Raman spectroscopic and TEM monitoring of selenite and selenate reduction by the bacterium Azospirillum thiophilum with the formation of selenium(0) nanoparticles: Effects of sulfate.

Microbial reduction of selenium oxyanions, highly soluble, mobile and toxic inorganic selenium compounds, to insoluble selenium nanoparticles (Se NPs) is a widely spread phenomenon which is of geochemical, environmental and biotechnological importance. While selenite bioreduction is known for a wide variety of microorganisms, selenate bioreduction is not so common and has mostly been documented for anaerobes, with merely a few reported cases related to aerobic or microaerobic conditions. In some biogenic Se NPs of microbial origin, the presence of sulfur was detected together with selenium in Se NPs, particularly when increased concentrations of sulfate were present in the medium. In this work, the bacterial strain Azospirillum thiophilum BV-S, isolated earlier from a sulfur-containing aqueous environment (Lavrinenko et al. (2010), https://doi.org/10.1099/ijs.0.018853-0), has been shown to reduce selenite to Se NPs also in the presence of 7 mM sulfate in aerobic conditions. Raman spectroscopy was used to monitor the crystallinity and composition of Se NPs formed within the bacterial biomass in the presence of selenite and in the resulting isolated Se NPs, and their spherical morphology was visualised using transmission electron microscopy (TEM). While Se NPs both within the biomass and after isolation gave a typical strong broadened band at 248 cm related to the stretching Se-Se vibrations in amorphous Se, Raman spectrum of the biomass grown with 1 mM selenite + 7 mM sulfate showed also a weaker band at 348 cm typical of the stretching Se-S mode. The absence of the latter band in Raman spectra of the isolated Se NPs indicates that the Raman-detected Se-S bonds most probably occur in intermediate substances such as selenodiglutathione (GS-Se-SG), an intermediary product in the Painter-type reaction of selenite reduction, which is known to undergo further enzymatic transformations in bacteria resulting in the formation of Se. Strain A. thiophilum BV-S has also been found, for the first time for bacteria of the genus Azospirillum, to be capable of reducing selenate (SeO) under static conditions (similar to microaerobic conditions common to habitats of many Azospirillum species in different environments), but not in aerobic conditions, with the formation of Se NPs. The latter, giving a reddish coloration to the bacterial biomass, were characterised by TEM as round-shaped electron-dense structures over bacterial cell images. Raman spectra of the bacterial biomass after cultivation with 5 mM selenate (with or without added sulfate) showed a similar single band at ca. 248 cm in amorphous Se NPs, although 5 mM sulfate slowed down their formation. However, no signs of sulfur covalently bound to Se were observed in Raman spectra in this case. These results indicate a possible negligible role of the selenodisulfide-involving pathway in selenate reduction by A. thiophilum with a noticeable possible interference of sulfate in the process of selenate uptake (i.e., a common transport route) by A. thiophilum cells.