Estudio metabólico y molecular de cultivos bacterianos degradadores de hidrocarburos aromáticos policíclicos (PAH) y resistentes a metales pesados

La contaminación mixta con PAH y metales pesados conlleva dificultades debido a la potencial toxicidad del metal, limitando la supervivencia solo a los microorganismos que toleren ambos contaminantes. El estudio de cepas degradadoras de PAH y resistentes a metales pesados promete proporcionar herramientas claves para el proceso de biorremediación. Este trabajo tuvo como objetivos la selección, identificación y caracterización de cepas degradadoras de PAH y resistentes a metales pesados aisladas a partir de suelos proveniente de la Antártida y de zonas aledañas a la ciudad de La Plata.La identificación a nivel molecular mostró que las cepas antárticas A, B y C, filogenéticamente relacionadas al género Pseudomonas, presentaron alta resistencia al CdCl$latex _{2}$, usualmente presentes en suelos co-contaminados

Draft whole-genome sequence of Sphingobium sp. 22B, a polycyclic aromatic hydrocarbon- degrading bacterium from semiarid Patagonia, Argentina

Sphingobium sp. 22B is a polycyclic aromatic hydrocarbon-degrading strain isolated from Patagonia, Argentina, with capabilities to withstand the environmental factors of that semiarid region. The draft genome shows the presence of genes related with responses to carbon starvation and drying environmental conditions.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Draft whole-genome sequence of Sphingobium sp. 22B, a polycyclic aromatic hydrocarbon- degrading bacterium from semiarid Patagonia, Argentina

Sphingobium sp. 22B is a polycyclic aromatic hydrocarbon-degrading strain isolated from Patagonia, Argentina, with capabilities to withstand the environmental factors of that semiarid region. The draft genome shows the presence of genes related with responses to carbon starvation and drying environmental conditions.Fil: Madueño, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Macchi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Morelli, Irma Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Coppotelli, Bibiana Marina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Effect of petrochemical sludge concentrations on microbial communities during soil bioremediation

Qualitative and quantitative changes of microbial communities in soil microcosms during bioremediation were determined throughout one year. The soil was contaminated with 0%, 2.5%, 5%, 10% (wt/wt) of petrochemical sludge containing polynuclear aromatic hydrocarbons. We analyzed the hydrocarbon concentration in the microcosms, the number of cultivable bacteria using CFU and most probable number assays, the community structure using denaturing gradient gel electrophoresis, and the metabolic activity of soil using dehydrogenase activity and substrate-induced respiration assays. After one year of treatment, the chemical analysis suggested that the hydrocarbon elimination process was over. The biological analysis, however, showed that the contaminated microcosms suffered under long-term disturbance. The number of heterotrophic bacteria that increased after sludge addition (up to 108-109 cells ml-1) has not returned to the level of the control soil (2-6 × 107 cells ml-1). The community structure in the contaminated soils differed considerably from that in the control. The substrate-induced respiration of the contaminated soils was significantly lower (≈10-fold) and the dehydrogenase activity was significantly higher (20-40-fold) compared to the control. Changes in the community structure of soils depended on the amount of added sludge. The species, which were predominant in the sludge community, could not be detected in the contaminated soils.Facultad de Ciencias ExactasLaboratorio de Biodegradación Microbiológica de Hidrocarburo

Monitoring the impact of bioaugmentation with a PAH-degrading strain on different soil microbiomes using pyrosequencing

The effect of bioaugmentation with Sphingobium sp. AM strain on different soils microbiomes, pristine soil (PS), chronically contaminated soil (IPK) and recently contaminated soil (Phe) and their implications in bioremediation efficiency was studied by focusing on the ecology that drives bacterial communities in response to inoculation. AM strain draft genome codifies genes for metabolism of aromatic and aliphatic hydrocarbons. In Phe, the inoculation improved the elimination of phenanthrene during the whole treatment, whereas in IPK no improvement of degradation of any PAH was observed. Through the pyrosequencing analysis, we observed that inoculation managed to increase the richness and diversity in both contaminated microbiomes, therefore, independently of PAH degradation improvement, we observed clues of inoculant establishment, suggesting it may use other resources to survive. On the other hand, the inoculation did not influence the bacterial community of PS. On both contaminated microbiomes, incubation conditions produced a sharp increase on Actinomycetales and Sphingomonadales orders, while inoculation caused a relative decline of Actinomycetales. Inoculation of most diverse microbiomes, PS and Phe, produced a coupled increase of Sphingomonadales, Burkholderiales and Rhizobiales orders, although it may exist a synergy between those genera; our results suggest that this would not be directly related to PAH degradation.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones Industriale

Monitoring the impact of bioaugmentation with a PAH-degrading strain on different soil microbiomes using pyrosequencing

The effect of bioaugmentation with Sphingobium sp. AM strain on different soils microbiomes, pristine soil (PS), chronically contaminated soil (IPK) and recently contaminated soil (Phe) and their implications in bioremediation efficiency was studied by focusing on the ecology that drives bacterial communities in response to inoculation. AM strain draft genome codifies genes for metabolism of aromatic and aliphatic hydrocarbons. In Phe, the inoculation improved the elimination of phenanthrene during the whole treatment, whereas in IPK no improvement of degradation of any PAH was observed. Through the pyrosequencing analysis, we observed that inoculation managed to increase the richness and diversity in both contaminated microbiomes, therefore, independently of PAH degradation improvement, we observed clues of inoculant establishment, suggesting it may use other resources to survive. On the other hand, the inoculation did not influence the bacterial community of PS. On both contaminated microbiomes, incubation conditions produced a sharp increase on Actinomycetales and Sphingomonadales orders, while inoculation caused a relative decline of Actinomycetales. Inoculation of most diverse microbiomes, PS and Phe, produced a coupled increase of Sphingomonadales, Burkholderiales and Rhizobiales orders, although it may exist a synergy between those genera; our results suggest that this would not be directly related to PAH degradation.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones Industriale

Effect of petrochemical sludge concentrations on microbial communities during soil bioremediation

Qualitative and quantitative changes of microbial communities in soil microcosms during bioremediation were determined throughout one year. The soil was contaminated with 0%, 2.5%, 5%, 10% (wt/wt) of petrochemical sludge containing polynuclear aromatic hydrocarbons. We analyzed the hydrocarbon concentration in the microcosms, the number of cultivable bacteria using CFU and most probable number assays, the community structure using denaturing gradient gel electrophoresis, and the metabolic activity of soil using dehydrogenase activity and substrate-induced respiration assays. After one year of treatment, the chemical analysis suggested that the hydrocarbon elimination process was over. The biological analysis, however, showed that the contaminated microcosms suffered under long-term disturbance. The number of heterotrophic bacteria that increased after sludge addition (up to 108-109 cells ml-1) has not returned to the level of the control soil (2-6 × 107 cells ml-1). The community structure in the contaminated soils differed considerably from that in the control. The substrate-induced respiration of the contaminated soils was significantly lower (≈10-fold) and the dehydrogenase activity was significantly higher (20-40-fold) compared to the control. Changes in the community structure of soils depended on the amount of added sludge. The species, which were predominant in the sludge community, could not be detected in the contaminated soils.Facultad de Ciencias ExactasLaboratorio de Biodegradación Microbiológica de Hidrocarburo

Insights into the mechanisms of desiccation resistance of the Patagonian PAH-degrading strain Sphingobium sp. 22B

AimTo analyze the physiological response of Sphingobium sp. 22B to water stress.Methods and resultsThe strain was grown under excess of carbon source and then submitted to low (60RH) and high (18RH) water stress conditions for 96 h. Quantification of trehalose, glycogen, polyhydroxybutyrate (PHB), and transmission electron microscopy (TEM) was studied. Genes linked with desiccation were searched in Sphingobium sp. 22B and Sphingomonas ?sensu latu? genomes and their transcripts were quantified by Real-Time PCR. Results showed that, in absence of water stress, strain 22B accumulated 4.76± 1.41% of glycogen, 0.84± 1.62% of trehalose and 44.9± 6.4% of PHB per cellular dry weight. Glycogen and trehalose were mobilized in water stresses conditions, this mobilization was significantly higher in 60RH in comparison to 18RH. Gene treY was upregulated 6-fold change in 60RH relative to 18RH. TEM and quantification of PHB revealed that PHB was mobilized under 60RH condition accompanied by the downregulation of the phbB gene. TEM images showed an extracellular amorphous matrix in 18RH and 60RH. Major differences were found in the presence of aqpZ and trehalose genes between strain 22B and Sphingomonas genomes.ConclusionStrain 22B showed a carbon conservative metabolism capable of accumulation of three types of endogenous carbon sources. The strain responds to water stress by changing the expression pattern of genes related with desiccation, formation of an extracellular amorphous matrix and mobilization of the carbon sources according to the degree of water stress. Trehalose, glycogen and PHB may have multiple functions in different degrees of desiccation. The robust endowment of molecular responses to desiccation shown in Sphingobium sp. 22B could explain its survival in semiarid soil.Significance and Impact of the studyUnderstanding the physiology implicated in the toleration of the PAH-degrading strain Sphingobium sp 22B to environmental desiccation may improve the bioaugmentation technologies in semiarid hydrocarbons contaminated soils.Fil: Madueño, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Coppotelli, Bibiana Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Festa, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Alvarez, H. M.. Universidad Nacional de la Patagonia "San Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; ArgentinaFil: Morelli, Irma Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentin

Insights into the mechanisms of desiccation resistance of the Patagonian PAH-degrading strain Sphingobium sp. 22B

Aim: To analyze the physiological response of Sphingobium sp. 22B to water stress. Methods and results:The strain was grown under excess of carbon source and then submitted to low (60RH) and high (18RH) water stress conditions for 96 h. Quantification of trehalose, glycogen, polyhydroxybutyrate (PHB), and transmission electron microscopy (TEM) was studied. Genes linked with desiccation were searched in Sphingobium sp. 22B and Sphingomonas "sensu latu" genomes and their transcripts were quantified by Real-Time PCR. Results showed that, in absence of water stress, strain 22B accumulated 4.76± 1.41% of glycogen, 0.84± 1.62% of trehalose and 44.9± 6.4% of PHB per cellular dry weight. Glycogen and trehalose were mobilized in water stresses conditions, this mobilization was significantly higher in 60RH in comparison to 18RH. Gene treY was upregulated 6-fold change in 60RH relative to 18RH. TEM and quantification of PHB revealed that PHB was mobilized under 60RH condition accompanied by the downregulation of the phbB gene. TEM images showed an extracellular amorphous matrix in 18RH and 60RH. Major differences were found in the presence of aqpZ and trehalose genes between strain 22B and Sphingomonas genomes. Conclusion: Strain 22B showed a carbon conservative metabolism capable of accumulation of three types of endogenous carbon sources. The strain responds to water stress by changing the expression pattern of genes related with desiccation, formation of an extracellular amorphous matrix and mobilization of the carbon sources according to the degree of water stress. Trehalose, glycogen and PHB may have multiple functions in different degrees of desiccation. The robust endowment of molecular responses to desiccation shown in Sphingobium sp. 22B could explain its survival in semiarid soil.Significance and Impact of the studyUnderstanding the physiology implicated in the toleration of the PAH-degrading strain Sphingobium sp 22B to environmental desiccation may improve the bioaugmentation technologies in semiarid hydrocarbons contaminated soils.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Assessing interactions, predicting function, and increasing degradation potential of a PAH-degrading bacterial consortium by effect of an inoculant strain

A natural phenanthrene-degrading consortium CON was inoculated with an exogenous strain Sphingobium sp. (ex Sp. paucimobilis) 20006FA yielding the consortium called I-CON, in order to study ecological interactions into the bacterial community. DGGE and proteomic profiles and analyses by HTS (High-Throughput Sequencing) technologies demonstrated inoculant establishment and changes on CON composition. Inoculation increased degradation efficiency in I-CON and prevented intermediate HNA accumulation. This could be explained not only by the inoculation, but also by enrichment in Achromobacter genus at expense of a decrease in Klebsiella genus. After inoculation, cooperation between Sphingobium and Achromobacter genera were improved, thereby, some competition could have been generated, and as a consequence, species in minor proportion (cheaters), as Inquilinus sp. and Luteibacter sp., were not detected. Sequences of Sphingobium (corresponding to the inoculated strain) did not vary. PICRUSt predicted a network with bacterial phylotypes connected with enzymes, showing functional redundancy in the phenanthrene pathway, with exception of the first enzymes biphenyl-2,3-diol 1,2-dioxygenase and protocatechuate 4,5-dioxygenase that were only encoded in Sphingobium sp. This is the first report where a natural consortium that has been characterized by HTS technologies is inoculated with an exogenous strain in order to study competitiveness and interactions.Fil: Macchi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Festa, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Vega Vela, Nelson E.. Pontificia Universidad Javeriana; Colombia. Universidad de Bogotá Jorge Tadeo Lozano; ColombiaFil: Morelli, Irma Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Coppotelli, Bibiana Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentin