmental factors affecting the activity of the microorganisms, that is, the growth rate, the first is to find the medium environment in which the microorganisms are growing. However, as important as the medium environment are factors such as temperature, pH and dissolved oxygen.

The effect of pH on the growth of microorganisms is just as important as the effect of temperature. Therefore, maintenance is also required along with periodic pH measurements.

As the pH sensor, a glass electrode type sensor is used, and the measuring principle uses a phenomenon in which a potential difference proportional to the difference occurs when two kinds of solutions having different pHs exist on both sides of the glass thin film. The pH sensor should be calibrated with pH standard solution before entering the reaction experiment. Before calibration, organic or inorganic matters on the surface of the electrode should be removed, and it should be calibrated with 3-point calibration method to calibrate all three pH4, 7, 10.

la. DO measuring device

Oxygen is one of the most important factors in the growth of aerobic microorganisms. In general, in the culture of microorganisms, oxygen is present in the form of dissolved oxygen which is diffused into the culture medium from the air bubbles by aeration and agitation. Therefore, the growth of microorganisms for the decomposition of PCBs is greatly influenced by the dissolved oxygen concentration by aeration and agitation conditions. The dissolved oxygen concentration in the reactor is measured using a DO electrode. DO measuring electrode should be calibrated before use. After zero setting, saturation setting should be done.

DO electrodes should also be careful not to touch the bottom of the membrane, the oxygen-permeable membrane at the bottom of the electrode, and to avoid damage. In addition, if the membrane is blocked by foreign material, it should be cleaned carefully. The electrolyte solution inside should be replaced when the reaction is slow.

hemp. Agitator

Stirring device is very important, such as the oxygen supply necessary for the growth of microorganisms and the mixing of the insulating oil and PCBs containing PCBs to promote the movement of metabolites from the cells, and to help effectively cool the heat generated during the reaction process. The stirrer basically consists of a motor that generates rotational force, a stirring shaft that transmits this rotational force to an impellar, and a stirrer that actually causes stirring.

The stirring force of the stirrer must be driven by a motor installed outside the reactor, and in order to maintain the sterilization state of the reactor, the inside and outside of the reactor are blocked so that foreign substances do not flow from the outside. The stirrer is made of Teflon material.

bar. Thermostat

Microbial growth is the result of a series of chemical reactions, which means that the growth of microorganisms is greatly affected by temperature. In general, the reaction rate is increased about 2 times as the temperature rises by 10 ℃, the growth rate of the microorganism is observed to increase with increasing temperature. However, the width of the temperature at which the growth rate is increased is very narrowly observed.

This is because the growth of microorganisms is a biochemical reaction unlike general chemical reactions, so there is an optimum temperature for the reaction. The optimum temperature for microbial growth depends on the target microorganism.

Microorganisms are more sensitive to temperature than mortality than growth rates and require thorough control. Therefore, in the reactor, the cooling water for temperature control is supplied to the outer jacket of the reactor.

Underneath the reactor is a heating plate. This transfers heat into the reactor. In the temperature sensor, the temperature is measured and the temperature rises above the preset temperature, and the cooling water is circulated through the jacket to lower the temperature. When the temperature drops, the heating plate is operated to increase the temperature.

four. Chiller

The energy delivered to the stirrer during the operation of the reactor is transferred to the reactant to generate a large amount of heat, and the microorganisms dissipate a large amount of heat even during the growth, which requires a cooling device to effectively remove the generated heat. Done.

In this reactor, the coolant flows through the jacket inside the reactor and is cooled.

Ah. Other device

In addition, as an ancillary device for bioprocessing, there is an Anti Foam Sensor that removes bubbles, an Air pump for injecting air, an Air flow controller for adjusting the air volume, and a condenser that cools and returns moisture in the generated gas.

3. Establishment of reactor and establishment of optimum conditions

The reactor was installed in the laboratory of Chuncheon Bio Industry Promotion Institute. The inside of the laboratory is capable of maintaining and managing temperature and humidity, supplying coolant smoothly, and equipped with various laboratory equipment and safety protection equipment necessary for the experiment. Since this experiment requires a long-term reaction test, the preliminary operation and safety inspection of the reactor were carried out according to the procedure before entering the experiment. The preliminary operation was performed by putting water in the GX and GR reactors and washing the electrical safety test and equipment error test.

GX connects computer and communication line to acquire and test data. As a result of the preliminary operation for 4 days, it discovered electrical instability and equipment error and installed the single power supply electrically.In terms of equipment, it was possible to stably install the reactor by replacing parts inside the reactor. Could have its own optimum conditions.

After the reactor was installed, the reactor was named and managed by GX-1, GX-2, GR-1, and GR-2, respectively. In addition, liquid waste collection boxes and solid waste collection boxes were installed to prevent leakage of each waste and collect and store it.

Section 2 Analysis Methods and Selection of Analysis Institutions

1. Selection of analytical methods

After the PCBs-containing insulating oil was mixed with the microorganisms, the analysis was commissioned to an authorized analytical institution to confirm the decrease in PCBs concentration in the insulating oil through stirring and culturing.

Analysis of PCBs is largely shown in Table 11 below.

Analysis method	Sample amount (ml)	Analysis unit price (KRW 10,000)	Analysis period (days)	Contents
Simple analysis method (L2000DX method)	40	About 4 ~ 5	Immediately	-Immediate measurement-Precision analysis if more than 5ppm-Only above and below the standard
Precision Analysis GC-ECD Method	20	About 20	About 30	-Current standard certification method-Analysis value: Decimal point display
Improved GC-ECD Method	20	About 14	About 30	-Improvement of pretreatment method of precision analysis method-Analysis value: Integer display
HR-MS method	5 to 20	About 200 ~ 300	About 60	-Currently, 209 isomers cannot be analyzed.- 17 to 27 isomers are analyzed.

In this project, since we are trying to measure the resolution according to the change of PCBs concentration, not the designated waste standard, the simplified analysis method is not meaningful. It became. Therefore, the analysis was carried out using the HR-MS method for the concentration reduction of PCBs and the concentration change of the isomers of PCBs. However, the analysis of all samples is not a situation that can be analyzed by the HR-MS method, so that the correlation between the two methods by using the HR-MS method and the GC-ECD method can be analyzed and linked.

end. Precision analysis GC-ECD method (Gas Chromatography-Electron Capture Detector)

-It is used to check the decrease of PCBs concentration of microorganisms over time.

-Established and requested a sample analysis plan, but changed the plan to suit the situation inside the microorganism and reactor.

-The number of samples per time should be according to the number of reactors, but proportionally according to the situation, and corrected and supplemented according to the change of the sample.

I. HR-MS method (High Resolution-Mass Spectrometer)

-It is used to check the change of concentration according to each isomer of PCBs. (17 ~ 27 species)

-Established and requested a sample analysis plan, but changed the plan to suit the situation inside the microorganism and reactor.

-It was also requested to check the dioxin content of the final product (by-product).

2. Selection of analytical institutions

PCBs concentration analysis should be commissioned by a national accredited agency and technical cooperation (including consultation and evaluation) with the analytical organization should increase the reliability of the analysis and degradation process. Therefore, after analyzing the accredited certification bodies, information on each certification body was collected and an analysis institution was selected. The GC-ECD method is' Labfrontier 'and' Pohang Industrial Science Research Institute (RIST) 'and the HR-MS method is' Pohang Industrial Science Research Institute (RIST). 'Was selected.

Section 3 Sampling Insulating Oil Samples Containing PCBs

1. On-site sampling of insulating oil containing PCBs

On April 3, 2008, 10 l of insulating oil containing high concentration PCBs and 7 l of insulating oil containing low concentration PCBs were collected as a field sample embedded in a commercial transformer with the cooperation of Kangwon Branch of KEPCO.

Each sample was collected by KEPCO Kangwon Branch from the first Chonbuk National University Chemical Safety Management Research Center. The samples were selected from 339.58 ppm of insulating oil containing high concentration PCBs and 60.54 ppm of insulating oil containing low concentration PCBs. At the time of collection, various protective equipments were worn and collected according to the guidance of the person in charge. The prepared brown glass sample bottles were wiped about 3 times with insulating oil to be collected, and then samples were taken to be representative. Various wastes generated during the storage were put in plastic, sealed and stored. After collecting the sample, various items such as name, number, place, date, collector, method, and quantity of the sample were recorded in the collected sample bottle.

end. Harvesting tools

When sampling the insulating oil containing PCBs, various safety protection items were worn for safe collection and various sampling tools were used to prevent leakage of the insulating oil containing PCBs. The collection tools were disposed of after use and disposed of in plastic to be sealed and stored.

I. Insulation oil storage and handling with PCBs

The collected insulating oil sample was sealed and stored in the dark. In principle, the sample should be stored in a safe place without direct sunlight of 0 ~ 4, but due to the characteristics of the experiment, it should be stored in consideration of safety avoiding direct sunlight at room temperature in order to control it in the same way as the field sample. In the management of samples, it was necessary to check for leakage and stability at a designated time every day, and thoroughly managed to prevent contact with outsiders. In addition, it was to record the abnormality in the sample record management ledger, and if any abnormality occurred, immediately report and take measures according to the procedure.

Section 4 Cultivation of PCBs Degrading Strains

1. Selection of strains

In selecting strains for degradation of PCBs in insulating oil, a microbial community selected from the NBC2000 bacterial community and the EBC1000 bacterial community was used. Specifically, 12 strains of Cy106, Cy100, Cy101, Cy102, Cy103, Cy104, Cy107, Tnh, EBC106, W-24, EBC107 and NZ2001 were included.

2. Cultivation of Strains

The medium used for strain culture was Luria-Bertani nutrient medium (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 10 g of sodium chloride (NaCl)). / 1 liter of demineralized water, and incubated at an optimum temperature of incubator temperature of 30 ° C. and shaking at 115 rpm.

Section 5 Bioresolution Reproducibility Test of Insulating Oil Containing PCBs

1.Reproduction test of biodegradation of insulating oil containing PCBs

Selection of sample analysis method and analysis institute, formation of optimum conditions through preliminary operation of reactor, and after completion of field sampling and culture of strain containing PCBs, inserting PCBs containing insulating oil into reactor and adding microorganisms Bioresolution reproducibility experiments were conducted. Procedures for the operation and use of the reactor were carried out according to the procedure.

Before putting the PCBs-containing insulating oil sample into the reactor, wear protective equipment and pre-start, clean, and dry the main reactors (GX-1, GX-2) after the high concentration of PCBs-containing insulating oil (GX-1) and low concentration. After carefully inserting 4 liters of insulating oil (GX-2) containing PCBs, 20 ml of GC-ECD analysis oil was sampled from each insulating oil, and accessories such as pH measuring device and DO measuring device were calibrated and installed. The total volume of the reactor (GX) is 8l, and the working volume (about 70% of the total volume) is about 5 ~ 6l, so 4l of insulating oil was added, and the preliminary reactor (GR) had a total volume of 5l, and the working volume was Since it is about 3 ~ 4l, 3l of insulating oil was added.

2. Setting Optimum Condition of Reactor

In the biodegradation experiment of PCBs-containing insulating oil using microorganisms, the conditions of the reactor were set to a temperature of 25 ° C. and a stirring speed of 200 rpm as the optimum conditions set forth in the existing patent. In addition, the interval of data acquisition was set in units of 10 minutes so that data such as temperature, pH, DO, and stirring speed could be acquired. The injection of air was set at 0.5-1 l / min through the air pump roll.

Section 6 Sampling and Requesting Analysis

1. Sample Collection Method

In order to request for analysis by sampling the sample inside the reactor according to the procedure of the waste process test method, samples were collected by using disposable protective pipettes. At the time of sampling, the pipette was washed once with insulating oil inside the reaction tank so as to have representativeness of the sample.

The sampling procedure was carried out according to the procedure, and the waste generated after sampling was processed and stored in the waste collection box.

2. Request for analysis

The sample name, collection date, number, collector, collection place, and sample volume were recorded in the sample collection bottle and collected in the sample analysis request book. In addition, in order to prevent breakage and leakage during the request for analysis, the sample bottle was firmly fixed using styrofoam, and placed in an ice box containing an ice pack. In addition, it was fixed directly so as not to leak from the inside and then directly requested.

SECTION 7 Hazard Assessment and Other Experiments

1.Measurement of reactant gas

end. Outline of Reactor Tank Gas Measurement

The by-products generated during the experiments to prove the efficiency of biodegradation of PCBs-contaminated insulating oil using microbial purification technology for PCBs contained in insulating oil are considered as liquid by-products and gaseous by-products. Reactor-generated gas measurement experiments were conducted on gaseous components to evaluate gaseous by-products and their impact on the environment.

For experiments with microbial PCBs, air is supplied into the reactor and released through the sparger through the condenser. Therefore, the gas components emitted by the mechanism by the microorganism in the reaction tank was to be measured.

The measurement items are volatile organic carbons (VOCs) that can be volatilized in the insulating oil itself, chlorine behavior through decomposition of PCBs, and chlorine gas (Cl ₂ ) which can estimate the generation of harmful gas, metabolism of carbon and carbon Gas components were measured for three items of carbon dioxide (CO ₂ ), which can be seen in the behavior.

Currently, there are limitations on the items that can be measured by precision analysis equipment (GC-MS, etc.), and it is difficult to measure the continuous gas generation.

end. VOCs measuring equipment is shown in Table 12 below.

EntryRAE (VOCs Measurement)
Measuring range	0 to 999 ppm
Resolution	1 ppm
Response time	10sec
sensor	Photo-ionization sensor
Date of Calibration	2008. 08. 26
Calibration organization	RAE KOREA
Serial NO.	180-000833

I. Cl ₂ measuring equipment is shown in Table 13 below.

VRAE (Cl 2 measurement)
Measuring range	0-10 ppm
Resolution	0.1 ppm
Response time	60sec
Detection method	electrochemical for toxic gases
	catalytic for combustible gases
Date of Calibration	2008. 08. 26
Calibration organization	RAE KOREA
Serial NO.	170-102277

All. CO ₂ measuring equipment is shown in Table 14 below.

MultiRAE-IR (CO 2 measurement)
Measuring range	0 ~ 20000ppm
Resolution	10 ppm
Response time	30sec
sensor	NDIR
Date of Calibration	2008. 08. 26
Calibration organization	RAE KOREA
Serial NO.	080-901072

I. Experiment method

The calibrated gas measurement equipment was prepared and one of the four lines of the reactor feedline was left open. If the line is not open, the pressure inside the reactor may be damaged due to the difference between the amount of air flowing into the reactor and the gas sampling rate of the gas measurement equipment, which may cause damage to the reactor or the gas measurement equipment. In the open part, the air inside the reactor is forced out or flows out due to the pressure. In the actual experiment, it was confirmed that when one line was left open, the sampling rate of the gas measuring device was smaller than the gas supply amount of the reaction tank so that the air inside the reaction vessel was released.

In addition, gas measurement equipment with a filter was connected to the sparger part at the end of the condenser, and the connection part was closed with paraffin tape to prevent gas leakage.

With reference to the reaction rate of each item, VOCs and Cl ₂ were measured for 1 hour per reactor, in the case of CO ₂ was measured continuously for 20 minutes and each result was recorded by hand.

Each reactor was measured by changing the items, and in the case of CO ₂ items, CO ₂ was measured for indoor and outdoor.

2. Dioxin Measurement

Dioxin analysis was commissioned on the sample inside the reactor as well as the measurement of emissions, which is an environmentally friendly requirement for biodegradation experiments of PCBs. Dioxin analysis was analyzed by HR-MS method, and commissioned after collection by the same method as the existing sampling in the reactor.

3. Check the total number of microorganism

After the microorganisms had sufficiently cultured 12 kinds of PCBs degradation strains in advance, the strains were identified by the CFU / ml colony counting method on a plate medium.

4. Other experiment

According to the degradation of PCBs, some microorganism strains were additionally inoculated, and GC-ECD analysis values by microorganisms were analyzed by analyzing insulating oils containing non-PCBs and non-PCBs insulating oils. The interference effect of was observed.

Section 8 Experimental Results and Discussion

1. Microbial Reactor Operation Data

Data such as temperature, pH change, DO change, and stirring speed appearing in the reaction tank (GX) were averaged for two weeks. Experimental conditions were maintained at pH 6 ~ 8, temperature 24 ~ 25 ℃, agitator rotation speed 200RPM, oxygen concentration of the reactor was maintained at 80 ~ 90% of the initial level (Table 15 and Figure 1).

	High Concentration (GX-1)				Low concentration (GX-2)
parking	pH	Temperature (℃)	DO (%)	RPM	pH	Temperature (℃)	DO (%)	RPM
Week 1 & 2	8.21	24.02	88.95	199.77	7.82	25.02	95.14	200.00
Weeks 3 and 4	5.36	25.72	90.21	199.90	6.95	25.74	94.04	199.90
Week 5,6	6.16	25.69	85.25	200.00	6.69	25.77	89.45	199.90
Week 7, 8	6.27	25.66	81.36	200.00	6.43	25.81	89.35	200.00
Week 9,10	6.49	25.50	76.45	199.78	6.49	25.45	89.30	199.78
Week 11,12	6.95	25.64	73.76	200.00	6.37	25.72	93.12	200.00
Week 13,14	6.85	25.37	79.36	199.70	6.47	25.40	91.78	199.80
Week 15,16	7.10	25.54	81.81	200.00	6.85	25.18	89.07	200.00
Week 17,18	6.64	25.56	84.25	200.00	6.46	25.73	92.30	200.00
Week 19,20	6.57	25.48	73.90	200.00	6.56	25.69	93.30	200.00
Week 21,22	6.48	25.56	84.41	200.00	6.41	25.71	92.54	200.00
Week 23,24	6.03	25.72	77.24	200.00	6.44	25.77	93.06	200.00

1A to 1D are graphs of changes in pH, temperature, DO, and stirring speed of a reaction tank.

2. Changes in microbial growth over time in microbial reactors

end. Microbial Turbidity Changes in Reactors

After inserting the PCBs containing insulating oil into the reactor, the microbial turbidity was observed with the reaction time. Insulating oil containing PCBs showed yellow and transparent color, slight turbidity appeared in light yellow immediately after microorganism injection, and after about 70 days, microbial community was observed on the surface of the reactor, and after about 160 days, More turbidity was observed and more microbial communities were formed on the surface of the reactor.

Experimental results were the same as in Figs. 2a to 2d. 2A to 2D show microbial turbidity changes of reaction tanks over time.

I. Growth range of microbial strain

Early inoculating microorganisms essentially include Cy106, and microbial communities including Cy100, Cy101, Cy102, Cy103, Cy104, Cy107, Tnh, EBC106, W-24, EBC107, and NZ2001 up to 10 ⁹ CFU / ml for each strain. After incubation and inoculation, the colony counting CFU / ml on the plate medium showed an average of 5.8x10 ⁷ ～ 1.2x10 ⁸ / ml for the initial reaction, depending on the duration of the reactor (GX1, GX2, GR1) As shown in the figure, the number of microorganisms continued to grow up to a maximum of 6.2x10 ⁹ -1.5x10 ¹⁰ / ml on average (Table 14).

The average number of microorganisms in the reactor maintained 100-fold multiplication in terms of reproducibility of microbial biodegradation results of PCBs contaminated soil and PCBs insulated oil published in the data of Patents 10-588305, PCT / KR2005 / 001238, EP1745214. Resistance to PCBs and proliferation of microorganisms for biodegradation (FIGS. 2A-2D).

Table 16 below shows the increase in the number of microbial cells over the reaction time between the microorganism and the PCBs-containing insulating oil.

Inoculation (CFU / ml)	Initial reaction (CFU / ml)	In case of sustained reaction (CFU / ml)
10 9	5.8 × 10 7 ～ 1.2 × 10 8	6.2 × 10 9 to 1.5 × 10 10

3. PCBs Degradation Rate Analysis Results

end. GC-ECD Analysis Results

By requesting analysis of PCBs-containing insulating oil sample according to the microbial mixing reaction time, the results as shown in Table 17-18 were obtained. As a result of analysis, the PCBs concentration was increased and decreased with time, and the analysis results were different for each analytical institution, so the degradation rate of PCBs could not be confirmed by GC-ECD analysis.

Table 17 shows changes in PCBs concentration (main reactor-GX) over time.

Sampling date (D + elapsed date)	GX-1 (high concentration) (ppm)		GX-2 (low concentration) (ppm)
	Rapfrontier	RIST	Rapfrontier	RIST
04.22 (0)	415	-	79	-
04.24 (D + 2)	530	-	80	-
05.08 (D + 16)	415	-	51	-
05.26 (D + 34)	570	-	77	-
06.10 (D + 49)	520	-	71.5	-
06.24 (D + 63)	322.5	-	39	-
07.10 (D + 79)	410	511.98	37.5	44.52
07.17 (D + 86)	-	544.78	-	52.68
07.21 (D + 90)	160	-	51	-
07.23 (D + 92)	-	356.01	-	45.47
08.12 (D + 112)	-	388.19	-	56.24
09.09 (D + 140)	330		69	25.28
	(Middle Floor) 321	-	(Upper floor) 43	-
	-	-	(Middle floor) 40	-
09.16 (D + 147)	310	465.51	40	22.19

Table 18 shows changes in PCBs concentration (preliminary reactor-GR) over time.

Sampling date (D + elapsed date)	GR-1 (high concentration) (ppm)		GR-2 (ppm) (high concentration control)
	Rapfrontier	RIST	Rapfrontier	RIST
07.09 ((D + 1)	335	449.78	325	369.24
07.09 After mixing (D + 1)	385	495.05	-	-
07.17 (D + 9)	-	464.75	-	436.99
07.21 (D + 14)	370	-	270	-
07.23 (D + 16)	-	330.91	-	314.75
08.12 (D + 35)	-	364.58	-	394.42
09.09 (D + 63)	370	462.42	384	-
	316	-	-	-
	316	-	-	-
09.16 (D + 70)	310	480.14	295	-

As shown in Table 17-18, the rate of degradation of PCBs from the initial concentration to the final concentration varies considerably between analytical institutions, and it is assumed that the interference effect of microbial metabolites is likely. For example, when the microbial strain was added to the insulating oil containing PCBs, the PCBs concentration increased from 415ppm to 530ppm, and then increased to 6.5ppm after 14 days of injecting microorganisms into 0.7ppm of PCBs (MOC-1). (Table 19 below). Table 19 is a result of analyzing the results of the non-PCBs experiment by GC-ECD, the peak pattern according to each result is shown in Figures 4a to 4b.

Sample Name	Remarks	Concentration (ppm)	Analytical Institution
MOC-1	Non-PCBs insulating oil itself	0.7	Rapfrontier
MOM-1	Non-PCBs Insulating Oil + Microbial Culture	6.5

I. HR-MS analysis result

In the HR-MS analysis, the concentration of the insulating oil sample containing the high concentration PCBs itself, the microorganism reaction in the insulating oil containing the high concentration PCBs of the preliminary reactor 73 days, and the microbial reaction in the insulating oil containing the high concentration PCBs of the main reactor 150 days Samples were collected and analyzed three times by the Pohang Institute of Industrial Science (RIST).

The HR-MS analysis shown in Table 21 shows PCBs 118 (2,3 ', 4,4', 5-PeCB), PCBs 123 (2 ', 3,4,4', The concentration of 5-PeCB) isomers was increased by 2 to 5 times, but the low chlorine PCBs of 1 to 3 chloride PCBs were 100% decomposed. Most PCBs were degraded with an average of 60-100%, and the concentrations of coplanar PCBs with high toxic equivalent concentrations also tended to decrease.

HR-MS analysis showed that PCBs with 27 initial concentrations of about 24 ppm were 9.4 ppm, which was reduced by 60% after microbial degradation. Coplanar PCBs, 12 toxic PCBs, decreased from the initial 25,646 pg-TEQ / g to 1,526 pg-TEQ / g after microbial degradation. After 150 days, the toxic equivalent concentrations of the 12 Coplanar PCBs were reduced by approximately 94.04% (Table 20).

Table 20 shows changes in PCBs concentration over time.

Sample Name	Sampling date (D + elapsed date)	Contents	27 concentrations (degradation rate)	12 concentrations (degradation rate)	Toxicity equivalent concentration (pg-TEQ / g)
MHX-1	4.22	High concentration PCBs insulating oil	23.83 ppm	20.88 ppm	25646.14-
MHR-1	9.19 (D + 73)	High concentration GR-1	21.36 ppm (10.36%)	20.70 ppm (0.86%)	6036.69 (76.46%)
MHX-2	9.19 (D + 150)	High concentration GX-1	9.45 ppm (60.34%)	8.88 ppm (57.47%)	1526.46 (94.04%)

Table 21 shows the concentration analysis results for each isomer compared to the initial HR-MS method.

Total concentration (27 species)	Toxicity Equivalence (WHO 2005TEF)	Chlorine Substitution	MHX-1 (initial)	MHX-2 (D + 150)	Initial preparation
			Concentration (pg / g)	Concentration (pg / g)	Reduction Concentration (pg / g)	% Decomposition
PCB1		One	0.00	0.00	0.00	-
PCB3			8808.93	0.00	-8808.93	100.00
PCB4			0.00	0.00	0.00	-
PCB15		2	579684.54	0.00	-579684.54	100.00
PCB19		3	70475.86	0.00	-70475.86	100.00
PCB37			705234.91	0.00	-705234.91	100.00
PCB54		4	1955.85	1257.96	-697.89	35.68
PCB77	0.0001		192681.23	74356.47	-118324.76	61.41
PCB81	0.0003		80241.39	24905.52	-55335.87	68.96
PCB104		5	0.00	197.27	197.27	-
PCB105	0.00003		7018089.60	2704419.67	-4313669.93	61.47
PCB114	0.00003		692079.40	303527.80	-388551.60	56.14
PCB118	0.00003		164941.47	928454.26	+763512.79	-462.90
PCB123	0.00003		269106.64	981782.23	+712675.59	-264.83
PCB126	0.1		46757.38	12483.75	-34273.63	73.30
PCB155		6	0.00	0.00	0.00	-
PCB156	0.00003		6755316.41	2256769.35	-4498547.06	66.59
PCB157	0.00003		859659.60	307098.35	-552561.25	64.28
PCB167	0.00003		3081491.46	935468.30	-2146023.16	69.64
PCB169	0.03		677689.56	0.00	-677689.56	100.00
PCB188		7	9214.04	2830.38	-6383.66	69.28
PCB189	0.00003		1038437.83	354938.41	-683499.42	65.82
PCB202		8	661675.67	238378.85	-423296.82	63.97
PCB205			312425.50	113564.51	-198860.99	63.65
PCB206		9	489043.61	175489.76	-313553.85	64.12
PCB208			85839.30	31085.26	-54754.04	63.79
PCB209		10	34651.13	2606.98	-32044.15	92.48
Sum			23835501.31	9449615.08		60.35

All. Byproduct Hazard Assessment Results

Dioxin concentration of the liquid sample in the reaction tank was measured by the HR-MS instrumental analysis of the Pohang Institute of Science and Technology (Table 22).

In the microbial treatment of PCBs, 209 PCBs homologues can be easily converted into 209 dioxin forms due to their interaction with microbial metabolites when reacting with microorganisms in molecular structure. Representative toxic byproducts. In the experiment, the dioxin analysis of the sample inside the reactor showed a concentration of 0.5∼0.7ng-TEQ / g as shown in Table 20, which was lower than 3ng-TEQ / g, the legal limit of incineration ash. In addition, no harmful gases were detected in the experimentally measured emissions.

Table 22 shows the results of dioxin analysis in the reactor liquid sample.

Sample name (D + elapsed date)	Dioxin concentration (ng-TEQ / g)	Analytical Institution
GR-1 (D + 73)	0.697	RIST
GX-1 (D + 150)	0.517

Table 23 shows the measurement results of the reaction gas generated from the reactor.

division	Outdoor (ppm)	Laboratory (ppm)	Reactor (ppm)
CO2	496	700	713 ～ 844
Volatile Organic Carbons (VOCs)	-	-	N.D
Chlorine Gas (Cl2)	-	-	N.D

Section 9 Summary and Conclusion

-Biological treatment technology is applied to process high concentration (530ppm) and low concentration (80ppm) PCBs materials in insulating oil.

-The experiment was carried out using 17L of insulating oil (high concentration: 10L, low concentration: 7L) embedded in the pole transformer in KEPCO Kangwon branch.

The strains used in the experiments essentially include Cy106, using a mixed strain of Cy100, Cy101, Cy102, Cy103, Cy104, Cy107, Tnh, EBC106, W-24, EBC107 and NZ2001.

-Experimental conditions were pH 6-8, reactor temperature 24-25 ℃, stirring speed 200RPM and DO concentration kept saturated (80-90%).

-As a result of analysis by HR-MS, some PCBs concentration increased after 150 days of experiment, but TEQ toxicity of 12 coplanar-PCBs decreased by 94% (1526.46 pg-TEQ / g) and PCBs concentration decreased by 57%. Appeared. The degradation rate of 27 PCBs, including non-toxic PCBs isoforms, was reduced by 60% (Table 24).

Table 24 shows changes in PCBs concentrations (assay HR-MS) with treatment duration.

division	Initial (D + 0)	D + 150	% Decomposition
Total concentration (27 species) (ppm)	23.83	9.44	60.35
Total concentration (12 kinds) ※ (ppm)	20.88	8.88	57.47
Toxicity concentrations (12 species) (pg-TEQ / g)	25,646.14	1,526.46	94.05

Note) Analysis organization: Pohang Institute of Industrial Science

HR-MS Model: Thermo Trace GC Ultra-DFS

※: Coplanar-PCBs

-As a result of analyzing PCBs concentration change by GC-ECD method, the final concentration was decreased compared to the initial concentration.

The population of PCBs degrading microorganisms increased from 10 ⁸ CFU / ml to 10 ¹⁰ CFU / ml with the reaction time. This suggests that the degradation of PCBs by microorganisms is due to the catalytic reaction of enzymes that occur in microbial metabolism, and the active growth of microorganisms can be evaluated as an objective indicator that the degradation of PCBs is actively progressing.

-In the presence of harmful by-products from biological treatment of PCBs using microorganisms, dioxin components in liquid samples were evaluated, and 0.697ng-TEQ / g and 0.517ng-TEQ / g in GR-1 and GX-1 reactors, respectively. , Which is less than 25% of the legal emission limit of 3 ng-TEQ / g.

Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

[Accession number]

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC10623BP

Deposit date: 20040416

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC0652BP

Trust Date: 19990811

[Revision 13.02.2019 under Rule 26]

[Revision 13.02.2019 under Rule 26]

Claims (13)

1. In the PCBs processing method of insulating oil containing Polychlorinated Biphenyls (PCBs),

   Administering a PCBs treated bacterial community,

   The PCBs treated bacterial community is Bacillus sp. Cy106 strain, Pseudomonas sp. Cy100 strain, Brebundimonas vesicularis Cy101 selected from NBC2000 bacterial community with accession number KCTC 10623 BP. Strain, Brebundimonas vesicularis Cy102 strain, Brebunundimonasvesicularis Cy103 strain, Bacillus stearothermophilus Cy104 strain, Bacillus sp. Cy107 strain, Capital One or more of Pseudomonas aeruginosa Tnh strain, W24 strain which is an oil degradation gram negative bacterium and Nz2001 strain which is a sulfur strain; At least one of Bacillus cereus EBC106 strain and Pseudomonas sp. EBC107 strain selected from EBC1000 bacterial community with an accession number of KCTC 0652 BP; .
2. The method of claim 1,

   The PCBs treated bacterial community is Bacillus sp. Cy106 strain, Pseudomonas sp. Cy100 strain, Brebundimonas vesicularis Cy101 strain, Brebundimonas vesicularis (Brevundimonas vesicularis) Cy102 strain, Brebundimonas vesicularis Cy103 strain, Bacillus stearothermophilus Cy104 strain, Bacillus sp. Cy107 strain, Pseudomonas aeruginosa Tnh strain, oil A method for processing PCBs of insulating oil, comprising a W24 strain, a degraded Gram negative bacterium, a Nz2001 strain, a sulfur strain, a Bacillus cereus EBC106 strain, and a Pseudomonas sp. EBC107 strain.
3. The method of claim 1,

   The PCBs treatment bacterial community is to be administered after culturing the cell number 10 ⁹ CFU / ml or more, PCBs treatment method of insulating oil.
4. The method of claim 3, wherein

   The culture is incubated for 4 to 5 days with bacto-yeast extract in Luria-Bertani nutrient medium, PCBs treatment method of insulating oil.
5. The method of claim 1,

   The method of treating the PCBs of the insulating oil further comprises biodegrading after administering the PCBs treatment bacterial community, PCBs processing method of insulating oil.
6. The method of claim 5,

   The biodegradation step

   Temperature conditions of 24 ° C. to 26 ° C .;

   pH conditions of pH 5.3 to 8.5; And

   DO conditions of dissolved oxygen of 73% to 95%; Decomposing while satisfying any one or more of the conditions, Method for processing PCBs of insulating oil.
7. The method of claim 6,

   The biodegradation step

   Temperature conditions of 24 ° C. to 25 ° C .;

   pH conditions of pH 6-8; And

   DO conditions with a dissolved oxygen concentration of 80% to 90%; Decomposing while satisfying any one or more of the conditions, Method for processing PCBs of insulating oil.
8. The method of claim 6,

   The step of biodegradation further comprises the conditions of stirring using a stirrer with a rotational speed of 150 rpm to 250 rpm during biodegradation, PCBs processing method of insulating oil.
9. The method of claim 5,

   The biodegrading step has a biodegradation time of more than 50 days, PCBs processing method of insulating oil.
10. The method of claim 9,

    The biodegrading step has a biodegradation time of 70 days to 200 days, PCBs processing method of insulating oil.
11. The method of claim 1,

    The insulating oil is an insulating oil contained in the transformer, PCBs processing method of the insulating oil.
12. The method of claim 1,

    The method reduces the concentration of PCBs in insulating oil to 50 ppm or less.
13. The method of claim 11,

    Wherein the method reduces the concentration of PCBs in insulating oil to 50 ppm or less and at the same time generates dioxin below a concentration of 3 ng-TEQ / g.

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