Time-Resolved Examination of Fungal Selenium Redox Transformations

Abstract

Selenium (Se) is both a micronutrient required for most life and an element of environmental concern due to its toxicity at high concentrations, and both bioavailability and toxicity are largely influenced by the Se oxidation state. Environmentally relevant fungi have been shown to aerobically reduce Se(IV) and Se(VI), the generally more toxic and bioavailable Se forms. The goal of this study was to shed light on fungal Se(IV) reduction pathways and biotransformation products over time and fungal growth stages. Two Ascomycete fungi were grown with moderate (0.1 mM) and high (0.5 mM) Se(IV) concentrations in batch culture over 1 month. Fungal growth was measured throughout the experiments, and aqueous and biomass-associated Se was quantified and speciated using analytical geochemistry, transmission electron microscopy (TEM), and synchrotron-based X-ray absorption spectroscopy (XAS) approaches. The results show that Se transformation products were largely Se(0) nanoparticles, with a smaller proportion of volatile, methylated Se compounds and Se-containing amino acids. Interestingly, the relative proportions of these products were consistent throughout all fungal growth stages, and the products appeared stable over time even as growth and Se(IV) concentration declined. This time-series experiment showing different biotransformation products throughout the different growth phases suggests that multiple mechanisms are responsible for Se detoxification, but some of these mechanisms might be independent of Se presence and serve other cellular functions. Knowing and predicting fungal Se transformation products has important implications for environmental and biological health as well as for biotechnology applications such as bioremediation, nanobiosensors, and chemotherapeutic agents.

Figure 1

Net dry biomass weights (mg) for A. alternata (top panel) and P. sporulosa (bottom panel) grown in the presence of 0.1 mM Se(IV), 0.5 mM Se(IV), or no added Se. Points are averages of replicate cultures, where bars represent the range of measured values for the two replicates. Points where no bars are visible have ranges smaller than the vertical point size. Dashed lines from time point zero are inferred, as the actual biomass at the start of the experiment was too low to be measured (∼0 mg at t = 0).

Figure 2

Aqueous Se(IV) content (μmol) remaining in solution over time for A. alternata (squares) and P. sporulosa (triangles) grown in the presence of 0.1 mM Se(IV) (light blue) and 0.5 mM Se(IV) (dark blue). Gray diamonds represent fungi grown in the absence of Se (biological controls). Points are shown as the average of the replicate cultures, where bars represent the range of measured values for the two replicates.

Figure 3

Relative percentages of Se pools (aqueous, solid, volatile) throughout 32 days of growth for A. alternata (left panels) and P. sporulosa (right panels) in media supplemented with (a, b) 0.1 mM Se(IV) and (c, d) 0.5 mM Se(IV), respectively.

Figure 4

Relative abundance (%) of solid-associated Se species, as determined by linear combination fitting of Se K-edge EXAFS spectra, throughout 32 days of growth for A. alternata and P. sporulosa in media supplemented with 0.1 mM Se(IV) (top panels) or 0.5 mM Se(IV) (bottom panels). Selenium species shown represent those fractions >5% of total Se species. Missing data points for t = 32 days were due to flask contamination.

Figure 5

Transmission electron microscopy (TEM) images of cross sections through fungal hyphae of A. alternata (a, b) and P. sporulosa (c, d, e) showing electron dense SeNPs. (a) Black arrows highlight extracellular and intracellular SeNPs. (b) Extracellular aggregates and filaments of small SeNPs were characteristic for A. alternata. (c) Spherical nature of larger, individual SeNPs produced by P. sporulosa. (d) SeNPs embedded in the cell wall and (e) aligned along the interior of the cell membrane (black arrow) suggest that these particles could be expelled outside the cell.