Effect of Cr(VI) on biofilm development in Pseudomonas alcaliphila 34

Pseudomonas alcaliphila 34 is a highly chromate resistant and biofilm producing bacterium that was isolated from a Cr(VI)-polluted soil. A deeply characterization of the bacterium planktonic cultures was performed by a high-throughput technique, Phenotype MicroArrays (PM)-Biolog, which can test up to 2000 cellular phenotypes simultaneously. In order to examine the influence of Cr(VI) on bacterial biofilm, Pseudomonas alcaliphila 34 was used as a model. For biofilm analysis, the bacterium was grown in mineral medium (TMM) supplemented with gluconate and different concentrations of Cr(VI) into 96 wells polistirene microplates. After 24 hours cultures were removed by the wells and biofilm was quantified by cristal violet assay. In order to quantify the activity of biofilm, in a separate experiment, tetrazolium dye was used. The results showed that Cr(VI) concentration ranging from 5 to 25 mM enhanced bacterium biofilm production compared to the control condition [0 mM Cr(VI)]. On the contrary metabolic activity of biofilm decreases with Cr(VI) concentration, suggesting that Cr(VI) enhances formation of biofilm in which cells are mainly metabolic inactive. In order to confirm this result, biofilms of P. alcaliphila 34 grown in TMM added with gluconate or TMM added with gluconate and 6.25 mM of Cr(VI) were stained with Syto19 and propidium iodide and analysed by confocal laser scanning microscopy. The analysis revealed that biofilm grown in presence of Cr(VI) was formed mainly by dead cells while in not treated biofilm both dead and alive cells could be detected. Extracellular DNA (eDNA) is one of the main polymers contributing to the biofilm structure. Therefore, in order to assess the role of eDNA in biofilm structure of P. alcaliphila 34, cultures not treated and treated with Cr(VI) at different concentration were grown in presence and absence of DNase A and subsequently biofilm production was measured by cristal violet assay. Obtained results showed that for all biofilm samples tested biofilm was significantly decreased in cultures treated with DNase suggesting that eDNA has a key role in the formation of P. alcaliphila biofilm both in presence and absence of Cr(VI). The research was supported by the MIUR (PRIN2008)