Selenite reduction by the rhizobacterium Azospirillum brasilense, synthesis of extracellular selenium nanoparticles and their characterisation.

Microbial reduction of selenium oxyanions has attracted attention in recent years. In this study, an original and simple method for the synthesis of extracellular selenium nanoparticles (Se NPs) of relatively uniform size has been developed using strains Sp7 and Sp245 of the ubiquitous plant-growth promoting rhizobacterium Azospirillum brasilense, both capable of selenite (SeO32-) reduction. In addition, a reliable purification protocol for the recovery of the Se NPs has been perfected, which could be applied with minor modifications to cultures of other microbial species. Importantly, it was found that, by changing the conditions of bacterial reduction of selenite, extracellularly localised Se NPs can be obtained using bacteria which would otherwise produce intracellular Se NPs. In particular, bacterial cultures grown up to the end of the logarithmic growth phase, washed free of culture medium and then incubated with selenite, were used to obtain extracellular Se NPs. Their sizes depended on the initial selenite concentration (∼25-80 nm in diameter at 50-10 mM selenite, respectively). The Se NPs obtained were characterised by transmission electron microscopy (TEM), dynamic light scattering, as well as Raman and UV-vis spectroscopies. Their zeta potential was found to be negative (ca. minus 21-24 mV). Bacterial selenite reduction was also studied in the presence of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP). In this case, TEM indicated the formation only of intracellular selenium crystallites. The data show that the formation of extracellular Se NPs requires normal bacterial metabolic activity, while CCCP evidently blocks the membrane export of Se0 nuclei.