CN100451112C - Process for fixation of white rot fungus for inhibiting varied bacteria growing in non-sterilizing environment - Google Patents
Process for fixation of white rot fungus for inhibiting varied bacteria growing in non-sterilizing environment Download PDFInfo
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- CN100451112C CN100451112C CNB2005101256779A CN200510125677A CN100451112C CN 100451112 C CN100451112 C CN 100451112C CN B2005101256779 A CNB2005101256779 A CN B2005101256779A CN 200510125677 A CN200510125677 A CN 200510125677A CN 100451112 C CN100451112 C CN 100451112C
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- 241000233866 Fungi Species 0.000 title claims abstract description 42
- 241000894006 Bacteria Species 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000001954 sterilising effect Effects 0.000 title claims abstract description 20
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 9
- 229920005830 Polyurethane Foam Polymers 0.000 claims abstract description 17
- 239000011496 polyurethane foam Substances 0.000 claims abstract description 17
- 241000222393 Phanerochaete chrysosporium Species 0.000 claims abstract description 16
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 claims abstract description 6
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 238000012360 testing method Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 28
- 235000015097 nutrients Nutrition 0.000 claims description 25
- 239000002609 medium Substances 0.000 claims description 22
- 230000000593 degrading effect Effects 0.000 claims description 10
- 230000000844 anti-bacterial effect Effects 0.000 claims description 5
- 239000004677 Nylon Substances 0.000 claims description 3
- 229910000831 Steel Inorganic materials 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 239000003365 glass fiber Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000012423 maintenance Methods 0.000 claims description 3
- 229920001778 nylon Polymers 0.000 claims description 3
- 239000010959 steel Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 230000000452 restraining effect Effects 0.000 claims description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 9
- 230000015556 catabolic process Effects 0.000 abstract description 7
- 238000004042 decolorization Methods 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 2
- 239000000969 carrier Substances 0.000 abstract 3
- 238000009630 liquid culture Methods 0.000 abstract 2
- 230000002349 favourable effect Effects 0.000 abstract 1
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 21
- 238000011160 research Methods 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 10
- 239000002351 wastewater Substances 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000222355 Trametes versicolor Species 0.000 description 4
- 238000011169 microbiological contamination Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000001408 fungistatic effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 125000000751 azo group Chemical group [*]N=N[*] 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011217 control strategy Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- IEQIEDJGQAUEQZ-UHFFFAOYSA-N phthalocyanine Chemical compound N1C(N=C2C3=CC=CC=C3C(N=C3C4=CC=CC=C4C(=N4)N3)=N2)=C(C=CC=C2)C2=C1N=C1C2=CC=CC=C2C4=N1 IEQIEDJGQAUEQZ-UHFFFAOYSA-N 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 239000000985 reactive dye Substances 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- AAAQKTZKLRYKHR-UHFFFAOYSA-N triphenylmethane Chemical compound C1=CC=CC=C1C(C=1C=CC=CC=1)C1=CC=CC=C1 AAAQKTZKLRYKHR-UHFFFAOYSA-N 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention belongs to the field of microorganism application and relates to a method for immobilizing white rot fungi capable of inhibiting the growth of sundry bacteria in non-sterilizing environment. In the method, phanerochaete chrysosporium is used as the white rot fungus on the basis of liquid culture media capable of inhibiting the growth of bacteria, inert carriers, namely polyurethane foam carriers, with the best effect on inhibiting sundry bacteria are filtered out by way of orthogonal experiments, polyurethane foam is made into two triangular prisms, and the white rot fungus is immobilized by way of adding the polyurethane foam carriers into the liquid culture media. Through utilizing the structural features of the polyurethane foam, combining with the culture media for inhibiting the growth of bacteria, and taking contrast tests, other sundry bacteria are not infected during cultivation, stable decolorization ratio which is as high as 95% or more is achieved at the stage of active dye degradation, and the results are basically consistent with that obtained in sterilizing environment. The present invention has the advantages of convenient use, cheap and facile material for bacteriostasis, favourable effect on inhibiting sundry bacteria, and reduced operation cost in reality due to sterilization conditions.
Description
Technical field
The invention belongs to the using microbe field, particularly a kind of process for fixation of white rot fungus that suppresses varied bacteria growing at non-sterilized environment when non-sterilized environment is cultivated and use white rot fungus degrading active dye.
Background technology
Along with developing rapidly of dyestuffs industries, the dyestuff that uses has reached tens thousand of kinds at present.Waste water containing dye is the unmanageable trade effluents of generally acknowledging both at home and abroad from a plurality of industrial trades such as weaving, printing and dyeing, dyestuff manufacturings.
At present, handle in the research, many novel procesies, new technology have occurred, as membrane technique and high-level oxidation technology at waste water containing dye.These new technologies, novel method can be decoloured to the waste water containing dye of complexity, also can reach the high processing effect.But there is a common shortcoming in these technologies, are exactly that construction investment and running cost are quite high, and actual engineering is difficult to bear.Therefore, although also there are problems in biological treatment, because its low cost makes it remain the best method that solves the waste water containing dye pollution problem.
In recent years, manyly studies show that white-rot fungi (White rot fungi) is very promising microorganism in waste water containing dye is handled, wherein studying and discussing at most is Phanerochaete chrysosporium (Phanerochaete chrysosporium).Phanerochaete chrysosporium has non-specific, as to need not substrate for induction enzyme because of it, to many organic pollutants and dissimilar synthetic dyestuff (as azo, tritane, phthalocyanine pigment) have the degradation capability of wide spectrum, and it can thoroughly be degraded to CO with the dyestuff of various synthetic by secreted special degradation enzyme system and other mechanism
2And H
2O.From the later stage eighties, just there is the scientific research personnel to study abroad and uses the various synthetic dyestuff of white rot fungus degrading, prove its multiple dyestuff of under strict sterilising conditions, can degrading.Domestic just have the minority scientific research personnel to begin one's study white-rot fungi to the degraded of reactive dyestuffs in the middle and later periods nineties, and recent years, this research was in rising trend, but also have very big gap with comparing abroad.Yet, no matter domestic still external, the research of white-rot fungi dye wastewater treatment using is all also rested on the laboratory study stage, the case that is applied in the actual engineering does not have substantially.What restricts it and uses in actual engineering so? white-rot fungi is different from bacterium, and it belongs to eukaryotic microorganisms, and the speed of growth is very slow.Therefore, in case reaction system has bacterium to enter, bacterium will fight for nutritive substance in the substratum with white-rot fungi, because the reproduction speed of bacterium is fast more a lot of than fungi, like this, bacterium will be preponderated in reaction system, and white-rot fungi nutrition for want of will stop growing, and then influence born of the same parents' secretion of degradation enzyme system outward, make the entire treatment system lose the function of degradation of dye.Hence one can see that, has only the microbiological contamination problem that solves the white rot fungus degrading system, and white rot fungus degrading dyestuff technology is applied in the actual engineering.The sterilization means that employing is adopted in laboratory study solve the microbiological contamination problem, obviously in actual engineering, be impracticable, because it will improve the running cost of technology greatly, even abroad, the microbiological contamination problem that adopts the sterilization means to solve in the sewage treatment process also is beyond example.Have realized that abroad the microbiological contamination problem is the industrialized bottleneck problem of restriction white-rot fungi treatment technology, and take up to research and solve this problem recent years, obtained some achievements in research.
But; by the retrieval Chinese and foreign documents; only find the antibacterial technology when the white rot fungus degrading dyestuff is used in two pieces of researchs; protect the peroxidase of white-rot fungi Trametes versicolor and extracellular generation for employing polyvinyl alcohol embedding method for one and avoid germ attack, thereby reach continuous bio-oxidation dyestuff Poly R-478.But the problem of entrapping method is, because the continuous growth of white-rot fungi can be earned brokenly the polyethylene bead gradually, mycelium is come out, influence fungistatic effect (Leidig E, et al.Biotransformation of Poly R-478by continuous cultures of PVAL-encapsulated Trametes versicolor undernon-sterile conditions.Bioprocess Eng, 1999,21:5-12.).Another piece of writing is for hanging down the pH value by control, the control strategy of white-rot fungi Trametes versicolor at the non-sterilized environment degrading active dye used in nitrogen restriction substratum and natural carrier research, only rely on low pH of control and limit nitrogen substratum to be difficult to obtain secular fungistatic effect (Judy A but mention in the literary composition, et al.Competition strategies forthe decolorization of a textile-reactive dye with the white-rot fungiTrametes versicolor under non-sterile conditions.Biotechnilogy andBioengineering, 2003,82 (6): 736-744.).In addition, more than two kinds of antibacterial technology all be to adopt Trametes versicolor bacterial classification, suppress assorted bacterium method research and adopt Phanerochaete chrysosporium (Phanerochaetechrysosporium) to carry out non-sterilized environment, except that this seminar, yet there are no other researchist's reports.
To sum up, how to cultivate white-rot fungi and use the research of its degrading active dye also relatively more preliminary, also do not have at present and can cultivate the method that white-rot fungi also can effectively suppress varied bacteria growing at non-sterilized environment about non-sterilized environment.
Summary of the invention
The purpose of this invention is to provide in physical environment (non-sterilized environment) Phanerochaete chrysosporium cultivation and follow-up down a kind of process for fixation of white rot fungus that suppresses varied bacteria growing at non-sterilized environment in the reactive dyestuffs degradation process.It is characterized in that this method comprises:
1) bacterial classification is selected: white-rot fungi adopts Phanerochaete chrysosporium (Phanerochaete chrysosporium),
2) liquid nutrient medium is selected the bacteria growing inhibiting culture medium of the white rot fungus degrading active dye in non-sterilized environment of early stage invention for use, this antibacterial substratum can cultivate through sterilization white-rot fungi after 5 days on non-sterilized environment decolouring reactive dyestuffs stage good restraining bacterium.
3) carrier: the fixation of white rot fungus carrier is to adopt the 250mL Erlenmeyer flask, the 100mL liquid nutrient medium carries out orthogonal test and selects, filter out from stainless (steel) wire, nylon wire, glass fiber mesh and polyurethane foam and have the best inert support-polyurethane foam carrier that presses down assorted bacterium effect, the carrier size is 1.0 * 1.0 * 1.0cm
3, the quality of carrier is 1.2g, carrier be shaped as the triangular prism structure.
4) culturing process: polyurethane foam carrier is put into pot boil 30min, the oven dry back takes by weighing 1.2g tri-prismoid polyurethane foam carrier with one of percentage balance, it is rendered in the 250mL Erlenmeyer flask that contains 100mL liquid nutrient medium (Tsing-Hua University's Environmental Science and Engineering based environment simulation and the development of pollution control state key joint laboratory), this moment, the carrier piling height exceeded liquid level, be in non-submerged state, carrier and liquid nutrient medium are put into Autoclave together at 100~120 ℃ of 20~40min that sterilize down, after sterilization finishes, filter adding 100mg/L VITMAIN B1 solution 1ml with syringe and needle-based strainer (sterilization of band film) to the 100ml liquid nutrient medium, and the final concentration of VITMAIN B1 is 1mg/L in the maintenance liquid nutrient medium; Then, will be grown in 35~40 ℃ of aseptic access liquid nutrient mediums of Phanerochaete chrysosporium BKM-F-1767 spore equivalent on the PDA flat board, inoculum size is 1 * 10
5Individual spore/ml puts into Erlenmeyer flask temperature and is 35~38 ℃ constant temperature shaking table again, and rotating speed is made as 160rpm, nature (non-sterilization) air conditions down cultivation carry out the cultivation of white-rot fungi at non-sterilized environment, between incubation period 5 days.
5) to the degraded of reactive dyestuffs, non-sterilization was cultivated after 5 days, under non-sterilized environment, in the Erlenmeyer flask that contains white-rot fungi and liquid nutrient medium, add Reactive Brilliant Red K-2BP solution (1000mg/L) 3ml that sterilized is handled, keeping the concentration of Reactive Brilliant Red K-2BP in the interior solution of Erlenmeyer flask is 30mg/L, 24 hours time.
Described PDA flat board is the uniform mixing liquid of 200g/L murphy juice, 20g/L glucose and 20g/L agar.
The invention has the beneficial effects as follows in method of the present invention is not only between 5 days incubation periods and do not infect other assorted bacterium, and the efficient decolorizing effect of percent of decolourization more than 95% to Reactive Brilliant Red K-2BP, the basically identical as a result that its percent of decolourization and sterilized environment obtain have been obtained to stablize, reach in the reactive dyestuffs degradation period.Therefore, the present invention is easy to use, and antibacterial material price is cheap, be easy to get, it is effective to press down assorted bacterium, lignin degradation production of enzyme height is handled waste water containing dye for the practical engineering application white-rot fungi possibility is provided, and reduced the running cost of bringing because of sterilising conditions in practice.
Embodiment
The invention provides in physical environment (non-sterilized environment) Phanerochaete chrysosporium cultivation and follow-up down a kind of process for fixation of white rot fungus that suppresses varied bacteria growing at non-sterilized environment in the reactive dyestuffs degradation process.This method adopts Phanerochaete chrysosporium (Phanerochaete chrysosporium) as white-rot fungi, the fixation of white rot fungus carrier is to adopt the 250mL Erlenmeyer flask, the 100mL liquid nutrient medium carries out orthogonal test and selects, from stainless (steel) wire, nylon wire, glass fiber mesh and polyurethane foam, filter out and have the best inert support-polyurethane foam carrier that presses down assorted bacterium effect, polyurethane foam is cut into 1.0 * 1.0 * 1.0cm
3Cubes breaks it into two from diagonal lines then, makes it become two tri-prismoids, prepares the tri-prismoid carrier with this.Polyurethane foam carrier is put into pot boil 30min, the oven dry back takes by weighing 1.2g tri-prismoid polyurethane foam carrier with one of percentage balance, and it is rendered to (250mL) in the Erlenmeyer flask that contains the 100mL liquid nutrient medium.This moment, the carrier piling height exceeded liquid level, was in non-submerged state.The liquid nutrient medium that is adopted is the bacteria growing inhibiting culture medium (seeing Tsing-Hua University's Environmental Science and Engineering based environment simulation and the patent of invention 200410073821.4 of polluting control state key joint laboratory) of invention in early stage.Carrier and liquid nutrient medium are put into Autoclave together at 113 ℃ of 30min that sterilize down.After sterilization finishes, use syringe and needle-based strainer (sterilization of band film) to filter to the 100ml liquid nutrient medium and add 1ml 100mg/L VITMAIN B1 solution, the final concentration of VITMAIN B1 is 1mg/L in the maintenance liquid nutrient medium.Then, will be grown in the aseptic access liquid nutrient medium of Phanerochaete chrysosporium BKM-F-1767 spore equivalent on 37 ℃ of PDA flat boards (200g/L murphy juice, 20g/L glucose and 20g/L agar), inoculum size is 1 * 10
5Individual spore/ml.Erlenmeyer flask is put into temperature again and be 37 ℃ constant temperature shaking table, rotating speed is made as 160rpm, in the cultivation down of nature (non-sterilization) air conditions.Non-sterilization was cultivated after 5 days, add Reactive Brilliant Red K-2BP solution (1000mg/L) 3ml that sterilized is handled under non-sterilized environment in the Erlenmeyer flask that contains white-rot fungi and liquid nutrient medium, keeping the concentration of Reactive Brilliant Red K-2BP in the interior solution of Erlenmeyer flask is 30mg/L.Behind the 24h, non-sterilized environment adds the percent of decolourization of Reactive Brilliant Red K-2BP in 3 Erlenmeyer flasks of Reactive Brilliant Red K-2BP all more than 95%; Add Reactive Brilliant Red K-2BP with sterilized environment and compare, the two percent of decolourization is basic identical.And to cultivate and decolouring during nutrient solution carry out microscopy, only found on the 5th day in decolouring that the minute quantity yeast was arranged, the decolouring system is not impacted.
Claims (1)
1. one kind is suppressed the process for fixation of white rot fungus of varied bacteria growing at non-sterilized environment, it is characterized in that this method comprises:
1) bacterial classification is selected: white-rot fungi adopts Phanerochaete chrysosporium Phanerochaete chrysosporium,
2) liquid nutrient medium is selected the bacteria growing inhibiting culture medium of existing white rot fungus degrading active dye in non-sterilized environment for use, this antibacterial substratum can cultivate through sterilization white-rot fungi after 5 days on non-sterilized environment decolouring reactive dyestuffs stage good restraining bacterium;
3) carrier: the fixation of white rot fungus carrier is to adopt the 250mL Erlenmeyer flask, the 100mL liquid nutrient medium carries out orthogonal test and selects, filter out from stainless (steel) wire, nylon wire, glass fiber mesh and polyurethane foam and have the best polyurethane foam carrier that presses down assorted bacterium effect, the carrier size is 1.0 * 1.0 * 1.0cm
3, the quality of carrier is 1.2g, carrier be shaped as the triangular prism structure;
4) culturing process: polyurethane foam carrier is put into pot boil 30mi n, the oven dry back takes by weighing 1.2g tri-prismoid polyurethane foam carrier with one of percentage balance, it is rendered in the 250mL Erlenmeyer flask that contains the 100mL liquid nutrient medium, this moment, the carrier piling height exceeded liquid level, be in non-submerged state, carrier and liquid nutrient medium are put into Autoclave together at 110~120 ℃ of 20~40min that sterilize down, after sterilization finishes, filter adding 100mg/L VITMAIN B1 solution 1ml with syringe and needle-based band film sterilising filter to the 100ml liquid nutrient medium, and the final concentration of VITMAIN B1 is 1mg/L in the maintenance liquid nutrient medium; Then, will be grown in 35~38 ℃ of aseptic access liquid nutrient mediums of Phanerochaete chrysosporium BKM-F-1767 spore equivalent on the PDA flat board, inoculum size is 1 * 10
5Individual spore/ml puts into Erlenmeyer flask temperature and is 35~38 ℃ constant temperature shaking table again, and rotating speed is made as 160rpm, cultivates under the natural air condition and carries out the cultivation of white-rot fungi at non-sterilized environment, between incubation period 5 days;
5) to the degraded of reactive dyestuffs, non-sterilization was cultivated after 5 days, adding concentration under non-sterilized environment in the Erlenmeyer flask that contains white-rot fungi and liquid nutrient medium is the Reactive Brilliant Red K-2BP solution 3ml of the sterilized processing of 1000mg/L, keeping the concentration of Reactive Brilliant Red K-2BP in the interior solution of Erlenmeyer flask is 30mg/L, and the time is 24 hours.
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CN101165164B (en) * | 2007-09-30 | 2011-06-29 | 清华大学 | Application of knotting thread for filamentous fungi breeding and breeding method for filamentous fungi |
CN101698534B (en) * | 2009-11-12 | 2011-08-31 | 上海交通大学 | Method for optimizing stability of aerobic granular sludge |
CN109111054A (en) * | 2018-09-30 | 2019-01-01 | 北京东方园林环境股份有限公司 | A kind of organic pollution aquifer dystopy Ecosystem restoration system and ecological restoring method |
CN111826288B (en) * | 2020-03-18 | 2021-10-19 | 广东省生态环境技术研究所 | Method for degrading tricresyl phosphate by using Phanerochaete chrysosporium and application of method |
CN113087282B (en) * | 2021-03-02 | 2023-11-03 | 商丘师范学院 | Sewage treatment device containing immobilized microorganism filler |
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白腐真菌培养条件对其分泌木质素降解酶的影响. 高大文等.中国环境科学,第25卷第5期. 2005 |
白腐真菌培养条件对其分泌木质素降解酶的影响. 高大文等.中国环境科学,第25卷第5期. 2005 * |
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