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WO2019151799A1 - Procédé de traitement de biphényles poly-chlorés à l'aide de micro-organismes - Google Patents

Procédé de traitement de biphényles poly-chlorés à l'aide de micro-organismes Download PDF

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WO2019151799A1
WO2019151799A1 PCT/KR2019/001360 KR2019001360W WO2019151799A1 WO 2019151799 A1 WO2019151799 A1 WO 2019151799A1 KR 2019001360 W KR2019001360 W KR 2019001360W WO 2019151799 A1 WO2019151799 A1 WO 2019151799A1
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pcbs
strain
insulating oil
concentration
conditions
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이성기
이성호
송미경
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    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10G—CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION; RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
    • C10G32/00—Refining of hydrocarbon oils by electric or magnetic means, by irradiation, or by using microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10G—CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION; RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
    • C10G2300/00—Aspects relating to hydrocarbon processing covered by groups C10G1/00 - C10G99/00
    • C10G2300/10—Feedstock materials
    • C10G2300/1037—Hydrocarbon fractions
    • C10G2300/1062—Lubricating oils

Definitions

  • the present invention relates to a method for treating polychlorinated biphenyls (PCBs) using certain microorganisms.
  • PCBs PolyChlorinated Biphenyls
  • 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.
  • PCBs Polychlorinated Biphenyls
  • insulating oils such as transformers and condensers used in power plants.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • PCBs polychlorinated Biphenyls
  • PCBs of insulating oil containing PCBs (Polychlorinated Biphenyls)
  • 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.
  • 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.
  • EBC107 strains Provide a method.
  • 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.
  • 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.
  • 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.
  • the step of biodegrading is a temperature condition of 24 °C to 26 °C; 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.
  • the step of biodegradation is a temperature condition of 24 °C to 25 °C; 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.
  • 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.
  • the step of biodegrading has a biodegradation time of 50 days or more, to provide a method for processing PCBs of insulating oil.
  • the step of biodegrading has a biodegradation time of 70 days to 200 days, to provide a method for processing PCBs of insulating oil.
  • the insulating oil is an insulating oil contained in a transformer, to provide a method for processing PCBs of insulating oil.
  • 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.
  • 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).
  • the variable includes all values within the described range including the listed endpoints of the range.
  • 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.
  • 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.
  • 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.
  • PCBs of insulating oil containing PCBs (Polychlorinated Biphenyls)
  • the step of administering a 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.
  • 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.
  • EBC107 strains Provide a method.
  • 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.
  • Cy100 Pseudomonas 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").
  • 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.
  • 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.
  • 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.
  • PCBs can be effectively decomposed.
  • 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.
  • the step of biodegrading is a temperature condition of 24 °C to 26 °C; 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.
  • the step of biodegradation is a temperature condition of 24 °C to 25 °C; 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.
  • 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.
  • a stirrer with a speed of 190 rpm to 210 rpm during biodegradation may be used.
  • PCBs can be effectively decomposed.
  • the step of biodegrading has a biodegradation time of 50 days or more, to provide a method for processing PCBs of insulating oil.
  • the biodegradation time is satisfied, PCBs can be effectively decomposed.
  • the step of biodegrading has a biodegradation time of 70 days to 200 days, to provide a method for processing PCBs of insulating oil.
  • the biodegradation time may be 150 days to 170 days.
  • the insulating oil is an insulating oil contained in a transformer, to provide a method for processing PCBs of insulating oil.
  • 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.
  • 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.
  • concentration of dioxins may be 0.5 to 0.7 ng-TEQ / g concentration.
  • 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.
  • 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.
  • 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.
  • 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.
  • API Kits API20E, API20NE, API50CH and API50CHB purchased from bioMerieux (bioMerieux sa 69280 Marcy I'Etoile / France). To Table 5).
  • 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.
  • 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.
  • 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.
  • 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]
  • 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
  • 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.
  • 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.
  • 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. 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;
  • 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;
  • Bacillus sp. Bs100 Taric acid
  • 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;
  • waste acid waste alkali solid medium
  • waste alkali waste alkali, medium formed by adding only 1.5% of agar (agar) to medium, pH neutralization
  • chlorine compound solid medium BB 20 in chlorine compound liquid medium
  • 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.
  • 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.
  • 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 °C, 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.
  • 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.
  • 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
  • EBC106 is 3 mm
  • EBC107 is about 1.2mm brown colony
  • EBC108 is about 1mm yellow colony
  • EBC109 is about 2mm double circle.
  • 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.
  • 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
  • EBC108 is Gram-positive bacteria.
  • EBC 100 EBC 101 EBC 103 Gram strain - - - Catalase + Oxidase - Urease + Citrase utilizat. + Glucose utilizat. + V-P test + Lysine decarboxylase + Ornithine decarboxylase -
  • EBC 100 EBC 101 EBC 103 Inositol + + Arabinos + + + Mannitol + + + Rhamnos + + + + Glucose + + + + Sorbitol + + + + ⁇ -cyclodextrin - - - dextrin + + - Glycogen - Adonitol + + D-Arabitol + + Cellobiose + + D-fructose + + + L-fucose + + + + D-galactose + + + + ⁇ -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 + + +
  • FAMEs fatty acid methyl esters
  • This fused capillary column (HP 19091B-102) was used.
  • the carrier gas is hydrogen
  • the column head pressure is 10 psi
  • the split ratio is 100: 1
  • 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 ⁇ l.
  • 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.
  • EBC100 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.
  • the method of separating individual strains from bacterial community EBC1000 is as follows.
  • EBC106 Genus Bacillus
  • EBC107 Gram-negative bacteria
  • EBC108 Gram-positive bacteria
  • 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
  • EBC107 can be distinguished by the morphological features of brown colony, EBC108 is yellow colony.
  • 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 is light brown
  • EBC108 is colorless
  • EBC109 is red (Dictionary of Microbiology and Molecular Biology, 2nd, Paul Singleton Diana Sainsbury 1987).
  • 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.
  • a minimum medium 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.
  • Tamol-SN (ppm) Degradation rate (mg / l / h) 500 1.3 1000 3.8 2000 5.2 4000 4.0
  • PCP contaminated digestive phenolic compound
  • PCP Digestive Phenolic Compounds
  • 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.
  • Liflus GX and Liflus GR models of fermenters from Biotron 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.
  • the main reactor Liflulus 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).
  • 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.
  • 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.
  • DO dissolved oxygen
  • pH control measurement
  • 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.
  • the first is to find the medium environment in which the microorganisms are growing.
  • factors such as temperature, pH and dissolved oxygen.
  • 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.
  • Oxygen is one of the most important factors in the growth of aerobic 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.
  • 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.
  • Microbial growth is the result of a series of chemical reactions, which means that the growth of microorganisms is greatly affected by temperature.
  • the reaction rate is increased about 2 times as the temperature rises by 10 °C, the growth rate of the microorganism is observed to increase with increasing temperature.
  • the width of the temperature at which the growth rate is increased is very narrowly observed.
  • 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.
  • the reactor Underneath the reactor is a heating plate. This transfers heat into the reactor.
  • the temperature sensor 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.
  • 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.
  • the coolant flows through the jacket inside the reactor and is cooled.
  • an Anti Foam Sensor that removes bubbles
  • an Air pump for injecting air
  • an Air flow controller for adjusting the air volume
  • a condenser that cools and returns moisture in the generated gas.
  • 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.
  • the reactor was named and managed by GX-1, GX-2, GR-1, and GR-2, respectively.
  • liquid waste collection boxes and solid waste collection boxes were installed to prevent leakage of each waste and collect and store it.
  • 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.
  • 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.
  • 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.
  • the collected insulating oil sample was sealed and stored in the dark.
  • 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.
  • it was necessary to check for leakage and stability at a designated time every day, and thoroughly managed to prevent contact with outsiders.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • samples were collected by using disposable protective pipettes.
  • 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.
  • 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.
  • 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.
  • 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.
  • VOCs volatile organic carbons
  • CO 2 carbon dioxide
  • VOCs measuring equipment is shown in Table 12 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
  • 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.
  • 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.
  • 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.
  • the strains were identified by the CFU / ml colony counting method on a plate medium.
  • 1A to 1D are graphs of changes in pH, temperature, DO, and stirring speed of a reaction tank.
  • 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 shows the increase in the number of microbial cells over the reaction time between the microorganism and the PCBs-containing insulating oil.
  • Table 17 shows changes in PCBs concentration (main reactor-GX) over time.
  • Table 18 shows changes in PCBs concentration (preliminary reactor-GR) over time.
  • 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).
  • Table 20 shows changes in PCBs concentration over time.
  • Table 21 shows the concentration analysis results for each isomer compared to the initial HR-MS method.
  • 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).
  • Table 22 shows the results of dioxin analysis in the reactor liquid sample.
  • Table 23 shows the measurement results of the reaction gas generated from the reactor.
  • 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.
  • Table 24 shows changes in PCBs concentrations (assay HR-MS) with treatment duration.
  • 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.

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Abstract

La présente invention concerne un procédé de traitement de biphényles polychlorés (PCB) dans une huile isolante contenant des PCB, dans lequel la biodégradation peut être réalisée par l'administration d'une certaine cellule microbienne et la culture de celle-ci dans certaines conditions, de telle sorte que les PCB de l'huile isolante peuvent être efficacement traitées.
PCT/KR2019/001360 2018-02-01 2019-01-31 Procédé de traitement de biphényles poly-chlorés à l'aide de micro-organismes Ceased WO2019151799A1 (fr)

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CN114164139A (zh) * 2021-10-27 2022-03-11 福州大学 一株耐碱耐铬的蜡样芽孢杆菌及其应用
CN118834799A (zh) * 2024-08-08 2024-10-25 安徽大学 一株蜡样芽孢杆菌cy-1及其应用和降解剂

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CN114164139B (zh) * 2021-10-27 2023-03-10 福州大学 一株耐碱耐铬的蜡样芽孢杆菌及其应用
CN118834799A (zh) * 2024-08-08 2024-10-25 安徽大学 一株蜡样芽孢杆菌cy-1及其应用和降解剂

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