Development, validation and application of molecular method based on extraction, amplification and sequencing of the rRNA for the identification of biofilm-forming bacteria on the surface of the reverse osmosis membranes. / Desenvolvimento, validação e aplicação de método molecular baseado na análise do rRNA para a identificação das bactérias formadoras de biofilme metabolicamente ativas na superfície das membranas de osmose reversa.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

A method based on extraction of rRNA, followed by RT-PCR of 16S rRNA, cloning and ARDRA was optimized and validated for identification of bacteria active in biofilms. The method was first tested with artificial three-membered consortia. Cloning and RT did not lead to significant changes in the composition of the artificial consortia, but a reduction in cycle number in the PCR reaction from 30 to 10 was necessary for limiting the distortion in the proportion of amplicons relative to that of the templates. Analysis of real biofilms revealed that clones from active organisms occurred in frequencies >2% in the clone library, but reproducibility of analysis was unsatisfactory, probably due to factors such as the spatial heterogeneity of colonization of biofilms by microbes, PCR bias and more than one ARDRA clone per organism. Storage of biofilm samples at -20 °C for 2 months did not lead to important changes in composition. Very different clone profiles were obtained in the analysis of the same biofilm sample with the optimized method and with a kit (Mo Bio) for extraction of RNA.

ASSUNTO(S)

reverse osmosis "biofouling" biofilmes biofouling rrna 16s biofilms biotechnology osmose reversa comunidades microbiana microbial communities 16s rrna metabolismo (atividade) metabolism (activity) biotecnologia

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