INVESTIGADORES
LOZADA mariana
congresos y reuniones científicas
Título:
Combining culture dependent and independent approaches to characterize bioremediation potential at Córdova Cove, a chronically polluted site of Northern Patagonia
Autor/es:
ISAAC, PABLO; MARCOS, MAGALÍ; DI MARZIO, WALTER; LOZADA, MARIANA; DIONISI, HEBE; FERRERO, MARCELA
Lugar:
San Miguel de Tucumán, Tucumán, Argentina
Reunión:
Congreso; VII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE DEL BICENTENARIO; 2011
Resumen:
Polycyclic aromatic hydrocarbon (PAH)-bioremediation potential was assessed in intertidal sediment samples collected from Córdova Cove, a chronically polluted site located in Northern Patagonia, using culture dependent and independent methods.We estimated the relative abundance of various genes encoding PAH dioxygenases by qPCR, in DNA extracted directly from the sediment samples. We quantified the following genes: phnA1 (Cycloclasticus spp.), nahAc (Pseudomonas spp.), phnAc (b-Proteobacteria) as well as A, B, C and D dioxygenase genes, which are novel gene groups that we determined to be abundant in sediments from Southern Patagonia and for which no cultured representative is known. Additionally, we quantified bacterial 16S rRNA genes using a degenerate primer set. Cycloclasticus phnA1 genes were abundant in the most polluted sediment sample (8.104 ± 3.104 copies/mg sediment DNA, vs. ca. 108 copies for the 16S rRNA gene). Their abundance was found to be two orders of magnitude higher in sediments with twice the concentration of 3-ring PAHs. The other analyzed genes were also detected, although below quantification limit for this technique. These results suggest that Cycloclasticus, a cosmopolitan marine obligate hydrocarbonoclastic bacterium, plays an important role in the biodegradation of low molecular weight PAHs in coastal sediments of Northern Patagonia too. On the other hand, culture-dependent methods consisted in the isolation of bacterial strains by enrichment on ONR7 synthetic medium using crystals or vapors of naphthalene, phenanthrene or pyrene as carbon and energy source. After two months of incubation, colonies were purified by multiple streaking or by transferring the hydrocarbon crystal on ONR7 plates with PAH vapor. This resulted in the isolation of twelve gram-negative bacteria, four of them based on theirability to form biofilms on the crystal surface. All twelve isolates presented unique RFLP and ITS amplification patterns. These isolates are being identified by 16S rRNA gene sequencing. The isolates were tested for their ability to produce indigo from indole, a functional assay suitable for some oxygenase enzymes. Only one of the 12 isolates (naph1) produced blue color on solid media in the presence of indole. Similar results were obtained for the molecular screening of the isolates. PCR amplification of oxygenase genes involved in PAH metabolic pathways (nahAc naphthalene dioxygenase gene and c12 and c23 catechol dioxygenase genes from Pseudomonas spp.), were used to assess the presence of archetypical naphthalene degradation genes. The expected amplicons were found only in naph1 strain. None of the isolates showed the presence of phnA1 genes, suggesting that although very abundant in the sediments, Cycloclasticus could not be obtained in culture by this method. Considerable efforts are being carried out in the laboratory in order to isolate this fastidious microorganism from the sediments.