Biorremediación de suelos contaminados con Hidrocarburos Policíclicos Aromáticos: una visión molecular

El limitado conocimiento sobre la diversidad, dinámica y funcionalidad de las comunidades microbianas durante los procesos de biorremediación hace difícil clarificar la contribución biológica a la efectividad del proceso, considerando que los microorganismos son los mayores responsables de un proceso que sin dudas resulta útil para el manejo de problemas de contaminación. En particular en las estrategias de bioaumento, la supervivencia de los microorganismos inoculados se encuentra íntimamente relacionada con la competencia por los recursos con la población microbiana nativa del sitio contaminado; por lo tanto resulta crítico no solo entender la fisiología del inóculo sino también como afecta la estructura y la función de la comunidad microbiana del suelo a la cual se está introduciendo. Con el objetivo de contribuir al mejoramiento de los conocimientos básicos relacionados con la ecología microbiana y la efectividad de estrategias de bioaumento, aplicadas a la biorremediación de suelos contaminados con PAH, se orientó el trabajo de tesis, en primer lugar, a obtener y caracterizar un consorcio bacteriano degradador de fenantreno para ser utilizado en estrategias de bioaumento y aplicar estrategias “ómicas” para estudiar su dinámica funcional durante la degradación de fenantreno en medio líquido. El consorcio, (CON) obtenido de un suelo crónicamente contaminado con PAH, presentó la capacidad de degradar el 59% del fenantreno suplementado a los 7 días de incubación. La composición de CON se estudió por métodos dependientes e independientes de cultivo. Por métodos dependientes de cultivo, se lograron aislar cinco cepas bacterianas y se estudió la degradación del hidrocarburo tanto en cultivos puros de las mismas, encontrándose que solo la cepa AM (afiliada al género Sphingobium sp.) mostró la capacidad de degradar fenantreno, como en consorcios definidos (cultivos mixtos), encontrando una mayor eficiencia de degradación que el consorcio natural y que la cepa AM. De los métodos independientes de cultivo utilizados, la pirosecuenciación del gen 16S rRNA nos permitió conocer la composición de CON en términos de abundancia relativa de cada uno de los miembros. La caracterización catabólica de CON se llevó a cabo con un enfoque metagenómico y metaproteómico. Mediante la construcción de una biblioteca metagenómica a través de un screening funcional se encontraron clones con secuencias de genes codificantes de enzimas de la ruta de degradación de compuestos aromáticos que se afiliaron al orden Burkholderiales, revelando la presencia de al menos otra cepa con capacidad de degradar PAH en CON. Analizando el metaproteoma del consorcio mediante electroforesis bidimensional se identificaron proteínas pertenecientes a dos órdenes presentes en CON, Sphingomonadales y Burkholderiales, confirmando que se encontraron metabólicamente activos durante la degradación de fenantreno. En segundo lugar se estudió el impacto de la inoculación con diferentes formulaciones bacterianas sobre la estructura y dinámica de comunidades bacterianas de un suelo prístino contaminado con fenantreno y de un suelo crónicamente contaminado con PAH (proveniente de un proceso de Landfarming de un residuo petroquímico) mediante pirosecuenciación del gen 16S rRNA, a partir del DNA total de cada suelo. Además se correlacionaron los cambios en la estructura y dinámica de las comunidades bacterianas con la efectividad del proceso de biorremediación. Para ello se llevaron a cabo ensayos en microcosmos de los suelos mencionados y se inocularon diferentes formulaciones, entre las que se encontraron el consorcio natural (CON) y la cepa degradadora AM. En el suelo recientemente contaminado se podría afirmar que la estrategia de bioaumento llevada a cabo con el consorcio natural y la cepa degradadora Sphingobium sp. AM no solo mejoró la degradación del contaminante, reduciendo el tiempo de adaptación de la comunidad microbiana, sino que además generó un impacto positivo sobre la diversidad del sistema. Sin embargo, en un suelo crónicamente contaminado, las estrategias de inoculación lograron aumentar la diversidad de la comunidad bacteriana de un suelo crónicamente contaminado con hidrocarburos, sin incrementar la degradación de los contaminantes, donde la comunidad nativa ya habría sufrido la presión de selección ejercida por la contaminación y se encontró adaptada a la presencia de hidrocarburos.Facultad de Ciencias Exacta

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

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

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

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

Integrating Shotgun Metagenomics, 16s Rrna Gene Metabarcoding and Culture Approaches: A Better Outlook for Functional Profiling of a Pah-Contaminated Soil

Understanding bacterial diversity and function is critical for designing bioremediation strategies. This research aimed to assess chronically hydrocarbon contaminated soil bacterial diversity and their aromatic compound degradation (ACD) potential by integrating shotgun metagenomic, 16S rRNA gene metabarcoding and culture approaches. While soil metabarcoding showed dominance of Proteobacteria, metagenomics indicated that 99,5% of the sequences were taxonomically assigned to Streptomycetales order and that almost all genes related to ACD were assigned to the latter. To inspect other phyla contribution to ACD, a functional prediction was delved, and two culture approaches were used. PICRUSt2 revealed that ACD pathways were mostly found in Alphaproteobacteria, Actinobacteria and Gammaproteobacteria classes. An enrichment culture (r-EFP) was obtained with pyrene as sole carbon and energy source and a bacterial strain (S19P6), identified as a member of Mycolicibacterium genus, was isolated. Both cultures demonstrated the ability to degrade more than 90% of the supplemented pyrene after 21 days of incubation. 16S rRNA gene metabarcoding and shotgun metagenomics approaches in r-EFP indicated predominance of Proteobacteria Phylum and the presence of genes responsible for the degradation of ACD mostly assigned to the predominant phyla. Complementing different methodologies enable the recognition of the metabolic potential of soil Proteobacteria related to ACD.Fil: 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: Granada, 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: Irazoqui, José Matías. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Cuadros Orellana, Sara. Universidad Catolica de Maule; ChileFil: Quevedo, Claudio. Universidad Catolica de Maule; ChileFil: 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: 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; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentin

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

Treatment of Cutaneous Tumors with Topical 5% Imiquimod Cream

INTRODUCTION: There are various approaches to the treatment of cutaneous tumors; one of them is treatment with imiquimod, a synthetic toll-like receptor agonist with a low molecular weight that offers a topical, noninvasive, and non-surgical therapeutic option. The main objective of our study was to provide data on 89 patients who used a 5% imiquimod cream for the treatment of cutaneous tumors at the Cutaneous Oncology Group of the Dermatology Department of Hospital das Clinicas from 2003 to 2008. MATERIALS AND METHODS: Here, we present our experience in the treatment of 123 cutaneous tumors of various types, including basal cell carcinoma (BCC), squamous cell carcinoma (SCC), Bowen's disease, erythroplasia of Queyrat, Paget's disease, and trichoepithelioma, with 5% imiquimod cream from 2003 to 2008 in the Cutaneous Oncology Group of the Dermatology Department of Hospital das Clinicas. Patients were divided into two separate groups according to their diagnosis and comorbidities; these comorbidities included epidermodysplasia verruciformis, xeroderma pigmentosum, albinism, basal cell nevus syndrome, Brooke-Spiegler syndrome, HIV, chronic lymphocytic leukemia, B-cell lymphoma, and kidney transplantation. Treatment duration, response to imiquimod, follow-up, recurrence, and local and systemic reactions associated with use of the drug were analyzed. Epidemiological data were obtained and cure rates were calculated. RESULTS: The ratio of women to men was 1.28:1, and the mean age was 63.1 years. Tumors were located mainly on the face, back, trunk, and legs. For patients with comorbidities, the overall cure rate was 38%. These specific patients demonstrated cure rates of 83.5% for superficial BCC and 50% for Bowen's disease. Aggressive BCC and superficial and nodular BCC did not present a good response to treatment. Trichoepitheliomas and nodular BCC showed a partial response, and erythroplasia of Queyrat showed a complete response. For patients without comorbidities, the overall cure rate was 73%. For these patients, the cure rates were 85.7% for superficial and nodular BCC, 88% for superficial BCC, 57% for Bowen's disease, 50% for nodular BCC, and 50% for aggressive BCC. One SCC lesion demonstrated a complete response, and tumors caused by Paget's disease and erythroplasia of Queyrat presented a partial response. None of the tumors considered as clinically cured recurred. Thirty-seven lesions demonstrated no response to imiquimod. Having a cutaneous comorbidity, high-risk tumors such as mixed aggressive BCC (sclerodermiform or micronodular), nodular BCC, or Bowen's disease, and presenting no local reaction to imiquimod were considered as risk factors for a worse prognosis. We demonstrate that patients with no response to imiquimod, even when they demonstrated no local reaction, can undergo another cycle of six weeks of imiquimod treatment and show a complete response. The healing pattern led to good cosmetic outcomes, and the side effects were tolerable. CONCLUSIONS: Our experience confirms imiquimod as an effective treatment option for several types of cutaneous tumors, especially in patients without the cutaneous comorbidities cited above and with low-risk tumors. Imiquimod has a relatively low cost compared to other therapeutic options and can be delivered via ambulatory care to patients with surgery contraindications, and its side effects are tolerable