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Isolation of a selected microbial consortium from a contaminated site soil capable of degrading the pesticides methyl parathion and chlorpyrifos
New Biotechnology · Volume 25S · September 2009
conformational changes necessary for catalysis with a lower energy demand. Owing to their high catalytic activity at low temperatures, low thermostability and unusual specificities, cold-active enzymes offer novel opportunities for biotechnological exploitation. The enormous potential of biocatalysts for the production of enantiopure products of industrial interest remains to be exploited, especially in the area of enantioselective alkene reductions, due to their marked regio- and stereo-selectivity. One of the emerging reductive chemistry is the asymmetric reduction of alpha,beta-unsaturated carbonyl compounds through microbial whole-cells rich in enoate reductase (ER), a flavin-dependent enzyme belonging to the ‘old yellow enzyme’ (OYE) family. Even though massive screenings for yeasts as reducing catalysts have been carried out so far, there is still a need for new biocatalysts able to performing highly stereoselective reactions. Consequently, the potential of yeast biocatalysis appears to be still far from being fully explored. In this study we report the results of a screening on facultative psychrophilic yeasts (belonging to the species Cryptococcus gilvescens and Cryptococcus gastricus) for new cold-adapted enoate reductases using (4S)-(+)-carvone as a model substrate. The preliminary results obtained using growing cells demonstrate that the ERs activities are apparently expressed to a greater extent in psychrophilic yeasts with respect to the mesophilic ones. In particular, C. gastricus exhibited the better biocatalytic performances. The results obtained by using other catalytic forms (resting or lyophilized cells) will be presented. doi:10.1016/j.nbt.2009.06.267
ABSTRACTS
of methyl parathion and chlorpyrifos, each one at a concentration of 150 mg/L, 100% degradation of methyl parathion and 48% of chlorpyrifos were carried out in 144 hours. When 6 g/L of glucose or peptone were used as an additional carbon source, 60% of 1200 mg/L of methyl parathion, 100% of 150 mg/L of methyl parathion and chlorpyrifos were degraded in 72 hours. When degradation of 150 mg/L of methyl parathion was reached, the number of microorganisms was 1 × 109 UFC/ml. The methyl parathion concentration that inhibited 50% of the microbial population was 700 mg/L. In the methyl parathion degradation tests in soil samples of Moravia, gathered at 5, 10, 20 and 30 m of depth, using the microorganisms isolates and glucose or sugar cane as an additional carbon source, in a relation of 98:2, was verified the degradation of 100% of 150 mg/L of methyl parathion and 100% of 150 mg/L of chlorpyrifos, independent of the origin depth of the samples. Those soil samples that were not inoculated did not manage to degrade 100% of neither of both pesticides. This verifies the efficiency of the isolated microorganisms to be used in bioremediation. Toxicity analysis was performed using the method of bacterial luminescence, which descended to 90% at the end of the degradation of methyl parathion. doi:10.1016/j.nbt.2009.06.268
2.1.022 Decolorization of Reamzol Brillant Blue Royal and Drimaren Blue CL-BR by white rot fungi E.A. Erkurt 1,∗ , A. Unyayar 1 , H. Kumbur 1 , H. Atacag Erkurt 2 1
2.1.021 Isolation of a selected microbial consortium from a contaminated site soil capable of degrading the pesticides methyl parathion and chlorpyrifos N.J. Pino Rodríguez ∗ , G.A. Pe˜nuela Mesa Universidad de Antioquia, Medellin, Colombia
Although the pesticide methyl parathion is forbidden in many countries, in Colombia it is used for some crops, like cotton. With this study, we wanted to determine the capacity to degrade high concentrations of toxic organic pollutants of isolated microbial consortiums from the soil of a site (Moravia, Medellín, Colombia), in which were deposited any type of solid remainders, and was closed in 1984. In the future it is expected to do a bioremediation in said place, using original microbial consortiums from the same site; this study was proved the degradation the methyl parathion, the influence of the pesticide concentration, the crossed effect with other pesticides, and the influence of nutrients in the pesticide degradation. Three microbial consortiums able to degrade methyl parathion were identified, which have the bacteria Acinetobacter sp., Pseudomonas putida, Bacillus sp., Pseudomonas aeruginosa, Citrobacter freundii, Stenotrophomonas sp., Flavobacterium sp., Proteus vulgaris, Pseudomonas sp., Acinetobacter sp., Klebsiella sp. and Proteus sp. In liquid culture, 150 mg/L of methyl parathion were degraded 100% at 96 hours and 27.5% of 1200 mg/L were degraded in the same time. When carrying out crossed effect tests of degradation
2
University of Mersin, Mersin, Turkey Cyprus International University, Cyprus
Decolorization of Remazol Brillant Blue R, Drimaren Blue CL-BR by using three white rot fungi named as Pleurotus ostreatus, Coriolus versicolor and Funalia trogii was investigated in this study. Decolorization studies were continued for 48 hours under static conditions at 30◦ C and pH 5.0. pH, dry mycelium weight, dye concentration, laccase activity, peroxidase activity and protein content were analyzed, the enzyme responsible for decolorization was detected and kinetic parameters were calculated for different dye concentrations. During 48 hours incubation period, pH alternated between 5.0 and 4.75. Maximum and minimum decolorization was obtained by F. trogii and P. ostreatus, respectively. Both dyes at all concentrations were found to be toxic for P. ostreatus growth, whereas only Drimaren Blue CL-BR above 60 mg/L was found to be toxic for C. versicolor growth. Laccase and peroxidase activities were increased by increasing dye concentrations. Maximum and minimum laccase activities were detected in decolorization media of F. trogii and P. ostreatus respectively. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the enzyme responsible for decolorization. Gels of SDS-PAGE were stained with both dyes and colorless zones were obtained where the molecular weight of proteins were approximately 56 kDa. Occurrence of orange color by the oxidation of laccase substrate named as guaicol after staining the colorless zones by guaicol, revealed that the enzyme responsible for decolorization is laccase. At the end of this study, kinetic paramwww.elsevier.com/locate/nbt S53
conformational changes necessary for catalysis with a lower energy demand. Owing to their high catalytic activity at low temperatures, low thermostability and unusual specificities, cold-active enzymes offer novel opportunities for biotechnological exploitation. The enormous potential of biocatalysts for the production of enantiopure products of industrial interest remains to be exploited, especially in the area of enantioselective alkene reductions, due to their marked regio- and stereo-selectivity. One of the emerging reductive chemistry is the asymmetric reduction of alpha,beta-unsaturated carbonyl compounds through microbial whole-cells rich in enoate reductase (ER), a flavin-dependent enzyme belonging to the ‘old yellow enzyme’ (OYE) family. Even though massive screenings for yeasts as reducing catalysts have been carried out so far, there is still a need for new biocatalysts able to performing highly stereoselective reactions. Consequently, the potential of yeast biocatalysis appears to be still far from being fully explored. In this study we report the results of a screening on facultative psychrophilic yeasts (belonging to the species Cryptococcus gilvescens and Cryptococcus gastricus) for new cold-adapted enoate reductases using (4S)-(+)-carvone as a model substrate. The preliminary results obtained using growing cells demonstrate that the ERs activities are apparently expressed to a greater extent in psychrophilic yeasts with respect to the mesophilic ones. In particular, C. gastricus exhibited the better biocatalytic performances. The results obtained by using other catalytic forms (resting or lyophilized cells) will be presented. doi:10.1016/j.nbt.2009.06.267
ABSTRACTS
of methyl parathion and chlorpyrifos, each one at a concentration of 150 mg/L, 100% degradation of methyl parathion and 48% of chlorpyrifos were carried out in 144 hours. When 6 g/L of glucose or peptone were used as an additional carbon source, 60% of 1200 mg/L of methyl parathion, 100% of 150 mg/L of methyl parathion and chlorpyrifos were degraded in 72 hours. When degradation of 150 mg/L of methyl parathion was reached, the number of microorganisms was 1 × 109 UFC/ml. The methyl parathion concentration that inhibited 50% of the microbial population was 700 mg/L. In the methyl parathion degradation tests in soil samples of Moravia, gathered at 5, 10, 20 and 30 m of depth, using the microorganisms isolates and glucose or sugar cane as an additional carbon source, in a relation of 98:2, was verified the degradation of 100% of 150 mg/L of methyl parathion and 100% of 150 mg/L of chlorpyrifos, independent of the origin depth of the samples. Those soil samples that were not inoculated did not manage to degrade 100% of neither of both pesticides. This verifies the efficiency of the isolated microorganisms to be used in bioremediation. Toxicity analysis was performed using the method of bacterial luminescence, which descended to 90% at the end of the degradation of methyl parathion. doi:10.1016/j.nbt.2009.06.268
2.1.022 Decolorization of Reamzol Brillant Blue Royal and Drimaren Blue CL-BR by white rot fungi E.A. Erkurt 1,∗ , A. Unyayar 1 , H. Kumbur 1 , H. Atacag Erkurt 2 1
2.1.021 Isolation of a selected microbial consortium from a contaminated site soil capable of degrading the pesticides methyl parathion and chlorpyrifos N.J. Pino Rodríguez ∗ , G.A. Pe˜nuela Mesa Universidad de Antioquia, Medellin, Colombia
Although the pesticide methyl parathion is forbidden in many countries, in Colombia it is used for some crops, like cotton. With this study, we wanted to determine the capacity to degrade high concentrations of toxic organic pollutants of isolated microbial consortiums from the soil of a site (Moravia, Medellín, Colombia), in which were deposited any type of solid remainders, and was closed in 1984. In the future it is expected to do a bioremediation in said place, using original microbial consortiums from the same site; this study was proved the degradation the methyl parathion, the influence of the pesticide concentration, the crossed effect with other pesticides, and the influence of nutrients in the pesticide degradation. Three microbial consortiums able to degrade methyl parathion were identified, which have the bacteria Acinetobacter sp., Pseudomonas putida, Bacillus sp., Pseudomonas aeruginosa, Citrobacter freundii, Stenotrophomonas sp., Flavobacterium sp., Proteus vulgaris, Pseudomonas sp., Acinetobacter sp., Klebsiella sp. and Proteus sp. In liquid culture, 150 mg/L of methyl parathion were degraded 100% at 96 hours and 27.5% of 1200 mg/L were degraded in the same time. When carrying out crossed effect tests of degradation
2
University of Mersin, Mersin, Turkey Cyprus International University, Cyprus
Decolorization of Remazol Brillant Blue R, Drimaren Blue CL-BR by using three white rot fungi named as Pleurotus ostreatus, Coriolus versicolor and Funalia trogii was investigated in this study. Decolorization studies were continued for 48 hours under static conditions at 30◦ C and pH 5.0. pH, dry mycelium weight, dye concentration, laccase activity, peroxidase activity and protein content were analyzed, the enzyme responsible for decolorization was detected and kinetic parameters were calculated for different dye concentrations. During 48 hours incubation period, pH alternated between 5.0 and 4.75. Maximum and minimum decolorization was obtained by F. trogii and P. ostreatus, respectively. Both dyes at all concentrations were found to be toxic for P. ostreatus growth, whereas only Drimaren Blue CL-BR above 60 mg/L was found to be toxic for C. versicolor growth. Laccase and peroxidase activities were increased by increasing dye concentrations. Maximum and minimum laccase activities were detected in decolorization media of F. trogii and P. ostreatus respectively. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the enzyme responsible for decolorization. Gels of SDS-PAGE were stained with both dyes and colorless zones were obtained where the molecular weight of proteins were approximately 56 kDa. Occurrence of orange color by the oxidation of laccase substrate named as guaicol after staining the colorless zones by guaicol, revealed that the enzyme responsible for decolorization is laccase. At the end of this study, kinetic paramwww.elsevier.com/locate/nbt S53