KR20110108957A - Microbial agents using bamboo active carbon as a biocarrier - Google Patents

Abstract

In order to achieve the above object, the present invention is a microorganism using the bamboo activated carbon as a culture carrier, characterized in that the culture culture of the oil-decomposed mixed bacteria (CSMix-1) cultured from oil contaminated soil using bamboo activated carbon as a culture carrier Formulation is a technical subject. Thereby, there is an advantage of culturing the microbial preparation using bamboo activated carbon as a culture carrier of the oil lysing mixed bacteria, thereby purifying (sea) soil contaminated with crude oil and the like.

Description

Microbial agents using bamboo active carbon as a biocarrier}

The present invention relates to a microbial agent used to purify a contaminated marine soil, using bamboo activated carbon as a culture carrier of the oil decomposition mixture to promote the growth of the oil decomposition mixture bacteria, and effectively clean up the marine soil contaminated with crude oil The present invention relates to a microbial preparation using bamboo activated carbon as a culture carrier.

Due to industry and population growth, oil consumption is increasing rapidly. It is true that the frequency of leakage accidents is also increasing rapidly. In the event of an oil spill, part of the removal is eliminated by control, but most remain in the environment, resulting in complex decomposition by physical, chemical and biological mechanisms.

The oil pollution purification technology developed and used up to now has many problems. For example, physical effluent treatment alone can not expect complete oil removal effect, it is expensive to control, and the use of chemical oil dispersant causes the problem of secondary pollution due to toxicity.

In addition, there is a method of washing the soil using a chemical synthetic emulsifier as a method of washing, but the removal by washing rather than the complete decomposition of the contaminated oil has a problem of secondary treatment, thereby researching biological restoration technology Is actively underway and is being applied to actual pollution sites.

In general, biological restoration techniques of oil-contaminated soils can be divided into biostimulation techniques for supplying nutrients and air and bioaugmentation techniques for injecting adapted hydrocarbon-degrading microorganisms. In general, natural ecosystems show a diversity of hydrocarbon-degrading microorganisms, but in the absence of oil inflow, the number of degrading microorganisms is small. Therefore, microorganisms isolated from oil contaminated areas are best adapted to the contaminated site, exhibit high activity, and can minimize the occurrence of secondary pollution, which can be useful for bioaugmentation techniques, which are biological treatment techniques of oil contaminated soils. have.

Recently, alcanivorax spp ., Marinobacter sp., Neptunomonas sp., And Oleiphilus spp. sp.) and Oleispira sp. have been reported. In addition, Alcanivorax sp. ( Alkanivorax species) was found to be the dominant species of oil-contaminated seawater. And, there honaeng bacteria and is ssalra sound tooth species to decompose the oil (Thalassolituus sp.), Cycloalkyl-class tea kusu species (Cycloclasticus sp.) And Lodge O bakteo species (Roseobacter sp.) Are reported in 4 ℃, these seasonal It is thought to contribute to the purification of oil contaminated by the sea without much influence.

As such, it is known that the microbial agent used in the existing marine oil pollution treatment is mainly used to select and produce several excellent decomposition microorganisms. One important consideration in bioinoculation is to increase the density of decomposed microorganisms in contaminated areas. To date, microorganisms have been used to fix gravel, granular clay and glass beads. However, these studies did not consider the effects of microbial growth and immobilization of oil on bioavailability.

The present invention is to solve the above problems, to provide a microbial preparation using the bamboo activated carbon as a culture carrier to effectively cleanse the marine soil contaminated with crude oil by producing a microbial preparation using the bamboo activated carbon as a culture carrier of the oil decomposition mixture bacteria For that purpose.

In order to achieve the above object, the present invention, the bamboo activated carbon characterized in that for using the culture medium mixed cultured using bamboo activated carbon as a culture carrier of the oil decomposition mixture (CSMix-1) enriched from oil contaminated soil The microbial preparation used as the culture carrier is the technical subject.

In addition, it is preferable to further add an excipient to the microbial preparation, and the excipient is preferably made of bamboo activated carbon capable of adsorbing oil pollutants and inorganic nutrients, and pitmos, elvan minerals and humic substances.

Here, the microbial agent, 37 to 50 parts by weight of bamboo activated carbon, 75 to 100 parts by weight of peat moss, 37 to 50 parts by weight of ganban pore minerals, and 0.5 to Humic material as excipients based on 100 parts by weight of the mixed culture solution. It is preferred to include 2.5 parts by weight.

In addition, when the microbial agent is utilized, an oxygen producing chemical (OPC) and a surfactant may be further added, and the oxygen generating agent may be any one of MgO, CaO, CaO ₂ and Ca (OH) ₂ or It is preferable to mix and use these.

The present invention by the above-mentioned means for solving the problem has the effect of culturing the microbial preparation using the bamboo activated carbon as a culture carrier of the oil decomposition mixture bacteria, it is possible to purify the marine soil contaminated with crude oil and the like.

Figure 1-a diagram showing the oil resolution of the oil lysate mixed bacteria used in the preparation of microbial preparations according to the present invention.
Figure 2-After culturing by adding lyolysis mixed bacteria and diesel oil and bamboo activated carbon according to the present invention, and showing the result of diluting the culture solution.
Figure 3 shows the degree of purification of crude oil contaminated sea soil by inoculation of the microorganism preparation (MA-1) according to the present invention.
Figure 4 is a view showing the degree of purification of crude oil contaminated sea soil by inoculation of the microorganism preparation (MA-2) according to the present invention.
Figure 5 is a view showing the monitoring of the changes in the microbial community during the biological purification of crude oil contaminated sea soil by inoculation of the microorganism preparation (MA-2) according to the present invention.

The present invention relates to a microbial preparation that is mixed and cultured using bamboo activated carbon activated at a high temperature as a culture carrier of the superior oil decomposition mixture microorganisms, wherein the marine soil contaminated with crude oil by preparing the microbial preparation by adding an excipient thereto. It is used to purify the water. Here, the bamboo activated carbon promotes the propagation of the oil lysate mixed bacteria, and the microbial agent significantly promotes oil decomposition in the presence of an excipient such as a surfactant or an oxygen generator to more effectively remove the marine soil contaminated with crude oil. Purified.

Hereinafter, with reference to the accompanying drawings will be described an embodiment of the present invention.

Soil samples used in the present invention are the soils of the West Coast intertidal zone contaminated with crude oil, and are mainly representative of clay soils (polluted area A) and sandy soils (polluted area B). Sampling was conducted with soil 60 cm deep from the surface layer. 2mm sieving was carried out to remove large particles of soil, gravel and foreign matter.

<Isolation of oil-combining mixed bacteria and enrichment of bioaugmentation by enrichment culture>

Thickening was carried out to separate the oil lysed bacteria from the oil contaminated soil. Fill 100 ml of SM1 medium (pH 7.5) in a 250 ml Erlenmeyer flask, sterilize for 15 minutes at 121 ° C, cool, add 5 g of sample, and add 500 µl of commercial diesel oil (5,000mg / L) as a carbon source and shake at 27 ° C. The incubator was incubated at 190 rpm. After each week, 5 ml of fresh medium was transferred to a fresh culture, and the process was repeated four times. In this process, two representative consortiums were selected through the oil digestion test.

<Collective analysis of oily-mixed bacteria through clonal library formation>

16S rDNA was cloned from the PCR amplification product using TA Cloning kit (Solgent, Korea) for the cluster analysis of the oil lysates. To construct the clone library, PCR amplification products were loaded on 1.5% agarose gel, cut into bands of about 1,500bp, purified using LaboPass Gel Extraction kit (CosmoGen, Korea), and then T-Blunt vector. Ligation was carried out in (Solgent, Korea), and the plasmid was added to Escherichia coli DH5α (RBC Korea, Korea) and confirmed in selective medium. These clones were grown in LB medium containing 50 μg / ml kanamycin and the resulting colonies were used for 16S rDNA analysis. At least 50 colonies were selected and rearranged in LB + kanamycin medium to extract plasmids, and then sequenced.

Preparation of Hydrolyzed Microbial Formulations

Mix the two final selected oil lysates "C" and "S" in a 2L Erlenmeyer flask containing SM1 (500-600 ml; containing 1-2% (w / w) bamboo activated carbon) for at least 1 week After cultivation, this mixed culture solution (CSMix-1) was used to prepare a microbial microbial formulation. In this case, the bacterial density reached 10 ⁸ to 10 ^9.5 (CFU / ml). The used bamboo activated carbon has a specific surface area of 1,300 m ² / g, has excellent CO ₂ adsorption capacity, and was used as a culture carrier for the oil decomposition bacteria.

Further, an excipient is further added to the microbial mixed culture cultured on the bamboo activated carbon, which may adsorb oil pollutants (such as aliphatic hydrocarbons and aromatic hydrocarbons) and inorganic nutrients (Mg ++, Ca ++, etc.) to 100 parts by weight of the mixed culture medium. Bamboo activated carbon (other than Eulji Entec product), peat moss, peat moss 75 ~ 100 parts by weight, 37 ~ 50 parts by weight, Machwang pore minerals (Davistone Co., Ltd. Songda pebble, etc.) 37 ~ 50 parts by weight And a humic substance (Gumus K et al., GreenVortex Co., Ltd.) (0.5 ~ 2.5 parts by weight) to prepare a microbial preparation (water content 30 ~ 40%) was used to clean the contaminated soil. As a preferred embodiment, 80 parts by weight of peat moss, 40 parts by weight of bamboo activated carbon, 40 parts by weight of mach mineral, and 1.2 parts by weight of humic material are used with respect to 100 parts by weight of the mixed culture, and the water content of the final microbial preparation is the total mixed culture solution. To 35 parts by weight.

<Installation of Oil Purification Device by Biological Inoculation>

Purification by bioaugmentation of crude oil contaminated oceans was carried out with 1 kg of contaminated soil (points A and B) in a polypropylene beaker (2L). The initial total petroleum hydrocarbon (TPH) concentration of the contaminated soil was 5,000 ~ 6,000ppm, and the density of oil decomposition bacteria was 10 ⁷ (CFU / g soil) at the time of inoculation of microorganisms. Did not do it. The experiment was carried out for 5 weeks under the condition of maintaining the soil water content of 30 ~ 40% at room temperature (25 ℃).

<Measuring oil decomposition rate>

Sampling was carried out once a week for 7 weeks, and three samples were taken from each reactor and mixed as samples. First, the collected sample was placed in 5 ml of 4 ml vials (National Scientific Company, USA) and dried at room temperature for 24 hours. In order to remove residual moisture of the dried sample, 1.5 times of anhydrous sodium sulfate (Na ₂ SO ₃ ) to the sample was added and mixed. 10 ml of a mixture of acetone and hexane in a 1: 1 ratio was injected into the sample from which moisture was removed, followed by mixing for 2 minutes, and allowed to stand for 12 hours. Dissolve the supernatant, which was left still, in a 1 ml aliquot of the screw cap vial (Agilent, USA) to GC-FID [Agilent Technologies 6890

Network System; HP-5 capillary column (50 mx0.2mmx0.33μm)] was analyzed for TPH concentration. Analytical conditions stayed at 80 ° C. for 2 minutes, and the temperature was raised to 8 ° C./min, and stopped at a final temperature of 320 ° C. for 10 minutes. At this time, the temperature of the injector and the detector was set to 300 ℃ and 320 ℃, respectively. Carrier gas was used for nitrogen, and the injection amount was 1 μl.

<Measurement of the number of viable bacteria mixed bacteria>

The measurement of the number of viable biodegradable bacteria was carried out by diluting the culture solution with 10 ^-3 , 10 ^-4 , 10 ^-5 , and 10 ^-6 , smearing on SM1 agar medium, and then soaking diesel oil in a filter paper strip. Lids were placed on the lids and fed in a vapor form to count the lysophilic bacteria grown after at least one week. Microbial community analysis by DNA extraction and PCR-DGGE technique PCR-DGGE and clone library techniques were used to measure microbial community changes. DNA was extracted using a Fast DNA extraction kit (MPbio, USA), and PCR reaction was performed using DNA obtained from a sample to confirm microbial diversity. Primary PCR was performed by adding 1xPCR buffer, 20 mM MgCl ₂ , 40 mM dNTP mixture, each primer (1 μM), template DNA and 0.5U Taq polymerase in 50μl using 27F and 1541R. Reaction conditions were first denatured at 94 ° C for 5 min, followed by 30 cycles of 45 sec denaturation at 94 ° C, 45 sec annealing at 56 ° C, 45 sec extension at 72 ° C, and final extension at 72 ° C for 7 min. Was performed. Each PCR product was identified on 1% agarose gel after ethidium bromide staining. Secondary PCR was performed using the amplified product of the primary PCR as a template. The primers used were 341F-GC and 786R with 40 GC-clamps. The PCR mixture for the secondary PCR is the same as that used for the primary PCR, and the PCR reaction conditions used are denatured DNA at 94 ° C. for 5 minutes, 45 sec denaturation at 94 ° C., 45 sec annealing at 60 ° C., and 72 ° C. 45 sec extension at and final extension for 7 min at 72 ° C. The annealing temperature was initially performed at 60 ° C and 20 cycles were decreased by 0.5 ° C per cycle, and then 15 cycles were performed at 50 ° C to complete the touch-down PCR. DGGE was performed using the product of the obtained secondary PCR. PCR products were subjected to DGGE using the Dcode ^™ System (Bio-Rad, USA). Denaturing gradient gels were prepared by adding 10% polyacrylamide (37.5: 1 = acrylamide: bisacrylamide) to form gradients of urea and formamide modifiers from 40% to 70%. 30 μl of PCR amplification products were loaded onto the gel thus prepared and electrophoresed at 60 ° C. and 85 V for 20 hours in 1 × TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0). After electrophoresis, the gel was stained with 1% ethidium bromide and checked by UV light.

<Base sequence analysis>

Each band was selected to recover DNA fragments present at different locations on the denaturing gradient gel, cut and added to 50 μl of the third DW and left at 4 ° C. overnight to obtain supernatant. The DNA recovered from the band was reamplified using 341F-GC and 786R primers as templates, and the PCR product was purified by PCR purification kit (QIAGEN, Germany) and then cloned using T-Blunt vector. This plasmid was added to E. coli DH5α (RBC Korea, Korea) and confirmed in selective medium. These clones were grown in LB medium containing 50μg / ml kanamycin and the resulting colonies were used for 16S rDNA analysis. The selected colonies were rearranged in LB + kanamycin medium to extract plasmids, and then sequenced. The determined nucleotide sequence was analyzed by BLAST search using GenBank database of NCBI (www.ncbi.nlm.nih.gov).

<Analysis Result>

-Selection of oil lysis mixed bacteria through enrichment culture

The oil degradation bacteria were selected through enrichment culture using diesel oil or crude oil as the only carbon source. After enrichment in this process, the degradation rate and growth rate of TPH were measured to secure microbial communities "S" and "C" containing excellent oil (diesel) degradation bacteria. In case of "C", naphthalene decomposability was better than "S".

However, "S" showed better decomposition power in diesel oil than "C". Therefore, these two mixed bacteria were mixed and cultured to prepare a microbial preparation.

-Identification of oil lysed bacteria

As a result of colony formation and colonization analysis of the oil lysed bacteria by the smear method, the halomonas species were found in the diesel oil decomposition community "S". sp.) (Appendix Patent Microorganism Depository, Microorganism Name: Halomonas sp. Sok1-1, Microbial Accession No .: KACC91531P) was dominant species. In addition, the diesel decomposition cluster "C" refers to Thalassospira species. sp.) (Appendix Patent Microbial Acquisition, Microorganism Name: Thalassospira sp. Ch1-2, Microbial Accession No .: KACC91530P) and mixed these two communities in diesel oil and secured CSMix-1 to produce microbial preparations.

-Oil-degrading ability of oil-combining bacteria used in the preparation of microbial preparations

In order to compare the oil-degrading performance of the oil-combining bacteria used in the preparation of microbial preparations used for crude oil-contaminated soil purification and the individual bacteria that make up them, 200ml SM1 medium was used, and each treatment was unique to 5,000 mg / L diesel oil. It was added as a carbon source and incubated at 25 ° C. for 7 days with shaking (190 rpm). The microbial community "S" showed a degradation rate of about 70% compared to the control group, but the microbial community "C" showed a resolution of 55% (FIG. 1). However, the microbial community "C" was observed to accumulate yellow degradation products (estimated semialdehydes) during the culture. Therefore, the resolution will be more remarkable when time passes. On the other hand, when cultured by mixing the microbial community "S" and microbial community "C" showed a significant degradation rate of about 75%. Therefore, it is assumed that the microbial community "S" and the microbial community "C" exhibited mutual synergy in the decomposition of oil (crude oil).

Effect of Bamboo Activated Carbons on the Growth of LDLB

In order to increase the density of the decomposed microorganism per unit culture volume, bamboo activated carbon was added as a carrier, and then cultured. After culturing the oil-dissolved mixed bacterium CSMix-1 with diesel oil (5000 mg / L) and 1% (w / w) of bamboo activated carbon for 7 days, the culture solution was diluted with phosphate buffer and plated on SM1 solid medium for 7 days. The culture results are as shown in FIG. In this case, the bamboo activated carbon added spheres (FIGS. 2C and D) showed several orders of magnitude increase in density compared to the control (FIGS. 2A and B), and when the treated carbon treated 1.5%, the density increased by more than ten times. The reason why the addition of activated carbon increases the density of decomposed microorganisms is that activated carbon can be used as a habitat for microbial cells, minerals contained in activated carbon can promote the growth of decomposed microorganisms, and adsorption of oil whose activated carbon is a substrate. It can be provided to microorganisms continuously, which is believed to be the cause.

-Cleaning of Crude Oil Soil by Inoculation of Microorganisms

Microbial preparations MA-1 and MA-2 were prepared from sedimentary oil degradation bacteria (CSMix-1) isolated from the sediments collected from two crude oil contaminated areas (A and B areas). . The microbial preparations MA-1 and MA-2 were then treated with contaminated soils in areas A and B, respectively, to carry out a purification test.

First of all, the soil in area A was viscous soil, and when the initial TPH concentration (5,200 mg / L) was treated 35 days after treatment, only 71% of the microbial agent (MA-1) was treated (1,500 mg / L) (FIG. 3). However, simultaneous treatment with MA-1 and surfactant Tween 80 resulted in 42% purification. This somewhat lower purification effect was due to the fact that the soil was more difficult to ventilate than sandy soil as clay, and the concentration of treated Tween 80 was 5%, which was relatively high, causing some degree of inhibition of soil microbial activity. Seems to be.

Therefore, in the case of purifying clay soil, treatment of surfactant and oxygen generator in proper concentration is expected to contribute greatly to the improvement of purification efficiency. Therefore, in the case of purifying clay soil, treatment with surfactant (1% or less) and oxygen generator (MgO, CaO, CaO ₂ or Ca (OH) ₂ substance) of appropriate concentration according to soil conditions is purified. It is expected to contribute greatly to the improvement of efficiency.

On the other hand, the soil in area B was sand soil, and when the TPH concentration (5,200 mg / L) at the beginning of the test was treated with microbial agent (MA-2) after 35 days of treatment, about 73% of purification effect was recognized (FIG. 4). . However, microorganisms (MA-2) include oxygen releasing (producing) compounds; When 10% of ORC or OPC] was added, the purification effect of about 90% was recognized. This superior purification effect seems to be due to the air permeability of sandy soil and the treatment of oxygen generator. On the other hand, simultaneous treatment of T-2 with MA-2 and surfactant Tween 80 resulted in about 44% of purification effect, suggesting that the inhibitory effect of biodegradation by slightly higher concentration of surfactant was observed.

Therefore, as described above, the treatment of the surfactant and oxygen producing chemical (OCPC) at an appropriate concentration may be greatly contributed to the improvement of the purification efficiency according to the soil condition. The density of diesel oil decomposed bacteria before and after purification by MA-2 treatment showed 10 ⁷ (CFU / g) in 35 days after purification compared to 10 ⁶ (CFU / g soil), the density at the beginning of purification. It was found that there was proliferation of degrading bacteria. This is expected to promote the purification of microorganisms prepared by using indigenous microorganisms separated from the soil or similar environment to be cleaned, especially oxygen generators (MgO) and other oxygen generators CaO ₂ , CaO, Ca The treatment of (OH) ₂ and the like and other measures to improve breathability (such as high pressure air injection and cultivation) suggest that significant purification can be achieved.

Monitoring of community changes of microorganisms during biopurification

The microbial community changes were investigated by PCR-DGGE technique in the purification process of marine oil B contaminated with crude oil (FIG. 5). The microbial community pattern at the beginning immediately after treatment was similar in general, but showed a clear change in the community after 5 weeks of treatment. Dominant species at the beginning were band 1, band 2 and band 3, respectively, such as Marinobacter sp., Roseovarius sp., And Erythrobacter seohaensis sp. Similar to marine microorganisms, especially Marinobacter sp. And Erythrobacter seohaensis sp. Have recently been actually isolated from tidal flats on the west coast. Therefore, the sample at the start of the treatment was found to have the advantage of microorganisms generally present in the ocean. On the other hand, in the control group, Roseobacter sp. And Sulfitobacter sp. Were predominantly microorganisms in the control group after 35 days, but Thalassospira sp in the band 10 of the MA-2 treatment group. It was identified as RP4 (99% similarity), which had been separated from Spain's crude oil contaminated coast.

In the isolated colony "C" in the present invention, rice larassospira species ( Thalassospira) The presence of sp.) has been confirmed by clone library analysis. Band 11 of the treatments of MA-2 and Tween 80 was found to have 100% similarity with Halomonas sp. HA-T, which has been reported as a marine microorganism that degrades TBT (NCBI accession no. AB104435). In addition, Halomonas sp. Is known to show better oil degradation than co-culture with Alcanivorax sp.

The microbial species when the microbial agent MA-2 and the oxygen generator are co-treated with bands 12, 13, 14 and 15 of FIG. 5 are Rhodobacter sp. And Idiomarina homiensis sp. homiensis sp.), and Uncultured Paracoccus sp., although the former two do not seem to be directly related to oil degradation, but in the case of Paracoccus sp., PAH (anthracene, phenanthrene, fluorene , fluoranthene, chrysene, and pyrene) and are known to use cresol compounds and n-alkanes as the only carbon sources.

Therefore, in the case of active oil degradation as a whole (MA-2 treatment and MA-2 + Tween 80 treatment), oil-degrading bacteria can be confirmed by PCR-DGGE, but oil decomposition is almost completed first (MA-2 + Oxygen generators) are thought to have relatively active activities of coexisting non-oil-degradable microorganisms. One interesting thing is that, as mentioned above, if Paracoccus sp. Decomposes PAH, it is hardly degradable PAH (anthracene, phenanthrene, fluorene, fluoranthene, chrysene, and pyrene) that may remain at the end of crude oil degradation. It is thought to be related to the decomposition of.

As described above, the present invention was obtained by incorporating microbial community, which is an aliphatic and aromatic decomposition function microbial community, to obtain a mixed culture solution CSMix-1 cultured after inoculation into a medium containing bamboo activated carbon. It is trying to purify the marine quality contaminated with crude oil manufactured. Purification of crude oil showed more than 90% removal effect after 5 weeks of treatment with MA-2 with oxygen generator, and high concentration of surfactant inhibits the action of degrading microorganisms. Verification is required and the use of appropriate aeration methods (oxygen generator administration, mechanical aeration, etc.) is required to purify the clay soil.

Institute of Agricultural Biotechnology KACC91530 20100209 Institute of Agricultural Biotechnology KACC91531 20100209

Claims (6)

A microbial agent using bamboo activated carbon as a culture carrier, characterized by using a mixed culture medium mixed with bamboo activated carbon as a culture carrier of oil-decomposed mixed bacteria (CSMix-1) enriched from oil contaminated soil. The microorganism preparation according to claim 1, further comprising an excipient in the microbial preparation. The microorganism preparation according to claim 2, wherein the excipient comprises bamboo activated carbon capable of adsorbing oil pollutants and inorganic nutrients, and bamboo activated carbon as a culture carrier. . The method of claim 3, wherein the microbial agent,
Bamboo, characterized in that containing 37 to 50 parts by weight of bamboo activated carbon, 75 to 100 parts by weight of peat moss, 37 to 50 parts by weight of mach mineral, and 0.5 to 2.5 parts by weight of humic material based on 100 parts by weight of the mixed culture solution. Microbial preparation using activated carbon as a culture carrier. The microbial preparation according to any one of claims 1 to 4, wherein an oxygen producing chemical (OPC) and a surfactant are further added when the microbial preparation is utilized. . [Claim 5] The microorganism preparation according to claim 4, wherein the oxygen generating agent is one of MgO, CaO, CaO ₂ and Ca (OH) ₂ or a mixture thereof.

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