A Comparative Study of the Synthesis and Characterization of Biogenic Selenium Nanoparticles by Two Contrasting Endophytic Selenobacteria

Abstract

The present study examined the biosynthesis and characterization of selenium nanoparticles (SeNPs) using two contrasting endophytic selenobacteria, one Gram-positive (Bacillus sp. E5 identified as Bacillus paranthracis) and one Gram-negative (Enterobacter sp. EC5.2 identified as Enterobacter ludwigi), for further use as biofortifying agents and/or for other biotechnological purposes. We demonstrated that, upon regulating culture conditions and selenite exposure time, both strains were suitable "cell factories" for producing SeNPs (B-SeNPs from B. paranthracis and E-SeNPs from E. ludwigii) with different properties. Briefly, dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) studies revealed that intracellular E-SeNPs (56.23 ± 4.85 nm) were smaller in diameter than B-SeNPs (83.44 ± 2.90 nm) and that both formulations were located in the surrounding medium or bound to the cell wall. AFM images indicated the absence of relevant variations in bacterial volume and shape and revealed the existence of layers of peptidoglycan surrounding the bacterial cell wall under the conditions of biosynthesis, particularly in the case of B. paranthracis. Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray (EDS), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) showed that SeNPs were surrounded by the proteins, lipids, and polysaccharides of bacterial cells and that the numbers of the functional groups present in B-SeNPs were higher than in E-SeNPs. Thus, considering that these findings support the suitability of these two endophytic stains as potential biocatalysts to produce high-quality Se-based nanoparticles, our future efforts must be focused on the evaluation of their bioactivity, as well as on the determination of how the different features of each SeNP modulate their biological action and their stability.

Keywords: Bacillus paranthracis; Enterobacter ludwigii; biofortification; biogenic nanoparticles; selenite; selenium.

Figure 1

TEM images of cells of Bacillus sp. E5 (a1,a2) and Enterobacter sp. EC5.2 (b1,b2) after 6 h of growth in the presence of 5 mM Na₂SeO₃. Red arrows indicate SeNPs located on the cell surface, whereas green arrows indicate SeNPs located in the surrounding medium. Purple frames show components actively secreted by bacterial cells. Blue frames on image (a2) show nanorods (SeNRs). Images (a3,b3) show bacterial cells grown without selenite.

Figure 2

AFM images of Bacillus sp. E5 (a) and Enterobacter sp. EC5.2 (b) after 6 h of growth in the absence (a1,a2,b1,b2) or in the presence (a3,a4,b3,b4) of 5 mM Na₂SeO₃. The first column of each bacterial strain shows amplitude images (a1,a3,b1,b3), whereas topography images are shown in the second column (a2,a4,b2,b4). Red arrows indicate SeNPs attached to the cell wall, whereas green arrows indicate SeNPs located in the surrounding medium. The white arrows correspond to the collapsed cell envelope (see details in main text). The blue arrows correspond to bacterial filaments.

Figure 3

TEM observation of the purified SeNPs produced by Bacillus sp. E5 (a) and Enterobacter sp. EC5.2 (b) showing their spherical shape. The size distribution of SeNPs produced by Bacillus sp. E5 (c) and Enterobacter sp. EC5.2 (d).

Figure 4

Characterization of SeNPs synthesized by Bacillus sp. E5 (B-SeNPs) and Enterobacter sp. EC5.2 (E-SeNPs). Two-dimensional AFM topographical image of B-SeNPs (a) and E-SeNPs (b). The graphics below (c,d) show the AFM histogram of B-SeNP and E-SeNP diameters.

Figure 5

XRD pattern of SeNPs synthesized using Bacillus sp. E5 (B-SeNPs) and Enterobacter sp. EC5.2 (E-SeNPs).

Figure 6

Raman spectrum at 532 nm excitation wavelength of SeNPs synthesized by Bacillus sp. E5 (B-SeNPs) and Enterobacter sp. EC5.2 (E-SeNPs) and incubated with 5 mM of sodium selenite within 6 h. The signal of Se–Se vibrations was observed at 254 cm⁻¹.

Figure 7

FTIR spectrum of bacterial cells compared to the one obtained from purified SeNPs (a,b) and the FTIR spectrum of both SeNPs synthesized by Bacillus sp. E5 (B-SeNPs) and Enterobacter sp. EC5.2 (E-SeNPs) (c).

Figure 8

XPS spectra: (a) B-SeNPs and (b) E-SeNPs.