Investigation on Bacterial Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01 and Stenotrophomonas maltophilia SeITE02 through Spectroscopic and Metabolomics Analyses, with Characterization of Biogenic Selenium Nanoparticles (Bio-SeNPs)

Bacillus mycoides SeITE01 and Stenotrophomonas maltophilia SeITE02 are environmental bacterial isolates that rely on detoxification processes to transform selenite (SeO32-), a highly toxic and bioavailable chemical species of selenium, into insoluble and virtually nontoxic elemental selenium (Se0) with the formation of biogenic selenium nanoparticles (Bio-SeNPs). In the last decade, Bio-SeNPs have attracted attention for their interesting applications in the nanotechnology, industrial and medical fields not only due to their special physico-chemical features, but also for their attractive antimicrobial activities and anticancer properties. These worthwhile biotechnological traits are related to the presence on Bio-SeNPs of an external organic coating, whose composition and role are mostly unknown and currently under investigation. In the first part of this thesis, FTIR (Fourier Transform Infrared) spectroscopy was applied to study the SeO32- bio-reduction process analysing the bio-molecular composition of both SeITE01 and SeITE02 cells. The analysis was conducted during the diverse cellular growth phases and in different conditions, namely untreated (growth without the presence in the medium of sodium selenite Na2SeO3) and Se-treated (exposure to the stress factor SeO32-). Moreover, along with FTIR spectroscopic analyses, the biogenic intracellular and extracellular SeNPs bio-produced by the bacterial strains and collected with two extraction methods (vacuum filtration and pelleting processing) were examined also using DLS (Dynamic Light Scattering) measurements and TEM (Transmission Electron Microscopy) imaging. In the second part of this research, metabolomics was used to investigate the biological reduction and effect of Na2SeO3 on SeITE01 and SeITE02 cells by a LC-MS (Liquid Chromatography Mass-Spectrometry) approach. Both intracellular and extracellular metabolites and their concentration fluxes during a defined time course and in response to the exposure of bacterial cells to SeO32- were studied. From the results obtained with all the investigation techniques it was possible to observe and underline two distinct behaviors and trends assumed by the bacterial strains. SeITE01 cells showed substantial changes when exposed to SeO32-, activating a series of macromolecular responses and biochemical pathways to defend the cells against the toxic action of both the oxyanion and the Se nanostructures. Moreover, different FTIR spectral trends were acquired with regard to the organic coatings present on the surfaces of the biogenic SeNPs synthesized by this bacterium, and the most marked differences were recorded according to their different localization (intracellular or extracellular). The Bio-SeNPs production in this Gram-positive microorganism was also described by the data collected with the DLS and the zeta-potential measurements. The analyzed Bio-SeNPs showed an average dimension between 637 and 393 nm for the intracellular Bio-SeNPs, and 147 nm for the extracellular ones. Low value of negative potentials suggest a lower stability of these nanostructures in solution. On the contrary, the Gram-negative bacterium SeITE02 did not present drastic changes in the macromolecular composition after SeO32- exposure, and the variations recorded were mainly due to the maintenance of vital cellular functions. DLS data revealed an average size of 315 nm for the intracellular Bio-SeNPs and 160 nm for the extracellular ones, whilst negative potential values at or below -30 mV were recorded, indicating a remarkable stability of these biogenic Se nanostructures and a lower tendency to form aggregates and thus to precipitate. Thus, the results obtained in the course of this investigation revealed two distinct attitudes and responses on the part of the two microorganisms SeITE01 and SeITE02 to the SeO32- exposure and Bio-SeNPs production.