Investigation on reduction selenium oxyanions, an enigmatic reaction in the biogeochemical selenium cycle.

Selenium (Se) is a naturally occurring metalloid element, which is essential to human and other animal health in trace amounts but is harmful in excess (Barceloux, 1999).Of all the elements, selenium has one of the narrowest ranges between dietary deficiency (<40μg day−1) and toxic levels (>400μg day−1) (WHO 1996), which makes it necessary to carefully control intakes by humans and other animals, hence, the importance of understanding the relationships between environmental exposure and health. Because diet is the most important source of selenium in humans, understanding the biogeochemical controls on the distribution and mobility of environmental selenium is key to the assessment of selenium-related health risks (Raab, 2000). High selenium concentrations are associated with some phosphatic rocks, organic-rich black shales, coals, and sulfide mineralization, and with anthropogenic sources of industrial and agricultural activities (Fernandez-Martez et al., 2009; Vinceti et al, 2001). However, health outcomes are not only dependent on the total selenium content of rocks and soils but also on the amount of selenium taken up into plants and animals—the bioavailable selenium. In the environment, selenium occurs in a variety of oxidation state: in particular the water soluble oxyanions selenite (SeO32-) and selenate (SeO42-) are the predominant Se species in aerobic environment and they are also the forms with the highest toxicity level (Zannoni et al., 2008; Shrift, 1964). Among these, selenite is the most toxic inorganic selenium. (Frankenberger et al., 1998; Jayaweera et al.,1996) The lifetime of selenite in soils is closely associated with the microbial activity. In particular, the process of selenite reduction to Se(0) is of great significance for its bioremediation and geochemical cycles (Ma et al., 2007;). A wide variety of microorganisms can reduce selenite to elemental selenium in aerobic and anaerobic condition (Li et al., 2013; Hunter et al., 2008; Hunter et al., 2007; Mishra et al., 2011; Blum et al., 1998; Lortie et al., 1992). In a few microorganisms the reduction of SeO32- can even serve as a respiratory process; though, in the majority of microorganisms studied SeO32- reduction has no apparent respiratory function (Pierru et al., 2006). In biotic reduction pathways, several types of bacteria have been reported to play a role in the detoxification of Se by reducing selenate or selenite to elemental Se (Ike et al., 2000; Tomei et al., 1995).Neverthlesses, the reduction aerobic processes indicated as “detoxification reduction” by which the microbial reduction of selenite is accomplished are poorly understood. Aim of this PhD work is entire in the understanding of the aerobic reduction mechanisms in the biogeochemical cycle of selenium and to shed a new light on the biological “detoxification mechanism” activated by Stenotrophomonas spp strains, improving the knowledges of genetics and biochemistry involved in the selenium pathway of this microorganism. In the present work, Stenotrophomonas maltophilia SelTE02, a strain isolated from the rhizosphere of the selenium hyperaccumulator Astragalus bisulcatus, was analyzed. The strain was able to reduce selenite to elemental selenium in aerobic conditions: this ability offers the possibility of exploitation of this strain in remediation protocols for the treatment of selenium-bearing wastewaters (Di Gregorio et al., 2005). In the first phase of this project, strain SelTE02 was compared with other four environmental strains of Stenotrophomonas spp isolated from contaminated soils. The strains analyzed, in addition to SelTE02, were: - A16: isolated from soil collected from the Ex-SLOI area in Trento-Nord and was obtained by enrichment cultures added with organic lead. - AW, B, T: isolated from soil collected from Scarlino industrial site and were obtained by enrichment cultures added with arsenite. The physiological characterization of these five Stenotrophomonas spp. strains involved the measurement of LD50 (lethal dose 50), selenite reduction efficiency and elemental selenium production. The strain SelTE02 showed the best performance in the selenite reduction efficiency and elemental selenium productionIn addition, in each one of the strains, we observed a gap between selenite reduction and elemental selenium production: in fact, the elemental selenium production rate is always lower than selenite consumption rate and the maximum gap between elemental selenium production and selenite consumption is at 24 hours (except for strain A16), during early stationary phase.The different rate between the elemental selenium production compared to the disappearance of selenite was related not only with the number of microbial cells but also is depending on the growth phases. The selenium nanoparticles formation was investigated with using Transmission Electron Microscope (TEM). The TEM images of the time course show the presence of extremely rare Se particles before 11h from the culture start, according with the biochemical findings. The number of selenium nanoparticles increase over time and the pictures show that reach a maximum number already at 24 h of the growth, in the early stationary phase. Moreover, the TEM analysis indicate the presence of the selenium nanoparticles only in the external part of the cells, suggesting that the nanoparticles are probably form outside the cell. In the second part of the work, the esopolisaccharides (EPS) production and the correlation with the selenite reduction were investigated in the five strains. Strain A16, the most tolerant to selenite, is also the strain that produces the highest amount of exopolysaccharides, while strain SelTE02, the less tolerant isolate, is the strain that produces the lowest quantity of EPS. The positive correlation between the production of EPS and the selenite resistance was confirmed from the analysis of the LD50 data corroborating an interaction between the production of EPS and resistance of all strains to selenite oxyanions. The EPS role in the reduction was performed with EPSs extract from the five strains in “in vitro” reduction assay where the reduction activity has been highlighted because the red selenium precipitate. EPSs extract of A16, SeITE02 and AW strains were able to reduce selenite to elemental selenium forming red nanoparticles while EPS extracts of strain B and T did not show any activity. For the strain SeITE02 at least two different reduction mechanisms it could be postulated to reduce the toxic selenite oxianions to non toxic elemental selenium: inside the cell by the cellular enzymatic machinery and an ancillary catalytic activity implemented by the EPS outside of the cells. The strain B forms EPS unable to form red elemental selenium in vitro: but the correlation between EPS and resistance is clear for this strain too. In this strain nanoparticles are detected inside the cells and seems that the selenium nanoparticles are growing in the cells. For this reason it could be hypothesized a selenite reducing mechanism exclusively inside the cell and the EPS could contribute to protect cells from hostile environments maybe binding a significant amounts of toxic selenite. In the last part of the work, in order to go deeper in the heart of the selenite reaction, proteomic analysis of the cytoplasmic protein fraction of Stenotrophomonas maltophilia strain SelTE02 was carried out, to investigate the enzyme(s) involved in the selenite reduction: the most interesting suggestion, arising from the proteomic analysis, concerns the identification of an enzyme involved in the mercury cycle. This finding has opened the way to new research that explores the possible interaction between mercury and mechanism for the reduction of selenite. Finally, the whole genome of Stenotrophomonas maltophilia SeITE02 was performed and analyzed to detect known enzymes correlated with selenite reduction and with the proteomic results.