RESUMO
The Río de la Plata, one of the most important estuarine environments in South America that sustains valuable fisheries, is affected by PAH contamination associated with oil industry and port activities. A total of 95 bacteria with potential to degrade phenanthrene were obtained from water samples using traditional culture methods. PCR-RFLP analysis of 16S rDNA partial fragments was used as a screening tool for reducing the number of isolates during diversity studies, obtaining 42 strains with different fingerprint patterns. Phylogenetic analysis indicated that they were affiliated to 19 different genera of Gamma- and Alpha-Proteobacteria, and Actinobacteria. Some of them showed an efficient phenanthrene degradation by HPLC (between 83% and 97%) and surfactant production (between 40% and 55%). They could be an alternative for microbial selection in the degradation of PAHs in this estuarine system. In order to detect and monitor PAH-degrading bacteria in this highly productive area, rDNA amplicons of the 33 isolates, produced by PCR real time, were tested by the high-resolution melting (HRM) technique. After analyzing the generated melting curves, it was possible to accurately distinguish nine patterns corresponding to eight different genera. HRM analysis allowed a differentiation at the species level for genera Pseudomonas, Halomonas and Vibrio. The implementation of this method as a fast and sensitive scanning approach to identify PAH-degrading bacteria, avoiding the sequencing step, would mean an advance in bioremediation technologies.
Assuntos
Bactérias , Hidrocarbonetos Policíclicos Aromáticos , Biodegradação Ambiental , Filogenia , RNA Ribossômico 16SRESUMO
Rhodococcus opacus PD630 was investigated for physiological and morphological changes under water stress challenge. Gluconate- and hexadecane-grown cells were extremely resistant to these conditions, and survival accounted for up to 300 and 400 days; respectively, when they were subjected to slow air-drying. Results of this study suggest that strain PD630 has specific mechanisms to withstand water stress. Water-stressed cells were sensitive to the application of ethanol, high temperatures and oxidative stress, whereas they exhibited cross-protection solely against osmotic stress during the first hours of application. Results indicate that the resistance programme for water stress in R. opacus PD630 includes the following physiological and morphological changes, among others: (1) energetic adjustments with drastic reduction of the metabolic activity ( approximately 39% decrease during the first 24 h and about 90% after 190 days under dehydration), (2) endogenous metabolism using intracellular triacylglycerols for generating energy and precursors, (3) biosynthesis of different osmolytes such as trehalose, ectoine and hydroxyectoine, which may achieve a water balance through osmotic adjustment and may explain the overlap between water and osmotic stress, (4) adjustments of the cell-wall through the turnover of mycolic acid species, as preliminary experiments revealed no evident changes in the thickness of the cell envelope, (5) formation of short fragmenting-cells as probable resistance forms, (6) production of an extracellular slime covering the surface of colonies, which probably regulates internal and external changes in water potential, and (7) formation of compact masses of cells. This contributes to understanding the water stress resistance processes in the soil bacterium R. opacus PD630.