Delayed formation of zero-valent selenium nanoparticles by Bacillus mycoides SeITE01 as a consequence of selenite reduction under aerobic conditions

Abstract

Background: Selenite (SeO32-) oxyanion shows severe toxicity to biota. Different bacterial strains exist that are capable of reducing SeO32- to non-toxic elemental selenium (Se0), with the formation of Se nanoparticles (SeNPs). These SeNPs might be exploited for technological applications due to their physico-chemical and biological characteristics. The present paper discusses the reduction of selenite to SeNPs by a strain of Bacillus sp., SeITE01, isolated from the rhizosphere of the Se-hyperaccumulator legume Astragalus bisulcatus.

Results: Use of 16S rRNA and GyrB gene sequence analysis positioned SeITE01 phylogenetically close to B. mycoides. On agarized medium, this strain showed rhizoid growth whilst, in liquid cultures, it was capable of reducing 0.5 and 2.0 mM SeO32- within 12 and 24 hours, respectively. The resultant Se0 aggregated to form nanoparticles and the amount of Se0 measured was equivalent to the amount of selenium originally added as selenite to the growth medium. A delay of more than 24 hours was observed between the depletion of SeO32 and the detection of SeNPs. Nearly spherical-shaped SeNPs were mostly found in the extracellular environment whilst rarely in the cytoplasmic compartment. Size of SeNPs ranged from 50 to 400 nm in diameter, with dimensions greatly influenced by the incubation times. Different SeITE01 protein fractions were assayed for SeO32- reductase capability, revealing that enzymatic activity was mainly associated with the membrane fraction. Reduction of SeO32- was also detected in the supernatant of bacterial cultures upon NADH addition.

Conclusions: The selenite reducing bacterial strain SeITE01 was attributed to the species Bacillus mycoides on the basis of phenotypic and molecular traits. Under aerobic conditions, the formation of SeNPs were observed both extracellularly or intracellularly. Possible mechanisms of Se0 precipitation and SeNPs assembly are suggested. SeO32- is proposed to be enzymatically reduced to Se0 through redox reactions by proteins released from bacterial cells. Sulfhydryl groups on peptides excreted outside the cells may also react directly with selenite. Furthermore, membrane reductases and the intracellular synthesis of low molecular weight thiols such as bacillithiols may also play a role in SeO32- reduction. Formation of SeNPs seems to be the result of an Ostwald ripening mechanism.

Figure 1

Neighbour-joining tree inferred through MEGA 5.0 software [61] based on the sequences of 16S rRNA gene, showing the phylogenetic relationship of strain SeITE01 and related species. Bootstrap values are shown for nodes that had >50% support in a bootstrap analysis of 1000 replicates. The scale bars indicate the number of substitutions per nucleotide position.

Figure 2

Neighbor-joining tree inferred through MEGA 5.0 software [61] based on the sequences of GyrB gene, showing the phylogenetic relationship of strain SeITE01 and related species. Bootstrap values are shown for nodes that had >50% support in a bootstrap analysis of 1000 replicates. The scale bars indicate the number of substitutions per nucleotide position.

Figure 3

Growth of Bacillus SeITE01 on agarized medium in absence (A) and presence (B) of 2.0 mM selenite.

Figure 4

Time courses of bacterial growth, SeO₃²⁻ depletion, and Se⁰ formation by B. mycoides SeITE01, in presence of (A) 0.5, and (B) 2.0 mM SeO₃²⁻. Each curve shows means based on the results of three experiments. Minor ticks (5-hours range) are inserted in the Time axis.

Figure 5

TEM micrographs and EDAX spectra of B. mycoides SeITE01 cultures grown in presence of 2 mM SeO₃²⁻ registered at different incubation times. 12 (A and B) and 24 (C and D) hours. Arrows point electron-dense particles (indicated by number 1 and 2), whose corresponding EDAX spectra are given on the bottom of the micrographs (E and F).

Figure 6

SEM micrographs and EDAX spectra of B. mycoides SeITE01 cultures grown in absence (A), or in presence of 2.0 mM SeO₃²⁻, at increasing incubation times: 6 (B), 24 (C), and 48 (D) hours, in panel (E) and (F) are shown EDAX spectra corresponding to control and 24 hours SeITE01 culture, respectively. SE and BSE stand for Secondary Electrons and Back-Scattered Electrons signal, respectively.

Figure 7

Time dependence of the UV–vis spectrum of SeNPs collected at different incubation times: (blue line) 6 h, (red line) 24 h, and (green line) 48 h.

Figure 8

Selenite reduction assay carried out on protein fractions (shown at the top) and on supernatant, boiled and not boiled, (shown at the bottom) of SeITE01 liquid cultures. All tests were done in duplicate (indicated by roman numbers), with addition of 2.0 mM SeO₃²⁻ and 2.0 mM NADH. Three negative controls were set up: without protein fractions or supernatant, without selenite, without NADH.

Figure 9

Hypothesis of SeNPs formation in Bacillus mycoides SeITE01. A - Synoptic schematization of proposed biogenesis mechanisms of zero-valent selenium nanoparticles in Bacillus mycoides SeITE01. (1) Cytosolic precipitation of SeO₃²⁻ as Se⁰ nanoparticles due either to the possible activity of LMW thiols including bacillithiol (BSH) or to the Trx/TrxRed system. (2) Intracellular selenite reduction and formation of SeNPs as a consequence of presumable activity of membrane reductases. (3) Release of intracellularly generated SeNPs after cell lysis. (4) Membrane reductases may even catalyze extracellular selenite precipitation. (5) Peptides and other compounds carrying thiol groups may be released from the bacterial cell and directly react with selenite. (6) Evidence of the formation of SeNPs by Bacillus mycoides SeITE01 culture supernatant spiked with SeO₃²⁻ only after NADH addition can be due to the presence of extracellular proteins capable of mediating selenite precipitation once provided with reducing equivalents. (7) Nascent SeNPs are inherently unstable due to their high surface area and therefore tend to grow and increase their average size to attain a lower-energy state by means of an Ostwald ripening mechanism. B - Suggested mechanism of selenite detoxification in Bacillus sp. involving Brx-like proteins, according to [55].