Reduction of selenite to Se(0) nanoparticles by filamentous bacterium Streptomyces sp. ES2-5 isolated from a selenium mining soil
Tan et al. Microb Cell Fact
Reduction of selenite to Se(0) nanoparticles by filamentous bacterium Streptomyces sp. ES2-5 isolated from a selenium mining soil
Yuanqing Tan 0
Rong Yao 0
Rui Wang 0
Dan Wang 0
Gejiao Wang 0
Shixue Zheng 0
0 State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University , Wuhan 430070 , People's Republic of China
Background: Selenium (Se) is an essential trace element in living systems. Microorganisms play a pivotal role in the selenium cycle both in life and in environment. Different bacterial strains are able to reduce Se(IV) (selenite) and (or) Se(VI) (selenate) to less toxic Se(0) with the formation of Se nanoparticles (SeNPs). The biogenic SeNPs have exhibited promising application prospects in medicine, biosensors and environmental remediation. These microorganisms might be explored as potential biofactories for synthesis of metal(loid) nanoparticles. Results: A strictly aerobic, branched actinomycete strain, ES2-5, was isolated from a selenium mining soil in southwest China, identified as Streptomyces sp. based on 16S rRNA gene sequence, physiologic and morphologic characteristics. Both SEM and TEM-EDX analysis showed that Se(IV) was reduced to Se(0) with the formation of SeNPs as a linear chain in the cytoplasm. The sizes of the SeNPs were in the range of 50-500 nm. The cellular concentration of glutathione per biomass decreased along with Se(IV) reduction, and no SeNPs were observed in different sub-cellular fractions in presence of NADPH or NADH as an electron donor, indicating glutathione is most possibly involved in vivo Se(IV) reduction. Strain ES2-5 was resistant to some heavy metal(loid)s such as Se(IV), Cr(VI) and Zn(II) with minimal inhibitory concentration of 50, 80 and 1.5 mM, respectively. Conclusions: The reducing mechanism of Se(IV) to elemental SeNPs under aerobic condition was investigated in a filamentous strain of Streptomyces. Se(IV) reduction is mediated by glutathione and then SeNPs synthesis happens inside of the cells. The SeNPs are released via hypha lysis or fragmentation. It would be very useful in Se bioremediation if Streptomyces sp. ES2-5 is applied to the contaminated site because of its ability of spore reproduction, Se(IV) reduction, and adaptation in soil.
Actinobacteria; Glutathione; Selenium nanoparticles (SeNPs); Intracellular deposition; Export system; Aerobe
Selenium (Se) is an essential trace element for the
adequate and healthy life of human, animal, bacterium and
other living systems and has an uneven distribution in
the Earth’s crust [
]. Today, selenium is well recognized
to play fundamental roles on several physiological
functions in diverse organisms, such as biosynthesis of
selenocysteine (Sec), the 21st amino acid with specific UGA
stop codon, and many selenoenzymes including formate
dehydrogenase, thioredoxin reductase, and glutathione
]. In human, either Se excess or
deficiency results in more than 20 kinds of symptoms such
as growth retardation, endemic diseases, impaired bone
metabolism and risk of diabetes [
]. Events of selenium
toxicity in human have been reported in Enshi, Hubei
province of China and in Indian Punjab [
selenium contamination requires bioremediation
initiatives especially in those geographic locations.
Phylogenetically diverse microorganisms are involved in the
transformation of selenium from one oxidation state to
another and thus play a pivotal role on the selenium
biogeochemical cycle [
4, 7, 8
]. Numerous bacteria are able
to reduce the toxically soluble forms of Se(VI)/Se(IV) to
less-toxic insoluble Se(0), visible as red-colored
nanoparticles (SeNPs) [
]. The biosynthesized SeNPs have
been found applications in various fields including
medicine as antimicrobial, antioxidant and anticancer agents
], biosensors [
] and environmental
Se(IV)-reducing bacteria generate SeNPs under
aerobic and anaerobic conditions. Anaerobic Se(IV)-reducing
bacteria encompassed many species such as Thauera
], Aeromonas salmonicida [
nonsulfur bacteria [
] and Shewanella oneidensis MR-1 [
Aerobic Se(IV)-reducing bacteria included diverse
species such as Rhizobium sp. B1 [
maltophilia SeITE02 [
seleniipraecipitans CA5 [
], Duganella sp. and Agrobacterium sp. [
Comamonas testosteroni S44 [
] and Bacillus mycoides
]. Therefore, the most Se(IV)-reducing bacteria were
distributed in alpha-, beta-, gamma-,
delta-proteobacteria and Firmicutes.
Selenium nanoparticles were formed not only under
aerobic and anaerobic conditions, but also appeared in
the cytoplasm, periplasm and/or outside the cells in
different bacteria [
4, 9, 10, 13, 14, 24, 29, 31
], implying the
various mechanisms of Se(IV)-reduction in diverse
microbes. One of mechanisms linking redox
precipitation of both elemental sulfur and elemental selenium
was observed outside sulfate-reducing bacterial cells
. The intracellular Se(IV) reduction was usually
driven by reduced thiols such as glutathione (GSH) via
the Painter reaction in Rhodospirillum rubrum,
Escherichia coli and Bacillus mycoides [
28, 30, 33, 34
diverse enzymes were responsible for Se(IV) reduction to
SeNPs. The periplasmic nitrite reductase was involved in
Se(IV) reduction in T. selenatis  and Rhizobium
], while fumarate reductase catalyzed
Se(IV) reduction in Shewanella oneidensis [
addition, glutathione reductase and thioredoxin reductase in
Pseudomonas seleniipraecipitans [
reductase in Bacillus selenitireducens [
] and hydrogenase in
Clostridium pasteurianum [
] were potentially involved
in Se(IV) reduction. However, so far no gene product or
enzyme solely responsible for Se(IV) reduction in aerobic
bacteria has been identified in vivo.
In addition, the efflux system by which Se(0) or
SeNPs deposits were exported from inside the cells to
the extracellular environment still remains unknown.
It was suggested that SeNPs were released into the
medium via a rapid expulsion process [
] or elemental
Se(0) was transported out of the cell where the SeNPs
were formed [
]. The large sizes of SeNPs were also
possibly released by cell lysis [
] or vesicular
In this study, we isolated a filamentous
actinobacterium ES2-5 from a selenium mining soil in Enshi, Hubei
province of China. The process of Se(IV) reduction
leading to biosynthesized SeNPs under aerobic condition was
investigated using scanning electron microscopy (SEM),
transmission electron microscopy (TEM) and electron
dispersion spectroscopy (EDX). Evidences were provided
for the SeNPs formation to be mainly in the cytoplasm of
cells and then released through hyphal lysis or
fragmentation. The possible mechanism of Se(IV) reduction was
Characteristics and taxonomic identification of the strain
Strain ES2-5 was isolated from a selenium mine soil
in Hubei province, China. The acidic soil (pH 4.7) had
38 mg kg−1 of total Se content and 119 mg kg−1 of total
Cr content. Accordingly, the resistance of strain ES2-5
to Se(IV), Cr(VI) and other heavy metals was
determined in 1/10 TSA plates. The minimal inhibitory
concentrations (MIC) of Se(IV) and Cr(VI) were 50 and
80 mM, respectively. In contrast, the MICs of Zn(II)
(1.5 mM), Cu(II) (0.2 mM), As(III) (0.05 mM) and
Sb(III) (0.08 mM) were lower than that of Se(IV) and
Cr(VI). In addition, strain ES2-5 has the ability to
produce lecithinase and H2S when it grew in TSB. It was
positive for motility and hemolytic reaction, whereas it
was negative for utilization of citrate and hydrolysis of
gelation and tyrosine.
The 16S rRNA gene sequence of strain ES2-5 (1487 bp)
revealed highest similarities to that of Streptomyces
siamensis KC-038T (98.77 %), S. kanamyceticus NBRC
13414T (98.63 %), S. olivochromogenes NBRC 3178T
(98.63 %), S. aureus NBRC 100912T (98.56 %), S.
spirocerticilatus NBRC 12821T (98.43 %) and S.
albiflavescens n20T (98.39 %). Phylogenetic analyze using the
neighbor-joining method showed that strain ES2-5 fell in
the same cluster with S. siamensis KC-038T (AB773848)
and S. albiflavescens n20T (KC771426C) (Fig. 1).
Moreover, strain ES2-5 formed grey, floury colonies on 1/10
TSA plates (Fig. 2a), with well growing substrate
mycelia, aerial hypha and sporophores. Consequently, strain
ES2-5 was characterized as Streptomyces sp. based on
the phylogenetic, morphologic and some physiologic
85 Streptomyces sanglieri NBRC 100784T AB249945
99 Streptomyces atratus NRRL B-16927T DQ026638
Streptomyces pulveraceus LMG 20322T AJ781377
Streptomyces violascens NBRC 12920T AB184246
Streptomyces spiroverticillatus NBRC 12821T AB249921
Streptomyces umbrinus NBRC 13091T AB184305
Streptomyces kanamyceticus NBRC 13414T AB184388
Streptomyces aureus NBRC 100912T AB249976
96 Streptomyces durmitorensis MS405T DQ067287
Streptomyces siamensis KC-038T AB773848
Streptomyces albiflavescens n20T KC771426
Streptomyces olivochromogenes NBRC 3178T AB184737
Streptomyces chartreusis NBRC 12753T AB184839
Streptomyces similanensis KC-106T AB773850
Fig. 1 Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences using MEGA software version 5, showing the phylogenetic
relationship of strain ES2-5 and related type strains. Bootstrap values >50 % based on 1000 replications are shown at branch nodes. Bar, 0.002 substitutions
per nucleotide position
Filamentous Streptomyces sp. ES2‑5 was able to reduce
Se(IV) to SeNPs under aerobic condition
Streptomyces sp. ES2-5 was not able to grow under
anaerobic condition, indicating it is an obligate aerobe.
Streptomyces sp. ES2-5 formed reddish colonies after
7 day’s incubation on 1/10 TSA plates amended with
10.0 mM selenite (Fig. 2b). The stained mycelia were
observed in situ by light microscopy after 3 day’s
incubation, the red-colored selenium particles were scattered
away from mycelia or distributed as bean chains
attaching on the mycelial surface (Fig. 2d). After 3 day’s
incubation in 1/10 TSB broth, the mycelia were harvested
and observed by SEM. Surprisingly, the selenium
particles did not attach on the surface of mycelia but located
in the mycelia as mature beans in pods (Fig. 2f ). TEM of
ultra-thin sections also revealed the common presence of
intracellular Se(0) particles when mycelia were grown on
Se(IV) (Fig. 3b–d). It was clear that the sizes of
intracellular SeNPs varied from 50 to 500 nm and small SeNPs
may aggregate into bigger particles. Dark, fine-grained
nanoparticles were observed by EDX spectra which
indicated that these nanoparticles were composed entirely
of selenium as the expected emission peaks for selenium
at 1.37 (Fig. 3e, f ), 11.22 and 12.49 keV (data not shown)
corresponding to the SeLα, SeKα, and SeKβ transitions,
respectively, but EDX peaks for C, K, O, P, Cl and Ca
were also produced, suggesting that these elements were
in cytoplasm of cells.
The growth and capability of Streptomyces sp.
ES2-5 to transform selenite to elemental selenium
were tested in 1/10 TSB broth with the addition of
1.0 mM selenite (Fig. 4). On selenite-exposed cultures
the growth was delayed with respect to controls, but
after 24 h the biomass decreased gradually in a similar
way. The formation of red cell suspension of
elemental selenium started after 16 h of exposure to selenite.
Streptomyces sp. ES2-5 was unable to reduce Se(IV)
to elemental selenium completely. It was only able to
reduce 1.0 mM Se(IV) to 0.5 mM slowly and smoothly
during 52 h incubation in 1/10 TSB broth under
Mechanism of Se(IV) reduction to Se(0) nanoparticles by Filamentous Streptomyces sp. ES2‑5
To help understand how Se(IV) is reduced, the ability
of vitro Se(IV) reduction by cultural supernatant and
different cellular fractions was determined. When
cultural supernatant without cells was mixed with Se(IV),
the reduction of Se(IV) to red-colored precipitation and
decrease of Se(IV) concentrations were not observed,
indicating the reduction of Se(IV) is processed in cells.
Moreover, neither red-colored precipitation nor decrease
of Se(IV) concentrations appeared in the cytoplasmic
fraction or in cell membrane fraction with NADPH or
NADH as electron donors. These results suggest that
NADPH or NADH dependent reductase and reduced
chemicals are not involved in vitro Se(IV) reduction.
Consequently, the concentrations of glutathione (GSH)
per biomass in cells (intracellular) and in cultural broth
(extracellular) were determined when Streptomyces sp.
ES2-5 grew in 1/10 TSB broth at 1.0 mM concentration
In selenite-exposed cultures the intracellular GSH
content per biomass was lower than in controls during first
24 h of incubation (Fig. 5a). While, the extracellular GSH
content showed an opposite pattern (Fig. 5b). After 24 h
the intercellular and extracellular GSH contents in
selenite-exposed cultures were similar to controls.
Although the reduction of Se oxyanions to Se(0)
nanoparticles by microorganisms has been known for some
4, 9, 10, 13, 14, 24, 29, 38, 39
], the SeNP-synthetic
process and Se(IV)-reducing mechanism of filamentous
bacteria have not been examined previously. In this case
we found a typical actinomycete, Streptomyces sp. ES2-5,
has ability to reduce Se(IV) to Se(0) and forms SeNPs in
cells. TEM and EDX analyses showed that red-colored
SeNPs accumulated in the hyphae with a diameter range
of 50–500 nm. These bigger particles were aggregated by
small SeNPs and then arranged along with hyphal
cytoplasm as particle chains (Figs. 2, 3). It does not seem
possible that these large Se(0) particles in the cytoplasm
could have been derived from primary cytoplasmic
synthesis and met cellular assimilation. Such a system for
reduction of Se(VI) to Se(0) would be a detoxification
mechanism. This mechanism could result in an
incomplete selenite reduction under oxic growth conditions
during a limited time frame (Fig. 4), which is consistent
with a previous study in C. testosteroni [
]. Moreover, the
large Se(0) particle chains could be extremely unmatched
for hyphae, and thus the particle chains should be
released only upon hyphal lysis or fragmentation (Fig. 2d,
f ). This could be very easy for filamentous bacteria due to
the hyphal extending and branched growth. Similarly, the
release of large Se(0) particles from cytoplasm via cell lysis
can be observed in single-celled bacteria such as Bacillus
] and B. selenitireducens [
]. In comparison
with single-celled bacteria, it seems that Streptomyces sp.
ES2-5 was lack of the mechanism of SeNP size control. In
most cases, the diameters of SeNPs were <300 nm [
size of SeNPs was about 200 nm even in filamentous fungi
]. In Streptomyces sp. ES2-5, the size of larger SeNPs
reached 500 nm (Fig. 3). The large Se(0) particles may also
been formed by the aggregation of small particles during
the movement of the cytoplasmic flow, which cannot be
processed in single-celled bacteria and in eukaryotic cells
with functional zoning.
As the diverse reducing mechanisms of Se oxyanions,
it is very different from the reduction of As (such as ars
cluster) and S (dsr cluster) associated with a certain
enzymatic system in various bacteria [
]. There could be
multiple Se-reducing pathways in a strain, e.g., at least
two selenite reductases in P. seleniipraecipitans or in R.
]. In Shewanella oneidensis MR-1,
only 60 % selenite was reduced by reductase FccA [
suggesting that more pathways are responsible for Se(IV)
reduction in a bacterial strain. Among these reducing
determinants, reduced thiols could be involved in Se(IV)
reduction at more or less extent. Three bacterial groups
produce thiols encompassing glutathione (GSH) in
proteobacteria, bacillithiol (BSH) in Firmicutes and
mycothiol (MSH) in actinobacteria [
]. As a result, it is not
surprising that the most Se reducing bacteria distributed
in these thiols-rich groups. Although a few studies on
Se(IV) reduction in actinobacteria [
Se(IV)reducing actinobacteria would be examined in the future.
In this study, no red-colored precipitation was
observed in different sub-cellular fractions in presence
of NADPH or NADH as electron donors, suggesting
NADPH or NADH dependent Se(IV) reductase was not
responsible for Se(IV) reduction compared with
previous studies [
]. Moreover, the intracellular
concentration of GSH (could be MSH, an analog of GSH) per
biomass decreased with Se(IV) reduction (Fig. 5), i.e.,
the reduced intracellular GSH was consumed to reduce
Se(IV). In contrast, there were more extracellular thiols
in broth amended with selenite than in control (Fig. 5b),
indicating extracellular GSH was not involved in Se(IV)
reduction because of its oxidized state under aerobic
condition. Accordingly, the actinobacteria specific MSH
(analog of GSH) was possibly involved in Se reduction in
Streptomyces sp. ES2-5. Although the red-colored Se(0)
nanoparticles are confirmed by Se(IV) reduction, this fact
does not exclude the possibility that there are additional
reduced products such as selenides because Streptomyces
sp. ES2-5 has the ability to produce H2S. Se(IV) or Se(0)
also might be reduced to Se(-II) via metabolic pathway of
Many aerobic Se-oxyanions reducing bacteria were
isolated from Enshi where soil has a high content of
]. Diversely aerobic Se-oxyanions reducing
bacteria were also collected from different terrestrial soils
4, 13, 14, 29, 30
]. This is different from the aquatic
environments where anaerobic bacteria are responsible for
Se(VI)/Se(IV) reduction. In anaerobic bacteria, Se(VI)/
Se(IV) reduction is able to process on the surface of cells
which is similar to Fe(III)’s reduction in S. oneidensis
]. In contrast, it is a great challenge in aerobic
bacteria to reduce Se-oxyanions on surface of cells due to
oxygen prior to accept the electrons than Se(IV), or other
reducing determinants under oxidized stress in
extracellular environment. Consequently, reduction is mainly
processed in cells and then Se(0)/SeNP is exported or
released by cell lysis (in this case).
Recent studies showed SeNPs synthesized by
Streptomyces spp. had the anticancer activity [
]. It implies
the potential application of SeNPs from
actinobacteria. In addition, bioremediation of contaminated soils
needs aerobic microbes and anaerobic microbes.
Streptomyces sp. ES2-5 not only has ability to reduce soluble
Se(IV) into insoluble and less toxic SeNPs and to
produce branched hyphae and countless spores, but also can
adapt to selenite or chromate contaminated condition. Its
ability of reproduction and adaptation in soil would be
useful in Se/Cr bioremediation if Streptomyces sp. ES2-5
was applied to the contaminated site together with other
aerobic and anaerobic Se(IV)-reducing bacteria.
A filamentous bacterium Streptomyces sp. was involved
in reduction of Se(IV) to elemental SeNPs arranged in
linear chains in cells under aerobic condition. The
synthetic process of SeNPs and mechanism of Se(IV)
reduction were proposed. The sizes of intracellular SeNPs
varied from 50 to 500 nm and small SeNPs may aggregate
into bigger particles. The cellular concentrations of GSH
per biomass decreased along with Se(IV) reduction and
Se(IV)-reduction did not occur in different sub-cellular
fractions, showing that Se(IV) reduction was most
possibly mediated by GSH in the cytoplasm, and thus the
SeNPs were released via cell lysis or fragmentation.
The isolation, morphological and partial biochemical
characters of strain ES2‑5
The strain ES2-5 was isolated from a selenium mine
soil (30°17′54″ N, 109°28′16″ E) with 38 mg kg−1 of
total Se content in Hubei province, China and by serial
dilutions of the sample on 1/10 TSA (tryptic soy agar,
pH 7.3, Difco) containing 1 mM sodium selenite. After
2 days of incubation at 28 °C, colonies that developed a
reddish color on the initial isolation plates were
transferred to fresh media for further isolation, research and
Strain ES2-5 was inoculated in 1/10 TSA plates
supplemented with 10 mM sodium selenite. The plates
without sodium selenite were served as controls. Then, the
medium in plates was inserted with sterile glass slides
at a diagonal angle and placed at 28 °C. After 3 days of
cultivation, the cultures on the glass slide were fixed and
stained using crystal violet for 2 min and then washed by
water. After air-dried, the samples were observed using
Lecithinase enzyme activity, motility, hemolytic
reaction, anaerobic growth, utilization of citrate, hydrolysis of
gelation and tyrosine were tested using the conventional
method. Production of H2S was tested according to the
method described in [
16S rRNA gene sequencing and phylogenetic tree
The nearly-full 16S rRNA gene sequence of strain ES2-5
was amplified using 16S rDNA universal primers 27F
and 1492R following the genomic DNA extraction. The
accurate sequence of PCR product was acquired by
sequencing after T-A cloning with a pGEM-T Easy vector
(Promega). The 16S rRNA gene sequence was compared
with sequences available in the EzTaxon-e server [
and aligned with its close relatives using the CLUSTAL_X
]. Neighbor-joining tree was reconstructed
using MEGA version 5.0 software [
]. Distances were
calculated based on Kimura’s two-parameter method [
and bootstrap analysis was performed according to 1000
]. The 16S rRNA gene sequence was
registered as accession KF885787 in the GenBank database.
Growth, selenite resistance and reduction, and glutathione
In order to determine the minimal inhibitory
concentrations (MICs), strain ES2-5 was inoculated in 1/10 TSA
plates with different concentrations of Se(IV) (0, 1, 5, 10,
20, 50, 100, 150 mM) and Cr(VI), Zn(II), Cu(II), As(III)
and Sb(III) at 28 °C.
The growth curve was determined by inoculating strain
ES2-5 into 100 ml 1/10 TSB broth supplemented
without or with 1.0 mM sodium selenite at 28 °C with
shaking at 160 rpm. Cultures were taken at 4 h intervals and
centrifuged at 6000×g, 5 min. The supernatants (a) were
used to determine concentrations of extracellular GSH
and selenite. The pellet was washed twice with phosphate
buffer saline (PBS, pH 7.2) and re-suspended in the same
buffer. Then, the suspension was sonicated for 3 min and
centrifuged at 6000×g and 4 °C for 5 min. The
supernatants (b) were collected and used to measure the
concentrations of intracellular GSH and totally cellular proteins.
The determination for the content of GSH was performed
by using a fluorescence-based method as described in
]. The detailed process was realized as follows. 10 mM
naphthalene-2,3-dicarboxaldehyde (NDA) was dissolved
in dimethylsulfoxide (DMSO) and 50 mM Tris–HCl (pH
10.0). The NDA reagent reacts with amino and sulfhydryl
groups of GSH to form an adduct, which can be
measured by fluorescence signal (λ exc at 472 nM and λ em at
528 nM). The standard curve of the relationship between
concentrations of GSH and the value of fluorescence
signal was measured by using 100, 200, 300, 400 and 500 nM
reduced GSH standard solution. The GSH concentrations
of samples were calculated according to standard curve
and detected fluorescence value. The biomass of strain
ES2-5 was tested by measuring the contents of total
cellular proteins of samples using Coomassie brilliant blue
G-250 method with Bovine Serum Albumin (BSA) as
]. Selenite concentrations in the
supernatants (a) were measured by HPLC-HG-AFS (Beijing Tian
Instruments Co., Ltd., China) [
Selenite reduction activity assays in cultural supernatant
and cellular fractions
In order to determine the ability of vitro Se(IV)
reduction by cultural supernatant and different cellular
fractions, the culture was grown to log phase and centrifuged
at 6000×g, 5 min. The cultural supernatant was collected
and filtered by a filtration with 0.2 µm disks. The pellet
was washed twice with phosphate buffer saline (PBS, pH
7.2) and resuspended in the same buffer for sonication.
After sonication for 3 min, the cell lysate was centrifuged
at 6000×g for 5 min to remove the cell debris. Then, the
soluble supernatant was centrifuged at 20,000×g for
60 min to separate the cytoplasmic fraction and
membrane fraction. Selenite reductase activity was
determined using the following reaction mixture [
cultural supernatant, cytoplasmic or membrane fraction;
sodium selenite (final concentration 0.2 mM); NADPH
or NADH (final concentration 0.2 mM). The reaction
mixture was incubated at 28 °C for 24 h. Reaction
mixture without addition of cultural supernatant,
cytoplasmic or membrane fraction served as controls. Selenite
concentrations in the reaction mixture were measured
by HPLC-HG-AFS (Beijing Titan Instruments Co., Ltd.,
Scanning electron microscopy (SEM)
Strain ES2-5 was grown in TSB supplemented without
or with 1.0 mM sodium selenite at 28 °C, 160 rpm. After
3 days of cultivation, cells were centrifuged (6000 rpm,
10 min, 4 °C) and scanning electron microscopic
observation was performed on the processed samples.
Samples processing involves washing, fixing and drying of
cells. Harvested cells were washed thrice with phosphate
buffer saline (PBS, pH 8.0). Fixation was conducted with
2.5 % glutaraldehyde (24 h, 4 °C). Cells were washed again
with PBS. Fixed cells were dehydrated through a series of
alcohol dehydration steps (30, 50, 70, 80, 90 and 100 %)
and finally freeze dried and sputter coated. The samples
were then viewed using SEM (JSM-6390 JEOL JAPAN).
Samples collected from the culture without addition of
selenite were regarded as controls.
Transmission electron microscopy (TEM) and SeNP analysis
with energy dispersive X‑ray (EDX)
To obtain ultra-thin sections for TEM and EDX analysis,
harvested cells through above-mentioned method were
fixed using 2 % v/v glutaraldehyde in 0.05 M sodium
phosphate buffer (pH 7.2) for 24 h and were then rinsed three
times in 0.15 M sodium cacodylated buffer (pH 7.2) for
2 h. The specimens were dehydrated in graded series of
ethanol (70, 96 and 100 %) transferred to propylene oxide
and embedded in Epon according to standard procedures.
The sections, approximately 80 nm thick, were cut with an
ultrathin E (Reichert Jung) microtome and collected on
copper grids with Formvar supporting membranes. The
sections were stained with uranyl acetate and lead citrate
and then TEM-EDX (JEM2100F JAPAN) was performed.
EDX: electron dispersion spectroscopy; MIC: minimal inhibitory
concentration; SEM: scanning electron microscopy; SeNPs: selenium nanoparticles; TEM:
transmission electron microscopy.
SZ and GW designed the experiments. YT and RY conducted the experiments.
DW and RW assisted to the GSH determination. SZ and YT analyzed the results
and wrote the manuscript. GW reviewed and revised the manuscript. All
authors have read and approved the final manuscript.
We thank Dr. Qin at the Electronic Microscope Center of Huazhong
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the
This work was supported by the National Natural Science Foundation of China
(31470227) and the fund of the Tobacco Company of Enshi, Hubei Province, P.
1. Hatfield DL , Berry MJ , Gladyshev VN . Selenium: Its molecular biology and role in human health . 3rd ed. London: Springer science+business media; 2012 .
2. Soboh B , Pinske C , Kuhns M , Waclawek M , Ihling C , Trchounian K , Trchounian A , Sinz A , Sawers G. The respiratory molybdoselenoprotein formate dehydrogenases of Escherichia coli have hydrogen:benzyl viologen oxidoreductase activity . BMC Microbiol . 2011 ; 11 : 173 .
3. Shaw FL , Mulholland F , Gall GL , Porcelli I , Hart DJ , Pearson BM , Van Vliet AHM. Selenium-dependent biogenesis of formate dehydrogenase in Campylobacter jejuni is controlled by the fdhTU accessory genes . J Bacteriol . 2012 ; 194 : 3814 - 23 .
4. Nancharaiah YV , Lens PNL . Ecology and biotechnology of seleniumrespiring bacteria . Microbiol Mol Biol Rev . 2015 ; 79 : 61 - 80 .
5. Winkel LH , Johnson CA , Lenz M , Grundl T , Leupin OX , Amini M , Charlet L . Environmental selenium research: from microscopic processes to global understanding . Environ Sci Technol . 2012 ; 46 : 571 - 9 .
6. Combs JF Jr. Selenium in global food systems . Br J Nutr . 2001 ; 85 : 517 - 47 .
7. Dowdle PR , Oremland RS . Microbial oxidation of elemental selenium in soils lurries and bacterial cultures . Environ Sci Technol . 1998 ; 32 : 3749 - 55 .
8. Stolz JF , Basu P , Santini JM , Oremland RS . Arsenic and selenium in microbial metabolism . Annu Rev Microbiol . 2006 ; 60 : 107 - 30 .
9. Kessi J , Ramuz M , Wehrli E , Spycher M , Bachofen R . Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum . Appl Environ Microbiol . 1999 ; 65 : 4734 - 40 .
10. Oremland RS , Herbel MJ , Blum JS , Langley S , Beveridge TJ , Ajayan PM , Sutto T , Ellis AV , Curran S. Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria . Appl Environ Microbiol . 2004 ; 70 : 52 - 60 .
11. Kessi J . Enzymic systems proposed to be involved in the dissimilatory reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus . Microbiology . 2006 ; 152 : 731 - 43 .
12. Hunter WJ , Kuykendall LD . Reduction of selenite to elemental red selenium by Rhizobium sp . strain B1 . Curr Microbiol . 2007 ; 55 : 344 - 9 .
13. Bajaj M , Schmidt S , Winter J . Formation of Se (0) Nanoparticles by Duganella sp . and Agrobacterium sp. isolated from Se-laden soil of North-East Punjab, India . Microb Cell Fact . 2012 ; 11 : 115 - 20 .
14. Lampis S , Zonaro E , Bertolini C , Cecconi D , Monti F , Micaroni M , J.Turner R , S. Butler C , Vallini G. Selenite biotransformation and detoxification by Stenotrophomonas maltophilia SeITE02: novel clues on the route to bacterial biogenesis of selenium nanoparticles . J Haz Mat . 2016 . http://dx.doi. org/10.1016/j.jhazmat. 2016 . 02 .035.
15. Forootanfara H , Mahboubeh A , Maryam N , Mitra M , Bagher A , Ahmad S. Antioxidant and cytotoxic effect of biologically synthesized selenium nanoparticles in comparison to selenium dioxide . J Trace Elem Med Biol . 2014 ; 28 ( 1 ): 75 - 9 .
16. Hariharan H , Al-harbi N , Karuppiah P , Rajaram S . Microbial synthesis of selenium nanocomposite using Saccharomyces cerevisiae and its antimicrobial activity against pathogens causing nosocomial infection . Chalcogenide Lett . 2012 ; 9 : 509 - 15 .
17. Yang F , Tang Q , Zhong X , Bai Y , Chen T , Zhang Y , Li Y , Zhang X . Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles . Int J Nanomedicine . 2012 ; 7 : 835 - 44 .
18. Yazdi MZ , Mahdavi M , Varastehmoradi B , Faramarzi MA , Shahverdi AR . The immunostimulatory effect of biogenic selenium nanoparticles on the 4T1 breast cancer model: an in vivo study . Biol Trace Elem Res . 2012 ; 149 : 22 - 8 .
19. Wang T , Yang L , Zhang B , Liu J . Extracellular biosynthesis and transformation of selenium nanoparticles and application in H2O2 biosensor . Colloids Surf. B . 2010 ; 80 : 94 - 102 .
20. Zhang J , Zhang S , Xu J , Chen H. A new method for the synthesis of selenium nanoparticles and the application to construction of H2O2 biosensor . Chin Chem Lett. 2004 ; 15 : 1345 - 8 .
21. Losi M , Frankenberger W. Reduction of selenium oxyanions by Enterobacter cloacae SLD1a-1: isolation and growth of the bacterium and its expulsion of selenium particles . Appl Environ Microbiol . 1997 ; 63 : 3079 - 84 .
22. Jiang S , Cuong T , Lee J , Duong H , Han S , Hur H . Mercury capture into biogenic amorphous selenium nanospheres produced by mercury resistant Shewanella putrefaciens . Chemosphere . 2012 ; 87 : 621 - 4 .
23. Fellowes J , Pattrick R , Green D , Dent A , Lloyd J , Pearce C . Use of biogenic and abiotic elemental selenium nanospheres to sequester elemental mercury released from mercury contaminated museum specimens . J Hazard Mater . 2011 ; 189 : 660 - 9 .
24. DeMoll-Decker H , Macy JM . The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium . Arch Microbiol . 1993 ; 160 : 241 - 7 .
25. Hunter WJ , Kuykendall LD . Identification and characterization of an Aeromonas salmonicida (syn Haemophilus piscium) strain that reduces selenite to elemental red selenium . Curr Microbiol . 2006 ; 52 : 305 - 9 .
26. Li DB , Cheng YY , Wu C , Li WW , Li N , Yang ZC , Tong ZH , Yu HQ . Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm . Sci Rep . 2014 ; 4 : 3755 .
27. Antonioli P , Lampis S , Chesini I , Vallini G , Rinalducci S , Zolla L , Righetti PG . Stenotrophomonas maltophilia SeITE02, a new bacterial strain suitable for bioremediation of selenite-contaminated environmental matrices . Appl Environ Microbiol . 2007 ; 73 : 6854 - 63 .
28. Hunter WJ . Pseudomonas seleniipraecipitans proteins potentially involved in selenite reduction . Curr Microbiol . 2014 ; 69 : 69 - 74 .
29. Zheng S , Su J , Wang L , Yao R , Wang D , Deng Y , Wang R , Wang G , Rensing C . Selenite reduction by the obligate aerobic bacterium Comamonas testosteroni S44 isolated from a metal-contaminated soil . BMC Microbiol . 2014 ; 14 : 204 - 16 .
30. Lampis S , Zonaro E , Bertolini C , Bernardi P , Butler CS , Vallini G . Delayed formation of zero-valent selenium nanoparticles by Bacillus mycoides SelTE01 as a consequence of selenite reduction under aerobic conditions . Microb Cell Fact . 2014 ; 13 : 106 - 11 .
31. Hunter WJ . A Rhizobium selenitireducens protein showing selenite reductase activity . Curr Microbiol . 2014 ; 68 : 311 - 6 .
32. Hockin SL , Gadd GM . Linked redox precipitation of sulfur and selenium under anaerobic conditions by sulfate-reducing bacterial biofilms . Appl Environ Microbiol . 2003 ; 69 : 7063 - 72 .
33. Painter EP . The chemistry and toxicity of selenium compounds with special reference to the selenium problem . Chem Rev . 1941 ; 28 : 179 - 213 .
34. Kessi J , Hanselmann KM . Similarities between the abiotic reduction of selenite with glutathione and the dissimilatory reaction mediated by Rhodospirillum rubrum and Escherichia coli . J Biol Chem . 2004 ; 279 : 50662 - 9 .
35. Afkar E , Lisak J , Saltikov C , Basu P , Oremland RS , Stolz JF . The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10 . FEMS Microbiol Lett . 2003 ; 226 : 107 - 12 .
36. Yanke LJ , Bryant RD , Laishley EJ . Hydrogenase (I) of Clostridium pasteurianum functions a novel selenite reductase . Anaerobe . 1995 ; 1 : 61 - 7 .
37. Tomei FA , Barton LL , Lemanski CL , Zocco TG , Fink NH , Sillerud LO . Transformation of selenate and selenite to elemental selenium by Desulfovibrio desulfuricans . J Ind Microbiol . 1995 ; 14 : 329 - 36 .
38. Ahmad MS , Yasser MM , Sholkamy EN , Ali AM , Mehanni MM . Anticancer activity of biostabilized selenium nanorods synthesized by streptomyces bikiniensis strain Ess_amA- 1 . Int J Nanomed. 2015 ; 10 : 3389 - 401 .
39. Ramya S , Shanmugasundaram T , Balagurunathan R . Biomedical potential of actinobacterially synthesized selenium nanoparticles with special reference to anti-biofilm, anti-oxidant, wound healing, cytotoxic and antiviral activities . J Trace Elem Med Bio . 2015 ; 32 : 30 - 9 .
40. Vetchinkina E , Loshchinina E , Kursky V , Nikitina V . Reduction of organic and inorganic selenium compounds by the edible medicinal Basidiomycete Lentinula edodes and the accumulation of elemental selenium nanoparticles in its mycelium . J Microbiol . 2013 ; 51 : 829 - 35 .
41. Venceslau SS , Stockdreher Y , Dahl C , Pereira IAC . The “bacterial heterodisulfide” dsrc is a key protein in dissimilatory sulfur metabolism . BBABioenergetics . 2014 ; 1837 : 1148 - 64 .
42. Fahey RC . Glutathione analogs in prokaryotes . BBA-Bioenergetics . 1830 ; 2013 : 3182 - 98 .
43. Yao R , Wang R , Wang D , Su J , Zheng S , Wang G . Paenibacillus selenitireducens sp. nov., a selenite-reducing bacterium isolated from a selenium mineral soil . Int J Syst Evol Microbiol . 2014 ; 64 : 805 - 11 .
44. Xiang W , Wang G , Wang Y , Yao R , Zhang F , Wang R , Wang D , Zheng S. Paenibacillus selenii sp. nov., isolated from selenium mineral soil . Int J Syst Evol Microbiol . 2014 ; 64 : 2662 - 7 .
45. Li X , Kot W , Wang D , Zheng S , Wang G , Hansen LH , Rensing C . Draft genome sequence of Se(IV)-reducing bacterium Pseudomonas migulae ES3-33 . Gen Announc. 2015 ; 3 ( 3 ): e00406 - 15 .
46. Dong XZ , Cai MY . Determinative manual for routine bacteriology . Beijing: Scientific Press; 2001 .
47. Kim OS , Cho YJ , Lee K , Yoon SH , Kim M , Na H , Park SC , Jeon YS , Lee JH . Introducing EzTaxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species . Int J Syst Evol Microbiol . 2012 ; 62 : 716 - 21 .
48. Thompson JD , Gibson TJ , Plewniak F , Jeanmougin F , Higgins DG . The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools . Nucleic Acids Res . 1997 ; 25 : 4876 - 82 .
49. Tamura K , Peterson D , Peterson N , Stecher G , Nei M , Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods . Mol Biol Evol . 2011 ; 28 : 2731 - 9 .
50. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences . J Mol Evol . 1980 ; 16 : 111 - 20 .
51. Felsenstein J . Confidence limits on phylogenies: an approach using the bootstrap . Evolution . 1985 ; 39 : 783 - 91 .
52. Lewicki K , Marchand S , Matoub L , Lulek J , Coulon J , Leroy P . Development of a fluorescence-based microtiter plate method for the measurement of glutathione in yeast . Talanta . 2006 ; 70 ( 4 ): 876 - 82 .
53. Van Wilgenburg MG , Werkman EM , Van Gorkom WH , Soons JB . Criticism of the use of Coomassie Brilliant Blue G-250 for the quantitative determination of proteins . J Clin Chem Clin Biochem . 1981 ; 19 : 301 - 4 .
54. Li J , Wang Q , Zhang SZ , Qin D , Wang GJ . Phylogenetic and genome analyses of antimony-oxidizing bacteria isolated from antimony mined soil . Int Biodeter Biodegr . 2013 ; 76 : 76 - 80 .